A simple flow-cytometry method to evaluate peripheral blood contamination of bone marrow aspirates.

Bone marrow (BM) aspirates used for flow-cytometry (FCM) studies are usually obtained from a second aspiration, as the primary aspirate is used for morphological assessment. For this reason, the FCM samples unavoidably contain some blood; although, good-quality samples contain only a small amount. It is of utmost importance to assess the quality of samples prior to FCM analysis; yet, contamination with peripheral blood (PB) is not evaluated in most laboratories, possibly because the methods available are either qualitative or too complex for daily practice. Here, we propose a simple FCM method to quantitatively evaluate PB contamination in BM aspirates, by analyzing the percentage of plasma cells and CD34+ cells - two cell populations nearly absent from PB - and CD10+ granulocytes, which comprise the majority of the PB granulocyte population. We analyzed these three populations in 122 BM aspirates from subjects without hematological disease, and identified samples with PB contamination by performing a hierarchical cluster analysis. A discriminant analysis yielded a function, which we named the PB contamination index (PBCI). This index value gives a quantitative indication about the degree of hemodilution of a given sample. A threshold was identified that discriminates low-quality samples. The method and the threshold proved to be useful in BM aspirates infiltrated with malignant cells, with the exception of cases where hematological disease altered two of the three parameters included in the index. We have easily implemented the PBCI calculation in our daily routine, and find it very helpful for an accurate interpretation of FCM results in a large proportion of BM specimens. Limitations of the technique are discussed.

Relatively high levels of anti-HLA antibodies (measured by Luminex Single-Antigen bead assay) are required to mediate inhibition of lymphocyte proliferation induced by sera from alloimmunized renal patients.

BACKGROUND: Rejection of transplanted organs is caused by alloimmune responses, primarily against HLA molecules. Anti-donor HLA antibodies are associated with antibody-mediated rejection (AMR) and poor graft outcome. Because of clinical interest in detecting these antibodies, new technologies have recently been introduced to increase the sensitivity of detection. The Luminex Single-Antigen (LSA) bead assay may yield new information, but it must be validated against biological and clinical data. MATERIAL AND METHODS: Based on previously published data regarding the in vitro effects of anti-HLA antibodies on lymphocytes, we measured the effect on lymphocytes of sera from patients on the transplant waiting list who had high titers of anti-HLA antibodies. Anti-CD3-mediated lymphocyte activation was studied in the presence of whole serum from these patients. Changes in lymphocyte proliferation, measured by carboxyfluorescein succinimidyl ester (CFSE) labeling, were detected, and these changes correlated with the level of anti-HLA antibodies. RESULTS: Whole serum containing anti-HLA antibodies inhibited lymphocyte proliferation; this effect correlated with the level of antibodies, as measured by LSA. This inhibitory effect was HLA-specific, as shown by adsorption experiments. We also found that relatively high levels of anti-HLA antibodies were necessary to induce changes in an in vitro model of lymphocyte proliferation. CONCLUSIONS: Our results demonstrate the clinical utility of detecting anti-HLA antibodies by LSA.

Predictive value of the Luminex single antigen panel for detecting flow cytometry cross-match positivity.

Anti-human leukocyte antigen (HLA) antibodies are a major cause of allograft loss. Solid-phase immunoassays, notably Luminex technology, have lately begun to replace traditional techniques for detecting these antibodies. This platform, however, carries some restrictions in the type of antibodies it detects. For this reason, results using these new technologies must be correlated with results using traditional techniques that have proven clinical significance. We have correlated flow cytometry cross-match (FCXM) outcomes with results from Luminex assays. Serum samples from patients awaiting transplantation who had known anti-HLA antibodies as detected by Luminex were incubated with lymphocytes expressing (a) 1 of the HLA antigens detected by the sera or (b) several of them. Of the 169 T-cell FCXMs we performed, in 92 cases the target cell expressed only 1 of the HLA antigens detected by the serum. The results obtained correlated well with Luminex data (r = 0.84). A cutoff mean fluorescence intensity value of 6,500 for the Luminex single antigen assay yielded a sensitivity of 85% and specificity of 82% for detecting a positive FCXM. In the other 77 cases, the target cell expressed 2 or more of the HLA antigens detected by the serum. In this situation, the same cutoff proved a useful tool for differentiating negative from positive FCXMs.