Practical Immunocytochemical Identification of Human Blood Cells

Abstract

A practical immunocytochemical method of demonstrating surface antigens of human blood cells on air-dried smears or other cytologic preparations has been developed. This method uses monoclonal antibodies as the primary antibodies and calf intestinal alkaline phosphatase as the enzymatic indicator. Combined staining with cytochemical stains for myeloperoxidase or nonspecific esterase on the same slide is also possible when needed. These methods are very useful for accurate identification of human blood cells on the commonly available clinical specimens and are very helpful in the diagnosis and classification of various hematologic neoplasms, including chronic lymphocytic leukemias, acute leukemias, and related diseases.

New Developments in Immunochemistry with Immunoalkaline Phosphatase Methods

“…These are direct, indirect, alkaline phosphatase-antialkaline phospha tase (APAAP) and variations of an avidinbiotin complex (ABC-AP) system (table I) [7][8][9][20][21][22][23][24], In the direct method the pri mary antibody reacting with the antigen is directly linked with the enzyme indicator. This method is seldom used because of its low sensitivity and also because it requires that each primary antibody be conjugated to the enzyme.…”

“…This method is seldom used because of its low sensitivity and also because it requires that each primary antibody be conjugated to the enzyme. In the indirect method the en zyme indicator is linked to antibodies (sec ondary antibodies) specific to the immuno globulin of the animal species in which the primary antibodies are produced [7,8,20]. This method is more versatile and sensitive than the direct method.…”

“…We have found that methods using naphthol-AS-phosphate as substrate with either fast red violet LB salt [8,13], fast blue BBN [28,34], or fast red KL salt [35] are most satisfactory ( fig. 1, 2).…”

“…The major concern here is pres ervation of cell morphology and sensitivity of staining. Of the several fixatives we have examined [8,20], a buffered formalin-ace tone mixture is the most satisfactory [8,20]. The addition of 0.1% Triton X-100 to this fixative not only will decrease nonspecific staining, but also will enhance penetration of reagents into the cells and therefore increase staining intensity [28].…”

Enrichment and characterization of dendritic cells from human bronchoalveolar lavages

“…Antigens on (un)fractionated BAL cells were detected by either an immunoenzymatic labelling using immune eomplexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes; Dakopatts, Glostrup, Denmark) according to Li et al [25] or an immunoperoxidase method using diaminobenzidinetetrahydrochloride (Sigma Chemical Co., St Louis, MO) as substrate as described in Havenith et al [13]. The MoAbs 9.3F10 (anti-HLA-DR/DQ; ATCC; [26]) and EBM 11 (anti-CD68; Dakopatts; [27]) were used.…”