Comparison of Three Procedures for Cytochemical Detection of Leukocyte Alkaline Phosphatase Activity in Dogs With Leukemia

BACKGROUND: Assessment of alkaline phosphatase (ALP) activity in cytologic smears is used as a phenotyping tool for hematopoietic and solid tissue neoplasms in dogs. Different procedures that vary in substrate-dye combinations, fixative, incubation times, and nuclear counterstains are available for detection of ALP activity. It is unknown if these procedures provide comparable results for phenotyping tumors in the same animal. OBJECTIVE: To compare results obtained with three ALP cytochemical procedures in blasts in blood and tissue aspirate smears from dogs with previously diagnosed leukemia: naphthol-AS-MX phosphate/fast blue (fast blue), 5-bromo-4-chloro-3-indoxyl-phosphate/nitroblue tetrazolium (BCIP/NBT), and naphthol-AS-BI-phosphate/fast red violet LB (fast red). METHODS: Smears of blood, bone marrow, and lymph node from 54 dogs prospectively enrolled in a multi-institutional study on acute leukemia were stained and assessed. One observer counted the percentage of positive blasts and quantified staining intensity on a scale of 1-3. Cases were classified as acute myeloid leukemia, lymphoblastic leukemia/lymphoma, acute leukemia of ambiguous lineage, or acute lineage-negative leukemia based on flow cytometric analysis and myeloperoxidase cytochemical staining. RESULTS: The fast blue procedure yielded a significantly higher median percentage of ALP-positive blasts (48%) than the BCIP/NBT (46%) or fast red (42%) procedure despite similar median staining intensities. The proportion of samples that would have been classified as ALP-positive was similar (40/54 fast blue, 39/54 BCIP/NBT, and 37/54 fast red). CONCLUSIONS: Our results indicate that the three procedures can be used interchangeably for determining ALP activity in blasts of dogs with leukemia.