WO2018048586A1

WO2018048586A1 - Improved reagent and method for identification and enumeration of basophils

Info

Publication number: WO2018048586A1
Application number: PCT/US2017/046961
Authority: WO
Inventors: Chiranjit Deka; Jay R. STRINGER
Original assignee: Chiranjit Deka
Priority date: 2016-09-06
Filing date: 2017-08-15
Publication date: 2018-03-15

Abstract

A lytic reagent composition and methods for identification of basophils in a flow cytometer are disclosed. In embodiments, the reagent used in the analysis includes an antihistamine compound. In embodiments, the antihistamine may be ketotifen fumarate. The flow cytometer may be configured as an automated hematology analyzer that measures light scatter to identify basophils and other white cell populations, including lymphocytes, neutrophils, eosinophils and monocytes.

Description

IMPROVED REAGENT AND METHOD FOR IDENTIFICATION AND ENUMERATION OF BASOPHILS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 62/383,967, filed September 06, 2016, the contents of which are incorporated by reference in its entirety.

STATEMENT REGARDING GOVERNMENT SUPPORT

[0002] This work was funded in part by the National Institutes of Health SBIR Grant Number 1R43 All 13897-01

FIELD

[0003] The present invention relates to improvements in methods and reagents for the identification and enumeration of basophils in a blood sample analyzed on a flow cytometer or a hematology analyzer.

BACKGROUND OF THE INVENTION

[0004] The present invention relates to improvements in methods and reagents for the identification and enumeration of basophils in a blood sample. The invention is useful for differentiating basophils from four other leukocyte populations, namely, neutrophil, eosinophil, lymphocytes and monocytes in a blood sample. The invention is particularly suitable for use in a semi-automated or automated flow cytometer or an automated hematology analyzer. In this document, the terms flow cytometer and automated hematology analyzer will be used interchangeably, and the term flow cytometer will be used to mean any one of either a semi- automated flow cytometer, and automated flow cytometer, and an automated hematology analyzer. [0005] Automated hematology analyzers are flow cytometers that use the basic principles of flow cytometry and on which the sample processing steps are automated. Automated hematology analyzers identify and enumerate blood cells and measure many different blood cell parameters in a human whole blood sample, including the percentage and absolute numbers of major sub-populations of leukocytes (white blood cells or WBC). Hematology analyzers that identify and enumerate the five WBC sub-populations, namely, the lymphocytes, monocytes, neutrophils, eosinophils and basophils, are called 5-Part differential hematology analyzers. In a typical 5 -part differential hematology analyzer, to count the leukocytes, the erythrocytes (red blood cells or RBCs) in a whole blood sample are first lysed by adding a lytic reagent to the blood sample and causing the remaining leukocytes in the sample to flow seriatim, substantially in a single file, through a narrow aperture or cell interrogation zone in a flow cell, subjecting each cell to an external energy, either a light beam or electrical field, or both. While passing through the interrogation zone, several different measurements are simultaneously made on each blood cell to measure each cell's unique response to the said external energy. Typical measured responses used in prior arts include light scatter, Coulter DC volume, radio frequency (RF) electrical conductivity, polarization, and/or fluorescence. In some instruments, in addition to the lytic reagent, other reagents are also used to induce differential biochemical processes between the different leukocyte sub-populations. The greater the number of measurements and reagents needed to identify these five leucocyte sub-populations, the greater the complexity and size of the instrument and its cost are. Hence, methods that can enumerate all five leucocyte populations with fewer parameters and fewer reagents and reaction steps are desirable.

