Immunohistochemical Detection of Hairy Cell Leukemia in Paraffin Sections: The Role of Pax5 and CD103 Double Staining to Improve Specificity and Sensitivity.

Context: - In hematopathology practice, abnormal expression of CD103 on B cells is detected by flow cytometry in hairy cell leukemia (HCL) and, in combination with other phenotypic and morphologic findings, provides diagnostic specificity and sensitivity. Immunostaining on paraffin sections makes it possible to perform immunophenotyping in situ. However, normal bone marrow contains CD103-positive cells, which are not B cells, making it difficult to be certain about low-level involvement by HCL.

Accurate assessment of cell count and viability with a flow cytometer.

Background: In this study we developed a method to measure cell concentration and viability in specimens received in flow cytometry and cytogenetics laboratories.

Methods: Specimens are stained with a vital fluorescent dye, SYTO13, the cell impermeant viability dye, 7-AAD, and a leukocyte marker, CD45. After the addition of an internal calibrator microsphere, FLOW-COUNT, the flow cytometer is capable of measuring the viability of nucleated cells, giving a general assessment of leukocyte populations and measuring their concentration.

Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLL.

Background: In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity.

Methods: B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.