Detection of ZAP70 Negative B-CLL Patients Using the Beckman Coulters Positional Parameter Technology and RPD (Research Population Data) Analysis.
Authors: Deutsch Varda , Herishanu Yair , Polliack Aaron , Naparstek Elizabeth , Meyuhas Dina , Kay Sigi 
Published: April 2020

Abstract
B-CLL, the most common type of leukemia in adults, is heterogeneous with a varying clinical course ranging from indolent to aggressive disease. CLL patients with mutated Ig V(H) genes have a good prognosis while those with non-mutated Ig V(H) status and expression of the ZAP70 protein tyrosine kinase generally have aggressive disease and shorter survival. Testing for IgV(H) mutations is not routinely performed due to cost and ZAP70 testing is still technically difficult. As these tests remain generally inappropriate for B-CLL patient screening, we tested the capacity of the new Beckmann Coulter LH750 blood analyzers to provide additional morphometric information on the CLL cell population. These analyzers use a highly informative positional parameter technology - VCS (Volume, Conductivity, Scatter) technology, and automated RPD analysis which provide the Mean and Standard Deviation of Volume, Conductivity and Scatter (VCS) for each of the main WBC types (neutrophils, lymphocytes, monocytes and eosinophils), thereby enabling the detection of abnormal WBC populations associated with various hematological and non-hematological disorders. We attempted to determine if the RPD generated could differentiate between the ZAP70pos and ZAP70neg B-CLL subgroups as determined by high resolution quantitative ZAP70 flow cytometry analysis (1). This study included 30 newly diagnosed untreated CLL patients. CBC was performed on all samples using automated leukocyte differentials in the Beckmann-Coulter LH750. Statistical analysis was performed with Medcalc® 8.1.1.0. Results show that there was no correlation between the WBC number, ZAP70pos expression (found in 59% of the cases) and lymphocyte volume, scatter, or conductivity or SD of these parameters. However, differences in the automated morphology of the Zap70neg compared with Zap70pos CLL lymphocytes was detected. When compared to normal lymphocyte volume (85.8±14.5), the Lymphocyte Mean Volume (MLYV) was lower in the ZAP70neg samples (77.7±17.6) (p&lt;0.05), while the ZAP70pos samples had increased volumes (87.5±15.8), similar to values reported in other myeloproliferative states. The conductivity of the ZAP70neg cells was reduced (110±15.0) below normal (117±11.5) (p=0.038), while the ZAP70pos cells had conductivity similar to the control group. Higher levels of lymphocyte apoptosis were clearly observed in the ZAP70neg samples by VCS. When using ROC (receiver operating curve analysis) to detect the potential ZAP70neg cases, the cut off proposed is MLYV &lt; 76 with a sensitivity of 69.2% and a specificity of 70% AUC 0,650. Herein we propose an entirely new approach which may provide a novel diagnostic screening approach to enable identification of CLL patients who do not require further complex testing. This data is derived directly from the CBC of the Beckman-Coulter LH750 analyzers at no extra cost. As it will be necessary to analyze many more cases from different laboratories, an international working group has been formed in an attempt to confirm these early observations.