CA2951558A1

CA2951558A1 - Methods, devices, and systems for sample analysis

Info

Publication number: CA2951558A1
Application number: CA2951558A
Authority: CA
Inventors: Daniel Young; Elizabeth Holmes
Original assignee: Theranos Inc
Current assignee: Labrador Diagnostics LLC
Priority date: 2014-06-11
Filing date: 2015-06-10
Publication date: 2015-12-17
Also published as: EP3155420A2; MX2016016234A; EP3155420A4; WO2015191716A2; WO2015191716A3

Abstract

Methods, devices, and systems for analyzing biological samples for the presence of an analyte by an initial assay, and, contingent upon the results of the initial assay, by a subsequent assay are provided. A sample may be diluted. A portion of a sample may be retained for use in such a subsequent assay. Initial assay results may be used to determine, for example, one or more of: whether or not a subsequent assay is performed; which subsequent assay is performed; the method of performing a subsequent assay; the order of performance of a sequence of subsequent assays; the steps, or order of steps, performed in a subsequent assay; the timing of the performance of a subsequent assay; the choice of a reagent used in a subsequent assay; the detection method used in a subsequent assay; and other particulars of assays may be contingent on the results of a prior assay.

Description

METHODS, DEVICES, AND SYSTEMS FOR SAMPLE ANALYSIS
BACKGROUND
[00011 Rapid and accurate detection and identification of analytes and pathogens in biological samples is useful in the diaposis of diseases and disorders in subjects. However, such analysis may be a mufti-step process, and it may be advantageous, or may be required, that different assays be performed in sequence. Assay devices and systems may be configured for one, but not all, of the assays required for a complete analysis.
100021 Accordingly, improved methods, devices, and systems for analysis of biological samples are required.
INCORPORATION BY REFERENCE
[0003] All publications, patents, and patent applications mentioned in this specification are herein incomorated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.
SUMMARY
[0004] Methods, devices, and systems for the analysis of biological samples are provided which may be configured to perform multiple assays on a sample or samples, comprising an initial assay or multiple initial assays, and further comprising a subsequent assay or subsequent assays, the performance of, and/or order of performance of, such subsequent assay or assays being contingent on the results of such initial assay(s). Thus, a subsequent assay or such subsequent assays may be performed in a contingent manner, wherein the performance of a subsequent assay depends on the results of one or more prior assay. In embodiments, whether or not a subsequent assay is performed at all may be contingent on the results of a prior assay. In embodiments, the order of performance of subsequent assays, or of steps in a subsequent assay, may be contingent on the results of a prior assay. In embodiments, the choice of a subsequent assay, from among a plurality of possible subsequent assays, may be contingent on the results of a prior assay.
In embodiments, the method of performing a subsequent assay may be contingent on the results of a prior assay. In embodiments, the timing of the performance of a subsequent assay may be contingent on the results of a prior assay. In embodiments, the choice of a reagent used in a subsequent assay may be contingent on the results of a prior assay. In embodiments, the choke of a method of detection used in a subsequent assay may be contingent on the results of a prior assay. In embodiments, the choice of whether or not to use a second biological sample in a subsequent assay may be contingent on the results of a prior assay in which a first biological sample was assayed. In embodiments, the choice of whether or not to obtain a second biological sample may be contingent on the results of a prior assay of a first biological sample. In embodiments, the choice of whether or not to obtain a second biological sample for use in a subsequent assay may be contingent on the results of a prior assay of a first biological sample.
[00051 in embodiments, analysis of a biological sample may comprise assaying the sample, or a portion of the sample, for the presence of an analyte. An analyte is the subject of an analysis. An analyte may be a natural constituent of a biological sample, or may be an element, compound, material, or cell not normally found in a biological sample that may be the subject of analysis. An analyte may comprise a chemical coinpound, e.g., a small molecule, a protein, a nucleic acid, or other compound, present in the sample.
An analyte may comprise a physical or chemical characteristic of a sample, or of a portion of the sample. An analyte may comprise a marker or physical or chemical characteristic of a cell or virus in a sample, or in a portion of the sample. An analyte may comprise a cell or virus, or portion thereof, in a sample, or in a portion of the sample.
100061 Accordingly, methods, devices, and systems are disclosed herein. In embodiments, a method of testing a biological sample comprises:
performing an initial assay for the presence of an analyte in said biological sample, whereby an initial result is obtained, wherein said initial assay may provide a negative result indicating that the presence of said analyte is not detected in the biological sample, or may provide a positive result indicating that the presence of the analyte is detected in the biological sample; and determining further testing of said biological sample contingent on said initial result, wherein if the initial result is negative, then no subsequent assay is performed on said biological sample; and wherein if the initial result is positive, then a subsequent assay is performed on said biological sample.
[00071 In embodiments, a method of testing a biological sample comprises:
performing an initial assay in a device for an analyte in said biological sample, whereby an initial result is obtained, wherein said initial assay may provide a negative result indicating that the presence of said analyte is not detected, or is detected at a normal level, in

-2-the biological sample, or may provide a positive result indicating that the presence of the analyte is detected, or is detected at an abnormal level, in the biological sample; and determining further testing of said biological sample contingent on said initial result, wherein if the initial result is negative, then no subsequent assay is performed on said biological sample; and wherein if the initial result is positive, then a subsequent assay is performed in said device on said biological sample.
[00081 In embodiments, such subsequent assay may be a type of assay selected from the group consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays. In embodiments, the subsequent assay may comprise an assay of a different type than the initial assay type. In embodiments, such subsequent assays may comprise assays that are more sensitive for the detection of said analyte than said initial assay. In embodiments, the analyte to be detected by such subsequent assays may comprise a different analyte than the analyte to be detected by said initial assay.
[00091 In embodiments, such an initial assay may comprise the use of a detector to obtain said initial result, wherein said detector is selected from an optical detector, a pH
detector, an electrochemical detector, a temperature sensor, an ion-sensitive electrode, a radiation detector, and other detectors. In embodiments, such a subsequent assay may comprise the use of a detector to obtain a further result, wherein said detector is selected from an optical detector, a pH detector, an electrochemical detector, a temperature sensor, an ion-sensitive electrode, a radiation detector, and other detectors.
[001.01 Methods disclosed herein comprise protocols for performance of a sequence of assays. In embodiments, protocols may include contingent assays, whose performance is dependent on the results of prior assays, wherein a result of a first assay, if it meets a criterion, triggers, alters, or prevents the performance of a subsequent assay. In embodiments, such a subsequent assay is automatically performed if the results of an initial assay, or other prior assays, neet the criterion or criteria. In embodiments, such a subsequent assay may not be performed in the absence of prior results which meet the criterion. In embodiments, the performance of a subsequent assay may be contingent on two, or three, or more criteria.
Protocols include contingent sequences of assays, where the performance of a subsequent assay is determined by the outcome of an assay performed prior to the performance of the subsequent assay. In embodiments, a protocol including one or more contingent sequences of assays may be requested at the time the assays are ordered. In embodiments, a protocol including one or more contingent sequences of assays may be determined at the time the assays are ordered. In embodiments, a protocol including one or more contingent sequences

-3-of assays may be requested at the time the assays are ordered, and the particulars of the sequence, and of the contingent steps, may be determined at a time after the time the assays are ordered.
[00111 In embodiments, a protocol including one or more assays, whose performance or sequence of performance is contingent on the result of an initial assay, may include instructions, or a protocol, for the acquisition of a biological sample. In embodiments, such a protocol may direct or require that a biological sample of sufficient volume or amount be acquired in order to provide sufficient biological sample for the performance of a contingent assay, if the contingent assay is needed. In embodiments, such a protocol may direct or require that a biological sample be divided into aliquots for the performance of a contingent assay, if the contingent assay is needed. In embodiments, such a protocol may direct or require that a biological sample be diluted, in order to provide sufficient biological sample for the performance of a contingent assay, if the contingent assay is needed. In embodiments, such a protocol may direct or require that a biological sample, or portion or dilution thereof, be retained for use in the performance of a contingent assay, if the contingent assay is needed.
[0012] In embodiments, an assay, and a protocol regarding such an assay, may utilize a diluted sample (e.g., a diluted biological sample). In embodiments, e.g., where the performance or sequence of performance of an assay is contingent on the result of an initial assay, an assay may include instructions, or a protocol, for the dilution of a biological sample.
In embodiments, a protocol including one or more assays, whose performance or sequence of performance is contingent on the result of an initial assay, may include instructions, or a protocol, for the dilution of a biological sample. In embodiments, such dilution may provide a diluted sample diluted by at least about 10-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 30-fold as compared to the original sample;
or may provide a diluted sample diluted by at least about 50-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 100-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 1000-fold, or more, as compared to the original sample. In embodiments, such a protocol may direct or require that a biological sample, or portion thereof, including diluted portions thereof, be retained for the performance of a contingent assay, if the contingent assay is needed.
[0013] In embodiments, a contingent assay is automatically run if the contingent assay is indicated on the test order or other instruction, regardless of the results from any initial assay. Such contingent assay(s) may be run concurrently with or even before results are available from any initial assay(s). Although such alternative protocols may result in

-4-additional usage of reagent(s), diluent(s), and/ or other materials, the workflow of parallel, concurrent, or continuous processing may be beneficial in some situations, such as but not limited excess system availability while processing the initial assays or waiting for initial assays to complete.
[0014] In embodiments, an initial assay may comprise a less sensitive assay, and a subsequent assay may comprise a more sensitive assay; in embodiments, performance of a subsequent assay may be contingent on the results of the initial assay. For example, an initial assay may comprise a nucleic acid assay performed under a first condition, and a subsequent assay may comprise a nucleic acid assay performed under a second condition, where the second condition comprises more stringent nucleic acid assay conditions than the first condition (e.g., the second condition comprises a higher temperature than the first condition, or the second condition comprises a lower ionic strength than the first condition, or the second condition comprises a denaturing agent (such as formamide) not present, or present at a lower concentration, in the first condition). In embodiments, an initial assay that comprises a nucleic acid assay may be perfomied under moderately stringent conditions, and a subsequent assay may comprise a nucleic acid assay performed under high stringent conditions.
[0015] In embodiments, an initial assay may comprise a first type of assay, and a subsequent assay, contingent on the results of the initial assay may comprise a second type of assay; for example, an initial assay may comprise an antibody-based assay, and a subsequent assay may comprise a nucleic acid assay.
[0016] Accordingly, Applicant further discloses methods of testing a biological sample in a device, comprising: performing an initial assay in said device for an analyte in said biological sample, whereby an initial result is obtained, wherein said initial assay may provide a negative result indicating that the presence of said analyte is not detected, or is detected at a normal level, in the biological sample, or may provide a positive result indicating that the presence of the analyte is detected, or is detected at an abnormal level, in the biological sample; performing further testing of said biological sample, wherein a subsequent assay is performed in said device on said biological sample regardless of the results of said initial assay; and reporting the results of said subsequent assay of said biological sample contingent upon the results of said initial assay.
[0017] In embodiments, the methods further comprising reporting the results of said initial assay. In embodiments, the results of said subsequent assay are not reported if said initial assay provides a negative result, and wherein the results of said subsequent assay are

-5-

6 reported if said initial assay provides a positive result. In embodiments, the results of said subsequent assay are not reported if said initial assay provides a positive result, and wherein the results of said subsequent assay are reported if said initial assay provides a negative result.
100181 In embodiments, the subsequent assay is an assay for the same analyte as said initial assay, and the subsequent assay is a more sensitive assay than said initial assay.
[0019] In embodiments, the biological sample upon which the subsequent assay is performed is obtained before the results of said initial assay are obtained.
In embodiments, the initial assay and the subsequent assay are performed on portions of the same biological sample. In embodiments, at least one of said portions of said biological sample is a diluted portion of the biological sample. In embodiments, the subsequent assay comprises an assay of a type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays. In embodiments, the subsequent assay comprises a different type of assay than the initial assay. In embodiments, the analyte to be detected by said subsequent assay comprises a different analyte than the analyte detected by said initial assay. In embodiments, the initial assay comprises measurement of an analyte, and said subsequent assay comprises a crometric assay.
[0020] The methods, compositions, devices, and systems provide rapid tests, which require only small biological samples, and thus provide advantages over other methods, compositions, assays, devices, and systems. Devices and systems disclosed herein are configured to perform such rapid assays which require only small amounts of sample, such as only small amounts of sample, urine, sputum, tears, material obtained from. a nasal swab, throat swab, cheek swab, or other biological sample. Accordingly, the methods, devices, and systems provide rapid tests, which require only small biological samples, and thus provide advantages over other methods, compositions, assays, devices, and systems.
100211 In embodiments, a biological sample may comprise a sample selected from blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
100221 In embodiments, such an initial assay and such a subsequent assay may be performed on different biological samples. In embodiments, the biological sample upon which the subsequent assay is performed may be obtained before the results of said initial assay are obtained. In embodiments, the biological sample upon which the subsequent assay may be performed is obtained after the results of said initial assay are obtained.
[0023] Accordingly, Applicant discloses herein methods in which a subsequent assay is performed within a short amount of time from the time at which the biological sample tested by the subsequent assay was accepted within the device. In embodiments, the initial assay and the subsequent assay are each performed on at least a portion of the same biological sample; in such embodiments, the subsequent assay is performed within a short amount of time from the time at which an initial biological sample was accepted within the device.
[0024] In embodiments, a subsequent assay is performed if an initial assay is performed. In embodiments of methods disclosed herein, if the result of said initial assay performed in a device is positive, then a subsequent assay is performed on a biological sample in the same device within a short amount of time from the time of accepting a sample within said device, wherein said short am.ount of time is a time prior to the performance of the subsequent assay. In embodiments, the same sample is used for the performance of both the initial assay and the subsequent assay, or portions of the same sample are used for the performance of both the initial assay and the subsequent assay. Such a short amount of time may be, for example, a short amount of time consisting of about 3 hours or less; about 2 hours or less; about 1 hour or less; about 50 minutes or less; about 45 minutes or less; about 40 minutes or less; about 35 minutes or less; about 30 minutes or less; about 25 minutes or less; about 20 minutes or less; about 15 minutes or less; about 10 minutes or less; about 5 minutes or less; about 4 minutes or less; about 3 minutes or less; about 2 minutes or less; and about 1 minute or less.
[0025] In embodiments, the biological sample upon which the subsequent assay is performed is obtained after the results of said initial assay are obtained; in such embodiments, the subsequent assay is performed within a short amount of time from the time at which a further biological sample was accepted within the device. Such a further biological sample may be obtained following the time at which an initial biological sample is obtained. In embodiments, a further biological sample is obtained from a subject during a single session in which biological samples are obtained. In embodiments, a further biological sample is obtained from a subject during a second, or subsequent, session following the session during which the initial biological sample was obtained. In embodiments, a second, or subsequent, session may follow the session during which the initial biological sample was obtained by a

-7-time period of about 10 minutes or less; or about 20 minutes or less; or about 30 minutes or less; or about 40 minutes or less; or about 50 minutes or less; or about one hour or less; or about two hours or less; or about three hours or less; or other time period.
[0026] The assays and methods disclosed herein may be performed on a device, or on a system, for processing a sample. The assays and methods disclosed herein can be readily incorporated into and used in an automated assay device, and in an automated assay system.
In embodiments, a device as disclosed herein may be suitable for the detection, identification, or measurement of an analyte or characteristic in a biological sample. In embodiments, a device as disclosed herein may be an automated assay device, suitable for automated detection, identification, or measurement of an analyte or characteristic in a biological sample. Such automated detection, identification, or measurement of an analyte or characteristic in a biological sample may include, for example, detection, identification, or measurement of an analyte or characteristic in a biological sample according to a protocol performed by the automated assay device; a processor may be used in the performance of assays according to such a protocol. The automated detection, identification, or measurement of a characteristic in a biological sample may include the automated detection, identification, or measurement of a characteristic of a cell in the biological sample, such as may be detected, identified, or measured, e.g., by a cytometric assay, and may include the automated detection, identification, or measurement of a morphological characteristic of a cell in the biological sample.
[0027] In embodiments, a device for assaying a sample for the presence of analyte in a sample may comprise: a fluid handling system for transporting at least a portion of a biological sample; and a detector effective to detect or measure an analyte or characteristic.
In embodiments, a detector may comprise one or more of an optical detector, a pH sensor, an electrochemical detector, a radiation detector, a temperature sensor, and other sensors. An optical detector may comprise one or more of a camera, a photomultiplier, a photodiode, a spectrophotometer, and other optical elements. In embodiments, a detector may comprise an imaging device or imaging system, and may include optical elements such as lenses, filters, mirrors, gratings, polarizers, and other optical elements. In embodiments, a device for assaying a sample for the presence of analyte in a sample may comprise a system for transporting at least a portion of a satnple. In embodiments, a device for assaying a sample for the presence of analyte in a sample may comprise a system for transporting a reagent. In embodiments, a system for transporting at least a portion of a sample, or for transporting a reagent, may comprise a fluid handling system. In embodiments, a fluid handling system of a

-8-device for assaying a sample for the presence of analyte in a sample may be configured to transport at least a portion of a biological sample and also be configured to transport a reagent. In embodiments, a device for assaying a sample for the presence of analyte in a sample may be configured to dilute at least a portion of a sample with a reagent. In embodiments, a device for assaying a sample for the presence of analyte in a sample may be configured to mix a reagent with at least a portion of a sample.
[0028] In embodiments, a device comprising a fluid handling system for transporting at least a portion of a biological sample may comprise a means for contacting a biological sample; or may comprise a means for accepting a biological sample; or may comprise means for storing a biological sample. In embodiments, a biological sample may be provided by a cartridge. In embodiments, reagents for assays to be performed on a biological sample may be provided by a cartridge. In embodiments, all reagents required for the assays to be performed on a biological sample may be provided by a cartridge. In embodiments, consumables, including pipette tips, mixing vessels, cuvettes, and other containers, implements, and materials, may be provided by a cartridge; in embodiments, all such materials required for the assays to be performed on a biological sample may be provided by a cartridge. In embodiments, a cartridge may hold a container in which a biological sample is contained. A container configured to hold a biological sample may be configured to store a biological sample, and may be configured to allow access to said biological sample by a fluid handling system. Access by a fluid handling system to a biological sample may be effective to allow transport of at least a portion of the biological sample; to allow mixing of the biological sample; to allow addition of a reagent to at least a portion of the biological sample;
or to allow division of said biological sample into two or more portions. In embodiments, a cartridge may hold a biological sample, and reagents for use in the assays to be performed on the sample; in embodiments, a biological sample and all reagents required for the assays to be performed on a biological sample may be provided by a single cartridge. In embodiments, a cartridge may hold a biological sample, reagents, and consumables, including pipette tips, mixing vessels, cuvettes, and other containers, implements, and materials for use in the assays to be perfomied on the sample; in embodiments, a single cartridge may carry a biological sample, and all reagents and all consumables required for the assays to be performed on a biological sample.
[0029] In embodiments, systems are provided which include devices configured to detect the presence of an analyte in a biological sample. In embodiments, systems are provided which include devices configured to detect the presence of an analyte in a biological

-9-sample, and a cartridge containing reagents for assays. In embodiments, systems are provided which include devices configured to detect the presence of an analyte in a biological sample, and a cartridge containing reagents for assays, and consumable items for assays. In embodiments, a device or system may further comprise a communication assembly, which may comprise a display element and/or a communication element effective to report the results of said detection and/or measurement. In embodiments, a communication assembly, such as a display element and/or communication element, may be suitable for two-way communication. In embodiments, a communications assembly may be configured to communicate data obtained from assaying a sample. In embodiments, a device for assaying a sample for the presence of analyte in a sample may comprise other elements and assemblies, including, without limitation, a heating assembly, a cooling assembly, a sonicator, and other elements and assemblies.
[0030] For example, systems as disclosed herein may include a communication assembly for transmitting or receiving a protocol based on the analyte to be detected or based on other analytes to be detected by the device or system. In embodiments, an assay protocol may be changed based on results previously obtained from a sample from a subject, or based on results previously obtained from a different sample from the subject. In embodiments, a communication assembly may comprise a channel for communicating information from said device to a computer, said wherein said channel is selected from a computer network, a telephone network, a metal communication link, an optical communication link, and a wireless communication link. In embodiments, systems as disclosed herein may transmit signals to a central location, or to an end user, and may include a communication assembly for transmitting such signals. Systems as disclosed herein may be configured for updating a protocol as needed or on a regular basis.
100311 Accordingly, Applicant discloses devices configured to measure an analyte in a biological sample according to a method disclosed herein. Devices configured to measure analytes in a biological sample according to a method disclosed herein may be configured to measure analytes from a biological sample that comprises no more than about 1000 IA, of sample, or no more than about 500 td, of sample, no more than about 250 p.L.
of sample, or no more than about 200 p.L of sample, or no more than about 150 1i.1, of sample, or no more than about 1001.tL of sample, or no more than about 75 RI, of sample, or no more than about 50 tiL of sample, or no more than about 40 L of sample, or no more than about 30 iu.L of sample, or no more than about 25 1.i1. of sample, or no more than about 20 p.L. of sample, or no more than about 15 I, of sample, or no more than about 10 p.L of sample, or no more than

-10-about 5 j.tL of sample, or no more than about 4 IA, of sample, or no more than about 3 AI, of sample, or no more than about 2 !IL of sample, or no more than about 1 j.tL of sample, or less.
As used herein, the phrase "pL of sample" refers to the volume, in pi., of a sample;
accordingly, a biological sample that comprises no more than about 100 j.t.L
of sample has a volume of no more than about 100 lit. Such devices (e.g., devices configured to measure an analyte in a biological sample according to a method disclosed herein) may be configured to measure an analyte in a biological sample in less than about one hour, or, in embodiments, in less than about 40 minutes, or in less than about 30 minutes.
[00321 Devices disclosed herein may be configured to perform an assay for the measurement of a first analyte and also to perform an assay for the measurement of a second analyte in the biological sample. In embodiments, performance of an assay for the measurement of a second analyte in the biological sample may be contingent on the results of an assay for the measurement of a first analyte in the biological sample. For example, devices disclosed herein may be configured to perform an assay for the measurement of an analyte and also to perform an assay comprising the measurement of a morphological characteristic of a blood cell in the blood sample. Devices disclosed herein may be configured to perform an assay for the measurement of a first analyte and also to perform an assay comprising the measurement of another blood analyte, e.g., a vitamin, a hormone, a drug or metabolite of a drug, or other analyte. Such devices may be configured wherein the assays, or the order of performance of assays, that are performed by said device may be altered by communication with another device.
[0033] Applicant also discloses systems comprising a device as disclosed herein. In embodiments, the system comprises a device that is configured to perform an assay for the measurement of a first analyte and also to perform an assay for the measurement of another analyte in the blood sample. In embodiments, the system comprises a device that is configured to perform an. assay for the measurement of an analyte and also to perform an assay for the measurement of a morphological characteristic of a blood cell in the blood sample. In embodiments of such a system, assays, or the order of performance of assays, that are performed by said device may be altered by communication with another device.
[00341 This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter.

-11-BRIEF DESCRIPTION OF THE DRAWINGS
[0035] Fig. 1 shows representative images of blood cells from a sample of whole blood, illustrating various types of images that may be obtained using different imaging techniques and dyes.
10036j Fig. 2 shows a representative composite image of several cell-types in whole blood, including images of a monocyte, a lymphocyte, an eosinophil, and a neutrophil.
[0037] Fig. 3A shows a plots of monocytes identified and quantified by the cytometric assays described herein.
[0038] Fig. 3B shows a plots of ba,sophils identified and quantified by the cytometric assays described herein.
[0039] Fig. 3C shows a plots of lymphocytes identified and quantified by the cytomettic assays described herein.
[0040] Fig. 3D shows a plots of neutrophils and eosinophils identified and quantified by the cytometric assays described herein.
[00411 Fig. 4A shows a plot demonstrating that cytometric methods as disclosed herein identify different cell types consistent with such identification by other methods. Fig.
4A shows plots of numbers of white blood cells ("WBCs") from blood samples.
[0042] Fig. 413 shows a plot demonstrating that cytometric methods as disclosed herein identify different cell types consistent with such identification by other methods. Fig.
4B shows plots of numbers of red blood cells ("RBCs) from blood samples.
[0043] Fig. 4C shows a plot demonstrating that cytometric methods as disclosed herein identify different cell types consistent with such identification by other methods. Fig.
4C shows plots of numbers of platelets from blood samples.
[0044] Fig. 4D shows a plot demonstrating that crometric methods as disclosed herein identify different cell types consistent with such identification by other methods. Fig.
4D shows plots of numbers of neutrophils from blood samples.
[0045] Fig. 4E shows a plot demonstrating that cytometric methods as disclosed herein identify different cell types consistent with such identification by other methods. Fig.
4E shows plots of numbers of monocytes from blood samples.
[0046] Fig. 4F shows a plot demonstrating that cytometric methods as disclosed herein identify different cell types consistent with such identification by other methods. Fig.
4F shows plots of numbers of lymphocytes from blood samples.

-12-[0047] Fig. 5A. shows a first side of an order sheet including lstings of test panels and of reflex tests according to the methods disclosed herein.
[0048] Fig. 5B shows a second side of an order sheet including listings of test panels and reflex tests according to the methods disclosed herein.
[00491 Fig. 6A shows a further example of an order sheet listing individual tests, test panels, test groupings, and including lists of reflex tests according to the methods disclosed herein.
[0050] Fig. 6B shows shows a listing of panel components and capped price offerings for the list shown in Fig. 6A.
[0051] Fig. 7A shows a further example of an order form listing individual tests, test panels, test groupings, and including lists of reflex tests according to methods disclosed herein.
[0052] Fig. 7B shows a listing of panel components and capped price offerings for the list shown in Fig. 7A. CPT codes are listed for the panels and panel components.
[0053] Fig. 7C shows a further listing of panel components and capped price offerings for the list shown in Fig. 7A. CPT codes are listed for the panels and panel components.
[0054] Fig. 8A shows a further example of an order form listing individual tests, test panels, test groupings, and including lists of reflex. tests according to methods disclosed herein.
[0055] Fig. 8B shows a listing of panel components and capped price offerings for the list shown in Fig. 8A. CPT codes are listed for the panels and panel components.
DETAILED DESCRIPTION
[00561 Description and disclosure of examples of reagents, assays, methods, kits, devices, and systems which may use, or be used with, methods, devices, and systems disclosed herein may be found, for example, in U.S. Patent 8,088,593; U.S.
Patent 8,380,541;
U.S. Pat. App. Ser. No. 13/769,798, filed February 18, 2013; U.S. Pat. App.
Ser. No.

