Conservative Whole Blood Extended Differential Method.

Abstract

The neutrophilic precursors (excluding bands), blast cells and erythroblasts normally reside in the bone marrow. The presence of any of these precursors as well as atypical/variant lymphocytes in peripheral blood is indicative of a diseased or disordered state. Therefore, it is of clinical importance to identify and enumerate these atypical cell populations. In general, commercial hematology systems do not have the ability to enumerate all of the nucleated atypical cells that are traditionally identified by manual differential. However, the manual differential is labor intensive and can be highly variable between qualified readers resulting in significant cost and quality assurance issues for the laboratory. We have developed a unique fluorescent based Extended Differential method that uses a minimum number of monoclonal antibodies in combination with a cell permeant metachromatic nucleic acid dye to detect circulating atypical cells. Data will be presented comparing the results of this approach with the manual differential. The current study was comprised of 579 clinical samples containing a variety of atypical cell types as determined by morphological examination. Analysis of difference plots comparing the Extended Differential method with the manual differential revealed good agreement between the percent of immature granulocytes (IGs) and the number of nucleated red blood cells (NRBCs) per 100 white blood cells (WBCs). The same analysis applied to the determination of blast percent exposed a greater frequency of disparate results. These disparate results were further investigated using a comprehensive monoclonal antibody based reference method incorporating a variety of specificities including anti-CD45 and anti-CD34. The data demonstrated that cells classified as atypical/variant lymphocytes or smudge cells by morphological examination were classified as blasts utilizing CD45 and/or CD34 expression with the Extended Differential and flow reference methods. In addition to the difference analyses, the results obtained with manual differential and Extended Differential methods were subjected to Receiver Operating Characteristics (ROC) analysis in order to determine the overall analytical sensitivity and specificity using a threshold of 1% or greater as the criteria for the presence of atypical cells. The ROC analysis demonstrated areas under the curve (AUC) of 0.9743, 0.9831 and 0.9643 for IGs, blasts and NRBCs, respectively showing good agreement for the presence or absence of the respective atypical cell populations (p < 0.0001).