WO2007145942A2

WO2007145942A2 - A rapid, semi-automated method to detect respiratory virus infected cells in nasopharyngeal samples using direct fluorescent antibody

Info

Publication number: WO2007145942A2
Application number: PCT/US2007/013234
Authority: WO
Inventors: Nathan Chapman; James Class; Mark Carle Connelly; Jimmy Page; Galla Chandra Rao; Leon W.M.M. Terstappen
Original assignee: Immunivest Corporation
Priority date: 2006-06-08
Filing date: 2007-06-05
Publication date: 2007-12-21
Also published as: WO2007145942A3

Abstract

Tests for rapid viral detection which rely on antigen detection vary considerably in their sensitivity and the turn-around-time (TAT) for a result. Point of care (POC) tests have a 15- 20 min TAT and are easy to use. POC tests find utility in physician's offices, microbiology labs and in virology labs during the second and third shift. However, the sensitivity of these tests is widely reported to be low 1,2, particularly out of season. Direct specimen testing using Cytospin3,4 or smear preparations has been reported to be more sensitive but requires 60-90 min, is tedious to perform, currently lacks standardization, leads to relatively large numbers of specimens with insufficient number of cells to conduct the test (QNS>20%), and requires a medium complex lab with skilled technologists performing and interpreting the fluorescent staining results. The present invention describes a method which combines the TAT of the POC tests with the higher sensitivity and ability to assess the specimen quality available with direct specimen testing.

Description

A RAPID, SEMI-AUTOMATED METHOD TO DETECT RESPIRATORY VIRUS INFECTED CELLS IN NASOPHARYNGEAL SAMPLES USING DIRECT FLUORESCENT ANTIBODY

Nathan Chapman, James Class, Mark Carle Connelly, Frank A.W. Coumans, Jimmy Page, Galla Chandra Rao, and Leon W.W.M. Terstappen

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a non-provisional application, which is incorporated by reference herein and claims priority, in part, of US Provisional Application No. 60/811,837, filed 08 June 200^Λ..

FIELD OF THE INVENTION

This invention relates generally to the detection and diagnosis of viral infections as used in automated fluorescence microscopy. The novel counting and imaging techniques are particularly applicable to rapid viral detection which rely on antigen recognition. Further, the present invention is applicable in the analysis of specific sample characteristics such as, but not limited to the presence of influenza A, Influenza B and Respiratory Syncytial Virus.

BACKGROUND OF THE INVENTION The enumeration of absolute levels of cells and their subsets in body fluids is of primary importance in determining the state of health of human beings and mammals in general. The primary analytical platform for performing such analyses is flow cytometry in which the specimen is either injected directly or after prior enrichment in rare cell analysis. Flow cytometry and similar complex analytical systems remain largely inaccessible for routine clinical use in resource-poor countries due to high instrument and reagents costs, lack of technical support, lack of robustness requiring frequent service, and the need for AC power. There is a clear need for simpler, more compact and less expensive systems also operable with emergency DC battery power and preferably exhibiting comparable performance characteristics. Jn addition to the above-cited full sized flow cytometry systems available from

Becton Dickinson and Beckman-Coulter, these vendors also sell scaled down less expensive versions, which still suffer from the other cited limitations. Similar limitations apply to the compact CyFlow® from Partec GmbH, (Munster, Germany) and to the Guava Personal Cytometer (Burlingame, CA). US Patent 6,097,485 (assigned to Integrated Wave Guides, Brookings, SD) discloses an ultra-miniature personal flow cytometer (pFCM) claimed to be of lower cost, but still exhibiting rather complex, electronic circuitry, optical designs, data reduction, all of which contribute to unacceptable complexity for a third world setting. All these systems use the flow concept, which obviously complicates the instrumental design. These scaled down versions of flow cytometry systems do not meet the clear need for a truly simple, compact, rugged, battery-operable and affordable cell analyzer.

Among the numerous clinical applications for a simple cell analyzer, counting of CD4 cells in HFV, granulocytes and platelets in patients treated with chemotherapy, and leukocytes in blood bags are most important. The current systems and methods for cell analysis have some significant disadvantages. They generally require sophisticated techniques, which involve the use of instruments that are expensive both in terms of initial cost and maintenance as well as requiring highly trained personnel. This makes the conventional systems unsuitable for use in laboratories of resource-poor countries. Therefore, a low-cost, easy-to- use method, for example, for CD4 cell enumeration is needed. Such a method may serve as a compact alternative to the current cell analysis systems that would be suitable for physician practices, bedside testing, or in open field settings with the ability to count rare cells in each condition. Further enumerating white cells in, for example, blood bags by a rapid, inexpensive means, instead of using flow cytometry where the analysis time is very lonn. The invention described herein meets the criteria above. The invention uses a CCD camera to image samples. Object detection algorithms are performed on the captured image to count the number of target entities present in a sample.