13/769,779, filed February 18, 2013; U.S. Pat. App. Ser. No. 13/769,817, filed February 18, 2013; U.S. Pat. App. Ser. No. 13/769,818, filed February 18, 2013; U.S. Pat.
App. Ser. No.
13/769,820, filed February 18, 2013; U.S. Pat. App. Ser. No. 13/244,947 filed Sept. 26, 2011;
International Patent Application PCT/US2012/057155, filed September 25, 2012;
U.S.
Application Serial No. 13/244,946, filed September 26, 2011; U.S. Patent Application 13/244,949, filed September 26, 2011; U.S. Patent Application 61/805,900, filed March 27, 2013; U.S. Patent Application 62/058,632, filed October 1, 2014; U.S. Patent Application 62/011,016, filed June 11, 2014; U.S. Patent Application 61/805,900, filed March 27, 2013;
International Patent Application PCT/US2014/032071, filed March 27, 2014; and U.S.
Application Serial No. 13/945,202, filed July 18, 2013, the disclosures of all of which patents and patent applications are hereby incorporated by reference in their entireties.
DEFINITIONS
[0057] Before the present methods, devices, and systems are disclosed and described, it is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. It is also to be understood that the present disclosure provides explanatory and exemplary descriptions and examples, so that, unless otherwise indicated, the devices, systems, and methods disclosed herein are not limited to the specific embodiments described herein.
[0058] it must be noted that, as used in the specification and the appended claims, the singular forms "a," "an" and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a salt" refers to a single salt or mixtures of different salts, and the like.
[0059] In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:
[0060] Acronyms and abbreviations, such as "min" (minute), "sec" (second), and so forth, have their customaiy meanings.
[0061] As used herein, the terms "automated assay device" and "automated assay system" refer to devices and systems which, once started in operation, are capable of running substantially without human control or intervention, and may be run according to one or more protocols which govern the particulars of the operation of the device or system. automated assay devices and systems are typically configured to facilitate the collection of a sample; to prepare a sample for an assay (e.g., a clinical test); to effect a chemical reaction between a sample and one or more reagents; to produce, detect, or measure a chemical or physical characteristic or process in a sample; to observe, image, detect, identify, or measure a chemical or physical characteristic in a sample; and, in general, to perform assays and obtain assay results from a sample (e.g., a biological sample). An automated assay device may be configured to process a sample; to obtain data from a sample; to transmit data obtained from a sample; to analyze data from a sample; and to perform other operations on and with a sample (e.g., a biological sample). An automated assay device may be configured to communicate with another device, or a laboratory, or an individual affiliated with a

-14-laboratory, to analyze data obtained from a sample (e.g., a biological sample). As used herein, an automated assay device or system may be equivalently termed a sample processing device or system, a sample analysis device or system, an automatic assay device or system, an automatic sample processing device or system, an automatic sample analysis device or system, or other such terms.
[0062] As used herein, the terms "consumable" and "consumables" refer to implements and materials useful in the performance of assays, and include pipette tips, mixing vessels, cuvettes, and other containers, implements, and materials which may be used during an assay for the detection or measurement of an analyte or characteristic in or of a biological sample. An automated sample analysis device or system may use consumables during the performance of an assay. Consumables may be carried on a cartridge, and may be provided for use by an automated sample analysis device, e.g., for use in the performance of an assay.
[0063] As used herein, the term "assay" and its grammatical equivalents refers to tests, measurements, observations, and other experimental procedures which may be applied to a sample for detection of an analyte, identification of an analyte, and measurement of the amounts of an analyte in a sample. Assays may be physical assays which detect, identify, or measure a physical property of a sample; assays may be chemical assays, which detect, identify, or measure a chemical property of a sample, or perform chemical reactions in or with a sample; and include assays which use optical, electrical or electronic, chemical, or other means of detection and measurement.
[0064] As used herein, the terms "test" and "tests" and grammatical equivalents thereof are used interchangeably with the term "assay", and refer to measurements, observations, and other experimental procedures which may be applied to a sample for detection of an analyte, identification of an analyte, and measurement of the amounts of an analyte in a sample.
100651 As used herein, the terms "panel", "assay panel", and grammatical equivalents refers to two or more assays that may be ordered, and that may be performed, together as a group, on a sample or samples for detection of an analyte, identification of an analyte, and measurement of the amounts of an analyte in a sample, or for the detection of two or more analytes, or identification of two or more analytes, or measurements of the amounts of two or more analytes in a sample or samples. The assays grouped together in a panel are typically, although not necessarily, directed to markers or characteristics indicative of the same disease or disorder (e.g., to markers or characteristics indicative of celiac disease), or are directed to

-15-

16 markers or characteristics indicative of the same condition (e.g., to markers or characteristics indicative of pregnancy, or of cardiovascular disease, or of liver disorders), or are directed to markers or characteristics indicative of similar diseases or disorders (e.g., to markers or characteristics indicative of a respiratory disease or a digestive system disease or disorder), or are directed to markers or characteristics indicative of diseases or disorders having similar symptoms (e.g., to markers or characteristics indicative of a viral infection, a bacterial infection, a blood disease or disorder, or a thyroid disease or disorder), or are directed to markers or characteristics indicative of diseases or disorders having similar causes or risk factors (e.g., sexually transmitted diseases), or are directed to markers or characteristics that may occur together in a subject (e.g., to markers or characteristics indicative of a plurality of infectious diseases).
100661 As used herein, an "analyte" is the subject of an assay or analysis, the presence or amounts of which are to be determined by the performance of the assay. An analyte may be a natural constituent of a biological sample; an analyte may be free in a fluid sample or solution, or may be bound to another compound (e.g., to a carrier protein), may be present on a cell, or may be present in a cell; an analyte may be a compound not normally found in a biological sample, such as a drug; a metabolite of a drug or other compound;
an infectious agent (e.g., a virus, bacteria, or other foreign organism or material); a toxin; or other compound, element, cell, or cellular marker that may be the subject of analysis. An analyte may comprise a cell, or virus, or portion thereof; and may comprise a physical or chemical characteristic of a cell, virus, tissue, or portion thereof.
100671 An assay for an analyte is a test or procedure directed at detecting the presence of that analyte, or determining the amount of that analyte, or identifying that analyte, or characterizing that analyte, or otherwise obtaining information about that analyte, in a sample.
100681 As used herein, the term "characteristic" refers to a dimension, property, marker, or other attribute of a sample or component of a sample (e.g., a biological sample); in embodiments, characteristics may include the presence or distribution of markers, such as identifying markers, on a cell; characteristics may include morphological characteristics of a cell, whether before treatment or after treatment of that cell;
characteristics may include physical characteristics (e.g., size, volume, shape, viscosity, mechanical properties, or other physical characteristics) including optical characteristics (e.g., color, reflectance, absorbance, polarization, light scattering, or other optical characteristics;
characteristics may include chemical characteristics, including, e.g., presence or concentration of elements or compounds, pH, chemical reactivity, and other characteristics; and other characteristics. Such characteristics may be detected, identified, measured, or otherwise assayed on a sample as a whole, or on components of a sample (e.g., cells within a biological sample).
[0069] An assay for a characteristic is a test or procedure directed at detecting the presence of that characteristic, or identifying that characteristic, or quantifying that characteristic, or characterizing that characteristic, or otherwise obtaining information about that characteristic, in a sample (e.g., a biological sample).
[0070] An assay for an analyte or characteristic is a test or procedure directed at detecting the presence of that analyte or characteristic, or determining the amount of that analyte or that characteristic, or identifying that analyte or characteristic, or characterizing that analyte or characteristic, or otherwise obtaining information about that analyte or characteristic, in a sample (such as a biological sample).
[0071] As used herein, the term "negative result" and grammatical equivalents thereof refers to a result of an assay in which the presence of the target analyte is not detected, or is detected at a normal level, in the biological sample.
[0072] As used herein, the term "positive result" and grammatical equivalents thereof refers to a result of an assay in which the presence of the target analyte is detected, or is detected at an abnormal level, in the biological sample.
[00731 As used herein, the terms "reflex test" and "reflex assay" refer to an assay (equivalently, a test) the performance of which is contingent on a result of a previous test (which may be termed an "initial test" or an "initial assay"). A reflex assay may be termed a subsequent assay (e.g., when performed following an. initial assay). Whether or not the reflex test is performed may be determined by a result of an initial test. The timing of the performance of a reflex test may be determined by a result of an initial test.
The order of performance of a reflex test, or reflex tests, may be determined by a result of an initial test [00741 As used herein, a "subsequent test" may be a reflex test. For example, where a subsequent test is performed only upon obtaining a particular result, or one of a particular group of results, then that subsequent test may be a reflex test. For example, where a subsequent test is reported to a subject (or to a clinican, doctor, or other health care professional regarding that subject), only upon obtaining a particular result, or one of a particular group of results, then that subsequent test may be a reflex test.
[0075] As used herein, the term "contingent" refers to dependence on a prior event, result, condition, or state. Thus, where an act is contingent on a criterion (e.g., the occurrence of an act; the outcome of a test; the existence of a state or condition; or the satisfaction of a

-17-criterion of any kind) the perfomiance of that act is conditional upon the criterion, and that act will occur or be performed, or not, or will occur or be performed in a particular way, depending upon that criterion. For example, one form of reflex test is one in which a positive result from a general test triggers an automatic test of a more specific nature; for example, an influenza reflex test which is positive for the presence of influenza virus may automatically trigger a more specific test which will identify the particular strain of influenza present in a sample from the subject, or will determine whether or not a particular strain of influenza is present in a sample from the subject.
[0076] As used herein, the term "contingent assay" refers to an assay the performance of which (or failure to perform) depends on a prior event or condition. For example, a contingent assay may be one that is performed only if the result of a prior assay satisfies a criterion (or criteria); a contingent assay may be one that is not performed if the result of a prior assay satisfies a criterion (or criteria); a contingent assay may be one that is performed in one way if the result of a prior assay satisfies a criterion (or criteria), but is performed in a different way if the criterion (or criteria) is (are) not met. Thus, a single contingent assay may depend upon a single criterion; or, a single contingent assay may depend upon multiple criteria. Multiple contingent assays may depend upon a single criterion; or, multiple contingent assays may depend upon multiple criteria.
100771 Similarly, a step in an. assay may be a "contingent step", i.e., one that is contingent upon the occurrence or outcome of a prior assay, or a prior step of an assay (including the assay in which a contingent step occurs).
[0078] As used herein, the acronym "CPT" refers to "current procedural terminology"
and the term "CPT code" refers to the code used to identify a diagnostic, treatment, or other clinical procedure. Current Procedural Terminology codes ("CPT codes") are used to describe clinical procedures, such as medical, surgical, and diagnostic procedures. Thus, CPT
codes identify a procedure, including procedures used to determine a diagnosis, while other codes may be used to identify diagnoses. CPT codes are often required in order to obtain reimbursement for a procedure by insurance companies and other third-party payers. For example, CPT codes may be required in order to obtain. reimbursement from a government agency (e.g., in order to obtain Medicare or Medicaid reimbursement).
[0079] As used herein, the acronym "ICD" refers to "International Classification of Diseases" and the term "ICD code" refers to the code used to identify a particular disease or condition. International Classification of Diseases codes ("ICD codes") are revised periodically; current revisions of ICD codes which are still typically used include ICD-9 and

-18-ICD-10. Thus, in contrast to CPT codes, 1CD codes typically identify a disease, and not a particular procedure associated with such a disease. However, the ICD codes have also been modified to include procedures as well as diagnoses; thus, ICD-9-CM and ICD-10-CM
include diagnosis codes, and ICD-9-PCS and ICD-10-PCS include procedure codes.
ICD
codes are used to describe a disease or disorder suffered by a subject, and may be used or required in order to obtain reimbursement for a procedure by insurance companies and other third-party payers. For example, ICD-9-CM codes may be required in order to obtain reimbursement from a government agency (e.g., in order to obtain Medicare or Medicaid reimbursement).
[00801 Abbreviations and acronyms used herein, including in the figures, are those in common usage in the art. The abbreviations and acronyms as used herein typically identify biomarkers, biochemical or physiological quantities or measurements, assays for those biomarkers, or specific assays or assay types. Such abbreviations and acronyms include "Ab"
which stands for antibody; "Ag" which stands for antigen; "ABO/RhD" stands for a blood-typing test to determine type A, B, or O and Rhesus factor positive or Rhesus factor negative (Rh+ or Rh-); "ALP" is an acronym for alkaline phosphatase; "ALT" is an acronym for alanine aminotransfera,se; "ASP" is an acronym for aspartase aminotransferase;
"BUN" is an acronym for blood urea nitrogen; "CBC" is an acronym for complete blood count;
"CONF"
refers to a "coinfinriatory test", i.e., a subsequent test used to confirm a possible fmding from an initial test; each confirmatory test is specific for the initial test which it is designed to confirm (for example, "HW W/CONF" indicates that a positive HIV test reflexes to a confirmatory differential test between HIV-1 and HIV-2); "CRP" is an acronym for C-reactive protein; "DGP" is an acronym for deamidated gliadin peptide; "DHEA"
(which stands for dehydroepiandosterone) and "DHEA-S" (which stands for dehydroepiandosterone sulfate); "Diff' refers to a "differential" test, and indicates a test which is used to distinguish (differentiate) between two or more similar (or often found together) markers (e.g., "HIV-1/2 Antibody Differentiation", an assay used to distinguish between HIV-1 and HIV-2) or to provide further and more detailed information (e.g., "CBC w/ Auto Differential WBC" refers to a complete blood emit with automatic differential measurements of white blood cells);
"DNA" is an acronym for deoxyribonucleic acid; "EMA" is an acronym for endomesial antibodies; "ESR" is an acronym for erythrocyte sedimentation rate; "FSH" is an acronym for follicle stimulating hormone; "GGT" is an acronym for gamma glutamyltransferase;
"hbA.lc" stands for hem.oglobin Al C; "HBsAb" stands for hepatitis B surface antigen;
"hCG" is an acronym for human chorionic gonadotropin; "HDL" is an acronym for high-

-19-density lipoprotein; "Hep C" stands for hepatitis C; "HIV" stands for human.
immunodeficiency virus; "HLA-DQ typing" refers to assays to determine HLA-DQ
surface antigens (DQ antigens are of the human leukocyte antigen (HLA) class of major histocompatibility (MHC) class II antigens) which may be relevant to transplantation, and to autoimmune and other disorders; "ItsCRP" is an acronym for high sensitivity C-reactive protein assay; "HSV" is an acronym for herpes simplex virus (including, e.g., forms 1 and 2, HSV1 and HSV2); "LDL" is an acronym for low-density lipoprotein; "LH" is an acronym for luteinizing hormone; "PSA" is an acronym for prostate specific antigen; "PT"
is an acronym for prothrombin time; "PT/INR" is an acronym for prothrombin time/international normalized ratio; "PTT" is an acronym for partial thromboplastin time; "Qual"
stands for qualitative; "Quant" stands for quantitative; "RBC" is an acronym for red blood cell; "RNA"
is an acronym for ribonucleic acid; "RPR" stands for rapid plasma reagin; "Sed Rate" stands for sedimentation rate; "T3" stands for tri-iodothyronine; "T4" stands for thyroxine; "TIBC"
is an acronym for total iron binding capacity; "TP" is an acronym for treponema pallidum;
"FPO" is an acronym for thyroid peroxidase; "TSH" is an acronym for thyroid stimulating hormone; "tTG" is an acronym for tissue transglutaminase; "Vv'BC" is an acronym for white blood cell.
[0081] The phrase "Reflex to culture" indicates that, where the results of a test indicate or require a subsequent test (a "reflex" test, e.g., for confirmation, for quantification, or for further specification of the target detected, or for other purpose), that reflex testing includes a culture assay, e.g., an assay in which a portion of a sample is placed in a culture medium or culture dish and maintained under culture conditions for further testing.
[0082] The phrase "Reflex to culture & susceptibility" indicates that, where the results of a test indicate or require a subsequent test (a "reflex" test, e.g., for confirmation, for quantification, or for further specification of the target detected, or for other purpose) that reflex testing includes a culture assay and a susceptibility assay (i.e., an assay in which different anti-microbial agents are applied to culture assays to determine which anti-microbial agents would be most effective in combatting an infection identified by the culture assay).
[0083] The term "random" as used, for example, in. the phrase "Microalbumin/Creatinine Urine Random" indicates that the (urine) sample may be taken at any time during the day or night.
[0084] The term "Complete" in the phrase "Urinalysis, Complete" indicates that microscopy is used in the analysis of a urine sample in addition to the other automated aspects of the urinalysis (e.g., chemical and optical examination).

-20-[00851 The terms "antibody" and "antibodies" refer to complete antibodies and to functional (i.e., specifically binding) fragments and variants thereof.
Antibody may be abbreviated "Ab". An antibody specifically binds to a target antigen; an antigen may be abbreviated "Ag" (e.g., "HBsAg" indicates hepatitis B surface antigen).
Antibodies may be identified by the target molecule to which they specifically bind; for example, an "anti-HIV"
antibody binds HIV virus particles with high specificity, and an "anti-CD41"
antibody binds CD41 antigens with high specificity. As used herein, "IgA" refers to antibodies of the immunoglobulin A type; "IgD" refers to antibodies of the immunoglobulin D
type; "IgE"
refers to antibodies of the immunoglobulin E type; "IgG" refers to antibodies of the immunoglobulin G type; and "IgM" refers to antibodies of the itnmunoglobulin M
type.
[00861 As used herein, the term "antibody-based assay" refers to an assay for an analyte which may be found in a biological sample, using specific binding of the analyte by an antibody, antibody fragment, or antibody mimetic (e.g., immtmoadhesin, aptamer, or other construct which specifically binds to a target with little or no cross-reactivity with other compounds) to detect the presence of, and, if desired, quantify the amounts of, an analyte in a sample. Thus, as used herein, the term "antibody-based assay" includes immunoassays (including ELISAs), receptor-based assays, and other assays which utilize specific binding between a receptor and a ligand to detect, identify, or quantify an analyte. A
Western blot may be termed an antibody-based assay.
100871 An aptamer is a nucleic acid molecule capable of binding to a target molecule.
The nucleic acid may be a deoxyribonucleic acid, a ribonucleic acid, a linked peptide nucleic acid, or other nucleic acid, analog, or derivative thereof. The generation and use of aptamers is known in the art; see, e.g., U.S. Patent No. 5,475,096. Aptamers may be used with, or in place of, antibodies in assays in which require binders which specifically bind to target molecules in a sample (e.g., "antibody-based assays").
100881 The term "immunoadhesin" designates an antibody-like molecule which combines the binding specificity of a heterologous protein (an "adhesin") with the effector functions of immunoglobulin constant domains. Structurally, the immtmoadhesins comprise a fusion of an amino acid sequence with the desired binding specificity which is other than the antigen recognition and binding site of an antibody (i.e., is "heterologous"), and an immunoglobulin constant domain sequence. The adhesin part of an immtmoadhesin molecule typically is a contiguous amino acid sequence comprising at least the binding site of a receptor or a ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG4 subtypes,

-21-IgA. (including IgA-1 and IgA.-2), IgE, IgD or IgM. Immunoadhesins reported in the literature include fusions of the T cell receptor (Gascoigne et al. Proc. Natl. Acad.
Sci. USA 84:2936-2940 [1987]), CD4 (Capon et al., Nature 337:525-531 [1989]), CD44 (Aniffo et al., Cell 61:1303-1313 [1990]), and IgE receptor alpha (Ridgway et al., J. Cell. Biol.
115:abstr. 1448 [1991]). For the production of immunoglobulin fusions see also U.S. Pat. No.
5,428,130 issued Jun. 27, 1995. Immunoadhesins may be used with, or in place of, antibodies in assays in which require binders which specifically bind to target molecules in a sample (e.g., "antibody-based assays").
[0089] As used herein, the term "cytometric assay" refers to an assay which detects, or identifies, or quantifies cells or particles in a sample, typically by optical means (e.g., by microscopy). For example, cells or particles may be counted in a sample, or a field of view within a sample, to provide a number, or density, or other numerical value regarding cells or particles in a sample. Size, or optical intensity, or other characteristic of cells or particles in a sample may be measured or characterized. Cells in a sample assayed in a cytometric assay may be labeled, or otherwise treated, to enhance their identification or to ease the differentiation between cells and cell types. Thus, for example, cell surface markers may be labeled and optically identified by exposure of cells to antibodies or antibody fragments directed at particular cell surface antigens, where the antibodies or antibody fragments are labeled with fluorescent dyes or other identifiable markers. In addition to antibodies and antibody fragments (and mimetics thereof), aptamers, immunoadhesins, nucleic acid molecules, nucleic acid mimetics, dyes, and other probes or labels specific for cellular features, organelles, molecules, or characteristics may also be used to identify and characterize cells in a cytometric assay.
[0090] As used herein, "measurement of a morphological characteristic" of a cell or particle, and grammatical variants thereof, refers to a cytometric assay directed at identifying, quantifying, or characterizing the size, shape, or other physical characteristic of a cell, particle, or group of cells or particles in a sample. Observation, measurement, or characterization of the appearance of a cell or particle is one type of measurement of a morphological characteristic. Measurement or characterization of the size of a cell or particle may refer to the measurement or characterization of the diameter, cross-sectional area, volume, or other characteristic of a cell or particle. Measurement or characterization of the shape of a cell or particle may refer to the measurement or characterization of the symmetry (or asymmetry), or presence or number of protrusions, smoothness (or roughness) of a cellular surface, or other characteristic of a cell or particle. Measurement or characterization

-22-of brightness, or other optical property, across a dimension of a cell or particle, comprises measurement of a morphological characteristic of a cell or particle. Other morphological characteristics may also be observed, measured and characterized.
[0091] The word "label" or "marker" or the phrases "detectable label" and "marker moiety" when used herein refer to a detectable compound or composition which is conjugated directly or indirectly to the antibody so as to generate a "labeled" antibody. The label may be detectable by itself (e.g. radioisotope labels or fluorescent labels) or, in the case of an enzymatic label, may catalyze chemical alteration of a substrate compound or composition which is detectable (a label may be, e.g., a dye, a fluorescent moiety, a luminescent moiety, a chemiluminescent moiety, an enzymatic label, a magnetic label, a paramagnetic label, a contrast agent, a nanoparticle, a radioisotope, an epitope tag, biotin, streptavidin, or a quencher). Dyes include, for example, fluorescent dyes that intercalculate with double-stranded DNA include, for example, SYBR GokIrm, SYBR Green ITm, SYBR
GreenlIrm, ethidium bromide, BlueViewTM, methylene blue, DAPI, and acridine orange.
Further fluorescent dyes include, for example, CAL Fluor Gold, CAL Fluor Orange, Quasar 570, CAL Fluor Red 590, CAL Fluor Red 610, CAL Fluor Red 610, CAL Fluor Red 635, Quasar 670 (Biosearch Technologies), VIC, NED (Life Technologies), Cy3, Cy5, Cy5.5 (GE
Healthcare Life Sciences), Oyster 556, Oyster 645 (Integrated DNA
Technologies), LC red 610, LC red 610, LC red 640, LC red 670, LC red 705 (Roche Applies Science), Texas red, FAM, TET, HEX, JOE, TMR, and ROX. Quenchers that may be used include, for example, DDQ-I, DDQ-II (Eurogentec), Eclipse (Epoch Biosciences), Iowa Black FQ, Iowa Black RQ
(Integrated DNA Technologies), BHQ-1, BHQ-2, BHQ-3 (Biosearch Technologies), QSY-7, QSY-21 (Molecular Probes), and Dabcyl.
[0092] As used herein, the term "small molecule" refers to a compound, typically a non-polymeric organic compound, that is smaller than a typical protein.
Examples of small molecules include acetylsalicylic acid (aspirin), caffeine, cholesterol, vitamin D, and other molecules. A small molecule typically has a molecular weight below about 500 Daltons. As used herein, small molecules may be small organic molecules, and may be small inorganic molecules.
[0093] As used herein, the term "general chemistry assay" refers to an assay for an element or compound which may be found in a biological sample, using chemical or physical reactions to detect the presence of, and, if desired, quantify the amounts of, an analyte in a sample. As used herein, a "general chemistry assay" may be directed at detecting a small molecule analyte (including ions and elements such as sodium or potassium), or directed at a

-23-chemical characteristic of a sample (e.g., pH, 02 saturation, or other sample characteristic determined by chemical means).
[0094] As used herein, the term "nucleic acid assay" refers to an assay for an nucleic acid (whether deoxyribonucleic acid (DNA) or ribonucleic acid (RNA)) which may be found in a biological sample; such assays typically use hybridization between a probe and the target nucleic acid to detect the presence of, and, if desired, quantify the amounts of, an analyte in a sample. Nucleic acid assays include, for example, Northern blots, Southern blots, polymerase chain reaction (PCR) assays of all types, including, e.g., PCR, reverse transcriptase PCR (RT-PCT), quantitative PCR (qPCR) (see, e.g. U.S. Patent No. 4,683,2020, and other assays.
Nucleic acid assays may utilize thermal cycling; nucleic acid assays include isothermal assays which do not require thermal cycling, or include steps which do not require thermal cycling (such as, e.g., loop-mediated isothermal amplification ("LAMP") as described, e.g., in U.S. Patent No. 6,410,278), and other isothermal methods (see, e.g., International Patent Application PCT/US2014/030034). Nucleic acid assays, including nucleic acid assays discussed herein, may be performed at one or more levels of stringency. For example, nucleic acid assays, including nucleic acid assays discussed herein, may be performed at high stringency (e.g., under "stringent conditions" or "high stringency conditions"). For further example, nucleic acid assays, including nucleic acid assays discussed herein, may be performed at moderate stringency (e.g., under "moderately stringent conditions"). For further example, nucleic acid assays, including nucleic acid assays discussed herein, may be performed at low stringency (e.g., under "low stringent conditions").
[0095] "Stringency" of hybridization reactions is readily detenninable by one of ordinary skill in the art, and generally is an empirical calculation dependent upon probe, length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures.
Hybridization generally depends on the ability of denatured DNA. or RNA to rea3nneal when complementary strands are present in an environment below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995).

-24-[009 "Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 500C.; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1. % Fico1110.1%

polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5 with 750 mM
sodium chloride. 75 mM sodium citrate at 42 C.; or (3) employ 50% formamide, 5 x SSC
(0.75 M
NaCI, 0.075 M sodium citrate). 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ps/m1), 0.1%
SDS, and 10% dextran sulfate at 42 C. with washes at 42 C. in 0.2 x SSC
(sodium chloride/sodium citrate) and 50% formamide at 55 C., followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55 C.
[0097] "Moderately stringent conditions" may be identified as described by Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) less stringent than those described for hioghly stringent conditions. An example of moderately stringent conditions is overnight incubation at 37 C. in a solution comprising: 20% Formamide, 5 x SSC (150 mM NaC1, 15 mM
trisodium citrate). 50 mM sodium phosphate (pH 7.6). 5 x Denhardt's solution.
10% dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50 C. The skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.
[0098] "Low stringent conditions" may include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and % SDS) even less stringent than those for moderately stringent conditions; such conditions include lower temperatures (temperatures lower than 37 'C, such as, e.g., 32 C, or 30 'C, or lower) and higher salt concentrations than are typically used for moderately stringent conditions.
[0099] The terms "blood" and "whole blood" refer to blood as it exists within an animal and as directly obtained from a subject in a blood sample. Blood contains red blood cells, white blood cells, proteins such as albumin, globulins, and clotting factors, salts, water, and other constituents.
[00100] The terms "plasma" and "blood plasma" refer to the liquid portion of blood (e.g., a blood sample) that remains after the removal of blood cells. R.ed blood cells and

-25-white blood cells may be removed by centrifugation of a blood sample, leaving plasma above the pelleted cells in the bottom of the centrifuge tube. Plasma retains blood clotting factors, and is obtained from anti-coagulated blood samples.
[001011 The terms "serum" and "blood serum" refer to the liquid portion of blood that remains after blood is allowed to clot, and the clot is removed. Serum differs from plasma in that serum lacks clotting factors: since clotting requires fibrin, thrombin, and other proteins, which form and remain part of a blood clot, serum lacks these proteins while plasma contains them.
1001021 As used herein, a "finger-stick" refers to: i) the act of making a small puncture in the skin of a subject, allowing a small amount (e.g., a droplet, or one, two, or a few drops) of blood to flow and become available for collection; ii) the puncture itself;
and iii) the blood collected thereby. Blood may be liberated in a finger-stick, for example, by use of a lancet or other sharp implement effective to pierce the skin of a subject. Typically, only a small amount of blood is collected in this way (e.g., the amount of blood may be about 250 ILL or less, or about 2004, or less, or about 150 lit or less, or about 100 j.iJ or less, or about 50 L.
or less, or about 25 1.11 or less, or about 15 1.: or less, or about 10 j.iL
or less, or about 101.11 or less, or about 5 uL or less, or about 3 lit or less, or about I ttL or less). Blood from a finger-stick may be collected, e.g., by needle, syringe, capillary tube, or other method. Blood from a finger-stick may be collected for transport to another location; for storage prior to use or analysis; for immediate use; or for a combination of the same.
[001031 As used herein, the term "biological sample" refers to a fluid, tissue, or other material collected from a subject. Examples of biological samples can include but are not limited to, blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions. Biological samples may include nasopharyngeal wash, or other fluid obtained by washing a body cavity or surface of a subject, or by washing a swab following application of the swab to a body cavity or surface of a subject. Nasal swabs, throat swabs, stool samples, hair, finger nail, ear wax, breath, and other solid, semi-solid, or gaseous samples may be processed in an extraction buffer, e.g., for a fixed or variable amount of time, prior to their analysis. The extraction buffer or an aliquot thereof may then be processed similarly to other fluid samples if desired. Examples of tissue samples of the subject may

-26-include but are not limited to, connective tissue, muscle tissue, nervous tissue, epithelial tissue, cartilage, cancerous sample, or bone. The sample may be obtained from a human or animal. The sample may be obtained from a mammal, vertebrate, such as murines, simians, humans, farm animals, sport animals, or pets. The sample may be obtained from a living or dead subject. The sample may be obtained fresh from a subject or may have undergone some form of pre-processing, storage, or transport.
[00104] As used herein, "portion" and "portions" (e.g., of biological samples) include, without limitation, aliquots (which may be of equal volume, or may be of unequal volume);
dilutions (in which a sample, or a portion thereof, is mixed with a diluent to form a dilution of the sample); fractions (e.g., a fraction of whole blood, such as serum, or plasma, or a pellet formed by centrifugation); and combinations thereof.
REFLEX TESTING
[00105] Methods, devices, and systems disclosed herein may be used to provide and perform a "reflex test" or "reflex assay" in which a second assay is, or subsequent assays are, performed contingent on the results of an initial test or tests. When used as a verb, "reflex"
refers to the performance of a subsequent test pursuant to performing, or to obtaining the results of, an initial test. Thus, where the results of an antibody assay indicate that a subject has antibodies to a disease-causing organism, a reflex algorithm may dictate the performance of a nucleic acid assay to detect the presence of, or to quantify the am.ount of, that disease-causing organism in a sample obtained from the subject. In such an example, the nucleic acid assay is termed a "reflex test" or "reflex assay" with respect to the antibody assay. In such an example, the antibody assay is said to "reflex" to the nucleic acid assay.
[00106] In embodiments, a reflex test may be performed upon obtaining negative results from an initial test. For example, a negative result may indicate that a subject does not suffer from a suspected disease or condition that is suggested by a particular symptom, and indicates that a different assay directed at a different suspected disease or condition should be performed. In such embodiments, a reflex test for the different suspected disease or condition may be performed, as a result of obtaining the negative results from the initial test.
[001071 in embodiments, a reflex test may be performed upon obtaining positive results from an initial test. For example, an initial test may be, e.g, an HIV
test. In such an example, positive results of an antibody-based assay for HIV (e.g., an H1V-1 test, or an HIV-2 test) on a sample from a subject may trigger the testing of a sample from the same subject using a nucleic acid assay for HIV (e.g., a nucleic acid assay for HIV-1 test, or a nucleic acid assay for 1-IIV-2 test). In embodiments, a positive result in an antibody-based HIV assay for

-27-HIV may trigger testing of a sample from the same subject in a nucleic acid assay. In embodiments, a positive result in an initial nucleic acid assay for HIV may trigger testing of a sample from the same subject by a subsequent, higher stringency nucleic acid assay for HIV.
In embodiments, a positive result in an antibody-based HIV assay (e.g., an ELISA or other assay) may trigger testing of a sample from the same subject by Western blot (also an antibody-based assay) for HIV. In embodiments, a positive result in an antibody-based HIV
assay may trigger testing of a sample from the same subject in a cytometric assay. In embodiments, a positive result in a nucleic acid assay for HIV may trigger testing of a sample from the same subject in a cytometric assay. In embodiments, a positive result in an antibody-based HIV assay for HIV may trigger testing of a sample from the same subject in a general chemistry assay. In embodiments, a positive result in a nucleic acid assay-based HIV assay for HIV may trigger testing of a sample from the same subject in a general chemistry assay.
In embodiments, a positive result in a cytometric assay indicative of symptoms of HIV may trigger testing of a sample from the same subject in an antibody-based assay, or a nucleic acid assay, or in a general chemistry assay, or a combination of one or more of these assays. In embodiments, a positive result in any assay indicative of symptoms of HIV may trigger testing of a sample from the same subject in a cytometric assay, an antibody-based assay, or a nucleic acid assay, or in a general chemistry assay, or a combination of one or more of these assays.
[00108] It will be understood that the embodiments discussed above are exemplary, using HIV as an example, but that a biological sample may also be assayed and analysed analogously for any disease or condition by the methods, devices, and systems disclosed herein.
[00109] For a further example, an initial assay may comprise a complete blood cell count test, comprising testing a blood sample for white blood cells, where a white blood cell count outside the normal range (e.g., below about 2000 cells per microliter (RL) for males older than 12 years of age is outside the normal range) may trigger a reflex test comprising further testing of the blood of the subject. In embodiments, such further testing of the blood may be an automatic test performed by a cytometer on a device as disclosed herein. For example, such a reflex cytometric test may comprise identifying and/or quantifying white blood cells in the sample by cell type, using a cytometer. Such a cytometer test may be an automatic cytometer test, and may not require the intervention of any person to perform the cytometric assay. In embodiments, such further testing of the blood may be a "Manual" test performed by a trained person. In embodiments, such a reflex test comprising further testing

