Enzyme-enhanced immunocytochemical detection of cytoplasmic proteins in human leukocytes sorted by surface antigens using anti-CD antibody microarray

Anti-cluster-of-differentiation (anti-CD) antibody microarray is an attractive multiplex analogue to leukocyte immunocytochemistry for surface markers. The transparent plastic microarray support permits to sort leukocytes by their surface lineage-specific markers making them available for subsequent high-resolution morphology examination. The combined data on the pathologic cells’ immunophenotype, morphology and cytochemistry from the microarray is sufficient to suggest preliminary diagnosis in many leukemia types. However, in some cases additional staining for internal markers is required, i.e. in T cell acute leukemia the T cell origin of the blasts can only be proved by the presence of cytoplasmic CD3 (cytCD3). Here we describe a new protocol of immunocytochemical detection of internal proteins in mononuclear cells captured by mouse anti-CD antibodies on the microarray surface. The protocol uses primary mouse FITC-conjugated (FITC – fluorescein isothiocyanate) antibodies against the target, secondary anti-FITC antibodies conjugated with alkaline phosphatase and colorimetric alkaline phosphatase substrate, BCIP/NBT. We show on normal leukocytes and leukemic cell lines that the protocol is sensitive and specific. The percentages of cytCD3-positive cells determined by this method in bone marrow aspirates of two patients with T cell acute lymphoblastic leukemia are in excellent agreement with flow cytometry results. This method expands the diagnostic capabilities of anti-CD antibody microarray.