Utility of Cytochemical and Flow Cytometry Detection of Alkaline Phosphatase for Differential Diagnosis of <scp>CD34</scp> + Acute Leukaemia in Canines

Alkaline phosphatase (ALP) enzymatic activity has been proposed as a marker for distinguishing canine acute leukaemia (AL) subtypes (i.e., myeloid vs. lymphoid). However, ALP enzymatic activity has not been fully evaluated in CD34+ AL. Determine whether ALP enzymatic activity can differentiate CD34+ AL subtypes in dogs and distinguish CD34+ AL from CD34- haematopoietic tumours in tissue/effusion samples. Peripheral blood from 64 dogs with CD34+ AL, 10 with B cell chronic lymphocytic leukaemia (CLL), and 10 healthy controls were prospectively evaluated for ALP enzymatic activity via cytochemical staining; a subset also underwent ALP detection by flow cytometry (FC). Archived cytology slides from 67 tissue/effusion specimens, including 27 CD34+ AL, 22 T cell lymphomas, and 18 B cell lymphomas, were retrospectively assessed. CD34+ AL cases were categorised as acute myeloid leukaemia (AML), acute lymphoid leukaemia (ALL) or acute unclassifiable leukaemia (AUL) by established FC criteria. ALP positivity was defined as > 3% ALP+ neoplastic cells, which was selected based on receiver operating characteristic (ROC) curve analysis. Cytochemical ALP activity was detected in 61/64 (95.3%) CD34+ AL cases, with no significant differences between AML, ALL, and AUL subtypes (p > 0.05). All lymphoma and B cell CLL cases were ALP-negative. FC-based ALP analysis showed poor concordance with cytochemistry, and the correlation between %CD34 + ALP+ cells and %ALP+ neoplastic cells was weak (Spearman's ρ = 0.25). While ALP enzymatic activity is present in most CD34+ AL cases, it does not reliably differentiate CD34+ AL subtypes via cytochemistry. However, ALP may help distinguish CD34+ AL from B and T cell lymphomas. FC-based ALP analysis is not a reliable marker for CD34+ AL classification.