- Email: [email protected]
Laboratory methods in clinical transplantation
7.
8.
9.
I0.
11.
375-382. In B. Serrou and C. Rosenfeld (eds.), Human lymphocyte differentiation. Its application to cancer. North-Holland Pub. Co., Amsterdam. Howard, A. R., and L. Graham. 1976. Rosette formation by foetal liver and spleen cell incubated with theophylline. Clin. Exp. Immunol. 23: 279-284. Larson, S., et al. 1978. Theimmunopharmacology of vindesine (VND) in man. Proc. ASCO 19:405 (abstract C. 396). Limatibul, S., et al. 1978. Theophylline modulation of E-rosette formation: An indicator of T cell maturation. Clin. Exp. Immunol. 33: 503-513. Moretta, L., et al. 1977. Human T cell subpopulation: Help and suppression by T cells bearing receptor for IgM or IgG. J. Exp. Med. 146: 184-200. Opelz, G., etal. 1975. Autologous
Laboratory
Methods
13.
14. 15.
16.
17.
18.
19.
20.
Elsevier North-Holland Biomedical Press, Amsterdam. In press. Serrou, B., et al. 1980. In vivo and in vitro modulation of the immune response by human fibroblast interferon (HFI). Int. J. lmmunopharmacol. 2:159 (abstract). Serrou, B., D. Cupissol, and C. Rosenfeld. 1980. Immune imbalance and immune modulation in solid tumor patients. New insights. In G. Mathe, G. Bonadonna, and S. Salmon (eds.), Adjuvant therapies of cancer. Springer-Verlag, Heidelberg. In press. Wierda, D., and T. L. Pazdernlr. 1979. Suppression of spleen lymphocyte mitogenesis in mice injected with platinum compounds. Eur. J. Cancer 15:1013-1023. Yehuda, Z. P., et al. 1979. New approaches to the evaluation of immunomodulation by thymic hormones. Ann. N.Y. Acad. Sci. 332: 160-171.
in C l i n i c a l T r a n s p l a n t a t i o n
T. Mohanakumar, Ph.D. Thomas M. Ellis, Ph.D. Claude Duvall, B.S. H. M. Lee, M.D. Tissue Typing Laboratories Departments of Surgery and Microbiology Medical College of Virginia Virginia Common wealth University Richmond, Virginia 23298 There is no doubt that the human leukocyte antigen (HLA) system or region has a decisive influence on transplantation, as demonstrated by the observations that a) excellent resuits are obtained in HLA identical sibling transplants, and b) accelerated rejection o f allografts occurs when performed in the presence of specific sensitization to the donor antigens. After the H L A workshop held at Torino in 1967, it was realized that the H L A system is so complex that the procurement of H L A well-matched cadaver kidneys required regional and national exchange o f organs. For this reason, a central register o f all potential recipients in a region waiting for cadaver transplant was established. The register contains the relevant particulars such as ABO and HLA types, antibody status, and age. When
62
12.
stimulation of human lymphocyte subpopulations. J. Exp. Med. 142: 1327-1333. Rey, A., et al. 1980. Human autologous rosettes. III. Pharmacology of autologous rosette-forming cells. Submitted for publication. Rucheton, M., et al. 1980. Human autologous rosettes. IV. Further characterization of autologous rosettes forming cells. Submitted for publication. Serrou, B., et al. 1979. Regulation of human suppressor cell function by thymosin. Biomedicine 31" 89. Serrou, B., et al. 1980. In vivo and in vitro modulation of helper, suppressor and NK activities by human interferon. J. Clin. Hematol. Oncol. In press. Serrou, B., et al. 1980. Phase I-Evaluation of bestatin in patients bearing advanced solid tumors. In W. D. Terry (ed.), Immunotherapy of cancer: present status of trials in man.
the organs become available, and ABO and H L A typings have been carried out, using a computer facility, the central register is searched for the most suitable recipient. For example, through nonprofit organizations such as Southeastern Organ Procurement Foundation and the United Network for Organ Sharing, we can now obtain computer matching for 136 transplant centers in the USA comprising about 4,500 potential recipientS. Testing for ABO compatibility, HLA-A, B, C, D / D R compatibility, antibodies to HLA-A, B, C, and DR antigens, and HLA-A, B, C, and DR crossmatching are currently the primary procedures used in identifying the donor/recipient compatibility. We will limit discussion here to the techniques o f H L A typing and crossmatching against antigens coded by HLA-A, B, C, and DR. w e shall broadly divide the procedures into two categories based solely upon the current methodologies~ a) matching by serologic techniques for the determination of antigens coded for by the HLA-A, B, and C loci as well as the identification of HLA-DR antigens on isolated B lymphocytes, and b)
matching by cellular techniques for the definition of antigens coded for HLA-D locus employing the mixed lymphocyte culture (MLC) assay and its variants. Matching by Serologic Techniques H L A - A , B, and C typing Ten ml o f heparinized (25 U heparin/ml blood) blood are obtained by venipuncture and passed through a syringe lightly packed with 1.0-I .5 g of washed nylon in order to remove tile platelets. Alternatively, defibrinated blood samples can be employed. In some laboratories, the blood is then incubated with carbonyl iron to remove the phagocytic cell population. The blood sample is then diluted 1:3 with either barbital buffer or medium (Hanks balanced salt solution), and the lymphocytes are isolated by centrifugation in Ficoll-Hypaque density gradient. The final cell preparation is washed three times with barbital buffer or medium and adjusted to 2-2.5 x 106/ml for microcytotoxicity test. In the standard NIH microcytotoxicity procedure (8), 1 ~ul of cell suspension is incubated for 30 min (28°C) in