-28-of the blood may comprise both an automatic test performed by a cytometer on a device and a "Manual" test performed by a trained person.
MOM] For a further example, a positive test for hepatitis may trigger the further testing of a sample from the same subject. In embodiments, an initial test for hepatitis may comprise a test for analytes indicative of compromised liver function, e.g., increased serum aminotransferase levels, or increased alkaline phosphatase levels, or increased gamma-glutamyl transpeptidase levels, or increased bilirubin, or other blood marker which may be altered by hepatitis. In embodiments, such further testing of a sample from the subject may comprise an antibody-based assay. In embodiments, such further testing of a sample from the subject may comprise a nucleic acid assay. In embodiments, such further testing of a sample from the subject may comprise an automatic test performed by a cytometer on a device as disclosed herein.
[001111 For a further example, a positive test for hepatitis C may trigger the further testing of a sample from the same subject. In embodiments, an initial test for hepatitis C may comprise a test for the presence of antibodies to hepatitis C virus. In embodiments, such further testing of a sample from the subject may be a nucleic acid assay for the presence of nucleic acids indicative of hepatitis C virus. In embodiments, an initial test for hepatitis C
may comprise an antibody-based assay for the presence of epitopes (antibody targets) indicative of the presence of hepatitis C virus in the sample. In embodiments, such further testing of a sample from the subject may be a nucleic acid assay for the presence of nucleic acids indicative of hepatitis C virus. In embodiments, an initial test for hepatitis C may comprise a test for the presence of nucleic acids indicative of hepatitis C
virus. In embodiments, such further testing of a sample from the subject may be a nucleic acid assay for the presence of antibodies to hepatitis C virus.
[001121 For a further example, a positive test for the presence of syphilis may trigger the further testing of a sample from the same subject. In embodiments, an.
initial test for the presence of syphilis may comprise a nucleic acid test for the presence of nucleic acids indicative of syphilis bacteria. In embodiments, a subsequent test for the presence of syphilis may comprise a higher stringency nucleic acid test for the presence of syphilis bacteria (as compared to the stringency of the initial nucleic acid test for syphilis). In embodiments, a subsequent test for the presence of syphilis may comprise a Western blot test for the presence of syphilis bacteria.
[00113] In embodiments, an initial test for the presence of syphilis may comprise a test for the presence of antibodies to syphilis bacteria. In embodiments, such further testing of a

-29-sample from the subject may be an automatic test performed by a cytometer on a device as disclosed herein. In embodiments, an automatic test performed by a cytometer on a device as disclosed herein may be a cytometric test using darkfield illumination of a blood sample from the patient, in order to determine and/or quantify the presence of syphilis bacteria. In embodiments, such further testing may be a "Manual" test performed by a trained person.
[00114] An initial assay may comprise an assay for the presence of hepatitis B. For example, a sample obtained from a pregnant female may be tested for hepatitis B surface antigen (an antibody-based assay). If this initial test is found to be positive for the presence of hepatitis B surface antigen, a nucleic acid reflex test may be performed for a more sensitive and specific test for the hepatitis B virus; the reflex test may also provide quantification of the viral load in the sample. The nucleic acid reflex test may be performed on a different sample than the antibody-based hepatitis B surface antigen assay; this different sample may be a portion of the original sample, obtained by dividing the original sample into portions, and retained for future use if needed; this different sample may be a separate sample, obtained at the same, or nearly the same time, as the original sample, and retained for future use if needed; or this different sample may be a sample obtained after obtaining the original sample (e.g., after determination of the result of the initial test).
[00115] For a further example, a sample obtained from a pregnant female may be tested for hepatitis B surface antigen (an antibody-based assay). If this initial test is found to be positive for the presence of hepatitis B surface antigen, a reflex assay for the presence of syphilis may be performed. Such a reflex syphilis assay may be, for example, a nucleic acid reflex test for the presence of nucleic acids indicative of the presence of syphilis bacteria in the sample. In embodiments, such a reflex syphilis assay may be an antibody-based reflex test for the presence of proteins indicative of the presence of syphilis bacteria in the sample. In embodiments, such a reflex syphilis assay may be, for example, a cytometric assay for the presence of syphilis bacteria in the sample. The reflex test may be performed on a different sample than the antibody-based hepatitis B surface antigen assay; this different sample may be a portion of the original sample, obtained by dividing the original sample into portions, and retained for future use if needed; this different sample may be a separate sample, obtained at the same, or nearly the same time, as the original sample, and retained for future use if needed; or this different sample may be a sample obtained after obtaining the original sample (e.g., after determining the result of the initial test).
[00116] For a further example, a urine sample may be assayed for the presence of blood, for the presence of nitrite, for the presence of more than trace amounts of protein, or

-30-for the presence of white blood cells (e.g., leukocytes) in the urine. In embodiments, an initial assay performed on a urine sample may be an antibody-based assay. If the results of such an antibody-based assay are found to be positive, then a reflex test may be performed, in which a sample of urine is subjected to cytometric testing. Such cytometric testing may be automated, or may be manual (i.e., involving observation of the sample by a technician using a microscope, or a camera, or both), or a combination of these. The cytometric reflex test may be performed on a different sample than the antibody-based initial assay; this different sample may be a portion of the original sample, obtained by dividing the original sample into portions, and retained for future use if needed; this different sample may be a separate sample, obtained at the same, or nearly the same time, as the original sample, and retained for future use if needed; or this different sample may be a sample obtained after obtaining the original sample (e.g., after determining the result of the initial test).
1001171 For a further example, a urine sample may be assayed for the presence of blood, for the presence of nitrite, for the presence of more than trace amounts of protein, or for the presence of white blood cells (e.g., leukocytes) in the urine, as discussed above. In embodiments, an initial assay performed on a urine sample may be an antibody-based assay.
If the results of such an antibody-based assay are found to be positive, then a reflex test may be performed, in which an assay for the presence of bacteria is performed, using a sample of urine. In embodiments, such an assay for the presence of bacteria in the urine comprises a bacterial culture, in which urine is applied to a substrate suitable for the culture of bacteria and the substrate incubated under conditions conducive to bacterial growth.
Such a bacterial culture reflex test may be performed on a different sample than the antibody-based initial assay; this different sample may be a portion of the original sample, obtained by dividing the original sample into portions, and retained for future use if needed; this different sample may be a separate sample, obtained at the same, or nearly the same time, as the original sample, and retained for future use if needed; or this different sample may be a sample obtained after obtaining the original sample (e.g., after determining the result of the initial test).
[00118] In embodiments, after providing a biological sample to a device, all assays are performed automatically by a device or system, e.g., by a device or system as disclosed herein. In embodiments, after providing a single biological sample to a device, all assays are performed automatically by a device or system, e.g., by a device or system as disclosed herein. In embodiments, after providing a plurality of biological samples to a device, all assays are performed automatically by a device or system, e.g., by a device or system as disclosed herein. In embodiments, after providing a biological sample to a device, an initial

-31-assay is performed automatically by a device or system, e.g., by a device or system. as disclosed herein, and a further sample is requested; upon loading such further sample in a device, at least one subsequent assay is performed automatically on the subsequent sample by a device or system, e.g., by a device or system as disclosed herein. In embodiments, after providing a biological sample to a device, an initial assay is performed automatically by a device or system, e.g., by a device or system as disclosed herein, and a further sample is requested; upon loading such further sample in a device, all subsequent assays are performed automatically on the subsequent sample by a device or system, e.g., by a device or system as disclosed herein.
[00119] In embodiments, after providing a biological sample to a device, an initial assay is performed automatically by a device or system, e.g., by a device or system as disclosed herein, and a further sample is obtained automatically; at least one subsequent assay is performed on such subsequent sample automatically by a device or system, e.g., by a device or system as disclosed herein. In embodiments, after providing a biological sample to a device, an initial assay is perfonned automatically by a device or system, e.g., by a device or system as disclosed herein, and a further sample is obtained automatically;
all subsequent assays are performed on such subsequent sample automatically by a device or system, e.g., by a device or system as disclosed herein.
[00120] Reflex testing may be indicated, or may be suggested, or may be performed in response to an assay result. For example, the results of a first assay A may be determinative of whether or not a second assay B should be run; in this example, assay A is the initial assay, and assay B, contingent on the results of assay A., is the reflex assay.
Reflex testing may be indicated where the results of a first assay A determine that a second assay B
should be run (e.g., in this case, the results of assay A indicate that assay B should be run). Reflex testing may be suggested where the results of a first assay A determine that a second assay B should be run (e.g., in this case, where the results of assay A indicate that assay B
should be run, a display, a message, an alert, or a report, etc., may be provided (e.g., to the subject, to a health care professional, or other suitable party) suggesting that assay B should be run). Reflex testing may be perfonned where the results of a first assay A determine that a second assay B
should be run (e.g., in this case, the results of assay A. indicate that assay B should be tun).
Where an assay B is run, the results of that assay may be provided (e.g., in a display, message, alert, or report) to the subject, a health care professional, or other suitable party.
[00121] Thus, in embodiments, reflex testing may be performed in response to an assay result. For example, the results of a first assay A may be determinative of whether or

-32-not a second assay B should be run; in this example, assay A is the initial assay, and assay B, contingent on the results of assay A, is the reflex assay. Thus, for example, when an assay A
is ordered, a cartridge may be pre-loaded with reagents required by assay A, and also pre-loaded with reagents necessary for assay B. If the result of assay A meets a predefmed criterion initiating the reflex assay, then assay B is run with the same sample in the device. The device protocol is planned to account for the possibility of running the reflex test (i.e., the necessary reagents are loaded into the cartridge, sufficient sample is obtained to perform both assay A and assay B, and sufficient sample is retained in reserve for running assay B if needed).
[00122] In embodiments, some protocol steps of assay B may be performed before the results for assay A are complete. For example, sample preparation can be completed in advance on the device.
[00123] In embodiments, a protocol including one or more assays, whose performance or sequence of performance is contingent on the result of an initial assay, may include instructions, or a protocol, for the dilution of a biological sample. In embodiments, such dilution may provide a diluted sample diluted by at least about 10-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 20-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 30-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 50-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 100-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 200-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 300-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 500-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 1000-fold as compared to the original sample; or may provide a diluted sample diluted by more than about 1000-fold as compared to the original sample. In embodiments, such a protocol may direct or require that a biological sample, or portion thereof, including diluted portions thereof, be retained for the performance of a contingent assay, if the contingent assay is needed.
[001241 In embodiments, a sample of sufficient size may be obtained from the subject sufficient to perform assay A and to perform assay B. In embodiments, a sample may be obtained from the subject, and a portion of the sample may be diluted with a reagent (e.g., a diluent such as water, saline, a buffered solution, or other diluent) in order to provide sufficient volume of sample to perform one of assay A or assay B with undiluted sample, and

-33-to provide sufficeint diluted sample to perform one of assay A or assay B with diluted sample. In embodiments, a sample may be obtained from the subject, and all or a portion of the sample may be diluted with a reagent (e.g., a diluent such as water, saline, a buffered solution, or other diluent) in order to provide sufficient volume of diluted sample to perform assay A and to perform assay B. In embodiments, a sample may be obtained from the subject, used to perform assay A, and stored in order to be available for the performance of assay B if indicated. In embodiments, if the performance of assay B is indicated, assay B
is performed on the stored portion. In embodiments, if the performance of assay B is indicated, assay B is performed on a diluted sample, or on a diluted portion of a sample, where the stored portion is an undiluted portion, and the undiluted portion is diluted prior to performing assay B. In embodiments, if the performance of assay B is indicated, assay B is performed on a diluted sample, or on a diluted portion of a sample, where the stored portion is an undiluted sample, or undiluted sample portion.
[001251 In embodiments, a sample may be obtained from the subject, and divided into two portions, one of which may be used to perfomi assay A, and the other of which may be stored in order to be available for the performance of assay B if indicated.
In embodiments, if the performance of assay B is indicated, assay B is performed on the stored portion. In embodiments, both portions of a sample divided into two portions (one of which may be used to perform assay A, and the other of which may be used to perfomi assay B) may be treated equivalently. In embodiments, the portions of a sample divided into two portions (one of which may be used to perform assay A, and the other of which may be used to perform assay B) may be treated differently from each other.
[001261 In embodiments, a reflex assay may be performed with a second sample from the patient. In such embodiments, a second sample may be obtained from the subject following the satisfaction of the criterion or criteria by the results of assay A. In embodiments, a result of assay A may trigger a request for a second, or a subsequent, sample.
In embodiments, a request for a second, or a subsequent, sample may comprise a message, e.g., a message displayed on the device, a message displayed on an interface, a message sent to an electronic address (e.g., an email, or an internet posting, or a tweet, or other address of an electronic messaging system), a message to the subject, or to an operator of a device, or to a health-care provider, or to a payer (e.g., an insurance company), or more than one of these messages, or to more than one of these individuals or entities.

-34-1001271 A second or subsequent sample may comprise same type of sample as the initial sample. A second or subsequent sample may comprise a different type of sample than the initial sample.
[00120] Alternatively, in such embodiments, a second sample may be obtained from the subject prior to the satisfaction of the criterion or criteria by the results of assay A; for example, two samples may be obtained from a subject at the same time, or may be obtained at different times prior to obtaining the results from the initial assay A. In embodiments, both samples may be treated equivalently. In embodiments, both samples may be treated differently from each other.
[001291 Accordingly, Applicant discloses a method of testing a small-volume biological sample in a device, wherein said small-volume biological sample has a volume of less than about 250 microliters (IL), the method comprising: placing said small-volume sample within the device; dividing said small-volume biological sample into at least a first and a second portion; retaining said second portion within the device for use in a subsequent assay; perfonning, in said device on said first portion or aliquot thereof, an initial assay for detecting or measuring an analyte or characteristic of the sample, whereby an initial result is obtained from the first portion, wherein: i) said initial result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected or measured in the biological sample, or is detected or measured at a normal level in the biological sample, or ii) said initial result may comprise a positive result indicating that the presence of the analyte or characteristic is detected or measured in the biological sample, or is detected or measured at an abnormal level in the biological sample; determining whether or not said initial result is a positive result or a negative result; determining, contingent on whether or not said initial result is a positive result or a negative result, whether or not to perform a subsequent assay;
and i) performing said subsequent assay on said second portion or aliquot thereof if the initial result is positive for an assay requiring further testing for a positive result, or if the initial result is negative for an assay requiring further testing for a negative result; or ii) otherwise not performing said subsequent assay.
1001301 in embodiments of such methods, the performance of a subsequent assay is begun pursuant to the initial result within a short amount of time after placing the small-volume sample within the device, wherein said short amount of time is about two hours or less. In embodiments the performance of a subsequent assay is begun within a short time comprising about three hours, or about two hours, or about one hour, or about 45 minutes, or about 30 minutes, or about 20 minutes, or about 15 minutes, or about 10 minutes, or about 5

-35-minutes, or about 4 minutes, or about 3 minutes, or about 2 minutes, or about 1 minute, or less.
[001311 In embodiments of such methods, the performance of a subsequent assay is completed within a short amount of time after placing the small-volume sample within the device, wherein said short amount of time is about two hours or less. In embodiments the performance of a subsequent assay is completed within a short time comprising about three hours, or about two hours, or about one hour, or about 45 minutes, or about 30 minutes, or about 20 minutes, or about 15 minutes, or about 10 minutes, or about 5 minutes, or about 4 minutes, or about 3 minutes, or about 2 minutes, or about 1 minute, or less.
[001321 In embodiments of such a method, the first portion of the small-volume sample is diluted prior to performance of the initial assay. In embodiments of such a method, the second portion of the small-volume sample is diluted prior to performance of the subsequent assay. In embodiments of such a method, both the first portion and the second portion of the small-volume sample are diluted prior to performance of the initial and the subsequent assays, respectively.
[001331 In embodiments of such a method, the first portion of the small-volume sample is diluted prior to performance of the initial assay. In embodiments of such a method, the second portion of the small-volume sample is diluted prior to performance of the subsequent assay. In embodiments of such a method, both the first portion and the second portion of the small-volume sample are diluted prior to performance of the initial and the subsequent assays, respectively.
[001341 in embodiments of the methods disclosed herein, a subsequent assay may comprise an assay for detecting or measuring the same analyte or characteristic as an initial assay, and such a subsequent assay may be more sensitive for the detection or measurement of the analyte or characteristic than the initial assay. In embodiments of the methods disclosed herein, the analyte or characteristic to be detected or measured by a subsequent assay may comprise a different analyte or characteristic than the analyte or characteristic detected or measured by an initial assay. In embodiments of the methods disclosed herein, an initial assay may comprise an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays, and a subsequent assay may comprise a different type of assay than said initial assay. In embodiments of the methods disclosed herein, an initial assay may comprise a nucleic acid assay, and a subsequent assay may comprise an assay selected from an antibody-based assay, a general chemistry assay, and a cytometric assay. in embodiments of

-36-the methods disclosed herein, an initial assay may comprise an antibody assay, and a subsequent assay may comprise an assay selected from a nucleic acid assay, a general chemistry assay, and a cytometric assay. In embodiments of the methods disclosed herein, an initial assay may comprise a general chemistry assay, and a subsequent assay may comprise an assay selected from a nucleic acid assay, an antibody assay, and a cytometric assay. In embodiments of the methods disclosed herein, an initial assay may comprise a cytometric assay, and a subsequent assay may comprise an assay selected from a nucleic acid assay, an antibody assay, and a general chemistry assay. In embodiments where an initial assay comprises a cytometric assay, the cytometric assay may comprise the detection or measurement of a morphological characteristic of a cell. In embodiments where an initial assay comprises detection or measurement of an analyte, a subsequent assay may comprise a cytometric assay for the detection or measurement of a characteristic of the sample; in embodiments, such a cytometric assay may comprise detection or measurement of a morphological characteristic of a cell in the sample.
1001351 in embodiments of the methods disclosed herein, the methods may further comprise reporting the results of said initial assay of said biological sample. In embodiments, the results of an initial assay are reported prior to the performance of said subsequent assay.
In embodiments of the methods disclosed herein, the methods may further comprise reporting the results of said subsequent assay. In embodiments of the methods disclosed herein, the methods may further comprise reporting the results of an initial assay and of a subsequent assay. In embodiments of the methods disclosed herein, the results of an initial assay and of a subsequent assay may be reported together. In embodiments of the methods disclosed herein, the results of a subsequent assay are not reported if: i) said initial assay provides a negative result, and said negative result does not require performance of a subsequent assay, or ii) said initial assay provides a positive result, and said positive result does not require performance of a subsequent assay.
1001361 In embodiments of the methods disclosed herein, a small-volume biological sample may comprise a volume of less than about 200 microliters (IL), or of less than about 150 tdõ or of less than about 100 iLtIõ or of less than about 75 ILL, or of less than about 50 ttl.õ
or of less than about 40 pt, or of less than about 30 RL, or of less than about 20 pl., or of less than about 10 !IL, or of less than about 5 !IL, or less.
[001371 In embodiments of the methods disclosed herein, a small-volume biological sample may comprise a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine,

-37-gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
[00138] Applicant discloses herein methods of testing a small-volume biological sample in a device, wherein said small-volume biological sample has a volume of less than about 250 microliters (RI), the methods comprising: placing said small-volume sample within the device; dividing said small-volume biological sample into at least a first and a second portion; performing, in said device on said first portion or aliquot thereof, a first assay for detecting or measuring an analyte or characteristic of the sample, whereby a first result is obtained from the first portion, wherein said first result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected or measured in the biological sample, or is detected or measured at a normal level in the biological sample, or may comprise a positive result indicating that the presence of the analyte or characteristic is detected or measured in the biological sample, or is detected or measured at an abnormal level in the biological sample; performing, in said device on said second portion or aliquot thereof, a second assay for detecting or measuring an analyte or characteristic of the sample, whereby a second result is obtained from the second portion; determining whether or not said first result is a positive result or a negative result; determining, contingent on whether or not said first result is a positive result or a negative result, whether or not to report said second result of said second assay; and i) reporting the first result and the second result if the first result is positive for an assay requiring reporting of said second result pursuant to a positive first result, or if the first result is negative for an assay requiring reporting of said second result pursuant to a negative first result; or ii) otherwise reporting the first result and not reporting the results of said second assay. In embodiments of the methods disclosed herein., a second assay may comprise an assay for detecting or measuring the same analyte or characteristic as a first assay, and wherein the second assay is more sensitive for the detection or measurement of the analyte or characteristic than the first assay. In embodiments of the methods disclosed herein, the analyte or characteristic to be detected or measured by a second assay may comprise a different analyte or characteristic than the analyte or characteristic detected or measured by a fug assay. In embodiments of the methods disclosed herein, the first assay may comprise an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and

-38-cytometric assays, and said second assay comprises a different type of assay than said first assay. In embodiments of the methods disclosed herein, a cytometric assay may comprise the detection or measurement of a morphological characteristic of a cell. In embodiments of the methods disclosed herein, a first portion the small-volume sample may be diluted prior to performance of a first assay, or a second portion the small-volume sample may be diluted prior to performance of a second assay, or both. In embodiments of the methods disclosed herein, a small-volume biological sample may comprise a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
1001391 Applicant further discloses devices and systems suitable for the practice of the methods disclosed herein. Accordingly, Applicant discloses devices for testing a biological sample, wherein the device is configured to perform an initial assay and a subsequent assay according to the method disclosed herein (e.g., according to the methods disclosed above, and according to other methods disclosed herein). In embodiments, a device for testing a biological sample may be configured to perform antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays. In embodiments, a device for testing a biological sample may be configured to test a blood sample. In embodiments, a system as disclosed herein may comprise a device for testing a biological sample, e.g., a device as disclosed herein. In embodiments, a system as disclosed herein may comprise a device for testing a biological sample that is configured to perform antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays (including, e.g., detection, identification, or measurement of a morphological characteristic of a cell).
In embodiments, a system as disclosed herein may comprise a device for testing a biological sample that is configured to test a blood sample.
1001401 Applicant discloses herein methods of testing a small-volume biological sample in a device, wherein said small-volume biological sample has a volume of less than about 250 microliters (gL), the method comprising: placing said small-volume biological sample and a cartridge comprising reagents within the device; dividing the sample into at least a first and a second portion; retaining said second portion on said cartridge within the device for use in a subsequent assay; performing, in the device on said first portion or aliquot

-39-thereof, an initial assay for detecting, identifying, or measuring an analyte or characteristic of the sample, wherein said initial assay is performed using only reagents from said cartridge, whereby an initial result is obtained from the first portion, wherein: i) said initial result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected, identified, or measured in the sample, or is detected, identified, or measured at a normal level in the sample, or ii) said initial result may comprise a positive result indicating that the presence of the analyte or characteristic is detected, identified, or measured in the sample, or is detected, identified, or measured at an abnormal level in the sample;
determining whether or not the initial result is a positive result or a negative result;
determining, contingent on whether or not the initial result is a positive result or a negative result, whether or not to perform a subsequent assay; and automatically performing said subsequent assay on the second portion or aliquot thereof if the initial result is positive for an assay requiring further testing for a positive result, or if the initial result is negative for an assay requiring further testing for a negative result, wherein the subsequent assay is performed in the device using only reagents from said cartridge. In embodiments, the small-volume biological sample has a volume of less than about 200 microliters (p.L), or less than about 150 microliters (pL), or less than about 100 microliters (11L), or less than about 75 microliters (1.11..), or less than about 50 microliters (1.IL), or less than about 40 microliters (IL), or less than about 30 microliters (1.1.E.,), or less than about 25 microliters (1IL), or less than.
about 15 microliters (1.IL), or less than about 10 microliters (pL), or less than about 5 microliters (u1,), or less. In embodiments, the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasophaTyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
[00141] In embodiments, the sample is provided in a sample container that is disposed on said cartridge during placement of the sample within the device, and, following dividing the sample into at least a first and a second portion, the second portion is retained in the sample container (on the cartridge within the device) for use in the subsequent assay. In embodiments, the sample is provided in a sample container that is disposed on the cartridge during placement of the sample within the device, and, following dividing the sample into at least a first and a second portion, the second portion is retained in a second container that is

-40-disposed on the cartridge within the device. In embodiments, the second container is a dedicated container designated for holding the second portion on the cartridge prior to the performance of the subsequent assay. In embodiments, the second container is a dedicated container designated to contain the second portion during the performance of the subsequent assay.
[00142] In embodiments of the methods of testing a small-volume biological sample in a device, the automatic performance of the subsequent assay is begun within a short amount of time after placing the sample within the device, wherein the short amount of fime is about three hours or less, or about two hours or less, or about one hour or less, or about 45 minutes or less, or about 30 minutes or less, or about 20 minutes or less, or about 10 minutes or less, or about 5 minutes or less, or is a shorter amount of time.
[001431 In embodiments of the methods of testing a small-volume biological sample in a device, the automatic performance of the subsequent assay is completed within a short amount of time after placing the sample within the device, wherein the short amount of time is about three hours or less, or about two hours or less, or about one hour or less, or about 45 minutes or less, or about 30 minutes or less, or about 20 minutes or less, or about 10 minutes or less, or about 5 minutes or less, or is a shorter amount of time.
[001441 In embodiments of the methods of testing a small-volume biological sample in a device, the cartridge contains all consumables used in the performance of the initial assay and of the subsequent assay.
[001451 In embodiments of the methods of testing a small-volume biological sample in a device, the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or the second portion of the sample is diluted by at least about 10-fold prior to, or during, the automatic performance of the subsequent assay, or both.
In embodiments, following dilution, the volume of the diluted portion is less than about 500 microliters (1.L), or is less than about 400 pi., or is less than about 300 tdõ or is less than about 250 pL, or is less than about 200 pt, or is less than about 100 !IL, or is less than about 50 glõ or is less than about 40 pL, or is less than about 30 L, or is less than about 20 microliters iaL.
[001461 In embodiments, the first portion of the sample is diluted by at least about 30-fold prior to, or during, the performance of the initial assay, or the second portion of the sample is diluted by at least about 30-fold prior to, or during, the automatic performance of the subsequent assay, or both. In embodiments, following dilution, the volume of the diluted portion is less than about 500 microliters (gL), or is less than about 400 ML, or is less than

-41-about 300 pl.õ or is less than. about 250 pL, or is less than about 200 pt., or is less than about 100 pL, or is less than about 50 pL, or is less than about 40 pL, or is less than about 30 p,L, or is less than about 20 microliters p.L.
[001471 In embodiments of testing a small-volume biological sample in a device, the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or the second portion of the small-volume sample is diluted by at least about 100-fold prior to, or during, the automatic performance of the subsequent assay, or both. In embodiments, following dilution, the volume of the diluted portion is less than about 500 microliters (pL), or is less than about 400 pL, or is less than about 300 pt, or is less than about 250 !IL, or is less than about 200 p.L, or is less than about 100 p,L, or is less than about 50 pL, or is less than about 40 L, or is less than about 30 pL, or is less than about 20 microliters p.L.
[00148] In embodiments of the methods of testing a small-volume biological sample in a device, the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays. In embodiments, the subsequent assay comprises a different type of assay than the initial assay. In embodiments, cytometric assays may comprise the detection, identification, or measurement of a morphological characteristic of a cell.
[00149] In embodiments of the methods of testing a small-volume biological sample in a device, the subsequent assay comprises an assay for detecting, identifying, or measuring: a) the same analyte or characteristic as the initial assay, wherein the subsequent assay is more sensitive for the detection, identification, or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay. In embodiments in which the initial assay and the subsequent assay are different assays, i) the initial assay comprises a nucleic acid assay, and the subsequent assay comprises an assay selected from an antibody-based assay, a general chemistry assay, and a cytometric assay; or ii) the initial assay comprises an antibody assay, and the subsequent assay comprises an assay selected from a nucleic acid assay, a general chemistry assay, and a cytometric assay; or iii) the initial assay comprises a general chemistry assay, and the subsequent assay comprises an assay selected from a nucleic acid assay, an antibody assay, and a cytometric assay; or iv) the initial assay comprises a cytometric assay, and the subsequent assay comprises an assay selected from a nucleic acid assay, an antibody assay, and a general chemistry assay. In embodiments where either an initial assay or a subsequent assay comprise a cytometric assay, the cytometric assay may

-42-comprise the detection, identification, or measurement of a morphological characteristic of a cell. In embodiments, the initial assay may comprise the detection, identification, or measurement of an analyte, and the subsequent assay may comprise a cytometric assay for the detection, identification, or measurement of a morphological characteristic of a cell.
1001501 In embodiments of the methods of testing a small-volume biological sample in a device, the methods further comprise reporting the results of the initial assay of the sample.
In embodiments, the results of the initial assay are reported prior to the performance of the subsequent assay. In embodiments, methods of testing a small-volume biological sample in. a device comprise reporting the results of the initial assay of the sample and further comprise reporting the results of the subsequent assay. In embodiments, the results of the initial assay and of the subsequent assay are reported together.
1001511 Applicant discloses herein methods of testing a small-volume biological sample in a device, wherein said sample has a volume of less than about 250 microliters (pL), the method comprising: placing the sample and a cartridge comprising reagents within the device; dividing the sample into at least a first and a second portion, and retaining at least said second portion on said cartridge prior to performing assays on said portions; performing, in said device on the first portion or aliquot thereof, a first assay for detecting, identifying, or measuring an analyte or characteristic of the sample using only reagents from said cartridge, whereby a first result is obtained from the first portion, wherein said first result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected or measured in the sample, or is detected or measured at a normal level in the sample, or may comprise a positive result indicating that the presence of the analyte or characteristic is detected or measured in the sample, or is detected or measured at an abnormal level in the sample; performing, in the device on the second portion or aliquot thereof, a second assay for detecting, identifying, or measuring an analyte or characteristic of the sample using only reagents from said cartridge, whereby a second result is obtained from the second portion;
determining whether or not said first result is a positive result or a negative result;
determining, contingent on whether or not the first result is a positive result or a negative result, whether or not to report said second result of said second assay;
reporting the first result; and reporting the second result only if: i) the first result is positive for an assay requiring reporting of the second result pursuant to a positive first result, or ii) if the first result is negative for an assay requiring reporting of the second result pursuant to a negative first result. In embodiments, the methods comprise retaining both said first and said second portions on said cartridge prior to performing assays on said portions. In embodiments, the

-43-small-volume biological sample has a volume of less than about 200 microliters (AL), or less than about 150 microliters (4), or less than about 100 microliters (AL), or less than about 75 microliters (AL), or less than about 50 microliters (AL), or less than about 40 microliters (AL), or less than about 30 microliters (AL), or less than about 25 microliters (AL), or less than about 15 microliters (AL), or less than about 10 microliters (4), or less than about 5 microliters (AL), or less. In embodiments, the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
[00152] In embodiments of these methods, the second assay comprises an assay for detecting, identifying, or measuring: a) the same analyte or characteristic as the first assay, and wherein the second assay is more sensitive for the detection or measurement of the analyte or characteristic than the first assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the first assay. In embodiments of these methods, the first assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays. In embodiments, a cytometric assay may comprise the detection, identification, or measurement of a morphological characteristic of a cell. In embodiments, the second assay comprises a different type of assay than the first assay. In embodiments, the cartridge contains all consumables used in the performance of the first assay and of the second assay.
[00153] In embodiments, the first portion of the sample is diluted prior to, or during, the performance of the first assay, or wherein the second portion of the sample is diluted prior to, or during, the performance of the second assay, or both. In embodiments, the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or the second portion of the sample is diluted by at least about 10-fold prior to, or during, the automatic performance of the subsequent assay, or both. In embodiments, following dilution, the volume of the diluted portion is less than about 500 microliters (AL), or is less than about 400 AL, or is less than about 300 AL, or is less than about 250 AL, or is less than about 200 AL or is less than about 100 glõ or is less than about 50 AL, or is less than about 40 AL, or is less than about 30 AL, or is less than about 20 microliters AL.

