Bhawna Jha

Leishman and Giemsa stain: a new reliable staining technique for blood/bone marrow smears.

Peripheral blood and bone marrow smear examination is an important basic tool for the diagnosis of different haematological conditions including haematological malignancies. We created a newer modification of the conventional Leishman and Giemsa stains as Leishman and Giemsa (L&G) stain and compared the efficacy and reliability of this stain with conventional stains. The study was performed to evaluate the staining efficacy, feasibility, time and cost of L&G stain over the conventional Leishman and Giemsa stains.

Downgraded Lymphoma: B-Chronic Lymphocytic Leukemia in a Known Case of Diffuse Large B-Cell Lymphoma--De Novo Occurrence or Transformation.

Low-grade indolent lymphomas can be transformed into high-grade aggressive lymphomas [1,2,3,4]. Very few cases of transformation of high/intermediate-grade lymphoma to low-grade lymphoma have been reported in the literature [5,6]. This may arise through transformation of the original clone or may represent a new neoplasm resulting from additional genetic mutations that alter the growth rate, growth pattern, and sensitivity to treatment. A 57-year-old male diagnosed with diffuse large B-cell lymphoma (DLBCL) (non-germinal center B-cell type) in 2002 completed 6 cycles of CHOP followed by radiotherapy. In 2006, 18Ffluorodeoxyglucose (FDG) positron emission tomography/computed tomography (PET/CT) showed no active disease. In 2007 there was recurrence in the left obturator and external iliac nodes. Lymph node biopsy done outside our facility showed CD20+ B-cell lymphoma. The patient was advised to undergo intensive chemotherapy, but was lost to follow-up. In 2010, the patient came to our hospital with bilateral firm non-tender inguinal and right axillary lymphadenopathy without any organomegaly. 18F PET/CT revealed heterogeneous uptake in the left paraaortic, retrocaval, precaval, and bilateral internal iliac nodes. A previous diagnostic lymph node biopsy was reviewed, showing diffuse infiltration of large atypical cells, positive for CD20, CD30, MUM1, and Bcl2 with a Ki67 index of 80% and negative for CD3, CD5, and CD10, which was consistent with DLBCL (Figure 1A). Biopsy of the paraaortic mass revealed sheets of small lymphoid cells, which were positive for CD20, CD5, and CD23 and negative for CD3 and cyclin D1 with a low Ki67 index, suggestive of small-cell lymphoma (Figure 1B). 18F PETCT was repeated after 1 year, showing multiple FDG-avid cervical, supraclavicular, mediastinal, axillary, abdominal, and pelvic lymphadenopathies (Figure 1C). After 10 months, hemoglobin was 90 g/L, total leukocyte count was 21.1x109/L, and platelet count was 40x109/L. Peripheral blood smear showed 84% abnormal lymphoid cells, which were immunopositive for CD19, CD5, CD23, CD22 (dim), CD200, and CD20 with lambda light chain restriction and negative for CD10, FMC7, CD38, IgM, and CD103, confirming the diagnosis of chronic lymphocytic leukemia (CLL) (Figure 1D). The patient was started on a fludarabine, cyclophosphamide, and rituximab (FCR) regimen. After 6 cycles of FCR, he was in complete remission and was started on rituximab maintenance therapy. Letters to the Editor

Disseminated Histoplasmosis in an Immunocompetent Host Presenting as Pancytopenia with Bilateral Adrenal Masses