I ~ulof various specific HLA antisera placed under oil in the microcytotoxicity trays (Falcon or Cook plastic tissue culture trays). Five ju I of rabbit complement is then added to each well and incubated for another hour at 28°C. Eosin and formalin are then added, and the percentage of viable cells is determined by phase microscopy. Dead cells take up the dye and appear large and dark. A modification of the NIH technique (1) was designed by Dr. D. B. Amos at Duke University that avoids the anticomplementary activities of the excess HLA-typing sera present in the test wells. This procedure calls for washing the unbound antibody by a quick snap of the plate (generally known as flickifig) after the initial 30 minute incubation with cells. The remainder of the procedure is basically similar to the NIH method but uses trypan blue instead of eosin as a vital dye. Usually excess complement is flicked off before adding trypan blue. This procedure, although requiring more time, has proven to be more sensitive than the NIH method. A third procedure with even greater sensitivity was developed by Johnson et al. (4). In this method, after the incubation of lymphocytes and antisera, cells are washed by flicking, and 1/~1 of titered antihuman immunoglobulin reagent is added for one to three minutes before the rabbit complement. Both the Amos modified and the antiglobulin lymphocytotoxicity techniques are much more sensitive than the standard NIH procedure and are often employed for crossmatching. A general flow diagram of tissue typing procedures in common usage is shown in Figure 1. HLA-A, B, and C antigens can also be defined using isolated platelets and a battery of complement-fixing antibodies to HLA antigens. But it is important that platelets be allowed to mature for at least 24 hours before typing is attempted. Because of this and other reasons (such as the observation that some antigens express themselves in rather low concentrations on the platelet surface in comparison to lymphocytes), platelet complement fixation has been of limited value as a method to identify
HLA-A, B, and C antigens. H L A - D R typing The realization that both the blocking of MLC reaction by some antiHLA sera and the extra reactivity of some sera to certain cell types were manifestations of immune responseassociated antigenic reactivity (Ia) in humans was largely responsible for the success of the search for the human equivalent of the mouse Ia antigens, which culminated in the definition at the Seventh International Histocompatibility Workshop of the allelic series of HLA-DR determinants. The tissue distribution of Ia antigens in both man and mouse is still being investigated, but there is overwhelming evidence that the antigens detected in humans are on B lymphocytes, monocytes, and activated T lymphocytes or T cells grown in the presence of
conditioned medium. However, they are not detected on normal peripheral blood lymphocytes, which are usually composed of about 80°7o T cells. Because of this, definition of HLA-DR specificities requires selective enrichment of B lymphocytes from the total lymphocyte populations. Recently, several methods for the enrichment of B lymphocytes have been described. Selective depletion of Tlymphocytes, using their ability to form rosettes with sheep erythrocytes and subsequently collecting the nonrosetting cell population that consists mostly of B lymphocytes, has been widely used. Although typing can be done successfully using this method, the number of contaminating nonrosette-forming cells (other than B lymphocytes) in the preparation remains a major problem. Hence,
LIVING RELATED T R A N S P L A N T S RECIPIENT
DONOR
7
mphocytes
Typing for HLA-A, -B. oC. & -DR (microcytotoxicity)
Percent Reactive Antibodies (microcytotoxicity)
Stimulation Index (mixed lymphocyte culture)
Cross match Against Donor (microcytotoxicity)
homozygOuS typing cells T Cells
Typing for HLA-D ~"~'~
B Cells #'~ . ~ M o n o c y t e s • ~Endothelial
primed lymphocyte typing
CADAVERIC
DONOR
Cells
TRANSPLANTS
RECIPIENT (lymph node, spleen)
Typing for HLA-A. -B, -C, & -DR (mictocytotoxicity)
Percent Reactive Antibodies (microcytotoxic~ty) Cross match Against Donor (microeytotoxicity)
T Cells B Cells #"~ ~,,~Monocytes • " ~ Endothelial Cells
Fig. 1. A flow diagram of tissue typing procedures used for matching recipients and donors of living related and cadaveric kidney allografts. (Question marks designate crossmatch targets that may influence renal allograft survivaL)
63
other investigators have used positive B-lymphocyte enrichment methods, using either the presence of immunoglobulin on the B-lymphocyte surface (2) or the ability of B lymphocytes to adhere to nylon wool (10). Using these methods, enough B lymphocytes can be obtained to perform HLA-DR typing from about 20-30 ml of peripheral blood. Most of the sera that contain HLADR antibodies also possess antiHLA-A, B, and C activity, since they are mostly derived from the same source as the HLA-A, B, and C typing sera, namely, multiparous women. Because of this, it is a prerequisite that the antisera used in HLA-DR typing should first be absorbed with pooled platelets to remove all the anti-HLA-A, B, and C activity. Microcytotoxicitytechniques, either the standard NIH method or the Amos modified method described earlier, can be used to define HLADR antigens. Experience shows that longer periods of incubation, both before and after the addition of complement, are required for the cytotoxic reaction to occur with B lymphocytes than for HLA-A, B, and C typing of peripheral blood lymphocytes. Thus, the cells and serum are incubated at room temperature for one hour instead of 30 minutes, and incubation with complement is also extended to two hours at room temperature. The complement should also be pretested for its natural cytotoxicity towards B cells, and only nontoxic complement should be used in the HLA-DR typing. In addition to microcytotoxicity, a two-color fluorescence test for B cells has been described (9). In this assay, the cells are first incubated with fluorescein-labeled antihuman IgG prior to reacting with B-cell antisera and complement. The dead B cells are stained with red fluorescent ethidium bromide; with the appropriate use of filters, it is possible to detect the green fluorescence of fluorescein-labeled B cells while the red fluorescence of the ethidium bromide simultaneously identifies dead B cells. The main advantage of this technique is that it avoids the need to separate B lymphocytes from the total tymphocytes
