Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic control antibodies.

INTRODUCTION: Expression of ZAP-70 by chronic lymphocytic leukemia (CLL) is associated with more aggressive disease and can help differentiate CLL using mutated immunoglobulin heavy chain variable genes (VH) from cases expressing unmutated VH genes. However, flow cytometric detection of ZAP-70 in CLL shows considerable variability and may be of questionable significance because most laboratories cannot correlate their results to clinical outcome or VH mutational data. METHODS: Seventy cases of CLL were evaluated for ZAP-70 using a previously optimized staining procedure and two different methods to eliminate nonspecific background staining. One method, not previously reported, used isotypic control antibodies, where the concentrations were adjusted/optimized so that normal B-cells stained negatively for ZAP-70. The other used ZAP-70 stained peripheral blood B-cells from normal donors. The percentages of ZAP-70 stained CLL cells above the two thresholds were compared. RESULTS: Concentrations of isotypic control antibodies had to be increased from manufacture's recommendations to insure normal B-cells were ZAP-70 negative. ZAP-70 levels among the CLL cases formed a bimodal distribution using the optimized isotypic control threshold, with 30 having low values (0-32% positive) and 40 high values (60-99% positive). In contrast, a continuous distribution was obtained with the ZAP-70 stained B-cell threshold. VH mutational status strongly correlated with the optimized control values as 29/30 low ZAP-70 cases had mutated VH genes and 37/40 high ZAP-70 cases used unmutated VH genes. CONCLUSIONS: Use of an optimized isotypic control threshold could increase the reliability of flow based ZAP-70 detection and correlates well with VH mutational status.

Immunomagnetic bead separation of mononuclear cells from contaminating granulocytes in cryopreserved blood samples.

Density gradient centrifugation usually allows efficient separation of mononuclear cells from granulocytes using fresh human blood samples. However, we have found that with cryopreserved blood samples, density gradient centrifugation fails to separate granulocytes from mononuclear cells and have explored using immunomagnetic anti-CD15 microbeads as an alternate method to separate these cell populations. Using cryopreserved blood samples from 10 healthy donors we have shown that granulocytes express a significantly higher level of CD15 antigen than monocytes and lymphocytes, which allows for their efficient separation from mononuclear cells using anti-CD15 microbeads. This procedure is critical for purification of individual cell populations from cryopreserved leukocyte samples and could also potentially be applied to avoid granulocyte contamination of mononuclear cells isolated from stored blood and from patients with sepsis or thermal injury.

Flow cytometric detection and serotyping of enterovirus for the clinical laboratory.

Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1-4 after inoculation. Flow cytometry results for echovirus 6, 9, 11, and 30 and coxsackievirus B1 correlated with IFA in all cases. Coxsackievirus B1 and echovirus 30 infections were detected 1 day earlier by flow cytometry than IFA. Flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems.

Small B-cell neoplasms with typical mantle cell lymphoma immunophenotypes often include chronic lymphocytic leukemias.

Mantle cell lymphoma (MCL) and chronic lymphocytic leukemia (CLL) are CD5+ small B-cell neoplasms (SBCNs) with overlapping features. Flow cytometric immunophenotyping is often used to help differentiate CLL from MCL, and a characteristic CLL phenotype is considered essentially diagnostic. However, previous studies have not specifically examined how well a typical MCL immunophenotype distinguishes MCL from CLL. We identified 28 cases of SBCN with typical flow cytometry-determined MCL immunophenotypes consisting mostly of peripheral blood and bone marrow specimens. Fluorescence in situ hybridization analysis indicated that 57% (16/28) had t(11;14) translocations consistent with MCL, while 32% (9/28) lacked t(11;14) translocations but harbored other cytogenetic abnormalities commonly found in CLL. There were no significant morphologic or immunophenotypic differences between the t(11;14)-positive and t(11;14)-negative cases. Our findings suggest that many blood-based SBCNs with typical MCL immunophenotypes likely represent cases of phenotypically atypical CLL, which would have important clinical implications.

Whole-genome scanning by array comparative genomic hybridization as a clinical tool for risk assessment in chronic lymphocytic leukemia.

Array-based comparative genomic hybridization (array CGH) provides a powerful method for simultaneous genome-wide scanning and prognostic marker assessment in chronic lymphocytic leukemia (CLL). In the current study, commercially available bacterial artificial chromosome and oligonucleotide array CGH platforms were used to identify chromosomal alterations of prognostic significance in 174 CLL cases. Tumor genomes were initially analyzed by bacterial artificial chromosome array CGH followed by confirmation and breakpoint mapping using oligonucleotide arrays. Genomic changes involving loci currently interrogated by fluorescence in situ hybridization (FISH) panels were detected in 155 cases (89%) at expected frequencies: 13q14 loss (47%), trisomy 12 (13%), 11q loss (11%), 6q loss (7.5%), and 17p loss (4.6%). Genomic instability was the second most commonly identified alteration of prognostic significance with three or more alterations involving loci not interrogated by FISH panels identified in 37 CLL cases (21%). A subset of 48 CLL cases analyzed by six-probe FISH panels (288 total hybridizations) was concordant with array CGH results for 275 hybridizations (95.5%); 13 hybridizations (4.5%) were discordant because of clonal populations that comprised less than 30% of the sample. Array CGH is a powerful, cost-effective tool for genome-wide risk assessment in the clinical evaluation of CLL.

Optimization of flow cytometric measurement of ZAP-70 in chronic lymphocytic leukemia.

BACKGROUND: The goal of this study was to optimize a cell staining procedure for flow cytometric detection of zeta-chain associated protein-70 (ZAP-70). Our specific objectives were to improve antibody selection criteria, identify a cell permeabilization procedure better tailored to ZAP-70 analysis, as well as to establish objective criteria to control antigen stability. METHODS: Sequentially titrated 2F3.2-FITC, 1E7.2-FITC, and 1E7.2-Alexa Fluor 488 anti-ZAP-70 antibodies were used to stain normal B and T cells and Scatchard analysis was applied to calculate K(d) and B(max) values from saturation curves of specific binding. ZAP-70 staining was compared in cells permeabilized with two commercially available kits, Triton X-100, and a custom saponin procedure. RESULTS: Normal B-cells were found to provide an excellent measure of nonspecific staining while varying ZAP-70 antibodies and concentrations. Comparing Scatchard analyses of specific T-cell binding revealed that 1E7.2-Alexa Fluor 488 had the highest binding affinity of the tested anti-ZAP-70 antibodies and was the best choice. The highest levels of ZAP-70 fluorescence occurred when cells were permeabilized using a noncommercial saponin procedure. Decrease of chronic lymphocytic leukemia cell viability correlated with diminished ZAP-70 expression; when viability was lower than 95% the percentage of bright positive samples was significantly decreased, indicating a possibility of false-negative results. CONCLUSIONS: The efficiency and reliability of flow cytometric detection of ZAP-70 can be optimized by using Scatchard analysis to help select the most effective antibodies and antibody concentrations that maximize specific to nonspecific binding, by using a "custom" ZAP-70 permeabilization procedure, and by better controlling antigen stability by measuring cell viability.