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IgA anti-donor antibodies in liver transplantation
133
Abstracts
P727
ELISA DETECTION OF SPECIFIC ANTIBODY TO HLA CLASS I ANTIGENS IN TRANSPLANT PATIENTS WITH PANEL REACTIVE ANTIBODY (PRA) BY A LEUKOCYTE ANTIGEN TRAY (LAn
GuixiWang.ChristineTarsitani,Steve Takemura,Rui Pei, An-NaLiu, Jar-HowLee OneLambda,Inc. Los Angeles,CA., U.S.A. A micro-ELISAwas performedon a panelofClass I antigenspurified DIRECTLY bound onTERASAKI trays. from 42 selectedEBV cell lines, and Specificitywas confirmed byreactivitywitha setof84 IgG allosera from commerciallyavailableHLA Class I typingtrays. Parallel results were (LCT) and LAT. All 52 HLA specificities obtained bylymphocytotoxicity L AT. Out of3,528 rxns, 97%ofthe (18 A- and 35 B- locus) were called by TP (374/385) were recognized. 81%ofthe tail reactions (440/540) were cross-reacting groups (CREGs). The optimumtiter was five totwenty-fold higher in theLAT, Sera from 300 transplant patients fromclinicalcenters 3 were tested by LAT and by LCT, and the%PRA was compared in the twoassays. A correlationcoefficientofr = 0.8 was obtained between theL AT and the non-Hl.A standardcytotoxicitytest. Certain sera could be shown to contain antibody(LCT+, LAT-) and others noncomplement-binding anti -HLA antibody(LAT+, LCT-) when adsorbed with platelets and retested in the appropriate assay.Antigenspecificity,when present, could clearly be demonstrated by theL AT assay.
P729
lRIPLE FLUORESCENCE fLOW CYTOMETRY CROSSMATCH (FCXM3) FOR THE SIMULTANEOUSIDENTIF1CATIONOFANTI-CD3ANDANTI-CDl9ANTIBODIES Puglisi G, *MascarettiL, Sioh V, *ScalamognaM, SircbiaG Centro Trasfusionalee di Immunologiadei Trapianti, *Servizio Autonomo Prelievo di Tessuti,OspedaleMaggiorePoliclinico, via F. Sforza35,20122 Milano, Italy The flowcytometrycrossmatchis moresensitivethan thecomplementdependent assay thoughsome doubtsstill persist on the clinical relevanceof the identifiedantibodiesin organtransplantation,Ourreference techniqueforeseesa doublefluorescenceassay wbich identifies anti-CD3 and anti-CD19 antibodiesseparately.In this studyweinvestigatedthe feasabilityof using 3fluorochromes(fluoresceinFlTC, pbycoerythrinPE and peridinin cbloropbyll protein PerCP) for thesimultaneousdetectionof CD3 andCDI9antibodiesby comparing our standard technique with the FCXM3 which was performed as follows: 300,000lympbocyteswereincubatedfor 30' at 22" C with30~1 of serumsample, washed twice with PBS with 0.1% sodium azide and 1% FCS then incubated with 21l\J.1 of adequatelydiluted anti-humanIgG-FITC F(ab'),(Tago) for 15'a14" C in the dark, washed with PBSand finallyincubatedwith 5~1 of anti-CD3-PeICP and5111 of anti-CDI9-PE at 4"C in thedarkfor 20', Analysiswasperformedwith a FACScan(BectonDickinson) daily monitoredwith Calibrite beads; electroniccompensationwasused to correctoverlapof the 3 fluorochromes'emission spectra. 50 sera of patients onthe waiting list for kidney usnsplantationand adequatecontrols werestudied.For CD3 antibodies,concordancyrate was 100% since43samples werenegativeand 7 positive with bothmethods.For CDI9 antibodiesconcordancyrate was94% since for 3samplesthe FCXM' was negativeand standard was positive.In conclusion, the FCXM3 provedto be a reliable technique which allows to save time and reagents (especially sera and cells). The simultaneous identification of CD3 and CD19 antibodiesis more informative on the specificity of the detectedantibodies;an antibodywhicb reacts with both CD3 and CD19 is more probably anti-HLA than one whichreacts only with one lymphocyte subset. In the ligbt ofthese resultswe have adoptedtheFCXM' as thestandardoperatingprocedurein our laboratory.
