Enumeration of Lymphocyte Populations in Whole Peripheral Blood with Alkaline Phosphatase-labelled Reagents. A Method for Routine Clinical Use

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.