Peripheral Blood Smear Analysis Research Articles

Development of a Robust Algorithm for Detection of Nuclei and Classification of White Blood Cells in Peripheral Blood Smear Images.

Peripheral Blood Smear analysis plays a vital role in diagnosis of many diseases such as leukemia, anemia, malaria, lymphoma and infections. Unusual variations in color, shape and size of blood cells indicate abnormal condition. We used a total of 117 images from Leishman stained peripheral blood smears acquired at a magnification of 100X. In this paper we present a robust image processing algorithm for detection of nuclei and classification of white blood cells based on features of the nuclei. We used novel image enhancement method to manage illumination variations and TissueQuant method to manage color variations for the detection of nuclei. Dice similarity coefficient of 0.95 was obtained for nucleus detection. We also compared the proposed method with a state-of-the-art method and the proposed method was found to be better. Shape and texture features of the detected nuclei were used for classifying white blood cells. We considered classification of WBCs using two approaches such as 5-class and cell-by-cell approaches using neural network and hybrid-classifier respectively. We compared the results of both the approaches for classification of white blood cells. Cell-by-cell approach offered 1.4% higher sensitivity in comparison with the 5-class approach. We obtained an accuracy of 100% for lymphocyte and basophil detection. Hence, we conclude that lymphocytes and basophils can be accurately detected even when the analysis is limited to the features of nuclei whereas, accurate detection of other types of WBCs will require analysis of the cytoplasm too.

A prolonged microscopic observation improves detection of underpopulated cells in peripheral blood smears.

We evaluated an extended time in the microscopic review in samples in which the potential clinical information could be increased with respect to those that could be achieved with the usual laboratory methodologies. We used samples containing nucleated red blood cells in a small amount and cytopenic samples. For these purposes for each peripheral blood smear, the timing of eye-count differential was increased up to 20min, regardless of the final number of cells which could be counted. In addition, an automated system for digital analysis of peripheral blood smears was employed and the number of cells counted was brought up to 1000 leukocytes. In both manual and automatic light microscopy extended observation, we obtained more diagnostic information in respect to those with routine or standard methods. Both automated and manual increase systems of the timing for microscopic review are useful tools to find diagnostic information that otherwise would be lost using normal and standard procedures. So, these methods should be used especially when there is a higher pre-test probability for discovery of pathological cells.

Pilot Study on the Performance of a New System for Image Based Analysis of Peripheral Blood Smears on Normal Samples.

Image analysis based automated systems aiming to automate the manual microscopic review of peripheral blood smears have gained popularity in recent times. In this paper, we evaluate a new blood smear analysis system based on artificial intelligence, Shonit™ by SigTuple Technologies Private Limited. One hundred normal samples with no flags from an automated haematology analyser were taken. Peripheral blood smear slides were prepared using the autostainer integrated with an automated haematology analyser and stained using May-Grunwald-Giemsa stain. These slides were analysed with Shonit™. The metrics for evaluation included (1) accuracy of white blood cell classification for the five normal white blood cell types, and (2) comparison of white blood cell differential count with the automated haematology analyser. In addition, we also explored the possibility of estimating the value of red blood cell and platelet indices via image analysis. Overall white blood cell classification specificity was greater than 97.90% and the precision was greater than 93.90% for all the five white blood cell classes. The correlation of the white blood cell differential count between the automated haematology analyser and Shonit™ was found to be within the known inter cell-counter variability. Shonit™ was found to show promise in terms of its ability to analyse peripheral blood smear images to derive quantifiable metrics useful for clinicians. Future enhancement should include the ability to analyse abnormal blood samples.

SCD-101: A New Anti-Sickling Drug Reduces Pain and Fatigue and Improves Red Blood Cell Shape in Peripheral Blood of Patients with Sickle Cell Disease

Background: Sickle cell disease (SCD) is the most common inherited blood disorder in the United States, caused by polymerization of a mutated form of hemoglobin (HbS). HbS can form long polymers inside red blood cells (RBCs) that affect RBC shape and adhesion, resulting in RBC destruction, acute and chronic pain, inflammation and cumulative organ damage. SCD-101 is a botanical drug with in vitro and in vivo anti-sickling activity. The mechanism by which SCD-101 inhibits sickling is unknown; however, it does not bind directly to Hb or change the affinity of Hb for oxygen. We recently completed a dose-escalation clinical study of SCD-101 in adults with sickle cell disease in steady state, and other clinical studies are on-going.Methods: The initial prospective, open-label, Phase 1B dose-escalation study enrolled homozygous (SS) sickle cell patients and S/beta0 thalassemia patients ages 18-55 with baseline Hb levels 6.0-9.0 g/dL and hemoglobin F ≤10%. Admission within 30 days, transfusion within 90 days, and hydroxyurea within 6 months were exclusions. SCD-101 was dosed orally for 28-days with a 14-day follow-up visit. Doses administered during the dose escalation study were 550 mg, 1100 mg, 2200 mg and 4400 mg BID. The primary endpoint was safety, and the secondary endpoints were mean change in Hb, Hct, percent reticulocytes, LDH, indirect bilirubin, CRP, PROMIS fatigue questionnaire score, and percent circulating partially oxygenated sickle cells (POSCs) in venous whole blood fixed in 2% glutaraldehyde without exposure to air. A repeat cohort study is ongoing with dose adjusted to 2750 mg TID, a 6-minute walk test added as a functional endpoint and measurement of inflammatory cytokines by ELISA to explore the effect of SCD-101 on inflammation.Results: As of August 4, 2016, 26 patients with sickle cell disease have been enrolled, 20 patients have completed the 28-day dose-escalation study and 3 were discontinued before receiving their first dose of SCD-101. Three patients are enrolled in the 28-day repeat cohort study. There have been no dose reductions or interruptions due to drug-related effects. SCD-101 was generally well-tolerated with mild-moderate bloating and flatulence at the highest dose on a BID schedule in the dose-escalation study, which were not observed with a TID schedule in the repeat cohort study. There were no significant changes in safety laboratory tests and no significant changes in hemoglobin, hematocrit, LDH, unconjugated bilirubin, or reticulocytes. No significant changes were observed in renal, hepatic or other safety chemistries or electrocardiograms. SCD-101 participants treated at higher doses reported a decrease in chronic pain and fatigue after 7-14 days, which returned post-treatment when measured on the 14-day follow-up visit. Participants at the two highest doses reported an increase in exercise capacity and an improvement in sleep. Two participants with ankle ulcers, enrolled at the two highest doses, showed improved healing. Analysis of peripheral blood smears revealed an improvement in the shape of circulating RBCs. There was no change in inflammatory cytokines in the first 7 days in the repeat cohort study, though the Day 28 data may show a different result.Conclusions: SCD-101 is a promising new drug for the treatment of sickle cell disease, based on the results from the studies reported here. SCD-101 was well tolerated over an 8-fold dose range, and dose-dependent anti-sickling effects on RBC were observed, though without significant changes in hemolysis. Clinical benefits included reduced chronic pain and fatigue, improved sleep and improved ulcer healing. While the identity of the active substance(s) in SCD-101 and the mechanism(s) of action are unknown, SCD-101 has a direct anti-sickling effect on RBCs, compatible with an intracellular or membrane effect on RBCs. We hypothesize that the observed effects could be explained by increased vascular flow or increased oxygen delivery or a reduction in inflammation. A placebo-controlled crossover study will be completed in 2016 including a 6-minute walk test as a primary clinical endpoint, and a multi-site Phase 2 study is planned for 2017. As a botanical drug, SCD-101 may be a useful treatment for sickle cell disease worldwide. [Display omitted] [Display omitted] [Display omitted] DisclosuresSwift:Global Blood Therapeutics: Equity Ownership; Mast Therapeutics: Equity Ownership; SCD Development: Employment, Equity Ownership.

