| Abstract|| |
To determine the specificity of flow cytometry based assay for HLA antibody screening prior to transplantation, we evaluated 11 positive sera that were tested by both Flow Panel Reactive Assay (PRA) TM class I and class II specific beads after initial FlowPRA TM . HLA specificity was compared with previous data from the complement dependent cytotoxicity (CDC) testing of these samples. We found that five samples (22%) were more HLA class II specific (DR- or DQ-) by FlowPRA TM than CDC. One sample, negative by FlowPRA TM screening, was shown to be HLA class I and class II reactive by FlowPRA TM -specific test. Class I specificity was only defined by CDC in this sample. Two other samples, shown to be HLA class I and class II reactive by FlowPRA TM screening and FlowPRA TM -specific test, were found to be HLA class I reactive only in the first sample and HLA class II reactive only in the second sample by CDC. Our study suggests that FlowPRA TM -specific test has higher specificity and sensitivity than CDC in identification of HLA class II specificity than CDC. Nevertheless, FlowPRA TM -specific test failed to identify precisely the HLA specificity in samples with broad-spectrum specificity, such as those that have HLA specific antibodies directed against large number of shared epitopes. A software protocol for specificity analysis might help overcome this problem. Also, studies involving larger number of samples are required to validate our findings.
Keywords: HLA, Antibodies, Flow cytometry, Transplantation, Complement, Cytotoxicity.
|How to cite this article:|
Kuda E, Al-Wahadneh A. Comparison of Flow Panel Reactive Assay (PRA) TM Specific Test with Complement Dependent Cytotoxicity (CDC) to Define the HLA Antibodies Specificity: A Preliminary Study. Saudi J Kidney Dis Transpl 2001;12:21-7
|How to cite this URL:|
Kuda E, Al-Wahadneh A. Comparison of Flow Panel Reactive Assay (PRA) TM Specific Test with Complement Dependent Cytotoxicity (CDC) to Define the HLA Antibodies Specificity: A Preliminary Study. Saudi J Kidney Dis Transpl [serial online] 2001 [cited 2020 May 29];12:21-7. Available from: http://www.sjkdt.org/text.asp?2001/12/1/21/33881
| Introduction|| |
Preformed HLA specific antibodies against donor antigens in patients awaiting renal transplant contraindicate transplantation.  It is not only the sensitivity, but also the specificity of these antibodies that is important. Transplantation must not be denied on the basis of the mere presence of nonspecific antibodies, since they may not affect transplant outcome. 
It is generally accepted for renal and thoracic organ transplantation that IgG antibodies directed against donor HLA-A or -B specificity and present at the time of transplant would cause hyperacute rejection.  Although less data are available, donor HLA-DR specific antibodies present in the recipient may also result in rejection.  The outcome differs between individuals and organs depending on antibody titer and the level of expression of HLA-DR. Thus, it may appear only after transplantation as a result of ischemic or immunological injury. 
There is little information on the role of antibodies to HLA-Cw or HLA-DQ specificities, in transplant failure. Renal transplant failure in a patient who had antibodies to HLA-Cw5, which was present in his donor, has been reported.  If a patient has antibodies to HLA-Cw or HLA-DQ, many centers consider it advisable to avoid organs from donors with these specificities, as they would be when the patient has antibodies to HLA-A, B and DR. 
If sera are carefully screened during patients' work-up for transplantation, then HLA specific antibodies can be defined, and consequently their crossmatch against a particular donor of known HLA type can be predicted.  Screening for HLA specific antibodies has traditionally been performed by the complement dependent cytotoxicity (CDC) assay.  More recently, Enzyme Linked Immunosorbent Assay (ELISA) and Flow Cytometry based assays have been developed for the detection and definition of alloantibodies. It is argued that laboratories which use flow cytometry to crossmatch patients prior to transplantation, should also perform HLA antibody screening using the flow cytometry based assay. 
In this report, we attempt to determine the specificity of the flow cytometry based assay for HLA antibody screening prior to transplantation. HLA specificity was compared with previous data from CDC testing of these samples.
| Material and Methods|| |
We evaluated 11 positive sera that were tested using Flow Panel Reactive Assay (PRA) TM class I and class II beads (a flow cytometry based screening test can screen alloantibodies using specific microbeads and differentiate between class I and class II HLA-specific antibodies). The HLA class I positive sera were tested for specificity using FlowPRA TM class I specificity beads, while the HLA class II positive sera were tested for specificity using FlowPRA TM class II specificity beads. HLA specificity was compared with the previous results of the CDC adopted with kind permission from Robson et al, Transplantation Laboratory, Manchester Royal Infirmary Hospital, Manchester U.K.
