TESTS IN FOCUS
STORIES AT A GLANCE
Juvenile Hyaline Fibromatosis
Dr Rimsha Imran, Hematology
Reticulocytes are immature red cells released from bone marrow that contain remnant cytoplasmic RNA and organelles like mitochondria and ribosomes. They mature in circulation over one-two days.
The reticulocyte count determines bone marrow erythropoietic activity in response to anemia. Reticulocytosis is seen in conditions like hemolytic anemias, hemorrhage, post-splenectomy and during recovery from anemia. They are visualized using supravital stains like methylene blue. On a blood film, reticulocytes (as highlighted by red arrows in the below figure) appear as pale blue containing dark blue reticular or granular material. It’s imperative to distinguish them from RBC inclusions like Howell-Jolly bodies, Heinz bodies, Pappenheimer bodies, and HbH inclusions during manual counting.
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Metachromatic Clues: A Diagnostic Challenge of Systemic Mastocytosis in a Patient with Bone Marrow Fibrosis
Dr Maria Owais and Dr Amna Qadri, Hematology
Case Details:
A 39-year-old married male from Quetta presented in September 2024 with complaints of severe back pain, intermittent undocumented fever and headache. CT scan of the abdomen and pelvis revealed hepatomegaly and multiple small lytic lesions involving the spine and pelvic bones (largest lesion measuring 11.0 mm in the right iliac bone).
His laboratory investigations for the workup of plasma cell neoplasm including serum protein electrophoresis, immunofixation and free light chain ratio revealed no monoclonal gammopathy. Bone marrow biopsy and a separate bone biopsy from the left iliac blade were taken to evaluate the lytic lesions further.
Histopathological analysis of the core of bone trephine and bone biopsy from the left iliac blade revealed marrow elements with clusters of histiocytic infiltration along with sheets of atypical cells with granular densely eosinophilic cytoplasm (Fig: A) and dense fibrosis (Fig: B). Immunohistochemical stains for plasma cells (CD138), epithelial cells (CKAE1/AE3) and Langerhans cells (CD1a) were negative. CD20 and CD3 were also negative in sheets of eosinophilic cells, while CD68 highlighted the admixed population of histiocytes (Fig: C). Hence a diagnosis of Myelofibrosis was favored.
As part of the further evaluation for Myelofibrosis, molecular testing—including JAK2 V617F and an extended MPN panel (MPL W515K/L, KIT, and CALR frameshift mutations)—was conducted. The results identified a KIT p.D816V (c.2447A>T) mutation in exon 17 of the C-KIT gene, with no other mutations detected, raising suspicion for an underlying Mastocytosis.
The bone marrow and bone biopsy from left iliac blade was reviewed, and CD117 immunohistochemical staining was performed, revealing positivity in clusters of atypical cells, confirming them as mast cells [Fig: D]. Further special staining showed strong metachromatic positivity for mast cell granules with toluidine blue, which further corroborated the diagnosis [Fig: E].
Discussion: Systemic Mastocytosis (SM) is a rare and heterogeneous group of clonal mast cell disorders classified under Myeloproliferative Neoplasms (MPNs). It is characterized by the abnormal accumulation of neoplastic mast cells in extracutaneous organs, most commonly the bone marrow, liver, spleen, lymph nodes, and gastrointestinal tract. 95% of the cases are driven by an activating mutation in the KIT gene, most frequently the KIT D816V mutation. This mutation leads to constitutive activation of the KIT receptor tyrosine kinase, promoting mast cell survival, proliferation, and accumulation.
Diagnosis relies on a combination of histopathological findings, immunophenotyping, and molecular analysis as outlined in the World Health Organization (WHO) classification. The detection of multifocal dense infiltrates of mast cells in the bone marrow, aberrant expression of CD25 and/or CD2, presence of KIT mutations, and positive special stains such as toluidine blue are critical diagnostic elements.
This case report highlights the diagnostic journey of a middle-aged patient who presented with non-specific systemic symptoms and skeletal pain and was ultimately diagnosed with Systemic Mastocytosis using an integrated approach, taking radiological, histopathological and molecular findings onboard. The case highlights the importance of a multidisciplinary diagnostic approach in patients with unexplained skeletal lesions and systemic symptoms.
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Integrating Molecular Testing in Diagnosing Rare Childhood Infections: A Case of Congenital Neutropenia
Dr Sana Hassan, Hematology
Congenital neutropenia is a rare yet life-threatening condition that often manifests early on in life with recurrent infections. Prompt diagnosis is essential to initiate appropriate management and prevent complications, including leukemic transformation due to acquisition of secondary mutations such as RUNX1 and many others. We recently encountered a rare but classic presentation of congenital neutropenia in our hospital AKUH.
A 5-month-old baby boy presented to the emergency room with cough, chest congestion, and a maculopapular rash on face and neck. The child had a history of multiple hospital admissions in the last 3 months of age, initially for bronchopneumonia, later for suspected meningitis and recurrent fevers. Family and birth history were unremarkable.
Initial CBC revealed Hb: 11.3 g/dL, WBC: 8.32 × 10⁹/L with ANC: <0.05 × 10⁹/L, ALC: 6.6 x 10⁹/L and Plt: 458 x 10⁹/L (Figure 1). Bone marrow biopsy showed maturation arrest at the promyelocyte stage.
Figure 1: Aspirate showing arrest at the promyelocyte stage.
The diagnostic clue came from persistent neutropenia and propensity to infections on multiple occasions with a normal total WBC count. The Bone marrow examination showed arrest at the promyelocyte stage and raised suspicion for congenital neutropenia. For further testing the child's Next Generation Sequence testing, were sent to Turkey that identified a pathogenic ELANE mutation, further confirming the diagnosis.
Persistent neutropenia with recurrent infections in infancy should prompt evaluation for congenital neutropenia. The Bone marrow examination revealed maturation arrest, and integrated molecular testing for ELANE mutation through NGS confirmed the diagnosis. Early detection is crucial, as it can be lifesaving by guiding G-CSF therapy and enabling timely stem cell transplantation in cases resistant to treatment.
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