Peripheral Smear Evaluation Research Articles

Ethylenediaminetetraacetic acid-induced pseudothrombocytopenia: The story of platelet clumps and report of three cases

Ethylenediamine tetra-acetic acid (EDTA) induced pseudo-thrombocytopenia is an infrequent phenomenon occurring when samples are processed in an automated analyzer using EDTA as the anticoagulant. We report three cases of Pseudothrombocytopenia wherein peripheral smear evaluation and rerun in separate Haematology analyzers helped us in reaching a conclusion. The widespread use of automated instruments has led to the identification of this phenomenon. However, confirmation of PTCP can only be done under microscopic examination of the slide.

Bio-net dataset: AI-based diagnostic solutions using peripheral blood smear images

Peripheral blood smear examination is one of the basic steps in the evaluation of different blood cells. It is a confirmatory step after an automated complete blood count analysis. Manual microscopy is time-consuming and requires professional laboratory expertise. Therefore, the turn-around time for peripheral smear in a health care center is approximately 3–4 hours. To avoid the traditional method of manual counting under the microscope a computerized automation of peripheral blood smear examination has been adopted, which is a challenging task in medical diagnostics. In recent times, deep learning techniques have overcome the challenges associated with human microscopic evaluation of peripheral smears and this has led to reduced cost and precise diagnosis. However, their application can be significantly improved by the availability of annotated datasets. This study presents a large customized annotated blood cell dataset (named the Bio-Net dataset from healthy individuals) and blood cell detection and counting in the peripheral blood smear images. A mini-version of the dataset for specialized WBC-based image processing tasks is also equipped to classify the healthy and mature WBCs in their respective classes. An object detection algorithm called You Only Look Once (YOLO) with a refashion disposition has been trained on the novel dataset to automatically detect and classify blood cells into RBCs, WBCs, and platelets and compare the results with other publicly available datasets to highlight the versatility. In short the introduction of the Bio-Net dataset and AI-powered detection and counting offers a significant potential for advancement in biomedical research for analyzing and understanding biological data.

Two cases of vitamin B12 deficiency in patients with beta-thalassemia trait: lessons in diagnosis

BackgroundCoexisting pathologies is not a new phenomenon and can cause diagnostic difficulties when they overshadow one another. They may alter the cytomorphology of cells and the machine readings to an extent that the actual diagnosis is missed. One such scenario is created by coexisting megaloblastic anemia and beta-thalassemia trait where the RBC indices and the hemoglobin chromatogram findings conform to neither of the two. Case presentationWe present two cases of anemia presenting with normocytic normochromic indices, increased red cell distribution width (RDW), elevated HbA2 levels and moderately high HbF levels in both of them and an additional low HbA1c in case 2. Upon evaluation of peripheral smear and biochemical tests, a non-immune hemolytic component was also identified with deficient Vit B12 levels in case 1 and highly elevated levels post-supplementation in case 2. ConclusionsTaking together the peripheral smear, erythrogram, chromatogram and biochemical findings, it was deduced that the alterations were caused by coexisting beta-thalassemia trait and megaloblastic anemia. Vit B12 deficiency was the cause behind hemolysis owing to intramedullary destruction of abnormal precursors. Upon supplementation in case 2, the immediate reticulocyte response was the reason behind spuriously low HbA1c.

Rituximab-induced severe acute thrombocytopenia in a patient with splenic marginal zone lymphoma.

Rituximab, which is widely used in the treatment of B-cell lymphoma, is a chimeric monoclonal antibody directed against the CD20 antigen. Rituximab has many side effects, mainly allergic and neurological. Rituximab may cause thrombocytopenia in the long term after administration. Rare cases of rituximab-induced acute thrombocytopenia have been reported in the literature. A 51-year-old female patient who was newly diagnosed with splenic marginal zone lymphoma received rituximab as first-line therapy. Petechiae occurred in the lower extremities on the day following rituximab administration. The blood test showed a severe drop in the platelet count from 112,000/μL to 5000/μL. Blood peripheral smear evaluation confirmed severe thrombocytopenia. There was no change in hemoglobin or white blood cell levels. After the diagnosis of rituximab-induced acute thrombocytopenia, thrombocyte suspension was administered due to the risk of bleeding. Close clinical and laboratory observations were made. The platelet count began to rise gradually in the following period. Before the second week of rituximab administration, the platelet count was 122,000/μL. No complications developed after premedication and slow rituximab administration, and subsequent treatments were continued in the same way. Rituximab has widespread use, especially in malignancies and autoimmune diseases. Like many monoclonal antibodies, rituximab has several side effects. Thrombocytopenia is a long-term side effect associated with rituximab, and rituximab-induced severe acute thrombocytopenia has been rarely reported. Therefore, it should be kept in mind that severe acute thrombocytopenia may develop after rituximab administration.

