Genomic analyses reveal recurrent mutations in epigenetic modifiers and the JAK-STAT pathway in Sézary syndrome.

Sézary syndrome (SS) is an aggressive leukaemia of mature T cells with poor prognosis and limited options for targeted therapies. The comprehensive genetic alterations underlying the pathogenesis of SS are unknown. Here we integrate whole-genome sequencing (n=6), whole-exome sequencing (n=66) and array comparative genomic hybridization-based copy-number analysis (n=80) of primary SS samples. We identify previously unknown recurrent loss-of-function aberrations targeting members of the chromatin remodelling/histone modification and trithorax families, including ARID1A in which functional loss from nonsense and frameshift mutations and/or targeted deletions is observed in 40.3% of SS genomes. We also identify recurrent gain-of-function mutations targeting PLCG1 (9%) and JAK1, JAK3, STAT3 and STAT5B (JAK/STAT total ∼11%). Functional studies reveal sensitivity of JAK1-mutated primary SS cells to JAK inhibitor treatment. These results highlight the complex genomic landscape of SS and a role for inhibition of JAK/STAT pathways for the treatment of SS.

A limited plasma cell flow cytometry panel with reflex CD138 immunohistochemistry is an optimal workflow process for evaluating plasma cell neoplasms in bone marrow specimens.

OBJECTIVES: To determine the optimal workflow combination of flow cytometry (FC) and immunohistochemistry tests for efficient and cost-effective evaluation of plasma cell (PC) neoplasms (PCNs) in bone marrow (BM) specimens. METHODS: Various workflow combinations of immunohistochemistry and FC for 4,031 BM specimens worked up for PCNs were compared. Turnaround time (TAT), immunohistochemistry usage, technical charges, and addendum/amendment rates were compared between periods to determine the optimal workflow combination. RESULTS: Five distinct workflow periods were identified, with varying combinations of full or limited FC panels for assessing PC clonality and CD138/κ/λ immunohistochemistry for PC quantification. Replacement of full FC with limited FC was associated with significant reductions in TAT and number of immunostains performed per case. Elimination of immunohistochemistry on cases determined to be polyclonal by FC also resulted in significant reductions in immunohistochemistry usage and significant cost savings. CONCLUSIONS: Assessment of PC clonality using a limited FC panel followed by reflex CD138 immunohistochemistry on cases that are monoclonal by FC provides an optimal and cost-effective workflow for evaluating PCNs in BM samples.

Recurrent reciprocal RNA chimera involving YPEL5 and PPP1CB in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common form of leukemia in adults in the Western hemisphere. Tumor-specific chromosomal translocations, characteristic findings in several human malignancies that directly lead to malignant transformation, have not been identified in CLL. Using paired-end transcriptome sequencing, we identified recurrent and reciprocal RNA chimeras involving yippee like 5 (YPEL5) and serine/threonine-protein phosphatase PP1-beta-catalytic subunit (PPP1CB) in CLL. Two of seven index cases (28%) harbored the reciprocal RNA chimeras in our initial screening. Using quantitative real-time PCR (q real-time PCR), YPEL5/PPP1CB and PPP1CB/YPEL5 fusion transcripts were detected in 97 of 103 CLL samples (95%) but not in paired normal samples, benign lymphocytes, or various unrelated cancers. Whole-genome sequencing and Southern blotting demonstrated no evidence for a genomic fusion between YPEL5 and PPP1CB. YPEL5/PPP1CB chimera, when introduced into mammalian cells, expressed a truncated PPP1CB protein that demonstrated diminished phosphatase activity. PPP1CB silencing resulted in enhanced proliferation and colony formation of MEC1 and JVM3 cells, implying a role in the pathogenesis of mature B-cell leukemia. These studies uncover a potential role for recurrent RNA chimeras involving phosphatases in the pathogenesis of a common form of leukemia.

A quantitative allele-specific PCR test for the BRAF V600E mutation using a single heterozygous control plasmid for quantitation: a model for qPCR testing without standard curves.

We describe a novel method for mutant allele quantitation using allele-specific PCR. The method uses a heterozygous plasmid containing one wild-type and one mutant sequence as a calibrator that is run at a single concentration, with each quantitative allele-specific PCR run. PCR data from both calibrator alleles, together with predetermined PCR efficiencies, are used to quantitate the mutant allele burden in unknown specimens. We demonstrate the utility of this method by using it to calculate BRAF V600E allele frequencies in cases of hairy-cell leukemia and show that it generates data that are comparable to those obtained via allele quantitation using conventional standard curves over a wide range of allelic ratios. This method is not subject to errors that may be introduced in traditional standard curves as the result of variations in pippetting or errors in the calculation of the absolute copy numbers of standards. Furthermore, it simplifies the workflow in the clinical laboratory and would provide significant advantages for efforts to standardize clinical quantitative PCR testing.

