A Single Tube Antibody Panel for Differential Count by Flow Cytometry.
Authors: Bjornsson Sven , Wahlstrom Saga , Bernevi Ingela , Simonsson Per 
Published: October 2019

Abstract
Introduction
Several techniques are used for classification of leukocytes, i.e. microscopy, cell counters and flow cytometry. A skilled microscopist will classify at least 14 cell classes in bone marrow or blood. We have designed a one tube immunophenotyping panel for leukocyte classification and validated it against microscopy. Multiparameter flow cytometry provide at least seven measurements on each cell passing through the laser beam. Our goal was to use the seven parameters available in our flow cytometer to provide a reliable differential count using only one tube.
Materials and Methods
The FL-1 to FL-4 channels was used for the antibodies CD36-FITC, CD203-PE, CD138-PE, CD45-ECD, CD16-Pcy5 and CD56-Pcy5. FL-5 was used for the DNA-stain DRAQ5. We used a lyse no wash method to ensure minimal loss of fragile cells and 40000 events were aquired a on a Beckman Coulter FC500 flow cytometer with live gating on DRAQ5-positive cells to aquire only nucleated cells. Sequential gating was used to classify cells according to immunophenotypes given in Table 1. Peripheral blood samples collected in EDTA-tubes were analyzed on the Beckman Coulter LH750 cell counter. Cell counter differentials containing >2% erythroblasts (NRBC) was used to select 79 pathological samples included in the study. An extended microscopic evaluation following the NCCLS-H20A protocol was performed on two different slides using Cellavision DiffMaster™ Octavia digitilized microscopy.
Results
There was a linear correlation within each cell class between flow cytometry and morphology. The correlation coefficients (R-values) given in table 1 seem reasonable for cellular classification. There was a very good correlation between flow cytometric and morhpological classification of immature red cells (NRBC). There was, however, a low correlation for immature granulocytes and basophilic granulocytes. Immature granulocytes was defined as CD16-negative granulocytes in flow cytometry and exceeded the sum of promyelocytes, myelocytes and metamyelocytes in microscopy in all cases. Basophilic granulocytes was defined as CD203-positive events in flow cytometry, and as granulocytes with basophilic granula in microscopy. Flow cytometry basophil values were either lower or higher then microscopical values in the lower measuring range i.e. <1,5% basophils. Table 1
Conclusions
Flow cytometry is often used to detect malignant cells but may as well be used to classify normal cells in blood and bone marrow. The use of antibodies will allow accurate classification even if cellular morphology is pertuberated by disease or drug treatment but the extensive antibody panels commonly used are expensive and labour intensive. The seven parameters available in one single tube in a modern cytometer seem to be enough for reliable leucocyte classification even in difficult pathological samples. The main discrepancy between the methods seems to be in the classification of immature granulocytes and basophils.
Table 1 Cell Phenotype R-value (flow/morpholgy) N = 79 1 Monocytes CD36+++/SSC+/CD45++ 0,8686 2 Erythroblasts (NRBC) CD36++/CD45+ 0,9560 3 Neutrophiles, all stages SSC+++/CD45++ 0,9473 4 Neutrophiles, band + segm SSC+++/CD45++ /CD16+++ 0,8445 5 Immature granulocytes (promyelo/myelo/metamyelo) SSC+++/CD45++/CD16− 0,6975 6 Eosinophiles SSC+++/CD45+++/CD16− 0,8532 7 Plasma cells CD138++/CD45− - 8 Blasts CD36−/CD16−/CD203−/CD138−/CD45++/SSC− 0,8033 9 Basophiles CD203+/CD45++/SSC+ 0,6038 10 Lymphocytes CD203−/CD138−/CD45+++/SSC− 0,9741 11 NK-cells CD16+/CD56+/CD45+++/SSC− -