[0006] Of the five leukocytes sub-populations, the basophils are the most difficult to differentiate from the other cell types by light scatter measurements. This is because light scatter signals intensities from the basophils tend to overlap with the light scatter signals from some of the monocytes and lymphocytes, which causes the basophils to be difficult to identify as a separate population. To further complicate matters, the number of basophils in a blood sample generally tend to be much lower than the average number of lymphocytes and monocytes, and they are often not tightly clustered. This makes it harder to distinguish them in a light scatter gram in which light- scatter measurements from two or more detectors are plotted against each other. This problem is discussed in the article by Terstappen et. al., Cytometry 9:39-43 (1988), where a scattergram obtained by comparing light scatter measurements at two different angular ranges, l°-2.6° and 3°-l 1°, was used to identify the three major populations of white blood cells, namely, the lymphocytes, monocytes and granulocytes (which, in this case, included the eosinophil and neutrophil sub-populations). In this scattergram, the basophils were not resolvable as an independent cluster although a general region or "gated" area was tentatively assigned by the authors where the basophils were purported to appear. However, it was the conclusion of the authors of the article that this gated area was insufficient to obtain a reliable basophil count.

[0007] Other different approaches to the challenge of identifying and enumerating basophils have been disclosed in the prior art. For example, in U.S. Pat. No. 5,125,737, Rodriguez et. al., the problem of overlapping lymphocyte and basophil sub-populations was addressed by using a measurement parameter termed "opacity". In this method, in addition to light scatter, two electrical parameters, the DC impedance and RF conductivity, were measured on each blood cell. Opacity was defined as the ratio of a cell's DC impedance (volume) to its RF conductivity. While useful in resolving the basophils from other leukocytes, this approach requires a complex flow cell, a high voltage source, complex electrical circuits to create the DC and RF energy field within the cell-interrogation zone, and complex circuitry to detect changes in the DC and RF currents. Owing to signal-to-noise issues, this approach is difficult to implement and is expensive to manufacture.

[0008] Another approach for differentiating basophils, used in the. H* 1 Hematology Analyzer manufactured by Technicon, Inc., employed a two-step chemical process to count the basophils after differentially lysing the other leukocyte sub-populations. The two sequential chemical processes increased the total time for the assay, the complexity of automating the two reactions added to the instrument's cost and size, and additional reagents added to the consumable cost per test. Another approach, disclosed by Hubl et al. [J. Clin. Lab. Anal. 10: 177- 183 (1996)] describes the use of double staining with fluorescence-labeled monoclonal antibodies to identify basophils. Other special methods, such as staining of heparin within the basophils at low pH and in the presence of lanthanum ions, have also been used to resolve basophils [Gilbert et. al., Blood, 46:279-286 (1975)]. These prior art approaches are complex and expensive and not suitable to automated hematology analysis.

[0009] Deka et. al. (US6232125) disclosed a method for enumeration basophils in an apparatus comprising light scatter and DC measurements, wherein a flow cell similar to the one used previously by Rodriguez et. al. (US 5,125,737) were used. In this method, a population comprising lymphocytes and basophils were identified by comparing DC and light scatter, and the basophils were subsequently identified by light scatter measurements in 4 different angular ranges and combining them in complex mathematical transformations. The used to transformation based on multiple light scatter measurements can make the results less reliable as the calibration factors used in the transformation can vary from instrument to instrument, subject to slight differences in optical alignments.

[0010] Koester et al described a method for identifying basophils by staining the cells with the dye Astrazon Orange (Basic orange 21) and measuring the fluorescence from the stained cells. Syouhei and Sakata et. al (US 5,039,613) taught a method for identifying five sub-populations of leukocytes, including basophils, using fluorescence dyes to differentially stain the various cells. However, using a fluorescence dye adds to the complexity of the protocol and cost of the instrument.

[0011] Montero- Julian et al. (US 7, 101,678 B l) describes a method for identifying basophils using monoclonal antibodies. Reagents containing monoclonal antibodies are expensive and not suitable for routine hematology analysis. [0012] Uchihashi et al. (US 7943383) describes a method in which two separate reagents and two reaction steps are used to enumerate basophils. In the first reaction step, 4 different population of white blood cells (lymphocytes, monocytes, eosinophils and neutrophils) are enumerated. The second reaction step is used to identify basophils. This is complex and requires expensive fluidic automation to implement.

