Analytical and biological considerations in the measurement of cell-associated CCR5 and CXCR4 mRNA and protein.
The accurate measurement of T cell-associated CC chemokine receptor type 5 (CCR5) and CXC chemokine receptor type 4 (CXCR4) expression, including expression of CCR5 and CXCR4 mRNA as an immune measure of immunologic response to highly active antiretroviral therapy (HAART) and newer agents, including entry inhibitors, is essential. Previous studies have reported alterations in lymphocyte cell membrane CCR5 expression that were related to blood collection and cell separation media. Clinical trials often require the transport of specimens to central laboratories for evaluation, resulting in significant time delays between specimen procurement and analysis. This study shows that CCR5 expression on naïve and memory T cells is influenced by blood collection media and specimen age. Peripheral blood collected in Streck Vacutainer tubes containing a cell stabilizer and fixative was found to improve detection of CCR5 expression compared to specimens collected in K2 EDTA anticoagulant. The selection of flow cytometry gating strategies for the identification of naïve and memory T-helper cells can also significantly influence the sensitivity of detection of CCR5 expression. Procedural methods are described that allow for the optimal measurement of naïve and memory T-helper cell CCR5 and CXCR4 expression as well as the quantitation of CCR5 and CXCR4 mRNA.
Campbell
D E
DE
Division of Allergy and Immunology, The Children's Hospital of Philadelphia, 34th St. and Civic Center Blvd., Philadelphia, PA 19104, USA. campbelld@email.chop.edu
Lai
J P
JP
Tustin
N B
NB
Riedel
E
E
Tustin
R
R
Taylor
J
J
Murray
J
J
Douglas
S D
SD
D005434:Flow Cytometry / Q000379:methods; D007109:Immunity; D008722:Methods; D012333:RNA, Messenger / Q000032:analysis*; D019713:Receptors, CCR5 / Q000032:analysis* / Q000235:genetics; D019718:Receptors, CXCR4 / Q000032:analysis* / Q000235:genetics; D013601:T-Lymphocytes / Q000737:chemistry
D012333:RNA, Messenger; D019713:Receptors, CCR5; D019718:Receptors, CXCR4
JAMA. 2006 Aug 16;296(7):815-26
16905787
J Immunol Methods. 2003 Aug;279(1-2):199-207
12969561
Clin Diagn Lab Immunol. 2003 Nov;10(6):1123-8
14607877
N Engl J Med. 2002 Aug 8;347(6):385-94
12167680
Ann Intern Med. 2001 Jul 3;135(1):17-26
11434728
Proc Natl Acad Sci U S A. 2006 Sep 19;103(38):13950-5
16966601
Pediatr Infect Dis J. 2008 Aug;27(8):749-52
18664987
Mol Pharmacol. 2008 Mar;73(3):789-800
18096812
Science. 2005 Nov 11;310(5750):1025-8
16284180
Annu Rev Med. 2007;58:445-59
16958560
AIDS Res Hum Retroviruses. 2007 Mar;23(3):415-26
17411375
J Virol. 2006 Jan;80(2):802-9
16378982
J Infect Dis. 2009 Dec 1;200(11):1724-8
19874179
Drugs. 2009 May 29;69(8):1059-75
19496631
J Antimicrob Chemother. 2009 Sep;64 Suppl 1:i37-40
19675018
J Biol Chem. 2006 Nov 17;281(46):35446-53
16963439
N Engl J Med. 2008 Oct 2;359(14):1429-41
18832244