Detection of terminal deoxynucleotidyl transferase (TdT)-positive leukemia cells by an immunoperoxidase staining

Area of Science:

• Hematology
• Immunohistochemistry
• Cell Biology

Background:

• Terminal deoxynucleotidyl transferase (TdT) is a key marker in lymphoid malignancies.
• Accurate detection of TdT is crucial for leukemia diagnosis and subtyping.
• Existing detection methods may have limitations in accessibility or specificity.

Purpose of the Study:

• To establish and validate an optimized immunoperoxidase staining protocol for TdT detection in leukemic cells.
• To compare the reliability of immunoperoxidase staining with established methods like immunofluorescence and biochemical assays.
• To investigate the subcellular localization of TdT-associated peroxidase activity in different leukemic subtypes.

Main Methods:

• Systematic optimization of fixation (methanol/hydrogen peroxide) and blocking (normal goat serum) steps.
• Utilizing specific anti-TdT antibodies and appropriate negative controls (non-immune rabbit IgG).
• Comparison of results with immunofluorescence (IF) and biochemical assays for TdT presence and percentage of positive cells.

Main Results:

• Optimized protocol effectively eliminated endogenous peroxidase and reduced nonspecific staining.
• Immunoperoxidase staining results showed high concordance with IF and biochemical assays for TdT detection.
• Peroxidase+ granules were observed in the nucleus of TdT+ acute lymphoblastic leukemia (ALL) blasts.
• Some chronic myeloid leukemia (CML) blast crisis cases exhibited cytoplasmic and nuclear peroxidase+ granules, suggesting shared phenotypes with thymocytes.

Conclusions:

• Immunoperoxidase staining is a reliable, easy-to-perform method for TdT assessment in leukemic cells using bright-field microscopy.
• The method aids in differentiating leukemic subtypes and understanding TdT expression patterns.
• Observed cytoplasmic localization in CML blast crisis suggests potential phenotypic overlap with TdT+ thymic populations.