-44-[001541 in embodiments, the first portion of the sample is diluted by at least about 30-fold prior to, or during, the performance of the initial assay, or the second portion of the sample is diluted by at least about 30-fold prior to, or during, the automatic performance of the subsequent assay, or both. In embodiments, following dilution, the volume of the diluted portion is less than about 500 microliters (ttL), or is less than about 400 L, or is less than about 300 gL, or is less than about 250 pL, or is less than about 200 ttL, or is less than about 100 pL, or is less than about 50 L, or is less than about 40 gL, or is less than about 30 AL, or is less than about 20 microliters ILL.
[001551 In embodiments, the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or the second portion of the sample is diluted by at least about 100-fold prior to, or during, the automatic performance of the subsequent assay, or both. In embodiments, following dilution, the volume of the diluted portion is less than about 500 microliters (pL), or is less than about 400 L, or is less than about 300 ML, or is less than about 250 L, or is less than about 200 tiL, or is less than about 100 utõ or is less than about 501.11.õ or is less than about 40 gl.õ or is less than about 30 utõ or is less than about 20 microliters lit.
[00156] Applicants disclose herein devices for testing a small-volume biological sample, wherein said devices are configured to perform an initial assay and a subsequent assay according to the methods disclosed herein. In embodiments, a device disclosed herein is configured to perform antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays. In embodiments, a device disclosed herein is configured to test the small-volume biological sample, wherein the sample is selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.In embodiments, a device disclosed herein is configured to test a blood sample.
[001571 Applicants disclose herein devices for testing a small-volume biological sample, wherein said systems comprise a device as disclosed herein. In embodiments, a system disclosed herein is configured to perform antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays. In embodiments, the systems comprise a device as disclosed herein, wherein the device is configured to test a small-

-45-volume biological sample, where the small-volume biological sample is a sample as disclosed herein. In embodiments, the systems comprise a device as disclosed herein, wherein the device is configured to test a blood sample.
1001581 The assays and methods disclosed herein may be performed on a device, or on a system, for processing a sample. The assays and methods disclosed herein can be readily incorporated into and used in device for processing a sample, or a system for processing a sample, which may be an automated assay device, or may be an automated assay system.
Such a device, and such a system, may be useful for the practice of the methods disclosed herein. For example, a device may be useful for receiving a sample. A device may be useful for preparing, or for processing a sample. A device may be useful for performing an assay on a sample. A device may be useful for obtaining data from a sample. A device may be useful for transmitting data obtained from a sample. A device may be useful for disposing of a sample following processing or assaying of a sample.
1001591 A device may be part of a system, a component of which may be an automated assay device. A device may be an automated assay device. An automated assay device may be configured to facilitate collection of a sample, prepare a sample for a clinical test, or effect a chemical reaction with one or more reagents or other chemical or physical processing, as disclosed herein. An automated assay device may be configured to obtain data from a sample.
An automated assay device may be configured to transmit data obtained from a sample. An automated assay device may be configured to analyze data from a sample. An automated assay device may be configured to communicate with another device, or a laboratory, or an individual affiliated with a laboratory, to analyze data obtained from a sample.
1001601 An automated assay device may be configured to accept a sample from a subject, either directly or indirectly. A sample may be, for example, a blood sample (e.g., a sample obtained from a fingerstick, or from veniptmcture, or an arterial blood sample), a urine sample, a biopsy sample, a tissue slice, stool sample, or other biological sample; a water sample, a soil sample, a food sample, an air sample; or other sample. A blood sample may comprise, e.g., whole blood, plasma, or serum. An automated assay device may receive a sample from the subject through a housing of the device. The sample collection may occur at a sample collection site, or elsewhere. The sample may be provided to the device at a sample collection site.
1001611 Examples of samples may include various fluid or solid samples. In some instances, the sample can. be a bodily fluid sample from the subject. The sample can be an

-46-aqueous or gaseous sample. In some instances, solid or semi-solid samples can be provided.
The sample can include tissues and/or cells collected from the subject. The sample can be a biological sample.
1001621 Any volume of biological sample may be obtained from a subject.
Examples of sample volumes that may be obtained from a subject include, but are not limited to, volumes of about: 10 mL or less, 5 inL or less, 3 mL or less, 1 microliter (pL, also "uL"
herein) or less, 500 gL or less, 300 pi, or less, 250 gi, or less, 200 pi, or less, 170 j.iL or less, 150 gL or less, 125 gL or less, 100 p.L or less, 75 p.L or less, 50 p.L or less, 25 p.L or less, 20 gL or less, 15 gL or less, 10 gL or less, 5 gL or less, 3 gL or less, 1 gL or less, 500 nL or less, 250 nL or less, 100 nL or less, 50 nL or less, 20 nL or less, 10 nL or less, 5 nL or less, 1 nL or less, 500 pL or less, 100 pi, or less, 50 pL or less, or 1 pL or less.
In embodiments, a biological sample may have a volume of: about 250 gL or less; or about 200 pL
or less; or about 150 gL or less; or about 100 gL or less; or about 75 pL or less; or about 50 pL or less;
or about 40 pL or less; or about 30 j.tL or less; or about 20 gt, or less; or about 10 pi, or less;
or about 5 pL or less; or about 4 gL or less; or about 3 pi, or less; or about 2 pL or less; or about 1 pL or less. The amount of sample may be about a drop of a sample. The amount of sample may be the amount collected from a pricked finger or fingerstick. The amount of sample may be the amount collected from a microneedle or a venous draw. Any volume, including those described herein, may be provided to the device.
1001631 in embodiments, a biological sample may include cells.
[001641 In embodiments of each and any of the methods disclosed herein, a sample, such as a biological sample, may be delivered to an automated assay device or system by a cartridge. In embodiments, a biological sample may be contained within a sample collection device, such as a vial, a tube, a jar, a specimen bottle, or other container or device suitable for holding or transporting a biological sample. In embodiments, a sample collection device may be or comprise a small volume container. In embodiments, a sample collection device may be or comprise a small volume container having a holding capacity of about 250 giõ or less, or about 200 gL or less, or about 150 gL or less, or about 100 gL or less, or about 50 gL or less, or about 25 gL or less. In embodiments, a sample collection device may be or comprise a nanotainer' vessel. In embodiments, the biological sample delivered by a cartridge is a processed biological sample. In embodiments, the biological sample delivered by a cartidge is a processed biological sample, and, following delivery of the processed biological sample, the processed biological sample is mixed with a reagent within an automated assay device or

-47-system.. In em.bodiments, the biological sample delivered by a cartridge is a processed biological sample, and the processed biological sample is mixed with a reagent prior to detection of the presence or absence of a target analyte or characteristic in the processed biological sample within an automated assay device or system. In embodiments, the biological sample delivered by a cartridge is a diluted biological sample. In embodiments, the biological sample delivered by a cartridge is a diluted biological sample, and, following delivery of the diluted biological sample, the diluted biological sample is mixed with a reagent within an automated assay device or system. In embodiments, the biological sample delivered by a cartridge is a diluted biological sample, and the diluted biological sample is mixed with a reagent prior to detection of the presence or absence of a target analyte or characteristic in the diluted biological sample within an automated assay device or system.
100165] In embodiments of each and any of the methods disclosed herein, the biological sample delivered by a cartridge is an undiluted biological sample.
In embodiments, the biological sample delivered by a cartridge is an undiluted biological sample and the biological sample is diluted within an automated assay device or system. In em.bodiments, the biological sample delivered by a cartridge is an undiluted biological sample and the biological sample is diluted within an automated assay device or system prior to analysis of the biological sample. In embodiments, the biological sample delivered by a cartridge is an undiluted biological sample and the first step applied to the biological sample within the automated assay device or system is a dilution step. In embodiments, such a first step is performed within an automated assay device or system. In embodiments, the biological sample delivered by a cartridge is an undiluted biological sample, and the biological sample is diluted and then mixed with a reagent prior to detection of the presence or absence of a target analyte or characteristic in the diluted biological sample within an automated assay device or system.
100166] In embodiments, an assay, and a protocol regarding such an assay, may utilize a diluted sample (e.g., a diluted biological sample). In embodiments, e.g., where the performance or sequence of performance of an assay is contingent on the result of an initial assay, an assay may include instructions, or a protocol, for the dilution of a biological sample.
In embodiments, such dilution may provide a diluted sample diluted by at least about 10-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 20-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 30-fold as compared to the original sample; may provide a diluted sample diluted by at least about 50-fold as compared to the original sample; or may provide a diluted sample

-48-diluted by at least about 100-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 200-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 300-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 500-fold as compared to the original sample; or may provide a diluted sample diluted by at least about 1000-fold as compared to the original sample; or may provide a diluted sample diluted by more than about 1000-fold as compared to the original sample. In embodiments, such an assay, or a protocol regarding such an assay, may direct or require that a biological sample, or portion thereof, including diluted portions thereof, be retained for the performance of a contingent assay, if the contingent assay is needed.
100167] In embodiments of each and any of the methods disclosed herein, a cartridge may contain a biological sample; a reagent unit; and an assay unit. In embodiments, a cartridge may contain a biological sample; a reagent unit; an assay unit; and a calibration unit. In embodiments, a cartridge may contain a biological sample; a reagent unit; an assay unit; a calibration unit; and a pipette. In embodiments, a cartridge may contain a biological sample; a reagent unit; an assay unit; a calibration unit; and a vessel. In embodiments, a cartridge may contain a biological sample; a reagent unit; an assay unit; a calibration unit; a pipette; and a vessel. A cartridge may comprise an identifying mark, or label, or other identification.
1001681 it will be understood that, in embodiments, a cartridge may contain one or more biological samples; one or more reagent units; one or more assay units;
one or more calibration units; one or more pipettes; and may contain combinations thereof.
In embodiments, a cartridge may include a vessel, or a plurality of vessels, in addition to biological samples, reagent units, assay units, calibration units, pipettes, and combinations thereof.
1001691 In other embodiments, a cartridge may lack a biological sample; for example, in embodiments, a biological sample may be provided to an automated assay device or system separately from a cartridge, where the cartridge lacking a biological sample may contain one or more reagent units, one or more assay units, one or more calibration units, one or more pipettes, one or more vessels, and combinations thereof.
[00170] In some embodiments, an automated assay device may be configured to accept or hold a cartridge. In some embodiments, an automated assay device may comprise a cartridge. The cartridge may be removable from the automated assay device. In some embodiments, a sample may be provided to the cartridge of the automated assay device.

-49-Alternatively, a sample may be provided to another portion of an automated assay device.
The cartridge and/or device may comprise a sample collection unit that may be configured to accept a sample.
1001711 A cartridge may include a sample, and may include reagents for use in processing or testing a sample, disposables for use in processing or testing a sample, or other materials. Following placement of a cartridge on, or insertion of a cartridge into, an automated assay device, one or more components of the cartridge may be brought into fluid communication with other components of the automated assay device. For example, if a sample is collected at a cartridge, the sample may be transferred to other portions of the automated assay device. Similarly, if one or more reagents are provided on a cartridge, the reagents may be transferred to other portions of the automated assay device, or other components of the automated assay device may be brought to the reagents. In some embodiments, the reagents or components of a cartridge may remain on-board the cartridge.
In some embodiments, no fluidks are included that require tubing or that require maintenance (e.g., manual or automated maintenance).
[00172] In embodiments, a cartridge may include a biological sample, or may include two or more biological samples. In embodiments, a biological sample is from a subject, and multiple biological samples may be from a single subject. In alternative embodiments, multiple biological samples may be from multiple subjects. A cartridge that has one or more identifier that is readable by the devke. The device may include an automated lancing cartridge. The cartridge may be disposable. One or more disposable component may be used to collect a sample from a subject. The disposable component may provide the sample to a non-disposable device. Alternatively, the disposable component may be the automated assay device.
(00173) In embodiments, a cartridge may include a reagent or a plurality of reagents, and may be configured to allow delivery of said reagent or reagents to said device. A
cartridge may be configured to deliver a biological sample and a reagent to the device. A
cartridge may be configured to deliver a plurality of biological samples and a reagent to the device. A cartridge may be configured to deliver a biological sample and a plurality of reagents to the device. A cartridge may be configured to deliver a plurality of biological samples and a plurality of reagents to the device. Identification information may include subject identifying information, information based on signals generated related to the sample, information based on signals generated related to reactions performed with the sample,

-50-information based on signals detected related to the sample, information based on signals detected related to reactions performed with the sample, device identification information, cartridge identification information, component identifying information, and other information transmitted from the device.
[00174] A sample or reagent carried by a cartridge may be transferred to a device, such as an automated assay device. A sample or reagent may be transferred within a device. Such transfer of sample or reagent may be accomplished without providing a continuous fluid pathway from cartridge to device. Such transfer of sample or reagent may be accomplished without providing a continuous fluid pathway within a device. In embodiments, such transfer of sample or reagent may be accomplished by a fluid handling system (e.g., a pipette); for example, a sample, reagent, or aliquot thereof may be aspirated into an open-tipped transfer component, such as a pipette tip, which may be operably connected to a fluid handling system which transfers the tip, with the sample, reagent, or aliquot thereof contained within the tip, to a location on or within the automated assay device. The sample, reagent, or aliquot thereof can be deposited at a location on or within the automated assay device. Sample and reagent, or multiple reagents, may be mixed using a fluid handling system in a similar manner. One or more components of the cartridge may be transferred in an automated fashion to other portions of the automated assay device, and vice versa.
[00175] In embodiments of the devices, systems and methods disclosed herein, a fluid handling system may be used to transport and deliver a sample solution to a vessel, and to fill a vessel (either partially or fully) with a sample solution. In embodiments, a fluid handling system comprises a pipette. A pipette may be configured to accept a pipette tip, e.g., to mount and transport a pipette tip attached to the pipette. In embodiments, a pipette comprises a nozzle configured to accept a pipette tip. A pipette may be configured to aspirate a fluid, such as a sample solution, into a pipette tip attached to the pipette (e.g., a pipette tip which is attached to a nozzle of the pipette). In embodiments, a pipette may be configured to dispense a fluid, such as a sample solution, from a pipette tip attached to the pipette (e.g., to a nozzle of the pipette). A pipette may be configured to transmit force to a surface or component of a device. In embodiments, a pipette nozzle may contact a surface or component of a device, effective to transmit force to that surface or component. In embodiments, a pipette nozzle may contact a mating recess of a vessel, and, in embodiments, may engage a mating recess of a vessel. In embodiments, two, or more pipette nozzles may contact mating recesses of a vessel, and, in embodiments, may engage mating recesses of a vessel. In embodiments, a pipette of a fluid handling system may be movable, and is preferably movable in at least two, and more preferably in three dimensions (e.g., is movable in one, two, or all three of horizontally, laterally, and vertically).
1001761 A device, such as an automated assay device, may have a fluid handling system. A fluid handling system may perform, or may aid in performing, transport, dilution, extraction, aliquotting, mixing, and other actions with a fluid, such as a sample. In some embodiments, a fluid handling system may be contained within a device housing.
A. fluid handling system may permit the collection, delivery, processing and/or transport of a fluid, dissolution of dry reagents, mixing of liquid and/or dry reagents with a liquid, as well as collection, delivery, processing and/or transport of non-fluidic components, samples, or materials. The fluid may be a sample, a reagent, diluent, wash, dye, or any other fluid that may be used by the device, and may include, but not limited to, homogenous fluids, different liquids, emulsions, suspensions, and other fluids. A fluid handling system, including without limitation a pipette, may also be used to transport vessels (with or without fluid contained therein) around the device. The fluid handling system may dispense or aspirate a fluid. The sample may include one or more particulate or solid matter floating within a fluid.
1001771 In embodiments, a fluid handling system may comprise a pipette, pipette tip, syringe, capillary, or other component. The fluid handling system may have portion with an interior surface and an exterior surface and an open end. The fluid handling system may comprise a pipette, which may include a pipette body and a pipette nozzle, and may comprise a pipette tip. A pipette tip may or may not be removable from a pipette nozzle. In embodiments, a fluid handling system may use a pipette mated with a pipette tip; a pipette tip may be disposable. A tip may form a fluid-tight seal when mated with a pipette. A pipette tip may be used once, twice, or more times. In embodiments, a fluid handling system may use a pipette or similar device, with or without a pipette tip, to aspirate, dispense, mix, transport, or otherwise handle the fluid. The fluid may be dispensed from the fluid handling system when desired. The fluid may be contained within a pipette tip prior to being dispensed, e.g., from an orifice in the pipette tip. In embodiments, or instances during use, all of the fluid may be dispensed; in other embodiments, or instances during use, a portion of the fluid within a tip may be dispensed. A pipefte may selectively aspirate a fluid. The pipette may aspirate a selected amount of fluid. The pipette may be capable of actuating stirring mechanisms to mix the fluid within the tip or within a vessel. The pipette may incorporate tips or vessels creating continuous flow loops for mixing, including of materials or reagents that are in non-liquid form. A pipette tip may also facilitate mixture by metered delivery of multiple fluids simultaneously or in sequence, such as in two-part substrate reactions.
100178] The fluid handling system may include one or more fluidically isolated or hydraulically independent units. For example, the fluid handling system may include one, two, or more pipette tips. The pipette tips may be configured to accept and confine a fluid.
The tips may be fluidically isolated from or hydraulically independent of one another. The fluid contained within each tip may be fluidically isolated or hydraulically independent from one fluids in other tips and from other fluids within the device. The fluidically isolated or hydraulically independent units may be movable relative to other portions of the device and/or one another. The fluidically isolated or hydraulically independent units may be individually movable. A fluid handling system may comprise one or more base or support. A
base or support may support one or more pipette or pipette units. A base or support may connect one or more pipettes of the fluid handling system to one another.
1001791 An automated assay device may be configured to perform processing steps or actions on a sample obtained from a subject. Sample processing may include sample preparation, including, e.g., sample dilution, division of a satnple into aliquots, extraction, contact with a reagent, filtration, separation, centrifugation, or other preparatory or processing action or step. An automated assay device may be configured to perform one or more sample preparation action or step on the sample. Optionally, a sample may be prepared for a chemical reaction and/or physical processing step. A sample preparation action or step may include one or more of the following: centrifugation, separation, filtration, dilution, enriching, purification, precipitation, incubation, pipetting, transport, chromatography, cell lysis, cytometry, pulverization, grinding, activation, ultrasonication, micro column processing, processing with magnetic beads, processing with nanoparticles, or other sample preparation action or steps. For example, sample preparation may include one or more step to separate blood into serum and/or particulate fractions, or to separate any other sample into various components. Sample preparation may include one or more step to dilute and/or concentrate a sample, such as a blood sample, or other biological samples.
Sample preparation may include adding an anti-coagulant or other ingredients to a sample. Sample preparation may also include purification of a sample. In embodiments, all sample processing, preparation, or assay actions or steps are performed by a single device. In embodiments, all sample processing, preparation, or assay actions or steps are performed within a housing of a single device. In embodiments, most sample processing, preparation, or assay actions or steps are performed by a single device, and may be perfomied within a housing of a single device. In embodiments, many sample processing, preparation, or assay actions or steps are performed by a single device, and may be performed within a housing of a single device. In embodiments, sample processing, preparation, or assay actions or steps may be performed by more than one device.
[001801 An automated assay device may be configured to run one or more assay on a sample, and to obtain data from the sample. An assay may include one or more physical or chemical treatments, and may include running one or more chemical or physical reactions.
An automated assay device may be configured to perform one, two or more assays on a small sample of bodily fluid. One or more chemical reaction may take place on a sample having a volume, as described elsewhere herein. For example one or more chemical reaction may take place in a pill having less than femtoliter volumes. In an instance, the sample collection unit is configured to receive a volume of the bodily fluid sample equivalent to a single drop or less of sample or interstitial fluid. In embodiments, the volume of a sample may be a small volume, where a small volume may be a volume that is less than about 1000 pL, or less than about 500 pL, or less than about 250 pL, or less than about 150 pL, or less than about 100 !IL, or less than about 75 pL, or less than about 50 gL, or less than about 40 pL, or less than about 20 pi., or less than about 10 tdõ or other small volume. In embodiments, all sample assay actions or steps are performed by a single device. In embodiments, sample processing, preparation, or assay actions or steps may be performed by more than one device.
[001811 An automated assay device may be configured to perform a plurality of assays on a sample. In embodiments, an automated assay device may be configured to perform a plurality of assays on a single sample. A plurality of assays may be run simultaneously; may be run sequentially; or some assays may be run simultaneously while others are run sequentially. One or more control assays and/or calibrators (e.g., including a configuration with a control of a calibrator for the assay/tests) can also be incorporated into the device;
control assays and assay on calibrators may be performed simultaneously with assays performed on a sample, or may be performed before or after assays performed on a sample, or any combination thereof. In embodiments, many sample assay actions or steps, of a plurality of assays, are performed by a single device, and may be performed within a housing of a single device. In embodiments, sample processing, preparation, or assay actions or steps may be performed by more than one device.

1001821 in embodiments, devices, and systems and methods comprising or using such devices, may comprise a detector configured to detect analyte in a sample. A
detector may be, for example, an optical detector, such as a spectrophotometer, a photomultiplier, a charge-coupled device, a camera, or other device or system configured to detect a light-based signal indicative of the presence of a analyte. In embodiments, a detector may be configured to, or be effective to, detect a signal comprising chemiluminescence, luminescence, fluorescence, absorbance, transmittance, turbidity, a color change, or other change in light, whether emitted, transmitted, or absorbed, effective to signal the presence of a analyte in a sample. In embodiments, a detector may comprise an electrochemical detector, or a temperature sensor, or a pH sensor, or a radiation sensor, or an ion-sensitive electrode, or other sensor capable of detecting the presence of a analyte in a sample.
1001831 Methods for detecting analyte include assays for detecting nucleic acids (e.g., DNA or RNA), assays for detecting peptides and proteins (including glycoproteins), assays for detecting other pathogen-related molecules, complement fixation assays, hemagglutinafion assays (e.g., for influenza), and other assays. Methods for detecting nucleic acids may be termed "nucleic acid assays" and include polymerase chain reaction (pcm methods (including quantitative PCR (qPCR), reverse-transcriptase PCD (RT-PCR), "real-time" PCR, one-step PCR, two-step PCR, and other methods known in the art.
Methods for detecting peptides and proteins include "antibody-based assays" such as, e.g., enzyme immunoassays such as Enzyme-Linked ImmtmoSorbent Assays (ELISAs) and other assays utilizing antibodies or antibody fragments, complement-based reactions, measurement of absorbance of ultraviolet or other frequency of light, assays utilizing specific receptor-ligand interactions, and other assays known in the art. Assays for detecting other pathogen-related molecules include assays for bacterial sugars and lipids (e.g., bacterial lipopolysaccharide (LPS)), and other assays known in the art. A detector for use with such assays may be an optical detector, a pH detector, an electrochemical detector, a temperature sensor, an ion-sensitive electrode, a radiation detector, or other detector.
1001841 An automated assay device may be configured to detect one or more signals relating to the sample. An automated assay device may be configured to identify one or more properties of the sample. For instance, the automated assay device may be configured to detect the presence or concentration of one analyte or a plurality of analytes or a disease condition in the sample (e.g., in or through a bodily fluid, secretion, tissue, or other sample).
Alternatively, the automated assay device may be configured to detect a signal or signals that may be analyzed to detect the presence or concentration of one or more analytes (which may be indicative of a disease condition) or a disease condition in the sample.
The signals may be analyzed on board the device, or at another location. Running a clinical test may or may not include any analysis or comparison of data collected.
[001851 A chemical reaction or other processing step may be performed, with or without the sample. Examples of steps, tests, or assays that may be prepared or run by the device may include, but are not limited to immunoassay, nucleic acid assay, receptor-based assay, cytometric assay, colorimetric assay, enzymatic assay, electrophoretic assay, electrochemical assay, spectroscopic assay, chromatographic assay, microscopic assay, topographic assay, calorimetric assay, turbidmetric assay, agglutination assay, radioisotope assay, viscometric assay, coagulation assay, clotting time assay, protein synthesis assay, histological assay, culture assay, osmolarity assay, and/or other types of assays, centrifugation, separation, filtration, dilution, enriching, purification, precipitation, pulverization, incubation, pipetting, transport, cell lysis, or other sample preparation action or steps, or combinations thereof. Steps, tests, or assays that may be prepared or run by the device may include imaging, including microscopy, cytometry, and other techniques preparing or utilizing images. Steps, tests, or assays that may be prepared or run by the device may further include an assessment of histology, morphology, kinematics, dynamics, and/or state of a sample, which may include such assessment for cells.
[001861 A device may be capable of performing all on-board steps (e.g., steps or actions performed by a single device) prior to the performance of a reflex test in a short amount of time. For example, a device may be capable of performing all on-board steps on a single sample in a short amount of time prior to the performance of a reflex test. For example, the am.ount of time between the time of sample collection from a subject to the time of transmitting data and/or analysis prior to the performance of a reflex test may be a short amount of time. For example, the amount of time between the time of accepting or placing a sample within the device to the time of transmitting data and/or analysis from the device regarding such a sample may depend on the type or number of steps, tests, or assays performed on the sample. In embodiments, the amount of time between the time of accepting or placing a sample within the device to the time of beginning to perform a subsequent assay (following performance of an initial assay, and following determining that a subsequent test is to be performed), may be a short amount of time. In embodiemnts, the amount of time between the time of accepting or placing a sample within the device to the time of obtaining a result from the performance of a subsequent assay (following perfonna3nce of an initial assay, and following determining that a subsequent test is to be performed), may be a short amount of time. In embodiments, the amount of time between the time of accepting or placing a sample within the device to transmitting data and/or analysis from the device regarding such a sample, prior to the performance of a reflex test, may be a short amount of time. In such examples and embodiments, and in other examples and embodiments disclosed herein, a short amount of time may comprise about 3 hours or less, about 2 hours or less, about 1 hour or less, about 50 minutes or less, about 45 minutes or less, about 40 minutes or less, about 30 minutes or less, about 20 minutes or less, about 15 minutes or less, about 10 minutes or less, about 5 minutes or less, about 4 minutes or less, about 3 minutes or less, about 2 minutes or less, or about 1 minute or less.
1001871 A device may be configured to prepare a sample for disposal, or to dispose of a sample, such as a biological sample, following processing or assaying of a sample.
[001881 In embodiments, an automated assay device may be configured to transmit data obtained from a sample. In embodiments, an automated assay device may be configured to communicate over a network. An automated assay device may include a communication module that may interface with the network. An automated assay device may be connected to the network via a wired connection or wirelessly. The network may be a local area network (LAN) or a wide area network (WAN) such as the Internet. In some embodiments, the network may be a personal area network. The network may include the cloud. The automated assay device may be connected to the network without requiting an intermediary device, or an intermediary device may be required to connect an automated assay device to a network. An automated assay device may conununicate over a network with another device, which may be any type of networked device, including but not limited to a personal computer, server computer, or laptop computer; personal digital assistants (PDAs) such as a Windows CE device; phones such as cellular phones, smartphones (e.g., iPhone, Android, Blackberry, etc.), or location-aware portable phones (such as GPS); a roaming device, such as a network-connected roaming device; a wireless device such as a wireless email device or other device capable of communicating wireless with a computer network; or any other type of network device that may communicate possibly over a network and handle electronic transactions. Such communication may include providing data to a cloud computing infrastructure or any other type of data storage infrastructure which may be accessed by other devices.