Functional Characterization of a Novel GFI1B Mutation Causing Congenital Macrothrombocytopenia

▪Congenital macrothrombocytopenia (CMTP) is a heterogeneous group of disorders characterized by abnormally large platelets and low platelet counts. So far, genes responsible for CMTP have been identified in approximately half of the cases. The genes currently known are MYH9, GP1BA, GP1BB, GP9, ITGA2B, ITGB3, ACTN1 and other less frequent genes. However, in the remaining half, CMTP-causing genes are unknown. To identify genes causing CMTP, we first performed whole exome sequencing and target sequencing in 46 Japanese CMTP families and identified 1 family with a heterozygous mutation in GFI1B gene. Several groups have recently reported GFI1B as a causative gene for CMTP with alpha-granule deficiency and red cell anisopoikilocytosis. Here, we report on the identification of a novel mutation in GFI1B gene and its functional characterization.We performed whole exome sequencing and additional targeted sequencing in genetically undefined 46 CMTP families and identified 1 family with GFI1B variant (Figure), which co-segregated with CMTP. The proband (arrow in the Figure) of this family was a 15 years old boy with a history of easy bruising and platelet count of 70-115x109/L. His mother also had low platelet count, although details of her bleeding complications are unknown. Morphologic analysis of peripheral blood smear from the affected family members showed gray appeared enlarged platelets and red cell anisocytosis. Subsequent sequencing analysis confirmed a heterozygous point mutation (c.G814+1C, p.G272fsX274) at the splice donor site flanking exon 6 of GFI1B. GFI1B contains six zinc fingers (Znfs), of which Znf 3 to 5 are essential for DNA binding. The mutated transcript predicted a truncation of 58 C-terminal amino acids, which resulted in complete deletion of Znf 5. Since previous reports on GFI1B mutations have also described mutations of Znf 5 (p.H294fsX307 and p.Q287X mutations by Stevenson et al and Monteferrario et al, respectively), we performed biological analyses of these 3 patient-derived mutations affecting essential DNA-binding domain.GFI1B is an essential transcriptional factor for megakaryocyte and erythroid development. Therefore, we tested the effects of the GFI1b mutations on the transcriptional repression of firefly luciferase reporter constructs containing GFI1B promoter as a validated GFI1B target. Whereas wild-type GFI1B repressed the expression of reporter gene, the G272fsX274, H294fsX307 and Q287X mutants were unable to repress the expression. To test whether these patient-derived mutations acted in dominant-negative manner, we measured the effect of increasing the amount of mutant GFI1B cDNA, cotransfected into cells with a set amount of wild-type GFI1B cDNA. All 3 mutants inhibited gene repression mediated by wild-type GFI1B in a dose-dependent manner.To investigate biological effects of mutant GFI1B on proplatelet formation from megakaryocytes, we expressed the mutant GFI1B in mouse fetal liver cells (FLCs) by retrovirus-mediated gene transfer. FLCs harvested from embryonic day 13.5 embryos were transfected with pGCDNsamIRES/EGFP vector containing wild-type or the 3 patient-derived mutant GFI1B and were cultured with thrombopoietin. And we observed proplatelet formation of EGFP positive megakaryocytes. The number of proplatelet-producing megakaryocytes was reduced in mutant GFI1B transduced FLCs on day 3 and 4 after transfection, suggesting that mutant GFI1B inhibited the rate of proplatelet production from megakaryocytes. Next, we investigated the size and number of proplatelet tips from megakaryocytes, and found that proplatelet tips produced from G272fsX274, H294fsX307 and Q287X-transduced megakaryocytes were significantly reduced in numbers, but were increased in the size of the tips. This was compatible with decreased platelet count and enlarged platelet seen in GFI1B mutated patients. These findings indicated that patient-derived mutant GFI1B disrupted proplatelet formation of megakaryocyte.In summary, we have identified a novel GFI1B mutation that causes autosomal dominant macrothrombocytopenia with gray platelets and red cell anisocytosis. The mutant GFI1B acted in a dominant-negative manner over wild-type GFI1B. In addition, we first showed that the mutant-transduced FLCs produced abnormally enlarged proplatelet tips, which was reduced in numbers. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

GBT440, a Potent Anti-Sickling Hemoglobin Modifier Reduces Hemolysis, Improves Anemia and Nearly Eliminates Sickle Cells in Peripheral Blood of Patients with Sickle Cell Disease

