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Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer.

Björnsson, Sven LU ; Wahlström, Saga; Norström, Eva LU ; Bernevi, Ingela; O'Neill, Ulla; Johansson, Eva; Runström, Håkan and Simonsson, Per LU (2008) In Cytometry Part B: Clinical Cytometry 74B. p.91-103
Abstract
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... (More)
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. (c) 2007 Clinical Cytometry Society. (Less)
Please use this url to cite or link to this publication:
author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Cytometry Part B: Clinical Cytometry
volume
74B
pages
91 - 103
publisher
John Wiley & Sons
external identifiers
  • pmid:18061952
  • wos:000254724400002
  • scopus:42549115147
ISSN
1552-4949
DOI
10.1002/cyto.b.20382
language
English
LU publication?
yes
id
e0261248-ae1c-4923-ba13-48b8815fe3d6 (old id 1035587)
alternative location
http://www.ncbi.nlm.nih.gov/pubmed/18061952?dopt=Abstract
date added to LUP
2008-02-28 12:12:38
date last changed
2017-11-05 04:42:02
@article{e0261248-ae1c-4923-ba13-48b8815fe3d6,
  abstract     = {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. (c) 2007 Clinical Cytometry Society.},
  author       = {Björnsson, Sven and Wahlström, Saga and Norström, Eva and Bernevi, Ingela and O'Neill, Ulla and Johansson, Eva and Runström, Håkan and Simonsson, Per},
  issn         = {1552-4949},
  language     = {eng},
  pages        = {91--103},
  publisher    = {John Wiley & Sons},
  series       = {Cytometry Part B: Clinical Cytometry},
  title        = {Total nucleated cell differential for blood and bone marrow using a single tube in a five-color flow cytometer.},
  url          = {http://dx.doi.org/10.1002/cyto.b.20382},
  volume       = {74B},
  year         = {2008},
}