Pronase treatment improves flow cytometry crossmatching results.

Flow cytometry crossmatching (FC-XM) is the most sensitive cell-based method for detecting donor-specific antibodies in clinical organ transplantation. Unfortunately, background FC-XM reactivity is elevated in assays with B lymphocytes-partly because of nonspecific immunoglobulin binding by Fc receptors and B-cell surface immunoglobulins. To reduce the background reactivity in a B-cell FC-XM assay, we treated lymphocytes with pronase (1 mg/mL for 30 minutes). This treatment drastically reduced the presence of kappa light chains and Fc receptors (CD32b), while the concomitant decrease in CD19, CD20 and major histocompatibility complex (MHC) I and II expression on B-cells was acceptable. Higher pronase concentrations (>2 mg/mL) started to significantly affect CD19, CD20, MHC-I and -II expression on B-cells. In subsequent prospective experiments (on 42 donor cells tested with 102 sera), we found that pronase treatment was associated with a relative increase of the sensitivity and specificity in our B-cell FC-XM assay.

CD14+ CD16+ monocytes rather than CD14+ CD51/61+ monocytes are a potential cytological marker of circulating osteoclast precursors in multiple myeloma. A preliminary study.

INTRODUCTION: Osteolytic bone destruction is a major clinical problem in multiple myeloma patients. Osteoclasts can differentiate in vitro from bone marrow-resident monocyte progenitors, such as common monocyte progenitors, as well as circulating monocytes. Various types of monocytes, including osteoclast precursors, appear to circulate systemically. METHODS: We investigated the possibility of demonstrating, by in vitro differentiation and flow cytometry, a circulating osteoclast precursor population in multiple myeloma (MM) patients by studying the distribution of CD14(+/++) CD11b(+) CD51/61(+) and CD14(+/++) CD16(+/-) populations. RESULTS: Under short-term in vitro osteoclastic differentiation conditions, almost all CD14 monocytes acquired CD51/61 and CD16 expression. Flow cytometry studies failed to demonstrate a statistically significant increase in circulating CD14(+/++) CD11b(+) CD51/61(+) populations in 20 MM patients with osteolytic lesions. However, the minor circulating CD14(+/++) CD16(+) fraction was significantly increased in MM patients compared with healthy volunteers (109.3 ± 63.1/mm(3) vs. 65.3 ± 34.9/mm(3) ; P = 0.005), but with no correlation with markers of tumour burden. The CD14(+/++) CD16(+) to CD14(+/++) CD16(-) ratio was higher in MM patients. CONCLUSION: The circulating CD14(+/++) CD11b(+) CD51/61(+) fraction was not correlated with bone lesions in MM patients. However, CD14(+/++) CD16(+) monocytes may be a candidate marker. A larger study must be conducted to confirm these promising results for the diagnosis and follow-up of MM patients.