Jasmita Dass

Double heterozygous hemoglobin Q India/hemoglobin D Punjab hemoglobinopathy: Report of two rare cases.

Cation exchange high performance liquid chromatography (CE HPLC) provides an excellent tool for accurate and reliable diagnosis of various hemoglobin (Hb) disorders. HbQ India is a rare alpha chain variant that usually presents in the heterozygous state. Its presence in double heterozygous state with HbD Punjab is extremely rare. The double heterozygosity for α and β chain variants leads to formation of abnormal heterodimer hybrids, which can lead to diagnostic dilemmas. We report two rare cases of double heterozygous HbQ India/HbD Punjab where the hybrid Hb was seen to elute at retention time similar to HbC on CE HPLC. The first case had unconjugated hyperbilirubinemia at presentation; while, the second case was asymptomatic.

Evaluation of Surrogate Markers for Prediction of CD4 Counts in People Living with Human Immunodeficiency Virus/ Acquired Immunodeficiency Syndrome

India is a developing country where resources are limited. HIV/ AIDS is an ominous public health problem faced by our population and the affordability of patients for 3-6 monthly monitoring of CD4 counts becomes difficult for most patients. The intent of the study was to identify parameters on complete blood counts that can predict a CD4 count of 1250/μL is predictive of a CD4 count <200/μL with a sensitivity and specificity of 87.3% and 70.0% respectively. In addition, a haemoglobin value <11.15g/dL is also a good predictor of the CD4 count <200/μL. The combination of both Hb <11.15g/dl and ALC counter <1250/μL was like a confirmatory test with a specificity of 92.2% and a NPV of 79.5%. Hemoglobin and absolute lymphocyte counts obtained on automated cell counter are robust, cost-effective and easily available methods to follow up PLHA patients and patients on ART. These can effectively predict the CD4 count <200/μL and are especially useful in a developing country where the cost of these tests is one-fifth of flow cytometry.

Incidence of Antiphospholipid Antibodies in Patients With Immune Thrombocytopenia and Correlation With Treatment With Steroids in North Indian Population

Introduction: Antiphospholipid antibodies (APLAs) have been variably reported in 14% to 75% of patients with immune thrombocytopenia (ITP). There is lack of Indian data on incidence of APLA in ITP. Objective: We studied the incidence of APLA in patients with pediatric and adult Indian ITP. Materials and Methods: We prospectively studied 100 patients including acute (n = 37), persistent (n = 13), and chronic (n = 50) ITP. Male to female ratio was 1.22:1. Median age was 18 years (1.5-56). All patients underwent investigations for lupus anticoagulant (LA), anticardiolipin (aCL) immunoglobulin G (IgG) and IgM antibodies, and anti-β2 glycoprotein 1 (β2GP1) IgG and IgM antibodies. Patients with secondary ITP were excluded. Bleeding manifestations were recorded. Patients with acute and persistent ITP were assessed for steroid response. Response rates were compared between APLA-positive and APLA-negative patients. Results: Antiphospholipid antibodies were detected in ∼12% of patients with ITP: 8.1% (3 of 37) in acute, 0% (0 of 13) in persistent, and 18% (9 of 50) in chronic ITP. Anti-β2GP1 antibodies were most frequent (9%). Only 2 patients each were positive for anti-aCL antibodies and LA. Although platelet counts were significantly higher in APLA-positive patients, there was no significant difference in bleeding between the APLA-positive versus APLA-negative patients with ITP. There was also no significant difference in steroid response between APLA-positive and APLA-negative patients with acute/persistent ITP. In the short follow-up (median 8 months), none of the APLA-positive patients developed thrombosis. Conclusions: Incidence of APLA in Indian population was lower than reported in the West, which indicates that not all patients of ITP need to be subjected to these manifestations upfront at diagnosis.

Compound Heterozygous Hemoglobin D-Punjab/Hemoglobin D-Iran: A Novel Hemoglobinopathy

Cation exchange high performance liquid chromatography (CE- HPLC) is an excellent tool for the diagnosis of various hemoglobin (Hb) disorders. HbD-Punjab is an uncommon structural Hb variant seen in North-India. Rarely, a compound heterozygous state for HbD-Punjab with high HbA2 has been described. We describe an index case whose CE-HPLC showed a compound heterozygous state for Hb-Punjab/HbD-Iran which was confirmed by family study, acid and alkaline electrophoresis and beta gene sequencing. This case highlights the role of alkaline and acid electrophoresis to resolve common peaks that elute with HbA2 on CE-HPLC. To the best of our knowledge, this compound heterozygous state of HbD-Punjab with HbD-Iran has not been reported earlier.