Complement-dependent cytotoxicity (CDC)
This is currently the most widely used method for the detection of antibodies to HLA. It enables the detection and definition of complement-fixing IgG and also IgM antibodies, which may be directed against HLA or non-HLA targets, including autoantibodies. This test has some disadvantages. It requires large panels of viable lymphocytes in order to cover all HLA specificities, detects only complement fixing antibodies, detects autoantibodies that are proved to be irrelevant to transplant outcome, and its results are subjectively read. 
FlowPRA TM -specific HLA class I and class II tests
These tests consist of a panel of 32 FlowPRA TM -specific class I and class II beads; each is coated with different purified class I or class II antigens. The 32 different beads in each test are divided equally into four HLA groups. A serum sample must be tested separately with each group of beads to complete the 32 bead panel analysis for the HLA class I or class II antibodies in that serum. In the FlowPRA TM -specific testing procedure, each group of beads is incubated separately with the test serum, followed by staining with a fluorescene isothiocyanate (FITC) conjugated anti-human IgG antibody. Positive reaction shows fluorescene intensity (FL1) channel shift in contrast to a negative reaction. Each of the eight beads shows a different FL2 channel shift. By analyzing the FL1 versus FL2 dot plots, one can determine HLA specificity, which has both high specificity and reproducibility. It is a quick test and highly accurate, shows no false positive reactions due to non-HLA antibodies, needs no frozen panels to maintain and shows comprehensive analysis (i.e. it determines PRA and identifies HLA class I and class II specificities). ,,
The FlowPRATM -specific beads were gently vortexed prior to use. Five a l of each FlowPRA TM -specific bead groups (one through four for FlowPRA TM -specific class I or class II) was incubated separately with 20 Q l test serum, each in separate 5-ml test tubes at 22 0 C for 30 minutes. Ten times (X) wash buffer was diluted in distilled water to make a 1X solution. One ml of the washed buffer 1X was added to each tube of bead/serum solution, vortexed and centrifuged at 3000 rpm for 10 minutes and washed as described earlier. One μl per test of 100X FITC-conjugated goat anti-human IgG (Fcγ) was diluted with wash buffer to make a 1X solution. We added 100 Q l 1X FITC-conjugated goat anti-human IgG (Fcγ) to each tube of beads and incubated them for 30 minutes in the dark at 22 0 C, then we repeated the wash step. Afterwards, we added 0.5 ml 1X of wash buffer to each test tube. Samples were placed on MCL carousels in the following order: four negative controls followed by four positive controls, then the patients' sera. Subsequently, samples were run on EPICS XL a flow cytometer (Beckman Coulter). Using forward (FS) and side (SS) scatter plots, a gate was placed around the bead population [Figure - 1]A. FL2 histogram was obtained showing fluorescent intensity of different beads regions [Figure - 1]B. Using the positive and negative control sera, a cutoff line on FL1 vs. FL2 dot blot for each group of beads was set [Figure - 1]C. The populations located to the right of the gate were considered positive reactions [Figure - 1]D, and the populations located to the left of the gate were considered negative reactions [Figure - 1]E. HLA specificity was determined by entering the median % PRA into a FlowPRA-specific analysis sheet and the reaction pattern was analyzed.
| Results|| |
HLA class II specificity (DR- or DQ-) was defined in five (45%) samples (patients 5,79,11 in [Table - 1]) by FlowPRA TM -specific test but not by CDC [Table - 1]. Also, HLA class I and class II antigens were better defined in patients 11 and 7 in [Table - 1] by FlowPRA TM screening and FlowPRA TM specific tests than CDC; the two patients were found to be only HLA class I and HLA class II reactive respectively, by CDC.