P731
P728
PHOTOMETRIC DETECTION OF ANTI-HLA ANTIBODIES BY AN ENZYMELINKED IMMUNE-PHAGOCYTOSI5-INHIBITION-TEST (IPI) U. Cassens, H.S.P Garritsen,R. Kelsch, W. Sibrowski. Department of Transfusion Medicine, Muenster, Germany Anti-HLA antibodiesw~h HLA A, B. Cw and DR specificity are detectable by the immune phagocytosis inhibnion assay (IPI), which has been described as a sensitive and specific functional test [Neppert et al. Vox Sang. 1986). We performed a photometric evaluation of immune phagocytosis and compared this test with the microscopic IPI and the Iymphocytotoxicily test (LCT). Monocytes were purified from mononuclear leukocytes by adhesion technique and incubated with HLA antibody sera in microtitre plates, After incubation with anti-D RBC, phagocytosed erythrocytes were determined photomellically(haemoglobincatalyzed peroxidase-reaction). Autologous sera and 8 specific antibody sera were tested against 10 HLA-typed monocyte panels (322 crossmatches). Optical denshy of the negative control (pre tested AB serum) was defined 100%. The photomellic IPI was defined.pos~ive· with a percentage of negative control (PNC) < 30%. Results of photometric and microscopic IPI showed a good correlation (","0.89). All autologous sera yielded distinct negative results(mean PNC 91%). Antibody sera against corresponding antigens showed a mean PNC of 16% vs. 85% against non corresponding antigens (p<0.001). Two monoclonal antibodies against constant HLA-regions showed~ive results in all LCT and microscopic / photometric IPI tests. First tests with diluted, LCT-positive patient sera showed a distinct higher sen~iv~y olthe photometric IPI in comparison to the LCT. We conclude that the photometric IPI test represents a specific, sensitive and convenient method to detect cytotoxic as well as non-cytotoxic anti-HLA antibodies.
P730
EVALUATION OF A SIMPLIFiED ANTI-HUMANGLOBULIN AUGMENTED CYTOTOXICITY TECHNIQUE
A. Papassavas,' A. Germenis,' M. Spyropoulou-Vlachou,' A. Kostakis,' C. Stavropoulos-Giokas' 'Department of Immunology and National Tissue Typing Center, General Hospital of Athens "G. Gennimatas", and 'Transplant Unit, "Laikcn'' Hospital, Athens, Greece To simplify the cumbersome but extremely useful aati-humao globulin aug1994, 40 mented cytotoxicity technique (AHG-CDC) Steen et [.'1umImmuno! al (Suppll):136]proposed a modification consisting of the use of AHG diluted 1:50 in the complement To evaluate this modified AHG-CDC (m'AHG-CDC), we examined 200 sera samples obtained from 50 highly sensitized patients. In all samples, the under evaluation technique was performed along with both the complement-dependent cytotoxicity (CDC) and the conventional AHG-CDC. The same cell panel from 60 HLA-typed donors was used In all cases. The mean (% PRA) detected by m-AHG-CDC and percentage of panel reactive antibodies AHG-CDC was found exactly the same (83.34±8.07 a.id 83.48±8.07, respec(p
P732
PREDICTION OF ACCEPTABLE MISMATCHES BASED ON THE DETECTION OF PATIENTS' CREGs
IgA ANTI-DONORANTIBODIESIN LIVERTRANSPLANTATION
A. Kostakis,' A. Papassavas,' A. Germenis,' M. Spyropoulou-Vlachou,'
'Piazza A., Torlone N. Valeri M., Poggi E., Monaco 1., Romagnoli Tisone G., AdornoD., Casciani C.U.
