WO2008132755A1

WO2008132755A1 - A test kit for estimating cd4 + /cd8 + t-cells in a human blood sample

Info

Publication number: WO2008132755A1
Application number: PCT/IN2007/000171
Authority: WO
Inventors: Subhash Khimandas Hira; Sudhir Nanasaheb Kadam
Original assignee: Mahatma Gandhi Mission's University Of Health Sciences
Priority date: 2007-04-27
Filing date: 2007-04-27
Publication date: 2008-11-06

Abstract

A test kit for estimating CD4 + / CD 8 + T-cells in a human blood sample. It comprises at least one microscopic glass slide provided with at least four spots; saline wash buffer; reverse osmosis water; poly-L-Lysine solution; phosphate buffered saline; histopaque; RBC Lysis buffer; a carbonyl iron powder; antibody diluting fluid; glycine buffer; gluteraldehyde; primary antibody selected from Anti-CD4 monoclonal antibody, Anti-CD8 monoclonal antibody and Anti-CD5 monoclonal antibody; secondary antibody selected from Rabbit Anti-Mouse antibody and Swine Anti- Rabbit antibody; enzyme linked conjugate like Peroxidase Anti-Peroxidase antibody (PAP); Chromogen; substrate buffer; slide holding jar; haematoxylin; a mounting solution and a cover slip for the slide. Also a method of estimation of CD4 + / CD 8 + T-cells in a human blood sample by using the test kit comprising immobilizing T-cells isolated from the blood sample on all the spots of a microscopic slide provided with at least four spots and pre-treated with poly-L-lysine followed by fixing the T-cells with solution of glutaraldehyde. The T-cells are neutralized with glycine buffer; antibody diluting fluid is added onto the T-cells followed by primary monoclonal antibodies and secondary antibodies and finally enzyme linked conjugate. The T-cells are dyed with chromogen, stained with the haematoxylin. The stained and unstained T-cells on all the spots of the slide are counted under a microscope. Also use of the test kit for the CD4 + and CD8 + T-cells estimation.

Description

TITLE OF THE INVENTION

A test kit for estimating CD4 + /CD8 + T-cells in a human blood sample

Technical field:

This invention relates to a test kit for estimating CD4 + / CD 8 + T cells in a human blood sample.

This invention also relates to a method of estimating of CD4 + / CD8 + T cells in a human blood sample using the above test kit.

This invention also relates to use of the above test kit for checking immunity of a person and/or monitoring the changing composition of subsets of T-lymphocytes in peripheral blood of a person associated with conditions like autoimmune diseases, immunodeficiency states or organ transplantation rejection.

Background of the invention:

The two main subsets of T-Lymphocytes known as CD4 + and CD8 + are distinguished by their helper and suppressor functions, respectively. These are cells that determine immunity and capacity of an individual to fight diseases in general. The CD4 + lymphocytes and CD8 + lymphocytes generally comprise approximately 65% and 25-35 % of peripheral T-cells, respectively. Estimation of these subsets of T-lymphocytes is of utmost important in the diagnosis, treatment or control of diseases in general particularly autoimmune diseases, immunodeficiency states or organ transplantation rejection. The HIV epidemic is growing world over especially in countries like India. Among the persons infected with HIV, count of the CD4 + T-cells is one of the best indicators of disease progression, risk of opportunistic infections and response to therapeutic intervention.

Immunocapture techniques for estimation of CD4 + and CD8 + T-cells in human blood are known in antiretroviral therapy. One of the methods for estimation of CD4+ and CD8 + T- cells in human blood is Capcellia, an immunocapture assay based on an enzyme-linked immunosorbent assay (ELISA). Paramagnetic particles coated with CD4+ / CD8 + monoclonal antibodies are used to capture T-Lymphocytes in the whole blood. T-cell subsets are estimated using peroxidase labeled monoclonal antibodies specific for CD4 + and CD8 + T-cells. The absorbance value is proportional to the number of CD4 + / CD8 + T-cells. Estimation of CD4 + and CD8+ T-cells is carried out by Spectrophotometer based on the colorimetric signal given by the cells. A moderately skilled person and a moderately equipped laboratory are required to carry out the whole analysis. Microwell plates used for estimation in Capcellia are expensive. A Spectrophotometer costs around Rs. 5 Lacs and also requires periodic maintenance. Further Capcellia kits are being imported and are expensive and are not indigenously, easily or plentifully available.