[001891 An automated assay device may provide data regarding a sample to, e.g., a health care professional, a health care professional location, such as a laboratory, or an affiliate thereof. One or more of a laboratory, health care professional, or subject may have a network device able to receive or access data provided by the automated assay device. An automated assay device may be configured to provide data regarding a sample to a database.
An automated assay device may be configured to provide data regarding a sample to an electronic medical records system, to a laboratory information system, to a laboratory automation system, or other system or software. An automated assay device may provide data in the form of a report.
[001901 In embodiments, devices, and systems and methods comprising or using such devices, may comprise a controller. In embodiments, a controller may comprise a processor.
In embodiments, a controller may be connected to, and may control the operation of, components of a device; such components are typically disposed within a housing of the device. In embodiments, a controller may control the operation of a fluid handling system. In embodiments, a controller may control the operation of a detector. In embodiments, a controller may control the operation of any component or unit of the device.
Other components may include, for example, a camera, a chemistry assay unit, a nucleic acid assay unit, a heating unit, a communication unit, a protein chemistry unit, or other component or unit. In embodiments, a controller may control the operation of one or more components of a device according to a protocol. In embodiments, a protocol by which a controller controls the operation of any one or more component or unit of a device may be preprogrammed, e.g., may be resident on the device. In embodiments, a protocol by which a controller controls the operation of any one or more component or unit of a device may be obtained from another device, or from a user, or from a laboratory, or from a network, or from the cloud. In embodiments, a protocol by which a controller controls the operation of any one or more component or unit of a device may be updated, or may be updatable, according to information or instructions from another device, or from a user, or from a laboratory, or from a network, or from the cloud. In embodiments, a device may receive information, or instructions, or updates, or protocols, via a user interface. In embodiments, a device may receive information, or instructions, or updates, or protocols, via a communication assembly.
[001911 In embodiments, devices, and systems and methods comprising or using such devices, may comprise a display effective to provide a user with information regarding the operation of the device, information regarding the progress of an assay performed by the device, or information regarding the results of an assay performed by the device. In embodiments, a display may comprise a visual display, or may comprise a printed display, or may comprise an audio signal, which may include an audio signal understandable as speech by a user, or may comprise any combination or all of such displays. In embodiments, a display may comprise a user interface. In embodiments in which a display comprises a user interface, a device may receive, e.g., information, commands, protocols, or other input. For example, a user interface may communicate a request for a second, or subsequent, biological sample. For example, a user interface may communicate instructions regarding obtaining a second, or subsequent, biological sample.
[00192] In embodiments, devices, and systems and methods comprising or using such devices, may comprise a communication assembly effective to communicate with one or more of a user, another device, a laboratory, a network, the cloud, or other communication target. In embodiments, a communication assembly may provide a communication target with information regarding the operation of the device, information regarding the progress of an assay performed by the device, or information regarding the results of an assay performed by the device. In embodiments, a communication assembly may be configured to allow a device to receive, e.g., information, commands, protocols, or other input from an outside source, such as, e.g., a user, another device, a laboratory, a network, the cloud, or other communication source.
[00193] In embodiments, a protocol may include instructions regarding one or more of:
preparation of a sample; preparation of a plurality of samples; performing a chemical reaction; performing a plurality of chemical reactions; sequence of performing a plurality of chemical reactions; performing a clinical test; performing a plurality of clinical tests;
sequence of performing a plurality of clinical tests; detecting the presence of an analyte;
detecting the presences of a plurality of analytes; sequence of detecting the presences of a plurality of analytes; detecting the concentration of an analyte; detecting the concentrations of a plurality of analytes; sequence of detecting the concentrations of a plurality of analytes; pre-processing data; and sequence of steps in processing data. In embodiments, protocol information may be changed according to transmitted data obtained from said biological sample within said housing of said device according to said protocol.
[00194] Order forms, methods of ordering clinical assays, and methods of performing clinical assays using such order forms are disclosed herein. In embodiments, such order forms include listings of a plurality of assays which may be ordered, where these listings include prices for each of the assays which may be ordered. In embodiments, methods for ordering clinical assays include marking order forms which include listings of a plurality of assays which may be ordered, where these listings include prices for each of the assays which may be ordered. In embodiments, methods for performing clinical assays include ordering clinical assays using order forms which include listings of a plurality of assays which may be ordered, where these listings include prices for each of the assays which may be ordered, and performing the clinical assays. These clinical assays may be performed on samples obtained from subjects at a single sample acquisition session; such samples may be small volume samples, e.g., small volume samples of blood or urine.
[001951 Accordingly, Applicant discloses methods of ordering assays. In embodiments, a method of ordering a clinical assay to be performed by a clinical testing facility on a sample obtained from a subject, the method comprising: Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form; Marking the order form to indicate the name and other identifying information of said subject; Marking the order form to identify one or more selected assays of the plurality of assays that are ordered for performance; and Providing the marked order form to the clinical testing facility. In embodiments of these methods, the sample is a small-volume biological sample having a volume of less than about 250 microliters (4). In embodiments of these methods, the sample is a blood sample or a urine sample. In embodiments of these methods, the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device.
[001961 Further embodiments of these methods comprise ordering a plurality of assays comprising an initial assay and a subsequent assay, wherein the sample is provided in a sample container that is disposed on a cartridge during placement of the sample within an automated sample analysis device, wherein said sample is divided into portions, said portions comprising a first portion and at least a second portion, and wherein said second portion is retained in said sample container disposed on the cartridge within the device for use in the subsequent assay. In embodiments of these methods, the cartridge contains all consumables used in the performance of the initial assay and of the at least one subsequent assay.
[001971 In embodiments of these methods, the sample is a small-volume biological sample having a volume of less than. about 250 microliters (1.11.). In embodiments of these methods, the sample is a blood sample or a urine sample. In embodiments of these methods, the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device. Embodiments of these methods comprise ordering a plurality of assays comprising an initial assay and a subsequent assay, wherein the sample is provided in a sample container that is disposed on a cartridge during placement of the sample within an automated sample analysis device, wherein said sample is divided into portions, said portions comprising a first portion and at least a second portion, and wherein said second portion is retained in said sample container disposed on the cartridge within the device for use in the subsequent assay. In embodiments of these methods, the cartridge contains all consumables used in the performance of the initial assay and of the at least one subsequent assay.
[001981 Accordingly, Applicant discloses methods of performing assays. In embodiments, a method of performing a clinical assay by a clinical testing facility on a sample obtained from a subject, the method comprising: Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form; Marking the order form to indicate the name and other identifying information of said subject; Marking the order form to identify one or more selected assays of the plurality of assays that are ordered for performance;
Providing the marked order form to the clinical testing facility; Obtaining a sample from said subject during a single sample acquisition session; and Performing said one or mom selected assays. In embodiments of these methods, the sample is a small-volume biological sample having a volume of less than about 250 microliters (.1i.1). In embodiments of these methods, the sample is a blood sample or a urine sample. In embodiments of these methods, the one or more selected assays comprise at least two linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay, wherein the results of said initial assay affect the decision whether or not to perform said at least one subsequent assay, or affect the decision whether or not to report results of said at least one subsequent assay. In embodiments of these methods, the sample is provided in. a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device.
1001991 Further embodiments of these methods comprise performing a plurality of assays comprising an initial assay and a subsequent assay, wherein the sample is provided in a sample container that is disposed on a cartridge during placement of the sample within an automated sample analysis device, further comprising dividing said sample into portions, said portions comprising a first portion and at least a second portion, and wherein said second portion is retained in said sample container disposed on the cartridge within the device for use in the subsequent assay. In embodiments of these methods, the sample is obtained from said subject during a single sample acquisition session. In embodiments, the sample is a blood sample or a urine sample. In embodiments, the cartridge contains all consumables used in the performance of the initial assay and of the at least one subsequent assay.
1002001 Applicant discloses further methods of ordering assays. Applicant discloses a method of ordering at least an initial clinical assay and a subsequent clinical assay for performance by a clinical testing facility on a sample obtained from a subject, wherein said initial assay and said subsequent assay each comprise detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising: Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay, wherein the performance of said at least one subsequent assay is contingent upon the results of said initial assay; Marking the order form to indicate the name and other identifying information of said subject; M.arking the order form to indicate linked assays selected for performance, wherein said initial assay of said linked assays is to be performed on the sample or portion thereof, and initial assay results are to be obtained, wherein said initial results may comprise either: i) a negative result indicating that the presence of said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the presence of the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an. abnormal level in the sample;
Wherein said at least one subsequent assay is to be performed only if further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical information: i) if the results of said initial assay comprise a positive result, and such positive result indicates that; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information; and providing said marked order form to said clinical testing facility.
[00201] Applicant discloses further methods of ordering assays. Applicant discloses a method of ordering at least two clinical assays to be performed by a clinical testing facility on a sample obtained from a subject, said at least two clinical assays comprising linked assays, wherein linked assays comprise an initial assay and at least one subsequent assay, wherein said linked assays each comprise detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising: Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay; Marking the order form to indicate the name and other identifying information of said subject; Marking the order form to indicate linked assays that have been selected for performance; wherein said initial assay and said at least one subsequent assay are to be performed on the sample or a portion thereof, and results obtained and reported, wherein said initial results may comprise either: i) a negative result indicating that said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an abnormal level in the sample; Wherein the results of said at least one subsequent assay are to be reported, contingent upon the results of said initial assay, only when further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical information: i) if the results of said initial assay comprise a positive result indicate the presence of the analyte or characteristic, and such positive result indicates that further assay results would provide additional useful clinical information; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information; and providing said marked order form to said clinical testing facility.
[00202] Methods of ordering may further comprise methods as disclosed above, wherein the at least one subsequent assay comprises an assay for detecting, identifying, or measuring: a) the same analyte or characteristic as the initial assay, and wherein the at least one subsequent assay is more sensitive for the detection or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay. The methods may further comprise methods wherein the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays. The methods may further comprise methods wherein the at least one subsequent assay comprises a different type of assay than the initial assay. The methods may further comprise methods wherein said cytometric assay comprises the detection, identification, or measurem.ent of a morphological characteristic of a cell. The methods may further comprise methods wherein said cytometric assay comprises the detection, identification, or measurement of a morphological characteristic of a cell. The methods may further comprise methods wherein the initial assay comprises the detection, identification, or measurement of art analyte, and the subsequent assay comprises a cytometric assay for the detection, identification, or measurement of a morphological characteristic of a cell. The methods may further comprise methods wherein the first portion of the sample is diluted prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted prior to, or during, the performance of the at least one subsequent assay, or both. In embodiments, the sample is a small-volume biological sample having a volume of less than about 250 microliters (IL).
1002031 In embodiments, the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device, the methods further comprising: dividing the sample into portions prior to the performance of said initial assay, wherein said portions comprise a first portion and at least a second portion. In embodiments, the sample in said sample container is disposed on a cartridge during placement of the sample within said automated sample analysis device, and wherein said second portion is retained on said cartridge within the automated sample analysis device for use in the at least one subsequent assay. In embodiments, the second portion is retained in said sample container disposed on the cartridge within the automated sample analysis device for use in the performance of the at least one subsequent assay. In embodiments, the second portion is retained in a second container that is disposed on the cartridge within the automated sample analysis device. In embodiments, the second container is a dedicated container designated for holding the second portion on the cartridge prior to the performance of the at least one subsequent assay. In embodiments, the second container is a dedicated container designated to contain the second portion during the performance of the at least one subsequent assay.
1002041 In embodiments of such methods, the performance of the subsequent assay is begun within a short amount of time after placing the sample within the automated sample analysis device, wherein the short amount of time is about two hours or less.
In embodiments of such methods, the automatic performance of the subsequent assay is completed within a short amount of time after placing the sample within the automated sample analysis device, wherein said short amount of time is about one hour or less. In embodiments of such methods, the sample is provided on a cartridge, and wherein said cartridge contains all consumables used in the performance of the initial assay and of the subsequent assay. In embodiments of such methods, the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (IL). In embodiments of such methods, the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the small-volume sample is diluted by at least about 100-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (1.11..).
1002051 In embodiments of the method disclosed herein, the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
In embodiments, the subsequent assay comprises a different type of assay than the initial assay. In embodiments, cytomenic assays comprise the detection, identification, or measurement of a morphological characteristic of a cell. In embodiments, the subsequent assay comprises an assay for detecting, identifying, or measuring: a) the same analyte or characteristic as the initial assay, wherein the subsequent assay is more sensitive for the detection, identification, or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay. In embodiments, the sample comprises a volume of less than about 100 microliters (ttL). In embodiments, the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
[00206] In embodiments of the methods of ordering disclosed herein, the results of the initial assay of the sample are to be reported. In embodiments of the methods of ordering disclosed herein, the results of the initial assay are to be reported prior to the performance of the subsequent assay. In embodiments of the methods of ordering disclosed herein, the results of the at least one subsequent assay are to be reported. In embodiments of the methods of ordering disclosed herein, the results of the initial assay and of the at least one subsequent assay are to be reported together.
1002071 In embodiments of the methods of ordering, the linked assays comprising an initial assay and at least one subsequent assay are selected from the group of linked assays consisting of: An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising manual differential and smear review; An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising an assay for anemia; An initial assay comprising an acute hepatitis panel, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C; An initial assay comprising one or more of celiac panel assays selected from fgA
and HLA-DQ
typing, and a subsequent assay comprising one or more of celiac reflex panel assays selected from tTG, DGP, EMA, and EMA titer; An initial assay comprising a Hepatitis C
antibody assay, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C;
An initial assay comprising an HIV antibody screening assay, and a subsequent assay comprising a confirmatory assay for HIV; An initial assay comprising an HIV
antibody screening assay, and a subsequent assay comprising an assay for differentiating between HIV-1 antibodies and HIV-2 antibodies; An initial assay comprising an assay for treponema pallidum IgG, and a subsequent assay comprising a rapid plasma reagin (RPR) assay; An initial assay comprising a rapid plasma reagin (RPR) assay, and a subsequent assay comprising an assay for an assay for antibodies to treponema pallidum; An initial assay comprising a thyroid stimulating hormone (TSH) assay, and a subsequent assay comprising comprising one or more of an assay for tri-iodothyronine (T3), thyroxine (T4), and thyroid uptake.
1002081 Accordingly, Applicant discloses methods of performing assays. In embodiments, a method of performing assays comprises a method of performing.
by a clinical testing facility, at least an initial clinical assay and a subsequent clinical assay on a sample obtained from a subject, wherein said initial assay and said at least one subsequent assay each comprise detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising: Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay, wherein the performance of said at least one subsequent assay is contingent upon the results of said initial assay;
Marking the order form to indicate the name and other identifying information of said subject; Marking the order form to indicate linked assays selected for performance; Providing the marked order form to the clinical testing facility; Obtaining a sample from said subject during a single sample acquisition session; Performing said initial assay of said linked assays selected for performance, using said sample, or a portion thereof, for the performance of said initial assay, whereby an initial result is obtained from the sample or a portion thereof, wherein said initial result may comprise either: i) a negative result indicating that the presence of said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the presence of the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an abnormal level in the sample; and Performing said at least one subsequent assay only if further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical infomiation: i) if the results of said initial assay comprise a positive result, and such positive result indicates that further assay results would provide additional useful clinical information; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information. In embodiments, such a method further comprised performing said at least one subsequent assay using said sample, or a portion thereof, for the performance of said at least one subsequent assay.
[002091 Applicant discloses further methods of performing assays. In embodiments, a method of performing assays comprises a method of performing, by a clinical testing facility, at least two linked assays on a sample obtained from a subject, said linked assays comprising an initial assay and at least one subsequent assay, each of said linked assays comprising detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising: Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay; Marking the order form to indicate the name and other identifying information of said subject; Marking the order form to indicate linked assays that have been selected for performance; Providing the marked order form to the clinical testing facility; Obtaining a sample from said subject at a single sample acquisition session; Performing said initial assay on said sample, or a portion thereof, effective to obtain results of said initial assay; wherein said initial result may comprise either:
i) a negative result indicating that said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an abnormal level in the sample; Reporting the initial results;
Performing said at least one subsequent assay on said sample, or a portion thereof, effective to obtain results of said at least one subsequent assay; Reporting the results of said at least one subsequent assay only when further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical information: i) if the results of said initial assay comprise a positive result indicate the presence of the analyte or characteristic, and such positive result indicates that further assay results would provide additional useful clinical information; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information.
1002101 In embodiments of the methods of performing assays disclosed herein, the at least one subsequent assay comprises an assay for detecting, identifying, or measuring: a) the same analyte or characteristic as the initial assay, and wherein the at least one subsequent assay is more sensitive for the detection or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay.
10021.1] In embodiments, the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and crometric assays. In embodiments, the at least one subsequent assay comprises a different type of assay than the initial assay. In embodiments, the cytometric assay comprises the detection, identification, or measurement of a morphological characteristic of a cell. In embodiments, the initial assay comprises the detection, identification, or measurement of an analyte, and the subsequent assay comprises a cytometric assay for the detection, identification, or measurement of a morphological characteristic of a cell.

[002121 in embodiments of the methods of performing assays, the first portion of the sample is diluted prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted prior to, or during, the performance of the at least one subsequent assay, or both. In embodiments, the sample is a small-volume biological sample having a volume of less than about 250 microliters (p.I.).
[002131 In embodiments, the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device, further comprising: Dividing the sample into portions prior to the performance of said initial assay, said portions comprising a first portion and at least a second portion.
In embodiments, the sample in said sample container is disposed on a cartridge during placement of the sample within said automated sample analysis device, and wherein said second portion is retained on said cartridge within the automated sample analysis device for use in the at least one subsequent assay. In embodiments, said second portion is retained in said sample container disposed on the cartridge within the automated sample analysis device for use in. the performance of the at least one subsequent assay. In embodiments, said second portion is retained in a second container that is disposed on the cartridge within the automated sample analysis device. In embodiments, said second container is a dedicated container designated for holding the second portion on the cartridge prior to the performance of the at least one subsequent assay. In embodiments, the second container is a dedicated container designated to contain the second portion during the performance of the at least one subsequent assay.
[002141 In embodiments, the performance of the subsequent assay is begun within a short amount of time after placing the sample within the automated sample analysis device, wherein the short amount of time is about two hours or less. In embodiments, the automatic performance of the subsequent assay is completed within a short amount of time after placing the sample within the automated sample analysis device, wherein said short amount of time is about one hour or less. In embodiments, the sample is provided on a cartridge, and wherein said cartridge contains all consumables used in the performance of the initial assay and of the subsequent assay.
[00215] In embodiments, the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (RP. In embodiments, the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the small-volume sample is diluted by at least about 100-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters ( L). In embodiments, the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays. In embodiments, the subsequent assay comprises a different type of assay than. the initial assay. In em.bodiments, the cytometric assays comprise the detection, identification, or measurement of a morphological characteristic of a cell.
[00216] In embodiments, the subsequent assay comprises an assay for detecting, identifying, or measuring: a) the sam.e analyte or characteristic as the initial assay, wherein the subsequent assay is more sensitive for the detection, identification, or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay.
[00217] In embodiments of methods of performing assays, the sample comprises a volume of less than about 100 microliters (gL). In embodiments, the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
1002181 In embodiments, the methods of performing assays further comprise:
Reporting the results of the initial assay of the sample. In embodiments, the results of the initial assay are reported prior to the performance of the subsequent assay.
In embodiments, the methods comprise reporting the results of the subsequent assay. in embodiments, the results of the initial assay and of the subsequent assay are reported together.
[00219] In embodiments of the methods of performing assays, the linked assays comprising an initial assay and at least one subsequent assay are selected from the group of linked assays consisting of An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising manual differential and smear review; An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising an assay for anemia; An initial assay comprising an acute hepatitis panel, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C; An initial assay comprising one or more of celiac panel assays selected from IgA
and HLA-DQ typing, and a subsequent assay comprising one or more of celiac reflex panel assays selected from tTG, DGP, EMA, and EMA titer; An initial assay comprising a Hepatitis C antibody assay, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C; An initial assay comprising an HIV antibody screening assay, and a subsequent assay comprising a confirmatory assay for HIV; An initial assay comprising an HIV antibody screening assay, and a subsequent assay comprising an assay for differentiating between HIV-1 antibodies and HIV-2 antibodies; An initial assay comprising an assay for treponema pallidum IgG, and a subsequent assay comprising a rapid plasma reagin (RPR) assay; An initial assay comprising a rapid plasma reagin (RPR) assay, and a subsequent assay comprising an assay for an assay for antibodies to treponema pallidum; An initial assay comprising a thyroid stimulating hormone (TSH) assay, and a subsequent assay comprising comprising one or more of an. assay for tri-iodothyronine (T3), thyroxine (T4), and thyroid uptake.
[002201 In embodiments of the methods of performing assays, the sample is obtained in a single sample acquisition session, and wherein said sample comprises a volume sufficient for the performance of an initial assay and a subsequent assay. In embodiments, the sample is a small-volume sample having a volume of less than about 250 microliters (IL).
In embodiments, the sample is a small-volume sample having a volume of less than about 100 microliters (IL). In embodiments, the sample is a small-volume blood sample or a small-volume urine sample. In embodiments, the sample is a blood sample obtained by fingerstick.
[00221] Applicant further provides order forms (also termed "order sheet", "lab order form", "lab order sheet", "assay form", "assay sheet", "assay order form", "assay order sheet", "test form", "test sheet", "test order form", "test order sheet", "medical order form", "medical order sheet", "medical test fonn", "medical test sheet", and plurals and similar and related terms) configured for ordering clinical diagnostic assays for testing biological samples obtained from human subjects. Examples of order forms for ordering a clinical assay having features as disclosed herein are shown in Figs. 5A, 5B, and 6. In embodiments, the order forms for ordering a clinical assay provide lists of clinical assays with prices for each clinical assay. In embodiments, the list of clinical assays identifies each assay by at least one of: an assay name; an identifying acronym; an identifying number; an identifying alphanumeric designation; or combinations thereof. In embodiments, the order forms provide lists of panels of clinical assays with prices for each panel. In embodiments, the order forms provide lists of clinical assays with prices for each clinical assay, where the clinical assays that are listed include initial assays and corresponding subsequent assays, and corresponding prices, with explicit indications of the connection between such initial assays and corresponding subsequent assays. The corresponding prices listed include prices for the subsequent assays and for the initial assays; in embodiments, a combined price may be listed for an initial assay and a subsequent assay. In embodiments, prices are listed for panels of assays, which may include initial assays and subsequent assays.
[00222] In embodiments, the order forms provide lists of clinical assays with prices for each clinical assay, where the clinical assays that are listed include initial assays and corresponding subsequent assays, and prices for the initial and corresponding assays, with explicit indications of the connection between such initial assays and corresponding subsequent assays. The listed prices include prices for the subsequent assays and for the initial assays; in embodiments, a combined price may be listed for an initial assay and a subsequent assay.
[00223] in embodiments, the order forms include lists of assays performed together in panels; in embodiments, the order forms include explanations of the assays to be performed, including explanations of the panels or of the assays performed together in panels. In embodiments, some or all of the more commonly performed assays may be listed near to each other, or together, on the order forms. In embodiments, some or all of the assays listed on the order forms may be listed near to, or grouped together with, other assays which may commonly be ordered for patients exhibiting a common symptom or common symptoms. In embodiments, some or all of the assays listed on the order forms may be listed near to, or grouped together with, other assays which may commonly be ordered for patients sharing common risk factors. In embodiments, some or all of the assays listed on the order forms may be listed near to, or grouped together with, other assays which may commonly be ordered for patients sharing common age, sex, activity, or other characteristic. In embodiments, some or all of the assays listed on the order forms may be listed alphabetically.
[00224] In embodiments, the order forms include check boxes for selecting and indicating assays to be performed (including reflex assays which may be performed, continent upon the results of initial assays); lines, boxes, or other locations for providing patient identification and contact information; lines, boxes, or other locations for providing clinician information; lines, boxes, or other locations for providing payer information (e.g., patient payment information, insurance company information, or other information regarding payment); lines, boxes, or other locations for providing clinic information;
lines, boxes, or other locations for providing reimbursement or other code information (e.g., 1CD-9, ICD-9-CM, ICD-10, 1CD-10-CM, ICPM, DSM, CPT, or other code for classifying a disease, treatment, test, or for insurance reimbursement; etc.). In embodiments, the samples may include blood samples, urine samples, and other biological samples.
[002251 Applicant discloses a method of providing a clinical assay performed on a sample obtained from a subject, comprising: Providing an order form listing clinical assays and prices for each of said clinical assays; Selecting one or more clinical assays from the order form; Obtaining a sample from the subject; Performing the selected clinical assay or assays; and Collecting payment for the selected clinical assay or assays, where the payment is determined by the price for each clinical assay listed on the order form. The order form may comprise any order form discussed or shown herein. The clinical assay or assays may be performed according to any method disclosed herein. In embodiments, the clinical assays listed on the order form are listed in panels, and the order forms comprise prices for each panel. In embodiments, the prices for each panel comprise capped prices for at least one panel, wherein a capped price is a m.aximum price for any combination of individual clinical assays of a panel. In embodiments, the payment is collected, at least in part, from the subject, or from an insurance company, or from a government agency, or from the subject's employer, or any combination thereof.
[002261 Applicant discloses a further method of providing a clinical assay performed on a sample obtained from a subject, comprising: Providing an order form listing clinical assays, prices for each of said clinical assays, and CPT codes for each of said clinical assays;
Selecting one or more clinical assays from the order form; Obtaining a sample from said subject; Performing the selected clinical assays; and Collecting payment for the selected clinical assay or assays, where the payment is determined by the price for each clinical assay listed on the order form. The order form may comprise any order form described or shown herein. The clinical assay may be performed according to a method as disclosed herein. In embodiments, the clinical assays listed on the order form comprise clinical assays listed in panels, and the order forms comprise prices and a CPT code or CPT codes for each panel. In embodiments, the prices for each panel comprise capped prices for at least one panel, wherein a capped price is a maximum price for any combination of individual clinical assays of a panel. In embodiments, the payment is collected, at least in part, from the subject, or from an insurance company, or from a government agency, or from the subject's employer, or any combination thereof.
1002271 Applicant further discloses a method of doing business, comprising:
Providing an order form listing clinical assays and prices for each of said clinical assays; Selecting one or more clinical assays from the order form; Obtaining a sample from a subject; Performing the selected clinical assay or assays; and Collecting payment for the selected clinical assay or assays, where the payment is determined by the price for each clinical assay listed on the order form. The order form may comprise any order form discussed or shown herein. The clinical assays may be performed according to any method disclosed herein. In embodiments, the clinical assays listed on the order form comprise clinical assays listed in panels, and the order forms comprise prices for each panel. In embodiments, the prices for each panel comprise capped prices for at least one panel, wherein a capped price is a maximum price for any combination of individual clinical assays of a panel. In embodiments, the payment is collected, at least in part, from the subject, or from an insurance company, or from a government agency, or the subject's employer, or any combination thereof 1002281 Applicant further discloses a method of doing business, comprising:
Providing an order form listing clinical assays, prices for each of said clinical assays, and CPT codes for each of said clinical assays; Selecting one or more clinical assays from the order form;
Obtaining a sample from a subject; Performing the selected clinical assay or assays; and Collecting payment for the clinical selected assay or assays, where the payment is determined by the price for each clinical assay listed on the order form. The order form may comprise any order form discussed or shown herein. The clinical assays may be performed according to any method disclosed herein. In embodiments, the clinical assays listed on the order form comprise clinical assays listed in panels, and the order form comprises prices and CPT codes for each panel. In embodiments, prices for each panel comprise capped prices for at least one panel, wherein a capped price is a maximum price for any combination of individual clinical assays of a panel. In embodiments, the payment is collected, at least in part, from the subject, or from an insurance company, or from a government agency, or from the subject's employer, or any combination thereof.
1002291 Embodiments of the methods, assays, and order forms disclosed herein are further described in the following examples.
Example 1 ¨ HIV Assays [002301 Assays for the detection of human immunodeficiency virus (HIV) in a biological sample typically report negative results, i.e., no HIV is detected in the sample.
However, since detection of the virus in a biological sample is of such importance, and may have such dire consequences for the subject from whom the sample is obtained, Applicant discloses methods for performing confirmatory follow-up testing on the basis of positive results to an initial assay. In embodiments, such confirmatory follow-up testing may be performed prior to reporting positive assay results to a health-care provider or to a subject. In embodiments, such confirmatory follow-up testing may be performed concurrent with, or following, the reporting of positive assay results to a health-care provider or to a subject.
[00231] In embodiments, an anti-HIV antibody screening test may be performed on a biological sample obtained from a subject; for example, an anti-HIV antibody screening test may be performed on a sample of blood obtained from a subject. In embodiments, an anti-HIV antibody screening test may be performed on a sample of a bodily fluid obtained from a subject other than a blood sample; such a sample may be, for example, a sample of urine, sputum, semen, tears, interstitial fluid, a sample obtained from a nasal swab, a sample obtained from a throat swab, a sample obtained from a vaginal swab, or other sample. In embodiments, a sufficient amount of a biological sample is obtained so as to allow the automatic reflex testing of the sample, or of a portion of the sample, without need to obtain a further sample in order to perform a subsequent assay. In embodiments, only the amount of a biological sample needed for an initial assay or assays is obtained; in such an embodiment, a krther biological sample must later be obtained if needed in order to perform a subsequent assay contingent on the results of an initial assay.
002321 For example, an antibody-based assay may comprise contacting a sample with a substrate to which antibodies specific for a target antigen are bound. In embodiments of such an antibody-based assay, the sample may be mixed, or diluted, with a reagent containing a known amount of labeled conjugate, where the labeled conjugate binds the bound antibodies. For example, the labeled conjugate may be a conjugate comprising the target analyte covalently linked with a detectable label. The mixture of sample and reagent may be then added to a chamber containing the substrate to which the antibodies are bound, and left to incubate for a sufficient time for target analyte, and analyte-conjugate, to bind to the substrate. Following the incubation period, the chamber may be washed with a washing solution, in order to wash out any remaining unbound analyte and unbound analyte-conjugate. After washing, the amount of labeled conjugate bound to the substrate may be determined, and the amount of target artalyte in the sample determined. Such a determination may be made, e.g., by comparison with the amount of labeled conjugate bound in the absence of any target, and optionally by comparison with the amount of labeled conjugate bound in the presence of one or more known amounts of, or otherwise by comparison with control values, such as with a control curve. For example, after washing, a reagent allowing the detection of the label may be added to the chamber (e.g., where the label is an chemiluminescent label which emits light in the presence of a substrate, the reagent allowing the detection of the label may comprise the required substrate).
1002331 In embodiments, the anti-HIV antibody screening test may comprise an anti-HIV-1 antibody screening test. In embodiments, the anti-HIV antibody screening test may comprise an anti-HIV-2 antibody screening test. In embodiments, the anti-HIV
antibody screening test may comprise an anti-HIV-I and an anti-HIV-2 antibody screening test. In embodiments, the possible results of such an assay may comprise a result reporting that the sample is negative for the presence of HIV-1, and may comprise a result reporting that the sample is positive for the presence of HIV-1; in such embodiments, a normal result comprises a result reporting that the sample is negative for the presence of HIV-1. In embodiments, the possible results of such an assay may comprise a result reporting that the sample is negative for the presence of HIV-2, and may comprise a result reporting that the sample is positive for the presence of HIV-2; in such embodiments, a nomial result comprises a result reporting that the sample is negative for the presence of HIV-2.
1002341 As disclosed herein, where the result of such a test comprises a result that the sample is negative for the presence of HIV-1, or negative for the presence of HIV-2, no further HIV test is automatically performed. As disclosed herein, where the HIV test comprises testing for the presence of both HIV-1 and H1V-2, and the result of such a test comprises a result that the sample is negative for the presence of HIV-1 and is negative for the presence of H1V-2, no further HIV test is automatically performed.
1002351 As disclosed herein, where the result of such a test comprises a result that the sample is positive for the presence of HIV-1, or is positive for the presence of H1V-2, or is positive for the presence of both HIV-1 and HIV-2, a further HIV test is automatically performed. Such a further HIV test may be performed on the same sample, or on an additional sample. In embodiments where the further HIV test is performed on an additional sample, the additional sample may be, or may be obtained from, a sample that was originally obtained from the subject; or, in embodiments, a further sample may be obtained from the subject and used for the further HIV test.
[00236] in embodiments, where a further HIV test is automatically perfonned subsequent to a positive result obtained from an initial HIV test, the further HIV test may comprise a nucleic acid assay. For example, in embodiments, where a further HIV test is automatically performed subsequent to a positive result obtained from an initial HIV test, the further HIV test may comprise a Western Blot HIV test.
[00237] In embodiments, the possible results of such a further HIV test may comprise a result reporting that the sample is negative for the presence of HIV-1, or is negative for the presence of HIV-2, or is negative for the presence of both HIV-1 and HIV-2. In such embodiments, a normal result comprises a result reporting that the sample is negative for the presence of HIV. As disclosed herein, where the result of a further HW test comprises a result that the sample is negative for the presence of H1V-1, or negative for the presence of HIV-2, no further HTV test is automatically performed. Such results may be reported to a health-care provider, or to a subject, if appropriate, as indicating that the biological sample obtained from the subject is free of HIV.
1002381 However, in embodiments, results of such a further HIV test may comprise a result reporting that the sample is positive for the presence of HIV-1, or is positive for the presence of HIV-2, or is positive for the presence of both HIV-1 and HIV-2.
[00239] However, where the result of a further HIV test comprises a result that the sample is positive for the presence of HIV-1, or positive for the presence of HIV-2, such a result indicates that HIV is present in the biological sample obtained from the subject.
Accordingly, such results indicate that the subject may suffer from HIV. Such results may be reported to a health-care provider, or to a subject, if appropriate, as indicating that the biological sample obtained from the subject contains HIV.
[00240] Additional testing may be indicated in the event of a positive result for either HIV-1 or HIV-2. For example, since HIV is a disorder of the immune system, cytometric testing directed to blood, and particularly to white blood cells, in which the blood cells in a sample of blood obtained from a patient, may be performed contingent on positive HIV
results. Examples of cytometric tests are provided herein in a subsequent example.
Example 2 ¨ White Blood Cell Count Assays 1002411 in embodiments, an initial assay may be an assay for the determination of the white blood cell count of a sample of blood obtained from a subject. For example, white blood cell count may be obtained as part of a complete blood cell count. A
white blood cell count assay may determine that the white blood cell count of blood obtained from a subject falls outside a normal range, e.g., may be below about 2000 cells per microliter (gL). Where an initial white blood cell count assay result determines that the white blood cell count of a blood sample is outside of a normal range, a reflex blood test may be required. Accordingly, Applicant discloses methods for performing confirmatory follow-up testing on the basis of white blood cell count results that fall outside of a pre-determined range. In embodiments, such confirmatory follow-up testing may be performed prior to reporting the initial white blood cell count results to a health-care provider or to a subject.
1002421 For example a normal range for an adult male subject (e.g., a male subject over the age of 12) may be between 3200 white blood cells/RL and 10,600 white blood cells/RI,. If the result of an initial test is that a blood sample has a white blood cell count of less than 2000 white blood cells/RI, (e.g., a white blood cell count of 1800 cells/RL), then a reflex blood test may be performed. Such a reflex blood test may comprise cytometric examination of the blood of the subject, such as a cytometric examination of white blood cells in a sample of blood from the subject. In embodiments, the sample of blood examined in the reflex assay may be a portion of the sample of blood examined in the initial test, e.g., where a portion of the sample is retained for finther testing. In embodiments, the sample of blood examined in the reflex assay may be a sample of blood obtained at the same time as the sample of blood examined in the initial test, e.g., where a second blood sample is obtained and retained for further testing. In embodiments, a subsequent sample of blood may be obtained from the subject for reflex testing at a time after the time of obtaining the initial sample of blood. In embodiments, the subsequent sample of blood may be obtained following determination of the results of the initial assay.
1002431 As discussed in more detail in the following Example, an automatic cytometric assay may be used to identify, quantify, and classify white blood cells in a sample. Blood samples may be pre-treated so as to avoid interference by red blood cells and platelets, e.g., by causing swelling and lysis of red blood cells and platelets, allowing white blood cells to settle and attach to a substrate, or by other means. White blood cells may be contacted with one or more labels specific for cell markers, which are thus useful for identifying and classifying the white blood cells. Such labels may include labels, such as fluorescent labels, to ease the detection of the labels and of cells labeled thereby.
Example 3 ¨ Cytornetrv [002441 Assays and tests that may be contingent on the results of an initial test may include assays and tests that observe and describe cells in a biological sample, including assays and tests that identify cells, that determine the numbers of cells of one or more populations of cells, or that determine whether or not abnormal cells are present, or whether or not abnonnal numbers of a cell type or cell types are present. Such tests and assays may utilize cytometry.
[002451 Cytometry may include preparing and analyzing two-dimensional images of cells in. a biological sample, where the cells are labeled (e.g., with fluorescent, chemiluminescent, enzymatic, or other labels) and plated (e.g., allowed to settle on a substrate) and imaged by a camera. The camera may include a lens, and may be attached to or used in conjunction with a microscope. Cells may be identified in the two-dimensional images by their attached labels (e.g., from light emitted by the labels).
[002461 80 microliters of whole blood obtained from a fingerstick was loaded into a capped vessel preloaded with 2 mg/ml EDTA. The capped vessel was centrifuged at 1200 x g for 5 minutes, to separate the blood cells from the blood plasma.
Centrifugation of the capped vessel resulted in the separation of the blood sample in the capped vessel into two major components (from top of the capped vessel to the bottom): 1) blood plasma and 2) packed blood cells. This process ensures that no droplets of blood remain isolated, but coalesce with the main body of the liquid. In addition, this process separates the cells from elements of the plasma thus reducing metabolism and allowing for longer storage of the sample.
[002471 The centrifuged capped vessel was loaded into a cartridge containing multiple fluidically isolated reagents, tips, and a cytometry cuvette. The cartridge contained all the reagents required for the assay. The cartridge was loaded into a device equipped with at least a centrifuge, a pipette and a platfonn to load the cuvette. The pipette in the device has a plurality of nozzles, some nozzles being of a different size than some other nozzles.
[002481 Inside the device, a nozzle on the pipette was lowered on a cuvette carrier tool causing it to engage a corresponding hole on the carrier tool. This tool was subsequently moved to the cartridge and lowered on the cytometer cuvette. Pins on the tool were then able to engage corresponding holes on the cuvette and pick it up. The cuvette was transferred to a loading station elsewhere in the device.

