Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer.
BACKGROUND
Flow cytometry allows the use of several antibodies in addition to light scatter, and most flow cytometers will provide at least seven measurements on each cell passing through the laser beam. A skilled microscopist will classify at least 14 cell classes in bone marrow or blood. Our goal was to use the seven parameters available in our flow cytometer to provide a reliable differential count using only one tube.


METHODS
Peripheral blood samples were analyzed on the Beckman Coulter LH750 cell counter, and the flagging and messages from the cell counter were used to select normal or pathological samples. Samples without flags (N = 50), with >2% erythroblasts (N = 80), or with "Blast" or "Verify diff" flags (N = 54) were investigated. We used a lyse-no-wash method to ensure minimal loss of fragile cells with live gating on DRAQ5-positive cells to acquire only nucleated cells. The FL-1 to FL-4 channels were 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.


RESULTS
Using live gate acquisition on DRAQ5, we were able to classify total nucleated cells into 10 classes. We were unable to identify megakaryocytes, but platelets could be studied by rerunning the sample after dilution and gating on DRAQ5-negative CD36-posive events. Validation against digitized microscopy and cell counter showed linear correlations within each cell class with correlation coefficients that seem reasonable for cellular classification. The lowest correlation was found for basophil granulocytes. Flow cytometry detected twice as many immature neutrophils compared to microscopy.


CONCLUSIONS
We have designed a one-tube immunophenotyping panel for classification of total nucleated cells and platelets in blood or bone marrow. The seven parameters available in one single tube in our cytometer seem to be enough for reliable differential count even in difficult pathological samples. The analytical imprecision of the flow cytometer differential was much lower than that obtained with microscopy or cell counter differentials.
Department of Clinical Chemistry, University Hospital MAS, S 205 02 Malmö, Sweden. Sven.Bjornsson@skane.se
Sven
S
Björnsson
Saga
S
Wahlström
Eva
E
Norström
Ingela
I
Bernevi
Ulla
U
O'Neill
Eva
E
Johansson
Håkan
H
Runström
Per
P
Simonsson
D001772:Blood Cell Count / Q000295:instrumentation / Q000379:methods; D001854:Bone Marrow Cells / Q000145:classification / Q000166:cytology; D002467:Cell Nucleus; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods*; D006402:Hematologic Diseases / Q000097:blood / Q000175:diagnosis; D006801:Humans; D016130:Immunophenotyping / Q000295:instrumentation* / Q000379:methods*; D007958:Leukocyte Count / Q000295:instrumentation* / Q000379:methods*; D008853:Microscopy / Q000592:standards; D010976:Platelet Count / Q000295:instrumentation / Q000379:methods; D015203:Reproducibility of Results