C. stavrcpoutos-otckas'
J.,
'Department of Immunology and National Tissue Typing Center, General Hospital of Athens "G. Gennimatas", and 'Transplant Unit, "Laikon" Hospital, Athens, Greece
lstituto C.N.R. Tipizzazione Tissutale, L'Aquila and Clinica Chirurgica, Universit:l. Tor Vergata, Rome, Italy
This study was scheduled to search the possibility of the acceptable HLA-A and -B mismatches (AM) to be predicted on the basis of their CREGs in highly sensitized patients (HSP). The anti-human globulin augmented cytotoxicity technique, using a screening cell panel from 60 HLA-typed donors, was performed for the determination of the HLA alloantibodies in 50 HSP (PRA>60%). The defined HLA class I specificities of the alloantibodies, the specificities of the nonreactive cells as well as the class I HLA antigens of the patients were assigned to CREGs. Thereafter, the AM were detected using a cell panel in which almost all the HLA antigens composing the CREGs of previously nonreactive cells were represented. It was found that: (a) In all patients, the detected AM had an HLA class I specificity broader than lhat of the nonreactive cells. (b) Both the detected AM and the ciass I HLA specificities of the nonreactive cells were members of the same CREGs, which were the same with the CREGs of the class I HLA antigens of the corresponding patients. It is concluded that the determination of the AM in HSP can be easily and rapidly obtained by defining their CREGs. Furthermore, this approach can increase the number of the acceptable HLA-phenotypes by the HSP, resulting in a significant augmentation of the polential donor pool who could be suitable for their transplantation.
Since anti-HLA Abs were found to be strongly associated with long kidneytransplantsurvival,we investigatedfor the presenceof anti.donor
19A
19A
Abs by flowcytometry20 livertransplantpatients(LTx pts)usingdonorcells, as targets. Serawere monthly collectedduringthe 1st yearpost LTx and' were studied for the presence of anti-donorAbs (isotype IgG, 19M, IgA).' 4120 L Tx pts with clinical symptoms and histopathologicalevidence of rejectionrequiredantirejectiontreatment. Consideringthetime ofappearance ofthese Abs, we distinguished: Group A: 6/20 LTx pts had anti-donor Abs pre LTx; Group B: 9/20 LTx pts didn't develop anti-donorAbs; Group C: 5/20 LTx pts developed anti-donorAbs only after LTx. Correllatingthese data with graft outcomethe rejectionepisodes occuredin, 2/6 LTx pts ofGroup A, 2/9 LTx ofGroup Band 0/5 LTx pts ofGroup C. lOur dara suggest apossibleenhancing effect of IgA anti-donorAbs also in iLTx pts and this fact may be explaned by differenthypotheses(blockingof IIgG, or complement-mediatedfunctions,or T cells attachmentto graft, etc.). The lack ofrejectionin LTx pts who develop these Abs suggest animmune activationpathwaydistinctfromthat responsibleof graft rejection.
19A
19A
19A
Abstracts
P727
ELISA DETECTION OF SPECIFIC ANTIBODY TO HLA CLASS I ANTIGENS IN TRANSPLANT PATIENTS WITH PANEL REACTIVE ANTIBODY (PRA) BY A LEUKOCYTE ANTIGEN TRAY (LAn
GuixiWang.ChristineTarsitani,Steve Takemura,Rui Pei, An-NaLiu, Jar-HowLee OneLambda,Inc. Los Angeles,CA., U.S.A. A micro-ELISAwas performedon a panelofClass I antigenspurified DIRECTLY bound onTERASAKI trays. from 42 selectedEBV cell lines, and Specificitywas confirmed byreactivitywitha setof84 IgG allosera from commerciallyavailableHLA Class I typingtrays. Parallel results were (LCT) and LAT. All 52 HLA specificities obtained bylymphocytotoxicity L AT. Out of3,528 rxns, 97%ofthe (18 A- and 35 B- locus) were called by TP (374/385) were recognized. 81%ofthe tail reactions (440/540) were cross-reacting groups (CREGs). The optimumtiter was five totwenty-fold higher in theLAT, Sera from 300 transplant patients fromclinicalcenters 3 were tested by LAT and by LCT, and the%PRA was compared in the twoassays. A correlationcoefficientofr = 0.8 was obtained between theL AT and the non-Hl.A standardcytotoxicitytest. Certain sera could be shown to contain antibody(LCT+, LAT-) and others noncomplement-binding anti -HLA antibody(LAT+, LCT-) when adsorbed with platelets and retested in the appropriate assay.Antigenspecificity,when present, could clearly be demonstrated by theL AT assay.