64
prior to testing. Other techniques such as an antibody-dependent lymphocyte killing assay have also been employed occasionally to detect human Ia antigens. Several studies have shown a close relationship between HLA-DR and HLA-D locus antigens. This relationship is of great practical importance, since it means that in many cases the cumbersome MLC assay could be replaced by the much simpler serologic technique. Examination of the relationship between the HLA-D antigens assigned by MLC and serologically defined HLA-DR antigens showed a high correlation between the two. On the basis of this, HLA-DR antigens are given numbers corresponding to the HLA-D locus antigen with which they show this close association. Thus, HLA-DR-I is closely associated with HLA-Dwl, HLA-DR2 to HLA-Dw2, and so on. However, it should be pointed out that a number of discrepancies exist; in general, the serologic methods yield a broader definition than the MLC cell typing. Such factors as antigenic crossreactivity, difficulties associated with B-cell enrichment, as well as problems of complement toxicity and viability of B cells after lengthy manipulation, have to be considered as critical variables. So, it seems likely that many of
the apparent discrepancies between HLA-D and HLA-DR are caused by technical problems. It remains to be seen, however, whether sera produced by different methods and tested on various cell types will reveal the presence of Ia-like antigens coded for either within the HLA region or outside of it.
Clinical histocompatibility testing Most of the laboratories perform not only HLA typing of the serologically defined antigens but also screening to detect antibodies to HLA antigens that may be present in the serum. Serum samples are collected from patients awaiting renal transplants on a monthly basis (HLA antibody specificities and titers change frequently) and are tested against a panel of lymphocytes covering all the HLA-A, B, and C antigens. The results are computed and antibody activity is calculated on a percent basis that is usually referred to as percent reactive antibodies (PRA). This provides a basis for identifying the degree of sensitization of a given patient, which correlates with the ease with which a donor tissue can be matched-- the lower the PRA, the better the chance to find a negative crossmatch and, hence, the better the chance to find a donor. When crossmatching against a given donor, the most recent and most
Editors: Herman Friedman, Mario Escobar, and Noel Rose Editorial Committee: Charles D. Graber, Ph.D., Medical University of South Carolina; John R. Kateley, Ph.D., Edward W. Sparrow tlospital Association; Bruce S. Rabin, M.D., Ph.D., University of Pittsburgh School of Medicine; Robert F. Ritchie, M.D., Foundation for Blood Research, Maine; John L. Sever, M.D., Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health; Steven Specter, Ph.D., University of South Florida College of Medicine; Roy W. Stevens, Ph.D., New York State Health Department Laboratories; Norman Talal, M.D., VA Hospital and University of California Medical Center at San Francisco; Eng M. Tan, M.D., University of Colorado Medical Center; Gabriel Virella, M.D., Ph.D., Medical University of South Carolina. Subscription Rates in U.S. and Canada: one year $54.00; two years $103.00; three years $146.00. All other countries: one year $62.00; two years $118.50; three years $168.00. Single copies are $2.50 in U.S. and Canada; 52.90 in all other countries. Single issues are available in quantity (prices available upon request). Volume I (1980), 24 issues $46.50. All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada. Address correspondence regar~ling subscription to: Clinical hnmunology A'ewsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clinical hnnlunolog), A'ewsletter is published twice monthly. All rights, including that of translation into other languages, reserved. Photomechanical reproduction (photocopy, microcopy) of this newsletter or parts thereof without special permission of the publisher is prohibited.
reactive serum samples of the potential recipient are always to be used. Techniques of standard crossmatching are basically the same as described earlier for HLA-A, B, and C typing. However, in order to increase the sensitivity, most of the laboratories currently perform microcytotoxicity either by the Amos modified method with two or three washes or by the antiglobulin lymphocytotoxicity method described earlier. In addition to the standard crossmatch for HLA-A, B, and C antigens, several laboratories also perform crossmatches against separated donor T- and B-lymphocyte populations in order to differentiate anti-HLA-A, B, and C activity from anti-B-lymphocyte antigen activities. This is especially relevant since much of the crossmatching in cadaveric transplants is done using lymph node or splenic lymphocyte populations, which have a high percentage of B cells, so that some cytotoxicity (and, hence, positive crossmatch), occasionally could be directed to B-lymphocyte antigens. Several investigators have already shown that successful transplants can be performed across B-cell crossmatch (6, 7), and it has also been postulated that at least some of these anti-Bcell antibodies may function as enhancing antibodies (7). Thus, the role of antibodies to various subpopulations of cells in recipient serum remains to be clearly defined. Recent studies have already suggested a decisive role for antibodies directed towards organ-specific alloantigens, monocyte/endothelial cell antigens, etc., in human renal transplantation, and future crossmatching to some of these antigenic determinants prior to transplant may be feasible.