RITUXIMAB-INDUCED SEVERE ACUTE THROMBOCYTOPENIA IN A PATIENT WITH SPLENIC MARGINAL ZONE LYMPHOMA

Rituximab, which is widely used in the treatment of B-cell lymphoma, is a chimeric monoclonal antibody directed against the CD20 antigen. Rituximab has many side effects, mainly allergic and neurological. Rituximab may cause thrombocytopenia in the long term after administration. Rare cases with rituximab-induced acute thrombocytopenia have been reported in the literature. A 51-year-old female patient who newly diagnosed splenic marginal zone lymphoma recieved rituximab as first line therapy. Petechiae occurred in the lower extremities on the day following rituximab administration. The blood test showed a severe drop in the platelet count from 112,000/μL to 5,000/μL. Blood peripheral smear evaluation confirmed severe thrombocytopenia. There was no change in hemoglobin or white blood cell levels. After the diagnosis of rituximab-induced acute thrombocytopenia, thrombocyte suspension was administered due to the risk of bleeding. Close clinical and laboratory observations were made. The platelet count began to rise gradually in the following period. Before the second week of rituximab administration, the platelet count was 122,000/μL. Rituximab has a widespread use, especially in malignancies and autoimmune diseases. Like many monoclonal antibodies, rituximab has several side effects. Thrombocytopenia is a long-term side effect associated with rituximab, and rituximab-induced severe acute thrombocytopenia has been rarely reported. Therefore, it should be kept in mind that severe acute thrombocytopenia may develop after rituximab administration.

PB2212: VARIANT HAEMOGLOBIN ASSESSMENT AND CORRELATION WITH PHENOTYPIC PRESENTATION AT DIAGNOSIS IN SICKLE CELL DISEASE: A SINGLE CENTRE RETROSPECTIVE ANALYSIS OF CENTRAL GUJARAT COHORT

Background: Sickle cell disease (SCD) is caused by heritable point mutation at codon 6 of the human beta-globin gene widely distributed around the world. According to 2011 census, in India, there is a large tribal community of about 180 million. Among them18 million people carry the sickle cell trait and ~140,000 suffer from SCD. Gujarat State of India has a population of about 62 million, there are ~8.9 million tribal populations, among whom ~9, 00,000 people with Sickle cell Trait and ~70,000 people with SCD are expected. Though phenotypic heterogeneity of SCD is well known across the India, there is limited data on phenotypic presentation with variant haemoglobin correlation on large cohort from Central Gujarat. Aims: To correlate variant haemoglobin pattern with phenotypic presentation at diagnosis in SCD. Identify laboratory and clinical parameters to predict risk of complications in SCD. Methods: Five Hundred and seventy three patients registered at Haemato Oncology Care Centre (HOCC), Vadodara, Gujarat between August 2012 to December 2021, and diagnosed to have SCD were included in this study. This cohort represents tribal belt covering Bharuch,Narmada,Vadodara,Panchmahal,Dahod,Sabarkantha and Banaskantha districts of Central Gujarat. Diagnosis was based on clinical profile, complete blood counts (CBC) and peripheral smear evaluation along with variant haemoglobin assessment by High Performance Liquid chromatography / Capillary electrophoresis. Relevant Biochemistry investigations were done along with radiological assessment of hip joints in patients with specific symptoms.Osteonecrosis of Femur head (ONFH) was diagnosed according to Ficat and Arlet classification for ONFH. Recurrent Vaso occlusive episode(R-VOE) was labelled if there are >3 VOE/yr. Occasional VOE (O-VOE) labelled if <3 VOE/yr.Blood transfusion dependency was labelled if > 3 transfusion /yr required to maintain HB > 7 gm%.Splenomegaly was noted as palpable spleen below costal margin. Results: A total 573 SCD patients were evaluated. The median age was 20 years (range: 1-74) with 328 (57%) males and 245 (43%) females.HB was < 10 gm% in 378 (66%),HB <7 gm% in 78(14%),between7gm% to 10 gm% in 300(52.4%).315/378 (83%) had HBA <5%,Only 5/378 (1.32%)had Ferritin <20ng/ml.None had high indirect bilirubin with low HB. Transfusion dependency was found in 28(5%),HBA was 0%,<5%,6 to 10%,>10% in 12/28 (43%),7/28 (25%),6/28(21%),3/28(11%) respectively. R-VOE observed in 207 (36%),O-VOE in 259(45%),No VOE in 107(19%).Splenomegaly was found in 268(47%).HBF <10% in 86 (15%),10 to 20% in 258(45%)>20% in 229(40%).All Patients with no VOE 107(19%) had HBF >10% with palpable splenomegaly.ONFH was diagnosed on imaging in 108 (19%).101/108(93.5%) had HBF >10%.,25/108 (23%)had palpable splenomegaly. Summary/Conclusion: In our cohort High HBF with Splenomegaly was associated with no VOE. No protection for ONFH seen with high HBF or palpable spleen Regional increased prevalence of coinheritance of Thalassemia in SCD play important role in influencing clinical presentation and progression of complication like transfusion dependency and ONFH. In LMIC, Variant haemoglobin assessment with CBC, Retic count values can be used to predict probable coinheritance of Thalassemia; however DNA Mutation analysis on larger cohort can be done for validation. Region specific risk adapted optimization of treatment can modify natural history of disease