Another technique for estimation of CD4 + / CD8 + T-cells in human blood is based on flow cytometry. Flow cytometry is performed on liquid suspensions of cells from whole blood, bone marrow or body fluids or tissue cells incubated with fluorescent tagged antibodies directed against specific cell surface proteins. Fluorescent signal emitted by the T-cells is estimated by flow cytometer for CD4 + and CD8+ T-cells. A highly skilled person and a highly equipped laboratory are required to carry out the test. Glass tubes used for estimation in flow cytometry are expensive. A flow cytometry equipment costs around Rs. 25 lacs and also requires periodic maintenance. Further the equipment and reagents are imported and are expensive and are not indigenously, easily or plentifully available.

As the above test methods require moderately skilled technicians and moderately skilled laboratory set up or highly skilled technicians and highly equipped laboratories, they are not only expensive but also unaffordable and unfeasible, especially for poor people and in rural areas. Both the Capcellia and Flow cytometry methods use test samples in suspension. The T- cells are prone to deterioration while being estimated by spectrophotometer or cytometer and the test cells are to be discarded after estimation. Therefore, the test cells cannot be preserved for future reference as and when required. The above methods do not estimate CD5 + count which can act as a positive control and range fixer in order to indicate the correctness and accuracy of the methods. Therefore, it is difficult to check the correctness and accuracy of the Capcellia and Flow cytometry methods.

Ob j ects of the Invention

An object of the invention is to provide a test kit for estimating CD4 + / CD8 + T-cells in a human blood sample, which is cheap and can be used to estimate CD4+ /CD8 + T-cells accurately and reliably.

Another object of the invention is to provide a test kit for estimating CD4 + / CD8 + T-cells in a human blood sample, which requires only minimally equipped laboratory with simple microscope and persons with minimal training for the estimation. Another object of the invention is to provide a test kit for estimating CD4 + / CD8 + T-cells in a human blood sample, which is affordable to poor sections of society and suitable for rural areas.

Another object of the invention is to provide a test kit for estimating CD4 + / CD8 + T-cells in a human blood sample, which facilitates preservation of the test cells for future reference as and when required.

Another object of the invention is to provide a method for estimating CD4 + / CD 8 + T-cells in a human blood sample, which is accurate, reliable and cheap.

Another object of the invention is to provide a method for estimating CD4 + / CD 8 + T-cells in a human blood sample, which requires only minimally equipped laboratory with simple microscope and persons with minimal training for the estimation.

Another object of the invention is to provide a method for estimating CD4 + / CD 8 + T-cells in a human blood sample, which is affordable to poor sections of society and suitable for rural areas.

Another object of the invention is to provide a method for estimating CD4 + / CD8 + T-cells in a human blood sample, which allows the test cells to be preserved for future reference as and when required.

Another object of the invention is to provide use of a test kit for estimating CD4 + / CD8 + T- cells in a human blood sample accurately and reliably and in an inexpensive manner in order to check immunity of a person and / or monitor the changing composition of subsets of T- lymphocytes in peripheral blood of a person associated with conditions like auto immune diseases, immunodeficiency states or organ transplantation rejection.

Another object of the invention is to provide use of a test kit for estimating CD4 + / CD8 + T- cells in a human blood sample in order to check immunity of a person and / or monitor the changing composition of subsets of T-lymphocytes in peripheral blood of a person associated with conditions like auto immune diseases, immunodeficiency states or organ transplantation rejection, which requires only minimally equipped laboratory with simple microscope and persons with minimal training for the estimation.

Another object of the invention is to provide use of a test kit for estimating CD4 + / CD8 + T- cells in a human blood sample in order to check immunity of a person and / or monitor the changing composition of subsets of T-lymphocytes in peripheral blood of a person associated with conditions like auto immune diseases, immunodeficiency states or organ transplantation rejection, which is affordable to poor sections of society and suitable for rural areas.

