PMC8784545_01
Bone marrow biopsy showing absence of granulocytic series. The cellularity observed in the Hematoxylin-Eosin stain (A) is myeloperoxidase negative (B). LMO2 (C) and CD79a (D) showing that the observed cellularity corresponds mainly to red and plasma cells.
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Bone marrow biopsy showed the absence of granulocytic lineage with normal erythroid and megakaryocytic lineages ( Figure 1 ).
Blood immunophenotypic study of T lymphocytes. Flow cytometry was performed in peripheral blood with 50.000 total events acquired per tube (FACS Canto II, BD Biosciences). T lymphocytes were gated using CD3 antigen (A, B). Distribution of CD4+ (57%) and CD8+ (38%) populations were normal (C). Expression of PAN-T antigens CD2 (100%), CD5 (100%) and CD7 (90%) were normal (D-F).
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T lymphocytes, performed by flow cytometry, was normal ( Figure 2 ). The diagnosis of agranulocytosis was made.
Anti-neutrophil antibody test performed by immunofluorescence technique and flow cytometry reading. The patient's granulocytes were isolated and next incubated with polyvalent IgG+IgM antiglobulin (ATG) conjugated with fluorescein isothiocyanato (FITC). To detect the presence of antibodies bound to the granulocyte membrane, live (7-aminoactinomycin D negative) neutrophils were selected and the intensity of the FITC fluorescence was analyzed. (A) shows our patient's positive result. (B) illustrates a negative control. The study was completed after investigating the presence of free autoantibodies in the serum of the patient. In this indirect test, the patient's serum was incubated with donor granulocytes and ATG. Median fluorescence intensity was again analyzed by flow cytometry.
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However, no free anti-neutrophil antibodies were detected in the serum ( Figure 3 ).
Timeline of the evolution of the patient's neutrophil count from the diagnosis of Waldenstrom macroglobulinemia to the present. After 10 days of treatment with G-CSF, the patient maintained neutrophil count of 0 x 109/L. The initiation of MTP and IVIG achieved a fast and lasting recovery that was established in the next 48-72h. Abbreviations: CI, first cycle; C2, second cycle;D, day within the cycle from the start of treatment; RB:Rituximab-Bendamustine.
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At the last follow-up, 24 months after the agranulocytosis event and 20 months after steroid withdrawal, the patient remained in partial response and no additional episodes of neutropenia were observed ( Figure 4 ).