[002491 Next, inside the device, a larger nozzle of the pipette was lowered into the cartridge to engage a pipette tip stored in the cartridge. The pipette and tip together were then used to mix the cells and plasma in the capped vessel by positioning the pipette tip within the sample in the capped vessel and repeatedly aspirating material into and dispensing material from the tip. Once the cells were resuspended in the plasma so that the whole blood sample was thoroughly mixed, 5 microliters of the mixed whole blood was aspirated to provide an aliquot for measurements of properties of the blood sample. This 5 microliter aliquot was used for measurements directed to the red blood cells and platelets in the sample. As discussed below, a portion of the sample remaining after removal of this 5 microliter aliquot was used for measurements directed at white blood cells in the sample.
1002501 The 5 microliters of whole blood was dispensed into a vessel containing a mixture of phosphate buffered saline and 2% by weight of bovine serum albumin, to dilute the whole blood twenty-fold (resulting in 100 microliters of diluted sample).
After mixing vigorously, 5 microliters of this sample was transferred to another vessel containing a cocktail of labeling antibody reagents: anti-CD235a conjugated to alexa-fluor 647 (AF647), anti-CD41 and anti-CD61 conjugated to phycoerythrin (PE). The mixture was incubated for 5 minutes. Subsequently, 10 microliters of this mixture was mixed with 90 microliters of a buffer containing a zwitterionic surfactant at <0.1% by weight. The surfactant molecules modify bending properties of the red cell membrane such that all cells assume a stable spherical shape. This transformation is isovolumetric as the buffer used is isotonic with cytoplasm and no exchange of fluid can occur across the cell membrane. After incubating this for another 2 minutes, 30 microliters of this solution was mixed with a solution containing glutaraldehyde, a fixative and non-fluorescent beads of 10um diameter. The mixture had a final concentration of 0.1% glutaraldehyde and 1000 beads per microliter.
Glutaraldehyde rapidly fixes cells thus preventing cell lysis and other active biological processes.
[00251] The pipette then engaged a tip in the cartridge, aspirated 7 microliters (1IL) of the above mixture of and loaded the 7 1., into a channel within the cuvette placed on a platform with the carrier tool. After the mixture was loaded in into cuvette, the pipette aspirated 10 1.tL of mineral oil from a vessel in the cartridge, and placed a drop of mineral oil on both open ends of the loaded channel of the cuvette. Mineral oil was added to the ends of the open channel to prevent evaporation of liquid from the loaded cuvette channel. Next, the device-level sample handling apparatus engaged the cuvette carrier / cuvette combination, and transported the cuvette carrier / cuvette combination from the module containing the cartridge to the cytometry module of the device. At the cytometry module, the device-level sample handling apparatus placed the cuvette carrier / cuvette combination on the microscopy stage of the cytometry module. The time required for these operations, in addition to a 2 minute wait time allowed the swollen cells to settle to the floor of the cuvette prior to imaging.
1002521 After the cuvette carrier/cuvette was placed on the microscopy stage, the stage was moved to pre-determined location so that the optical system of the cytometer could view one end of the channel containing the sample. At this location, the optical system relayed images of the sample acquired with darkfield illumination from a ringlight. These images coupled with actuation of the optical system on an axis perpendicular to the plane of the cuvette were used to find the plane of best focus. Once focused, the optical system was used to acquire fluorescence images of the sample at different wavelengths, commensurate with the fluorophores that were being used. For example, to visualize red blood cells that had been labeled with anti-CD235 conjugated to alexa fluor 647, a red (630 nanometer (nm) wavelength) light source was used to excite the sample and wavelengths between 650nm and 700nm were used to image the sample. A combination of a polychroic mirror and a bandpass emission filter was used to filter out unwanted wavelengths from the optical signal. Since the cells had settled on the floor of the cuvette, images at a single plane of focus were sufficient to visualize all cells in the region.
1002531 Data from the images was processed by a controller associated with the automated assay device. The image processing algorithms employed here utilized fluorescence images of cells to detect them using a combination of adaptive thresholding and edge detection. Based on local intensity and intensity gradients, regions of interest (RoI) were created around each cell. Using darkfield images, beads in the sample were also identified and RUN were created around the beads. All the Rols in each field of view were enumerated and their intensity in each image of that field of view were calculated. The information output by the image processing algorithm consisted of shape or morphometric measurements and fluorescence and darkfield intensities for each Rol. This information was analyzed using statistical methods to classify each object as either a red blood cell (positive for CD235a, but negative for CD41/CD61), a platelet (positive for CD41/CD61 and negative CD235a) or a bead. The shape descriptors such as perimeter, diameter and circularity were used to calculate the volume of each red blood cell and platelet. Since the beads were added at a known concentration, the average ratio of beads to cells over the whole channel was used to calculate cell concentration in terms of cells/microliter. Based on the steps performed for processing the sample, this concentration was corrected for dilution to arrive at concentration of cells in the original whole blood sample. The following quantities were calculated from a sample: 1) number of red blood cells in the cuvette; 2) average volume of red blood cells in the cuvette;
3) red blood cell distribution width (RDW) of red blood cells in the cuvette;
4) number of platelets in the cuvette; and 5) average volume of platelets in the cuvette.
Based on these calculations, the following was calculated for the original blood sample.
Measured Value Result Exemplary Range Concentration of red blood cells (million cells per 4.8 4-6 microliter) Mean volume of red blood cells, femtoliter 88 80-100 red blood cell distribution width (RDW) (%) 12 11-14.6 Concentration of platelets (thousand cells per 254 150-400 microliter) Mean volume of platelets, femtoliter 10.4 7.5-11.5 1002541 After removal of the 5 LLL aliquot used for analysis of RBC and platelet information, the remaining 75 LLL of sample was used to analyze the white blood cell population of the whole blood sample. The remaining 75 uL of whole blood had also been mixed by repeatedly aspirating and dispensing the sample within the same the vessel by the pipette. Approximately 40 !IL of the remaining 75 FiL of mixed whole blood was aspirated into a pipette tip, and transferred by the pipette to a centrifuge tube in the cartridge. The centrifuge tube containing the blood sample was engaged by the pipette, and transferred to and deposited in a swinging bucket in a centrifuge within the module. The centrifuge was spun to provide 1200 x g for 3 minutes, separating the blood into EDTA-containing plasma as the supernatant and packed cells in the pellet.
[00255] After centrifugation, the centrifuge tube was removed from the centrifuge and returned to the cartridge. The plasma supernatant was rem.oved by the pipette and transferred to a separate reaction vessel in the cartridge. From a reagent vessel in the cartridge, 16 pt of resuspension buffer was aspirated by the pipette, and added to the cell pellet in the centrifuge tube. The pipette then resuspended the cell pellet in the resuspension buffer by repeatedly aspirating and dispensing the mixture in the centrifuge tube. Next, the pipette aspirated 21 microliters of the resuspended whole blood and added it to another vessel containing 2 microliters of anti-CD14-pacific blue and DRAQ5 , mixed, and incubated for 2 minutes.

Twenty microliters of this mixture was then added to 80 microliters of a lysis buffer. The lysis buffer is a solution of a gentle surfactant such a saponin in conjunction with a fixative such as paraformaldehyde. The detergent causes a large number of holes to be formed in the membranes of cells. Red blood cells, due to their unique membrane properties, are particularly susceptible to this hole formation and lyse completely, their contents leaking out into the liquid around. Presence of the fixative prevents unintentional lysis of the white blood cells. Platelets also remain unlysed. The purpose of this step is to remove red blood cells from the mixture as they outnumber white blood cells by about 1000:1. Platelets do not interfere with imaging and hence are irrelevant to this process. The lysis buffer also contained 10 uM
non-fluorescent beads at a known concentration.
[00256] After a 5 minute incubation, the vessel was spun again at 1200 x g for 3 minutes. The supernatant was aspirated by a pipette tip, removing the red blood cell ghosts and other debris, and deposited into a waste area in the cartridge.
Approximately 15 uL of liquid with packed white blood cells were present in the cell pellet.
1002571 in order to determine a rough approximation of the number of white blood cells present in the cell pellet, the pipette first resuspended the white blood cells in the vessel and then aspirated the liquid, transferred it to spectrophotometer in the blade The white blood cell suspension was illuminated with light at a wavelength of 632 nm, which is the excitation wavelength for alexa fluor 647 dye and DRAQ5 . The light emitted by the cell suspension was filtered by a 650 nm long pass filter and measured in the spectrophotometer. This measurement was correlated with previously generated calibration curve to estimate a rough concentration of white blood cells in the cell suspension. Typically, cell concentrations ranged from about 1000 cells per fiL to about 100,000 cells per IA¨ This estimate was used to calculate an appropriate dilution factor to ensure that the concentration of cells in the cuvette was constrained to within a two-fold range around a pre-defined target concentration. The purpose of this step was to ensure that cells are not present at too high or too low a density on the cuvette. If the cell density is too high, the accuracy of image processing algorithms is compromised, and if the cell density is too low, an insufficient number of cells are sampled.
1002581 Based on the dilution factor calculated in the above step, a diluent containing labeled antibodies against CD45 (pan-leukocyte marker), CD16 (neutrophil marker) and CD123 (basophil marker) was added to the cell suspension and mixed.
[002591 Once the cuvette in complex with cuvette carrier was placed on the cuvette carrier block, 10 Lt1 of the mixture of white blood cells resuspended in cytometry buffer was loaded into each of two channels in the cuvette. After the mixture was loaded into channels of the cuvette, the pipette aspirated 10 I of mineral oil from a vessel in the cartridge, and placed a drop of mineral oil on both open ends of both channels in the cuvette loaded with white blood cells.
[002601 Next, the device-level sample handling apparatus engaged the cuvette carrier /
cuvette combination, and transported the cuvette carrier / cuvette combination from the module containing the cartridge to the cytometry module of the device. At the cytometry module, the device-level sample handling apparatus placed the cuvette carrier / cuvette combination on the microscopy stage of the cytometry module. After the cuvette carrier /
cuvette was placed on the microscopy stage, the two channels of the cuvette containing white blood cells were imaged as described above for the RBC / platelet mixture.
[00261] Darkfield images of the white blood cells were used to count the numbers of cells in a field (as shown in Fig. IA). Cell surface markers were used to determine the cell type of individual white blood cells in an image; for example, CD14 marks monocytes;
CD123 marks basophils; CD16 marks neutrophils; and CD45-AF647 were used to mark all leukocytes (Figs. 1B-1E). The nuclear stain DRAQ5 was used to mark cell nuclei, and so to differentiate nucleate cells (such as white blood cells) from mature red blood cells, which have no nucleus.
00262] The image processing algorithms employed here utilized fluorescence images of cells to detect them using a combination of adaptive thresholding and edge detection.
Based on local intensity and intensity gradients, boundaries of regions of interest (RoI) were created around each cell. Using darkfield images, beads in the sample were also identified and Rol boundaries were created around the beads. All the Rols in each field of view were enumerated and their intensity in each image of that field of view were calculated. The information output by the image processing algorithm consisted of shape or morphometric measurements and fluorescence and darkfield intensities for each RoI. This information was analyzed using statistical methods to classify each object as a lymphocyte, monocyte, basophil, eosinophil, neutrophil or a bead. Based on enumeration of cells of different types, the corresponding bead count and the dilution ratio implemented during sample processing, an absolute concentration of cells per microliter of original whole blood was calculated. This was calculated for all white blood cells and each subtype, and reported as both absolute concentration (cells per ttL) and proportion CYO.
[00263] Examples of images and plots of results of such measurements are presented in Figs. 1, 2, and 3.

[002641 Fig. I shows representative images of blood cells from a sample of whole blood; these images were taken using different imaging techniques and dyes.
The image shown in Fig. IA was taken of cells from whole blood using dark-field illumination. The image shown in Fig. 1B was taken of cells from whole blood showing fluorescence from anti-CD14 antibodies labeled with PAC Blue dye; the fluorescent cells are monocytes. The image shown in Fig. IC was taken of cells from whole blood showing fluorescence from anti-CD123 antibodies labeled with PECy5 dye; the fluorescent cells are basophils.
The image shown in. Fig. ID was taken of cells from whole blood showing fluorescence from anti-CD16 antibodies labeled with PE dye; the fluorescent cells are neutrophils. The image shown in Fig.
1E was taken of cells from whole blood showing fluorescence from anti-CD45 antibodies labeled with AF647 dye; all leukocytes fluoresce under these conditions. The image shown in Fig. 1F was taken of cells from whole blood dyed with DRAQ5* to stain cell nuclei. Thus, leukocytes and platelets are stained and fluoresce under these conditions, but red blood cells (lacking nuclei) are not stained and do not fluoresce.
[002651 Fig. 2 shows a representative composite image of cell-types in whole blood from images acquired according to the methods disclosed herein. Images of a monocyte (labeled and seen in the upper left quadrant of the figure, with a reddish center surrounded by a blue-purple ring), a lymphocyte (labeled and seen in the center of the figure, with a bright red center surrounded by a dimmer red ring), an eosinophil (labeled and seen in the lower left quadrant of the figure, with a green center surrounded by a red border), and a neutrophil (labeled and seen in the lower right quadrant of the figure, with a green center surrounded by a yellow and green border) are shown in the figure.
[00266] It is of interest to identify and quantify various cell types found in such blood samples. There may be multiple ways to approach such a classification process, which, in some embodiments, may be considered as being a statistical problem for multi-dimensional classification. It will be understood that a wide variety of methods are available in the field to solve these types of classification problems. A particular embodiment of such an analysis is provided below.
[002671 Fig. 3 shows plots of various cell types identified and quantified by the cytometric assays described in this example. Fig. 3A shows a plot of spots (cells) by intensity of the marker FL-17 versus intensity of the marker FL-9 to identify monocytes.
Fig. 3B
shows a plot of spots (cells) by intensity of the marker FL-19versus intensity of the marker FL-I5to identify basophils. Fig. 3C shows a plot of spots (cells) by intensity of the marker FL-15 versus intensity of the marker FL-11 to identify lymphocytes. Fig. 3D
shows a plot of spots (cells) by intensity of the marker FL-15 versus intensity of the marker FL-9 to identify neutrophils and eosinophils.
100268] The initial identification of monocytes (9.6%, as shown in Fig. 3A) is used to guide the subsequent identification of basophils (0.68%, as shown in Fig. 3B).
The identification of monocytes and basophils as shown in Figs. 3A and 3B is used to guide the subsequent identification of neutrophils and eosinophils (68% neutrophils, 3.2% eosinophils, of the WBCs shown in Fig. 3D). Finally, lymphocytes are identified as shown in Fig. 3C
(93% of the WBCs plotted in Fig. 3D, corresponding to 18% of the cells in the original sample).
1002691 The present methods correlate well with other methods. Counts of white blood cells, red blood cells, and platelets were made with samples of EDTA-anti coagulated whole blood. The white blood cells were further counted to determine the numbers of neutrophils, nonocytes, and lymphocytes in the sample. In the measurements shown in Fig.
9, EDTA-anti coagulated whole blood samples were split into two, and one part of the samples were run on the system disclosed herein, using the methods disclosed herein. The other part of the samples was run on an Abbott CELL-DYN Ruby System (Abbott Diagnostics, Lake Forest, IL, USA), a commercial multi-parameter automated hematology analyzer. A comparison of the results obtained with both methods is shown in Fig. 4.
1002701 Fig. 4 shows plots demonstrating that cytometric methods as disclosed herein identify different cell types consistent with such identification by other methods. As shown in Figs. 4A-4C, the numbers of white blood cells ("WBCs", Fig. 4A), red blood cells ("RBCs", Fig. 4B) and platelets (Fig. 4C) measured by the present methods correlate well with the numbers of WBCs, RBCs, and platelets measured by other methods in corresponding aliquots of the same samples as were analyzed by the present methods. As shown in Figs.
4D-4F, the numbers of neutrophils, monocytes, and lymphocytes measured by either method were very similar, and correlated well with each other.
Example 4 ¨ Test Panels and Reflex Tests 1002711 As disclosed herein, the results of some tests may indicate that a further test, or fiwther tests, could be useful for a more complete understanding of the health status of a subject. As shown in the figures of this example, many test and test panels may be usefully associated with reflex tests useful in the diagnosis of a number of conditions, disorders, and health situations.

[002721 For example, Fig. 5A shows one side of an order form for ordering blood tests that includes listings of test panels and of reflex tests according to the methods disclosed herein. Fig. 5A includes lists many different tests which may be ordered for, and performed on samples of blood obtained from, a subject. These tests may be grouped together in panels, as shown in Fig. 5A. For example, a metabolic panel may include (but need not be limited to) the following tests: BMP Basic Metabolic Panel; CBC (complete blood count), no Diff;
CBC with Automatic Differential; Celiac Panel; Celiac Panel Reflex;
Chlamydia/Gonorrhea;
CMP - Comprehensive Metabolic Panel; Coagulation Panel with Reflex; Drug Panel;
Electrolytes Panel; Epstein-Barr (EBV) Antibody Panel; Hepatic Function Panel;
Hepatitis Panel, Acute; Lipid Panel; Obstetric Panel; Renal Function Panel; and STD
Panel. The termd "Diff" and "cliff" as used on an order form are abbreviations that refer to "Differentiation"
assays; similarly, without regard to capitalization of any letters, the terms "auto diff' and "manual diff' as used on an order form are abbreviations that refer to "automatic differentiation" assays and "manual differentiation" assays.
[002731 Depending on the results of the CBC with Automatic Differential, a reflex test may be indicated; such a reflex test may include a Manual Diff & Smear Review test (e.g., a blood test in which both a manual count of different types of blood cells (such as different WBC types, and an inspection of a blood smear (e.g., a manual or visual inspection of a blood smear) is performed), an Anemia Test, or both. Similarly, depending on the results of the celiac panel, a reflex test may be indicated; such a reflex test may include a celiac panel reflex test which provides more extensive or more sensitive testing, or both.
Similarly, depending on the results of the acute hepatitis panel a reflex test may be indicated; such a reflex test may include a hepatitis C test, a quantitative RNA test (e.g., for hepatitis C RNA), or both.
[00274] Also as shown in Fig. 5A, many other tests may be commonly ordered for subjects and performed on blood samples obtained from the subjects. Some of these tests include reflex tests where the results indicate that such further testing would be helpful. For example, as discussed above, and as indicated in Fig. 5A, a positive result of an HIV antibody screen (e.g., for HIV-I, HIV-2, or both) indicates, and may provoke, a reflex test for a confirmatory HIV test. Similarly, as discussed above, For example, as discussed above, and as indicated in Fig. 5A, a positive result of a syphilis screen indicates, and may provoke, a reflex test for an antibody test (treponema pallidum "TP antibody") test that may serve as a confirmatory syphilis test.

[002751 An abnormal result of a thyroid stimulating hormone (TSH) test indicates, and may provoke, a reflex test for a confirmatory thyroid panel of tests confirmatory of, more sensitive for, thyroid abnormalities or thyroid hormone deficiencies than the initial TSH
screen.
1002761 Further lists and explanations regarding such tests are provided in Fig. 5B. As indicated in Fig. 5B, a basic metabolic panel (BMP) includes tests for glucose, blood urea nitrogen (BUN), sodium (Na), potassium (K), chloride, (C1), carbon dioxide (CO2), and calcium (Calcium Total, indicative of the level of total calcium (i.e., in all forms) in the blood sample). A complete metabolic panel (CMP) inludes tests for glucose, blood urea nitrogen (BUN), sodium (Na), potassium (K), chloride, (CI), carbon dioxide (CO2), and calcium (Calcium Total) as in the BMP, and further includes tests for albumin, alkaline phosphatese (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (Bilinibin Total), and total protein (Protein Total).
1002771 As indicated in Fig. 5B, an electrolyte panel includes tests for Na, K, CI, and CO2.
1002781 As indicated in Fig. 5B, a lipid panel includes tests for cholesterol (CHOL), high density lipoprotein (HDL), low density lipoprotein (LDL), and triglycerides (Trig).
[002791 As indicated in Fig. 5B, a renal panel includes tests for glucose, BUN, creatinine, Na, K, Cl, CO2, Calcium Total, and albumin.
[002801 As indicated in Fig. 5B, a complete blood count (CBC) includes tests for hematocrit (HCT), hemoglobin (HGB), red blood cell count (RBC), white blood cell count (WBC), and platelet count. A blood test with differential, e.g., a WBC count with differential provides a count for each of several different types of white blood cells.
[002811 As indicated in Fig. 5B, a complete blood count (CBC) with reflex to anemia testing includes the CBC tests, and further includes (as reflex tests) tests for feffitin, folate, iron, total iron binding capacity (TIBC), vitamin B12, and a smear review test (inspection of a blood smear).
[00282] As indicated in Fig. 5B, a celiac panel includes tests for immunoglobulin A
(IgA), and test for HLA-DQ typing (a serotype test). If indicated by the results of the celiac panel, further testing (reflex testing) including tests for tissue transglutaminadase antibody (tTG), deamidated gliadin peptide (DGP), and endomysial antibody (EMA) may be performed. Depending on the results of the EMA test, a reflex test for EMA
titer may be performed.