Testing paradigms for rapid viral detection which rely on antigen detection vary considerably in their sensitivity and the tum-around-time (TAT) for a result. Point of care (POC) tests have a 15-20 min TAT and are easy to use. POC tests find utility in physician's offices, microbiology labs and in virology labs during the second and third shift. However, the sensitivity of these tests is widely reported to be low 1,2, particularly out of season. Direct specimen testing using Cytospin3,4 or smear preparations has been reported to be more sensitive but requires 60-90 min, is tedious to perform, currently lacks standardization, leads to relatively large numbers of specimens with insufficient number of cells to conduct the test (QNS>20%), and requires a medium complex lab with skilled technologists performing and interpreting the fluorescent staining results. A method

The present invention describes a method combines the TAT of the POC tests with the higher sensitivity and ability to assess the specimen quality using direct specimen testing. SUMMARY OF THE INVENTION

This invention describes a simple, rapid, and efficient method for the detection of antigen positive cells in direct specimens in conjunction with rapid, objective recognition on a semi-automated, compact electronic optical instruments, incorporating a fluorescence microscope.

Cells of epithelial origin are captured and probed for the presence of virus. The cells could be captured using either cellular or viral specific antigens as targets for enrichment. The combination of magnetic enrichment and viral specific antibody detection provide a means for high sensitivity direct detection of infected cells in naspharyngeal samples. Alternatively, an instrument based on EasCount™ (Immunicon Corporation) can be used which is an automated fluorescence microscope using two light emitting diodes (LED's at 470 nm and 530 ran as excitation lamps and emission filters, respectively). The excitation lamps and emission filters allow imaging of fluorescein for virally infected cells and longer wavelengths for obtaining the total cell count. The target cells are collected and immobilized substantially uniformly on the optically transparent surface of the chamber. Each field of view in and individual well is interrogated at both wavelengths and a CCD camera captures the images for analysis. The on-board computer uses an image analysis algorithm to determine what signals should be assessed as real events. The required number of cells for interrogation is imaged by moving the slide with a motorized stage.

The cells are counted based on their fluorescence intensity difference with the background. The optimal target cell concentration after sample preparation is between 10³ and 10⁷ per milliliter. The emitted fluorescence is imaged onto a CCD camera. Image analysis routines coded inside the system determine the number of cells present, and then the number of cells per unit volume is calculated. The development of the algorithms for image acquisition and data reduction required considerable laborious experimentation and optimization. This resulted in the present invention configuration that exhibits the excellent performance characteristics as described herein.

The LEDs are positioned to illuminate along the long axis of the cartridge at a mean angle of incidence of 45 degrees. The turret provides up to four wavelengths, depending on the intensity required to illuminate the specimen. The present configuration is for two different wavelengths. This ensures maximum illumination and light capture. The use of solid state illumination devices ensures that the light source will outlive the life of the instrument, providing a distinct advantage in field use. The filter changer operates through a slider crank having an eccentric bearing to align the individual filters. The slider crank is optimized for a small space and minimal expense. The preferred number of emission filters is two, but multiple filters are contemplated with the present application.

It is to be understood and appreciated that these discoveries, in accordance with the invention, are only illustrative of the many additional potential applications of the apparatus, methods and algorithms that may be envisioned by one of ordinary skill in the art, and thus are not in any way intended to be limiting of the scope of the invention. Accordingly, other objects and advantages of the invention will be apparent to those skilled in the art from the following detailed description, together with the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1. Flow chart of liquid processing protocol for direct specimens. The assay buffer used provides permeabilization of the cells for MAb access to viral antigens inside the cells. The specimen slide has 6 wells which hole approximately 8 ul each and an attached cover slip.

FTGURE 2: Schematic representations of optical and illumination arrangements. In (A), light from an LED is focused on the sample through a condenser, a set of filters and a 1OX objective. An image of the fluorescence of the cells is projected on and captured by a CCD camera. In (B), the light of two LED's is directly projected onto the sample. FIGURE 3: A cross-sectional representation of the improved magnetic cartridge holder mounted on observation stage. The "spring positioning" tabs are positioned along the longitudinal axis as shown.