▪Background: Sickle cell disease (SCD) is caused by polymerization of Hemoglobin S, resulting in red blood cell (RBC) sickling, RBC destruction, vaso-occlusive episodes and end-organ damage. No direct anti-polymerization, mechanism-based, preventive therapy is available. GBT440 is a novel small molecule hemoglobin modifier which increases hemoglobin oxygen affinity. In vitro and in vivo studies have shown it to be a potent and direct anti-sickling agent with high specificity for hemoglobin, and that 10-30% hemoglobin modification could be both safe (not compromising oxygen delivery) and effective (preventing HbS polymerization). We hypothesized that a potent antisickling hemoglobin modifier should rapidly interrupt RBC hemolysis, improve anemia and potentially become a safe and effective long-term disease-modifying therapy. We therefore explored safety, pharmacokinetics and pharmacodynamics and potential efficacy in healthy volunteers and SCD patients.Methods: This prospective, randomized, placebo-controlled, double blind, parallel group phase I/II study enrolled healthy volunteers (HV) ages 18-55, and homozygous HbSS SCD patients ages 18-60 with baseline Hb levels ³6 g/dL and ²10 g/dL and without vaso-occlusive crisis or transfusion within 30 days of screening. The study was conducted in two parts: part A tested single ascending doses and part B multiple ascending doses of study drug with 6:2 randomization (GBT440:placebo). Doses administered were: part A, HV cohorts 100mg to 2800 mg, SCD cohort 1000 mg; part B, HV cohorts 300 mg to 900 mg once daily for 15 days, SCD cohort 700 mg once daily for 28 days. The primary endpoint was safety. Secondary endpoints included pharmacokinetics (PK) and pharmacodynamics (PD). Clinical indices of hemolysis were prespecified as endpoints and are described for the SCD multiple dose cohort (700 mg QD for 28 days). Descriptive statistics were used to analyze data.Results: As of July 24 2015, 64 healthy volunteers (HVs) and 16 SCD patients had been enrolled. 54 healthy volunteers had completed the study, 2 were discontinued due to mild-moderate nonserious adverse events (headache, rash) and 8 were in follow-up; 8 SCD patients had completed Part A of the study and 8 were in Part B follow-up. No SCD patients were discontinued; one part B subject had a dose reduction from 700 mg to 400 mg (due to abdominal discomfort). GBT440 was generally well-tolerated, most adverse events were mild, there were no deaths, and there was one serious adverse event (AE) of acute painful crisis in a placebo subject. There were no AEs related to tissue hypoxia. GBT440 showed dose proportional PK, a terminal half-life of 1.5-3 days, high partitioning into RBCs (RBC:Plasma 60-90:1), and a dose dependent increase in hemoglobin oxygen affinity in both HV and SCD patients. In the multiple dose SCD cohort, all patients were evaluable at 28 days (age range 21-52 years; 2 were on hydroxyurea; 3 males/5 females). GBT440-treated patients showed increased hemoglobin, hematocrit and erythrocyte counts with corresponding decreases in LDH, unconjugated bilirubin, reticulocytes and erythropoietin levels with trends evident as early as day 8 compared to no changes in the placebo group (Figure 1 and Table 1). Analysis of peripheral blood smears revealed a marked reduction in sickle cells with GBT440 treatment.Conclusions: Single, oral, daily dosing with GBT440 was well tolerated across a wide dose range and demonstrated dose proportional and predictable PK and PD. GBT440 demonstrated proof of mechanism with a dose-dependent increase in hemoglobin oxygen affinity without causing tissue hypoxia. GBT440 demonstrated proof of concept in SCD patients with rapid reduction in RBC hemolysis and improved oxygen delivery to tissues as evidenced by reduced erythropoietin level, and a marked reduction in circulating sickle cells. These results support further clinical investigation of GBT440 as a potential disease-modifying therapy for SCD.Table 1Mean change to Day 28 (SEM)GBT440PLAHemoglogin (g/dL)0.8 (0.2)-0.5 (0.4)% Sickle cells in peripheral blood (%)-83 (9)19 (4)Erythrocyte count (1012/L)0.5 (0.1)-0.1 (0.2)Reticulocyte count (%)-2.2 (1)0.8 (1.6)LDH (% change from baseline)-12 (7)-9 (1)Unconjugated bilirubin (% change from baseline)-25 (10)-9.6 (16)Erythropoietin (U/L)-29 (16)21 (33)GBT = GBT440 treated subject; PLA = placebo subject; SEM = standard error of the mean [Display omitted] DisclosuresLehrer-Graiwer:Global Blood Therapeutics: Employment, Equity Ownership. Howard:Pfizer: Consultancy; Novartis: Consultancy, Other: Travel Grant; Aes-Rx: Consultancy. Layton:Agios: Consultancy; Novartis: Consultancy; Glaxo Smith Kline: Consultancy. Mant:Quintiles: Employment, Equity Ownership. Dufu:Global Blood Therapeutics: Employment, Equity Ownership. Hutchaleelaha:Global Blood Therapeutics: Employment, Equity Ownership. Koller:Global Blood Therapeutics: Employment, Equity Ownership. Oksenberg:Global Blood Therapeutics: Employment, Equity Ownership. Patel:Global Blood Therapeutics: Employment, Equity Ownership. Ramos:Global Blood Therapeutics: Employment, Equity Ownership.

Interpretation of Macrocytes in Peripheral Blood Smears

Background‘Macrocytes’ refers to larger erythrocytes with increased mean corpuscular volume (MCV) in routine blood smears. It can be either an incidental finding or may be associated with underlying disease. Aim of the studyto identify the underlying cause for macrocytosis and also to analyse the haematological features of macrocytosis in routine haemogram and peripheral blood smear. Resultsout of 89 adult patients, alcoholism was the major etiological factor of macrocytosis, followed by Vitamin B12 deficiency and drug intake. Isolated macrocytosis without anaemia was seen in 20 cases. Peripheral smear examination revealed hypersegmented neutrophils and macroovalocytes predominantly in megaloblastic conditions. ConclusionsMacrocytes on peripheral blood smears has to be evaluated even in absence of anaemia. Careful analysis of routine haemograms and peripheral blood smears assist in identifying the underlying cause for macrocytosis.