[002831 As indicated in Fig. 513, a coagulation profile panel includes tests for platelet count (auto platelet count), prothrombin time and international normalized ratio test (PT/INR), partial thromboplastin time (PTT), and fibrinogen activity.
[002841 As indicated in Fig. 5B, a drug panel includes tests for Amphetamines, Barbiturates, Benzodiazepines, Cocaine, Dolophine (methadone), Ecstasy, (MDMA), Marijuana (THC), Heroin (opiate screen), Methadone metabolite, Phencyclidine (PCP), Propoxyphene, and Tricyclic antidepressants.
[002851 As indicated in Fig. 513, an. Epstein-Barr Antibody (Epstein-Barr Ab) test includes tests for immunoglobulin G (IgG) antibodies to Epstein-Barr early antigen (EA), IgG antibodies to Epstein-Barr nuclear antigen (NA), IgG antibodies to Epstein-Barr viral capsid protein (VCA), and immunoglobulin M (1gM) antibodies to Epstein-Barr VCA.
[00286] As indicated in Fig. 5B, a hepatic function panel includes tests for albumin, ALP, ALT, AST, Bilirubin Direct, total bilirubin (Bilirubin Total), and total protein (Protein Total).
[002871 As indicated in Fig. 513, an. acute hepatitis panel (Hepatitis Acute) includes tests for hepatitis A virus (HAV), hepatitis B core antigen gibeAn), hepatitis B surface antigen (HBsAg), and hepatitis C virus (HCV).
[00288] As indicated in Fig. 5B, an obstetric panel includes tests for ABU
RhD, an antibody screen (Ab screen), a syphilis test (SYP), an. HBsAg test, a rubella antibody test (RUB IgG), and a complete blood count with differential (CBC w/diff).
[002891 As indicated in Fig. 5B, a sexually transmitted disease panel (STD
Panel) includes tests for gonorrhea and chlamydia (CT/GC), hepatitis B surface antigen (HbsAg), hepatitis C virus (HCV), human immunodeficiency virus 1 and 2 (HIV 1/2), herpes simplex virus 1 and 2 (HSV 1/2), and Syphilis rapid plasma regain test (RF1R).
[00290] As indicated in Fig. 5B, a Triage Panel includes tests for BMP, amylase, PT/INR, prothrombin time (PTO, a CBC with auto differential and with reflex to manual differential and smear reviews of the blood sample.
[002911 As indicated in Fig. 5B, a Thyroid stimulating hormone with reflex to Thyroid Panel (rsH w/ Reflex to Thyroid Panel) includes tests for free tri-iodothyronine (free T3), free thyroxine (free T4), and a thyroid uptake test.
[00292] As shown in Figs. 5A and 5B, tests offered may be listed alphabetically (e.g., tests listed under the headings "Common Tests A-G", Common Tests H-S", and "Common Tests T-Z)"), listed according to type of sample (e.g., tests listed under "Stool" and "Urine"), grouped under the heading "Common Panels", or other groups. A panel is a group of tests performed together; for example, as discusssed above, a basic metabolic panel (BMP) includes tests for glucose, blood urea nitrogen (BUN), sodium (Na), potassium (K), chloride, (C1), carbon dioxide (CO2), and calcium (Calcium Total, indicative of the level of total calcium (i.e., in all forms) in the blood sample). As illustrated in Fig. 5A, a lab order form as disclosed herein may include lines, spaces, boxes, or other locations for entering patient information, clinician information, insurance information, billing codes, and may include locations for particulars regarding testing (e.g., whether or not the tests are for a fasting patient; whether or not the test is urgent, or is routine; whether or not the test is to be performed repeatedly, and if so, at what frequency; and so forth). Clinician information may include clinician name, NPI number, address, and other clinician information.
As illustrated in Fig. 5A, a lab order form may provide a location for a signature (e.g., for the signature of the ordering clinician). A lab order form as illustrated in Fig. 5A may include a location to fill in a date after which the test order is no longer valid (e.g., "valid until"
location).
[002931 In one non-limiting example, test methodologies are embodied in the lab requisition form. In such an example, the design. of the lab requisition form includes but is not limited to having prices on it and the way the tests are laid out on the form which is in part an algorithm that says if a subject has a certain test and has certain results, one should run certain other tests.
1002941 In embodiments, the lab order form embodies algorithm-based testing across different assay and/or signal detection methodologies, such that many unique features incorporated into the layout of the lab requisition form. For example, one feature is the option of selecting whether all of the tests should be done by a micro-sample and by traditional phlebotomy. Optionally, some embodiments may designate which tests are done by micro-sample (e.g., a small-volume sample having a volume of about 250 or less) and which are done by other technique(s). Optionally, some may default to micro-sampling for most tests and only traditional phlebotomy using venipuncture for any remaining test(s) that require it due to technical or other limitations.
[002951 Another feature is an option to designate frequency (routine, urgent, standing order, and others) of testing. A still firther feature is a cash pay option, wherein a doctor or other lab reqisition ordering medical professional may opt to have the patient perform the test under cash pay, which essentially makes the test anonymous to health insurers.
This may be of particular interest to patients who may have a certain lifestyle, condition, or have certain health risks where the subject may desire to test on a certain. frequency (every quarter, every m)nth, annually, or some other frequency, including just once) that if such testing and/or testing frequency is known. to health insurers, may have a negative impact on their insurance coverage. A still further feature is that prices are also shown for a substantial number, or optionally, all of the tests on the form.
[00296] Normally, when one does reflex testing, one may use an antibody screen and then reflex to an RNA or other nucleic acid amplification-based screen. In this non-limiting example, the antibody test describes mainly the immune response but the quantitative nucleic acid test provides information and measurements regarding viral load and can more definitively show whether a subject has the disease or not.
[002971 In a still further embodiment of an algorithm-based lab order form, one may test for CBC and then reflex, per a reflex algorithm, to anemia testing. One example may have a celiac panel with the of selecting a celiac panel follow-up. Thyroid panel may algorithm to certain sub tests such as but not limited to T3, T4, or other sub tests related to thyroid function and hormones produced by, or which affect, the thyroid. In one embodiment, the back of the lab order form (or other location on the order form) describes what is in each of the panels. This ability to have a lab order form follow a pre-selected algorithm for follow-up testing on micro-sample from the same sample collection session can reduce unnecessary lab visits and unnecessary prescription associated with not having a follow-on order because one needs more tubes of blood are required in order to use traditional dedicated discrete analyzers and each of those analyzers requires its own tube of blood with its own dedicated preparatory reagents and/or dedicated discrete anti-coagulants.
Therefore, in a legacy model, the system requires more tubes of blood from the subject and this typically requires a follow-on draw, more visits, and one does not get the diagnosis until later.
[002981 A further example of a lab order form (an order sheet) including listings of individual tests, test panels, test groups, and reflex tests according to the methods disclosed herein is provided in Figs. 6A and 6B. As shown in Fig. 6A, tests offered may be grouped together with other tests directed to similar indications or organs (e.g., tests listed under the heading "Thyroid"), or grouped according to general applicability (e.g., tests listed under the heading "Reproductive Hormones"), listed alphabetically (e.g., tests listed under the headings "Alphabetical Tests (A-H)" and "Alphabetical Tests (H.-Z)"), listed according to type of sample (e.g., tests listed under "Stool" and "Urine"), or other groups. As illustrated in Fig.
6A, a lab order form as disclosed herein may include lines, spaces, boxes, or other locations for entering patient information, clinician information, insurance information, billing information, and may include locations for particulars regarding testing (e.g., whether or not the tests are for a fasting patient; whether or not the test is urgent, or is routine; whether or not the test is to be performed repeatedly, and if so, at what frequency; and so forth).
Clinician information may include clinician name, NPI number, address, and other clinician information. A lab order form as illustrated in Fig. 6A may include a location to fill in a date after which the test order is no longer valid (e.g., "valid until" location).
As illustrated in Fig.
6A, a lab order form may provide a location for a signature (e.g., for the signature of the ordering clinician). In addition, as illustrated in Fig. 6A, a lab order form as disclosed herein may include a QR code, a bar code, or other machine-readable code or feature.
Such a machine-readable feature may be effective to more readily enter, store, track, and retrieve patient information. Figure 6A also shows one embodiment of the lab order form wherein algorithm-based testing (designated in phantom by dotted rectangles) is shown wherein the option to include is select-able by the ordering physician. In this non-limiting example, this lab order form with algorithm-based testing differs in that additional testing is offered for tests that traditionally do not include an option to order such additional follow-up testing.
This may be due in part to limitations of traditional devices preventing such options from being physically possible due to sample size limitations or other reasons. As seen in Figure 6A, test code and pricing are both listed for each selectable line associated with a test or test panel. Figure 6A also shows that there may be at least four or more algorithm-based tests, wherein at least four categories (common panels, reproductive health, STI, other, etc...) each includes at least one algorithm-based test. Figure 6A also shows that there may be at least four or more algorithm-based tests, wherein each includes at least one follow-up test based on an detection technology different from detection technique used in the initial test.
1002991 Figure 6B shows an emboidment that may optoinally include a reverse side on the lab order form. In this non-limiting example, the reverse side shows details regarding assays that are part of in certain combination test panels. As seen in Figure 6B, there may also be test code and pricing information listed for the panel andior its sub components.
100300] Figs. 7A, 7B, and 7C provide yet further examples of an order form having features as disclosed herein. The first page of the order form illustrated in these figures is shown in Fig. 7A, and lists individual tests, test panels, test groupings, and including lists of reflex tests according to methods disclosed herein. A portion of the second page of the order form illustrated in Fig. 7 is shown in Fig. 7B, and lists panel components and capped price offerings for the list shown in Fig. 7A. CPT codes are listed for the panels and panel components. A further portion of the order form of Fig. 7, placed on the third page of the order form illustrated in Fig. 7, is shown in Fig. 7C, and lists panel components and capped price offerings for the list shown in Fig. 7A. CPT codes are listed for the panels and panel components.
[003011 Figs. 8A and 8B provide yet further examples of an order form having features as disclosed herein. The first page of the order form illustrated in these figures is shown in Fig. 8A, and lists individual tests, test panels, test groupings, and including lists of reflex tests according to methods disclosed herein. Fig. 8B shows the second page of the order form illustrated in Fig. 8A, and lists panel components and capped price offerings for the list shown in. Fig. 8A. CPT codes are listed for the panels and panel components.
CPT codes are listed for the panels and panel components. The diamond symbols indicate tests for which particular criteria may exist which may limit reimbursement by a subject's insurance.
Asterisks indicate tests for which price caps are offered for multiple tests within a panel grouping. As an additional advisory regarding reimbursement, the form also indicates that "Ordered reflex tests only performed when medically appropriate. For Medicare and Meidcaid patients physicians should only order those tests that are medically necessary for diaposis and treatment?"
[003021 The lab order forms illustrated in Figs. 5A and 5B, Figs. 6A and 6B, Figs. 7A., 7B, and 7C, and in Figs. 8A and 8B include prices for each test, including reflex tests, and for panels of tests. Providing price information for each test, and for combinations of tests (e.g., test panels) better allows clinicians and patients to understand the costs of tests that are contemplated. Providing price information for each test, and for combinations of tests (e.g., test panels) to weigh costs versus benefits of such tests; in the absence of such price information, it is difficult or impossible to evaluate the costs versus the benefits of such tests.
In this way, providing price information on a test-order form provides great benefit to patients, clinicians, patient's families, insurers, and others affected by testing and the costs thereof. Providing reflex test information, including reflex test costs, provides benefits by allowing better planning of testing, and may result in fewer patient visits being needed for the same number of tests, since a reflex test may be ordered along with an initial test, and so may obviate any need for a subsequent return visit by a subject after the results of an initial test are obtained. Providing reflex test infomiation, including reflex test costs, provides benefits in avoiding delay; the delay that may be avoided is the delay which would otherwise occur if a reflex test were ordered only after the results of an initial test were known, since a reflex test may be ordered along with an initial test, and so may eliminate any unnecessary delay.
Providing reflex test information, including reflex test costs, provides benefits by suggesting related tests to a clinician, patient, or insurer, and so provides better information and may prompt more extensive testing, thereby providing more detailed and extensive clinical information of benefit to the patient and others involved with the care of that patient (including insurers and others).
[003031 While the above is a complete description of the preferred embodiment as described herein, it is possible to use various alternatives, modifications and equivalents.
Therefore, the scope of the present invention should be determined not with reference to the above description but should, instead, be determined with reference to the appended claims, along with their scope of equivalents. Any feature, whether preferred or not, may be combined with any other feature, whether preferred or not. Although embodiments herein may be described in the context of a physical lab order form, some embodiments may embody such a form in an electronic format which may present all of the form at once like the physical form or provide portions of it at one time that the user navigates through, such as but not limited to one screen at a time, to complete the form. For these electronic format test order fonns, some embodiments may select the test available to select based in part on medical history of the subject. The appended claims are not to be interpreted as including means-plus-function limitations, unless such a limitation is explicitly recited in a given claim using the phrase "means for." It should be understood that as used in the description herein and throughout the claims that follow, the meaning of "a," "an," and "the"
includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein and throughout the claims that follow, the meaning of "in" includes "in" and "on" unless the context clearly dictates otherwise. Finally, as used in the description herein and throughout the claims that follow, the meanings of "and" and "or" include both the conjunctive and disjunctive and may be used interchangeably unless the context expressly dictates otherwise.
Thus, in contexts where the terms "and" or "or" are used, usage of such conjunctions do not exclude an "and/or" meaning unless the context expressly dictates otherwise.
[00304] This document contains material subject to copyright protection.
The copyright owner (Applicant herein) has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure, as they appear in the US
Patent and Trademark Office patent file or records, but otherwise reserves all copyright rights whatsoever. The following notice shall apply: Copyright 2013-2015 Theranos, Inc.

Claims

1. A method of testing a biological sample in a device, comprising:
performing an initial assay in said device for an analyte in said biological sample, whereby an initial result is obtained, wherein said initial assay may provide a negative result indicating that the presence of said analyte is not detected, or is detected at a normal level, in the biological sample, or may provide a positive result indicating that the presence of the analyte is detected, or is detected at an abnormal level, in the biological sample; and determining further testing of said biological sample contingent on said initial result, wherein if the initial result is negative, then no subsequent assay is performed on said biological sample; and wherein if the initial result is positive, then a subsequent assay is performed in said device on said biological sample.
2. The method of claim 1, wherein said subsequent assay comprises an assay of a type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
3. The method of claim 1 or claim 2, wherein said subsequent assay comprises a different type of assay than said initial assay.
4. The method of any of claims 1 to 3, wherein the analyte to be detected by said subsequent assay comprises a different analyte than the analyte detected by said initial assay.
5. The method of any of claims 1 to 3, wherein the initial assay comprises measurement of an analyte, and said subsequent assay comprises a cytometric assay.
6. The method of claim. 5, wherein said cytometric assay comprises a measurement of a morphological characteristic of a cell.
7. The method of claim. 6, wherein the measurement of a morphological characteristic of a cell comprises measurement of a morphological characteristic of a blood cell in a blood sample.
8. The method of any of claims 1 to 7, wherein said initial assay comprises use of a detector to obtain said initial result, wherein said detector is selected from an optical detector, a pH detector, an electrochemical detector, a temperature sensor, an ion-sensitive electrode, a radiation detector, and other detectors.

9. The method of any of claims I to 7, wherein said subsequent assay comprises the use of a detector to obtain a further result, wherein said detector is selected from an optical detector, a pH detector, an electrochemical detector, a temperature sensor, an ion-sensitive electrode, a radiation detector, and other detectors.
10. The method of any of claims 1 to 5, wherein said subsequent assay comprises an assay that is more sensitive for the detection of said analyte than said initial assay.
11. The method of any of claims 1 to 10, wherein said biological sample comprises a sample selected from blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
12. The method of any of claims 1 to 11, wherein said initial assay and said subsequent assay are performed on different portions of a single biological sample.
13. The method of claim 12, wherein at least one of said different portions of said single biological sample comprises a diluted biological sample.
14. The method of any of claims 1 to 11, wherein said initial assay and said subsequent assay are performed on different biological samples.
15. The method of claim 15, wherein said different biological samples are each the same type of biological sample.
16. The method of claim 15, wherein each of said different biological samples is a blood sample.
17. The method of claim 16, wherein each of said different blood samples is a different fraction of blood.
18. The method of claim 17, wherein said fractions of blood are selected from whole blood, serum, and plasma.
19. The method of claim 15, wherein said different biological samples are each different types of biological sample.
20. The method of claim 19, wherein said different types of biological samples are selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
21. The method of claim 1, wherein said subsequent assay is performed within a short amount of time from the time at which the biological sample tested by said subsequent assay was accepted within said device.
22. The method of claim 21, wherein said initial assay and said subsequent assay are each performed on at least a portion of the same biological sample.
23. The method of any of claims 1 to 21, wherein the biological sample upon which the subsequent assay is performed is obtained after the results of said initial assay are obtained.
24. The method of claim 23, wherein if the result of said initial assay performed in a device is positive, then a subsequent assay is performed in said device on said biological sample within a short amount of time from the time of accepting a sample within said device, wherein said short amount of time is a time prior to the performance of said subsequent assay.
25. The method of any of claims 21 to 24, wherein said short amount of time is selected from the group of short amounts of time consisting of about 3 hours or less; about 2 hours or less; about 1 hour or less; about 50 minutes or less; about 45 minutes or less; about 40 minutes or less; about 35 minutes or less; about 30 minutes or less; about 25 minutes or less;
about 20 minutes or less; about 15 minutes or less; about 10 minutes or less;
about 5 minutes or less; about 4 minutes or less; about 3 minutes or less; about 2 minutes or less; and about 1 minute or less.
26. A device for testing a biological sample, wherein said device is configured to perform an initial assay and a subsequent assay according to the method of any of claims 1 to 25.
27. The device for testing a biological sample of claim 26, wherein said subsequent assay comprises a cytometric measurement.
28. The device for testing a biological sample of claim 27, wherein said cytometric assay comprises a measurement of a morphological characteristic of a cell.
29. The device for testing a biological sample of claim 26, 27, or 28, wherein said biological sample comprises a blood sample.

30. A system for testing a biological sample comprising a device of any of claims 26 to 29.
31. The system of claim 27 for testing a biological sample, wherein said biological sample comprises a blood sample.
32. A method of testing a biological sample in a device, comprising:
performing an initial assay in said device for an analyte in said biological sample, whereby an initial result is obtained, wherein said initial assay may provide a negative result indicating that the presence of said analyte is not detected, or is detected at a normal level, in the biological sample, or may provide a positive result indicating that the presence of the analyte is detected, or is detected at an abnormal level, in the biological sample;
performing further testing of said biological sample, wherein a subsequent assay is performed in said device on said biological sample regardless of the results of said initial assay;
and reporting the results of said subsequent assay of said biological sample contingent upon the results of said initial assay.
33. The method of claim 32, further comprising reporting the results of said initial assay.
34. The method of claim 32 or 33, wherein the results of said subsequent assay are not reported if said initial assay provides a negative result, and wherein the results of said subsequent assay are reported if said initial assay provides a positive result.
35. The method of claim. 32 or 33, wherein the results of said subsequent assay are not reported if said initial assay provides a positive result, and wherein the results of said subsequent assay are reported if said initial assay provides a negative result.
36. The method of claim 35, wherein said subsequent assay is an assay for the same analyte as said initial assay, and wherein said subsequent assay is a more sensitive assay than said initial assay.
37. The method of any of claims 32 to 36, wherein the biological sample upon which the subsequent assay is performed is obtained before the results of said initial assay are obtained.
38. The method of any of claims 32 to 36, wherein the initial assay and the subsequent assay are performed on portions of the same biological sample.

39. The method of claim 38, wherein at least one of said portions of said biological sample is a diluted portion of the biological sample.
40. The method of any of claims 32 to 39, wherein said subsequent assay comprises an assay of a type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
41. The method of any of claims 32 to 39, wherein said subsequent assay comprises a different type of assay than said initial assay.
42. The method of any of claims 32 to 39, wherein the analyte to be detected by said subsequent assay comprises a different analyte than the analyte detected by said initial assay.
43. The method of any of claims 32 to 39, wherein the initial assay comprises measurement of an analyte, and said subsequent assay comprises a cytometric assay.
44. A method of testing a small-volume biological sample in a device, wherein said small-volume biological sample has a volume of less than about 250 microliters (µL), the method comprising:
placing said small-volume sample within the device;
dividing said small-volume biological sample into at least a first and a second portion;
retaining said second portion within the device for use in a subsequent assay;
performing, in said device on said first portion or aliquot thereof, an initial assay for detecting or measuring an analyte or characteristic of the sample, whereby an initial result is obtained from the first portion, wherein:
i) said initial result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected or measured in the biological sample, or is detected or measured at a norm.al level in the biological sample, or ii) said initial result may comprise a positive result indicating that the presence of the analyte or characteristic is detected or measured in the biological sample, or is detected or measured at an abnormal level in the biological sample;
determining whether or not said initial result is a positive result or a negative result;
determining, contingent on whether or not said initial result is a positive result or a negative result, whether or not to perform a subsequent assay; and i) performing said subsequent assay on said second portion or aliquot thereof if the initial result is positive for an assay requiring further testing for a positive result, or if the initial result is negative for an assay requiring further testing for a negative result; or ii) otherwise not performing said subsequent assay.
45. The method of claim 44, wherein the performance of said subsequent assay is begun pursuant to said initial result within a short amount of time after placing said small-volume sample within the device, wherein said short amount of time is about two hours or less.
46. The method of claim 45, wherein said short amount of time is about one hour or less.
47. The method of claim 44, wherein the performance of said subsequent assay is completed within a short amount of time after placing said small-volume sample within the device, wherein said short amount of time is about two hours or less.
48. The method of claim 44, wherein said first portion the small-volume sample is diluted prior to performance of said initial assay, or wherein said second portion the small-volume sample is diluted prior to performance of said subsequent assay, or both.
49. The method of claim 44, wherein said subsequent assay comprises an assay for detecting or measuring the same analyte or characteristic as said initial assay, and wherein said subsequent assay is more sensitive for the detection or measurement of the analyte or characteristic than. the initial assay.
50. The method of claim 44, wherein the analyte or characteristic to be detected or measured by said subsequent assay comprises a different analyte or characteristic than the analyte or characteristic detected or measured by said initial assay.
51. The method of claim. 44, wherein said initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays, and said subsequent assay comprises a different type of assay than said initial assay.
52. The method of claim. 51, wherein said initial assay comprises a nucleic acid assay, and said subsequent assay comprises an assay selected from an antibody-based assay, a general chemistry assay, and a cytometric assay.

53. The method of claim 51, wherein said initial assay comprises an antibody assay, and said subsequent assay comprises an assay selected from a nucleic acid assay, a general chemistry assay, and a cytometric assay.
54. The method of claim 51, wherein said initial assay comprises a general chemistry assay, and said subsequent assay comprises an assay selected from a nucleic acid assay, an antibody assay, and a cytometric assay.
55. The method of claim 51, wherein said initial assay comprises a cytometric assay, and said subsequent assay comprises an assay selected from a nucleic acid assay, an antibody assay, and a general chemistry assay.
56. The method of claim 55, wherein said cytometric assay comprises the detection or measurement of a morphological characteristic of a cell.
57. The method of claim 44, wherein the initial assay comprises detection or measurement of an analyte, and said subsequent assay comprises a cytometric assay for the detection or measurement of a characteristic of the sample.
58. The method of claim. 57, wherein said cytometric assay comprises detection or measurement of a morphological characteristic of a cell in the sample.
59. The method of claim. 44, wherein small-volume biological sample comprises a small-volume of less than about 100 microliters (µL).
60. The method of claim. 44, wherein said small-volume biological sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
61. The method of claim 44, further comprising:
Reporting the results of said initial assay of said biological sample.
62. The method of claim 61, wherein the results of said initial assay are reported prior to the performance of said subsequent assay.
63. The method of claim 44, further comprising:
Reporting the results of said subsequent assay.

64. The method of claim 44, further comprising:
Reporting the results of said initial assay and of said subsequent assay.
65. The method of claim 64, wherein the results of said initial assay and of said subsequent assay are reported together.
66. The method of claim 61, wherein the results of said subsequent assay are not reported if:
i) said initial assay provides a negative result, and said negative result does not require performance of a subsequent assay, or ii) said initial assay provides a positive result, and said positive result does not require performance of a subsequent assay.
67. A method of testing a small-volume biological sample in a device, wherein said small-volume biological sample has a volume of less than about 250 microliters (µL), the method comprising:
placing said small-volume sample within the device;
dividing said small-volume biological sample into at least a first and a second portion;
performing, in said device on said first portion or aliquot thereof, a first assay for detecting or measuring an analyte or characteristic of th.e sample, whereby a first result is obtained from the first portion, wherein said first result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected or measured in the biological sample, or is detected or measured at a normal level in the biological sample, or may comprise a positive result indicating that the presence of the analyte or characteristic is detected or measured in the biological sample, or is detected or measured at an abnormal level in the biological sample;
performing, in said device on said second portion or aliquot thereof, a second assay for detecting or measuring an analyte or characteristic of the sample, whereby a second result is obtained from the second portion;
determining whether or not said first result is a positive result or a negative result;
determining, contingent on whether or not said first result is a positive result or a negative result, whether or not to report said second result of said second assay; and i) reporting the first result and the second result if the first result is positive for an assay requiring reporting of said second result pursuant to a positive first result, or if the first result is negative for an assay requiring reporting of said second result pursuant to a negative first result;
or ii) otherwise reporting the first result and not reporting the results of said second assay.
68. The method of claim 67, wherein said second assay comprises an assay for detecting or measuring the same analyte or characteristic as said first assay, and wherein said second assay is more sensitive for the detection or measurement of the analyte or characteristic than the first assay.
69. The method of claim 67, wherein the analyte or characteristic to be detected or measured by said second assay comprises a different analyte or characteristic than the analyte or characteristic detected or measured by said first assay.
70. The method of claim 67, wherein said first assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays, and said second assay comprises a different type of assay than said first assay.
71. The method of claim. 70, wherein said cytometric assay comprises the detection or measurement of a morphological characteristic of a cell.
72. The method of claim. 67, wherein said first portion the small-volume sample is diluted prior to performance of said first assay, or wherein said second portion the small-volume sample is diluted prior to performance of said second assay, or both.
73. The method of claim 67, wherein said small-volume biological sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
74. A device for testing a biological sample, wherein said device is configured to perform an initial assay and a subsequent assay according to the method of claim 44.

75. The device of claim 74, configured to perform antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays.
76. The device of claim 74, configured to test a sample, wherein the sample is selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, carwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
77. The device of claim 74, configured to test a blood sample.
78. A system for testing a biological sample comprising a device of claim 74.
79. The system of claim. 78, wherein said device for testing a biological sample is configured to perform antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays.
80. The system of claim 78, wherein said device for testing a biological sample is configured to test a blood sample.
81. A method of testing a small-volume biological sample in a device, wherein said small-volume biological sample has a volume of less than about 250 microliters (µL), the method comprising:
placing said small-volume biological sample and a cartridge comprising reagents within the device;
dividing the sample into at least a first and a second portion;
retaining said second portion on said cartridge within the device for use in a subsequent assay;
performing, in the device on said first portion or aliquot thereof, an initial assay for detecting, identifying, or measuring an analyte or characteristic of the sample, wherein said initial assay is performed using only reagents from said cartridge, whereby an initial result is obtained from the first portion, wherein:

i) said initial result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected, identified, or measured in the sample, or is detected, identified, or measured at a normal level in the sample, or ii) said initial result may comprise a positive result indicating that the presence of the analyte or characteristic is detected, identified, or measured in the sample, or is detected, identified, or measured at an abnormal level in the sample;
determining whether or not the initial result is a positive result or a negative result;
determining, contingent on whether or not the initial result is a positive result or a negative result, whether or not to perform a subsequent assay; and automatically performing said subsequent assay on the second portion or aliquot thereof if the initial result is positive for an assay requiring further testing for a positive result, or if the initial result is negative for an assay requiring further testing for a negative result, wherein the subsequent assay is performed in the device using only reagents from said cartridge.
82. The method of claim 81, wherein the sample is provided in a sample container that is disposed on said cartridge during placement of the sample within the device, and wherein said second portion is retained in said sample container disposed on the cartridge within the device for use in the subsequent assay.
83. The method of claim 81, wherein the sample is provided in a sample container that is disposed on the cartridge during placement of the sample within the device, and wherein said second portion is retained in a second container that is disposed on the cartridge within the device.
84. The method of claim 83, wherein said second container is a dedicated container designated for holding the second portion on the cartridge prior to the performance of the subsequent assay.
85. The method of claim 83, wherein the second container is a dedicated container designated to contain the second portion during the performance of the subsequent assay.
86. The method of claim 81, wherein the automatic performance of the subsequent assay is begun within a short amount of time after placing the sample within the device, wherein said short amount of time is about two hours or less.

87. The method of claim 86, wherein the short amount of time is about one hour or less.
88. The method of claim 81, wherein the automatic performance of the subsequent assay is completed within a short amount of time after placing the sample within the device, wherein said short amount of time is about two hours or less.
89. The method of claim 88, wherein the short amount of time is about one hour or less.
90. The method of claim 81, wherein the cartridge contains all consumables used in the performance of the initial assay and of the subsequent assay.
91. The method of claim 81, wherein the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted by at least about 10-fold prior to, or during, the automatic performance of the subsequent assay, or both.
92. The method of claim 81, wherein the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the small-volume sample is diluted by at least about 100-fold prior to, or during, the automatic performance of the subsequent assay, or both.
93. The method of claim 91, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (µL).
94. The method of claim 92, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (µL).
95. The method of claim 81, wherein the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
96. The method of claim 95, wherein the subsequent assay comprises a different type of assay than the initial assay.
97. The method of claim 95, wherein said cytometric assays comprise the detection, identification, or measurement of a morphological characteristic of a cell.
98. The method of claim 81, wherein the subsequent assay comprises an assay for detecting, identifying, or measuring:

a) the same analyte or characteristic as the initial assay, wherein the subsequent assay is more sensitive for the detection, identification, or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay.
99. The method of claim 96, wherein:
i) the initial assay comprises a nucleic acid assay, and the subsequent assay comprises an assay selected from an antibody-based assay, a general chemistry assay, and a cytometric assay;
ii) the initial assay comprises an antibody assay, and the subsequent assay comprises an assay selected from a nucleic acid assay, a general chemistry assay, and a cytometric assay;
iii) the initial assay comprises a general chemistry assay, and the subsequent assay comprises an. assay selected from a nucleic acid assay, an antibody assay, and a cytometric assay;
or iv) the initial assay comprises a cytometric assay, and the subsequent assay comprises an assay selected from a nucleic acid assay, an antibody assay, and a general chemistry assay.
100. The method of claim 99, wherein the cytometric assay comprises the detection, identification, or measurement of a morphological characteristic of a cell.
101. The method of claim 81, wherein the initial assay comprises the detection, identification, or measurement of an analyte, and the subsequent assay comprises a cytometric assay for the detection, identification, or measurement of a morphological characteristic of a cell.
102. The method of claim. 81, wherein the sample comprises a volume of less than about 100 microliters (µL).
103. The method of claim. 81, wherein the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
104. The method of claim 81, further comprising:
Reporting the results of the initial assay of the sample.