Detection of Merkel cell polyomavirus in chronic lymphocytic leukemia T-cells.

Chronic lymphocytic leukemia/small cell lymphoma (CLL/SLL) is the most common B-cell leukemia/lymphoma, effecting >15,000 patients/year. There has been a proposed limited antigenic etiology, at least in some cases, of CLL/SLL based upon immunoglobulin heavy chain stereotypy found across unrelated cases, suggesting viral source may provide such antigenic stimulation. With an established epidemiological link between CLL/SLL and Merkel cell carcinoma (MCC), there has been some interest in investigating a possible leukemogenic role of Merkel cell polyomavirus (MCPyV), which is found in 80% of MCC cases. Recent studies have shown that MCPyV is present in lymphocytes in a small percentage of CLL/SLL cases, but the specific tropism for lymphocytes has not been well-established. In this study, we used quantitative PCR to investigate the presence of MCPyV in fluorescence activated cell sorted purified B- and T-cells from 23 CLL/SLL cases. Three of 23 cases (13%) had detectable MCPyV in T-cells, and none of the cases had detectable MCPyV in B-cells. These findings suggest that MCPyV may have tropism for T-cells in addition to previously reported neoplastic B-cells.

Whole-genome sequencing identifies recurrent somatic NOTCH2 mutations in splenic marginal zone lymphoma.

Splenic marginal zone lymphoma (SMZL), the most common primary lymphoma of spleen, is poorly understood at the genetic level. In this study, using whole-genome DNA sequencing (WGS) and confirmation by Sanger sequencing, we observed mutations identified in several genes not previously known to be recurrently altered in SMZL. In particular, we identified recurrent somatic gain-of-function mutations in NOTCH2, a gene encoding a protein required for marginal zone B cell development, in 25 of 99 (∼25%) cases of SMZL and in 1 of 19 (∼5%) cases of nonsplenic MZLs. These mutations clustered near the C-terminal proline/glutamate/serine/threonine (PEST)-rich domain, resulting in protein truncation or, rarely, were nonsynonymous substitutions affecting the extracellular heterodimerization domain (HD). NOTCH2 mutations were not present in other B cell lymphomas and leukemias, such as chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL; n = 15), mantle cell lymphoma (MCL; n = 15), low-grade follicular lymphoma (FL; n = 44), hairy cell leukemia (HCL; n = 15), and reactive lymphoid hyperplasia (n = 14). NOTCH2 mutations were associated with adverse clinical outcomes (relapse, histological transformation, and/or death) among SMZL patients (P = 0.002). These results suggest that NOTCH2 mutations play a role in the pathogenesis and progression of SMZL and are associated with a poor prognosis.

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.

A pyrosequencing-based test for detection and relative quantification of the BCR-ABL1 T315I point mutation.

AIMS: The BCR-ABL1 T315I mutation imparts resistance to tyrosine kinase inhibitors currently available for treatment of chronic myelogenous leukaemia. Thus, quantitative monitoring of the emergence and expansion of T315I-positive subclones may be clinically useful. The goals of this study were to retrospectively review the authors' experience with Sanger sequencing-based BCR-ABL1 kinase domain mutation testing, paying particular attention to the T315I mutation, and to develop an alternative test for relative quantification of T315I using pyrosequencing. METHODS: The performance of a new T315I pyrosequencing assay was evaluated. Total RNA was isolated from whole blood and reverse-transcribed. The resulting cDNA was subjected to an initial round of PCR across the BCR-ABL1 breakpoint followed by a second round to amplify the sequence flanking ABL1 codon 315. The final PCR product was pyrosequenced to detect and quantify the T315I point mutation. Additional experiments were carried out to determine the effects of background untranslocated ABL1 on assay sensitivity in samples with low tumour burden. RESULTS: The results show that T315I was the most commonly detected kinase domain mutation and was persistent in follow-up testing. All 26 specimens that tested positive by Sanger sequencing for the T315I mutation were also positive using the pyrosequencing test. Relative quantification data derived from pyrosequencing matched the approximate wild-type/mutant ratios found by Sanger sequencing. Serial dilution experiments show sensitivity to 5% mutant allele. The authors also quantitatively assessed the influence of untranslocated ABL1 in the sample background on the assay and found that it occurred at levels not likely to influence performance. CONCLUSION: The described test is useful for detection and relative quantification of the T315I point mutation in chronic myelogenous leukaemia in a sensitive, specific and reproducible manner.

Combined testing for CCAAT/enhancer-binding protein alpha (CEBPA) mutations and promoter methylation in acute myeloid leukemia demonstrates shared phenotypic features.

