Mike Keeney

An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia.

ZAP‐70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B‐cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP‐70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations.

Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection

Background: Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH. Methods: CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15 and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages. Results: Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R2 >0.99) with predicate values over a range (0.06% - 99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples. Conclusion: While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs.

User‐defined protein marker assay development for characterization of circulating tumor cells using the CellSearch® system

The majority of cancer‐related deaths result from metastasis, which has been associated with the presence of circulating tumor cells (CTCs). It has been shown that CTC cut‐off values exist that predict for poorer overall survival in metastatic breast (≥5), prostate (≥5), and colorectal (≥3) cancer based on assessment of 7.5 ml of blood. Development of the CellSearch® system (Veridex) has allowed for sensitive enumeration of CTCs. In the current study, protocols were developed and optimized for use with the CellSearch system to characterize CTCs with respect to user‐defined protein markers of interest in human blood samples, including the cancer stem cell marker CD44 and the apoptosis marker M‐30. Flow cytometry (FCM) experiments were initially carried out to assess expression of CD44 and M‐30 on MDA‐MB‐468 human tumor cells. Human blood samples were then spiked with MDA‐MB‐468 cells and processed with the appropriate antibody (CD44/M‐30) on the CellSearch. Detailed optimization of CD44 was carried out on the CellSearch using various antibody concentrations, exposure times, and cell lines with varying CD44 expression. Troubleshooting experiments were undertaken to explain observed discrepancies between FCM and CellSearch results for the M‐30 marker. After extensive optimization, the best CD44/M‐30 concentrations and exposure times were determined to be 1.5/3.5 μg/ml and 0.2/0.8 s, respectively. The percentage of CD44+ tumor cells was 99.5 ± 0.39% by FCM and 98.8 ± 0.51% by the CellSearch system. The percentage of M‐30+ tumor cells following paclitaxel treatment was 17.6 ± 1.18% by FCM and 10.9 ± 2.41% by CellSearch. Proper optimization of the CD44 marker was achieved; however, M‐30 does not appear to be a suitable marker for use in this platform. Taken together, the current study provides a detailed description of the process of user‐defined protein marker development and optimization using the CellSearch, and will be an important resource for the future development of protein marker assays by users of this platform.

Initial performance evaluation of the UniCel® DxH 800 Coulter® cellular analysis system.

The Beckman Coulter UniCel® DxH 800 is a hematology analyzer incorporating new electronic and mechanical design with advanced algorithm technology to perform CBC, white blood cell (WBC) differential, nucleated red blood cell (NRBC), and reticulocyte analysis. Evaluation of this instrument was performed in our 800‐bed tertiary care hospital and specifically centered upon the correlation of WBC, NRBC, and platelet (PLT) enumeration when compared to a predicate analyzer, the Coulter® LH 780, and flow cytometry (FCM) reference methods. Of particular interest were those samples with morphologically confirmed interference and extreme leukocytosis (evaluated with respect to red blood cell parameter correction). The sample set (n = 272) consisted of morphologically normal and hematologically abnormal patients. Correlation of the WBC, PLT, and NRBC showed r2 values of 0.994, 0.985, and 0.910 for the DxH 800 vs. FCM, respectively. The presence of interfering particles did not affect the accuracy of the DxH 800 with respect to WBC counts. The DxH 800 showed accurate PLT and NRBC counts in the clinically significant low range when compared to FCM. Compared to the LH 780, flagging rates were significantly reduced (NRBC flag), or equivalent (WBC, PLT flag) on the DxH 800. The DxH 800 demonstrated higher sensitivity and specificity for PLTs and NRBCs and achieved a lower NRBC false negative rate compared to the LH 780. The UniCel® DxH 800 represents a significant improvement to previous impedance analyzers in accurately detecting the presence of NRBCs at counts >1/100 WBC. Furthermore, it provides accurate PLT and WBC counts in the presence of interference and improved NRBC flagging efficiency when compared to the LH 780. Correction of red blood cell parameters is appropriate and accurate in cases of extreme leukocytosis.

Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection: Use of CD157 in Flaer-Based Assays

Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER‐based assays to detect granulocytes and monocytes lacking expression of GPI‐linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH.

