Accurate white blood cell differential by Alinity hq: A comparison with flow cytometry and manual differential.

INTRODUCTION: White blood cell (WBC) differential by flow cytometry can report a six-part WBC differential and enumerate blasts. Some modern hematology analyzers are also able to provide a six-part WBC differential (including immature granulocytes). Our goal was to compare the WBC differential obtained by the Abbott Alinity hq hematology analyzer to an 8-color single-tube flow cytometry method and to manual WBC differential. METHODS: Samples from 144 patients were tested with Alinity hq, flow cytometry, and microscopic WBC differential. The WBC count ranged from 1.22 to 359 × 109 /L, and 34 samples were flagged by the analyzer for abnormal WBC morphology. RESULTS: Strong concordance was demonstrated between Alinity hq and flow cytometry for all six components of the differential, with correlation coefficients ranging from 0.86 (basophils) to 1.00 (lymphocytes). Small, clinically insignificant positive difference was observed between Alinity hq and flow cytometry for mature and total neutrophils (2.51% and 1.85%) and eosinophils (0.14%), and small negative difference for immature granulocytes (-0.65%), lymphocytes (-0.61%), and basophils (-0.21%). No bias was detected between the Alinity hq and flow cytometry monocyte counts. Alinity hq and flow cytometry results agreed with the manual differential, apart from small, clinically insignificant differences. Alinity hq nucleated red blood cell concentrations were equivalent with the manual results (r = 0.95, slope = 1.16). The percentage of blasts by flow cytometry demonstrated good correlation and agreement with the manual count (r = 0.99, slope = 1.35). CONCLUSION: Alinity hq has produced accurate six-part WBC differential in this three-way comparison, equivalent to flow cytometry and morphological classification.

Reproducible diagnosis of chronic lymphocytic leukemia by flow cytometry: An European Research Initiative on CLL (ERIC) & European Society for Clinical Cell Analysis (ESCCA) Harmonisation project.

The diagnostic criteria for CLL rely on morphology and immunophenotype. Current approaches have limitations affecting reproducibility and there is no consensus on the role of new markers. The aim of this project was to identify reproducible criteria and consensus on markers recommended for the diagnosis of CLL. ERIC/ESCCA members classified 14 of 35 potential markers as "required" or "recommended" for CLL diagnosis, consensus being defined as >75% and >50% agreement, respectively. An approach to validate "required" markers using normal peripheral blood was developed. Responses were received from 150 participants with a diagnostic workload >20 CLL cases per week in 23/150 (15%), 5-20 in 82/150 (55%), and <5 cases per week in 45/150 (30%). The consensus for "required" diagnostic markers included: CD19, CD5, CD20, CD23, Kappa, and Lambda. "Recommended" markers potentially useful for differential diagnosis were: CD43, CD79b, CD81, CD200, CD10, and ROR1. Reproducible criteria for component reagents were assessed retrospectively in 14,643 cases from 13 different centers and showed >97% concordance with current approaches. A pilot study to validate staining quality was completed in 11 centers. Markers considered as "required" for the diagnosis of CLL by the participants in this study (CD19, CD5, CD20, CD23, Kappa, and Lambda) are consistent with current diagnostic criteria and practice. Importantly, a reproducible approach to validate and apply these markers in individual laboratories has been identified. Finally, a consensus "recommended" panel of markers to refine diagnosis in borderline cases (CD43, CD79b, CD81, CD200, CD10, and ROR1) has been defined and will be prospectively evaluated. © 2017 International Clinical Cytometry Society.

Validation of cell-based fluorescence assays: practice guidelines from the ICSH and ICCS - part IV - postanalytic considerations.

Flow cytometry and other technologies of cell-based fluorescence assays are as a matter of good laboratory practice required to validate all assays, which when in clinical practice may pass through regulatory review processes using criteria often defined with a soluble analyte in plasma or serum samples in mind. Recently the U.S. Food and Drug Administration (FDA) has entered into a public dialogue in the U.S. regarding their regulatory interest in laboratory developed tests (LDTs) or so-called home brew assays performed in clinical laboratories. The absence of well-defined guidelines for validation of cell-based assays using fluorescence detection has thus become a subject of concern for the International Council for Standardization of Haematology (ICSH) and International Clinical Cytometry Society (ICCS). Accordingly, a group of over 40 international experts in the areas of test development, test validation, and clinical practice of a variety of assay types using flow cytometry and/or morphologic image analysis were invited to develop a set of practical guidelines useful to in vitro diagnostic (IVD) innovators, clinical laboratories, regulatory scientists, and laboratory inspectors. The focus of the group was restricted to fluorescence reporter reagents, although some common principles are shared by immunohistochemistry or immunocytochemistry techniques and noted where appropriate. The work product of this two year effort is the content of this special issue of this journal, which is published as 5 separate articles, this being Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS - Part IV - Postanalytic considerations.

