Procedure for the simultaneous quantification, in a single measurement, of the major types of human lymphocytes and their subsets

Abstract: Procedure for the simultaneous quantification, in a single measurement, of the major types of human lymphocytes and their subsets. It includes: incubation of the sample with five different monoclonal antibodies conjugated with three different fluorochromes; measurement by flow cytometry of the three fluorescent emissions; and analysis of the results obtained by means of multiparametric analysis to determine the presence/absence of a certain fluorescence in a cell and its intensity in each cell.

Description: Although not exclusively, this invention is mainly related to a procedure for the simultaneous quantification of the major types of human lymphocytes (T, B, and NK cells) and their subsets (CD3+/CD4+,CD3+/CD8+, etc) up to a total of 12 lymphoid subpopulations by means of a quick cost effective, sensitive and specific single measurement capable of being analyzed in a flow cytometer equipped with a single laser for research or diagnostic, prognostic and therapeutic purposes. Lymphocytes are the cells responsible for Immunosurveillance as well as for the specificity of Immune defense In humans. The Immunophenotyping of lymphoid subpopulations by combining the use of monoclonal antibodies and flow cytometry provides objectives and sensitive measurements of both the distribution of the lymphoid subpopulations and their Intensity of positivity for a certain antigen. Both types of Information have rapidly attracted the attention of professionals from different areas of Medicine, such as Cell Biology, Immunology and Haematology, as the measurement of different lymphoid subsets has proved to be of great diagnostic and prognostic value in different pathologic conditions. Of all these applications, one whose usefulness should be pointed out is the that of the enumeration of CD4+ T-cells in those individuals who display serum antibodies against the human Immunodeficiency virus (HIV) responsible for the acquired Immunodeficiency syndrome (AIDS). This measurement represents at present one of the most important prognostic criteria that should be taken into account when analyzing these patients progress. The clinical relevance of this kind of measurement has largely contributed to the fact that over the past few years there has been an enormous increase in the need for appropiate quality control procedures when analyzing lymphoid subpopulations by flow cytometry in order to eliminate technical artifacts that may lead to erroneous results. As a result of these studies dealing with the standardization of lymphoid subset measurement using flow cytometry, several research groups, scientific associations and public institutions have published documents with recommendations and guidelines for everyone to follow when immunophenotyping human lymphocytes by means of flow cytometry. These documents recognize the need for systematic identification of: 1) the proportion and purity of the lymphocytes analyzed, i.e., the proportion of lymphocytes not included in the analysis as well as the proportion of the events analyzed that correspond to lymphocytes; and 2) the total number of T, B and NK cells as well as CD3+/CD4+ and CD3+/CD8+ T-cell subsets. This means that a minimal panel of monoclonal antibodies must be used which in turn means that a high number of measurements must be carried out per sample. As an example, in 1992 the CDC (Center for Disease Control, USA) recommended the use of five different measurements combining two monoclonal antibodies with different fluorochromes for the enumeration of CD4+ T-cells in HIV+ patients in order to fulfill the two objectives mentioned above: 1) CD45/CD14 for the study of the purity and the proportion of lymphocytes 2) CD3/CD19 for the identification of the total number of T-lymphocytes (CD3+/CD19-) and the total number of B-lymphocytes (CD3-/CD19+). 3) CD3/CD56 for the characterization of NK-cells (CD56+/CD3-). 4) CD3/CD4 for the identification of CD3+/CD4+ T-cells wich are related to helper/induction functions within the immune system. 