Matching by Cellular Techniques The most commonly studied cellular changes that occur upon sensitization to histocompatibility antigens are lymphocyte proliferation and lymphocyte-mediatedkilling. An important assumption in cellular typing is that each gene product is recognized by a separate clone of lymphocytes, and this concept of clonality has been supported byboth deletion
and addition experiments (3). All cellular typing techniques are essentially the measurements of events that occur in MLC reaction in which responding lymphocytes are cultured with viable x-irradiated or mitomycintreated stimulating cells (one-way MLC). After four to seven days in culture, the proliferative response of the responding cell is measured by incorporation of 3H-thymidine. Although the proliferative response is the most commonly used in cellular typing, there are several other events in this reaction that activate a lymphocyte population. The gene products responsible for this are often referred to as lymphocyte-activating determinants (LAD) and have been identified using two principal methods: negative typing and positive typing (3). In negative typing, the responding lymphocyte population contains a variety of clones that proliferate strongly when incubated with foreign LADs. Self-LADs can thus be identified by negative reactions (or low reaction) when stimulating and responding cells are LAD-identical. The most widely used negative typing makes use of stimulating cells that are homozygous for the D-locus (bomozygous typing cells [HTC] ). Responding cells that share either the homozygous or heterozygous form of the HLA-D allele in question will not respond (or respond insignificantly) to the HTC or stimulator. Twelve alleles have been identified using this method (HLADwI--HLA-DwI2). HTCs are usually found among the offspring of first cousin marriages where the chance of such an occurrence is one in four if both cousins are heterozygous. Several modifications of HTC typing have been advocated; a) lymphoblastoid cell lines, which are homozygous, as a continuous source of HTCs, b) prior sensitization of the responder to a pool of stimulator cells to reduce the latent period of the test, and c) using sperm as typing reagents instead of HTCs after serologic separation of sperms into two populations with HLA-haploid expression. The concept of positive cellular typing consists of specific clonal expansion by prior sensitization tO a
given LAD specificity. A commonly used positive typing method for detecting HLA-D antigens is the primed lymphocyte typing test (PLT). This test is based on the fact that cells stimulated in one-way MLC and left for 14 days will give a heightened secondary type proliferative response if restimulated with cells that share the HLA-D allele with the original stimulating donor. Reasonable correlation coefficients were noted in the seventh workshop between most of the PLT responses and the HLA-D antigen used for priming (3). However, its usefulness in clinical transplantation as well as in typing for non-HLA antigens has yet to be established. Since cellular typing techniques rely heavily on the interaction of various subpopulations of lymphoid as well as nonlymphoid cells, several technical as well as biologic problems still persist. Recent developments in the methodologies for the identification and separation of lymphocyte subpopulations and other mononuclear cells in the circulation should soon allow improvement in the accuracy of cellular typing as well as in their applicability in clinical histocompatibility laboratories. ACKNOWLEDGMENT"This work was supported by grants from the American Heart Association (79772) and the National Institutes of Health (AI-12822).
References l. Amos, D. B., el al. 1969. A simple microcytotoxicitytest. Transplantation 7:220-223. 2. Barker, C. R., C. P. Worman, and J. L. Smith. 1975. Purification and quantitation ofT- and B-lymphocytes by an affinity method. Immunology 29:765-777. 3. Bradley,B. A., and H. Festenstein. 1978. Cellular typing. Br. Med. Bull. 34:223-232. 4. Johnson, A. H., R. D. Rossen, and W. T. Butler. I972. Detection of alloantigens using a sensitiveantiglobulin microcytotoxicitytest. Tissue Antigens 2:215-226. 5. Mendes, N. F., et al. 1973. Technical aspects of the rosette tests used to detect human complement receptor (B) and sheep erythrocyte-binding(T)
65
lymphocytes. J. Immunol. 111:860867. 6. Mohanakumar, T., et al. 1979. Relationship of B-cell alloantibodies to renal allograft survival. Transplantation 27:273-278. 7. Morris, P. J., el al. 1977. Renal transplantation and a positive sero-
logical crossmatch. Lancet 1:12881291. 8. National lnstilute of Allergy and Infectious Diseases. 1979. Manual of tissue typing techniques. U.S. Public Health Service, Washington, D.C. 9. Van Rood, J. J., andJ. Van Leeawen. 1976. Simultaneous detection of two
cell populations by two-color fluorescence and application to the recognition of B-cell determinants. Nature 262:795-796. 10. Werner, C. ti., et al. 1977. Isolation of B- and T-lymphocytes by nylon fiber columns. Tissue Antigens 9:227229.
Invitation to Contributors
Case Reports
Reports documenting interesting case presentations are invited for possible publication in the Clinical hnnnmology Newsletter. Submitted case reports should be written concisely and should contain: 1. a brief clinical history summarizing the symptoms and course of the illness; 2. a description of immunological and other laboratory tests performed; and 3. results of these laboratory tests, as related to the clinical observations o f the patients and the outcome o f the disease process and therapy, if appropriate. References should be listed in alphabetical order and should contain complete identification of authors, date, article or chapter title, journal or book title, volume number and inclusive page numbers for journals, and publisher and place of publication for books. Letters Letters expressing opinion(s) or helpful technical ideas and procedures are encouraged. Each contribution should be written concisely and should present one major point. The Newsletter will attempt to publish as many letters as possible, especially those of interest to a wide range of biomedical scientists. Questions and Answers Questions about immunological techniques, laboratory procedures for immunology, and general problems in clinical immunology are invited and will be answered by the editorial board or by selected individuals in the field. Submission of Material Four copies of material submitted for publication in the Newsletter should be sent to:
Dr. H. M. Frobar
Clinical Intnnmology Newsletter G. K. Hall & Co. 70 Lincoln Street Boston, Massachusetts 02111
66
8.
9.
I0.
11.
