J Clin Pathol: first published as 10.1136/jcp.37.12.1343 on 1 December 1984. Downloaded from J Clin Pathol 1984;37:1343-1347 Immunoperoxidase detection of T and B cells in blood compared with conventional methods PK HUI*, JWM LAWTONt From the *Haematology and tImmunology Units, Department ofPathology, University ofHong Kong SUMMARY Peripheral blood T and B cells were enumerated in 26 normal adults by conventional immunological markers (erythrocyte rosette and surface membrane immunoglobulin) and by monoclonal markers (Ti 1 and B1) using both membrane immunofluorescence of cells in suspen- sion and immunoperoxidase staining of dried, fixed, cytocentrifuged cells after separation from blood by buoyant density centrifugation. The results of immunochemistry were determined inde- pendently from the results of erythrocyte rosetting and membrane immunofluorescence techni- ques. A high correlation was found between all three methods for enumerating T cells (erythro- cyte rosetting, Ti 1/immunoperoxidase, and Ti 1/indirect immunofluorescence). Correlation was less good between the results of the three methods for enumerating B cells; surface membrane immunoglobulin and Bi/indirect immunofluorescence techniques showed the highest correlation. The results suggested that B cells may be preferentially lost during the extensive cell washings entailed in the indirect immunofluorescence procedures Immunochemical methods, using mono- clonal antibodies, are useful for enumerating lymphocyte subsets in blood, particularly when permanent records are desired. copyright. Conventionally, peripheral blood T and B lympho- idase technique we have carried out a comparative cytes are identified by the sheep erythrocyte roset- study of the immunoperoxidase method and the ting technique and the demonstration of surface conventional immunological methods in the enum- membrane bound immunoglobulin, respectively.' eration of peripheral blood B and T cells. As part of The recent development of monoclonal antibodies this study we correlated results of immuno- directed against various lymphocyte surface antigens cytochemistry and immunofluorescence in detecting http://jcp.bmj.com/ has provided an additional and precise means of markers defined by two monoclonal antibodies and defining lymphocyte subsets.23 The typing of lym- compared the two methods in terms of practical phocytes by monoclonal antibodies is usually based convenience for routine clinical laboratory use. on the immunofluorescence technique, whereby the labelled cells are quantitated either by fluorescence Material and methods microscopy or by a fluorescence activated cell sor- 5 ter.4 These same lymphocyte surface markers may BLOOD SAMPLES on September 30, 2021 by guest. Protected also be shown by immunochemical techniques using Venous blood samples were obtained from 26 heal- air dried cell preparations.6 The immunoperoxidase thy adult volunteers. A 20 ml portion of each blood method is technically simpler than the sample was anticoagulated with heparin (10 U/ml) immunofluorescence technique; in the latter viable and sent to the immunology laboratory for rosetting cells must be labelled in suspension, which entails and immunofluorescence studies. A 3 ml portion of many time consuming cell washings. Moreover, each sample was anticoagulated in edetic acid and additional steps have to be taken in the sent to the haematology laboratory for immunofluorescence method to identify other cell immunoperoxidase studies. The two samples were types such as monocytes. The immunoperoxidase processed and the results were assessed indepen- method, in contrast, is performed on a relatively dently. small number of fixed dead cells. To explore the potential use of the immunoperox- PREPARATION OF CELLS Peripheral blood mononuclear cells from edetic acid Accepted for publication 22 August 1984 and heparin anticoagulated blood were obtained by 1343 J Clin Pathol: first published as 10.1136/jcp.37.12.1343 on 1 December 1984. Downloaded from 1344 Hui, Lawton buoyant density centrifugation over Ficoll-Hypaque serum albumin and suspended in buffered glycerol, (specific gravity 1-077).' For immunoperoxidase before reading membrane fluorescence under epi- staining the separated mononuclear cells were ultraviolet illumination. One hundred cells were diluted to 105 cells per millilitre in normal saline, counted. and then portions were centrifuged on to glass slides in a cytocentrifuge (Shandon-Elliott Cheshire, UK). For rosetting and immunofluorescence techniques, IMMUNOFLUORESCENCE METHOD FOR Ti 1 AND the harvested mononuclear cells were washed twice B1 MARKERS in Hank's balanced salt solution, adjusted to 4 x 106 The cell membrane epitopes defined by Ti 1 and B 1 cells per millilitre, and incubated with