Persistence and efficacy of second generation CAR T cell against the LeY antigen in acute myeloid leukemia.

In a phase I study of autologous chimeric antigen receptor (CAR) anti-LeY T-cell therapy of acute myeloid leukemia (AML), we examined the safety and postinfusion persistence of adoptively transferred T cells. Following fludarabine-containing preconditioning, four patients received up to 1.3 × 109 total T cells, of which 14-38% expressed the CAR. Grade 3 or 4 toxicity was not observed. One patient achieved a cytogenetic remission whereas another with active leukemia had a reduction in peripheral blood (PB) blasts and a third showed a protracted remission. Using an aliquot of In111-labeled CAR T cells, we demonstrated trafficking to the bone marrow (BM) in those patients with the greatest clinical benefit. Furthermore, in a patient with leukemia cutis, CAR T cells infiltrated proven sites of disease. Serial PCR of PB and BM for the LeY transgene demonstrated that infused CAR T cells persisted for up to 10 months. Our study supports the feasibility and safety of CAR-T-cell therapy in high-risk AML, and demonstrates durable in vivo persistence.

A risk-adapted protocol for delayed administration of filgrastim after high-dose chemotherapy and autologous stem cell transplantation.

INTRODUCTION: The routine use of recombinant human granulocyte-colony stimulating factor (rhG-CSF) after high-dose chemotherapy and autologous stem cell transplantation (auto-SCT) is associated with increased costs. We prospectively explored a strategy that used prophylactic delayed filgrastim only in patients with risk factors. PATIENTS AND METHODS: This sequential cohort analysis compared the outcomes of consecutive patients, treated on the risk-adapted protocol (RAP) (risk factors: prior febrile neutropenia; age >60 years; and CD34+ cell infused dose of <2 × 10(6/)/kg), who received filgrastim from day +6 after auto-SCT with a historical cohort (historical day-1 cohort [HD1]), who received filgrastim from day +1. RESULTS: Eighty-two patients were treated in the RAP cohort and compared with 115 patients in the HD1 cohort. There were no differences in median age (55 years) or median CD34+ cell dose (5.21 × 10(6)/kg [range, 2-62.2 × 10(6)/kg] vs. 5.24 × 10(6)/kg [range, 2.4-29.8 × 10(6)/kg]). Filgrastim was used for 6 fewer days in the RAP cohort (median 5 days [range, 0-11 days] vs. 11 days [range, 9-47 days]). There was a small absolute but significant difference in median time to neutrophil recovery in the HD1 cohort for the whole group, 10 days (range, 8-46 days) vs. 11 days (range, 9-22 days) (P = .03) and in patients with myeloma; 10 days (range, 9-14 days) vs. 11 days (range, 9-18 days) (P < .0001) as compared to the RAP cohort. There was no difference in median inpatient duration, 13 days (range, 10-26 days) vs. 12 days (range, 1-38 days) (P = .22) and 3-year survival (79% vs. 83% [P = .43]) between HD1 and RAP cohorts respectively. CONCLUSIONS: The use of a RAP to identify patients likely to benefit from prophylactic filgrastim is safe and results in cost savings. Patients with myeloma benefit from earlier introduction of filgrastim in terms of neutrophil recovery; this disease-specific observation is an important consideration for future studies.

Peripheral blood CD34+ cell enumeration as a predictor of apheresis yield: an analysis of more than 1,000 collections.