5) CD3/CD8 for the identification of CD3+/CD8+ T-cells witch are associated with suppresor/cytotoxic immunologic functions. Furthermore, EP-A-0 219 309 discloses a method of identifying subpopulations of blood cells which consists of using a monoclonal antibody specific for each for each subpopulation conjugated with one or two fluorochromes. The different antibodies are distinguishable by labelling the antibodies with different amounts of one or both fluorochromes. In an illustrative example, 7 subpopulations are identified using 9 different monoclonal antibodies and two fluorochroms (see example 8). Up till now, no procedure has been described that allows direct and simultaneous analysis of the total number of T, B, NK, CD3+/CD4+ and CD3+/CD8+ populations in a single measurement, eliminating the need to perform up to five different measurements using ten monoclonal antibodies in double-staining combinations. Thus one of the aims of this invention is to propose a solution for the simultaneous study, in a single measurement, of 12 lymphoid subsets, including all of the T, B, NK, CD3+/CD4+ and CD3+/CD8+ lymphocytes. Another aim of the invention is the simultaneous assessment of contamination by non-lymphocytes present in the elements analyzed. . The procedure of this invention consists of the use of a mixture of five monoclonal antibodies conjugated with three different fluorochromes: CD19 and CD8 conjugated with on fluorochrome (FL1), for instance fluorescein isothiocyanate (FITC); CD3 and CD56 and/or CD16 conjugated with another fluorochrome (FL2), such as phycoerythrin (PE); and CD4 conjugated with a third fluorochrome (FL3), such as PerCP™ (peridinin chlorophyll protein) tm or TriColor™ (phycoerythin/cyanine 5 fluorochrome tandem). Thus, the method claimed in the present invention is distinguished from EP-A-0 219 309 by the use of only five antibodies, which allow to identify more than 5 different subpopulations. Moreover, the antibodies are labelled with only one of three fluorochromes, while in the method in EP-A-0 219 309 two fluorochromes have to be used in varying amounts so that each antibody used is distinguishable from the others. Sample preparation, cell staining with monoclonal antibodies, selection of lymphocytes, as well as the calibration and adjustment of the flow cytometer are performed in accordance with widely described and recommended methods for the Immunophenotyping of lymphoid subpopulations. For the analysis of the results and for the exact quantifcation of each subpopulation, different software programs can be used, of these PAINT-A-GATE PLUS tm was especially useful. In the analysis both the negativity/positivity and the intensity of positivity for each monoclonal antibody are taken into account. An explanation of the procedure for the analysis of the results obtained from the measurement performed with the flow cytometer is described below. B-lymphocytes are the only major population of FL1+ lymphocytes (CD19+ or CD8+) that lacks the expression of CD3 and CD56 (and/or CD16) (FL2), thus being the only FL1+ / FL2-lymphocytes when considering the FL1/FL2 bidimensional space. In addition, among T, B, and NK cells, the CD19 antigen is specific for B-lymphocytes, being present in all these cells, which in turn are CD8-. Accordingly we can state that the FL1+ / FL2- cells correspond to the total number of B-cells. The separation between T (FL2+ because they are CD3+) and NK cells (FL2+ because they are CD56+ (and/or CD16 ⊕ ) / CD3-) is clearly established when the intensity of FL2 fluorescence/cell is considered. This is due to the fact that the intensity of CD3 fluorescence in lymphocytes is constantly different from that of CD56 (and/or CD16) fluorescence in NK cells when the same fluorochrome is used for the detection of both antigens by flow cytometry, there being no overlap between these two lymphoid populations in the axis that in this case reflects the amount of FL2. Thus, when the FL1 /FL2 bidimensional space is exclusively considered, total B (CD19+ /CD3- /CD56- and/or CD16 - ), T (CD3+ /CD19- /CD56 and/or CD16 ⊕ + or -) and NK cells (CD56+ (and/or CD16 ⊕ ) /CD3- /CD19-) are measured. Moreover, it allows us to determine the purity of the lymphocytes analyzed since the sum of total CD19+, CD3+ and CD56+ (and/or CD16 + ) /CD3- cells represents the total number of lymphocytes present in the sample analyzed and thus the CD19- /CD3- /CD56- and/or