A 44-year-old male presented with fever, progressive weight loss, and anorexia for 6 months. The laboratory results showed deranged renal function tests. Serum adrenocorticotropic hormone was high at 252 pg/mL (normal limit: <46 pg/mL), suggestive of primary adrenal insufficiency. Serum free light chains were elevated, kappa at 87.97 mg/L (reference range: 3.30-19.40 mg/L) and lambda at 91.77 mg/L (reference range: 5.71-26.30). Ultrasonography of the abdomen showed hepatosplenomegaly with space-occupying lesions in bilateral suprarenal regions, while endoscopy ultrasound-guided fine-needle aspiration showed necrotizing granulomatous inflammation. Work-up for tuberculosis and human immunodeficiency virus was negative. The hematological parameters showed pancytopenia. The bone marrow aspiration revealed round to oval organisms with crescent-shaped eccentric nuclei both extracellularly and intracellularly, inside the macrophages and osteoclastic giant cells (Figure 1A). Bone marrow biopsy showed the presence of intracellular and extracellular oval capsulated globose organisms spread throughout the marrow spaces (Figure 1B). Periodic acid-Schiff (PAS) staining showed these organisms as bright eosinophilic structures with clear halos around them (Figure 1C). Gomori methenamine silver (GMS) staining showed clusters of fungal yeasts, morphologically compatible with Histoplasma capsulatum (Figure 1D). The patient was started with intravenous amphotericin B followed by oral itraconazole. His condition improved with recovery of counts and improvement of renal function; he is currently doing well. Informed consent was obtained. Figure 1 A) Bone marrow aspirate showing numerous Histoplasma capsulatum inside the osteoclastic giant cell and macrophage (inset) (Giemsa, 100x), B) bone marrow biopsy showing Histoplasma (H&E, 100x), C) PAS staining showed yeast-like cells with bright ... Histoplasmosis is a fungal infectious disease caused by inhalation of spores of Histoplasma capsulatum. It may present as a self-limiting form or progressive disseminated disease. Disseminated histoplasmosis may affect almost all systems, including the reticuloendothelial system, lungs, gastrointestinal tract, renal tract, central nervous system, visual system, bone marrow, and adrenal glands [1]. Histoplasmosis presenting as a bilateral adrenal mass in an immunocompetent patient is rare. A high index of suspicion is required for the diagnosis of disseminated histoplasmosis in a patient with unexplained fever, as it may mimic other chronic illnesses or a neoplasm. The differential diagnoses that should be considered are tuberculosis, sarcoidosis, adrenal hemorrhage, metastatic carcinoma, and lymphoma. Despite extensive imaging, positron emission tomography scanning, and fine-needle aspiration biopsy, a definite diagnosis may not be reached [2]. Bone marrow examination is a useful diagnostic test to establish a diagnosis of disseminated histoplasmosis. In our case, a middle-aged immunocompetent patient presented with nonspecific symptoms and bilateral adrenal mass with insufficiency, the diagnosis of which was only possible with bone marrow examination. Conflict of Interest Statement The authors of this paper have no conflicts of interest, including specific financial interests, relationships, and/or affiliations relevant to the subject matter or materials included.

Prompt diagnosis and management of Epstein-Barr virus-associated post-transplant lymphoproliferative disorder and hemophagocytosis: A dreaded complication in a post-liver transplant child.

EBV‐associated PTLD is increasingly recognized as an important cause of morbidity and mortality in both solid organ and hematopoietic stem cell transplant recipients. Mortality rates due to PTLD and virus‐induced HLH are reported to be quite high. We report a case of EBV‐associated PTLD and HLH in a child after liver transplantation who was successfully managed due to timely intervention. This case highlights that measurement of EBV load by quantitative polymerase chain reaction assays is an important aid in the surveillance and diagnosis of PTLD and early detection of EBV‐induced PTLD, and aggressive treatment with rituximab is a key to survival in patients who have undergone liver transplantation.

Picking up myelodysplastic syndromes and megaloblastic anemias on peripheral blood: use of NEUT-X and NEUT-Y in guiding smear reviews.

Sir, Diagnosing myelodysplastic syndrome (MDS) poses a peculiar challenge as they mainly present as cytopenia(s), which can occur due to a myriad of hematological and nonhematological factors. On a routine complete blood count (CBC) flagged for cytopenia(s), evaluating neutrophilic dysplasia on light microscopy suffers from a multitude of problems such as pH of the stain, quality of staining, time gap in preparation of smear, interobserver variation, and morphological alterations encountered due to secondary causes such as drugs, nutritional, toxin. There is no single parameter or test available to diagnose MDS, and so an extensive workup is required. Apart from battery of tests required, there are other morphologic overlaps present which may further complicate the problem such as megaloblastic anemia (MA) and few subtypes of myeloproliferative neoplasm (MPN). MA cases can present with pancytopenic blood picture with dyspoiesis noted in one or more lineages, similar to that noted in early MDS patients, making the early diagnosis of MDS cases difficult. The macrocytosis in MA can be obscured by concomitant disorders that in themselves cause microcytosis although the leukocyte abnormalities are preserved, resulting in masked MA [1]. Another differential of macrocytosis is MDS, which can be suspected by the presence of hypogranular neutrophils and monocytosis. Thus, in such cases, peripheral smear alone is not of much help, and further invasive tests such as bone marrow examination and cytogenetics needs to be carried out. MPN, on the other hand, usually can be differentiated from MDS and MA cases morphologically, however, with few overlaps, as in cases of primary myelofibrosis in fibrotic phase, MDS/MPN group which includes chronic myelomonocytic leukemia (CMML), atypical CML, juvenile myelomonocytic leukemia, and MDS/MPNU. They may have some clinical, laboratory, or morphologic findings at the time of initial presentation that support a diagnosis of MDS more than MPN. One or more of the other lineages may exhibit ineffective proliferation so that such cases may present with cytopenia(s) as well [2]. Such cases can present as a challenge and need to be differentiated from early cases of MDS. Neutrophil dysplasia is a feature seen in MDS as well as in CMML, thus making it difficult for NEUT-X and NEUT-Y values to differentiate them [3]. As MA is also associated with structural abnormalities in RBC and WBC as seen in MDS, they were also included in the study to evaluate its possible role in NEUT-X and NEUT-Y We evaluated the role of parameters NEUT-X and NEUT-Y (Sysmex XE-2100; Sysmex corporation, Kobe, Japan) in detecting neutrophil dysplasia in suspected MDS, MA, and MPN cases. NEUT-X and NEUT-Y are the latest parameters for neutrophil structural and maturation, NEUT-X is the direct measurement of side scatter diffraction, corresponding to channel number, and is representative of the internal structure of the neutrophils. It correlates with hypogranularity of neutrophils and when taken into consideration with anemia is suggestive of an underlying MDS. NEUT-Y is the direct measurement of the fluorescence intensity [1]. This study included four groups: control, MDS, MPN, and MA group with the aim to compare the NEUT-X and NEUT-Y values among each group as well as to record whether these two parameters have any relationship between each other in individual groups. The control group includes 66 cases which came for routine check up with no complaints or comorbidities, 30 cases diagnosed as MDS, 56 cases of MPN, and 16 cases of MA. The cases were collected over a period of one and a half years (March 2012–August 2013). Inclusion criteria in the study were as follows: • All the cases were diagnosed using multiparametric approach