Myeloperoxidase deficiency: a single center experience

Aim: Myeloperoxidase (MPO) deficiency is the most common inherited defect of phagocytes. In this article, we aimed to reveal clinical characteristics of our patients with primary MPO deficiency.&#x0D; Material and Method: In our study, patients aged 0-18 years, who were consulted to Ankara City Hospital Pediatric Hematology Department between 1 October 2019 and 1 December 2021 due to neutropenia, were retrospectively examined. If a patient had neutropenia in the complete blood count and inconsistently normal neutrophil count in the peripheral blood smear formula it was accepted as pseudoneutropenia. Patients with pseuduneutropenia were included in the study.&#x0D; Results: Fifteen patients diagnosed with MPO deficiency were analyzed in the study. Nine of the patients were female, 6 were male, median age of the patients was 7 (0 – 17.5) years. The mean white blood cell (WBC) count of the patients was reported as 8219±2879/mm3, and the mean neutrophil count and percentage in the complete blood count printout was 33.30±15.88/mm3 and 0,74%±0, 94% respectively. The mean neutrophil count and percentage counted in the peripheral blood smear were 5186±1710 and 63.8%±10.59%, respectively. The mean LUC value on the complete blood count printout was 54.35%±19.47% (Normal range, 0-4%). In the flow cytometry evaluation of peripheral blood samples of the patients, it was observed that neutrophils were stained with CD33, CD13, CD16, CD11b monoclonal antibodies but not with MPO.&#x0D; Conclusion: Peripheral smear evaluation is important when investigating the etiology of neutropenia. Many hematology analyzers using the MPO staining technique are indicative of MPO deficiency by identifying large unstained cells that do not stain with MPO. In patients who present with recurrent infections and MPO deficiency, other reasons that may predispose to infections should be investigated.