[0013] Therefore, improved reagents and methods are needed to identify basophils in an automated hematology analyzer or a flow cytometer that are simple, rapid, and relatively inexpensive.

SUMMARY OF THE INVENTION

[0014] In view of the foregoing discussion, an object of the present invention is to provide an improved reagent and method for the identification and enumeration of basophils in whole blood; the said reagent and method should not require complex RF (opacity) measurements, or need the use of expensive monoclonal antibodies and fluorescent dyes, and should minimize the number of reaction steps. Preferably, the method must produce results that are direct and are not dependent on complex mathematical models.

[0015] One object of this invention is to obtain an improved reagents and method for the identification and enumeration of basophils in whole blood using light scatter measurements that requires no more than two light scatter detectors to resolve basophils from the other white blood cells, and in some embodiments no more than 3 light scatter detectors. Another object is to obtain reagents and method where the number of reagents and reaction steps are few, preferably eliminate the need to multiple reaction steps to identify the basophils.

[0016] Another object of this invention is to provide a light-scatter based method for improved identification of basophils in whole blood using reagents and methods that also allows one to simultaneously identify and enumerate other major leukocyte sub- populations, such as for example two or more of the group comprising neutrophils, eosinophils, lymphocytes and monocytes.

[0017] In one embodiment, the reagent and method of this invention identifies basophils and four other major leukocyte sub-populations including neutrophils, eosinophils, lymphocytes, and monocytes using 3 light scatter detectors. In embodiments, the reagent and method of this invention identifies five leukocyte sub- populations, including basophils, using only 2 light scatter detectors.

[0018] According to a preferred embodiment of the present invention, an enhanced light scatter resolution of the basophils relative to other leukocytes were attained by a method comprising the steps of (a) mixing an aliquot of whole blood sample with a reagent that contains a compound that enhances the differences in light scatter signals for basophils relative to other leukocyte cell types; (b) running the sample through a flow cell where the blood cells pass through a focused beam of light; (c) measuring the light scattered by the blood cells in the samples within at least two different angular ranges; (d) and comparing the said light scatter measurements and identifying the basophils.

[0019] Preferably, in one embodiment the compound that enhances the differences in light scatter signals for basophils relative to the other cell types. In one embodiment, this compound is an antihistamine. In a preferred embodiment, the said antihistamine compound is an HI antagonists, also called HI blockers. An example of an HI antagonist antihistamine compound is Ketotifen Fumarate, which has been used in one preferred embodiment of this invention. Ketotifen Fumarate is a salt of the compound Ketotifen with fumaric acid whose structure is shown on FIG. 3. Ketotifen is an antihistamine that has been previously used to treat allergic symptoms by blocking a certain natural substance (histamine). It is often used as a medication to prevent and treat itching of the eyes caused by allergies.

[0020] One skilled in the art may select other antihistamine compounds to distinguish basophiles from monocytes and lymphocytes in a manner similar to the one disclosed in this document, using a variety of other lytic reagents . Other examples of HI antagonists include, among others, Acrivastine, Azelastine, Bilastine, Brompheniramine, Carbinoxamine, Cyclizine, Dexchlorpheniramine, and Dimenhydrinate. Other type of antihistamines can also be used, for example, H2-anithistamines.

[0021] In a preferred embodiment, the said reagent containing a compound that enhances the differences in light scatter signals for basophils relative to the other cell types also lyses the red blood cells. Most preferably, the said lytic reagent containing the antihistamine compound also enables four other populations of the white blood cells, namely, lymphocytes, neutrophils, monocytes, and eosinophils, to be identified alongside the basophils.

[0022] The invention will be better understood from the ensuing detailed description of the preferred embodiments, reference being made to the accompanying drawings.

