Optimal primer selection for clonality assessment by polymerase chain reaction analysis. III. Intermediate and high-grade B-cell neoplasms.

Previous studies have reported that low-grade B-cell lymphoproliferative disorders have variable B-cell monoclonality detection rates by polymerase chain reaction (PCR) analysis. For instance, monoclonal B-cell populations from chronic lymphocytic leukemia/small lymphocytic leukemia and mantle cell lymphoma are most often readily amplified with a single primer pair, whereas follicular lymphomas and plasma cell neoplasms require alternative strategies to approach these higher diagnostic sensitivity standards. Because most published reports have not focused on the variation in PCR B-cell monoclonality detection among subtypes of intermediate and high-grade B-cell neoplasms, additional information is necessary to determine primer selection strategies and identify problematic tumor subtypes within this group. The current investigation, the third part in a series, was aimed at documenting the efficiency of B-cell monoclonality detection by PCR in 71 aggressive B-cell neoplasms of various types using a comprehensive approach. A predetermined panel of primer sets was used in an algorithmic fashion. Specifically, all samples were analyzed with the standard VH-FRIII/JH assay previously shown to have the highest efficiency of monoclonality detection within low-grade B-cell lymphoproliferative disorders. Negative samples were further evaluated with primer sets in the following order until a positive result was observed, or all primer sets were used: (1) bcl-2/JH, (2) VH-FRI family specific/JH, and (3) VH-FRI consensus/JH. Forty-three (61%) of the 71 B-cell neoplasms evaluated with VH-FRIII/JH showed monoclonal B-cell populations. Sequential use of the three reserve primer sets in samples negative with this initial primer pair resulted in an overall improvement in PCR detection from 61% to 82% (58 of 71 specimens) (P < .001). The VH-FRI family specific assay identified B-cell monoclonality in 11 (73%) of these 15 specimens and was the most productive reserve primer set. Individual categories exhibited the following initial (I) and final (F) PCR detection rates: acute lymphoblastic leukemia/lymphoblastic lymphoma, 11 specimens (I = 91% to F = 91%); small noncleaved cell lymphoma, 14 specimens (I = 79% to F = 86% [P > .25]); diffuse large cell lymphoma, 33 specimens (I = 52% to F= 85% [P < .005]) and large cell, immunoblastic lymphoma, 13 specimens (I = 38% to F = 62% [P < .01]). The authors have shown that comprehensive PCR analysis is capable of detecting B-cell monoclonality in a significant proportion of samples from each subtype of intermediate and high-grade B-cell neoplasm. The VH-FRIII/JH assay was an adequate initial primer set, but required augmentation with the reserve PCR assays to attenuate the false negative rate and improve diagnostic sensitivity. The B-cell clonality PCR assay is optimally used as a screening tool and when used in this fashion, the more laborious and time-consuming restriction fragment-Southern blot hybridization (RF-SBH) method for IgH gene rearrangement detection may be limited to a relatively small proportion of PCR-negative aggressive B-cell neoplasms.

Flow cytometric analysis of residual white blood cell concentration and platelet activation antigens in double filtered platelet concentrates.

Reduction of contaminating leukocytes in platelet products by filtration has been shown to decrease the incidence of human leukocyte antigen (HLA) alloimmunization. Nonetheless, prevention is not complete when using current techniques, and a significant number of patients continue to exhibit clinical refractoriness and to produce alloantibodies. Interest in preventing HLA alloimmunization and other complications of white blood cell (WBC) contamination of transfused cellular products has resulted in ongoing efforts to increase the efficiency of leukodepletion filters. As the efficiency of these filters increases, more accurate and precise methods for counting extremely low numbers of WBCs must be instituted to ensure quality control. We have validated a simple, rapid flow cytometric assay for quantitating low numbers of WBCs in platelet products. The assay is sensitive to a lower limit of 0.1 WBC/microliter without concentration of the platelet product sample and has an excellent correlation (R2 = 1.00) between calculated and expected WBC concentration over a range of 0.1 to 100.0 WBC/microliter. (R2 values over the concentration ranges of 0.1 to 1.0 WBC/microliter and 1.0 to 10.0 WBC/microliter were 0.988 and 0.996, respectively.) The intraassay coefficients of variation at WBC concentrations of 50.4/microliter, 0.9/microliter, and 0.1/microliter were 4%, 8%, and 18%, respectively. The flow cytometric counting technique was applied, in concert with a Nageotte chamber manual counting method, to the enumeration of residual WBCs in 20 apheresis and random donor platelet concentrates filtered through two leukodepletion filters sterile docked in series. A greater than four log10 WBC reduction capability was demonstrated when utilizing this double filtration procedure, and its clinical applicability is underscored by data that showed no statistically significant change in expression of activation-specific platelet antigens before versus after filtration.