Loss of function mutations in CCAAT/enhancer binding protein alpha (CEBPA) have been identified in acute myeloid leukemia (AML) and bi-allelic (double) CEBPA mutations are associated with improved prognosis in cases of cytogenetically normal-AML. In a subset of AML patients lacking CEBPA mutations, core promotor methylation of CEBPA has been described and is associated with a gene expression profile similar to the mutated cases including the expression of T cell associated genes such as CD7. However, the overall incidence and pattern of CEBPA mutations and core promoter methylation has not been thoroughly explored in a larger subset of AML with expression of CD7. Here we describe a simple and clinically deployable CEBPA promoter methylation test and the results of combined testing for CEBPA mutations and promoter methylation in 102 cases of AML, including 43 CD7+ cases. Overall, there were 5 methylated cases, 6 cases with double mutations, and 3 cases with single mutations. Significantly, 10 of 43 CD7+ cases (23%) had either methylated or double-mutated CEBPA. The CD7+ subset included all 5 methylated cases and 5 of the 6 cases with double mutations. All 3 cases with single mutations were CD7-. No case exhibited both hypermethylation and mutations. We find that promoter methylation accounts for half of those CD7+ cases with CEBPA dysregulating abnormalities. Furthermore, methylated cases and those with bi-allelic CEBPA mutations have similar phenotypic features including expression of CD7 and lack of co-incident NPM1 mutations. Our study suggests that methylation testing may be as important as mutation testing for identifying AML cases with CEBPA dysregulation and may be indicated in the routine prognostic workup of AML.

Clinical laboratory analysis of immunoglobulin heavy chain variable region genes for chronic lymphocytic leukemia prognosis.

Chronic lymphocytic leukemia (CLL) is the most common leukemia affecting adults in the western world. The clinical course of CLL is highly variable: cases that express mutated immunoglobulin heavy chain variable regions (IgV(H)) typically have a more indolent clinical course compared with those with unmutated IgV(H). The use of the V(H)3-21 variable region has also been found to confer a poor prognosis, independent of mutation status. Here we describe an assay for the identification of the expressed V(H) segment and its mutation status in CLL. This test uses whole blood-derived RNA and PCR primers annealing to the leader regions and the joining region segments. This approach allows more accurate determination of the IgV(H) mutation status relative to using framework region specific V(H) primers. An additional primer specific for the leader region of the V(H)3-21 segment is described and is shown to be necessary to identify this diagnostically important variable region. We successfully analyzed 99 of 103 samples, including five expressing the V(H)3-21 variable region. Approximately 5% of cases had complement determining region 3 sequences similar to previously reported cases, and overrepresentation of the V(H)1-69 segment was observed among unmutated cases. These results confirm the proper functioning and high success rate of this valuable prognostic for CLL designed for the use in a clinical laboratory setting.

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.

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.

Use of similar immunoglobulin VH gene segments by MALT lymphomas of the ocular adnexa.

Extranodal marginal zone lymphomas of mucosa-associated lymphoid tissue type (MALT lymphomas) develop from acquired reactive infiltrates directed against external or autoantigens. Although some European cases of ocular adnexal MALT lymphoma have been associated with Chlamydia psittaci infections, C. psittaci has not been detected in large studies of US-based cases. To evaluate whether the growth of US-based ocular adnexal MALT lymphomas may be promoted by a similar antigen, we identified and analyzed the expressed immunoglobulin VH genes in 10 cases. Interestingly, the VH genes in two cases used the same VH1 family V1-2 gene segment, and three cases used the same VH4 family V4-34 gene segment. The other five cases all used different gene segments V4-31, V5-51, V3-23, V3-30, and V3-7. All of the VH genes were mutated from germ line, with percent homologies ranging between 96.9 and 89.0%. The distribution of replacement and silent mutations within the VH genes was nonrandom consistent with the maintenance of immunoglobulin function and also strongly suggestive of antigen selection in the six VH genes with highest mutation loads. The CDR3 sequences in two of three VH-34 cases were the same size (15 amino acids) and had similar sizes in the two VH1-2 cases (18 and 16 amino acids). In conclusion, US-based MALT lymphomas of the ocular adnexa preferentially express a limited set of VH gene segments not frequently used by other MALT lymphomas and consistent with some recognizing similar antigens. Analysis of somatic mutations present within the VH genes is also consistent with antigen binding stimulating the growth of these lymphomas.

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.

Array CGH analysis of chronic lymphocytic leukemia reveals frequent cryptic monoallelic and biallelic deletions of chromosome 22q11 that include the PRAME gene.

