Fully Automated Identification of Dysplastic Neutrophil in Peripheral Blood Using the Positional Parameters Provided by the Coulter LH750 Analyzer.
Authors: Zini Gina , Garzia Mariagrazia , Di Mario Antonella , Ricerca Bianca Maria , Piccioni Paola , Tarnani Michela , d’Onofrio Giuseppe 
Published: April 2020

The hematology analyzer Coulter LH750 provides WBC differential counts using the VCS technology, which combines three simultaneous measurements (Volume, Conductivity, Scatter) to classify circulating blood cells. Cell volume and internal cell structure (opacity) are detected, respectively, by applying a low frequency direct current (DC) and a high frequency alternative (RF) current. The light scattered at different angles between 10° and 70°, on the other hand, is proportional to cell size, granularity, surface topography and reflectance. The system also provides for the main four WBC subtypes (neutrophils, lymphocytes, monocytes, eosinophils) a set of data called ‘positional parameters’ (PP). They express the mean value and the standard deviation (SD) of the three VCS measurements. We focused our study to the analysis of PP of the neutrophil population in normal and in pathological samples in order to: i) evaluate any correspondence with morphology and ii) determine the predictivity of changes in these parameters for the presence of morphological abnormalities. We analysed 100 samples from healthy subjects, 59 males and 41 females, age ranged from 2 years to 79 years, (25 children, 75 adults) to define our neutrophil reference PP ranges. We found a mean volume of 151,7 (SD ± 22,5), a mean opacity of 143,9 (SD ± 7,4) and a mean scatter of 142,19 (SD ± 13,1), without consistent differences due to age or sex. Our results do agree with those found in the literature. In normal samples we could not observe, as expected, any morphological abnormality at the microscope.

Thereafter we also analyzed 16 samples from patients with bacterial pneumonitis. Their PP showed a moderate increase in the mean volume (168,9 with SD ± 24,1), a normal value of opacity and a slight decrease of the mean scatter (138,2 with SD ±14.4). Microscope analysis showed granulocytes with normal morphology, sometimes with toxic granules and the presence of band forms and metamyelocytes above 7%. The analysis of the PP of 11 patients with chronic myeloid leukemia at diagnosis showed an increase in the mean volume (160,1) with a consistently highly SD (± 33,4); opacity (142,8 with SD ± 11,7) and scatter (143,9 with SD ± 12,3) were normal. At the microscope the morphology of neutrophils was normal, with the presence of circulating immature granulocytes (range 22–28%). Finally we analyzed samples from N 28 patients with myelodysplastic syndromes (14 RA, 5 RC, 9 RAEB Type 1). Circulating granulocytes showed a great increase in the mean volume with a wide dispersion (178,2 with SD ± 35,6 ), a decreased mean opacity (132 with SD ± 8,3) and very low mean scatter value (119,9 with SD ± 12,4). At microscope neutrophils showed characteristic dysplastic features, such as abnormal nuclear segmentation, chromatin clumping and hypogranularity. Our study demonstrates the Coulter LH750 PP are highly predictive of the presence of dysplastic neutrophils in the peripheral blood. We have found a peculiar data set (high volume with increased SD, low opacity and scatter with small SD) which is easily distinguishable from normal as well as from other types of neutrophil abnormalities, such as myloproliferative and bacterial diseases. These parameters are automatically available during the routine load of hematology laboratories and can be usefully utilized to screen and to early identify patients with mylodysplasia.