225-P A reproducible false positive screen for HLA antibodies by ELISA

S156

Abstracts

224-P

A RARE DRB1 ALLELE IDENTIFIED IN A CORD BLOOD UNIT AND THE MOTHER. Lucie Richard, Élise Trudel, Diane Fournier, Diane Roy, Marie-Claire Chevrier. Stem Cell and Reference Laboratory, HemaQuebec, Saint-Laurent, QC, Canada. Aim: In 2004 Hema-Quebec started a public cord blood bank with the goal of increasing its proportion of ethnic diversity. In 2011 the ethnic and mixed (multiple ethnicity) cord blood units of the Hema-Quebec cord blood bank (over 5000 units) reached more than 19% of the bank’s inventory, which represents more than twice the ethnicity of the stem cell donor registry. In parallel, a higher number of non frequent alleles and haplotypes can be observed in the ethnic and mixed cord blood units. We report here the identification of a rare DRB1 allele in both the cord blood and the mother from a Pakistan origin. Methods: The HLA A, B, C and DRB1 were first analysed by rSSO low resolution and then sequenced by SBT using PCR reactions of exon 2 and 3 for class I and exon 2 for class II. The new DRB1 allele could not be confirmed by SSP since no reactivity pattern was available for this new allele. Results: The HLA DRB1 SBT analysis of the cord blood unit demonstrated a modified sequence of the DRB1*15:02 in exon 2 with one substitution at position 239, replacing a C with a G. This modification was reported and identified in 2010 by the Chinese Marrow Donor Program and the new allele was subsequently named DRB1*15:47. The parents of the child who gave his cord blood were also analysed. The DRB1*15:47 allele was identified in the mother’s haplotype. When analysed by SSP, the mutation could not be seen, both the cord and the mother reacted as the more common DRB1*15:02 and would have been mistyped if not sequenced. Conclusions: The identification of rare alleles such as DRB1*15:47 among the cord blood units showed a good example of the HLA ethnic diversity of the Hema-Quebec cord blood bank. Attempts to HLA type the sibling of the mother are made to further depict the inheritance pattern of this rare allele through the family study.

225-P

A REPRODUCIBLE FALSE POSITIVE SCREEN FOR HLA ANTIBODIES BY ELISA. Benjamin A. Schwarz,1 Todd L. Astor,2 Donna Fitzpatrick,1 Susan L. Saidman.1 1Department of Pathology, Massachusetts General Hospital, Boston, MA, USA; 2Department of Medicine, Massachusetts General Hospital, Boston, MA, USA. Aim: Many laboratories use ELISA to rapidly screen for HLA antibodies. A false positive could delay or preclude transplant. Here we describe a case of a reproducible positive ELISA for HLA antibodies, whereas testing by Luminex gave negative results. Methods: HLA antibodies were assayed using ELISA and Luminex mix, panel, and single antigen beads (One Lambda). Results: A 45 year old woman with pulmonary fibrosis was evaluated for lung transplant. Her serum was negative for HLA antibodies by Luminex mix. Luminex testing 8 months prior showed class I cPRA of 40%, so sample was retested by stat ELISA. HLA class I ELISA was positive (PRA⫽63%) with apparent HLA-B7 and B8-CREG reactivity. Repeat ELISA testing on later specimens gave the same results, but Luminex panel was 0%. Luminex single antigen beads showed 2 weak specificities (HLA-B8 and Cw5). Testing by the vendor produced the same ELISA and Luminex results. The patient was treated with IVIG and rituximab for desensitization and listing was delayed. Class I PRA by ELISA was reduced to 7% over 1 month of treatment, allowing the patient to be listed. To determine why the ELISA and Luminex assays gave discrepant results, ELISA data was reanalyzed and OD values were sorted and graphed. No clear cutoffs differentiated positive and negative wells and values spanned the range from positive to negative controls, suggesting nonspecific reactivity. Testing with a different ELISA kit was consistent with nonspecific reactivity and flow crossmatches with panel cells were negative. Conclusions: In this case, positive ELISA results were reproducible between different sera and different labs, and desensitization was associated with a reduction in PRA. The reason for the ELISA reactivity is unclear, but the nonspecific nature was revealed by reanalyzing the data graphically. We suggest that this is an important way to analyze ELISA data rather than by preset cutoffs alone, and demonstrate the importance of using multiple methods for HLA antibody identification.