Chronic myeloid leukemia with p210 BCR–ABL and monocytosis

Chronic myeloid leukemia (CML) is characterized by splenomegaly, a high total leukocyte count, and cytomorphological features of granulocytic hyperplasia. The diagnosis is established by demonstration of the BCR–ABL fusion transcript by reverse transcriptase-polymerase chain reaction (RT-PCR). Monocytosis is, however, an uncommon feature of CML at presentation [1]. Here, we report a rare case of adult CML with monocytosis. A 37-year-old male presented with splenomegaly of two and a half years duration. On examination he was found to have hepatosplenomegaly. A complete hemogram showed anemia and a high total leukocyte count (hemoglobin 10 g%, total leukocyte count 19.6610/L, platelet count 200610/L). A differential leukocyte count on a peripheral smear showed 23% mature monocytes (Bl01Promono01My02 MMy08N46 L08M23E06B05) with an absolute monocyte count of 4.5 6 10/L [Figure 1(A)]. There were no dysplastic changes on the peripheral smear. Bone marrow aspiration smears were cellular and showed myeloid preponderance with left shift, with mature monocytes constituting 15%. Blasts and promonocytes constituted 13% (myelogram: Bl08Promono05My10MMy14N30L06M15E01B03NRBC08; myeloid:erythroid [M:E] ratio, 10.7:1). There was significant megakaryocytic dysplasia with megaloblastic change in the erythroid series and no significant dysplasia in the myeloid series. In view of the high absolute monocyte count in the peripheral blood (416 10/L), a diagnosis of chronic myelomonocytic leukemia (CMML) was considered, and RT-PCR for BCR– ABL and leukocyte alkaline phosphatase (LAP) test were advised. The LAP score was low (test 16, control 162). RT-PCR for BCR–ABL showed the p210 fusion transcript [Figure 1(B)]. Hence, the case was diagnosed as CML in accelerated phase. Chronic myeloid leukemia is associated with the Philadelphia chromosome t(9;22)(q34;q11), which results in the BCR–ABL fusion transcript. There can be three fusion transcripts based on the breakpoint in the BCR gene on chromosome 22: p210, p190, and p230 [2]. Monocytosis in CML is rare at presentation. The association of monocytosis with p190 fusion transcript-positive CML was described in 1994 by Melo et al. The authors suggested that this could be a missing link between CMML and CML, with morphological features between the two [3]. This association of monocytosis with p190 CML has been in seen in a few cases reported in the literature [4–6]. Verma et al. reported that p190 BCR–ABL fusion protein is associated with the minor breakpoint cluster region in chromosome 22, and is seen in *1% of cases of CML [5]. In their series, five out of nine patients with CML in chronic phase had monocytosis. This subtype has been associated with a poor response to tyrosine kinase inhibitors [5]. However, most cases of CML have a breakpoint in the major breakpoint cluster region (M-bcr) including introns 13 and 14, resulting in the 210 kDa (p210) fusion transcript [2]. In the present case, monocytosis was associated with the classic p210 BCR–ABL transcript. Two pediatric cases of CML with p210 in association with monocytosis have previously been reported [7]. One patient was a 19month-old boy and the other was a 9-month-old boy, both of whom were suspected to have juvenile myelomonocytic leukemia (JMML). However, this association has not been reported in adult cases of CML.

Consensus Statement of the Indian Academy of Pediatrics in Diagnosis and Management of Hemophilia

JustificationDespite having standard principles of management of hemophilia, treatment differs in various countries depending on available resources. Guideline for management of hemophilia in Indian setting is essential.ProcessIndian Academy of Pediatrics conducted a consultative meeting on Hemophilia on 18th September, 2016 in New Delhi, which was attended by experts in the field working across India. Scientific literature was reviewed, and guidelines were drafted. All expert committee members reviewed the final manuscript.ObjectiveTo bring out consensus guidelines in diagnosis and management of Hemophilia in India.RecommendationsSpecific factor assays confirm diagnosis and classify hemophilia according to residual factor activity (mild 5-40%, moderate 1-5%, severe <1%). Genetic testing helps in identifying carriers, and providing genetic counseling and prenatal diagnosis. Patients with hemophilia should be managed by multi-specialty team approach. Continuous primary prophylaxis (at least low-dose regimen of 10–20 IU/kg twice or thrice per week) is recommended in severe hemophilia with dose tailored as per response. Factor replacement remains the mainstay of treating acute bleeds (dose and duration depends on body weight, site and severity of bleed). Factor concentrates (plasma derived or recombinant), if available, are preferred over blood components. Other supportive measures (rest, ice, compression, and elevation) should be instantly initiated. Long-term complications include musculoskeletal problems, development of inhibitors and transfusion-transmitted infections, which need monitoring. Adequate vaccination of children with hemophilia (with precautions) is emphasized.

Utility of mean sphered cell volume and mean reticulocyte volume for the diagnosis of hereditary spherocytosis

ABSTRACT Introduction: Hereditary spherocytosis (HS) is the most common congenital hemolytic anemia, characterized by anemia, jaundice, and splenomegaly. The diagnosis of HS relies on symptoms of hemolysis, a family history of HS, and a positive laboratory test which is usually the osmotic fragility test (OFT). We conducted a study to assess the utility of mean corpuscular hemoglobin concentration (MCHC), mean corpuscular volume (MCV), mean sphered cell volume (MSCV), and mean reticulocyte volume (MRV) in the diagnosis of HS and if these are helpful in distinguishing cases of HS from immune hemolytic anemia. Methods: A total of 102 patients suspected to have HS were enrolled. In addition 10 cases of immune hemolytic anemia (IHA) were included in the study and performance of the above screening tests was evaluated. The diagnosis of HS was based on incubated OFT, eosin 5′-maleimide (EMA) dye binding test, and flowcytometric OFT. Results: A total of 29 patients were diagnosed as having HS. The sensitivity and specificity for diagnosis HS by MCHC > 35 g/dL was 44.82%, and ΔMCV−MSCV > 10 fL has a sensitivity and specificity of 82.75% and 95.9% for diagnosis of HS. Using an algorithm of ΔMCV−MSCV > 10 fL and ΔMRV−MSCV < 25, for the differentiation of HS from IHA had sensitivity of 68.9% and specificity of 98.8%.