105. The method of claim 104, wherein the results of the initial assay are reported prior to the performance of the subsequent assay.
106. The method of claim 104, further comprising:
Reporting the results of the subsequent assay.
107. The method of claim 106, wherein the results of the initial assay and of the subsequent assay are reported together.
108. A method of testing a small-volume biological sample in a device, wherein said sample has a volume of less than about 250 microliters (µL), the method comprising:
placing the sample and a cartridge comprising reagents within the device;
dividing the sample into at least a first and a second portion, and retaining at least said second portion on said cartridge prior to performing assays on said portions;
performing, in said device on the first portion or aliquot thereof, a first assay for detecting, identifying, or measuring an analyte or characteristic of the sample using only reagents from said cartridge, whereby a first result is obtained from the first portion, wherein said first result may comprise a negative result indicating that the presence of said analyte or characteristic is not detected or measured in the sample, or is detected or measured at a normal level in the sample, or may comprise a positive result indicating that the presence of the analyte or characteristic is detected or measured in the sample, or is detected or measured at an abnormal level in the sample;
performing, in the device on the second portion or aliquot thereof, a second assay for detecting, identifying, or measuring an analyte or characteristic of the sample using only reagents from. said cartridge, whereby a second result is obtained from the second portion;
determining whether or not said first result is a positive result or a negative result;
determining, contingent on whether or not the first result is a positive result or a negative result, whether or not to report said second result of said second assay;
reporting the first result; and reporting the second result only if:
i) the first result is positive for an assay requiring reporting of the second result pursuant to a positive first result, or ii) if the first result is negative for an assay requiring reporting of the second result pursuant to a negative first result.
109. The method of claim 108, wherein the second assay comprises an assay for detecting, identifying, or measuring:
a) the same analyte or characteristic as the first assay, and wherein the second assay is more sensitive for the detection or measurement of the analyte or characteristic than the first assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the first assay.
110. The method of claim 108, wherein the first assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
I I I . The method of claim 110, wherein the second assay comprises a different type of assay than the first assay.
112. The method of claim 110, wherein said cytometric assay comprises the detection, identification, or measurement of a morphological characteristic of a cell.
113. The method of claim 108, wherein the cartridge contains all consumables used in the performance of the first assay and of the second assay.
114. The method of claim 108, wherein the first portion of the sample is diluted prior to, or during, the performance of the first assay, or wherein the second portion of the sample is diluted prior to, or during, the performance of the second assay, or both.
115. The method of claim 114, wherein the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted by at least about 10-fold prior to, or during, the automatic performance of the subsequent assay, or both.
116. The method of claim 114, wherein the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted by at least about 100-fold prior to, or during, the automatic performance of the subsequent assay, or both.
117. The method of claim 115, wherein, following dilution, the volume of the diluted portion is less than about 200 microliters (µL).

118. The method of claim 116, wherein, following dilution, the volume of the diluted portion is less than about 200 microliters (µL).
119. The method of claim 108, wherein the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
120. A device for testing a small-volume biological sample, wherein said device is configured to perform an initial assay and a subsequent assay according to the method of claim 1.
121. The device of claim 120, configured to perform. antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays.
122. The device of claim 120, configured to test a sample, wherein the sample is selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
123. The device of claim 120, configured to test a blood sample.
124. A system. for testing a small-volume biological sample comprising a device of claim 120.
125. The system of claim 124, wherein said device is configured to perform antibody-based assays, nucleic acid-based assays, general chemistry assays, and cytometric assays.
126. The system of claim 124, wherein said device is configured to test a sample, wherein the sample is selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
127. The system of claim 124, wherein said device is configured to test a blood sample.
128. A method of ordering a clinical assay to be performed by a clinical testing facility on a sample obtained from a subject, the method comprising:
Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form;
Marking the order form to indicate the name and other identifying information of said subject;
Marking the order form to identify one or more selected assays of the plurality of assays that are ordered for performance; and Providing the marked order form to the clinical testing facility.
129. The method of claim 128, wherein the sample is a small-volume biological sample having a volume of less than about 250 microliters (µL).
130. The method of claim 128, wherein the sample is a blood sam.ple or a urine sample.
131. The method of claim 128, wherein the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device.
132. The method of claim 131, comprising ordering a plurality of assays comprising an initial assay and a subsequent assay, wherein the sample is provided in a sample container that is disposed on a cartridge during placement of the sample within an automated sample analysis device, wherein said sample is divided into portions, said portions comprising a first portion and at least a second portion, and wherein said second portion is retained in said sample container disposed on the cartridge within the device for use in the subsequent assay.
133. The method of claim 132, wherein said cartridge contains all consumables used in the performance of the initial assay and of the at least one subsequent assay.
134. A method of performing a clinical assay by a clinical testing facility on a sample obtained from a subject, the method comprising:

Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form;
Marking the order form to indicate the name and other identifying information of said subject;
Marking the order form to identify one or more selected assays of the plurality of assays that are ordered for performance;
Providing the marked order form to the clinical testing facility;
Obtaining a sample from said subject during a single sample acquisition session; and Performing said one or more selected assays.
135. The method of claim 134, wherein the sample is a small-volume biological sample having a volume of less than about 250 microliters (µL).
136. The method of claim 134, wherein the sample is a blood sample or a urine sample.
137. The method of claim 134, wherein said one or more selected assays comprise at least two linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay, wherein the results of said initial assay affect the decision whether or not to perform said at least one subsequent assay, or affect the decision whether or not to report results of said at least one subsequent assay.
138. The method of claim 134, wherein the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device.
139. The method of claim 138, comprising performing a plurality of assays comprising an initial assay and a subsequent assay, wherein the sample is provided in a sample container that is disposed on a cartridge during placement of the sample within an automated sample analysis device, further comprising dividing said sample into portions, said portions comprising a first portion and at least a second portion, and wherein said second portion is retained in said sample container disposed on the cartridge within the device for use in the subsequent assay.
140. The method of claim 139, wherein the sample is obtained from said subject during a single sample acquisition session.

141. The method of claim 139, wherein the sample is a blood sample or a urine sample.
142. The method of claim 139, wherein said cartridge contains all consumables used in the performance of the initial assay and of the at least one subsequent assay.
143. A method of ordering at least an initial clinical assay and a subsequent clinical assay for performance by a clinical testing facility on a sample obtained from a subject, wherein said initial assay and said subsequent assay each comprise detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising:
Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay, wherein the performance of said at least one subsequent assay is contingent upon the results of said initial assay;
Marking the order form to indicate the name and other identifying information of said subject;
Marking the order form to indicate linked assays selected for performance, wherein said initial assay of said linked assays is to be performed on the sample or portion thereof, and initial assay results are to be obtained, wherein said initial results may comprise either:
i) a negative result indicating that the presence of said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the presence of the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an abnormal level in the sample;
Wherein said at least one subsequent assay is to be performed only if further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical information:
i) if the results of said initial assay comprise a positive result, and such positive result indicates that; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information; and providing said marked order form to said clinical testing facility.
144. A method of ordering at least two clinical assays to be performed by a clinical testing facility on a sample obtained from a subject, said at least two clinical assays comprising linked assays, wherein linked assays comprise an initial assay and at least one subsequent assay, wherein said linked assays each comprise detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising:
Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay;
Marking the order form to indicate the name and other identifying information of said subject;
Marking the order form to indicate linked assays that have been selected for performance;
wherein said initial assay and said at least one subsequent assay are to be performed on the sample or a portion thereof, and results obtained and reported, wherein said initial results may comprise either:
i) a negative result indicating that said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an abnormal level in the sample;
Wherein the results of said at least one subsequent assay are to be reported, contingent upon the results of said initial assay, only when further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical information:

i) if the results of said initial assay comprise a positive result indicate the presence of the analyte or characteristic, and such positive result indicates that further assay results would provide additional useful clinical information; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information; and providing said marked order form to said clinical testing facility.
145. The method of claim 143 or claim 144, wherein the at least one subsequent assay comprises an assay for detecting, identifying, or measuring:
a) the same analyte or characteristic as the initial assay, and wherein the at least one subsequent assay is more sensitive for the detection or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay.
146. The method of claim 143 or claim 144, wherein the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
147. The method of claim 146, wherein the at least one subsequent assay comprises a different type of assay than the initial assay.
148. The method of claim 146, wherein said cytometric assay comprises the detection, identification, or measurement of a morphological characteristic of a cell.
149. The method of claim 146, wherein the initial assay comprises the detection, identification, or measurement of an analyte, and the subsequent assay comprises a cytometric assay for the detection, identification, or measurement of a morphological characteristic of a cell.
150. The method of claim 143 or claim 144, wherein the first portion of the sample is diluted prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted prior to, or during, the performance of the at least one subsequent assay, or both.

151. The method of claim 143 or claim 144, wherein the sample is a small-volume biological sample having a volume of less than about 250 microliters (µL).
152. The method of claim 143 or claim 144, wherein the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device, further comprising:
dividing the sample into portions prior to the performance of said initial assay, wherein said portions comprise a first portion and at least a second portion.
153. The method of claim 152, wherein the sample in said sample container is disposed on a cartridge during placement of the sample within said automated sample analysis device, and wherein said second portion is retained on said cartridge within the automated sample analysis device for use in the at least one subsequent assay.
154. The method of claim 152, wherein said second portion is retained in said sample container disposed on the cartridge within the automated sample analysis device for use in the performance of the at least one subsequent assay.
155. The method of claim 152, wherein said second portion is retained in a second container that is disposed on the cartridge within the automated sample analysis device.
156. The method of claim 155, wherein said second container is a dedicated container designated for holding the second portion on the cartridge prior to the performance of the at least one subsequent assay.
157. The method of claim 155, wherein the second container is a dedicated container designated to contain the second portion during the performance of the at least one subsequent assay.
158. The method of any of claims 152 to 157, wherein the performance of the subsequent assay is begun within a short amount of time after placing the sample within the automated sample analysis device, wherein the short amount of time is about two hours or less.
159. The method of any of claims 152 to 158, wherein the automatic performance of the subsequent assay is completed within a short amount of time after placing the sample within the automated sample analysis device, wherein said short amount of time is about one hour or less.

160. The method of any of claims 152 to 159, wherein said sample is provided on a cartridge, and wherein said cartridge contains all consumables used in the performance of the initial assay and of the subsequent assay.
161. The method of any of claims 152 to 160, wherein the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (µL).
162. The method of any of claims 152 to 161, wherein the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the small-volume sample is diluted by at least about 100-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (µL).
163. The method of any of claims 152 to 162, wherein the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
164. The method of claim 163, wherein the subsequent assay comprises a different type of assay than the initial assay.
165. The method of claim 163, wherein said cytometric assays comprise the detection, identification, or measurement of a morphological characteristic of a cell.
166. The method of any of claims 152 to 165, wherein the subsequent assay comprises an assay for detecting, identifying, or measuring:
a) the sam.e analyte or characteristic as the initial assay, wherein the subsequent assay is more sensitive for the detection, identification, or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay.
167. The method of any of claims 152 to 166, wherein the sample comprises a volume of less than about 100 microliters (µL).
168. The method of any of claims 143 to 167, wherein the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
169. The method of any of claims 143 to 168, wherein the results of the initial assay of the sample are to be reported.
170. The method of claim 169, wherein the results of the initial assay are to be reported prior to the performance of the subsequent assay.
171. The method of claim 169, wherein the results of the at least one subsequent assay are to be reported.
172. The method of claim 171, wherein the results of the initial assay and of the at least one subsequent assay are to be reported together.
173. The method of any of claims 143 to 172, wherein said linked assays comprising an initial assay and at least one subsequent assay are selected from the group of linked assays consisting of:
An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising manual differential and smear review;
An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising an assay for anemia;
An initial assay comprising an acute hepatitis panel, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C;
An initial assay comprising one or more of celiac panel assays selected from IgA and HLA-DQ typing, and a subsequent assay comprising one or more of celiac reflex panel assays selected from tTG, DGP, EMA, and EMA titer;
An initial assay comprising a Hepatitis C antibody assay, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C;
An initial assay comprising an HIV antibody screening assay, and a subsequent assay comprising a confirmatory assay for HIV;
An initial assay comprising an HIV antibody screening assay, and a subsequent assay comprising an assay for differentiating between H1V-1 antibodies and H1V-2 antibodies;

An initial assay comprising an assay for treponema pallidum IgG, and a subsequent assay comprising a rapid plasma reagin (RPR) assay;
An initial assay comprising a rapid plasma reagin (RPR) assay, and a subsequent assay comprising an assay for an assay for antibodies to treponema pallidum;
An initial assay comprising a thyroid stimulating hormone (TSH) assay, and a subsequent assay comprising comprising one or more of an assay for tri-iodothyronine (T3), thyroxine (T4), and thyroid uptake.
174. A method of performing, by a clinical testing facility, at least an initial clinical assay and a subsequent clinical assay on a sample obtained from a subject, wherein said initial assay and said at least one subsequent assay each comprise detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising:
Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay, wherein the performance of said at least one subsequent assay is contingent upon the results of said initial assay;
Marking the order form to indicate the name and other identifying information of said subject;
Marking the order form to indicate linked assays selected for performance;
Providing the marked order form to the clinical testing facility;
Obtaining a sample from said subject during a single sample acquisition session;
Performing said initial assay of said linked assays selected for performance, using said sample, or a portion thereof, for the performance of said initial assay, whereby an initial result is obtained from the sample or a portion thereof, wherein said initial result may comprise either:
i) a negative result indicating that the presence of said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the presence of the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an abnormal level in the sample; and Performing said at least one subsequent assay only if further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical information:
i) if the results of said initial assay comprise a positive result, and such positive result indicates that further assay results would provide additional useful clinical information; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information.
175. The method of claim 174, further comprising performing said at least one subsequent assay using said sample, or a portion thereof, for the performance of said at least one subsequent assay.
176. A method of performing, by a clinical testing facility, at least two linked assays on a sample obtained from a subject, said linked assays comprising an initial assay and at least one subsequent assay, each of said linked assays comprising detecting, identifying, or measuring an analyte or characteristic of said sample, the method comprising:
Providing an order form listing a plurality of assays which may be ordered, wherein the cost of each assay of said plurality of assays is provided on said order form, and wherein said plurality of assays includes linked assays, wherein said linked assays comprise an initial assay and at least one subsequent assay;
Marking the order form. to indicate the nam.e and other identifying information of said subject;
Marking the order form. to indicate linked assays that have been selected for performance;
Providing the marked order form to the clinical testing facility;
Obtaining a sample from said subject at a single sample acquisition session;
Performing said initial assay on said sample, or a portion thereof, effective to obtain results of said initial assay;
wherein said initial result may comprise either:
1) a negative result indicating that said analyte or characteristic is not detected, measured, or identified in the sample, or is detected, measured, or identified at a normal level in the sample, or ii) a positive result indicating that the analyte or characteristic is detected, measured, or identified in the sample or a portion thereof, or is detected, measured, or identified at an abnormal level in the sample;
Reporting the initial results;
Performing said at least one subsequent assay on said sample, or a portion thereof, effective to obtain results of said at least one subsequent assay;
Reporting the results of said at least one subsequent assay only when further assay results would provide additional useful clinical information, wherein further assay results would provide additional useful clinical information:
i) if the results of said initial assay comprise a positive result indicate the presence of the analyte or characteristic, and such positive result indicates that further assay results would provide additional useful clinical information; or ii) if the results of said initial assay comprise a negative result, and a negative result indicates that a more sensitive assay for detecting, measuring, or identifying the analyte or characteristics would provide additional useful clinical information.
177. The method of claims 174, 175, or 176, wherein the at least one subsequent assay comprises an assay for detecting, identifying, or measuring:
a) the same analyte or characteristic as the initial assay, and wherein the at least one subsequent assay is more sensitive for the detection or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay.
178. The method of claims 174, 175, or 176, wherein the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
179. The method of claim 178, wherein the at least one subsequent assay comprises a different type of assay than the initial assay.
180. The method of claim 178, wherein said cytometric assay comprises the detection, identification, or measurement of a morphological characteristic of a cell.
-121-.

181. The method of claim 178, wherein the initial assay comprises the detection, identification, or measurement of an analyte, and the subsequent assay comprises a cytometric assay for the detection, identification, or measurement of a morphological characteristic of a cell.
182. The method of claims 174, 175, or 176, wherein the first portion of the sample is diluted prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted prior to, or during, the performance of the at least one subsequent assay, or both.
183. The method of claims 174, 175, or 176, wherein the sample is a small-volume biological sample having a volume of less than about 250 microliters (µL).
184. The method of claims 174, 175, or 176, wherein the sample is provided in a sample container for performance of said assays by the clinical testing facility using an automated sample analysis device, further comprising:
Dividing the sample into portions prior to the performance of said initial assay, said portions comprising a first portion and at least a second portion.
185. The method of claim 184, wherein the sample in said sample container is disposed on a cartridge during placement of the sample within said automated sample analysis device, and wherein said second portion is retained on said cartridge within the automated sample analysis device for use in the at least one subsequent assay.
186. The method of claim 184, wherein said second portion is retained in said sample container disposed on the cartridge within the automated sample analysis device for use in the performance of the at least one subsequent assay.
187. The method of claim 184, wherein said second portion is retained in a second container that is disposed on the cartridge within the automated sample analysis device.
188. The method of claim 187, wherein said second container is a dedicated container designated for holding the second portion on the cartridge prior to the performance of the at least one subsequent assay.
189. The method of claim 187, wherein the second container is a dedicated container designated to contain the second portion during the performance of the at least one subsequent assay.

190. The method of any of claims 184 to 189, wherein the performance of the subsequent assay is begun within a short amount of time after placing the sample within the automated sample analysis device, wherein the short amount of time is about two hours or less.
191. The method of any of claims 184 to 190, wherein the automatic performance of the subsequent assay is completed within a short amount of time after placing the sample within the automated sample analysis device, wherein said short amount of time is about one hour or less.
192. The method of any of claims 184 to 191, wherein said sample is provided on a cartridge, and wherein said cartridge contains all consumables used in the performance of the initial assay and of the subsequent assay.
193. The method of any of claims 184 to 192, wherein the first portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the sample is diluted by at least about 10-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (µL).
194. The method of any of claims 184 to 193, wherein the first portion of the sample is diluted by at least about 100-fold prior to, or during, the performance of the initial assay, or wherein the second portion of the small-volume sample is diluted by at least about 100-fold prior to, or during, the performance of the subsequent assay, or both, wherein, following dilution, the volume of said diluted portion is less than about 200 microliters (µL).
195. The method of any of claims 184 to 194, wherein the initial assay comprises an assay of an assay type selected from the group of assay types consisting of antibody-based assays, nucleic acid assays, general chemistry assays, and cytometric assays.
196. The method of claim 195, wherein the subsequent assay comprises a different type of assay than the initial assay.
197. The method of claim 195, wherein said cytometric assays comprise the detection, identification, or measurement of a morphological characteristic of a cell.
198. The method of any of claims 184 to 197, wherein the subsequent assay comprises an assay for detecting, identifying, or measuring:

a) the same analyte or characteristic as the initial assay, wherein the subsequent assay is more sensitive for the detection, identification, or measurement of the analyte or characteristic than the initial assay, or b) a different analyte or characteristic than the analyte or characteristic detected, identified, or measured by the initial assay.
199. The method of any of claims 184 to 198, wherein the sample comprises a volume of less than about 100 microliters (µL).
200. The method of any of claims 174 to 199, wherein the sample comprises a sample selected from the group of biological sample types consisting of blood, serum, plasma, a throat swab, a nasal swab, a nasopharyngeal wash, saliva, urine, gastric fluid, cerebrospinal fluid, tears, stool, mucus, sweat, earwax, oil, a glandular secretion, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, ocular fluids, breath, hair, finger nails, skin, biopsy tissue, placental fluid, amniotic fluid, cord blood, lymphatic fluids, cavity fluids, sputum, pus, microbiota, meconium, breast milk, and other secretions or excretions.
201. The method of any of claims 174 to 200, further comprising:
Reporting the results of the initial assay of the sample.
202. The method of claim 201, wherein the results of the initial assay are reported prior to the performance of the subsequent assay.
203. The method of claim 201, further comprising:
Reporting the results of the subsequent assay.
204. The method of claim 203, wherein the results of the initial assay and of the subsequent assay are reported together.
205. The method of any of claims 174 to 204, wherein said linked assays comprising an initial assay and at least one subsequent assay are selected from the group of linked assays consisting of:
An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising manual differential and smear review;
An initial assay comprising a complete blood count with automated differential, and a subsequent assay comprising an assay for anemia;
An initial assay comprising an acute hepatitis panel, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C;

An initial assay comprising one or more of celiac panel assays selected from IgA and HLA-DQ typing, and a subsequent assay comprising one or more of celiac reflex panel assays selected from tTG, DGP, EMA, and EMA titer;
An initial assay comprising a Hepatitis C antibody assay, and a subsequent assay comprising a quantitative nucleic acid assay for Hepatitis C;
An initial assay comprising an HIV antibody screening assay, and a subsequent assay comprising a confirmatory assay for HIV;
An initial assay comprising an HIV antibody screening assay, and a subsequent assay comprising an assay for differentiating between HIV-1 antibodies and HIV-2 antibodies;
An initial assay comprising an assay for treponema pallidum IgG, and a subsequent assay comprising a rapid plasma reagin (RPR) assay;
An initial assay comprising a rapid plasma reagin (RPR) assay, and a subsequent assay comprising an assay for an assay for antibodies to treponema pallidum;
An initial assay comprising a thyroid stimulating hormone (TSH) assay, and a subsequent assay comprising comprising one or more of an assay for tri-iodothyronine (T3), thyroxine (T4), and thyroid uptake.
206. The method of any of claims 174 to 205, wherein said sample is obtained in a single sample acquisition session, and wherein said sample comprises a volume sufficient for the performance of an initial assay and a subsequent assay.
207. The method of claim 206, wherein said sample is a small-volume sample having a volume of less than about 250 microliters (µL).
208. The method of claim 206, wherein said sample is a small-volume sample having a volume of less than about 100 microliters (µL).
209. The method of claim 206, wherein said sample is a small-volume blood sample or a small-volume urine sample.
210. The method of claim 206, wherein said sample is a blood sample obtained by fingerstick.
211. A form for ordering a clinical assay, comprising: a list of clinical assays for ordering diagnostic assays for performance on a sample obtained from a subject, wherein said list identifies each assay and the price for performing said assay.

212. The form for ordering a clinical assay of claim 211, wherein the list of clinical assays identifies each assay by at least one of: an assay name; an identifying acronym; an identifying number; an identifying alphanumeric designation; or combinations thereof.
213. The form for ordering a clinical assay of claim 211 or claim 212, wherein the list of the clinical assays listed on the form include initial assays and corresponding subsequent assays, and the form includes prices for the initial and for the corresponding assays.
214. The form for ordering a clinical assay of claim 211, 212, or 213, comprising explicit indications of the connection between such initial assays and corresponding subsequent assays.
215. The form for ordering a clinical assay of any of claims 211 to 214, comprising a combined price listed for an initial assay and a subsequent assay.
216. The form for ordering a clinical assay of any of claims 211 to 215, comprising lists of assays performed together in panels.
217. The form for ordering a clinical assay of any of claims 211 to 216, comprising explanations of the assays to be performed.
218. The form for ordering a clinical assay of claim 217, comprising explanations of the panels or of the assays performed together in panels.
219. The form for ordering a clinical assay of any of claims 211 to 218, wherein some or all of the more commonly performed assays are listed near to each other, or are listed together.
220. The form for ordering a clinical assay of any of claims 211 to 219, comprising assays listed near to, or grouped together with, other assays, wherein the assays listed near to each other, or grouped together, comprise assays which may commonly be ordered for patients exhibiting a common symptom or common symptoms.
221. The form for ordering a clinical assay of any of claims 211 to 220, comprising assays listed near to, or grouped together with, other assays, wherein the assays listed near to each other, or grouped together, comprise assays commonly ordered for patients sharing common risk factors.
222. The form for ordering a clinical assay of any of claims 211 to 221, comprising assays listed near to, or grouped together with, other assays, wherein the assays listed near to each other, or grouped together, comprise assays commonly ordered for patients sharing common age, sex, activity, or other characteristic.
223. The form for ordering a clinical assay of any of claims 211 to 222, wherein some or all of the listed assays are listed alphabetically.
224. The form for ordering a clinical assay of any of claims 211 to 223, comprising check boxes for selecting and indicating assays to be performed, wherein said assays include reflex assays which may be performed, contingent upon the results of initial assays.
225. The form for ordering a clinical assay of any of claims 211 to 224, comprising lines, boxes, or other locations for providing one or more of: a) patient identification and contact information; b) clinician information; c) payer information; d) clinic information; e) reimbursement information; and f) code information.
226. The form for ordering a clinical assay of any of claims 211 to 225, wherein the sample is a blood sample.
227. The form for ordering a clinical assay of any of claims 211 to 226, wherein the sample is a urine sample.
228. A method of providing a clinical assay performed on a sample obtained.

from a subject, comprising:
Providing an order form listing clinical assays and prices for each of said clinical assays;
Selecting one or more clinical assays from the order form.;
Obtaining a sample from. said subject;
Performing the selected clinical assay or assays; and Collecting payment for the selected clinical assay or assays, where the payment is determined by the price for each clinical assay listed on the order form.
229. The method of claim 228, wherein said order form. comprises an order form of any of claims 211-227.
230. The method of claim 228 or 229, wherein said clinical assay is performed according to a method any of claims 128-210.

231. The method of any of claims 228 to 230, wherein said clinical assays listed on said order form comprises clinical assays listed in panels, and said order forms comprise prices for each panel.
232. The method of claim 231, wherein said prices for each panel comprise capped prices for at least one panel, wherein a capped price is a maximum price for any combination of individual clinical assays of a panel.
233. The method of any of claims 228 to 232, wherein said payment is collected, at least in part, from the subject.
234. The method of any of claims 228 to 232, wherein said payment is collected, at least in part, from an insurance company.
235. The method of any of claims 228 to 232, wherein said payment is collected, at least in part, from a government agency.
236. The method of any of claims 228 to 232, wherein said payment is collected, at least in part, from the subject's employer.
237. A method of doing business, comprising:
Providing an order form listing clinical assays and prices for each of said clinical assays;
Selecting one or more clinical assays from the order form;
Obtaining a sample from. a subject;
Performing the selected clinical assay or assays; and Collecting payment for the selected clinical assay or assays, where the payment is determined by the price for each clinical assay listed on the order form.
238. The method of claim 237, wherein said order form comprises an order form of any of claims 211-227.
239. The method of claim 237 or 238, wherein said clinical assay is performed according to a method an.y of claims 128-210.
240. The method of any of claims 237 to 239, wherein said clinical assays listed on said order form comprises clinical assays listed in panels, and said order forms comprise prices for each panel.

241. The method of claim 240, wherein said prices for each panel comprise capped prices for at least one panel, wherein a capped price is a maximum price for any combination of individual clinical assays of a panel.
242. The method of any of claims 237 to 241, wherein said payment is collected, at least in part, from the subject.
243. The method of any of claims 237 to 241, wherein said payment is collected, at least in part, from an insurance company.
244. The method of any of claims 237 to 241, wherein said payment is collected, at least in part, from a government agency.
245. The method of any of claims 237 to 241, wherein said payment is collected, at least in part, from the subject's employer.
246. A method of providing a clinical assay performed on a sample obtained.

from a subject, comprising:
Providing an order form listing clinical assays, prices for each of said clinical assays, and CPT or ICD codes for each of said clinical assays;
Selecting one or more clinical assays from the order form;
Obtaining a sample from said subject;
Performing the selected clinical assays; and Collecting payment for the selected clinical assay or assays, where the payment is determined by the price for each clinical assay listed on the order form.
247. The method of claim 246, wherein said order form comprises an order form of any of claims 211-227.
248. The method of claim 246 or 247, wherein said clinical assay is performed according to a method any of claims 128-210.
249. The method of any of claims 246 to 248, wherein said clinical assays listed on said order form. comprise clinical assays listed in panels, and said order forms comprise prices and CPT or -ICD codes for each panel.
-129-.

250. The method of claim 249, wherein said prices for each panel comprise capped prices for at least one panel, wherein a capped price is a maximum price for any combination of individual clinical assays of a panel.
251. The method of any of claims 246 to 250, wherein said payment is collected, at least in part, from the subject.
252. The method of any of claims 246 to 250, wherein said payment is collected, at least in part, from an insurance company.
253. The method of any of claims 246 to 250, wherein said payment is collected, at least in part, from a government agency.
254. The method of any of claims 246 to 250, wherein said payment is collected, at least in part, from the subject's employer.
255. A method of doing business, comprising:
Providing an order form listing clinical assays, prices for each of said clinical assays, and CPT or ICD codes for each of said clinical assays;
Selecting one or more clinical assays from the order form;
Obtaining a sample from a subject;
Performing the selected clinical assay or assays; and Collecting payment for the clinical selected assay or assays, where the payment is determined by the price for each clinical assay listed on the order form.
256. The method of claim 255, wherein said order form comprises an order form of any of claims 211-227.
257. The method of claim 255 or 256, wherein said clinical assay is performed according to a method any of claims 128-210.
258. The method of any of claims 255 to 257, wherein said clinical assays listed on said order form comprise clinical assays listed in panels, and said order form comprises prices and CPT or ICD codes for each panel.
259. The method of claim 258, wherein said prices for each panel comprise capped prices for at least one panel, wherein a capped price is a maximum price for any combination of individual clinical assays of a panel.

260. The method of any of claims 255 to 259, wherein said payment is collected, at least in part, from the subject.
261. The method of any of claims 255 to 259, wherein said payment is collected, at least in part, from an insurance company.
261. The method of any of claims 255 to 259, wherein said payment is collected, at least in part, from a government agency.
263. The method of any of claims 255 to 259, wherein said payment is collected, at least in part, from the subject's employer.