Genetic Spectrum of Inherited/Congenital Hemolytic Anemias in Indian Patients

IntroductionHemolytic anemias are a group of disorders caused by the premature destruction of red blood cells with reticulocytosis. Common causes of inherited/congenital hemolysis are hemoglobinopathies and thalassemia syndromes, red blood cell membrane, and enzyme disorders. Most of the common causes (thalassemia, glucose-6-phosphate dehydrogenase (G6PD) deficiency, hereditary spherocytosis, etc.) are diagnosed based on laboratory testing; however, for remaining causes laboratory tests are either inaccessible or cumbersome. We follow a stepwise diagnostic pipeline and red cell morphology is helpful with membrane disorders. Phenotypes vary from severe hemolysis (transfusion-dependent) to mild/asymptomatic patients. Undiagnosed haemolytic anemias are taken up for multi-gene panel-based targeted resequencing which is rapid, accurate, and cost-effective. The use of these panels expedites the diagnoses of inherited hemolytic anemias and is eventually helpful for evidence-based genetic counseling.ObjectivesThis study aimed to determine the genetic defects in inherited/congenital hemolytic anemias which remained unexplained after routine laboratory tests.MethodsSeventy-five families were enrolled based on the clinical and laboratory features of inherited/congenital hemolytic anemias. Common causes of inherited hemolysis are G6PD deficiency, hemoglobinopathies and thalassemia syndromes, autoimmune hemolytic anemias, hereditary spherocytosis, and pyruvate kinase (PK) deficiency were excluded on the basis of biochemical and molecular tests. DNA extraction was done QIAamp DNA Blood Mini Kit. Quantity and quality of DNA were verified using NanoDrop and Qubit Fluorometer respectively. DNA libraries were prepared using Amplicon custom panels for genes implicated in hemolytic anemias and sequenced on Illumina MiSeq Sequencer. Alignment and variant calling were done in Illumina Local run Manager and Variant annotation was done in Basespace VariantInterpretor. Sanger sequencing was done as orthogonal validation in the index case. Predictive testing was performed for the family members.ResultsAfter targeted resequencing of the total 75 index cases, 19 patients were found to have red blood cell enzymopathies, 15 patients had stomatocytosis, 13 had membranopathies and three patients had unstable hemoglobins. In 8 patients cause was not established either only heterozygous variant was found for autosomal recessive or due to the lack of samples of family members for screening. Seventeen cases remained unexplained even after next-generation sequencing.Out of 19 patients, unexpected PK deficiency was found in 12 patients and G6PD deficiency was found in 3 patients; despite the enzyme assay being normal in these cases. We also found 2 patients with glucose-6-phosphate isomerase deficiency. One case each with hexokinase deficiency and glutathione synthetase deficiency was found.Among 15 patients with stomatocytosis, 8 had Mediterranean stomatocytosis/macrothrombocytopenia (ABCG5/ABCG8). These 8 patients showed the presence of stomatocytosis along with giant platelets on peripheral smear evaluation. Of the remaining 7 cases , 2 were found to have overhydrated hereditary stomatocytosis (RHAG) and dehydrated Stomatocytosis/xerocytosis was found in 5 (PIEZO1/KCNN4).We also found 13 cases of hemolytic anemia to have a genetic defect in red blood cell membrane protein-coding genes. Of these 5 had probably pathogenic variants in the ANK1 gene, 5 had a pathogenic variant in SPTA1, 2 had SPTB 2, and 1 patient SLC4A1.We also encountered 3 cases of unstable hemoglobins where no abnormality was noted in Hb-HPLC patterns.A total of seven patients underwent splenectomy and are transfusion free.ConclusionsOur cohort of 75 families of hemolytic anemia of unexplained etiology showed a highly heterogeneous genetic spectrum. Of the total cases, the confirmed diagnosis was achieved in 67% of the patients. This approach of using a multi-gene panel is cost-effective and can provide a rapid and accurate diagnosis. Unexpected PK deficiency, G6PD deficiency, and unstable hemoglobins suggest that such cases can be missed. Providing accurate diagnosis in such cases provides evidence-based counseling and saves the families from inappropriate treatments. DisclosuresNo relevant conflicts of interest to declare.

Immune Thrombocytopenia: American Society of Hematology Guidelines, 2019

Immune Thrombocytopenia is the commonest cause of thrombocytopenia in young children. A thorough history, examination and peripheral smear evaluation is central to diagnosis. The recent American Society of Hematology guidelines 2019, has shed light on diagnosis and management based on latest available literature. We, herein, delineate the important aspects of these guidelines.

Laboratory approach to investigation of anemia with a focus on pyruvate kinase deficiency.

Anemia is a major health burden worldwide and affects approximately one-third of world's population. It is not a diagnosis; it is a manifestation of an underlying pathophysiology leading to either decreased hemoglobin (Hb), hematocrit (Hct), or red blood cells (RBCs). Iron deficiency anemia is still the most common cause of anemia worldwide. The symptoms are usually due to the underlying compensatory responses to decrease in oxygen delivery to the tissues. Laboratory investigation should start with complete blood count (CBC), reticulocyte count (RC), and peripheral smear evaluation. Further testing depends on these indices, that is, iron parameters and hemoglobinopathies/thalassemia evaluation in microcytic hypochromic anemia, vitamin B12, and folic acid level in macrocytic anemia. Increased RC denotes adequate bone marrow response and points toward hemolytic process and vice versa. Anemia diagnosis can be complex and confusing for the practicing physician. This review tries to give a practical simplistic approach to the diagnosis, focusing mainly on the basic parameters, that is, CBC, RC, and peripheral smear etc. Moreover, we have also tried to provide an update on the pyruvate kinase deficiency, as there has been recent exciting development in the management of these patients.