Prognostic Significance Of TP53mutations and Single Nucleotide Polymorphisms In Acute Myeloid Leukemia

BackgroundThe response to treatment and overall survival (OS) of patients with acute myeloid leukemia (AML) is variable, with a median OS ranging from several months to more than 10 years. Age at diagnosis, performance status (PS), and karyotype expression have long been established in prognostication. Loss of TP53, a tumor suppressor gene located on the short arm of chromosome 17, is one of the most frequent genetic abnormalities in human cancer and is one of the more promising prognostic markers for AML. Studies have shown that TP53 mutations are present in 5-25% of all AML patients, in 70% of those with complex karyotypes, and are associated with old age, chemotherapy resistance, and worse OS. Single nucleotide polymorphisms (SNPs), changes in DNA seen in an appreciable amount of the population, have been examined in AML and studies have suggested a possible correlation with worse outcomes. Using genetic sequencing, we set out to look at our own experience with AML, and hypothesized TP53mutations and SNPs would mimic the literature, occurring in a minority of patients, and conferring a worse OS. MethodsWe performed a pilot study of randomly selected, newly diagnosed AML patients at Mount Sinai Medical Center, diagnosed from 2005-2008 (n =10). Immunohistochemical (IHC) analysis of bone marrows and peripheral blood smears was assessed via DO-1 antibody on paraffin embedded tissue. Conventional cytogenetic analyses were performed on short-term cultured bone marrow and peripheral blood cells with the use of the GTG-banding technique. TP53PCR sequencing was performed using DNA from bone marrow smears using the Sanger sequencing platform and resolved by capillary electrophoresis. Analysis was performed using Mutation Surveyor software with confirmation of the variants using the COSMIC and dbSNP databases. Descriptive frequencies and median survivals were calculated for demographic information, prognostic factors, and treatment variables. A univariate analysis was performed. ResultsThe majority of patients in our pilot study were older than age 60 (80%), male (60%), Hispanic (60%), and had a poor PS (ECOG 2-3: 60%). Most patients had de-novo AML (50%) with an intermediate (50%) non-complex (70%) karyotype and a TP53 P72R SNP (50%). Fewer than half of these patients harbored TP53 mutations (40%). There was no significant difference in OS based on sex, AML history, risk-stratified karyotype, or TP53 mutation. There was a trend toward improved survival among patients younger than age 60 (11, 4 mo, p = 0.09), of Hispanic ethnicity (8, 1 mo, p = 0.11), and those not harboring P72R (8, 2, p = 0.10). There was a significant improvement in survival among patients with a better PS (28, 4 mo, p = 0.01) and those who did not have a complex karyotype (8, 1 mo, p = 0.03). Among patients with a TP53-mutation, there were a larger number of individuals who were younger than age 60 (25.0, 16.7%), who were male (75.0, 50.0%), had a good performance status (ECOG 0-1: 50.0, 16.7%), had de-novo AML (50.0, 66.7%), and who had an adverse karyotype (50.0, 33%). Patients with a P72R SNP were more often male (80, 40%) and had a worse PS (ECOG 2-3: 80, 40%) with AML secondary to MDS (60, 20%) and a complex karyotype (40, 0%). The most commonly observed TP53 mutation was a missense N310K (40%) and the most commonly observed SNP was P72R (100.0%). Patients with more than one TP53mutation had a worse clinical course than those with only a single mutation. ConclusionOur study demonstrated that poor PS and the presence of a complex karyotype were associated with a decreased OS. TP53 mutations were relatively uncommon, occurring more frequently in male patients with an adverse karyotype. Although there was no significant difference in survival between TP53 mutated and un-mutated patients, there was a trend toward worse OS among patients with a specific SNP. These results suggest that different TP53mutations and SNPs should not be treated the same, and that some may confer a worse prognosis than others. Larger studies are needed to validate these findings.Table 1PatientMutationSNPKaryotype and Gene AbnormalitiesOS (mo.)1-P72RTrisomy 8, deletion chromosome 16, trisomy 22, trisomy 9, CBFB gene rearrangement12P89SP72RUnknown43-P72RDeletion chromosome 20, trisomy 6, trisomy 22, isochromosome 21q, trisomy 11, MLL gene rearrangement14-P72RNormal115V272G-Normal48-P72R6C238G-inv(3), deletion chromosome 52N310K-7N310K-t(1,2), deletion chromosome 528 Disclosures:No relevant conflicts of interest to declare.

Clinical performance evaluation of the CellaVision Image Capture System in the white blood cell differential on peripheral blood smears

IntroductionDifferential counting and morphological analysis of peripheral blood smears is of great diagnostic importance to the clinician. For economic and time-saving reasons, automated imaging processes have been successfully introduced over...

Megaloblastic Anemia and Mitochondriopathy Caused by a Homozygous Mutation in Sideroflexin-4.

