The Convenience of Flow Cytometry for HLA-B*5701 Screening to Prevent Abacavir Hypersensitivity Reactions

Abstract

To the Editor: Abacavir (ABC) is a nucleoside reverse transcriptase inhibitor that is currently recommended for antiretroviral drug combinations.1 Hypersensitivity reactions to ABC (AHR) may arise during the first weeks of treatment, requiring an immediate and permanent discontinuation of the drug.2 A strong association between AHR and major histocompatibility complex (HLA) class I allele HLA-B*5701 has been established by several independent studies.3-7 Moreover, a double-blind, prospective, randomized study demonstrated that pharmacogenetic screening of HLA-B*5701 reduced the risk of AHR.8 On the basis of these observations, HLA-B*5701 screening is now recommended by official guidelines of antiretroviral treatment before ABC prescription.1 However, HLA-B*5701 screening is based on HLA typing assays such as DNA sequence-based typing or sequence-specific oligonucleotide (SSO) probe method with additional DNA sequencing for patients in whom the probe results were positive.7,8 These approaches are time consuming and expensive and may not be widely available. Moreover, according to recent pharmacoeconomical estimations, HLA-B*5701 screening is not cost effective.9 An approach to reduce costs, based on the screening of HIV reverse transcriptase for an escape mutation at reverse transcriptase codon 245 reported to be associated with HLA-B*5701, was suggested by some authors,10 but others found that this mutation did not accurately predict the presence of HLA-B*5701 and cannot be used for this purpose.11 Martin et al12 suggested that a rapid flow cytometry test could be employed for HLA-B*5701 screening strategies. This was based on the use of a monoclonal antibody (MAb) specific for HLA-B17 (a serologic antigen that can be split into HLA-B*5701-14 and -B58 alleles by molecular biology testing). After analysis of 84 HIV+ patients previously typed for B*5701 by sequence-based test, the authors suggested that flow cytometry could provide a more rapid and cost-effective method. Unfortunately, to our knowledge, no further studies on this strategy have been performed. We have evaluated the feasibility of such an approach, based on screening for HLA-B17 by flow cytometry, followed by confirmation of suspected positive or indeterminate cases through molecular biology techniques. With this objective in mind, samples from HIV-positive patients were evaluated blindly by the 2 methods. The costs of this approach were also evaluated and compared with that of a standard molecular HLA typing. In this study, 189 consecutive HIV-positive individuals from the Infectious Disease Department in Brescia who were eligible for ABC treatment were evaluated at our laboratory in Brescia both by flow cytometry using MAb specific for HLA-B17 and by molecular biology techniques. These analyses were performed blinded as to each other. The research was conducted in accordance with the Declaration of Helsinki. Flow cytometry analysis was performed as described by Martin et al,12 with slight modifications. Briefly, 100 μL of EDTA-anticoagulated peripheral blood were lysed and fixed with Immunoprep reagent system (Beckman Coulter, Fullerton, CA) and washed and incubated with 5 μL of a 1:10 diluted commercially available anti-B17 immunoglobulin (IgM) MAb (One Lambda Inc, Canoga Park, CA) or with an IgM irrelevant MAb (Beckman Coulter) and with 10 μL of a fluorescein isothiocyanate-conjugated IgG CD3 MAb (Beckman Coulter). After 30 minutes, cells were washed and incubated with 10 μL of PE-conjugated goat anti-mouse IgM polyclonal antibody (Beckman Coulter); after 30 minutes, cells were washed and analyzed with a FC500 flow cytometer (Beckman Coulter). CD3+ cells were gated, and PE fluorescence was evaluated, using the irrelevant IgM antibody to set a threshold excluding nonspecific fluorescence. Samples with fluorescence observed in >10% CD3+ cells were considered suspected positive. Occasionally, samples with a high aspecific binding of the control antibody were also considered indeterminate. HLA molecular typing was performed by polymerase chain reaction (PCR)-SSO reverse13 to obtain information at allele group HLA-B and by PCR sequence-specific primer (SSP)14 to define HLA-B*57 allele. Genomic DNA was isolated from peripheral blood by Bioborot EZ-1 workstation that performs fully automated nucleic acid purification using magnetic particles (Qiagen, Valencia, CA). PCR-SSO reverse is based on the reverse hybridization of DNA, obtained after amplification of the second to the fourth exon of HLA-B locus, with oligonucleotide probes immobilized on strips (INNO-LiPA HLA-B, Innogenetics, Gent, Belgium). PCR-SSP was performed using specific primer set that contains 5′ and 3′ primers for identifying the HLA-B*5701 to -B*5714 alleles (Olerup SSP HLA-B*57, GenoVision, West Chester, PA). To evaluate the costs associated with 2 different strategies (ie, screening with flow cytometry followed by confirmation by molecular biology in case of positive of indeterminate results by flow cytometry versus molecular biology in all cases), the official fees of the Regione Lombardia, Italy, were applied as follows: 35.12 EURO per test for flow cytometry vs. 64.92 EURO per test for DNA extraction + 145.68 EURO per test for genomic HLA-B typing. Among 189 samples, 168 (89%) resulted negative and 21 were positive or indeterminate by flow cytometry. All samples were blindly evaluated by molecular biology techniques. In all cases, classified as negative by flow cytometry, the absence of HLA-B*5701 antigen was confirmed. Ten of the 21 samples considered positive or indeterminate by flow cytometry were characterized as HLA-B*5701 positive by PCR-SSP, whereas HLA-B*5703 and HLA-B58 were found in 2 and 5 cases, respectively. Applying the official fees of the Regione Lombardia, the cost for 189 genomic HLA sequences would be 39,803.40 EURO, whereas the cost for an alternative strategy, based on screening of all samples by flow cytometry and confirmation by molecular biology only in the 21 suspected or indeterminate cases, would be 6637.68 + 4422.60 = 11,060.28 EURO (ie, cost reduction by 72.2%). In this study, flow cytometry test for HLA-B17 was used as a screening for HLA-B*5701. Because our main concern was false negative results that would expose patients to the risk of inappropriate prescription of ABC, the test was designed to be as more sensitive as possible. Therefore, a very conservative approach was adopted, suggesting confirmation in all cases in which flow cytometry was not indubitably negative. Even so, among 189 patients evaluated by flow cytometry testing, indeterminate or positive results requiring further analysis by molecular biology test were found only in 21 cases (11%). Because all samples were blindly evaluated by molecular techniques, and no false negative result for HLA-B*5701 was observed at flow cytometry, we are confident that no further more expensive and labour intensive molecular analyses are required when negative results are obtained by flow cytometry. In our experience, the described approach greatly accelerated pharmacogenomic screening and reduced its cost. Indeed, 89% of patients would receive the results of the test within 24 hours (whereas time to complete the molecular test in our laboratory is 15 days). Moreover, direct costs would be reduced by more than 70%. It should also be considered that flow cytometry laboratories are more widely available than molecular biology ones, and this will facilitate the application of the pharmacogenomic screening. Paolo Airo, MD* Mirko Scarsi, MSc* Alberto Malagoli, MSc* Graziella Carella, MSc* Ilaria Izzo, MD† Giampiero Carosi, MD† Carlo Torti, MD† *Rheumatology and Clinical Immunology Service, Spedali Civili of Brescia Brescia, Italy †Infectious Disease Department Spedali Civili and University of Brescia Brescia, Italy