P729
lRIPLE FLUORESCENCE fLOW CYTOMETRY CROSSMATCH (FCXM3) FOR THE SIMULTANEOUSIDENTIF1CATIONOFANTI-CD3ANDANTI-CDl9ANTIBODIES Puglisi G, *MascarettiL, Sioh V, *ScalamognaM, SircbiaG Centro Trasfusionalee di Immunologiadei Trapianti, *Servizio Autonomo Prelievo di Tessuti,OspedaleMaggiorePoliclinico, via F. Sforza35,20122 Milano, Italy The flowcytometrycrossmatchis moresensitivethan thecomplementdependent assay thoughsome doubtsstill persist on the clinical relevanceof the identifiedantibodiesin organtransplantation,Ourreference techniqueforeseesa doublefluorescenceassay wbich identifies anti-CD3 and anti-CD19 antibodiesseparately.In this studyweinvestigatedthe feasabilityof using 3fluorochromes(fluoresceinFlTC, pbycoerythrinPE and peridinin cbloropbyll protein PerCP) for thesimultaneousdetectionof CD3 andCDI9antibodiesby comparing our standard technique with the FCXM3 which was performed as follows: 300,000lympbocyteswereincubatedfor 30' at 22" C with30~1 of serumsample, washed twice with PBS with 0.1% sodium azide and 1% FCS then incubated with 21l\J.1 of adequatelydiluted anti-humanIgG-FITC F(ab'),(Tago) for 15'a14" C in the dark, washed with PBSand finallyincubatedwith 5~1 of anti-CD3-PeICP and5111 of anti-CDI9-PE at 4"C in thedarkfor 20', Analysiswasperformedwith a FACScan(BectonDickinson) daily monitoredwith Calibrite beads; electroniccompensationwasused to correctoverlapof the 3 fluorochromes'emission spectra. 50 sera of patients onthe waiting list for kidney usnsplantationand adequatecontrols werestudied.For CD3 antibodies,concordancyrate was 100% since43samples werenegativeand 7 positive with bothmethods.For CDI9 antibodiesconcordancyrate was94% since for 3samplesthe FCXM' was negativeand standard was positive.In conclusion, the FCXM3 provedto be a reliable technique which allows to save time and reagents (especially sera and cells). The simultaneous identification of CD3 and CD19 antibodiesis more informative on the specificity of the detectedantibodies;an antibodywhicb reacts with both CD3 and CD19 is more probably anti-HLA than one whichreacts only with one lymphocyte subset. In the ligbt ofthese resultswe have adoptedtheFCXM' as thestandardoperatingprocedurein our laboratory.
P731
P728
PHOTOMETRIC DETECTION OF ANTI-HLA ANTIBODIES BY AN ENZYMELINKED IMMUNE-PHAGOCYTOSI5-INHIBITION-TEST (IPI) U. Cassens, H.S.P Garritsen,R. Kelsch, W. Sibrowski. Department of Transfusion Medicine, Muenster, Germany Anti-HLA antibodiesw~h HLA A, B. Cw and DR specificity are detectable by the immune phagocytosis inhibnion assay (IPI), which has been described as a sensitive and specific functional test [Neppert et al. Vox Sang. 1986). We performed a photometric evaluation of immune phagocytosis and compared this test with the microscopic IPI and the Iymphocytotoxicily test (LCT). Monocytes were purified from mononuclear leukocytes by adhesion technique and incubated with HLA antibody sera in microtitre plates, After incubation with anti-D RBC, phagocytosed erythrocytes were determined photomellically(haemoglobincatalyzed peroxidase-reaction). Autologous sera and 8 specific antibody sera were tested against 10 HLA-typed monocyte panels (322 crossmatches). Optical denshy of the negative control (pre tested AB serum) was defined 100%. The photomellic IPI was defined.pos~ive· with a percentage of negative control (PNC) < 30%. Results of photometric and microscopic IPI showed a good correlation (","0.89). All autologous sera yielded distinct negative results(mean PNC 91%). Antibody sera against corresponding antigens showed a mean PNC of 16% vs. 85% against non corresponding antigens (p<0.001). Two monoclonal antibodies against constant HLA-regions showed~ive results in all LCT and microscopic / photometric IPI tests. First tests with diluted, LCT-positive patient sera showed a distinct higher sen~iv~y olthe photometric IPI in comparison to the LCT. We conclude that the photometric IPI test represents a specific, sensitive and convenient method to detect cytotoxic as well as non-cytotoxic anti-HLA antibodies.