AbstractAbstract 79Megaloblastic anemias are characterized by impaired DNA metabolism, often due to deficiencies in vitamin B12 or folate. Genes underlying hereditary forms of megaloblastic anemia not caused by vitamin B12 or folate deficiencies, however, remain largely unknown. Here we characterize a genetic deficiency in a patient with infantile-onset megaloblastic anemia, developmental delays, and a mitochondrial disorder of unknown etiology. Analysis of peripheral blood smears from the patient revealed hypersegmented neutrophils and erythroid macrocytes, classic features of megaloblastic anemias. The patient's vitamin B12 and folate levels are normal, eliminating their deficiency as potential causes of the disease.Whole-exome sequencing of the proband cDNA identified a homozygous, single nucleotide deletion (c.231delC) in Sideroflexin-4 (SFXN4), a predicted mitochondrial multi-spanning transmembrane protein. We experimentally verified the mitochondrial localization of SFXN4 using a combination of western analyses on mitochondrial lysates and confocal fluorescence immunohistochemistry. Using trypsin-sensitivity assays on isolated mitoplasts, we further determined the submitochondrial localization of SFXN4 to the inner mitochondrial membrane.Bioinformatic analyses predict that the mutation introduces a frame shift and a premature stop codon (p.Pro78Leufs*25), resulting in a severely truncated polypeptide. To determine whether the mutant mRNA were expressed in vivo, we used qRT-PCR to assess the steady state level of SFXN4 mRNA in cultured fibroblasts from the proband. qRT-PCR revealed a 92% reduction in SFXN4 expression, consistent with nonsense-mediated decay of the mutant transcript. Genotyping of the index patient and 3 generations of her nuclear family using both Sanger sequencing and allele-specific oligonucleotide hybridization showed that the mutant allele is inherited in an autosomal recessive manner (Fig. A), the result of a presumed founder effect. [Display omitted] We used complementary zebrafish and human fibroblast systems to model the megaloblastic anemia and mitochondrial disease in the patient, respectively. Using splice-blocking antisense morpholino oligomers (MO) targeting sfxn4, we induced a loss-of-function phenotype in zebrafish embryos (hereafter, referred to as “morphants”). qRT-PCR confirmed the efficient knockdown of sfxn4, as morphants have <10% sfxn4 mRNA. Knockdown of sfxn4 in transgenic, Tg(globin LCR:eGFP) zebrafish showed a gross reduction in GFP+ erythrocytes and hemoglobinized cells stained by o-dianisidine (Fig. B, top), while quantification of the red cell population by flow cytometry showed a 60% reduction in the red cell mass. To characterize the anemia, we performed cytospins of flow-sorted erythroid cells from sfxn4 morphants and analyzed their morphology. Wright staining revealed that sfxn4 morphants have red cells with large nuclei containing non-condensed chromatin (Fig. B, bottom), consistent with the features of megaloblastic anemia observed in the index patient. Enumeration of the nuclear: cytoplasmic area ratios showed that red cells from sfxn4 morphants have a nearly 3-fold increase in the ratio of nuclear to cytoplasmic size.We also investigated the mitochondrial disorder using patient fibroblasts, which showed a severe reduction in complex I (37%) and complex I+III (7%) activity. The over-expression of wild-type human SFXN4 in proband fibroblasts completely rescued the respiratory defect of complex I+III, while transfection of the mutant c.231delC SFXN4 construct failed to increase complex I+III activity. In a complementary strategy, the over-expression of wild-type SFXN4 cRNA from either zebrafish or human partially rescues the anemia in morphant embryos, validating their functional orthologous relationship.In summary, a recessive loss-of-function mutation in SFXN4, a previously uncharacterized gene, causes the megaloblastic anemia and mitochondrial disorder described in the index patient. Genetic complementation studies in patient fibroblasts and sfxn4-silenced zebrafish morphants validate the pathogenicity of the mutation. Our findings: (1) demonstrate the requirement of SFXN4 for mitochondrial homeostasis and erythropoiesis, and (2) establish SFXN4 as a new candidate gene for mitochondriopathies and megaloblastic anemias. Disclosures:No relevant conflicts of interest to declare.

Beta Thalassemia Trait: How the New Information Provided by the Routine Hematology Analysers May Help in Its Differential Diagnosis or Flagging

AbstractAbstract 5186 Introduction:Beta Thalassemia (β-thalassemia) is one of the more common hemoglobinopathies worldwide, being the heterozygous variant, called Beta Thalassemia Trait, a benign variant, but important to diagnose, for genetic counseling, trying to avoid the homozygous variant, called major. Diagnostic of Beta Thalassemia Trait:Classic testing for β-thalassemia includes: hematologic testing of red blood cell indices, peripheral blood smear (prewsence of target cells and RBC with basophilic stippling, etc.), and qualitative and quantitative hemoglobin analysis. Have been proposed too Discriminant functions, like the one published many years ago, by England and Fraser. Objective:Recently have been developed new parameters and information in the new automated hematology analyzer called DxH8008™ from Beckman Coulter as @MSCV, @RSF, @MAF, @ LHD% and many morphological parameters for RBC and Reticulocytes calles Cell Population Data. All this parameters may be used to create flagging for laboratory use only (LUO) or Research use only (RUO). The purpose of this study is to investigate the possible use or utility of this new information for the screening/flagging of Beta Thalassemia Trait. Patient and Methods:We have collected 30 patients with Beta Thalassemia Trait. All of them were confirmed by red cell morphology, Hgb Electroforesis, cromatography in liquid phase in human whole blood for the determination of Hemoglobin A2, F, A1c, and identification of abnormal hemoglobins and DNA analysis (DNA Analysis by GAP-PCR). We have compared these patients with a control group (184 individuals) and with other anemias (see Table 1).Table 1DiagnosticnNON BETA (control)184HbH (alfa thalasemia intermedia)129Alfa Thalassemia Heterozygous48Beta Thalassemia Trait (study group)30Hereditary Spherocytosis28Iron Deficiency Anaemia22Thalassemia intermedia (Beta-Thal Interm)18microdrepanocitosi (Beta-S)8Blackfan-Diamond Anemia3Dyserithropoietic Anaemia type I (CDA I)3Sickle cell anaemia3Piruvate-Kinase deficiency3IRIDA iron-refractory iron deficiency anemia3Fanconi's Anaemia2Dyserithropoietic Anaemia type II (CDA II)2Hyporegenerative anemia2Megaloblastic Anemia1Piruvate-Kinase deficiency Splenectomized1Hemoglobinopaty (Taybe) + α-thalassemia1Hemoglobinopaty hyperunstable (Hb Cagliari)1Hemoglobinopaty Koln1Acute Lymphoblastic Leukemia (ALL)1Pyropoikilocytosis1Evans's Syndrome1 Results:Using ROC analysis, the best parameters differentiating the Beta Thalassemia Trait from the normals were: MCV (AUC 1. 000), MRV (AUC 0. 999), @MAF(AUC 0. 999), @MCNRET (AUC 0. 997), RDW (AUC 0. 957), HGB (AUC 0. 915), RBC(AUC 0. 912).Using ROC analysis, the best parameters differentiating the Beta Thalassemia Trait from other anemias (excluding normals) were: RDW-SD (AUC 0. 937), DF Eng-Fra (AUC 0. 779), RDW (AUC 0. 766), RBC (AUC 0. 734) Disclosures:Simon-Lopez:Beckman Coulter: @LHD, @MAF, @RSF, @LHD, @MAF, @RSF Patents & Royalties, Employment. Di Gaetano:Instrumentation Laboratory spa: Work for a distributor of Beckman Coulter Instruments in Italy Other. Galanello:Ferrokin: Research Funding; Apopharma: Research Funding, Speakers Bureau; Novartis: Research Funding, Speakers Bureau.