DESCRIPTION OF THE DRAWINGS

[0023] FIG. 1 is a bivariate plot of light scatter measurements (IALS versus SSC) showing the basophil population resolved from the monocyte and lymphocytes on FIG 1 panel labeled 1A. The same scatter plot on the right, FIG. l panel labeled IB, shows a basophil gate within which the basophils were counted. In this sample, within this gate, the percent of basophil events counted were 2%, which matched closely with results obtained on a commercial hematology analyzer, which was 2.2%.

[0024] FIG.2 is a bivariate plot of light scatter measurements (IAL-versus-SSC) showing the basophil population resolved from the monocyte and lymphocytes (2A). The same scatter plot on the right (2B) shows the basophil gate within which the basophils were counted. In this sample, within this gate, the percent of basophil events counted was 0.04%, which closely matched the actual result, 0.05%, obtained on a commercial hematology analyzer.

[0025] FIG. 3 show the Chemical structure of Ketotifen Fumarate. DETAILED DESCRIPTION

[0026] An object of this invention is to obtain a reagent and a method that provides enhanced differentiation of basophils relative to the other cells in a whole blood sample analyzed on a flow cytometer without the use of RF, DC or fluorescence measurements. The said flow cytometer may be configured either as semi -automated flow cytometer or configured as a completely automated hematology analyzer. The distinction between a semi-automated flow cytometer and an automated hematology analyzer is well-known in the industry. References cited on this disclosure provide examples of various configurations of automated hematology analyzers and semi-automated flow cytometers. In this disclosure, the term "flow cytometer" will be used to mean, interchangeably, any one of a semi-automated flow cytometer, automated flow cytometer, and an automated hematology analyzer.

[0027] Another object of this invention is to obtain a reagent and a method that will provide an enhanced differentiation of basophils in a whole blood sample relative to the other cell populations analyzed on a flow cytometer using only light scatter signals.

[0028] According to a preferred embodiment of the present invention, an enhanced light scatter resolution of the basophils from the other white cell populations were attained by a method comprising the steps of:

(a) mixing an aliquot of whole blood samples with a reagent that contained an antihistamine compound;

(b) running the sample through a flow cell in a flow cytometer where the blood cells pass through a focused beam of light;

(c) measuring the light scattered by the blood cells in the samples within at least two different angular ranges;

(d) comparing the said light scatter measurements and identifying the basophils.

[0029] Preferably, the said reagent containing an antihistamine compound is also lytic reagent that lyses red blood cells. Most preferably, the said lytic reagent containing the antihistamine compound also enables four other populations of the white blood cells, namely, lymphocytes, neutrophils, monocytes, and eosinophils, to be identified alongside the basophils.

Example 1 :

[0030] 12 uL of whole blood was added to 250 uL of a lytic reagent containing 0.05% Ketotifen Fumarate and incubated for about 1 minute. In this example, the said lytic reagent containing the 0.05% Ketotifen Fumarate comprised a30 mM solution ofNaCl, 0.001% (w/v) of surfactant sodium dodecyl sulfate (SDS), 4.6mM K2HP04, 0.74mM KH2P04, and 0.1 % BS A in an aqueous solution at pH 7.5.

[0031] The sample mixture of blood and lytic reagent was then run on a flow cytometer. In the flow cytometer the blood cells were illuminated with a laser beam and the scattered light from the cells were detected in three different angular ranges, namely the forward angle where axial light loss (ALL) was measured, the side-scatter measuring scattered light in the direction orthogonal to the direction of the laser beam and the direction of the flow of the sample in the flow cell (SSC), and a third angular range between about 25 degrees and about 50 degrees relative to the laser beam, hereinafter called the intermediate angle light scatter (IALS). By comparing IALS and SSC measurements, the basophils 1 were identified from the other white cell populations as shown in this example, the SSC measure scattered light within a cone of around the orthogonal direction having a half-angle of about 30°. Fig. 1 A. On FIG. 1 A the other populations shown are the monocytes 2, lymphocytes 3, neutrophils 4 and eosinophils 5. In FIG. IB, the same data is shown with an oval shaped boundary 6, called the basophil gate, around the basophil population 1. In this sample, the method of this invention estimated the basophil percentage to be 2%. This matches closely the corresponding result, 2.2%, obtained by an independent reference method on a commercial hematology analyzer.

