WO2008132755A1 - Trousse d'essai permettant d'estimer les lymphocytes t cd4 + /cd8 + dans un prélèvement de sang humain - Google Patents

Trousse d'essai permettant d'estimer les lymphocytes t cd4 + /cd8 + dans un prélèvement de sang humain Download PDF

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Publication number
WO2008132755A1
WO2008132755A1 PCT/IN2007/000171 IN2007000171W WO2008132755A1 WO 2008132755 A1 WO2008132755 A1 WO 2008132755A1 IN 2007000171 W IN2007000171 W IN 2007000171W WO 2008132755 A1 WO2008132755 A1 WO 2008132755A1
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cells
slide
spots
antibody
molar
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PCT/IN2007/000171
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English (en)
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Subhash Khimandas Hira
Sudhir Nanasaheb Kadam
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Mahatma Gandhi Mission's University Of Health Sciences
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Priority to PCT/IN2007/000171 priority Critical patent/WO2008132755A1/fr
Publication of WO2008132755A1 publication Critical patent/WO2008132755A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5094—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for blood cell populations

Definitions

  • 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.
  • 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.
  • 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).
  • ELISA enzyme-linked immunosorbent assay
  • 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.
  • CD4 + and CD8+ T-cells are 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.
  • 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.
  • 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.
  • 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 10 6 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 mono
  • a method of estimating CD 4 + / CD 8 + T- cells in a human blood sample 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 10 6 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, 1:1
  • kits 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 10 6 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 monoclo
  • 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.
  • 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.
  • CD4 + % 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 following the
  • Absolute Lymphocyte count Total WBC count X % of lymphocyte. Manual counter or conventional technique is used to get % of Lymphocyte.
  • 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.
  • T-cells are comparable to those obtained from Capcellia and Flow cytometry.
  • 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.
  • 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.
  • blood samples were divided in to 2 equal groups, one group of each sample was used for Capcellia and the method of invention.
  • blood samples were again divided into 2 equal groups, one group of each sample was used for flowcytometry and the method of invention.
  • 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
  • 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 ⁇ l 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.
  • T-cell suspension 10 ⁇ l 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 ⁇ l of 25 % aqueous solution of gluteraldehyde on each spot of the slide.
  • 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 ⁇ l 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 ⁇ l 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 ⁇ l 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.
  • 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.
  • 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.

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Abstract

L'invention porte sur une trousse d'essai qui permet d'estimer les lymphocytes T CD4 + / CD 8 + dans un prélèvement de sang humain. La trousse de l'invention comprend : au moins une lame de verre microscopique munie d'au moins quatre taches d'échantillon; une solution tampon saline de lavage; de l'eau d'osmose inverse; une solution de poly-L-Lysine; une solution saline tamponnée au phosphate; de l'Histopaque; un tampon de lyse RBC; une poudre de fer carbonyle; un liquide de dilution d'anticorps; un tampon glycine; un gluteraldéhyde; un anticorps primaire choisi entre un anticorps monoclonal anti-CD4, un anticorps monoclonal anti-CD8 et un anticorps monoclonal anti-CD5; un anticorps secondaire choisi entre un anticorps de lapin anti-souris et un anticorps de porc anti-lapin; un conjugé lié à l'enzyme tel le peroxydase-anticorps antiperoxydase (PAP); un chromogène; un tampon substrat; un bocal porte-lame; de l'hématoxyline; une solution de montage et une lamelle de protection de la lame. L'invention concerne également un procédé qui permet d'estimer les lymphocytes T CD4 + / CD 8 + dans un prélèvement de sang humain à l'aide de la trousse d'essai précitée, selon lequel on immobilise les lymphocytes T isolés du prélèvement sanguin sur toutes les taches d'échantillon d'une lame microscopique munie de quatre taches au moins et prétraitée à la poly-L-lysine; on fixe les lymphocytes T au moyen de la solution de gluteraldéhyde; on neutralise les lymphocytes T avec le tampon glycine; on ajoute du liquide de dilution d'anticorps sur les lymphocytes T, ensuite sur les anticorps monoclonaux primaires et sur les anticorps monoclonaux secondaires et enfin sur le conjugué lié à l'enzyme; on teinte les lymphocytes T à l'aide du chromogène et on les colore à l'hématoxyline; et on compte sous un microscope les lymphocytes T colorés et non colorés sur toutes les taches de la lame. L'invention se rapporte en outre à l'utilisation de la trousse d'essai précitée pour l'estimation des lymphocytes T CD4 + et CD8 + T.
PCT/IN2007/000171 2007-04-27 2007-04-27 Trousse d'essai permettant d'estimer les lymphocytes t cd4 + /cd8 + dans un prélèvement de sang humain WO2008132755A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014080017A1 (fr) 2012-11-23 2014-05-30 Epiontis Gmbh Procédé épigénétique pour l'identification de sous-populations de lymphocytes t cd8+, en particulier de lymphocytes t cd8 alpha et bêta
US9063127B1 (en) 2013-12-11 2015-06-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
US10928405B2 (en) 2013-12-11 2021-02-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
CN112964877A (zh) * 2021-03-09 2021-06-15 河南赛诺特生物技术有限公司 一种用于鉴别套细胞淋巴瘤的免疫组化多重染色试剂盒及染色程序
CN113504092A (zh) * 2021-07-15 2021-10-15 河南赛诺特生物技术有限公司 一种幽门螺杆菌染色试剂盒及应用
CN114196627A (zh) * 2021-12-08 2022-03-18 黑龙江八一农垦大学 一种牛血液cd4+t淋巴细胞的分离方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010091A1 (fr) * 1994-09-29 1996-04-04 Merck & Co., Inc. Crible haute capacite pour immunoregulateurs
WO1996012963A1 (fr) * 1994-10-24 1996-05-02 Biometric Imaging, Inc. Procede de preparation d'un echantillon dans un capillaire de scintigraphie pour une recherche par immunofluorescence

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996010091A1 (fr) * 1994-09-29 1996-04-04 Merck & Co., Inc. Crible haute capacite pour immunoregulateurs
WO1996012963A1 (fr) * 1994-10-24 1996-05-02 Biometric Imaging, Inc. Procede de preparation d'un echantillon dans un capillaire de scintigraphie pour une recherche par immunofluorescence

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014080017A1 (fr) 2012-11-23 2014-05-30 Epiontis Gmbh Procédé épigénétique pour l'identification de sous-populations de lymphocytes t cd8+, en particulier de lymphocytes t cd8 alpha et bêta
US9063127B1 (en) 2013-12-11 2015-06-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
US9709556B2 (en) 2013-12-11 2017-07-18 Analiza, Inc. Devices and methods for determining and/or isolating circulating cancer cells
US10928405B2 (en) 2013-12-11 2021-02-23 Analiza, Inc. Devices and methods for determining and/or isolating cells such as circulating cancer or fetal cells
CN112964877A (zh) * 2021-03-09 2021-06-15 河南赛诺特生物技术有限公司 一种用于鉴别套细胞淋巴瘤的免疫组化多重染色试剂盒及染色程序
CN112964877B (zh) * 2021-03-09 2023-07-21 河南赛诺特生物技术有限公司 一种用于鉴别套细胞淋巴瘤的免疫组化多重染色试剂盒及染色程序
CN113504092A (zh) * 2021-07-15 2021-10-15 河南赛诺特生物技术有限公司 一种幽门螺杆菌染色试剂盒及应用
CN114196627A (zh) * 2021-12-08 2022-03-18 黑龙江八一农垦大学 一种牛血液cd4+t淋巴细胞的分离方法

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