We used BAC array-based CGH to detect genomic imbalances in 187 CLL cases. Submicroscopic deletions of chromosome 22q11 were observed in 28 cases (15%), and the frequency of these deletions was second only to loss of the 13q14 region, the most common genomic aberration in CLL. Oligonucleotide-based array CGH analysis showed that the 22q11 deletions ranged in size from 0.34 Mb up to approximately 1 Mb. The minimally deleted region included the ZNF280A, ZNF280B, GGTLC2, and PRAME genes. Quantitative real-time PCR revealed that ZNF280A, ZNF280B, and PRAME mRNA expression was significantly lower in the 22q11 deletion cases compared to non-deleted cases.

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.

Expression of T-plastin, FoxP3 and other tumor-associated markers by leukemic T-cells of cutaneous T-cell lymphoma.

Peripheral blood cells from 28 patients with leukemic cutaneous T-cell lymphoma including 25 patients with Sezary syndrome were evaluated for expression of regulatory T-cell-associated markers (FoxP3, CD25, CTLA-4, neurophilin-1), T-cell activation markers (CD28 and its ligands B7.1 and B7.2) and NK cell-associated markers (NKG2D and its ligands Mic-A and Mic-B) using real-time quantitative polymerase chain reaction. T-plastin served as a positive genetic marker, and its expression correlated to blood tumor burden. More than 90% of samples had transcripts for CD28 and Mic-B, but less than 30% of samples expressed FoxP3, CTLA-4 and CD25. Expression of Mic-B by neoplastic cells could provide another mechanism to inhibit anti-tumor immune responses. FoxP3 expression correlated with a poor prognosis. Although the underlying mechanisms accounting for this correlation remain unclear, the expression of the Foxp3 and CTLA-4 regulatory elements indicates that a subset of leukemic cases displays a regulatory T-cell phenotype.

Fatal non-transplant-related epstein-barr virus-associated atypical lymphoid proliferations in infants and children: a clinicopathologic study.

Most Epstein-Barr virus (EBV)-related infections in infants and children are asymptomatic or self-limited mild viral illnesses, but rare cases of a rapidly fatal disorder have been described. Failure of the cellular response to control EBV-related lymphoid proliferation leads to severe disease with multiple complications, including a fatal outcome or development of an EBV-driven, clonal lymphoid neoplasm. In this report we characterize 3 cases of fatal, nontransplant, or immunodeficiency-related EBV infection in very young children with immunophenotypic and molecular evidence of B/natural killer (NK)-T cell clonal expansion. An immunohistochemical staining panel included testing for B-cell antigen (CD20), and T/NK cell antigens including CD2, CD3, CD4, CD8, CD56, CD57, and TIA-1. T-cell and B-cell PCR clonality testing was performed on paraffin tissue specimens to identify clonal populations. The ages of these 3 patients ranged from 22 months to 4 years. Initial clinical presentations included pneumonia, abnormal liver function tests and fever, and lymphadenopathy. The 3 patients died within 17 to 72 days of presentation, and autopsy was performed on 1 patient. All cases demonstrated prominent atypical lymphoid or lymphohistiocytic infiltrates, and necrosis was present in 2 of the 3 cases. The atypical lymphocytes were positive for CD3 (cytoplasmic), CD2, CD8, TIA-1, and CD57 and negative for CD4. We molecularly identified B-cell clones in the 2 tested patients, who also showed evidence of hemophagocytosis. Fatal EBV infection is characterized by a morphologic spectrum with atypical lymphoid infiltrates and variable necrosis. Our molecular studies of these patients suggest a clonally-derived expansive process, most likely driven by EBV infection. Our results also suggest that development of clonality is associated with an aggressive clinical course and may be useful in predicting greater risk for fatal outcome. A high index of suspicion, coupled with appropriate serologic and molecular testing, aids in early recognition and diagnosis of these lymphoproliferative processes.

A new DNA-based test for detection of nucleophosmin exon 12 mutations by capillary electrophoresis.

Mutations in nucleophosmin (NPM1) exon 12 are thought to be the most common genetic event in acute myelogenous leukemia (AML) and to confer favorable clinical prognoses. In this report, we describe a simple molecular test for the detection of NPM1 exon 12 mutations in patients with AML using polymerase chain reaction amplification of genomic DNA followed by the analysis of amplification products by capillary electrophoresis. Mutations were reproducibly detected when present in at least 5% of cells, and all NPM1 exon 12 mutations reported to date in AML could be identified using this method. This method was successfully employed using paraffin-extracted DNA, allowing for the examination of archived clinical specimens, and the assay was validated by the direct sequencing of 33 patient samples. This sensitive test is straightforward to perform and provides important information that can influence both the clinical management and treatment options for many patients with AML.

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.