CD16 - elements are non-lymphoid cells or events. Staining with CD8-FL1 allows other T and NK cell subpopulations to be identified: 1) CD3+ /CD8+ T-lymphocytes; 2) CD3+ /CD8- T-lymphocytes; 3) CD56+ (and/or CD16 + ) /CD3-/CD8+ NK cells; and 4) CD56+ (and/or CD16 + ) /CD3-/CD8- NK cells. In addition, regarding CD3+ /CD8+ T-lymphocytes, CD3+ T-cells that express CD8 at In addition, regarding CD3+ /CD8+ T-lymphocytes, CD3+ T-cells that express CD8 at low intensity can be identified and separated from those that show strong CD8 expression. Staining with CD4 conjugated with a third fluorochrome (FL3) allows the identification of the subset of CD3+ /CD4+ T-lymphocytes and the possible presence or absence in the sample of CD3- /CD4+ cells lacking the CD19 antigen; the latter cells correspond to the contamination of monocytes. Finally, when the reactivities for CD3, CD8 and CD4 are simultaneously taken into account, another two T-cell subpopulations are identified: 1) CD3+ /CD4- /CD8- T-lymphocytes; and 2) CD3+ /CD4+ /CD8+ T-lymphocytes. The invention can be used for both normal and pathologic samples for all purposes in which the analysis of lymphoid populations is required, particularly for research, diagnostic and prognostic purposes as well as for therapeutic evaluation. As can be concluded from what has been previously explained, the fluorochromes used in FL1, FL2 and FL3 can be modified. Moreover, it is understood that this invention includes variations that omit a third fluorescence such as the CD19-FL1 /CD8-FL1/CD3-FL2 /CD56 (and/or CD16) -FL2 and the CD19-FL1 /CD4-FL1 /CD3-FL2 /CD56 (and/or CD16) -FL2 combinations. Among other advantages, an important decrease in the time, the costs and the variability of the analysis of lymphoyd subpopuations is obtained with this invention. In addition, it should be taken into consideration that its capacity to perform this five-fold combination provides detailed information about a total of 12 lymphoid subpopulations, this being of great interest in order to systematically determine the distribution of lymphoid subsets and their possible role in different pathologic conditions. Moreover, it allows the direct quantification of the non-lymphoid elements that are present in the analysis. Next, the invention will be illustrated by the following example, which not limitative of its possibilities: EXAMPLE 1.- Material and methods. Peripheral blood (PB) was obtained from 10 healthy volunteers in the presence of liquid EDTA (K3) by venipunction and maintained at room temperature until processed. The analysis of PB lymphoid subpopulations as performed within five hours using a whole lysed blood and direct immunofluorescence technique measured by flow cytometry. 2.- Sample preparation. Six test-tubes containing too µl of PB and different combinations of directly-conjugated monoclonal antibodies purchased from Becton/Dickinson (San José, CA, USA) were prepared for each of the samples. The specificities of the combinations of monoclonal antibodies used were as follows: 1) Hle-FITC (CD45)/LeuM3-PE (CD14)/Leu4-PerCP (CD3); 2) Leu4-FITC (CD3)/Leu3a-PE (CD4); 3) Leu4-FITC (CD3)/Leu2a-PE (CD8); 4) Leu4-FITC (CD3)/Leu12-PE (CD19); 5) Leu4-FITC (CD3)/Leu19-PE (CD56); 6) Leu12-FITC (CD19)/Leu2a-FITC (CD8)/Leu4-PE (CD3)/Leu19-PE (CD56)/Leu3a-PerCP (CD4). The last test-tube was referred to as the PANEL tube. For each of the samples a test-tube containing isotype-matched mouse Immunglobulins was used as a negative control in order, to establish the cursors for positivity. The test-tubes were slowly vortexed and incubated for 10-15 minutes at room temperature in the dark. Inmediately after this incubation period 2ml of FACS lysing solution (Becton/Dickinson) were added per tube, followed by 4-5 seconds of vortexing. Afterwards, the cells were incubated for another 10 minutes in the dark (room temperature). Samples were then centrifuged at 300 g for 5 minutes (4° C). The supernatant was aspirated and the cell pellet resuspended by vigurous vortexing (1-2 second). Then, 1ml of phosphate buffer saline solution (PBS) containing 1 g of albumin bovine (Sigma Chemicals, St Louis, MO, USA) was added to each tube. Another centrifugation was then performed (300 g, 5 minutes), the cells being finally resuspended in 0,5 ml of PBS containing 1 g of albumin bovine and stored at 4° C until they were analyzed in the flow cytometer. 