Massive splenomegaly: flow cytometry as a diagnostic tool for systemic mastocytosis

dominantly occurs in younger patients and men. Bone marrow and extra-nodal involvements are more frequent in this subtype than in other types of DLBCL [4]. Lymphoma misdiagnoses that delay the appropriate treatment can be catastrophic. The lack of response to infection treatments and alternative diagnoses should promptly improve differential diagnoses. Lymphoma should be considered in patients with bone and joint pain.

Concomitant Presence of CD5-Positive Diffuse Large B-Cell Lymphoma and Monoclonal B Cells with the “CLL Immunophenotype” - Is It Richter’s Transformation?

The presence of diffuse large B-cell lymphoma (DLBCL) with a concomitant unsuspected population of B cells with chronic lymphoid leukemia (CLL) phenotype is very rare with no antecedent history of CLL. This may represent cases of de novo Richter’s transformation or the coexistence of two neoplasms [1]. In cases where the monoclonal B-cell population does not exceed 5x109/L, this may represent DLBCL with concomitant monoclonal B-cell lymphocytosis (MBL) of the CLL phenotype. The coexistence of MBL of the CLL phenotype creates a diagnostic conundrum, especially in an unusual case of CD5+ DLBCL as it may be a de novo CD5+ DLBCL or Richter transformation [2]. The former has an aggressive course compared to de novo DLBCLs [3,4]. CLL has been known to occur synchronously or metachronously with hairy cell leukemia [5] and DLBCL [6]. We present here a case of clinically aggressive de novo CD5+ DLBCL with an unsuspected second population of CLL-like MBL detected on flow cytometry.

Multiple Auer Rod Like Inclusions in Multiple Myeloma

Multiple myeloma is characterized by clonal plasma cell proliferation associated with end organ damage. In addition to classical morphological variants, several different types of nuclear and cytoplasmic inclusions have been described. We have come across a case of multiple myeloma with numerous Auer rod like cytoplasmic inclusions, with morphology like faggot bodies. A 54 year old male presented with back pain since last 6 months with progressive weakness. There was no history of organomegaly, sternal tenderness, lymphadenopathy, gum hypertrophy or bleeding manifestations. The clinical examination revealed only anemia. No other finding was seen on physical examination. Lab investigations showed hemoglobin of 7.1 g/dL with adequate total leucocyte counts, differential counts and platelet counts. The peripheral smear showed presence of rouleax formation. A bone marrow aspirate smear revealed 72% atypical plasma cells with immature nuclear chromatin, poorly defined cytoplasmic borders, many binucleate and syncytial forms. In addition, almost all the cells showed numerous intracytoplasmic thin slender Auer rods like structures (Fig. 1). Many cells had bundles of these structures resembling faggot cells (Fig. 2). Cytochemistry for myeloperoxidase, Sudan black, periodic acid Schiff and nonspecific esterase stain was negative using adequate positive controls. Serum imunofixation electrophoresis showed presence of a monoclonal band along with IgG kappa clonality. Skeletal survey showed multiple lytic lesions in left humerous and left iliac bone. A final diagnosis of multiple myeloma was made. Auer rod like inclusions have been described previously in multiple myeloma, prolymphocytic leukemia, aplastic anemia, B cell acute lymphoblastic leukemia, chronic lymphocytic leukemia and hypogammaglobulinemia [1]. All cases published till date revealed that this phenomenon is primarily restricted to IgA or IgG and kappa secreting myelomas [2–5]. Our case also showed IgG-j-type light chain restriction. Auer rod-like inclusions in myeloma are composed of crystallized lysosomal enzyme depositions. However, the exact nature of these inclusions is not known. Morphologically it is difficult to differentiate these structures from Auer rods seen in immature myeloid cells. One of the methods is to perform myeloperoxidase staining which is classically negative in these crystalline structures since true Auer rods shows bright staining [1]. However, positive myeloperoxidase and nonspecific esterase (NSE) have been reported in these Auer rods like structure [2, 5]. Many ultrastructural differences have also been described in literature [5]. In view of these conflicting results it is advised to carefully analyze these inclusions to differentiate it from true Auer rods seen in myeloid progenitors. To conclude Auer rod like inclusions in a case of multiple myeloma is a rare finding and should never be analyzed in isolation. A panel of cytochemical tests, detailed history, examination, laboratory investigations and other biochemical tests are required to avoid misinterpretation and misdiagnosis. Disclaimers The identity of the patient is not disclosed here in this case.