Evaluation of role of mean corpuscular volume and RBC-histogram in analysing anaemias as a rapid method in comparison to peripheral smear evaluation

Objective: From ages, Peripheral smear evaluation is being considered as the gold standard for evaluation of anaemias. Various other parameters have been assessed for their role as a substitute for peripheral smear. The present study evaluates Mean Corpuscular Volume (hereby referred to as MCV) and RBC-histograms if they are reliable as substitutes for the smear evaluation. Materials &amp; Methods: This is a prospective study conducted at the department of Clinical Pathology, Bhaskar Medical College. A total of 129 cases diagnosed as having anaemia were included in the study. Initially each case was analysed based upon the cell counter values, RBC indices &amp; RBC histogram. A detailed peripheral smear evaluation was done later for each case. The results were compared and analysed statistically. Sensitivity, Specificity and Positive, Negative predictive values were calculated. Results: Female cases outnumbered the male patients. Microcytic hypochromic anaemia was the most common type (61%); MCV was able to detect 75% of cases of macrocytic anaemia and 76% of cases of microcytic hypochromic anaemia. In contrast, 65% of cases of dimorphicanaemia had MCV values in the normal range. RBC-histograms were able to detect 63% of cases, 83% of cases with macrocytic anaemia, only 41% of patients had the characteristic double-peak on histograms. Conclusion: Though MCV and RBC histograms can detect most of the cases of macrocytic anaemia and microcytic anaemias with accuracy, a case with normal MCV and increased RDW requires a peripheral smear examination as the findings were variable in dimorphic anaemia.

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English

Haemostatic Profile in Patients of Myeloproliferative Neoplasms-A Tertiary Care Centre Experience

Patients of MPN commonly present with abnormalities in laboratory coagulation tests that are consistent with hypercoagulable state. Some individuals with MPN exhibit a pattern of exclusive bleeding or thrombotic events; many others have both bleeding and thrombosis during the course of the disease. This study was undertaken to assess the haemostatic defects and platelet functions in patients of MPN. One year prospective study was conducted at a tertiary care centre in North India in Department of Pathology in collaboration with Department of Clinical Haematology. All recently diagnosed cases of MPN along with 30 age and sex matched controls were included. Patients on antiplatelet drugs, antimyeloproliferative treatment, vitamin K agonists or antagonists, OCPs, Platelet count <1,00,000/μl, high grade fever, liver disease, pregnancy were excluded from this study. All the patients underwent screening investigations like CBC, peripheral smear evaluation, BT, PT, aPTT, Protein C and S measurement (clot based assay) and aggregation studies with ADP (5μM) (Optical Aggregometry with AGGRO/LINK 8 software and CHRONOLOG 700 aggregometer). In present study, 50 cases were included. There was an occult prothrombotic state, suggested by significantly (p<0.001) reduced levels of Protein C and Protein S, but no patient presented with frank thrombosis while 8 out of 50 patients had haemorrhagic manifestations ranging from subdural haematoma to pin point petechial haemorrhages. Patients of CML-CP, ET, PV, PMF, MPN-NOS showed significantly reduced maximal aggregation with ADP (5μM) when compared to control (p<0.001). MPV also showed a statistically significant increase in these patients. Thrombohaemorrhagic complications significantly affect the morbidity and mortality of MPN patients. This can be assessed by the use of platelet aggregation studies, Protein C and S activities and other coagulation studies. Timely diagnosis of these prothrombotic/haemorrhagic states can decrease the morbidity in these patients.

