PMC4272951_01
Peripheral blood smear, bone marrow biopsy and aspirate. (A) Pancytopenia and atypical lymphoid cells were observed in peripheral
blood smear (Wright-Giemsa stain; magnification, x1,000). (B) Hemophagocytic histiocytes were frequently observed in bone marrow aspiration (Wright-Giemsa stain; magnification, x1,000). (C) Histiocytes were high in number in the biopsy section (Hematoxylin-Eosin stain; magnification x200). (D) CD68 immunization (magnification, x400).
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F1
Atypical lymphocytes were observed on peripheral blood smear (Fig. 1A). Hemophagocytic histiocytes were observed on bone marrow biopsy (Fig. 1B, C, and D).
Sequencing analysis, flow cytometry results, and single-strand conformational polymorphism (SSCP) analysis of the T cell receptor by using paraffin-embedded tissue samples. (A) Compound heterozygous mutations in the UNC13D gene were observed: C to T substitution at nucleotide -308 of intron 1, relative to the cDNA positioned between nucleotides 117 and 118 (118-308C>T). (B) G to C substitution at nucleotide -1 of intron 9, relative to the cDNA positioned between nucleotides 753 and 754 (754-1G>C). The CD3+/CD8+ T cells (red). (C) showed variable downregulation of CD5 or CD7 (lack of expression) compared with CD3+/CD4+ cells (blue, presumably normal T cells; D and E). (F) DNA was extracted from paraffin-embedded bone marrow tissue. Consensus primer for Vgamma1-8, Vgamma9-11 was used for amplification. PCR products were analyzed by SSCP, which separated DNA fragments according to nucleotide sequence. Well-defined, distinct bands were considered evidence of monoclonality. The two black arrows indicate the distinct monoclonal band at the level of the lowest smear in the polyclonal control (-). Monoclonality was observed in Vgamma1-8, and polyclonality was observed in Vgamma9-11.
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F2
Sequencing analysis revealed compound heterozygous mutations in UNC13D [c.118-308 C>T (;)754-1G>C] (Fig. 2A and B). Immunophenotyping using bone marrow aspirate revealed an abnormal population of CD8+ T cells with downregulated levels of CD5 or CD7 (14.61% of total events; Fig. 2C, D, and E). TCR gene rearrangement study using paraffin-embedded tissue revealed T cell monoclonality (Fig. 2F). The patient was treated with HLH-2004 chemotherapy and allogenic bone marrow transplantation.