[A study on the appropriate fixation for the procedures for the better preservation of cellular antigenicity and morphology of the blood smear in immunocytochemistry: an improvement of the immunostain technique using alkaline-phosphatase (ALP) as a labeling enzyme].

The authors previously reported a new coloration method which utilized hexazotized newfuchsin as a coupler for the immuno-enzyme-cytochemistry. This procedure used alkaline-phosphatase (ALP) as the labeling enzyme. The insolubility of the reaction product to organic solvents made it possible to prepare permanent slides. However, this suffered from several drawbacks, due to the fixation procedures, in the preservation of better morphology and antigenicity of the cell. The present study was undertaken to overcome such problems by modifying the fixation procedure. The study utilized twenty monoclonal and polyclonal antibodies commonly used in immunohematological staining. Various fixative solutions and timing of fixation were evaluated. The results indicated that; 1) the best fixative solution was a mixture of buffered paraformaldehyde (PFA) and acetone (10 ml 40% PFA solution, 10 ml pH 6.6 0.02 M phosphate buffer, 20 ml distilled water, 60 ml acetone, with pH adjusted to 6.6-7.4 with HCl) and 2) the fixation should be performed just before the immunostain. The results further showed that unstained smear slides, when freshly air dried and stored in a desicator, could maintain various differentiation antigens (CD2, 3, 4, 5, 8, 10, 14, 15, 16, 19, 25, L26, HLA-DR) for at least 4 weeks without any change in the immuno-reactivity. Thus, we conclude that this improved fixation procedure is an optimum fixative and should be used in routine application of the immunostain method for blood smears.