Another object of the invention is to provide use of a test kit for estimating CD4 + / CD8 + T- cells in a human blood sample in order to check immunity of a person and / or monitor the changing composition of subsets of T-lymphocytes in peripheral blood of a person associated with conditions like auto immune diseases, immunodeficiency states or organ transplantation rejection, which allows the test cells to be preserved for future reference as and when required. Detailed description

According to the invention there is provided a test kit for estimating CD 4 + / CD 8 + T-cells in a human blood sample, the test kit comprising at least one microscopic glass slide provided with at least four spots; saline wash buffer; reverse osmosis water; 0.005 to 0.03 % solution of poly-L-Lysine; phosphate buffered saline; histopaque of 12.5 % density; 0.3 molar RBC Lysis buffer; a carbonyl iron powder; antibody diluting fluid consisting of 0.172 molar phosphate buffered saline, 4-5 % gelatin and bovine serum albumin of 67 x 10⁶ MW; 0.01 to 0.05 molar glycine buffer; 22 to 30 % aqueous solution of gluteraldehyde; primary antibody selected from 1:100 - 1:300 Anti-CD4 monoclonal antibody, 1:100-1:300 Anti-CD8 monoclonal antibody and 1:400-1:600 Anti-CD5 monoclonal antibody; secondary antibody selected from 1:800 - 1:1000 Rabbit Anti-Mouse antibody and 1:50-1:500 Swine Anti-Rabbit antibody; enzyme linked conjugate like 1:10-1:50 Peroxidase Anti-Peroxidase antibody (PAP); 0.7 molar chromogen; 0.1 molar substrate buffer; slide holding jar; 1 molar haematoxylin; a mounting solution comprising phosphate buffer containing 25 % aqueous gluteraldehyde and a cover slip for the slide.

According to the invention there is also provided a method of estimating CD 4 + / CD 8 + T- cells in a human blood sample, the method comprising (i) immobilizing T-cells isolated from a human blood sample on all the spots of a microscopic slide provided with at least four spots and pre-treated with poly-L-lysine followed by fixing the T-cells on the spots with 22 to 30 % aqueous solution of glutaraldehyde; incubating the T-cells at 2 to 8° C; neutralizing the T-cells with 0.01 to 0.05 molar glycine buffer followed by aspirating them, adding antibody diluting fluid consisting of 0.172 molar phosphate buffered saline, 4-5 % gelatin and bovine serum albumin of 67 x 10⁶ MW onto the T-cells on all the spots; incubating the T-cells at 2 to 8° C; adding primary monoclonal antibodies selected from 1:100 - 1:300 Anti-CD4 monoclonal antibody, 1:100 - 1:300 Anti-CD8 monoclonal antibody and 1 :400 - 1 :600 Anti-CD5 monoclonal antibody to the T-cells on each of the three spots respectively; incubating the T-cells on all the spots at 2 to 8° C; washing the slide with phosphate buffered saline; adding secondary antibodies selected from 1:800 - 1:1000 Rabbit Anti-Mouse and 1:10 - 1:50 Swine Anti-Rabbit to the T-cells on all the four spots of the slide; incubating the T-cells on all the spots at 2 to 8° C; washing the slide with phosphate buffered saline; adding enzyme linked conjugate like peroxidase antiperoxidase antibody on T-cells on all the four spots of the slide; incubating the T-cells on all the spots at 2 to 8° C; washing the slide with phosphate buffered saline; introducing the slide in a holding Jar containing chromogen; washing the slide with a substrate buffer;

(ii) staining the T-cells on all the spots of the slide with the haematoxylin followed by applying a mounting solution and covering the spots with a cover slip and (iii) counting stained and unstained T-cells on all the spots of the slide under a microscope.