RAP-536 Promotes Terminal Erythroid Differentiation and Reduces Anemia in a Murine Model of Myelodysplastic Syndromes

AbstractAbstract 3796Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by peripheral blood cytopenias such as anemia, neutropenia or thrombocytopenia. Ineffective erythropoiesis due to increased proliferation and abortive maturation of precursors leads to severe anemia, the most common cytopenia observed in MDS syndromes. Despite elevated erythropoietin (EPO) and erythroid hyperplasia, MDS patients are often given recombinant EPO therapy to stimulate erythropoiesis. However, only a small proportion of patients respond to EPO therapy. Frequent blood transfusions as supportive care result in iron overloading and recently iron overloading is also linked to enhanced progression to AML. Therefore, alternative therapies are necessary to treat anemia in MDS patients.Signaling by members of the TGFβ superfamily are known regulators of erythropoiesis. We developed ACE-536, a ligand trap consisting of a modified activin receptor Type IIB extracellular domain linked to a human Fc domain. In vitro assays revealed that ACE-536 inhibits smad 2/3 ligands of the signaling pathway but not smad 1/5/8 ligands. Dose dependent studies using ACE-536 in mice, rats and monkeys revealed that ACE-536 treatment resulted in increased red blood parameters but did not affect other cell types. These data suggests that ACE-536 inhibits smad 2/3 phosphorylation modulating the expression of downstream genes involved in erythroid development pathway. BFU-E and CFU-E colony formation assays from bone marrow and spleen in mice following ACE-536 treatment revealed that ACE-536 did not affect the proliferation stages of erythropoiesis. In mice, terminal erythroid differentiation analysis by flow cytometry at 72hrs following RAP-536 (10mg/kg) treatment demonstrated decreased basophilic and increased ortho- and poly-chromatophilic erythroblasts and reticulocytes compared to VEH treatment. Cell cycle analysis of bone marrow and splenic erythroblasts counterstained with BrdU and 7-AAD after RAP-536 (10mg/kg, for 24 hours) or VEH treatment to EPO pre-treated (1500 units/kg, for 40 hours) mice (N=5/group) revealed that EPO+RAP-536 treatment resulted in significant decrease in S-phase and increase in G1/G2-phases of cell cycle compared to EPO+VEH treatment. In addition, EPO+RAP-536 treatment resulted in a greater increase in RBC parameters than either of the treatments alone. Together, these results demonstrate that ACE-536 increases red blood cell formation by promoting maturation of late stage erythroblasts.We then investigated the effect of ACE-536 on anemia in NUP98-HOXD13 (NHD13) transgenic murine model of MDS. NHD13 mice develop anemia, neutropenia and lymphopenia, with normal or hyper cellular bone marrow. A Majority of the mice die by 14 months due to severe pancytopenia or progression to acute myeloid leukemia. In this study, mice were divided into three groups based on age. Early (∼4 months old), mid (∼8 months old) and late stage (∼10 months) groups were randomized and dosed with either RAP-536 at 10 mg/kg or VEH twice per week for 6–8 weeks. NHD13 mice in each group had severe anemia characterized by reduced RBC, Hemoglobin and HCT and compared to wild-type littermates prior to treatment. Treatment of RAP-536 for 6–8 weeks significantly increased RBC parameters and reversed anemia at all stages. Peripheral blood smear analysis revealed no indication of increased leukemic progression due to RAP-536 treatment. Cell differential and flow cytometric evaluation of erythroid precursors from bone marrow demonstrated decreased erythroid precursors and hyperplasia after RAP-536 treatment compared to vehicle treated control.Our data demonstrate that RAP-536 can increase hematology parameters by enhancing maturation of terminally differentiated red blood cells. We have shown RAP-536 corrects ineffective erythropoiesis, decreases erythroid hyperplasia and normalizes myeloid: erythroid ratios without enhanced progression to AML in a murine MDS model. Therefore ACE-536 may represent a novel treatment for anemia associated with MDS, particularly in patients that are refractory to EPO therapy. ACE-536 has completed Phase I clinical trials in healthy human volunteers and Phase II study in MDS patients is planned. Disclosures:Suragani:Acceleron Pharma Inc: Employment, Equity Ownership. Li:Acceleron Pharma Inc: Employment, Equity Ownership. Sako:Acceleron Pharma Inc: Employment, Equity Ownership. Grinberg:Acceleron Pharma Inc: Employment, Equity Ownership. Pearsall:Acceleron Pharma Inc: Employment, Equity Ownership. Kumar:Acceleron Pharma Inc: Employment, Equity Ownership.

Marginal zone lymphomas in HIV patients

Incidence of Non-Hodgkin’s Lymphoma (NHL) remains high in the context of HIV infection. High-grade NHL are the sub-types commonly seen. Indolent lymphomas have been rarely described although several cases of follicular or marginal zone lymphoma (MZL) have been published. Among MZL, MALT lymphomas are predominant. We describe 3 cases of MZL with leukemic presentation. In our on-going cohort of 580 HIV-patients, 3 cases of MZL were identified. The following characteristic were recorded: clinical exam, thoracoabdominal CT-scan, cytological aspect of the peripheral blood smear, immunologic and cytogenetic analysis. There were 2 male and 1 female. Duration of HIV infection was 8, 13 and 18 when MZL was diagnosed. HCV serology was positive in 2 cases with pcr negativity in one case. In all cases, MZL was diagnosed during the occurrence of a mild hyperlymphocytosis: 5000, 6000 and 9000/mm3 respectively while hemoglobin and platelets remained normal. All patients were received HAART for more than 8 years and all have CD4 above 500/mm3 and viral load under 50 copies/ml. All patients were asymptomatic without peripheral adenopathy neither hepatosplenomegaly. On thoracoabdominal CT scan, only infracentrimetric adenopathy were detected. On peripheral smears, several lymphocytes with a villous aspect were detected for each patient. In all cases, immunologic phenotype by FACS was consistent with the diagnosis of MZL. Karyotype of the peripheral lymphocytes was normal in one case, revealed an isochromosome 3 in the second case and showed 46, XX, der(1)t(1;?), t(8;14;18)(q24;q32;q21) in the last case. After a follow-up of 2, 2 and 5 years, all patients are alive with a stable disease without chemotherapy. These data suggest that, as for non-HIV infected patients, MZL have an indolent course in HIV patients.

Standards and impact of hematopathology in myelodysplastic syndromes (MDS).

