Dual Immunohistochemistry Bone Marrow Staining Detects Hairy Cell Leukemia

Hairy cell leukemia (HCL) is a B-cell lymphoproliferative disorder characterized by distinct immunophenotype (positive for CD19, CD20, PAX5, CD22, CD11c, CD25, CD103, CD123, and CD200). Immunophenotypic analysis by flow cytometry (FC) is considered the gold standard for diagnosis of HCL.

However, both FC and immunohistochemistry (IHC) can be used to determine these markers. Although both trephine bone marrow biopsy and aspirate are vital for assessment of the extent of bone marrow infiltration, in some cases a cellular aspirate cannot be obtained because of extensive fibrosis (i.e. “dry tap”).


Hematologists at the US National Institute of Cancer (Bethesda, MD, USA) and their colleagues analyzed on 148 bone marrow biopsy specimens (123 male and 25 female patients; mean age, 59.8 years; range, 25-81 years) collected from patients evaluated for HCL between 2016 and 2017. Specimens were stained within 24 hours of collection with a panel of antibodies. Specimens were subsequently washed with phosphate-buffered saline and stained for 30 minutes at room temperature with antibody combinations in eight-color cocktails.

Multiparameter flow cytometry was performed using CD19, CD20, CD22, CD11c, CD25, CD103, CD123, surface light chains, CD5, and CD23. In parallel, bone marrow IHC was done using PAX5/CD103 and PAX5/tartrate-resistant alkaline phosphatase (TRAP) dual IHC stains Specimens were acquired on FACSCanto II (BD Biosciences, San Jose, CA, USA). The bone marrow biopsies were fixed in B-Plus fixative and decalcified in Rapid Cal Immuno (BBC Biochemical, Vernon, WA, USA) and paraffin embedded using Tissue Tek processor (Sakura Finetek, Torrance, CA, USA).

The scientists reported that the overall sensitivity of dual IHC stains was 81.4%, positive predictive value was 100%, and negative predictive value was 81.7%. All IHC-positive cases concurred with flow cytometry data, even when HCL burden was extremely low in the flow cytometry specimens (as low as 0.02% of all lymphoid cells). PAX5/CD103 dual IHC staining generated brown nuclear staining for PAX5 and red membranous and cytoplasmic staining for CD103. PAX5/TRAP dual IHC staining showed similar results for PAX5 and red membranous and cytoplasmic staining for TRAP.

The authors concluded that dual IHC staining is a sensitive tool for detecting HCL, even in cases with minimal disease involvement. All IHC-positive cases concurred with FC data, even when HCL burden was extremely low. Only 18.3% of dual IHC–negative cases were positive for low-level involvement by FC analysis. PAX5/CD103 dual IHC staining was slightly more sensitive than PAX5/TRAP dual IHC staining. The study was published in the March 2020 issue of the American Journal of Clinical Pathology.

Related Links:
US National Institute of Cancer
BD Biosciences
BBC Biochemical
Sakura Finetek