Abstract
An improved method is described for the enumeration of lymphocyte surface markers in whole peripheral blood using reagents labelled with alkaline phosphatase. A suspension of washed whole blood is exposed to the labelled reagents and then smeared on slides. Endogenous peroxidase in monocytes is detected by the diaminobenzidine reaction amplified by osmication, and this identifies more cells than are recognised as monocytes by morphological criteria in Romanovsky-stained films. Lymphocytes are identified as peroxidase-negative mononuclear cells and those binding alkaline phosphatase-labelled reagents are demonstrated by treating the smears with naphthol ASMX phosphoric acid and fast red TR salt. By avoiding the loss of lymphocytes which is inevitable in any procedure for isolation of mononuclear cells from the blood, and by permitting elimination of monocytes from the counts, this method enables the proportion and absolute number of different circulating lymphocyte populations to be accurately enumerated. In the peripheral blood of seventeen normal individuals alkaline phosphatase rabbit F(ab)'2 anti-human immunoglobulin stained the following numbers (mean +/- s.d.) of lymphocytes, 9-0 +/- 1-5%, 214 +/- 66/microliter (B cells), and specific rabbit anti-T-cell serum followed by alkaline phosphatase goat F(ab)'2 anti-rabbit immunoglobulin stained 77 +/- 3%, 1846 +/- 488/microliter (T cells). The method, which is applicable to any surface marker which can be detected on living cells in suspension with a soluble reagent, provides permanent preparations which are counted in an ordinary light microscope and permits the use of counterstaining to reveal cellular morphology. Provided that appropriate specific reagents are available it is therefore suitable for routine clinical application.
Full text
PDF





Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Avrameas S. Coupling of enzymes to proteins with glutaraldehyde. Use of the conjugates for the detection of antigens and antibodies. Immunochemistry. 1969 Jan;6(1):43–52. doi: 10.1016/0019-2791(69)90177-3. [DOI] [PubMed] [Google Scholar]
- Brown G., Greaves M. F. Enumeration of absolute numbers of T and B lymphocytes in human blood. Scand J Immunol. 1974;3(2):161–172. doi: 10.1111/j.1365-3083.1974.tb01244.x. [DOI] [PubMed] [Google Scholar]
- Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
- Li C. Y., Lam K. W., Yam L. T. Esterases in human leukocytes. J Histochem Cytochem. 1973 Jan;21(1):1–12. doi: 10.1177/21.1.1. [DOI] [PubMed] [Google Scholar]
- Lobo P. I., Horwitz D. A. An appraisal of Fc receptors on human peripheral blood B and L lymphocytes. J Immunol. 1976 Sep;117(3):939–943. [PubMed] [Google Scholar]
- Lobo P. I., Westervelt F. B., Horwitz D. A. Identification of two populations of immunoglobulin-bearing lymphocytes in man. J Immunol. 1975 Jan;114(1 Pt 1):116–119. [PubMed] [Google Scholar]
- Nakane P. K., Kawaoi A. Peroxidase-labeled antibody. A new method of conjugation. J Histochem Cytochem. 1974 Dec;22(12):1084–1091. doi: 10.1177/22.12.1084. [DOI] [PubMed] [Google Scholar]
- Pepys M. B., Sategna-Guidetti C., Mirjah D. D., Wansbrough-Jones M. H., Dash A. C. Enumeration of immunoglobulin-bearing lymphocytes in whole peripheral blood. Clin Exp Immunol. 1976 Oct;26(1):91–94. [PMC free article] [PubMed] [Google Scholar]
- Preud'homme J. L., Flandrin G. Identification by peroxidase staining of monocytes in surface immunofluorescence tests. J Immunol. 1974 Nov;113(5):1650–1653. [PubMed] [Google Scholar]
- Stage D. E., Avrameas S. Detection of anti-TNP antibody-forming cells (AFC) with TNP-enzyme and TNP-Fab anti-enzyme conjugates. J Immunol Methods. 1976 Mar;10(2-3):105–118. doi: 10.1016/0022-1759(76)90161-7. [DOI] [PubMed] [Google Scholar]
- Winchester R. J., Fu S. M., Hoffman T., Kunkel H. G. IgG on lymphocyte surfaces; technical problems and the significance of a third cell population. J Immunol. 1975 Apr;114(4):1210–1212. [PubMed] [Google Scholar]