FIGURE 4: Representation of the filter changer. An eccentric bearing positions the sliding crank, having two or more filters, in position with the light path. Microscope is removed.

FIGURE 5. Flow chart of liquid processing protocol for direct specimens. The assay buffer used provides permeabilization of the cells for MAb access to viral antigens inside the cells. The specimen slide has 6 wells which hole approximately 8 ul each and an attached coverslip. FIGURE 6. Image capture of a representative field of view in a well containing RSV infected clinical specimen. The sample was treated according to the protocol shown. Images are of the same field captured using the different LEDs and emission filters to enable enumeration of total and RSV infected cells. The top 2 panels (A and B) were captured using the Evan's Blue channel and the bottom 2 (C and D) using the fluorescein channel. The left hand images are the raw images captured by the CCD camera and the right hand images show the resulting boxing/counting of those images by the imaging algorithm. The total cell count can be used to establish the specimen adequacy and allow a QNS determination.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The technical terminology with reference to biological, clinical, electronic, mathematical and statistical expressions used herein conform to conventionally accepted definitions.

The terms "sample" or "specimen" are interchangeably used herein and refer to biological material obtained from nasopharyngeal (NP) samples. A sample includes viruses, bacteria, or other pathogens. A typical example of a biological specimen would be an NP swab drawn from a subject. As utilized herein the term "cells" refers to animal or plant cells, cellular bacteria, fungi, which are identifiable separately or in aggregates. For example, cells can be human red blood cells (RBC) and white blood cell (WBC) populations, cancer, or other abnormal cells. The terms "target" or "target population" refers herein to biological entities of interest that may be present in a biological specimen that is being analyzed. System Design

The different components of the apparatus (sometimes referred to herein by its project name, "EasyCount") are shown in Figures 1. The imaging part of the apparatus is based on an epi-illumination fluorescence microscope. The surface of the sample chamber is illuminated by light emitting diodes. The light emitted from the fluorescently-labeled cells at the inner surface of the chamber is collected by an objective and focused onto a CCD.

To select and separate the target cells of interest, for example, from a whole blood sample, they are immunomagnetically labeled with a target specific antibody conjugated to magnetic particles, ferrofluids or superparamagnetic particles, as disclosed in US patents

5,579,531 and 5,698,271 and US application 10/208,939, each of which are incorporated by reference herein. The magnetic particles are typically about ISOnm in diameter and consist of a magnetic iron oxide core surrounded by a first polymeric layer to which streptavidin is conjugated. Target-specific antibodies can then be coupled to streptavidin by means of biotinylated antibodies. However, superparamagnetic particles made from other ferromagnetic materials, for example nickel, of similar or larger sizes of up to about 5μm, can be similarly coated and used for magnetic labeling of target cells. Finally alternative binders, such as lectins and boronate derivatives, recognizing glycosidic receptors on target cells may also be used in lieu of or in addition to antibodies on such magnetic capture particles.

For example, if the cells of interest are the total leukocyte population, a pan-leukocyte CD45 monoclonal antibody can be used that binds substantially specifically to all leukocyte populations in the blood sample. The cell labeling reaction can be conducted in test tubes or vials and an aliquot transferred to the sample chamber. Alternatively, the chamber itself can be used for incubations of specimen volumes of up to about 200μl. The unbound nonmagnetic materials are readily removable in the supernatants after magnetic separation. To enhance magnetic labeling efficiency of target cells one can use magnetic incubation or infield incubation (PCT/USOO/02034, which is incorporated by reference herein). The Imaging system Fluorescent staining of leukocytes

In order to make the nucleated cells detectable, the sample is stained with acridine orange (AO; Molecular Probes, Inc., Eugene, OR), a vital dye that stains the nucleus of live cells as well as several constituents of the cytoplasm. Acridine orange has its absorption peak at 490nm, and emits at 520nm when bound to DNA. Other fluorescent dyes, such as Hoechst 33258, and Hoechst 33342 may be used. In general, any fluorescent dye that non-specifϊcally stains cells, cytoplasm, cellular nucleic material, or the nucleus itself can be used. These dyes are referred to herein as "non-specific fluorescent dyes." Also, any particle that can be attached to an antibody and detected by microscopy is considered in the present invention. In general, illumination in fluorescence microscopy is achieved by mercury arc or quartz-halogen lamps. In some microscopy systems, more expensive lasers are used for illumination. However, recent advances in semiconductor technology have lead to the development of high-brightness light emitting diodes that can compete with incandescent light sources and lasers. The advantages of using LEDs as light source are that they are relatively compact, inexpensive, and have a long lifetime without a need to replace. The spectral power distribution of a LED is fairly narrow, with half-bandwidths of about 20 to 50nm, depending upon the substrate material. LEDs produce highly saturated, nearly monochromatic light and are ideal for constructing the compact and inexpensive cytometer devices of this invention. Optics