According to the invention there is also provided use of a test kit for estimating CD4 + / CD8

+ T-cells in a human blood sample in order to check immunity of a person and/or monitor the changing composition of subsets of T-lymphocytes in peripheral blood of a person associated with conditions like auto immune diseases, immunodeficiency states or organ transplantation rejection, the kit comprising at least one microscopic slide provided with at least three spots; saline wash buffer; reverse osmosis water; 0.005 to 0.03 % solution of poly-L-Lysine; phosphate buffered saline; histopaque of 12.5 % density; 0.3 molar RBC Lysis buffer; a carbonyl iron powder; antibody diluting fluid consisting of 0.172 molar phosphate buffered saline, 4-5 % gelatin and bovine serum albumin of 67 x 10⁶ MW; 0.01 to 0.05 molar glycine buffer; 22 to 30 % aqueous solution of gluteraldehyde; primary antibody selected from 1:100 - 1:300 Anti-CD4 monoclonal antibody, 1:100-1:300 Anti-CD8 monoclonal antibody and 1:400-1:600 Anti-CD5 monoclonal antibody; secondary antibody selected from 1:800 - 1:1000 Rabbit Anti-Mouse and 1:50-1:500 Swine Anti-Rabbit; enzyme linked conjugate like 1:10-1:50 Peroxidase Anti-Peroxidase antibody (PAP); 0.7 molar chromogen; 0.1 molar substrate buffer; slide holding jar; 1 molar haematoxylin; a mounting solution being phosphate buffer containing 25 % aqueous gluteraldehyde and a covering slip for the slide.

The microscopic slide is pretreated with Poly-L-Lysine by first washing the spots with saline wash buffer followed by reverse osmosis water, introducing a 0.005 to 0.03 % solution of Poly-L-Lysine on each of the spots followed by incubating the slide at 2 to 8° C and washing the slide with a phosphate buffered saline.

The poly-L-Lysine adheres to the surface of the glass slide, leaving the cationic sites therein free to combine with anionic sites of the T-cells. Therefore, the T-cells are immobilized on the poly-L-Lysine coated slide. Proteins specific to CD4 + and CD8 + present on the T-cells are density specific antigens which can conjugate with the specific primary monoclonal antibody and subsequently with the secondary antibody and finally with peroxidase anti- peroxidase to give a pentameric structure which facilitates identification of the CD4 + and CD8+ cells.

The T-cells are isolated from a blood sample in a known manner, for instance, by centrifuging the blood sample in a heparinised vacutainer, collecting the buffy coat in a vial containing a carbonyl iron powder, suspending vial containing the T-cells in water bath at 37° C for 10 to 30 minutes followed by rubbing the vial on magnet to collect lymphocytes rich plasma from the opposite side of the magnet, diluting the plasma with phosphate buffered saline followed by transferring the diluted plasma into a glass tube containing Histopaque of 12.5 % density, centrifuging the lymphocytes in a centrifuge and collecting the T- lymphocytes as thin ring at the interface of plasma and histopaque. The isolated T-cells are suspended in phosphate buffer saline followed by centrifuging in a glass tube, removing the supernatant from the centrifuge, adding RBC lysis fluid to the glass tube and incubating the mixture at 37° C followed by centrifuging the glass tube and resuspending the T-cells in phosphate buffered saline to form suspension of T-cells.

The concentration of T-cells in the suspension is required to be in the range of 4 to 5 million T-cells /ml so as to give accurate CD4 + and CD8 + counts. Before isolation of T-cells, the blood sample is tested for total and differential count using conventional and simple laboratory techniques. This count primarily decides the baseline for calculating the absolute count of CD4+ and CD8+ T-cells. This also helps the technician to prepare suspension with specific concentration of the T-cell.

The stained CD4+ and CD8+ T-cells are counted under a microscope for 100-200 cells. This count gives % CD4 +, % CD8 + and % CD 5+ T-cells. CD5 + % acts as positive control for the entire estimation method i.e. its values decides the correctness and accuracy of the method. If the CD5 + % is more than 100% or total of CD4+ % and CD8+ % is more than that of CD5 %, then it is an indication that some error has occurred in the method. CD5 % is a range fixer and should be always above 80 % and total of CD4+ % and CD8+ % should always be less than that of CD5 +. The absolute count is calculated by the following the

formula :

Absolute lymphocyte count X (CD4+ % / CD8+ % )

Absolute Count of CD4+ / CD8+ =

100

Absolute Lymphocyte count = Total WBC count X % of lymphocyte. Manual counter or conventional technique is used to get % of Lymphocyte.

