Abstract
Blood is filtered for selective removal of leukocytes (WBC) to reduce the immunological and virological risks of transfusion. Exceedingly low numbers of residual WBC in leukodepleted blood cannot be enumerated by conventional hematologic methods. Therefore, we investigated the application of a DNA enzyme immunoassay (DEIA) for detecting a region of the HLA-DQ alpha gene following amplification by PCR. After hybridization with a specific probe coated onto the wells of a microtiter plate, the PCR-amplified DNA was detected by adding monoclonal antibodies to double-stranded DNA, enzyme tracer, and chromogen substrate for colorimetric measurement. The sensitivities of DEIA and radioisotopic liquid hybridization were similar in five sets of experiments performed with a known number of human WBC. The optical density and the number of spiked human WBC in the range of 1.0 to 0.05 cells per microliter showed good correlation in five calibration experiments performed with human WBC suspended in heterologous blood. Using a calibration curve for DEIA, we estimated the concentration of residual WBC in five individual units of erythrocytes passed through blood filters. The postfiltration WBC count was 1.6 WBC per microliter in one unit, while in four other units it was below the lower detection limit (< 0.05 WBC per microliter) of the DEIA. DEIA obviates the use of radioisotopes in PCR for detection of exceedingly low numbers of residual WBC in filtered blood.
Full Text
The Full Text of this article is available as a PDF (165.9 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Buffone G. J., Darlington G. J. Isolation of DNA from biological specimens without extraction with phenol. Clin Chem. 1985 Jan;31(1):164–165. [PubMed] [Google Scholar]
- Erlich H. A., Gelfand D., Sninsky J. J. Recent advances in the polymerase chain reaction. Science. 1991 Jun 21;252(5013):1643–1651. doi: 10.1126/science.2047872. [DOI] [PubMed] [Google Scholar]
- Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Loeffelholz M. J., Lewinski C. A., Silver S. R., Purohit A. P., Herman S. A., Buonagurio D. A., Dragon E. A. Detection of Chlamydia trachomatis in endocervical specimens by polymerase chain reaction. J Clin Microbiol. 1992 Nov;30(11):2847–2851. doi: 10.1128/jcm.30.11.2847-2851.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mantero G., Zonaro A., Albertini A., Bertolo P., Primi D. DNA enzyme immunoassay: general method for detecting products of polymerase chain reaction. Clin Chem. 1991 Mar;37(3):422–429. [PubMed] [Google Scholar]
- Masse M., Naegelen C., Pellegrini N., Segier J. M., Marpaux N., Beaujean F. Validation of a simple method to count very low white cell concentrations in filtered red cells or platelets. Transfusion. 1992 Jul-Aug;32(6):565–571. doi: 10.1046/j.1537-2995.1992.32692367203.x. [DOI] [PubMed] [Google Scholar]
- Rapier J. M., Villamarzo Y., Schochetman G., Ou C. Y., Brakel C. L., Donegan J., Maltzman W., Lee S., Kirtikar D., Gatica D. Nonradioactive, colorimetric microplate hybridization assay for detecting amplified human immunodeficiency virus DNA. Clin Chem. 1993 Feb;39(2):244–247. [PubMed] [Google Scholar]
- Rawal B. D., Davis R. E., Busch M. P., Vyas G. N. Dual reduction in the immunologic and infectious complications of transfusion by filtration/removal of leukocytes from donor blood soon after collection. Transfus Med Rev. 1990 Oct;4(4 Suppl 1):36–41. doi: 10.1016/s0887-7963(90)70240-4. [DOI] [PubMed] [Google Scholar]
- Rawal B. D., Schwadron R., Busch M. P., Endow R., Vyas G. N. Evaluation of leukocyte removal filters modelled by use of HIV-infected cells and DNA amplification. Blood. 1990 Nov 15;76(10):2159–2161. [PubMed] [Google Scholar]
- Rebulla P., Porretti L., Bertolini F., Marangoni F., Prati D., Smacchia C., Pappalettera M., Parravicini A., Sirchia G. White cell-reduced red cells prepared by filtration: a critical evaluation of current filters and methods for counting residual white cells. Transfusion. 1993 Feb;33(2):128–133. doi: 10.1046/j.1537-2995.1993.33293158044.x. [DOI] [PubMed] [Google Scholar]
- Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
- Sato H., Okochi K. Transmission of human T-cell leukemia virus (HTLV-I) by blood transfusion: demonstration of proviral DNA in recipients' blood lymphocytes. Int J Cancer. 1986 Mar 15;37(3):395–400. doi: 10.1002/ijc.2910370311. [DOI] [PubMed] [Google Scholar]
- Yang G., Garhwal S., Olson J. C., Vyas G. N. Flow cytometric immunodetection of human immunodeficiency virus type 1 proviral DNA by heminested PCR and digoxigenin-labeled probes. Clin Diagn Lab Immunol. 1994 Jan;1(1):26–31. doi: 10.1128/cdli.1.1.26-31.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yang G., Ulrich P. P., Aiyer R. A., Rawal B. D., Vyas G. N. Detection of hepatitis B virus in plasma using flow cytometric analyses of polymerase chain reaction-amplified DNA incorporating digoxigenin-11-dUTP. Blood. 1993 Feb 15;81(4):1083–1088. [PubMed] [Google Scholar]