PMID- 23201386
OWN - NLM
STAT- MEDLINE
DCOM- 20130524
LR  - 20191210
IS  - 1872-7905 (Electronic)
IS  - 0022-1759 (Linking)
VI  - 388
IP  - 1-2
DP  - 2013 Feb 28
TI  - Accurate measurement of peripheral blood mononuclear cell concentration using 
      image cytometry to eliminate RBC-induced counting error.
PG  - 25-32
LID - S0022-1759(12)00343-2 [pii]
LID - 10.1016/j.jim.2012.11.010 [doi]
AB  - Peripheral blood mononuclear cells (PBMCs) have been widely researched in the 
      fields of immunology, infectious disease, oncology, transplantation, 
      hematological malignancy, and vaccine development. Specifically, in immunology 
      research, PBMCs have been utilized to monitor concentration, viability, 
      proliferation, and cytokine production from immune cells, which are critical for 
      both clinical trials and biomedical research. The viability and concentration of 
      isolated PBMCs are traditionally measured by manual counting with trypan blue 
      (TB) using a hemacytometer. One of the common issues of PBMC isolation is red 
      blood cell (RBC) contamination. The RBC contamination can be dependent on the 
      donor sample and/or technical skill level of the operator. RBC contamination in a 
      PBMC sample can introduce error to the measured concentration, which can pass 
      down to future experimental assays performed on these cells. To resolve this 
      issue, RBC lysing protocol can be used to eliminate potential error caused by RBC 
      contamination. In the recent years, a rapid fluorescence-based image cytometry 
      system has been utilized for bright-field and fluorescence imaging analysis of 
      cellular characteristics (Nexcelom Bioscience LLC, Lawrence, MA). The Cellometer 
      image cytometry system has demonstrated the capability of automated concentration 
      and viability detection in disposable counting chambers of unpurified mouse 
      splenocytes and PBMCs stained with acridine orange (AO) and propidium iodide (PI) 
      under fluorescence detection. In this work, we demonstrate the ability of 
      Cellometer image cytometry system to accurately measure PBMC concentration, 
      despite RBC contamination, by comparison of five different total PBMC counting 
      methods: (1) manual counting of trypan blue-stained PBMCs in hemacytometer, (2) 
      manual counting of PBMCs in bright-field images, (3) manual counting of acetic 
      acid lysing of RBCs with TB-stained PBMCs, (4) automated counting of acetic acid 
      lysing of RBCs with PI-stained PBMCs, and (5) AO/PI dual staining method. The 
      results show comparable total PBMC counting among all five methods, which 
      validate the AO/PI staining method for PBMC measurement in the image cytometry 
      method.
CI  - Copyright (c) 2012 Elsevier B.V. All rights reserved.
FAU - Chan, Leo Li-Ying
AU  - Chan LL
AD  - Department of Technology R&D, Nexcelom Bioscience LLC, Lawrence, MA 01843, USA. 
      lchan@nexcelom.com
FAU - Laverty, Daniel J
AU  - Laverty DJ
FAU - Smith, Tim
AU  - Smith T
FAU - Nejad, Parham
AU  - Nejad P
FAU - Hei, Hillary
AU  - Hei H
FAU - Gandhi, Roopali
AU  - Gandhi R
FAU - Kuksin, Dmitry
AU  - Kuksin D
FAU - Qiu, Jean
AU  - Qiu J
LA  - eng
PT  - Comparative Study
PT  - Journal Article
PT  - Validation Study
DEP - 20121129
PL  - Netherlands
TA  - J Immunol Methods
JT  - Journal of immunological methods
JID - 1305440
RN  - 36015-30-2 (Propidium)
RN  - F30N4O6XVV (Acridine Orange)
SB  - IM
MH  - Acridine Orange/chemistry
MH  - Blood Cell Count/*methods
MH  - Erythrocytes/chemistry/*cytology
MH  - Humans
MH  - Image Cytometry/*methods
MH  - Leukocytes, Mononuclear/*chemistry/*cytology
MH  - Propidium/chemistry
EDAT- 2012/12/04 06:00
MHDA- 2013/05/28 06:00
CRDT- 2012/12/04 06:00
PHST- 2012/10/15 00:00 [received]
PHST- 2012/11/12 00:00 [revised]
PHST- 2012/11/13 00:00 [accepted]
PHST- 2012/12/04 06:00 [entrez]
PHST- 2012/12/04 06:00 [pubmed]
PHST- 2013/05/28 06:00 [medline]
AID - S0022-1759(12)00343-2 [pii]
AID - 10.1016/j.jim.2012.11.010 [doi]
PST - ppublish
SO  - J Immunol Methods. 2013 Feb 28;388(1-2):25-32. doi: 10.1016/j.jim.2012.11.010. 
      Epub 2012 Nov 29.