Abstract
Lymphocyte proliferation in response to mitogens, phytohemagglutinin (PHA), concanavalin A, pokeweed, and/or specific antigens has been the method of choice for in vitro diagnosis of cell‐mediated immune dysfunction. Recently, an assay to measure intracellular adenosine triphosphate (ATP) production in response to PHA has been developed that requires a shorter, overnight incubation. We compared a standard 5‐ to 7‐day lymphocyte mitogen stimulation assay utilizing tritiated thymidine (3H‐thy) incorporation to one in which ATP production in response to PHA by CD4‐positive cells is measured in a luminometer that requires only 18–24 hr. A total of 20 patient samples suspected of having decreased cell‐mediated immunity submitted for mitogen induced lymphocyte proliferation and 21 normal controls were tested in both assays. A comparison of these two methods has demonstrated that the screening ATP assay has a sensitivity at 24 hr of 100% in detecting decreased PHA induced lymphocyte proliferation at 5 days and a specificity of 85% in the samples obtained from normal controls. The data indicate that the ATP assay may be a useful screening tool for more rapid detection of blood samples with decreased cell‐mediated immune responses. However, a positive screen should always be confirmed by 3H‐thy uptake using mitogens and recall antigens like candida and tetanus. J. Clin. Lab. Anal. 21:265–270, 2007. © 2007 Wiley‐Liss, Inc.
Keywords: lymphocyte proliferation, cell‐mediated immunity, immunodeficiency, severe combined immunodeficiency disease (SCID), CD4 cells, adenosine triphosphate (ATP), luminescence
REFERENCES
- 1. Hill HR. Laboratory aspects of immune deficiency in children. Pediatr Clin North Am 1980;27:805–830. [DOI] [PubMed] [Google Scholar]
- 2. Puck JM. Primary immunodeficiency diseases. JAMA 1997;278: 1835–1841. [PubMed] [Google Scholar]
- 3. Shyur SD, Hill HR. Immunodeficiency in the 1990's. Pediatr Infect Dis J 1991;10:595–611. [DOI] [PubMed] [Google Scholar]
- 4. Shyur SD, Hill HR. Recent advances in the genetics of primary immunodeficiency syndromes. J Pediatr 1996;129:8–24. [DOI] [PubMed] [Google Scholar]
- 5. Kowalski R, Post D, Schneider MC, et al. Immune cell function testing: an adjunct to therapeutic drug monitoring in transplant patient management. Clin Transplant 2003;17:77–88. [DOI] [PubMed] [Google Scholar]
- 6. Sottong PR, Rosebrock JA, Britz JA, Kramer TR. Measurement of T‐lymphocyte responses in whole blood cultures using newly synthesized DNA and ATP. Clin Diagn Lab Immunol 2000;7:307–311. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7. Fleisher TA, Tomar RH. Introduction to diagnostic laboratory immunology. JAMA 1997;278:1823–1834. [PubMed] [Google Scholar]
- 8. Fletcher MA, Klimas N, Morgan R, Gjerset G. Manual of clinical laboratory immunology, 4th edition. 1994. p 213–219.
- 9. Ling NR, Kay JE, editors. Lymphocyte stimulation.Amsterdam: Elsevier North Holland Publishing Company; 1975; 398p. [Google Scholar]
- 10. Moller G. Lymphocyte activation by mitogens. Transplant Rev 1972;11:1–267. [DOI] [PubMed] [Google Scholar]
- 11. Schiff RI, Buckley RH, Gilbertson RB, Metzger RS. Membrane receptors and in vitro responsiveness of lymphocytes in human immunodeficiency. J Immunol 1974;112:376–386. [PubMed] [Google Scholar]
- 12. ImmuKnow™ Package Insert . Columbia, MD: Cylex® Incorporated; June, 2005. [Google Scholar]
- 13. Hooper E, Hawkins DM, Kowalski RJ, et al. Establishing pediatric immune response zones using the Cylex® ImmuKnow™ assay. Clin Transplant 2005;19:834–839. [DOI] [PubMed] [Google Scholar]
- 14. Hickling JK. Measuring human T‐lymphocyte function. Expert Rev Mol Med 1998;1998:1–20. [DOI] [PubMed] [Google Scholar]
- 15. Prince HE, Lape‐Nixon M. 1997. CD 69 expression reliably predicts the anti‐CD3‐induced proliferative response of lymphocytes from human immunodeficiency virus type 1‐infected patients. Clin Diag Lab Immun 1997;4:217–222. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Simms PE, Ellis TM. Utility of flow cytometric detection of CD 69 expression as a rapid method for determining poly‐ and oligoclonal lymphocyte activation. Clin Diag Lab Immun 1996;3:301–304. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17. Myers LA, Dhavalkumar DP, Puck JM, Buckley RH. Hematopoietic stem cell transplantation for severe combined immunodeficiency in the neonatal period leads to superior thymic output and improved survival. Blood 2002;99:872–878. [DOI] [PubMed] [Google Scholar]