polystyrene monoclonal antibodies were detected by indirect latex at 37°C for 20 min. immunofluorescence using a technique similar to that of direct immunofluorescence. After incubation ANTIBODIES with polystyrene latex the mononuclear cells were The two monoclonal reagents Ti 1 and B 1 were pur- adjusted to 10 x 106/ml. The monoclonal antibody chased from Coulter Corporation, Florida, USA. was added to 100 ,ul of the cell suspension to give a Ti 1 has a specificity against the erythrocyte receptor final dilution of 1/50. After incubation at 4°C for 30 protein8 and Bi has a major specificity directed min the cells were washed twice and the second against B lymphocytes.9 The second antibody, antibody (fluorescein isothiocyanate labelled anti- peroxidase conjugated goat antimouse IgG, was mouse immunoglobulin), diluted 1/20, was added to purchased from Tago, California, USA. Fluorescein the cell pellet. After further incubation at 4°C for 30 isothiocyanate conjugated rabbit antihuman Ig(G + min the cells were washed three times and sus- A + M) was purchased from Behringwerke AG, pended in FA mounting fluid for fluorescence mic- Marburg, West Germany. The second antibody for roscopy. indirect immunofluorescence was fluorescein In the erythrocyte rosette and immunofluores- isothiocyanate conjugated rabbit antimouse IgG cence techniques, monocytes were identified by (Miles-Yeda Ltd, Rehovot, Israel). their morphology, the presence of ingested poly- styrene particles, and the irregular, granular, non- copyright. ERYTHROCYTE ROSETTING TECHNIQUE specific surface membrane fluorescence. This was performed according to the method of Kaplan and Clark'° using amino-ethyl- thiouronium-bromide treated sheep erythrocytes. IMMUNOPEROXIDASE METHOD FOR T1I AND B I Sheep erythrocytes were not more than two weeks MARKERS old when used. Mononuclear cells were incubated Air dried cytocentrifuged preparations of mononuc- together with treated erythrocytes for 3h at 4°C and lear cells were fixed for 30 s in pH6-6 buffered after resuspension the number of T cells (erythro- formalin-acetone at 40C,612 after which the cells http://jcp.bmj.com/ cyte rosettes) was determined by counting a total of were stained for Ti1 and Bi by an indirect 200 cells in a wet preparation with toluidine blue to immunoperoxidase technique as described previ- stain nucleated cells. ously.6 In brief, smears were first incubated for 1 h with 100,lO of monoclonal antibody diluted 1/10 in IMMUNOFLUORESCENCE TO DETECT SURFACE PBS at pH 7-2. The smears were then washed in MEMBRANE IMMUNOGLOBULIN PBS for 5 min and incubated for 45 min with 100,u B cell surface membrane immunoglobulin was of peroxidase conjugated goat antimouse IgG on September 30, 2021 by guest. Protected detected by direct immunofluorescence" using diluted 1/15 in PBS. The smears were washed again fluorescein isothiocyanate conjugated rabbit anti- for 5 min in PBS. The peroxidase conjugate was human Ig (G + A + M). Separated and washed stained by a substrate solution containing blood mononuclear cells were incubated with poly- 3-amino-9-ethylecarbazole (Sigma) and hydrogen styrene latex at 37°C for 20 min and then left at peroxide.'3 Stained smears were counterstained in ambient temperature for 30 min; this procedure was Mayer's haematoxylin and mounted in glycerin jelly. to allow monocytes to ingest polystyrene (for later Slides were examined under oil immersion at x 1000 identification) and to allow adsorbed immunoglobu- magnification using a Leitz Laborlux microscope. lin to elute from the surface of lymphocytes. Cells Positively stained cells showed a reddish brown pre- were then spun down, the supernatant was removed, cipitate on the cell surface with the marginal area of and the conjugate (diluted 1/8) was added directly the cell more intensely stained. Two hundred cells to the cell pellet. The mixture was incubated on ice were examined and the percentage of positively for 30 min and the cells washed three times in phos- stained cells was determined. Monocytes were phate buffered saline (PBS) azide with 1% bovine excluded from the count by morphological criteria. J Clin Pathol: first published as 10.1136/jcp.37.12.1343 on 1 December 1984. Downloaded from Immunoperoxidase detection of T and B cells in blood compared with conventional methods 1345 TITRATION OF ANTIBODIES immunoperoxidase method (p < 0.01) and by the The working dilutions of antibodies used in both the erythrocyte rosetting technique (p < 0.01). No immunofluorescence and immunoperoxidase significant difference was found between the percen- methods wete determined by titrating the individual tage of erythrocyte receptor positive cells and the reagents in doubling dilutions. The working dilution Ti 1 positive cells by the immunoperoxidase