[0032] One skilled in the art would understand that in some embodiments a lytic reagent with other formulations, but still containing an antihistamine, can be optimized to provide enhancement in the identification of basophils. For example, in some embodiment, the lytic reagent may comprise other organic or inorganic compounds and be hypotonic or hypertonic, and may contain surfactants that are either anionic, non-ionic, zwitter-ionic or cationic. In some embodiment, for example, the lytic reagent can be optimized to work without the anionic surfactant sodium octyl sulfate (SOS).

[0033] One skilled in the art would further understand that in some other embodiments the half-angle of the SSC measurements may be varied to certain degrees. The angular range of the IALS detector can also be optimized to certain extent. In some embodiments, the ALL detector may be replaced by either a forward light scatter (FLS) detector or a DC impedance detector.

Example 2:

[0034] The method of Example- 1 was repeated for a sample with very low basophils. By comparing IALS and SSC measurements, the basophils 7 were identified from the other white cell populations as shown in FIG. 2A. On FIG. 2A the other populations shown are the monocytes 8, lymphocytes 9, neutrophils 10 and eosinophils 11. In FIG. 2B, the same data is shown with an oval shaped boundary 12, called the basophil gate, around the basophil population 7. In contrast to the samples described in Examplel, the number of event within the basophil gate 12 were low, about 0.04%. This matched closely the corresponding basophil percentage, 0.05%, obtained for this sample by an independent reference method on a commercial hematology analyzer.

Claims

WE CLAIM:

1. A reagent for improved identification of basophils in a whole blood sample in a flow cytometer, comprising a lytic reagent that lyses red blood cells,

wherein the said lytic reagent comprises an antihistamine compound, and wherein the said flow cytometer comprises a hematology analyzer.

2. A reagent according to claim 1, wherein the said antihistamine compound is Ketotifen Fumarate.

3. A reagent according to claim 1, wherein the lytic reagent comprises a hypotonic solution of alkali metal salt.

4. A reagent according to claim 1 , wherein the lytic reagent comprises a hypotonic solution of alkaline metal earth salt.

5. A method for identification of basophils comprising the steps of:

(i) mixing a measured volume of the lytic reagent of claim 1 with a measured volume of whole blood,

(ii) incubating the said mixture of lytic reagent and whole blood sample,

(iii) running the said sample mixture through a flow cell in a flow cytometer,

(iv) exposing the said sample in the flow cell to a beam of light,

(v) measuring light scatter signals from the cells at least two different angular ranges,

(vi) identifying the basophils from the said light scatter measurements.

6. A method for identification of basophils as in claim 5, wherein the said at least two different angular ranges comprise an SSC angular range of about 90° +/- about 30° and an IALS angular range selected from about 25° to about 50° relative to the direction of the said beam of light.

7. A method for identification of basophils as in claim 5, wherein the said at least two different angular ranges comprise an SSC angular range of about 90° +/- about 30° and an IALS angular range between about 35° and about 45° relative to the direction of the said beam of light.

8. A method for identification of basophils as in claim 5, wherein the said at least two different angular ranges comprise an SSC angular range of about 90° +/- about 30° and an IALS angular range between about 40° and about 45° relative to the direction of the said beam of light.

9. A method according to claim 5, wherein additionally at least a third light scatter measurement is performed, the said third light scatter measurement comprising one of measuring forward light scatter (FLS) within a range of about 0.5° to about 2° and measuring axial light loss (ALL) within the range of 0° to about 0.5°.

10. A method according to claim 6, wherein five different leukocyte populations are identified by comparing the light scatter signals from the first, second and third light scatter angles in various pair-wise combinations thereof, wherein the said 5 different leukocyte populations comprise lymphocytes, monocytes, neutrophils, eosinophils and basophils.