375-382. In B. Serrou and C. Rosenfeld (eds.), Human lymphocyte differentiation. Its application to cancer. North-Holland Pub. Co., Amsterdam. Howard, A. R., and L. Graham. 1976. Rosette formation by foetal liver and spleen cell incubated with theophylline. Clin. Exp. Immunol. 23: 279-284. Larson, S., et al. 1978. Theimmunopharmacology of vindesine (VND) in man. Proc. ASCO 19:405 (abstract C. 396). Limatibul, S., et al. 1978. Theophylline modulation of E-rosette formation: An indicator of T cell maturation. Clin. Exp. Immunol. 33: 503-513. Moretta, L., et al. 1977. Human T cell subpopulation: Help and suppression by T cells bearing receptor for IgM or IgG. J. Exp. Med. 146: 184-200. Opelz, G., etal. 1975. Autologous
Laboratory
Methods
13.
14. 15.
16.
17.
18.
19.
20.
Elsevier North-Holland Biomedical Press, Amsterdam. In press. Serrou, B., et al. 1980. In vivo and in vitro modulation of the immune response by human fibroblast interferon (HFI). Int. J. lmmunopharmacol. 2:159 (abstract). Serrou, B., D. Cupissol, and C. Rosenfeld. 1980. Immune imbalance and immune modulation in solid tumor patients. New insights. In G. Mathe, G. Bonadonna, and S. Salmon (eds.), Adjuvant therapies of cancer. Springer-Verlag, Heidelberg. In press. Wierda, D., and T. L. Pazdernlr. 1979. Suppression of spleen lymphocyte mitogenesis in mice injected with platinum compounds. Eur. J. Cancer 15:1013-1023. Yehuda, Z. P., et al. 1979. New approaches to the evaluation of immunomodulation by thymic hormones. Ann. N.Y. Acad. Sci. 332: 160-171.
in C l i n i c a l T r a n s p l a n t a t i o n
T. Mohanakumar, Ph.D. Thomas M. Ellis, Ph.D. Claude Duvall, B.S. H. M. Lee, M.D. Tissue Typing Laboratories Departments of Surgery and Microbiology Medical College of Virginia Virginia Common wealth University Richmond, Virginia 23298 There is no doubt that the human leukocyte antigen (HLA) system or region has a decisive influence on transplantation, as demonstrated by the observations that a) excellent resuits are obtained in HLA identical sibling transplants, and b) accelerated rejection o f allografts occurs when performed in the presence of specific sensitization to the donor antigens. After the H L A workshop held at Torino in 1967, it was realized that the H L A system is so complex that the procurement of H L A well-matched cadaver kidneys required regional and national exchange o f organs. For this reason, a central register o f all potential recipients in a region waiting for cadaver transplant was established. The register contains the relevant particulars such as ABO and HLA types, antibody status, and age. When
62
12.
stimulation of human lymphocyte subpopulations. J. Exp. Med. 142: 1327-1333. Rey, A., et al. 1980. Human autologous rosettes. III. Pharmacology of autologous rosette-forming cells. Submitted for publication. Rucheton, M., et al. 1980. Human autologous rosettes. IV. Further characterization of autologous rosettes forming cells. Submitted for publication. Serrou, B., et al. 1979. Regulation of human suppressor cell function by thymosin. Biomedicine 31" 89. Serrou, B., et al. 1980. In vivo and in vitro modulation of helper, suppressor and NK activities by human interferon. J. Clin. Hematol. Oncol. In press. Serrou, B., et al. 1980. Phase I-Evaluation of bestatin in patients bearing advanced solid tumors. In W. D. Terry (ed.), Immunotherapy of cancer: present status of trials in man.
the organs become available, and ABO and H L A typings have been carried out, using a computer facility, the central register is searched for the most suitable recipient. For example, through nonprofit organizations such as Southeastern Organ Procurement Foundation and the United Network for Organ Sharing, we can now obtain computer matching for 136 transplant centers in the USA comprising about 4,500 potential recipientS. Testing for ABO compatibility, HLA-A, B, C, D / D R compatibility, antibodies to HLA-A, B, C, and DR antigens, and HLA-A, B, C, and DR crossmatching are currently the primary procedures used in identifying the donor/recipient compatibility. We will limit discussion here to the techniques o f H L A typing and crossmatching against antigens coded by HLA-A, B, C, and DR. w e shall broadly divide the procedures into two categories based solely upon the current methodologies~ a) matching by serologic techniques for the determination of antigens coded for by the HLA-A, B, and C loci as well as the identification of HLA-DR antigens on isolated B lymphocytes, and b)
matching by cellular techniques for the definition of antigens coded for HLA-D locus employing the mixed lymphocyte culture (MLC) assay and its variants. Matching by Serologic Techniques H L A - A , B, and C typing Ten ml o f heparinized (25 U heparin/ml blood) blood are obtained by venipuncture and passed through a syringe lightly packed with 1.0-I .5 g of washed nylon in order to remove tile platelets. Alternatively, defibrinated blood samples can be employed. In some laboratories, the blood is then incubated with carbonyl iron to remove the phagocytic cell population. The blood sample is then diluted 1:3 with either barbital buffer or medium (Hanks balanced salt solution), and the lymphocytes are isolated by centrifugation in Ficoll-Hypaque density gradient. The final cell preparation is washed three times with barbital buffer or medium and adjusted to 2-2.5 x 106/ml for microcytotoxicity test. In the standard NIH microcytotoxicity procedure (8), 1 ~ul of cell suspension is incubated for 30 min (28°C) in
I ~ulof various specific HLA antisera placed under oil in the microcytotoxicity trays (Falcon or Cook plastic tissue culture trays). Five ju I of rabbit complement is then added to each well and incubated for another hour at 28°C. Eosin and formalin are then added, and the percentage of viable cells is determined by phase microscopy. Dead cells take up the dye and appear large and dark. A modification of the NIH technique (1) was designed by Dr. D. B. Amos at Duke University that avoids the anticomplementary activities of the excess HLA-typing sera present in the test wells. This procedure calls for washing the unbound antibody by a quick snap of the plate (generally known as flickifig) after the initial 30 minute incubation with cells. The remainder of the procedure is basically similar to the NIH method but uses trypan blue instead of eosin as a vital dye. Usually excess complement is flicked off before adding trypan blue. This procedure, although requiring more time, has proven to be more sensitive than the NIH method. A third procedure with even greater sensitivity was developed by Johnson et al. (4). In this method, after the incubation of lymphocytes and antisera, cells are washed by flicking, and 1/~1 of titered antihuman immunoglobulin reagent is added for one to three minutes before the rabbit complement. Both the Amos modified and the antiglobulin lymphocytotoxicity techniques are much more sensitive than the standard NIH procedure and are often employed for crossmatching. A general flow diagram of tissue typing procedures in common usage is shown in Figure 1. HLA-A, B, and C antigens can also be defined using isolated platelets and a battery of complement-fixing antibodies to HLA antigens. But it is important that platelets be allowed to mature for at least 24 hours before typing is attempted. Because of this and other reasons (such as the observation that some antigens express themselves in rather low concentrations on the platelet surface in comparison to lymphocytes), platelet complement fixation has been of limited value as a method to identify