The role of the peripheral blood (PB) CD34(+) cell count in predicting the CD34(+) cell yield in hematopoietic progenitor cell apheresis collections is well established. However, sometimes unexpectedly poor CD34(+) cell yields are obtained. To determine the effect, if any, of a range of factors on the ability of the PB CD34(+) count to predict collection CD34(+) cell count, we performed a retrospective analysis on consecutive hematopoietic progenitor cell apheresis collections between 2004 and 2008. Factors investigated included mobilization regimen, PB white blood cell count, body weight, and disease. After exclusion of collections involving apheresis complications, a total of 1,225 PB CD34(+) cell results with corresponding collection CD34(+) cell results from 458 patients were analyzed. Although differences in the median PB CD34(+) cell counts and collection CD34(+) cell counts were seen between mobilized collections with chemotherapy plus granulocyte colony-stimulating factor and those with granulocyte colony-stimulating factor alone, the predictive capability of the PB CD34(+) cell count for the collection CD34(+) cell yield remained similar. Although poorer collection efficiencies were observed in the myelodysplastic syndrome/myeloproliferative disorder diagnostic subgroup, our findings confirm that PB CD34(+) cell analysis remains a powerful and irreplaceable tool for predicting hematopoietic progenitor cell apheresis CD34(+) cell yield.

Extracorporeal photopheresis for the treatment of Sézary syndrome using a novel treatment protocol.

Various studies have reported that extracorporeal photopheresis is effective in producing meaningful responses in patients with Sézary syndrome. A single-center, 5-year retrospective analysis was performed on our patients with Sézary syndrome who received extracorporeal photopheresis using a novel protocol. Thirteen patients were treated with extracorporeal photopheresis consistently for a minimum of 2 months. All patients received a modified protocol of one treatment per week for 6 sessions, one session every 2 weeks for 6 sessions, and then one session per month. The overall response rate was 62%: two patients achieved a complete response and 6 patients achieved a partial response. The median time to response was 10 months. The 2- and 4-year predicted overall survivals were 82%. This study was limited by its retrospective nature and small sample size. Response and survival compare favorably with those of previous studies. Our modified treatment protocol appears to produce outcomes similar to the two-day protocol.

Negative selection of chronic lymphocytic leukaemia cells using a bifunctional rosette-based antibody cocktail.

BACKGROUND: High purity of tumour samples is a necessity for accurate genetic and expression analysis and is usually achieved by positive selection in chronic lymphocytic leukaemia (CLL). RESULTS: We adapted a bifunctional rosette-based antibody cocktail for negative selection of B-cells for isolating CLL cells from peripheral blood (PB). PB samples from CLL patients were split into aliquots. One aliquot of each sample was enriched by density gradient centrifugation (DGC), while the other aliquot of each sample was incubated with an antibody cocktail for B-cell enrichment prior to DGC (RS+DGC). The purity of CLL cells after DGC averaged 74.1% (range: 15.9 - 97.4%). Using RS+DGC, the purity averaged 93.8% (range: 80.4 - 99.4%) with 23 of 29 (79%) samples showing CLL purities above 90%. RNA extracted from enriched CLL cells was of appropriately high quality for microarray analysis. CONCLUSION: This study confirms the use of a bifunctional rosette-based antibody cocktail as an effective method for the purification of CLL cells from peripheral blood.

Patients with multiple myeloma treated with thalidomide: evaluation of clinical parameters, cytokines,angiogenic markers, mast cells and marrow CD57+ cytotoxic T cells as predictors of outcome.

BACKGROUND AND OBJECTIVES: In vitro studies suggest that thalidomide has an immunoregulatory role and alters the marrow microenvironment. We assessed laboratory and clinical parameters in patients with myeloma treated with thalidomide as potential prognostic markers and looked for changes with therapy. DESIGN AND METHODS: Seventy-five patients with relapsed/refractory myeloma received thalidomide in a phase II trial. Serial samples of platelet-poor plasma and bone marrow were tested for angiogenic cytokines including vascular endothelial growth factor (VEGF), marrow microvessel-density (MVD), mast cells and CD57+ cell expression. The effects of these parameters on response rate (RR), progression-free survival (PFS) and overall survival (OS) were analyzed. RESULTS: Elevated baseline VEGF predicted for a superior RR (p=0.018) and PFS. Elevated CD57+ cells also predicted superior PFS (p=0.012). MVD did not predict for RR, PFS or OS, but MVD and VEGF fell significantly in responders. Multivariate analysis identified that inferior OS was associated with age >65 years (p=0.017), raised lactate dehydrogenase (p=0.001), raised hepatocyte growth factor levels (p=0.012) and low pre-treatment CD57+ cells (p<0.001). INTERPRETATION AND CONCLUSIONS: Our findings support the suggestion that thalidomide has anti-angiogenic and immunomodulatory effects in myeloma. The preferred method for assessing angiogenesis is plasma VEGF levels and the assessment of CD57+ cells for patients with myeloma receiving novel immunomodulatory drugs should be further investigated.