P730
EVALUATION OF A SIMPLIFiED ANTI-HUMANGLOBULIN AUGMENTED CYTOTOXICITY TECHNIQUE
A. Papassavas,' A. Germenis,' M. Spyropoulou-Vlachou,' A. Kostakis,' C. Stavropoulos-Giokas' 'Department of Immunology and National Tissue Typing Center, General Hospital of Athens "G. Gennimatas", and 'Transplant Unit, "Laikcn'' Hospital, Athens, Greece To simplify the cumbersome but extremely useful aati-humao globulin aug1994, 40 mented cytotoxicity technique (AHG-CDC) Steen et [.'1umImmuno! al (Suppll):136]proposed a modification consisting of the use of AHG diluted 1:50 in the complement To evaluate this modified AHG-CDC (m'AHG-CDC), we examined 200 sera samples obtained from 50 highly sensitized patients. In all samples, the under evaluation technique was performed along with both the complement-dependent cytotoxicity (CDC) and the conventional AHG-CDC. The same cell panel from 60 HLA-typed donors was used In all cases. The mean (% PRA) detected by m-AHG-CDC and percentage of panel reactive antibodies AHG-CDC was found exactly the same (83.34±8.07 a.id 83.48±8.07, respec(p
P732
PREDICTION OF ACCEPTABLE MISMATCHES BASED ON THE DETECTION OF PATIENTS' CREGs
IgA ANTI-DONORANTIBODIESIN LIVERTRANSPLANTATION
A. Kostakis,' A. Papassavas,' A. Germenis,' M. Spyropoulou-Vlachou,'
'Piazza A., Torlone N. Valeri M., Poggi E., Monaco 1., Romagnoli Tisone G., AdornoD., Casciani C.U.
C. stavrcpoutos-otckas'
J.,
'Department of Immunology and National Tissue Typing Center, General Hospital of Athens "G. Gennimatas", and 'Transplant Unit, "Laikon" Hospital, Athens, Greece
lstituto C.N.R. Tipizzazione Tissutale, L'Aquila and Clinica Chirurgica, Universit:l. Tor Vergata, Rome, Italy
This study was scheduled to search the possibility of the acceptable HLA-A and -B mismatches (AM) to be predicted on the basis of their CREGs in highly sensitized patients (HSP). The anti-human globulin augmented cytotoxicity technique, using a screening cell panel from 60 HLA-typed donors, was performed for the determination of the HLA alloantibodies in 50 HSP (PRA>60%). The defined HLA class I specificities of the alloantibodies, the specificities of the nonreactive cells as well as the class I HLA antigens of the patients were assigned to CREGs. Thereafter, the AM were detected using a cell panel in which almost all the HLA antigens composing the CREGs of previously nonreactive cells were represented. It was found that: (a) In all patients, the detected AM had an HLA class I specificity broader than lhat of the nonreactive cells. (b) Both the detected AM and the ciass I HLA specificities of the nonreactive cells were members of the same CREGs, which were the same with the CREGs of the class I HLA antigens of the corresponding patients. It is concluded that the determination of the AM in HSP can be easily and rapidly obtained by defining their CREGs. Furthermore, this approach can increase the number of the acceptable HLA-phenotypes by the HSP, resulting in a significant augmentation of the polential donor pool who could be suitable for their transplantation.
Since anti-HLA Abs were found to be strongly associated with long kidneytransplantsurvival,we investigatedfor the presenceof anti.donor
19A
19A
Abs by flowcytometry20 livertransplantpatients(LTx pts)usingdonorcells, as targets. Serawere monthly collectedduringthe 1st yearpost LTx and' were studied for the presence of anti-donorAbs (isotype IgG, 19M, IgA).' 4120 L Tx pts with clinical symptoms and histopathologicalevidence of rejectionrequiredantirejectiontreatment. Consideringthetime ofappearance ofthese Abs, we distinguished: Group A: 6/20 LTx pts had anti-donor Abs pre LTx; Group B: 9/20 LTx pts didn't develop anti-donorAbs; Group C: 5/20 LTx pts developed anti-donorAbs only after LTx. Correllatingthese data with graft outcomethe rejectionepisodes occuredin, 2/6 LTx pts ofGroup A, 2/9 LTx ofGroup Band 0/5 LTx pts ofGroup C. lOur dara suggest apossibleenhancing effect of IgA anti-donorAbs also in iLTx pts and this fact may be explaned by differenthypotheses(blockingof IIgG, or complement-mediatedfunctions,or T cells attachmentto graft, etc.). The lack ofrejectionin LTx pts who develop these Abs suggest animmune activationpathwaydistinctfromthat responsibleof graft rejection.
19A
19A
19A