According to the invention the kit comprises all the reagents and accessories necessary for a technician with minimal training to perform the estimation of CD4 + and CD8 + T-cells in a human blood sample under a microscope, preferably a binocular microscope. Counting is easier with a binocular microscope. Glass slides used in the invention are much cheaper than microwell plates used in Capcellia or glass tubes used in flow cytometer. A microscope costs around Rs. 15,000/- to 25,000/- and requires hardly any maintenance. The method of estimation is very simple and can be easily carried out by a technician with minimal training.

It is indigenously developed and cheap and can be made easily and plentifully available and it requires only a minimally equipped laboratory. Therefore, the invention is economical and ideal and suitable and also affordable to poor sections of society and rural areas. The slide with test material, which is practically dry and covered, can be easily conveniently stored and used as a reference as and when required as historical record. Estimation of T-cells according to the invention is comparable to those obtained from Capcellia and Flow cytometry. The

CD5+ count acts as positive control to determine the correctness and accuracy of the method and also acts as range fixer, which is not used in Capcellia and Flow cytometry. Therefore, estimation of CD4 + and CD8 + T-cells according to the invention is accurate and reliable.

The following experimental example is illustrative of the invention but not limitative of the scope thereof.

Blood samples of different people were used for estimating CD4 + / CD 8 + T-cells by the method of the invention and by the Capcellia and Flow cytometry methods. In the first evaluation, blood samples were divided in to 2 equal groups, one group of each sample was used for Capcellia and the method of invention. In the second evaluation, blood samples were again divided into 2 equal groups, one group of each sample was used for flowcytometry and the method of invention.

Example

1. Isolation of T-cells from the blood samples :

Each of the blood samples was analyzed for total and differential count by conventional manual technique. Following this each blood sample containing 8 to 10 ml of blood was centrifuged in a heparinised vacutainer at 1500 rpm for 5 minutes. 2 ml of buffy coat formed above the red cell sediments was collected in a plastic vial containing a pinch of carbonyl iron powder and the vial was suspended in a water bath at 37° C for 15 minutes. The vial was rubbed on the magnet for 3 minutes in such way to bring the particle to the opposite side of the magnet. The lymphocyte rich plasma was collected in a glass tube from the opposite side of the magnet and diluted with double the volume of phosphate buffered saline. The diluted plasma was added into a tube containing 1 ml of histopaque of 12.5 % density to get maximum number of T-lymphocytes and centrifuged at 1500 rpm for 15 minutes. T-Lymphocytes were collected as a thin ring at the interface of plasma and histopaque in a separate tube. T-cells were suspended in 3 ml phosphate buffered saline followed by centrifuging the suspension at 1500 rpm for 5 minutes. The supernatant was removed followed by adding 2 ml of 0.3 molar

RBC lysis fluid to it and the suspension formed was incubated at 37° C for 5 minutes. The suspension was centrifuged at 1500 rpm for 5 minutes. The RBC lysis treatment was repeated and the centrifuge obtained was washed with 3 ml of phosphate buffered saline. T-lymphocytes were resuspended in 3 ml of phosphate buffered saline so as to adjust the cell count to 4-5 million cells per ml with the help of haemocytometer.

2. Pretreatment of microscopic glass slide :

A microscopic glass slide provided with 4 spots was taken. The spots were washed by adding a drop of water on each spot for 5 minutes and drained by tilting the slide. The spots were washed with one drop of saline wash buffer followed by reverse osmosis water six times. The slide was kept in a beaker containing 250 ml of reverse osmosis water on a magnetic stirrer. 10 μl of 0.01 % solution of poly-L-Lysine was put on each spots and incubated at 2-8° C for 1 hour. Each spot was washed with phosphate buffered solution five times to obtain microscopic slide pretreated with poly-L-Lysine.

