Immunophenotype of normal vs. myeloma plasma cells: Toward antibody panel specifications for MRD detection in multiple myeloma.

In recent years, several studies on large series of multiple myeloma (MM) patients have demonstrated the clinical utility of flow cytometry monitoring of minimal residual disease (flow-MRD) in bone marrow (BM), for improved assessment of response to therapy and prognostication. However, disturbing levels of variability exist regarding the specific protocols and antibody panels used in individual laboratories. Overall, consensus exists about the utility of combined assessment of CD38 and CD138 for the identification of BM plasma cells (PC); in contrast, more heterogeneous lists of markers are used to further distinguish between normal/reactive PCs and myeloma PCs in the MRD settings. Among the later markers, CD19, CD45, CD27, and CD81, together with CD56, CD117, CD200, and CD307, have emerged as particularly informative; however, no single marker provides enough specificity for clear discrimination between clonal PCs and normal PCs. Accordingly, multivariate analyses of single PCs from large series of normal/reactive vs. myeloma BM samples have shown that combined assessment of CD138 and CD38, together with CD45, CD19, CD56, CD27, CD81, and CD117 would be ideally suited for MRD monitoring in virtually every MM patient. However, the specific antibody clones, fluorochrome conjugates and sources of the individual markers determines its optimal (vs. suboptimal or poor) performance in an eight-color staining. Assessment of clonality, via additional cytoplasmic immunoglobulin (CyIg) κ vs. CyIgλ evaluation, may contribute to further establish the normal/reactive vs. clonal nature of small suspicious PC populations at high sensitivity levels, provided that enough cells are evaluated.

No significant prognostic value of normal precursor B-cell regeneration in paediatric acute myeloid leukaemia after induction treatment.

B-cell precursors (BCP) regeneration in bone marrow (BM) after induction chemotherapy is prognostic for good treatment response in adult acute myeloid leukaemia (AML). We detected BCP regeneration in 81% of 59 paediatric AML patients at first complete remission; this compared to 46% in an adult study. BCP regeneration did not correlate with outcome or minimal residual disease levels. In 36 healthy BM controls, BCP levels were significantly higher in children as compared to adults. Therefore, BCP regeneration does not reflect good response to treatment in paediatric AML, possibly due to the relatively high base-line levels of BCP in children.

Long-term stabilized blood samples as controls for flow cytometric HLA-B27 screening: a feasibility study.

BACKGROUND: Long-term stabilized blood samples are potentially useful as positive or negative procedure controls for flow cytometric HLA-B27 screening, and could serve as test samples in an external quality assessment (EQA) scheme. We evaluated long-term stabilized whole blood specimens as prepared for the UK NEQAS for Leucocyte Immunophenotyping EQA scheme (Sheffield, UK). METHODS: Peripheral blood samples were obtained from nine blood bank donors with known HLA-B typing. Short-term stabilization with Trans-FIXtrade mark was performed before shipment to Sheffield. Thereafter, long-term stabilization was performed. Commercially available HLA-B27 mAb were tested periodically between 1 week and 12 months on (i) fresh, (ii) short-term stabilized, and (iii) long-term stabilized blood samples using a stain, lyse, and wash technique. We compared the forward scatter (FSC), sideward scatter (SSC), and fluorescence signals of lymphocytes as a function of time. Furthermore, a pilot send-out with stabilized blood samples of four blood bank donors was distributed among the participants to the Benelux EQA scheme for HLA-B27 screening, and results were compared with historical EQA data obtained using nonstabilized blood samples from the same donors. RESULTS: There were no major effects on FSC and SSC characteristics of lymphocytes. Background fluorescence of stabilized samples increased and specific fluorescence of stabilized HLA-B27 positive samples decreased as compared with fresh samples. However, discrimination between the investigated HLA-B27 positive and HLA-B27 negative samples remained feasible poststabilization. In the pilot send-out, the results obtained with stabilized samples were less concordant than with the corresponding fresh samples due to variable quality of the stabilized samples. CONCLUSION: Long-term stabilized whole blood samples are potentially useful as true HLA-B27 positive and true HLA-B27 negative control cells for daily and longitudinal quality control of flow cytometric HLA-B27 screening. In the same way, long-term stabilized samples may be used for EQA purposes. However, these samples are currently not feasible for reagent validation purposes. Extensive quality control of long-term stabilized samples is necessary before distribution in multicenter surveys.

Flow cytometric screening for the HLA-B27 antigen on peripheral blood lymphocytes.

HLA-B27 is common to the entire group of seronegative spondyloarthropathies, and assessment of HLA-B27 is of diagnostic importance if any of these diseases are considered. Among the alternatives to traditional typing by the complement-dependent cytotoxicity assay, flow cytometric HLA-B27 screening is most widely used in general diagnostic laboratories, as it is simple, rapid, and cost effective. This unit describes screening for HLA-B27 on peripheral blood lymphocytes using more than one HLA-B27 monoclonal antibody to detect possible cross-reactivity with non-HLA-B27 antigens. Screening is hampered by the lack of true monospecific anti-HLA-B27 monoclonal antibodies. Cross-reactivities of anti-HLA-B27 with other HLA-B antigens have been reported. The authors recommend use of GS145.2 and FD705 antibodies, as this combination avoids most false-positive conclusions. Lyse-and-wash sample processing is employed. A multiparameter flow methodology is applied. Three to four parameters-forward scatter, side scatter, HLA-B27 expression, and, in some cases CD3 or B7 expression-are acquired.

Flow cytometric HLA-B27 screening: cross-reactivity patterns of commercially available anti-HLA-B27 monoclonal antibodies with other HLA-B antigens.

BACKGROUND: Some 50 clinical laboratories in the Benelux perform flow cytometric HLA-B27 screening and participate in the Benelux external quality assessment scheme operational since 1995. Results from this scheme indicate that cross-reactivity of HLA-B27 monoclonal antibodies (mAbs) is a major problem. METHODS: We analyzed cross-reactivity patterns of commercially available mAbs for HLA-B27 screening. Three clones of HLA-B27 mAb (ABC-m3, n = 3; FD705; and GS145.2) from five manufacturers were evaluated. Test cells were selected as to express HLA-B antigens with known serologic cross-reactions (HLA-B7, B12, B13, B16, B17, B22, B37, B40, B41, B42, B47, and B48). Cells without B27 cross-reactive antigens (B5, B8, B14, B15, B21, and B35) and cells positive for B27 were included as controls. All tests were performed and interpreted as recommended by the manufacturers. Cross-reactivity was defined as increased fluorescence intensity in comparison with the baseline reactivity observed with the corresponding immunoglobulin G isotype control mAb. RESULTS AND CONCLUSIONS: All mAbs tested showed cross-reactivity, ranging from weak (+/-) to strong (+), with different antigens and different degrees of intensity-ABC-m3: (+/-) B12, B16, B17, B41, B47, and B48 and (+) B7, B13, B22, B37, B40, and B42; GS145.2: (+/-) B13, B17, B22, B40, and B47 and (+) B7, B16, B37, B42, and B48; FD705: (+/-) B12, B13, B16, and B48 and (+) B17, B37, and B47. If one mAb had been used for HLA-B27 screening, ABC-m3 would have yielded nine false-positive B27 assignments, FD705 would have yielded seven, and GS145.2 would have yielded two. This problem largely canbe avoided by the combined use of two different mAb clones. The combination of FD705 and GS145.2 yielded the best results, with one false-positive HLA-B27 assignment among the 99 HLA-B27(-) samples of this highly selected panel.