The diagnosis, classification, and prognostication of patients with myelodysplastic syndromes (MDS) are usually based on clinical parameters, analysis of peripheral blood and bone marrow smears, and cytogenetic determinants. However, a thorough histologic and immunohistochemical examination of the bone marrow is often required for a final diagnosis and exact classification in these patients. Notably, histology and immunohistology may reveal dysplasia in megakaryocytes or other bone marrow lineages and/or the presence of clusters of CD34-positive precursor cells. In other cases, histology may reveal an unrelated or co-existing hematopoietic neoplasm, or may support the conclusion the patient is suffering from acute myeloid leukemia rather than MDS. Moreover, histologic investigations and immunohistology may reveal an increase in tryptase-positive cells, a coexisting systemic mastocytosis, or bone marrow fibrosis, which is of prognostic significance. To discuss diagnostic algorithms, terminologies, parameters, and specific issues in the hematopathologic evaluation of MDS, a Working Conference involving a consortium of US and EU experts, was organized in June 2010. The outcomes of the conference and resulting recommendations provided by the faculty, are reported in this article. These guidelines should assist in the diagnosis, classification, and prognostication in MDS in daily practice as well as in clinical trials.

Primary cutaneous CD4+/CD56+ hematodermic neoplasm (blastic NK‐cell lymphoma): a case report

A previously healthy 44‐year‐old Chinese man presented with a nonitching, painless, skin lesion on the right lateral abdomen of 1‐year duration. This lesion presented initially as a nodule of approximately 0.5 cm in diameter, which eventually grew to the size of an egg by August 2006. The patient had experienced no systemic symptoms, and his past medical and family history were noncontributory. Physical examination was normal except for the skin lesion. Peripheral lymph nodes were not palpable. Skin examination showed a lobulated, contusiform, brownish, infiltrated nodule with a wide base on the right lateral abdomen (Fig. 1).A lobulated, contusiform, brownish, infiltrated nodule with a wide base on the right lateral abdomenimage Histologic examination of a biopsy specimen taken from the lesion revealed the presence of a dense dermal lymphomatous infiltrate with a diffuse growth pattern, which extended into the subcutis. The Grenz zone was present. No signs of necrosis, karyorrhexis, hemophagocytosis, or angiocentric infiltrate were found (Fig. 2a). The tumor was composed of medium‐sized pleomorphic cells with dispersed chromatin and absent or indistinct nucleoli, resembling lymphoblasts. Frequent mitosis was seen (Fig. 2b).(a) Dense dermal lymphomatous infiltrate with a diffuse growth pattern. The Grenz zone was present. No signs of necrosis, karyorrhexis, hemophagocytosis, or angiocentric infiltrate were found (hematoxylin and eosin, ×40). (b) The tumor was composed of medium‐sized pleomorphic cells with dispersed chromatin and absent or indistinct nucleoli, resembling lymphoblasts. Frequent mitosis was seen (hematoxylin and eosin, ×400)image Immunohistochemical studies revealed the reactivity of more than 90% of the cells with CD56, CD4, and CD43 (Fig. 3a,b). Immunostaining for CD3, CD8, CD20, CD30, CD68, CD79a, CD99, T‐cell intracellular antigen‐1 (TIA‐1), Gallyas–Braak silver (GB), L26, Kp‐1, myeloperoxidase (MPO), and terminal deoxynucleotidyl transferase (TdT) was either weak or negative. Clonal rearrangement of the T‐cell receptor (TCR) was not detected by polymerase chain reaction (PCR). Epstein–Barr virus (EBV) staining of the skin biopsy specimen was negative.Immunohistochemical studies revealed the reactivity of more than 90% of the cells with CD4 (a) and CD56 (b) (SP, streptavidin‐perosidase, ×400)image Laboratory examinations, including complete blood examination with peripheral blood smear, liver enzymes, electrolyte levels, kidney function, antinuclear antibody, and urine analysis, were all normal. EBV antibodies were negative. Chest radiograph, nasal bone X‐ray, and bone marrow biopsy revealed no abnormal findings. Computed tomography of the nasal cavity, abdomen, and pelvis was normal. On the basis of the aforementioned clinical, histopathologic, and immunohistochemical findings, the patient was diagnosed with primary cutaneous CD4+/CD56+ hematodermic neoplasm [blastic natural killer (NK)‐cell lymphoma]. After receiving a diagnosis of a very aggressive malignancy, the patient refused chemotherapy until right groin lymphadenopathy occurred 6 months later. In February 2007, the lesion was excised and showed the same pathologic features, consistent with cutaneous blastic NK‐cell lymphoma. From March 2007, the patient received five courses of polychemotherapy with cyclophosphamide, adriamycin, vincristine, and prednisolone (CHOP). The lymphadenopathy disappeared after one cycle of chemotherapy. The patient received four more cycles of chemotherapy and no recurrence was observed over the following 6 months.

Imaging spectrum of EBV-infection in a young patient

A wide variety of atypical presentations with complications affecting multiple organ systems during acute infectious mononucleosis (IM) is described in the literature, with an increase in the number of teenagers who are susceptible to a severe case of the disease. We report a case of a 14-year-old girl with severe IM and acute abdominal pain. Ultrasonographic (US) evaluation showed a marked thickening of the gallbladder wall (GBW) with enlargement of some mesenteric lymph nodes. CT scan showed multiple enlarged lung nodules of various sizes and a small pleural and pericardial effusion; a hypodense solid mass of unknown etiology was detected in the anterior mediastinum, mimicking a malignant tumor. Hematological analysis of peripheral blood smear was performed to exclude neoplastic pathology. IM was identified as the only underlying disease. The patient was carefully monitored: clinical evaluation, laboratory analysis and US examination were repeated at weekly intervals, until recovery.