3. Estimation procedure

10 μl of T-cell suspension was added on each spots of microscopic slides pretreated with poly-L-Lysine and allowed the suspension to settle on the spots for 25 minutes at 2-8° C to immobilize the T-cells. The T-cells were fixed by adding 20 μl of 25 % aqueous solution of gluteraldehyde on each spot of the slide. The slide _{v ,}__, _

13 was incubated at 2-8° C for 7 min. One drop of 0.03 molar glycine buffer was added on each spot and aspirated immediately. 10 μl of antibody diluting fluid consisting of 0.172 molar phosphate buffered saline, 4-5 % gelatin and bovine serum albumin of 67 x 10⁶ MW was added immediately on each spot and incubated it at 2-8° C for 25 minutes. 10 μl of each 1:100-300 of reconstituted solution of Anti-CD4, 1:100-300 of reconstituted solution of Anti-CD8 and 1:400- 300 of reconstituted solution of Anti-CD 5 monoclonal antibodies were added onto the T-cells on each of the three spots respectively. The slide was incubated at 4° C for 16 hours. The slide was kept at 28° C for 15 minutes. Each spots was washed with phosphate buffered saline by putting one drop and sucking immediately using suction pump. 10 μl of 1:800-1:1000 freshly reconstituted rabbit anti-mouse was added onto the T-cells on all the four spots and incubated the slide at 4° C for 20 minutes. The spots were washed 5 times with phosphate buffer saline by putting the drop and sucking immediately. 10 μl of 1:50-500 of freshly reconstituted swine-anti-rabbit was added on the four steps and incubated the slide at 4° C for 20 minutes. The spots were washed 5 times with phosphate buffered saline by putting one drop and sucking immediately. 10 μl of 1:10-50 of freshly reconstituted Peroxidase Anti-peroxidase was added onto the T-cells on all the four spots of the slide and incubated the slide at 4° C for 20 minutes. The spots were washed 5 times with phosphate buffered saline by putting one drop and sucking immediately. The slide was kept in slide holding jar containing 25 ml of substrate chromogen for 30 min and washed with saline. The T-cells of the slide was stained with two drops of the haematoxylin dye for 10 seconds and was washed with distilled water. 0.5 ml of mounting solution comprising phosphate buffer containing 25 % aqueous gluteraldehyde was applied on each spot of the slide and the slide was covered with a cover slip. The slide was maintained dry by wiping out the excess mounting solution with a tissue paper. The stained and unstained cells per 100-200 cells on each spot on the slide were counted by using a simple microscope.

The tests results were as shown in the following tables:

Table 1

AIDS related complex = intermediate stage progression towards AIDS

Asymptomatic HTV+ = chronic infection with HTV (human immunodeficiency virus) during which there are no symptoms of HIV infection.

The table shows that the test results as obtained by the method of the invention are closer to the normal standard values corresponding to the clinical stages and are accurate and reliable.

Table 2

Claims

CLAIMS:

1. A test kit for estimating CD4 + / CD 8 + T-cells in a human blood sample, the test kit comprising at least one microscopic glass slide provided with at least four spots; saline wash buffer; reverse osmosis water; 0.005 to 0.03 % solution of poly-L-Lysine; phosphate buffered saline; histopaque of 12.5 % density; 0.3 molar RBC Lysis buffer; a carbonyl iron powder; antibody diluting fluid consisting of 0.172 molar phosphate buffered saline, 4-5 % gelatin and bovine serum albumin of 67 x 10⁶ MW; 0.01 to 0.05 molar glycine buffer; 22 to 30 % aqueous solution of gluteraldehyde; primary antibody selected from 1:100 - 1:300 Anti-CD4 monoclonal antibody, 1:100-1:300 Anti-CD8 monoclonal antibody and 1:400-1:600 Anti-CD5 monoclonal antibody; secondary antibody selected from 1 :800 - 1:1000 Rabbit Anti-Mouse antibody and 1:50- 1:500 Swine Anti-Rabbit antibody; enzyme linked conjugate like 1:10-1:50 Peroxidase Anti-Peroxidase antibody (PAP); 0.7 molar Chromogen; 0.1 molar substrate buffer; slide holding jar; 1 molar haematoxylin; a mounting solution comprising phosphate buffer containing 25 % aqueous gluteraldehyde and a cover slip for the slide.