Persistent Monotypic Plasma Cells with Absence of Neoplastic B Cell Component in a Treated Case of Waldenström Macroglobulinemia: A Sign of Residual Disease?

Waldenström macroglobulinemia (WM) is a rare indolent variant of non- Hodgkins lymphoma characterised by lymphoplasmacytic infiltration of bone marrow (BM) associated with a serum IgM paraprotein. The WHO classification states that the neoplastic cells of WM usually are positive for monotypic surface immunoglobulin light chain, IgM, CD19, and CD20 and are negative for CD5, CD10, and CD23. Serum monoclonal protein detection by serum protein electrophoresis and bone marrow aspirate and biopsy are required for WM diagnosis, monitoring and response assessment. Pathologist must dissuade themselves from making a hasty decision on calling a complete response in WM when neoplastic B cell component is absent. Evaluation of clonality of any residual plasma cells must be done in all cases of WM to evaluate the presence and extent of residual or persistent disease. Role of additional therapy targeted at these residual plasma cells in WM can be evaluated as tools for achieving complete remission. Herein, we present a case of WM with residual monotypic plasmacytosis in BM, without B lymphocytes after therapy.

Testicular swelling: a rare manifestation of chronic lymphocytic leukemia presenting with Richter's syndrome.

Richter syndrome (RS) represents the clinico-pathologic transformation of indolent lymphomas to an aggressive lymphoma, most commonly diffuse large B-cell lymphoma. Majority of the patients have a previous diagnosis of Chronic Lymphocytic Leukemia and the median time to transformation is 2-4 years. De novo RS is extremely uncommon. RS frequently arises in the lymph nodes or bone marrow and rarely presents with extra nodal involvement, common sites being the gastrointestinal tract, eye, central nervous system, lung and kidney. Involvement of testis by RS is extremely rare and we came across only one such reported case in the literature. We are reporting this case as our patient presented with de novo RS at an extremely uncommon extra nodal site, testis.

Mixed-phenotypic acute leukemia: cytochemically myeloid and phenotypically early T-cell precursor acute lymphoblastic leukemia

Early T-cell precursor acute lymphoblastic leukemia (ETP-ALL) is characterized by the specific immunophenotype of CD1a-, CD8-, and CD5weak/- with stem cell or myeloid marker expression [1]. This leukemia is associated with increased genomic instability, a high remission failure frequency, and hematological relapse. ETP-ALL patients also represent a distinct molecular T-ALL subgroup with a low frequency of NOTCH1 mutations and a high frequency of FLT3 mutations. ETP-ALL with FLT3 mutation has also been described as a new ETP-ALL subgroup with unique immunophenotypic features and high levels of CD2, myeloid antigen CD13, and CD117 expression, indicating a stem cell phenotype [2]. Mixed-phenotypic acute leukemia (MPAL), T/Myeloid subtype is rarely suspected from morphology, except in a small subset of cases, which have a distinct dual-blast population with both lymphoid and myeloid features. Therefore, final diagnoses of MPAL are based on flow cytometric immunophenotyping. Common cytogenetic abnormalities associated with MPAL include t(9;22) and MLL rearrangements, which may be restricted to B-Myeloid cases, whereas T-myeloid cases exhibit generally non-recurring chromosomal abnormalities [3]. Herein, we report a case of MPAL (T/Myeloid) with trisomy 4 and t(6;14)(q27:q22) in a 25-year-old man, the diagnosis of which was directed by the morphological presence of Auer rods and myeloperoxidase (MPO) staining, despite exhibiting a flow cytometric ETP-ALL phenotype.