Evaluation of Normal Reference Range of Schistocytes and Burr Cells in Healthy Adults

Introduction:Schistocytes are split red blood cells that indicate microangiopathic hemolytic anemia. Their presence in a peripheral smear is the hallmark for diagnosing thrombotic thrombocytopenic purpura (TTP). Schistocytes may also be seen in healthy individuals and in patients with other diseases such as preeclampsia, eclampsia, chronic renal failure, solid organ or bone marrow transplantation, and diabetic microangiopathy as well as in patients with a prosthetic heart valve. Burr cells (echinocytes) are red blood cells with short, evenly spaced spicules and preserved central pallor that is usually artifactual (observed in uremia and liver disease). There has been no clear definition of a reference range for schistocytes or burr cells in normal individuals and among patients with various diseases. A previous study by Burns et al. included only 40 normal individuals to analyze the presence of schistocytes. In this study, we analyzed peripheral smears of 148 normal individuals to detect the normal reference range of schistocytes and burr cells in healthy adults and also attempted to determine the number of RBCs that need to be evaluated to consider the evaluation satisfactory.Method:We evaluated 148 peripheral blood smears of blood samples obtained from apparently healthy ambulatory adults (>18 years of age) with normal hemoglobin to determine the presence of schistocytes and burr cells.Result:Schistocytes were observed in approximately 78% of normal individuals with a mean of 0.15% of all RBCs. This finding is different from that of a previous study by Burns et al. (3) where schistocytes were observed in 58% of normal individual. Burr cell were observed in 77% of normal individual with a mean of 0.051% of all RBCs.Table 1Analysis of collected data for schistocytesRBC CountMeanStd. Deviation95% CI (predicted values)10001.731.261.51-1.9320001.921.561.74-2.1430001.591.261.39-1.8040001.170.970.97-1.3750001.261.101.06-1.46Total1.541.28Table 2Analysis of collected data for burr cellsRBC CountMeanStd. Deviation95% CI (predicted values)10000.860.870.75-0.9820000.340.500.23-0.4530000.590.790.48-0.7040000.320.690.20-0.4350000.440.560.33-0.553Total0.510.72Discussion:TTP is an acute hematologic disorder involving different organ systems. The main pathophysiology of the disease is microvascular thrombosis due to increased platelet aggregation resulting in mechanical damage to erythrocytes. The diagnosis of TTP is predominantly based on characteristic clinical and laboratory findings. Therefore, observing schistocytes on peripheral smear is one of the key findings in the diagnostic process. A schistocyte is a fragmented erythrocyte characteristic of hemolysis or cell fragmentation. There is no clear definition of its appearance, shape, or size. Schistocyte recognition in the peripheral smear is largely based on clinician experience. Its mere presence is not exclusive of any pathological conditions only because it can also be observed in peripheral smears of a healthy patient. In the study by Burns et al., schistocytes were found in all patients with a mechanical heart valve, in 93% of patients with chronic renal failure, and in 58% of normal individuals. This finding is significantly different from that of our study where schistocytes were present in 87% of normal individuals. The reason of such a difference may be the extensive evaluation of each peripheral smear with the analysis of at least 5000 cells on each slide. Burr cells, also known as echinocytes, have a speculated border over the entire cell surface. Burr cells are commonly found in both end-stage renal disease and liver disease. In our study, Burr cells were found in 80% of healthy individuals although the numbers of cells are very small. One of the key findings in our analysis is determining the successful evaluation of peripheral smears for these two morphological variants of RBCs. Our data show that there is no significant change in the variation of the presence of schistocytes if 3000 erythrocytes are evaluated in a peripheral smear. For Burr cells, the analysis of 1000 erythrocytes in a high-power field appears sufficient. However, the drawback is that it is extremely time consuming to manually count such high numbers of RBCs and thus, the chance of an error is quite high. DisclosuresNo relevant conflicts of interest to declare.

Evaluation of Macrocytosis in Routine Hemograms

Macrocytosis, a condition in which erythrocytes are larger than normal manifests as an increase in mean corpuscular volume (MCV) more than 100fl. The aim of this study was to identify the underlying causes of macrocytosis, detected in routine hemograms and to evaluate the hematological features in different etiologies. This study included 178 adult patients whose detailed medical history was recorded, and Vitamin B12 assay, folate assay, thyroid function tests, liver function tests, complete blood counts and peripheral smear evaluation was performed. Alcoholism was identified as the etiological factor in 65 cases (36.5%), Vitamin B12 deficiency in 43 cases (24.1%) and drug related in 23 cases (12.9%). These three conditions accounted for 73.6% of macrocytosis. Other causes identified were folate deficiency, liver disease, Myelodysplastic syndrome, chronic renal failure and Aplastic anemia. In 41 cases, the cause of macrocytosis could not be explained. Anemia was observed in 95 cases (53.3%) being most common in Vitamin B12 deficiency. 9 cases (20.9%) of Vitamin B12 deficiency presented with isolated macrocytosis without anemia. It was observed that mean hemoglobin was lower and red cell distribution width (RDW) higher in megaloblastic conditions. Peripheral smear revealed hypersegmented neutrophils in 86% and macro-ovalocytes in 72% of the megaloblastic cases. Complete medical history, red cell parameters and peripheral blood smear are simple, inexpensive tools which assist in identifying the underlying cause of macrocytosis, particularly in resource limited settings. Macrocytosis needs to be evaluated even in the absence of anemia, as it may be the first clue to an underlying pathology.

