Pax5 Standardization for the B-Cell Lineage Assignment in Acute Leukemia by Flowcytometry.

Presenting author: Shubham R Pawar (MSc.) ([e-mail])

Corresponding author: [e-mail]

Background: PAX5 (B-cell specific nuclear protein) used in immunohistochemistry as B-lineage marker. In clinical settings, its expression by MFC has not been reported. In complicated cases, confident lineage assignment as per WHO criteria requires multiple markers. PAX5 expression was standardized by MFC, and its importance studied as a B-lineage marker in acute leukemia.

Materials and Methods: We standardized Anti-PAX5 (PE, clone-REA140) antibody staining and studied in the leukemic blasts using MFC. The permeabilization reagents used for evaluation were FACS Lyse (BD), Fix-&-Perm (Invitrogen), Foxp3-fixation-kit (eBiosciences), and true nuclear transcription-factor staining buffer-set (Biolegend).

Results: Best expression for anti-PAX5-antibody was obtained with Foxp3-fixation-kit (titer = 5 µL). Our cohort comprised of 21 acute myeloid leukemia (AML), 25 B-acute lymphocytic leukemia (ALL), and 10 (Mixed Phenotype Acute Leukemia and B/myeloid) patients. Normal B-cells and T-cells served as positive and negative controls ([Table 1]). Median n mean fluorescence intensity (MFI) of PAX5 in B-ALL and AML patients were calculated ([Table 1]). AML patients did not show PAX5 expression (nMFI < 3) except one t(8;21) translocated AML which showed aberrant CD19 expression. B-ALL cases showed strong PAX5 expression (nMFI > 7). PAX5 expression was homogenous in B-ALLs with median (SD): 58.9 (41.7) as compared to 101.5 (131.8) in AML. PAX expression was also noted in two CD10-negative pro-B-ALLs and four B/Myeloid MPAL patients.

Conclusion: We standardized and studied PAX5 expression by flow cytometry in clinical settings. Best permeabilization reagent was Foxp3-fixation-kit (eBioscience). PAX5 expression was highly specific for B-lineage and therefore could be used in challenging and difficult cases of acute leukemias for determination and confirmation of B-cell lineage.

Table 1 Median nMFI of PAX5 expression

PAX5	Negative control (T-cells)	Positive control (B-cells)	B-ALL blast	AML blast	MPAL blast (B/myeloid)
Median nMFI	1.84	5.7	14.7	₋0.62	12.44
Range	0.18–1.4	3.19–17.3	3.18–33.1	(₋2.4)–10.8	2.0–65.3

Abbreviations: ALL, acute lymphocytic leukemia; AML, acute myeloid leukemia; MFI, mean fluorescence intensity; MPAL, mixed phenotype acute leukemia.

No conflict of interest has been declared by the author(s).

Publication History

Article published online:
22 August 2022