Issue link: https://ahint.epubxp.com/i/574474
25 ASHI Quarterly Third Quarter 2015 S C I E N T I F I C C O M M U N I C A T I O N S We have been using the Halifax FCXM protocol for routine clinical testing since May 2011 The most obvious benefit of this protocol is the marked reduction in TAT, especially during deceased donor work ups on-call This allows the clinical team to finalize recipient selection and organize transplant logistics such as patient transport and OR time In situations where prospective crossmatches are required as part of an import donor work-up, faster TAT may translate into reduced cold ischemia time Importantly, we have also noted a reduction in on-call staff fatigue Finally, with the 96-well assay platform, we are not as limited in terms of the numbers of patient sera that can be tested with each donor crossmatch This allows us to use a more robust set of controls, which improves the assessment of assay performance During the optimization phase of the study we identified a number of factors that affect FCXM results The most notable was the effect of the number of target cells per reaction, which was inversely proportional to crossmatch reactivity For example, crossmatches performed with positive control sera against 1 25x10 5 target cells/rxn resulted in MCF shifts that were higher by 50-120 channels compared to crossmatches performed using 1 0x10 6 target cells/rxn Thus, the number of target cells used in a crossmatch assay may have a significant impact on crossmatch interpretation In our experience, the number of cells/rxn used varies from lab to lab In addition, some laboratories use different numbers of target cells in different crossmatches, depending on how many donor cells are available and how many recipient sera need to be tested Such heterogeneity in practice likely contributes to and explains both inter- and intra-laboratory crossmatch result variability Collectively, our study underscores that protocol optimization and standardization leads to improved testing quality This should be the goal and an integral part of any test development and validation An important limiting factor in crossmatch TAT is the cell isolation step Many labs continue to use a density gradient centrifugation method to isolate PBMC, which is labor intensive, time consuming and, depending on the age and quality of the blood sample, not particularly robust or reliable The purity of lymphocytes in enriched PBMC preparations is often poor and highly variable, which may affect crossmatch results In contrast, the lymphocyte isolation using the EasySep TM Direct technique 10 is rapid and consistently yields lymphocyte purity of more than 90% We adopted this method and incorporated it into the Halifaster FCXM protocol The correlation between the Halifax and Halifaster FCXM protocols, based on 101 parallel crossmatches, was excellent (r 2 value of 0 98) with a 97% concordance in crossmatch results The three false-positive reactions (one with Halifax and two with Halifaster protocol) were borderline T+/B- and would not lead to changes in crossmatch interpretation Implementation of the Halifaster protocol has had a very positive impact on our cell isolation and FCXM assay set up time Furthermore, we observed an improvement in events acquisition as well as time and ease of gating and analysis based on lymphocyte enrichment The Halifax protocol has recently been tested in a multicenter evaluation study involving 13 HLA labs in Canada The results showed that the Halifax protocol compares favorably with other methods and improves FCXM results by augmenting the signal:noise ratio and concordance of the results 11 Based on these findings, HLA labs in Canada have validated and adopted the Halifax protocol for routine clinical practice Since then, several labs in the U S , the U K , Brazil, and other countries have either adopted the protocol or are in the process of so doing We predict that broader application of this protocol will improve pre-transplant risk assessment precision, facilitate transplant allocation and improve patient care The authors gratefully acknowledge the technical assistance of the Halifax HLA Laboratory technologists, especially Mr. Geoff Peladeau (MLT), Geoff Adams (MLT), and Mrs. Kelly Heinstein (BSc, MLT, HLA lab supervisor). References 1 Garovoy MR, Rheinschmidt MA, Bigos M, Perkins HA, Colombe, B Flow cytometry analysis: a high technology crossmatch technique facilitating transplantation Transplant Proc. 1983;15:1939–1944 2 Bray RA, Tarsitani C, Gebel HM, Lee J Clinical cytometry and progress in HLA antibody detection Methods Cell Biol. 2011;103:285-310 3 Mahoney RJ, Ault KA, Given SR, et al The flow cytometric crossmatch and early renal transplant loss Transplantation. 1990;49(3):527–535 4 Ogura K, Terasaki PI, Johnson C, et al The significance of a positive flow cytometric crossmatch test in primary renal transplantation Transplantation. 1993;56(2):294–298 5 Karpinski M, Rush D, Jeffery J, et al Flow cytometric crossmatching in primary renal transplant recipients with a negative anti-human globulin enhanced cytotoxicity crossmatch J Am Soc Nephrol. 2001;12(12):2807-2814 6 Hamrick C, Lebeck L Flow cytometry T cell and B cell crossmatching ASHI Laboratory Manual 2000; 4th edition, VI B 4 1:1-5 7 Bray RA, Lebeck LK, Gebel HM The flow cytometric crossmatch Dual-color analysis of T cell and B cell reactivities Transplantation. 1989;48(5):834-840 8 Lobo PI, Spencer CE, Stevenson WC, McCullough C, Pruett TL The use of pronase-digested human leukocytes to improve specificity of the flow cytometric crossmatch Transpl Int. 1995;8(6):472-480 9 Taylor CJ, Kosmoliaptsis V, Sharples LD, et al Ten-year experience of selective omission of the pretransplant crossmatch test in deceased donor kidney transplantation Transplantation. 2010;89(2):185-93 10 EasySep direct human total lymphocyte isolation kit [Product Insert] StemCell Technologies Inc http://www stemcell com/ 11 Liwski R, Pochinco D, Tinckam K, Gebel H, Campbell P, Nickerson P Canada-wide evaluation of rapid optimized flow crossmatch (ROFCXM) protocol [abstract 30-OR] Human Immunol. 2012;73:S26 Corresponding Author Information Robert S Liwski, MD, PhD, FRCPC Medical Director, HLA Typing Laboratory Department of Pathology Dalhousie University 5788 University Avenue Halifax, Nova Scotia, Canada Robert liwski@nshealth ca