Geriatric Anemia: An Indian Perspective

Dear Sir, Reference to the article “Characteristics of anemia in the Elderly: A hospital based study in South India” recently published in your esteemed journal (2011 volume 27(1), pages 26–32). The authors have systematically approached anemia in the elderly Indian population, an area often neglected. The general symptoms of fatigue in these patients being often attributed to advancing age [1]. A very similar study was conducted at our hospital (St. John’s Medical College Hospital, Bangalore) and the findings published [2]. We would like to draw the comparisons between the two studies (refer Table 1). Table 1 Comparison of findings in two studies on geriatric anemia In our study all anemic patients with age above 60 years (n = 236) were included in the study over a 1 year period (2006–2007). All cases with features of any haematological malignancy were excluded unlike the referenced study. The reference range followed for definition of anemia was as advocated by the WHO (hemoglobin of 75 year age group [2]. Similar to the quoted study, we also found the most common type of anemia to be normocytic, followed by microcytic, macrocytic and dimorphic blood pictures. In addition to peripheral smear evaluation, a bone marrow examination was performed in 24.15% cases. Reactive erythroid hyperplasia was a common finding seen in 45.61% cases. Normoblastic maturation was noted in 59.64% cases, micronormoblastic maturation seen in 22.8% and megaloblastic maturation was noted in 12.28% cases. Features of Myelodysplasia was noted in 3 cases. Iron stores were assessed in all the marrow aspirates. Increased iron stores was noted in 56.14% (indicative of infection), while 0.07% cases had normal iron stores. Etiologically, nutritional anemias were the most common and seen in 77 cases (32.62%). Chronic renal failure was another common cause seen in 46 cases. These findings parallel the quoted study [2]. The authors in the said study have very effectively defined the iron deficient population into those with absolute iron deficiency (Ferritin <20 ng/ml) and mild to moderate iron deficiency (Ferritin 20–100 ng/ml). They have also highlighted the importance of investigating these patients with stool for occult blood and subjecting them to gastroscopy to rule out chronic blood loss and gastro intestinal pathologies which was a frequent finding in their study (22 upper GI and 5 colonic pathologic cases) [1]. In our study the bone marrow iron stores were used as an indicator of iron stores. Depleted iron stores were noted in 29.82% cases. Other parallel studies by Milman and Schultz-Larsen [3] reported lower prevalence of iron deficient patients (2.4% with depleted iron and 3.5% with small/minimal iron stores). As discussed the iron deficient status and anemia per se are seen more commonly in our country as seen in both the studies and other studies done in India. Prevalence of anemia in rural populations in Tamil Nadu is as high as 52% [4]. This could be explained due to lack of awareness in these individuals. In contrast, studies done in the United States (3rd national health and nutrition examination survey (NHANESIII)) to assess the prevalence of anemia in the elderly (aged >65 years), showed the prevalence to be approximately 10% [5], the rate being much lower to that in developing countries like India. In addition the NHANESIII study was done on non institutionalised individuals, unlike our studies (hospital based) [2]. Nutritional deficiencies contributed to 34%, anemia of chronic diseases (ACD) 20% and chronic renal failure (CRF) 8, 4% had both ACD and CRF. In the remaining 34% cases the cause was unexplained [5]. In our study 33 cases (14%) had unexplained anemia [2]. In conclusion, anemia in the elderly is a very prevalent condition in our country. Though nutritional anemias are still common in India, the finding of a normocytic anemia is the most frequently encountered type. As anemia may be only the presenting feature in these patients harbouring more serious pathologies, they have to be approached in a very systematic manner, in order to improve the quality of life and lifespan of the geriatric population.