Technical issues: flow cytometry and rare event analysis

Flow cytometry has become an essential tool for identification and characterization of hematological cancers and now, due to technological improvements, allows the identification and rapid enumeration of small tumor populations that may be present after induction therapy (minimal residual disease, MRD). The quantitation of MRD has been shown to correlate with relapse and survival rates in numerous diseases and in certain cases, and evidence of MRD is used to alter treatment protocols. Recent improvements in hardware allow for high data rate collection. Improved fluorochromes take advantage of violet laser excitation and maximize signal‐to‐noise ratio allowing the population of interest to be isolated in multiparameter space. This isolation, together with a low background rate, permits for detection of residual tumor populations in a background of normal cells. When counting such rare events, the distribution is governed by Poisson statistics, with precision increasing with higher numbers of cells collected. In several hematological malignancies, identification of populations at frequencies of 0.01% and lower has been attained. The choice of antibodies used in MRD detection facilitates the definition of a fingerprint to identify abnormal populations throughout treatment. Tumor populations can change phenotype, and an approach that relies on ‘different from normal’ has proven useful, particularly in the acute leukemias. Flow cytometry can and is used for detection of MRD in many hematological diseases; however, standardized approaches for specific diseases must be developed to ensure precise identification and enumeration that may alter the course of patient treatment.

Determination of optimal replicate number for validation of imprecision using fluorescence cell-based assays: proposed practical method.

Background: Assay validation includes determination of inherent imprecision across the reportable range. However specific practical guidelines for determinations of precision for cell based fluorescence assays performed on flow cytometers are currently lacking. Methods: Replicates of 10 or 20 measurements were obtained for flow cytometric assays developed for clinical IVD use, including neutrophil CD64 expression for infection/sepsis detection, fetal red cell enumeration for fetomaternal hemorrhage detection, human equilibrative nucleoside transporter 1 (hENT1) quantitation in leukocytes for possible correlation with drug responsiveness, and CD34+ hematopoietic stem cell (HSC) enumeration of apheresis products, using up to three different instrument platforms for each assay. For each assay, the mean, 95% confidence intervals of the mean (95%CI), standard deviation and coefficient of variation (CV) of sequential replicates were determined. Results: For all assays and most instrument platforms <5 replicates were found adequate to validate assay imprecision levels below the 5-10% CV for repeatability claimed by the manufacturers of these assays. Results plotted as a novel parameter derived from the 95%CI and the cumulative mean for replicates, termed variance factor (VF), provide a data driven means for determining optimal replicate numbers. Conclusions: The novel VF can provide information to guide the practical selection of optimal replicate numbers for validation of imprecision in flow cytometric assays. The optimal number of replicates was assay and instrument platform dependent. Our findings indicate 3-4 replicates are sufficient for most flow cytometric assays and instrument combinations, rather than the higher numbers suggested by CLSI guidelines for soluble analytes.

Determination of optimal replicate number for validation of imprecision using fluorescence cell-based assays: Proposed practical method: Determination of Precision for Cell-Based Fluorescence Assays

Assay validation includes determination of inherent imprecision across the reportable range. However, specific practical guidelines for determinations of precision for cell‐based fluorescence assays performed on flow cytometers are currently lacking.

Flow cytometry—Recognizing unusual populations in leukemia and lymphoma diagnosis

Flow cytometry is an invaluable technology in the examination of blood, bone marrow, tissue and body fluids for the presence or absence of hematological disease. It is used in both diagnostic and follow‐up testing, with an increasingly important role in the detection of very small residual disease populations (Minimal Residual Disease, MRD) However, flow cytometry immunophenotyping of leukemia and lymphoma is highly dependent on interpretation of results and with the increased complexity of 8‐10 color instruments routinely used in clinical laboratories, knowledge of disease‐defining populations is increasingly important as is recognizing normal and reactive patterns. This manuscript presents case studies with flow cytometric patterns encountered in routine screening of samples sent for leukemia and lymphoma immunophenotyping, focusing mainly on B‐cell disorders which may be missed or incorrectly interpreted by the laboratory (including a hematopathologist) performing the test. Case studies are used to illustrate our laboratorys standardized approach to the interpretation of flow cytometric data. In addition to a standardized approach, these cases emphasize the importance of interpretative skills of technologist and hematopathologists in recognizing abnormal patterns in detecting hematological malignancies.

Blastic Plasmacytoid Dendritic Cell Neoplasm: A Review of Diagnosis, Pathology, and Therapy

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare malignancy of dendritic cell precursors classified as a subset of acute myeloid leukemia according to WHO. It commonly presents with dermal infiltration of plasmatoid dendritic cells that have the morphologic appearance of medium-sized blasts with irregular nuclei, faint chromatin, at least one nucleolus per cell and scant cytoplasm, expressing a CD4+CD56+CD123+lin- immunophenotype. Patients typically have discoloured cutaneous lesions that grow in size, and the diagnosis of BPDCN is confirmed on skin biopsy showing the characteristic cells. Bone marrow involvement is a common feature of this neoplasm and is found in most patients at diagnosis. There is no consensus on the most appropriate treatment for BPDCN, with the neoplasm showing initial good response to high intensity chemotherapy but inevitable relapse into a more chemotherapy-resistant disease. Hematopoietic stem cell transplant in patients who achieve their first complete remission with chemotherapy is a promising therapeutic modality requiring a prospective clinical trial to evaluate its efficacy.