3.- Data acquisition and analysis. Measurements were performed on a FACScan flow cytometer (Becton/Dickinson) equipped with an argon ion laser tuned at 488 nm and 15 mWatts. The instrument as calibrated using CALIBRITE beads (Becton/Dickinson) and PB lymphocytes stained in three different tubes for: 1) CD3 (Leu4-PerCP); 2) CD4 (Leu3a-FITC)/CD8 (Leu2a-PE) 3) CD4 (Leu3a-FITC)/CD8 (Leu2a-PE)/CD3 (Leu4- peridinin chlorophyl protein). Regarding day-to-day calibration, an identical separation was obtained between the fluorescence peaks of the positive and negative lymphoid subpopulations for FL1 (CD4-FITC), FL2 (CD8-PE strong positive) and FL3 (CD3-peridinin chlorophyl protein), variability being in all cases lower than 1,5 %. Analysis was performed with the PAINT-A-GATE Plus software program (Becton/Dickinson) using its capacity for log transformation of side scatter (SSC) in order to obtain a better separation between the cell populations identified by SSC/FSC (side scatter/forward scatter). In order to establish the ideal FSC/SSC lymphocytes gate the CD45-FITC/CD14-PE (LeucoGATE) test-tube was used according to previous established guidelines. In all cases the percentage of CD45 negative, CD14 positive and CD45-dim positive cells inside the FSC/SSC lymphocyte gate as well as the percentage of lymphocytes not included in the gate were calculated. The percentage of positive cells for each antigen was calculated from the selected FSC/SSC lymphocyte gate, the sum of the percentage of CD3+, CD19+ and CD3-/CD56+ cells being considered as 100% of the lymphocytes. Comparison of individual variables was performed through analysis of variance (ANOVA) at a level of significance of 5% . The LSD Fisher test was used for comparisons between two groups. Pearson's coefficients of correlation were calculated for the relationship between results obtained with the PANEL tube and with the other test-tubes for %CD3+, %CD19+, %CD3-/%CD56+, %CD3+/%CD4+ and %CD3+/%CD8+.

Claims: Procedure for both the simultaneous quantification, in a single measurement, of 12 B, T and NK lymphoid subpopulations, including, among others, the total number of B, T and NK cells as well as CD3+/CD4+ and CD3+/CD8+ T-cell subpopulations, and the determination of contamination of the cells analyzed by monocytes and other non-lymphoid elements, characterized because it includes the the steps: a) Incubation of the sample with a mixture of monoclonal antibodies CD3, CD4, CD8, CD19 and CD56 conjugated with three different fluorochromes each monoclonal antibody being conjugated with one single fluorochrome; b) Measurement of the three different fluorescent emissions by means of flow cytometry; and c) Analysis of the results obtained using multiparametric analysis to determine the presence/absence and the intensity of fluoresence in each cell of a certain fluorochrome. Procedure as in claim 1, characterized in that in step (a) either normal or pathologic samples obtained "in vivo" or obtained after being stored or treated "in vitro" are used. Procedure as in claim 1, characterized in that it can use any kind of fluorochrome as well as any monoclonal antibody within each cluster of differentiation (CD). Procedure as in claim 1, characterized in that CD8 and CD19 monoclonal antibodies are conjugated with one fluorochrome (FL1); CD3 and CD56 are conjugated with another fluorochrome (FL2); and CD4 is conjugated with a third fluorochrome (FL3). Procedure as in claim 4, characterized in that the FL1 fluorochrome is fluorescein isothiocyanate; the FL2 fluorochrome is phycoerythrin; and the FL3 fluorochrome is Peridinin chlorophyl protein or phycoerythrin / cyanine 5 fluorochrome tandem. Procedure as in claim 1, characterized in that staining for the identification of NK cells can also be obtained by adding either the CD16 monoclonal antibody with an identical fluorochrome to that of CD3 and CD56 or by simply replacing CD56 with CD16.

Inventors: ORFAO DE MATOS CORREIA E VALE, ORFAO DE MATOS CORREIA E VALE, JOSE ALBERTO, Orfao de Matos Correia E Vale, José Alberto
Applicants: UNIV SALAMANCA, UNIVERSIDAD DE SALAMANCA
Application year: 1993
Main classification: G01N33
IPC codes: G01N33