Role of NEUT-X & NEUT-Y in picking up megaloblastic anaemia on peripheral blood & in differentiating from other macrocytic anaemia

Macrocytic anaemia refers to an anaemic state, in which red blood cells (RBC) are larger than normal, recognized by automated RBC indices and confirmed on peripheral blood smear. Macrocytosis is relatively common with a prevalence ranging from 1.7 to 3.6 per cent1. The causes are broadly categorized into megaloblastic and non-megaloblastic. The megaloblastic causes comprise nutritional deficiencies such as vitamin B12/folate deficiency. The prevalence of subnormal vitamin B12 concentration in elderly varies from 3 to 40.5 per cent depending on the cut-off used for defining deficiency of cobalamin level in serum2. The non-megaloblastic causes commonly include drugs, alcoholism, liver diseases and hypothyroidism along with primary bone marrow disorders such as myelodysplasia, leukaemia, multiple myeloma and aplastic anaemia1,3. The gold standard for differentiating between these two major categories of macrocytic anaemia is based on a comprehensive approach with clinical history, high mean corpuscular volume (MCV) and/or red cell distribution width (RDW), vitamin B12 levels, peripheral blood smear and bone marrow examination.

Histoplasma capsulatum in a peripheral blood smear in a non-HIV patient

Dear Editor, Histoplasma capsulatum in a peripheral blood smear (PBS) is a rare finding and is associated with disseminated and fulminant infection and poor prognosis. Most patients who develop disseminated histoplasmosis are immunosuppressed (e.g., AIDS, solid organ transplantation, immunosuppressive therapy (e.g., corticosteroids, tumor necrosis factor-alpha inhibitors)) or are at the extremes of age [1]. Only few cases of H. capsulatum in peripheral blood have been reported in HIV patients and solid organ transplant recipients [2–5]. Herein, we described a case of histoplasmosis in a peripheral blood smear in an elderly patient with no known comorbidity. A 72-year-old man presented with fever and fatigue which had been present for 10 days. On examination, he was febrile and there was mild hepatosplenomegaly. A procalcitonin assay was raised to 4.40 ng/mL (0.00–0.05), suggestive of severe sepsis. The PBS (Fig. 1) obtained on admission showed neutrophilic leucocytosis with left shift and toxic change. Many monocytes and neutrophils showed grossly distorted nuclei and numerous intracellular yeast-like organisms measuring 1 to 4 μm in diameter which were surrounded by a pseudocapsule caused by cytoplasmic shrinkage. Few extracellular organisms were also seen. These PBS findings are diagnostic of H. capsulatum. The infection load was high (28% of the monocytes and neutrophils contained these organisms). The intracellular burden ranged from 1 to 20 organisms. Blood culture could not be done and no treatment could be instituted since his condition rapidly deteriorated and he died of septic shock within 24 h of admission. H. capsulatum is acquired by the inhalation of conidia or mycelial fragments. The most important risk factor for disseminated histoplasmosis is immunosupression. Extremes of age have also been described as a risk factor [1]. In addition, chronic progressive disseminated histoplasmosis occurs uncommonly in older adults with no known immunosuppressing conditions [6]. The majority of patients are asymptomatic or present with non-specific symptoms as fever, fatigue, anorexia, and weight loss. Physical examination may show pallor, hepatosplenomegaly, lymphadenopathy, petechiae, and in some patients, skin or mucous membrane lesions as ulcers, nodules, or molluscum-like papules. The differential diagnosis in these patients includes malignancy, infection (e.g., tuberculosis, other fungal diseases), or inflammatory diseases (e.g., inflammatory bowel diseases, sarcoidosis). Diagnosing histoplasmosis is important as misdiagnosing inflammatory disease in these patients may lead to inappropriate and harmful treatment with corticosteroids. Histopathological examination of the tissue samples and antigen determination helps in rapid diagnosis. Serological tests for antibody to H. capsulatum and culture of the organism are less rapid methods but are useful in diagnosis of disseminated histoplasmosis. In few cases, it may involve the bone marrow and then usually presents with anemia, leukopenia, and thrombocytopenia. Rarely, the organisms can be seen within white blood cells on a peripheral blood smear. This case illustrates the potential utility of peripheral blood smears as a useful and rapid diagnostic test in early diagnosis of disseminated fungal infection. A high index of suspicion and careful examination of the peripheral blood smear is necessary for prompt diagnosis and early institution of therapy. * Bhawna Jha bhawnajha@hotmail.com