HLA-A, B, and C antigens. H L A - D R typing The realization that both the blocking of MLC reaction by some antiHLA sera and the extra reactivity of some sera to certain cell types were manifestations of immune responseassociated antigenic reactivity (Ia) in humans was largely responsible for the success of the search for the human equivalent of the mouse Ia antigens, which culminated in the definition at the Seventh International Histocompatibility Workshop of the allelic series of HLA-DR determinants. The tissue distribution of Ia antigens in both man and mouse is still being investigated, but there is overwhelming evidence that the antigens detected in humans are on B lymphocytes, monocytes, and activated T lymphocytes or T cells grown in the presence of
conditioned medium. However, they are not detected on normal peripheral blood lymphocytes, which are usually composed of about 80°7o T cells. Because of this, definition of HLA-DR specificities requires selective enrichment of B lymphocytes from the total lymphocyte populations. Recently, several methods for the enrichment of B lymphocytes have been described. Selective depletion of Tlymphocytes, using their ability to form rosettes with sheep erythrocytes and subsequently collecting the nonrosetting cell population that consists mostly of B lymphocytes, has been widely used. Although typing can be done successfully using this method, the number of contaminating nonrosette-forming cells (other than B lymphocytes) in the preparation remains a major problem. Hence,
LIVING RELATED T R A N S P L A N T S RECIPIENT
DONOR
7
mphocytes
Typing for HLA-A, -B. oC. & -DR (microcytotoxicity)
Percent Reactive Antibodies (microcytotoxicity)
Stimulation Index (mixed lymphocyte culture)
Cross match Against Donor (microcytotoxicity)
homozygOuS typing cells T Cells
Typing for HLA-D ~"~'~
B Cells #'~ . ~ M o n o c y t e s • ~Endothelial
primed lymphocyte typing
CADAVERIC
DONOR
Cells
TRANSPLANTS
RECIPIENT (lymph node, spleen)
Typing for HLA-A. -B, -C, & -DR (mictocytotoxicity)
Percent Reactive Antibodies (microcytotoxic~ty) Cross match Against Donor (microeytotoxicity)
T Cells B Cells #"~ ~,,~Monocytes • " ~ Endothelial Cells
Fig. 1. A flow diagram of tissue typing procedures used for matching recipients and donors of living related and cadaveric kidney allografts. (Question marks designate crossmatch targets that may influence renal allograft survivaL)
63
other investigators have used positive B-lymphocyte enrichment methods, using either the presence of immunoglobulin on the B-lymphocyte surface (2) or the ability of B lymphocytes to adhere to nylon wool (10). Using these methods, enough B lymphocytes can be obtained to perform HLA-DR typing from about 20-30 ml of peripheral blood. Most of the sera that contain HLADR antibodies also possess antiHLA-A, B, and C activity, since they are mostly derived from the same source as the HLA-A, B, and C typing sera, namely, multiparous women. Because of this, it is a prerequisite that the antisera used in HLA-DR typing should first be absorbed with pooled platelets to remove all the anti-HLA-A, B, and C activity. Microcytotoxicitytechniques, either the standard NIH method or the Amos modified method described earlier, can be used to define HLADR antigens. Experience shows that longer periods of incubation, both before and after the addition of complement, are required for the cytotoxic reaction to occur with B lymphocytes than for HLA-A, B, and C typing of peripheral blood lymphocytes. Thus, the cells and serum are incubated at room temperature for one hour instead of 30 minutes, and incubation with complement is also extended to two hours at room temperature. The complement should also be pretested for its natural cytotoxicity towards B cells, and only nontoxic complement should be used in the HLA-DR typing. In addition to microcytotoxicity, a two-color fluorescence test for B cells has been described (9). In this assay, the cells are first incubated with fluorescein-labeled antihuman IgG prior to reacting with B-cell antisera and complement. The dead B cells are stained with red fluorescent ethidium bromide; with the appropriate use of filters, it is possible to detect the green fluorescence of fluorescein-labeled B cells while the red fluorescence of the ethidium bromide simultaneously identifies dead B cells. The main advantage of this technique is that it avoids the need to separate B lymphocytes from the total tymphocytes
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prior to testing. Other techniques such as an antibody-dependent lymphocyte killing assay have also been employed occasionally to detect human Ia antigens. Several studies have shown a close relationship between HLA-DR and HLA-D locus antigens. This relationship is of great practical importance, since it means that in many cases the cumbersome MLC assay could be replaced by the much simpler serologic technique. Examination of the relationship between the HLA-D antigens assigned by MLC and serologically defined HLA-DR antigens showed a high correlation between the two. On the basis of this, HLA-DR antigens are given numbers corresponding to the HLA-D locus antigen with which they show this close association. Thus, HLA-DR-I is closely associated with HLA-Dwl, HLA-DR2 to HLA-Dw2, and so on. However, it should be pointed out that a number of discrepancies exist; in general, the serologic methods yield a broader definition than the MLC cell typing. Such factors as antigenic crossreactivity, difficulties associated with B-cell enrichment, as well as problems of complement toxicity and viability of B cells after lengthy manipulation, have to be considered as critical variables. So, it seems likely that many of
the apparent discrepancies between HLA-D and HLA-DR are caused by technical problems. It remains to be seen, however, whether sera produced by different methods and tested on various cell types will reveal the presence of Ia-like antigens coded for either within the HLA region or outside of it.