The light from an LED is collected by a condenser lens with a focal distance of 27mm, passes a short pass optical filter, focused at the sample plane. This optical configuration results in a homogeneous illumination of the sample area. The light emitted from the fluorescent cells collected at the underside of the glass surface of the chamber is collected by the objective (1-20X, NA 0.03-0.25), after which it is filtered by a band-pass or long pass filter and focused onto a high QE, high bit resolution (minimum 12 bits) CCD camera (DSI, Meade Instruments Corporation, Irvine, CA). Figure 2A shows the conventional epi-illumination mode. Figure 2B shows a direct side illumination of the viewing surface with one or more LEDs in a "floodlight" arrangement, which provides sufficient excitation energy, and may be a simpler and less expensive illumination mode.

The present invention improves upon the orientation of the LED with respect to the cartridge and the cell alignment. LED's are aligned along the longitudinal axis of the cartridge, ensuring maximum light intensity. Figure 3 depicts the advantage of orientating the components in a small area using a fixed distance between the specimen and objective lens

In addition, the present invention improves upon the positioning of the filter assembly. Figure 4 shows a general orientation of the emission filter set positioned in EasyCount. The filter changer is a sliding crank with an eccentric bearing to position the filter. This orientation provides for an inexpensive and compact device for switching between two or more filters. Camera

The CCD used in this set-up (DSI, Meade Instruments Corporation, Irvine, CA) where the image is retrieved from the camera by software and stored in a computer memory as 12/ 16-bit TIF images.

Image Processing and Analysis

Algorithms were developed to count the cells in the images obtained from the optical system. First, a model is presented to describe the cell images. Then, a method for spot detection in the images is introduced. Cells are enumerated based on size, intensity, uniformity, aspect ration, etc.

Example 1

Diagnosis of viral infection often involves Direct Fluorescent Antibody (DFA) and viral culture. DFA is faster but is not as sensitive as culture whereas culture may take 6 to 48 hrs for a result. CeIIT racks Technology, developed to detect ultra-rare tumor cells in circulation, was used to capture and detect virally infected cells present in nasopharyngeal samples.

Viral culture was done using standard methods using A549, Mink Lung, SKBr-3, and RMix (DHI) as viral host cell lines. Ferrofluids (FF) were prepared as colloidal suspensions of 200 nM magnetic particles coated with antibodies to specific antigen targets. Suspensions of cells were fixed with 80% acetone, washed, incubated for 10 min with antibodies, DAPI, and FF, then placed in a MagNests (Immunicon) for 10 min to affect magnetic mounting of the immuno-selected cells. Cells were imaged on a CellTracks Analyzer II, a four color fluorescence imaging system.

Flow cytometric analysis showed cultured cell lines and nasopharyngeal (NP) cells expressed the epithelial antigens Ep-CAM, Cytokeratin (CK), and MUC-1 on both virally infected and uninfected cells. Suggesting these antigens have a role in cell capture for detecting infected cells. Cultured cells were infected with Influenza A, fixed and serially diluted in buffer. Cells were captured using anti-CK FF and detected using anti-lnfluenza-A-FITC (anti-Flu A). At high concentrations 513 of 803 spiked cells were captured and at low cell numbers where samples contained 3.1 , 1.6, or 0.8 cells, 3, 0, and 1 cell respectively were detected. Similar results were seen when virally infected cells were spiked into UTM containing uninfected NP cells. Infected cells could aiso be captured using virally specific FF. Cultures were infected with influenza A or B at various M.O.I. Cells were harvested 4, 6, 8, and 22 hours post infection, fixed, stained and captured using anti-Flu A or anti-Flu B FF. Virus positive cells could be detected in as little as 6 hours post infection. Cells of epithelial origin may be captured and probed for the presence of virus. Cells could be captured using either cellular or viral specific antigens as targets for enrichment. The combination of magnetic enrichment and viral specific antibody detection may lead to the development of a rapid, simple, and high sensitivity method for direct detection of infected cells in NP samples. Example 2 The goal of this study demonstrates the feasibility of a simple, rapid, efficient, liquid processing format for detection of antigen positive cells in direct specimens in conjunction with rapid, objective recognition on a semi-automated fluorescence microscope. Cell Culture;

