The Utility of Extended White Blood Cell Differential by Flow Cytometry in Routine Hematology.

Abstract 4708

Today's Hematology instruments identify white blood cells (WBC) by their physical characteristics using various measurements such as Impedance, Conductivity and Light Scatter. A complete cell identification and enumeration is performed but is limited to only six cell types: neutrophils, lymphocytes, monocytes, eosinophils, basophils and NRBCs. Automation of the WBC manual differential is possible by flow cytometry for more accurate and automated information (van Lochem et al. 2004, M. Roussel et al. 2007, J. Feuillard et al. 2007).

The CytoDiff” tube is a 5-color / 6-marker panel that provides an extended cytometric differential for whole blood specimens. The CytoDiff” tube is comprised of: CD36-FITC, (CD2+CD294)-PE, CD19-ECD, CD16-PC5, and CD45-PC7 (Beckman Coulter, Inc. (BEC), For Investigational Use Only. The performance characteristics of this product have not been established).The peripheral blood (PB) specimens were lyzed using VersaLyse (BEC) to prevent selective loss of cells and to conserve light scatter characteristics. PB leukocytes were analyzed according to their immunofluorescence reactivity. At least 20,000 CD45+ events per sample were acquired on an FC500 Cytomics flow cytometer (BEC) and analyzed using CXP software (BEC). For each case with elevated numbers of leukocytes or with abnormal cells, additional analyses were performed using light microscopy and full panel extended flow cytometric immunophenotyping to identify lineages and the level of differentiation of the cells. PB was evaluated from 62 patients with different pathological conditions. Half the patients were females and half males, with adult median age 60. Four boys; median age 10.5, and two girls; median age 4.5 were also evaluated. The study was approved by The Pavlov State Medical University's Institutional Review Board, and the following results were obtained:

Two cases had no immunological abnormalities in WBC subsets. Extended immunophenotyping determined 13 lymphocytosis cases with more than 50% of nucleated cells (53.1–94.5%) to be B-lymphocytosis, with 12 B-ChLL and one PLL. One specimen with normal lymphocytes had high B-cells (45%) and extended immunophenotyping specified it as hairy cell leukemia.

Six cases were identified with B-blasts, nine with noTnoB (myelo) blasts and two with immature granulocytes. The analysis of these specimens using light microscopy, extended immunophenotyping and cytogenetics identified six B-cell precursors ALL, two cases of MDS transformation to AML, two relapses of AML with maturation, one new diagnosed case of AML with maturation, one case of AML with myelomonoblastic differentiation, three cases of residual disease after AML chemotherapy, one plasmoblastic leukemia (transformed after multiple myeloma) and one acute promyelocytic leukemia with t(15;17). Therefore, true positive blasts were identified in15 of 17 PB specimens using extended immunophenotyping. Two cases with immature granulocytes in PB were misclassified with the CytoDiff” tube but verified as plasmablastic and acute promyelocytic leukemia after bone marrow extended immunophenotyping and cytogenetics.

Nine cases had increased numbers of monocytes (14.2–32.0%). Three cases came from the surgery department, with more than 20% of monocytes being inflammatory CD16+. Two cases with multiple myeloma after chemotherapy and cytopenia recovery had 38.8% and 43.0% of monocytes being inflammatory CD16+. Another three cases (two with craniocerebral trauma, and one with phlegmona of mandibula) showed very low inflammatory monocytes (3.5–9.3%). There was one case with very high monocytes (32%) but none were inflammatory. The analysis of this specimen using light microscopy, extended immunophenotyping and cytogenetics identified promyelocytic leukemia.