2. A method of estimating CD4 + / CD 8 + T-cells in a human blood sample, the method comprising

(i) immobilizing T-cells isolated from a human blood sample on all the spots of a microscopic glass slide provided with at least four spots and pre-treated with poly-L-lysine followed by fixing the T- cells on the spots with 22 to 30 % aqueous solution of glutaraldehyde; incubating the T-cells at 2 to 8° C; neutralizing the T-cells with 0.01 to 0.05 molar glycine buffer followed by aspirating them, adding antibody diluting fluid consisting of 0.172 molar phosphate buffered saline, 4-5 % gelatin and bovine serum albumin of 67 x 10⁶ MW onto the T-cells on all the spots; incubating the T-cells at 2 to 8° C; adding primary monoclonal antibodies selected from 1:100 - 1:300 Anti-CD4 monoclonal antibody, 1:100 - 1:300 Anti-CD8 monoclonal antibody and 1:400 - 1:600 Anti-CD5 monoclonal antibody to the T-cells on each of the three spots respectively; incubating the T-cells on all the spots at 2 to 8° C; washing the slide with phosphate buffered saline; adding secondary antibodies selected from 1:800 - 1:1000 Rabbit Anti-Mouse and 1:10 - 1:50 Swine Anti-Rabbit to the T-cells on all the four spots of the slide; incubating the T-cells on all the spots at 2 to 8° C; washing the slide with phosphate buffered saline; adding enzyme linked conjugate like peroxidase antiperoxidase antibody on T-cells on all the four spots of the slide; incubating the T-cells on all the spots at 2 to 8° C; washing the slide with phosphate buffered saline; introducing the slide in a holding Jar containing chromogen; washing the slide with a substrate buffer;

(ii) staining the T-cells on all the spots of the slide with the haematoxylin followed by applying a mounting solution and covering the spots with a cover slip and

(iii) counting stained and unstained T-cells on all the spots of the slide under a microscope.

3. A method as claimed in claim 2, wherein the microscopic slide is pretreated with poly-L-Lysine by first washing the spots with saline wash buffer followed by reverse osmosis water, introducing a 0.005 to 0.03 % solution of poly-L-Lysine on each of the spots followed by incubating at 2 to 8° C and washing the slide with a phosphate buffered saline.

4. Use of a test kit for estimating CD4 + / CD8 + T-cells in a human blood sample in order to check immunity of a person and/or monitor the changing composition of subsets of T-lymphocytes in peripheral blood of a person associated with conditions like auto immune diseases, immunodeficiency states or organ transplantation rejection, the kit comprising at least one microscopic slide provided with at least four spots; saline wash buffer; reverse osmosis water; 0.005 to 0.03 % solution of poly-L-Lysine; phosphate buffered saline; histopaque of 12.5 % density; 0.3 molar RBC Lysis buffer; a carbonyl iron powder; antibody diluting fluid consisting of 0.172 molar phosphate buffered saline, 4-5 % gelatin and bovine serum albumin of 67 x 10⁶ MW; 0.01 to 0.05 molar glycine buffer; 22 to 30 % aqueous solution of gluteraldehyde; primary antibody selected from 1:100 - 1:300 Anti-CD4 monoclonal antibody, 1:100-1:300 Anti-CD8 monoclonal antibody and 1:400- 1:600 Anti-CD5 monoclonal antibody; secondary antibody selected from 1:800 - 1:1000 Rabbit Anti-Mouse and 1:50-1:500 Swine Anti-Rabbit; enzyme linked conjugate like 1:10-1:50 Peroxidase Anti-Peroxidase antibody (PAP); 0.7 molar Chromogen; 0.1 molar substrate buffer; slide holding jar; 1 molar haematoxylin; a mounting solution being phosphate buffer containing 25 % aqueous gluteraldehyde and a covering slip for the slide.