Clinical histocompatibility testing Most of the laboratories perform not only HLA typing of the serologically defined antigens but also screening to detect antibodies to HLA antigens that may be present in the serum. Serum samples are collected from patients awaiting renal transplants on a monthly basis (HLA antibody specificities and titers change frequently) and are tested against a panel of lymphocytes covering all the HLA-A, B, and C antigens. The results are computed and antibody activity is calculated on a percent basis that is usually referred to as percent reactive antibodies (PRA). This provides a basis for identifying the degree of sensitization of a given patient, which correlates with the ease with which a donor tissue can be matched-- the lower the PRA, the better the chance to find a negative crossmatch and, hence, the better the chance to find a donor. When crossmatching against a given donor, the most recent and most
Editors: Herman Friedman, Mario Escobar, and Noel Rose Editorial Committee: Charles D. Graber, Ph.D., Medical University of South Carolina; John R. Kateley, Ph.D., Edward W. Sparrow tlospital Association; Bruce S. Rabin, M.D., Ph.D., University of Pittsburgh School of Medicine; Robert F. Ritchie, M.D., Foundation for Blood Research, Maine; John L. Sever, M.D., Ph.D., National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health; Steven Specter, Ph.D., University of South Florida College of Medicine; Roy W. Stevens, Ph.D., New York State Health Department Laboratories; Norman Talal, M.D., VA Hospital and University of California Medical Center at San Francisco; Eng M. Tan, M.D., University of Colorado Medical Center; Gabriel Virella, M.D., Ph.D., Medical University of South Carolina. Subscription Rates in U.S. and Canada: one year $54.00; two years $103.00; three years $146.00. All other countries: one year $62.00; two years $118.50; three years $168.00. Single copies are $2.50 in U.S. and Canada; 52.90 in all other countries. Single issues are available in quantity (prices available upon request). Volume I (1980), 24 issues $46.50. All subscriptions are payable in advance. Foreign subscriptions are sent guaranteed air mail. First class postage paid in U.S. and Canada. Address correspondence regar~ling subscription to: Clinical hnmunology A'ewsletter, G. K. Hall & Co., 70 Lincoln St., Boston, Massachusetts 02111. Please include zip code in subscription address. The Clinical hnnlunolog), A'ewsletter is published twice monthly. All rights, including that of translation into other languages, reserved. Photomechanical reproduction (photocopy, microcopy) of this newsletter or parts thereof without special permission of the publisher is prohibited.
reactive serum samples of the potential recipient are always to be used. Techniques of standard crossmatching are basically the same as described earlier for HLA-A, B, and C typing. However, in order to increase the sensitivity, most of the laboratories currently perform microcytotoxicity either by the Amos modified method with two or three washes or by the antiglobulin lymphocytotoxicity method described earlier. In addition to the standard crossmatch for HLA-A, B, and C antigens, several laboratories also perform crossmatches against separated donor T- and B-lymphocyte populations in order to differentiate anti-HLA-A, B, and C activity from anti-B-lymphocyte antigen activities. This is especially relevant since much of the crossmatching in cadaveric transplants is done using lymph node or splenic lymphocyte populations, which have a high percentage of B cells, so that some cytotoxicity (and, hence, positive crossmatch), occasionally could be directed to B-lymphocyte antigens. Several investigators have already shown that successful transplants can be performed across B-cell crossmatch (6, 7), and it has also been postulated that at least some of these anti-Bcell antibodies may function as enhancing antibodies (7). Thus, the role of antibodies to various subpopulations of cells in recipient serum remains to be clearly defined. Recent studies have already suggested a decisive role for antibodies directed towards organ-specific alloantigens, monocyte/endothelial cell antigens, etc., in human renal transplantation, and future crossmatching to some of these antigenic determinants prior to transplant may be feasible.