Asymptomatic child heterozygous for hemoglobin S and hemoglobin Pôrto Alegre

Hemoglobin Pôrto Alegre (PA) is a rare hemoglobin resulting from a mutation in β9(A6)Ser → Cys. We describe an asymptomatic Puerto Rican female with combined heterozygosity for Hb PA and Hb S. Since birth, she has maintained normal hemoglobin, bilirubin, LDH levels, and reticulocyte count. Peripheral smear evaluation has revealed normal erythrocyte morphology with no changes suggestive of hemolysis. We conclude that the presence of Hb PA does not increase the risk of red blood cell sickling in patients who carry the Hb S mutation.

Critical evaluation of peripheral smear in cases of anemia with high mean corpuscular hemoglobin concentration in children: A series of four cases

Mean corpuscular hemoglobin concentration (MCHC), a parameter that is reported as a part of a standard complete blood count by automated analyzer, is a measure of the concentration of hemoglobin in a given volume of packed red blood cell. Values of MCHC significantly above reference range are not physiologically possible due to limitations on solubility of hemoglobin. The high MCHC can give us a clue to certain type of hemolytic anemia and necessitate critical evaluation of peripheral smear to reach a definitive diagnosis. Here we are presenting a series of four cases with raised MCHC, emphasizing the importance of systematic and meticulous examination of the peripheral smear to render a definitive diagnosis.

Cold Hemagglutinin Hemolysis and Hypercoagulable State Due to Mycoplasma Pneumoniae.

Abstract 5094 IntroductionMycoplasma pneumoniae is responsible for 7 to 20% of community-acquired pneumonia in the United States. Up to 25% of patients develop extra-pulmonary complications. Hemolytic anemia associated with multiple vascular thromboses is a rare but severe complication of Mycoplasma pneumoniae. We present a case of cold agglutinin-associated hemolysis and skin necrosis. Case PresentationAn 81 year-old male on Coumadin for atrial fibrillation who recently returned from a cruise, developed non productive cough without fever and dyspnea. He was treated with a course of azithromycin. One week later he developed bruising and skin de-pigmentation of the tips of the pinnae and tip of the nose. On presentation to the emergency department, his physical exam was unremarkable except for these finding and mild rhonchi over his right posterior chest. Laboratory workup revealed a Hemoglobin of 13.5 g/ dL, leukocytosis of 23,700 c/mm3 and platelet count of 57,000 c/mm3. Moderate renal insufficiency was also present with a creatinine of 2.02. Evaluation of peripheral smear revealed mild Schistiocytosis with anisopoikilocytosis. Within 1 week, hemoglobin dropped to 7.4, platelet count reached a nadir of 16,000. Hemolytic parameters included LDH of 647 IU/L with haptoglobin of 26 mg/dL, and reticulocyte count of 1.3%. Total complement levels were severely depressed at 4 U/ml (normal: 30 to 75 U/ml), while there was a slight decrease in C4 complement levels and the C3 levels were normal. Immunoglobulin levels were within limits and cryofibrogen was negative. ANA, anticardiolipin antibody, myeloperoxidase and p ANCA levels showed normal titers. A bone marrow biopsy showed hypercellular marrow with erythroid hyperplasia. Urine Strep. pneumoniae antigen was negative. Cold agglutinin titers were done, which were negative. Due to our strong suspicion for mycoplasma-related cold agglutinin hemolysis, we repeated cold agglutinin titers which turned positive very slowly on prolonged standing. The patient was treated with high dose prednisone and received multiple sessions of plasmapheresis which improved his condition and platelet counts returned to baseline with LDH and haptoglobin levels trending back to normal. DiscussionOur patient had a wide differential ranging from Coumadin induced skin necrosis, auricular perichondritis, frost bite, local infections, TTP/ HUS syndrome, and Wegner's granulomatosis. But the clinical presentation of superficial skin necrosis of peripheral skin along with the presence of low complement, recent chest infection, hemolytic anemia and thrombocytopenia with acute renal failure all contributed to our final diagnosis confirmed by the presence of slow reactive Coomb's direct anticoagulation test and supported by improvement with plasmapheresis and steroid therapy. The astute clinician should always remember mycoplasma infection in setting of acute onset hemolytic anemia and suspect it in recently treated chest and sinus infections. DisclosuresNo relevant conflicts of interest to declare.