However, CDC was more specific in defining HLA class I antigens than FlowPRA TMspecific test in some patients; thus patient 9 in [Table - 1] was proven to be HLA class I and class II negative by FlowPRA TM screening test and positive by FlowPRA TM -specific test, while HLA class I specificity was well defined only by CDC. In this patient, there were HLA specific antibodies directed against a large number of shared epitopes, so it was difficult to identify precise HLA specifity by FlowPRA TM screening and FlowPRA TM -specific tests.
| Discussion|| |
The FlowPRA TM -specific test is a recently introduced method. This is the first study using this technique to be carried out in our laboratory. All 11 sera were run over four hours. This short period is comparable to the CDC test (personal communication). FlowPRA TM -specific test defined more specificity than CDC. HLA class II (DR- or DQ-) specific antibodies were defined clearly in five samples by FlowPRA TMspecific test, while their presence was highly under-appreciated by CDC due to the presence of HLA class I specific antibodies. We found that FlowPRA TM -specific test defined more specificity and showed higher sensitivity than CDC in identification of HLA class II specificity. The presence of HLA class I specific antibodies rendered the CDC method unable to identify HLA class II specific antibodies in 45% of the patients. Furthermore, it took shorter time to run the test protocol of the FlowPRA TM -specific test than the CDC test. It was only a fourstep reaction in contrast to the time consuming CDC method.
Nevertheless, FlowPRA TM -specific test failed to identify precisely, the HLA specificity in blood samples with broad-spectrum specificity. We found that in patient 9 in [Table - 1], where HLA specific antibodies were directed against a large number of shared epitopes, it was difficult to identify the precise HLA specifity. This specificity was defined clearly by the CDC method with special pre-treatment of the sample, such as dilution. We believe that this may be considered a disadvantage in the application of FlowPRA TM -specific test. We predict that a software protocol for specificity analysis might overcome this problem.
Finally, one should realize that determination of percentage PRA is considered as a preliminary screening diagnostic test and should not be the sole basis for a clinical decision affecting the patient's treatment. A final crossmatch test is routinely required prior to transplant.
We conclude that FlowPRA TM -specific test has higher specificity and sensitivity than CDC in identification of HLA class II specificity than CDC. However, we still need more studies to define the sesitivity and specificity of this tool in defining the HLA antbodies in patients who are candidates for transplantation.
| Acknowledgement|| |
The authors would like to thank Dr. Susan Martin and Ms. Amanda Robson, in the Transplantation Laboratory in Manchester Royal Infirmary, UK where the study was established for their kind and highly appreciated help in the supervision of the laboratory work and their critical review of the manuscript.
| References|| |
|1.||Worthington JE, Langton A, Liggett H, Robson AJ, Martin S. A novel strategy for the detection and definition of HLAspecific antibodies in patients awaiting renal transplantation. Transpl Int 1998; 11Suppl 1:S372-6. |
|2.||Martin S, Harmer A. Cross-matching by lymphocytotoxicity and flow cytometry. Histocompatibility testing (eds): Bidwell J and Navarrete C. Imperial college press, UK 1998. (in press). |
|3.||Mohanakumar T, Rhodes C, MendezPicon G, Goldman M, Moncure C, Lee H. Renal allograft rejection associated with presensitization to HLA-DR antigens. Transplantation 1981;31:93-5. [PUBMED] |
|4.||Chapman JR, Taylor C, Ting A, Morris PJ. Hyperacute rejection of a renal allograft in the presence of anti HLA-Cw5 antibody. Transplantation 1986;42:91-3. [PUBMED] |
|5.||Harmer AW, Sutton M, Bayne A, Vaughan RW, Welsh KI. A highly sensitive, rapid screening method for the detection of antibodies directed against HLA class I and class II antigens. Transpl Int 1993;6:277-80. [PUBMED] |
|6.||Robson AJ, Langton JE, Worthington JE, Martin S. A comparison of flow cytometry screening methods. Eur J Immunogenetics 1999;26:43-80. |
|7.||Ten-Hoor GM, Coopmans M, Allebes WA. Specificity and Ig class of preformed allo-antibodies causing a positive crossmatch in renal transplantation. The implications for graft survival. Transplantation 1993; 56(2):298-304. |
|8.||Bray RA, Chapman PT, Sinclair DA, et al. The flow of cytometric PRA. Evaluation of antibody reactivity and specificity using cell pools based on CREGs. ASHI abstracts 1998. |
|9.||Bray RA, Cook DJ, Gebel HM. Flow cyto-metric detection of HLA alloantibodies using classI coated beads. ASHI abstract 1997. |
|10.||Murphy CL, Blair B, Gonzalez C, et al. Identification of HLA antibodies in renal transplant recipient using class I and class II flow PRA beads. ASHI abstracts 1998. |
Department of Pediatrics, King Hussein Medical Center, P.O. Box 540554, Amman
[Figure - 1]
[Table - 1]