R-Mix and Influenza A (Flu A), Influenza B (Flu B), and Respiratory Syncytial Virus (RSV) was chosen as a prototype cell culture/virus system to simulate a direct specimen scenario for automated detection purposes. Viruses were grown on R-Mix under standard conditions in duplicate. One plate was fixed, stained and counted to confirm the viral input. The cells from the replicate plate were released by scraping or trypsinization and cells pelleted by centrifugation and the supernatant removed. The cell pellets were then resuspended in universal transport medium (UTM) at -600,000 cells/mL. These cell suspensions were then treated as a mock clinical specimen for further testing. Cell Counting; Fixed and stained cells in multi-well plates were read on a fluorescence microscope by a trained technologist as a comparator for ACEit™. The prototype ACEit™ instrument is essentially an automated fluorescent microscope with cell counting capability. Liquid Processing Protocol; A rapid, efficient liquid processing protocol was developed as outlined in Figure 5.

This protocol allows concentration of the cells, a short, efficient staining step, and simple loading into the automated microscope.

Correlation curves for ACEit and fluorescence microscopy using R-Mix cells inoculated with the indicated viral dilutions of either Flu A, Flu B, or RSV, incubated, and the cells detached. The ACEit assay was able to detect each of the viruses at the lowest viral input (approximately 100 viruses/well).

As shown in Figure 6, Image capture of a representative field of view in a well containing RSV infected clinical specimen. The sample was treated according to the protocol shown. Images are of the same field captured using the different LEDs and emission filters to enable enumeration of total and RSV infected cells. The top 2 panels (A and B) were captured using the Evan's Blue channel and the bottom 2 (C and D) using the fluorescein channel. The left hand images are the raw images captured by the CCD camera and the right hand images show the resulting boxing/counting of those images by the imaging algorithm. The total cell count can be used to establish the specimen adequacy and allow a QNS determination.

The study shows that liquid processing of virus-infected cells can be used to produce a good MAb staining reacton from a mock clinical sample in less than 15 minutes. Further, reading specimens produced in this manner on ACEit™ provided a specific, sensitive and, importantly, objective determination. Both total cell counts and viral infectivity are able to be determined in a 1 minute per sample read time. The method has applicability to multiple viruses, having the potential to apply to clinical samples to yield valid results.

While certain of the preferred embodiments of the present invention have been described and specifically exemplified above, it si not intended that the invention be limited to such embodiments. Various modification may be made thereto without departing from the spirit of the present invention, the full scope of the improvements are delineated in the following claims.

Claims

We claim:

1. An semi-automated method for detecting and counting a viral infection, comprising: a. obtaining a biological sample from a subject; b. fixing cells in the biological sample; c. immuno-magnetically capturing fixed cells that express epithelial antigens; d. preparing a fluorescently-tagged sample such that a specific fluorescent dye is linked to one or more viral targets; e. illuminating said surface, such that said illumination will encompass said target population; f. capturing light emitted by said target population; and g. acquiring images through algorithms from said capturing such that said algorithms count the number of captured light-emitting sample components and relate said number to said fluid sample.

2. A method as claimed in claim 1 , wherein said biological sample is a nasopharygeal sample.

3. A method as claimed in claim 1 , wherein said fixing includes the addition of 80% acetone.

4. A method as claimed in claim 1 , wherein said capturing is anti-CK FF;

5. A method as claimed in claim 1 , wherein the fluorescent tag is from a group consisting of Influenza A, Influenza B, and Respiratory Syncytial Virus.

6. A method as claimed in claim 1 , wherein the number of cells expressing said tag correlates to a disease state.

7. An semi-automated method for detecting and counting a viral infection, comprising: a. obtaining a biological sample from a subject; b. fixing cells in the biological sample; c. preparing a fluorescently-tagged sample such that a specific fluorescent dye is linked to one or more viral targets; d. illuminating said surface, such that said illumination will encompass said target population; e. capturing light emitted by said target population; and f. acquiring images through algorithms from said capturing such that said algorithms count the number of captured light-emitting sample components and relate said number to said fluid sample.

8. A method as claimed in claim 1 , wherein said biological sample is a nasopharyngeal sample.

9. A method as claimed in claim 1 , wherein the fluorescent tag is from a group consisting of Influenza A Influenza B and Respiratory Syncytial Virus.

10. A method as claimed in claim 1 , wherein the number of cells expressing said tag correlates to a disease state.