Identification of circulating CD10 positive T cells in angioimmunoblastic T-cell lymphoma

In most cases of lymphomas with blood dissemination, the careful cytological analysis of peripheral blood smears provides a rapid orientation to diagnosis, even if the final subtyping is achieved by histology and eventually other techniques. Here, we evaluated if the analysis of blood smears may suggest the blood dissemination of angioimmunoblastic T-cell lymphoma (AITL) and if CD10 expression on neoplastic T cells, as recently reported on AITL, may contribute to the diagnosis. In all, 11 lymph nodes and six peripheral blood samples from 12 patients with AITL were studied using four-colour flow cytometry associated to histological, cytological and molecular data. According to previous results, a fraction of T cells expressed CD10 in 10/11 lymph nodes. Interestingly, all blood smears showed atypical lymphoid cells and a fraction of T cells expressed CD10 with a mean percentage of 18.75% (range 5.00-47.00%), regardless of lymphocytosis level and of rate of CD10 T cells in corresponding lymph node. In contrast, in all control samples (100), none CD10-positive T cell was identified. This is to our knowledge the first description of circulating CD10 neoplastic T cells in AITL. Therefore, they ought to be explored in further studies when aggressive lymphoma, in particular with lymphopenia and circulating atypical cells, is suspected.

Additional Environmental and/or Genetic Factors Are Required to Trigger TTP in ADAMTS13-Deficient Mice.

Thrombotic Thrombocytopenic Purpura (TTP) is a life threatening systemic illness characterized by the formation of platelet-rich thrombi in the microcirculation and the clinical pentad of fever, hemolytic anemia, thrombocytopenia, neurological symptoms, and renal dysfunction. TTP is associated with ultra-large von Willebrand Factor multimers (UL-VWF) in the circulation due to deficiency of the ADAMTS13 metalloprotease. ADAMTS13 gene mutations account for most if not all cases of familial TTP, and autoantibodies to ADAMTS13 underlie most cases of acquired TTP. To further explore the pathogenesis of TTP in vivo, ADAMTS13 deficient mice were generated by gene targeting to remove exons 1–6 which encode most of the protease domain of ADAMTS13. Although the resulting homozygous null mice (Adamts13−/−) have lost all specific VWF-cleaving protease activity (< 1% of control), embryonic development is normal, and the null mice are born at the expected Mendelian frequency. Analysis of baseline hematologic parameters and peripheral blood smears revealed no difference between Adamts13−/− mice and wild-type littermates, with no evidence for thrombocytopenia or microangiopathic hemolytic anemia. Pathologic survey of multiple tissues revealed only normal histology and no evidence for platelet or VWF-rich thrombi in the vasculature. Despite the absence of VWF-cleaving protease activity, plasma from wild-type and ADAMTS13-deficient mice exhibited identical VWF multimer size distributions. However, VWF multimers from both wild-type and Adamts13−/− mice were observed to be considerably larger than those from normal human plasma, and equivalent in size to UL-VWF seen in plasma from familial TTP patients. Challenge of Adamts13−/− mice by injection with endotoxin, and genetic crosses to mice with markedly elevated VWF levels (CASA/Rk), failed to induce findings consistent with TTP. However, treatment with verotoxin-2 (a bacterial endothelial toxin important in the pathogenesis of the hemolytic uremic syndrome), caused thrombocytopenia in 6 of 11 Adamts13−/− mice (vs. 3 of 11 wild-type controls, p < 0.07) and mortality at 6 days in 9 of 11 Adamts13−/− mice (vs. 6 of 11 controls, p < 0.02). Examination of peripheral blood from one of the Adamts13−/− mice at 8 days following verotoxin administration demonstrated marked microangiopathic changes. In conclusion, mice with targeted disruption of the Adamts13 gene do not develop TTP spontaneously, suggesting the requirement for additional environmental triggers or genetic modifiers. Though some humans with congenital ADAMTS13 deficiency have been reported to remain asymptomatic for many years, the increased size of the baseline VWF multimer distribution in mice may indicate a higher threshold for VWF-ADAMTS13 interaction which may be protective for TTP. Our results also suggest that microbial-derived toxins, or other sources of endothelial injury, may be one of the key environmental triggers responsible for the lack of spontaneous TTP findings in the ADAMTS13-deficient mice, and possibly for the intermittent symptoms seen in humans.

Human AML1/MDS1/EVI1 fusion protein induces an acute myelogenous leukemia (AML) in mice: a model for human AML.

The human t(3;21)(q26;q22) translocation is found as a secondary mutation in some cases of chronic myelogenous leukemia during the blast phase and in therapy-related myelodysplasia and acute myelogenous leukemia. One result of this translocation is a fusion between the AML1, MDS1, and EVI1 genes, which encodes a transcription factor of approximately 200 kDa. The role of the AML1/MDS1/EVI1 (AME) fusion gene in leukemogenesis is largely unknown. In this study, we analyzed the effect of the AME fusion gene in vivo by expressing it in mouse bone marrow cells via retroviral transduction. We found that mice transplanted with AME-transduced bone marrow cells suffered from an acute myelogenous leukemia (AML) 5-13 mo after transplantation. The disease could be readily transferred into secondary recipients with a much shorter latency. Morphological analysis of peripheral blood and bone marrow smears demonstrated the presence of myeloid blast cells and differentiated but immature cells of both myelocytic and monocytic lineages. Cytochemical and flow cytometric analysis confirmed that these mice had a disease similar to the human acute myelomonocytic leukemia. This murine model for AME-induced AML will help dissect the molecular mechanism of AML and the molecular biology of the AML1, MDS1, and EVI1 genes.

ひ破裂を併発した重症熱帯熱マラリアの１例

Pathological rupture of the spleen is a recognized but rare complication of malarial infection. This is a case report of a 59-year-old male patient with a pathological rupture of the spleen caused by Plasmodium falciparum infection. The patient first became febrile at 40°C 7 days after returning from Kenya. He was admitted to ourhospital with dysfunction of the renal, liver, and coagulation systems. Examination of a peripheral blood smear and DNA analysis of the blood sample using alle-specific PCR demonstrated a severe malarial parasitemia with P. falciparum alone. He was treated immediately with quinine, which completely removed the parasites by the 3rd day of admission. Nevertheless, repeated ultrasonography revealed a rapidly enlarging spleen, and he suddenly fell into hemorrhagic shock due to rupture of the spleen on the 4th hospital day. The spleen with a tear was removed because of uncontrolled bleeding. Histopathological examination revealed features of atrophy of the lymphoid follicles, dilated sinusoidal space, and prominent erythrophagocytosis. He recovered from severe malaria and was discharged without any disability. It is important to anticipate that malaria can be a primary cause of pathological rupture of the spleen, and we should also pay attention to rapid hyperplasia of the spleen, even after parasitemia has disappeared.