Matching by Cellular Techniques The most commonly studied cellular changes that occur upon sensitization to histocompatibility antigens are lymphocyte proliferation and lymphocyte-mediatedkilling. An important assumption in cellular typing is that each gene product is recognized by a separate clone of lymphocytes, and this concept of clonality has been supported byboth deletion
and addition experiments (3). All cellular typing techniques are essentially the measurements of events that occur in MLC reaction in which responding lymphocytes are cultured with viable x-irradiated or mitomycintreated stimulating cells (one-way MLC). After four to seven days in culture, the proliferative response of the responding cell is measured by incorporation of 3H-thymidine. Although the proliferative response is the most commonly used in cellular typing, there are several other events in this reaction that activate a lymphocyte population. The gene products responsible for this are often referred to as lymphocyte-activating determinants (LAD) and have been identified using two principal methods: negative typing and positive typing (3). In negative typing, the responding lymphocyte population contains a variety of clones that proliferate strongly when incubated with foreign LADs. Self-LADs can thus be identified by negative reactions (or low reaction) when stimulating and responding cells are LAD-identical. The most widely used negative typing makes use of stimulating cells that are homozygous for the D-locus (bomozygous typing cells [HTC] ). Responding cells that share either the homozygous or heterozygous form of the HLA-D allele in question will not respond (or respond insignificantly) to the HTC or stimulator. Twelve alleles have been identified using this method (HLADwI--HLA-DwI2). HTCs are usually found among the offspring of first cousin marriages where the chance of such an occurrence is one in four if both cousins are heterozygous. Several modifications of HTC typing have been advocated; a) lymphoblastoid cell lines, which are homozygous, as a continuous source of HTCs, b) prior sensitization of the responder to a pool of stimulator cells to reduce the latent period of the test, and c) using sperm as typing reagents instead of HTCs after serologic separation of sperms into two populations with HLA-haploid expression. The concept of positive cellular typing consists of specific clonal expansion by prior sensitization tO a
given LAD specificity. A commonly used positive typing method for detecting HLA-D antigens is the primed lymphocyte typing test (PLT). This test is based on the fact that cells stimulated in one-way MLC and left for 14 days will give a heightened secondary type proliferative response if restimulated with cells that share the HLA-D allele with the original stimulating donor. Reasonable correlation coefficients were noted in the seventh workshop between most of the PLT responses and the HLA-D antigen used for priming (3). However, its usefulness in clinical transplantation as well as in typing for non-HLA antigens has yet to be established. Since cellular typing techniques rely heavily on the interaction of various subpopulations of lymphoid as well as nonlymphoid cells, several technical as well as biologic problems still persist. Recent developments in the methodologies for the identification and separation of lymphocyte subpopulations and other mononuclear cells in the circulation should soon allow improvement in the accuracy of cellular typing as well as in their applicability in clinical histocompatibility laboratories. ACKNOWLEDGMENT"This work was supported by grants from the American Heart Association (79772) and the National Institutes of Health (AI-12822).
References l. Amos, D. B., el al. 1969. A simple microcytotoxicitytest. Transplantation 7:220-223. 2. Barker, C. R., C. P. Worman, and J. L. Smith. 1975. Purification and quantitation ofT- and B-lymphocytes by an affinity method. Immunology 29:765-777. 3. Bradley,B. A., and H. Festenstein. 1978. Cellular typing. Br. Med. Bull. 34:223-232. 4. Johnson, A. H., R. D. Rossen, and W. T. Butler. I972. Detection of alloantigens using a sensitiveantiglobulin microcytotoxicitytest. Tissue Antigens 2:215-226. 5. Mendes, N. F., et al. 1973. Technical aspects of the rosette tests used to detect human complement receptor (B) and sheep erythrocyte-binding(T)
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lymphocytes. J. Immunol. 111:860867. 6. Mohanakumar, T., et al. 1979. Relationship of B-cell alloantibodies to renal allograft survival. Transplantation 27:273-278. 7. Morris, P. J., el al. 1977. Renal transplantation and a positive sero-
logical crossmatch. Lancet 1:12881291. 8. National lnstilute of Allergy and Infectious Diseases. 1979. Manual of tissue typing techniques. U.S. Public Health Service, Washington, D.C. 9. Van Rood, J. J., andJ. Van Leeawen. 1976. Simultaneous detection of two
cell populations by two-color fluorescence and application to the recognition of B-cell determinants. Nature 262:795-796. 10. Werner, C. ti., et al. 1977. Isolation of B- and T-lymphocytes by nylon fiber columns. Tissue Antigens 9:227229.
Invitation to Contributors
Case Reports
Reports documenting interesting case presentations are invited for possible publication in the Clinical hnnnmology Newsletter. Submitted case reports should be written concisely and should contain: 1. a brief clinical history summarizing the symptoms and course of the illness; 2. a description of immunological and other laboratory tests performed; and 3. results of these laboratory tests, as related to the clinical observations o f the patients and the outcome o f the disease process and therapy, if appropriate. References should be listed in alphabetical order and should contain complete identification of authors, date, article or chapter title, journal or book title, volume number and inclusive page numbers for journals, and publisher and place of publication for books. Letters Letters expressing opinion(s) or helpful technical ideas and procedures are encouraged. Each contribution should be written concisely and should present one major point. The Newsletter will attempt to publish as many letters as possible, especially those of interest to a wide range of biomedical scientists. Questions and Answers Questions about immunological techniques, laboratory procedures for immunology, and general problems in clinical immunology are invited and will be answered by the editorial board or by selected individuals in the field. Submission of Material Four copies of material submitted for publication in the Newsletter should be sent to:
Dr. H. M. Frobar
Clinical Intnnmology Newsletter G. K. Hall & Co. 70 Lincoln Street Boston, Massachusetts 02111
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