ALBERT

Description: Iron loading in patients with thalassemia intermedia occurs slowly and is mainly due to increased gastrointestinal iron absorption secondary to chronic anemia, while in patients with thalassemia major iron overload is faster and secondary to the chronic transfusion therapy. Moreover, iron accumulation in thalassemia intermedia is prevalent in parenchymal cells, while in thalassemia major iron derived from red cell breakdown firstly accumulates in the reticulo-endothelial cells and subsequently in the parenchymal cells. Although heart disease represents the main determinant of survival in beta thalassemias, the cardiac complications are different in the two clinical forms, that are thalassemia major and thalassemia intermedia. In this study we evaluated liver and iron overload in patients with thalassemia intermedia. We have studied 8 patients with thalassemia intermedia with a mean age of 36 ± 12 years. All these patients were homozygotes for the beta zero 39 non-sense mutation (C→T) and were never transfused or had received only sporadic transfusions (less than 10 blood units throughout their life). Myocardial iron (heart T2*, Anderson et al 2001), echocardiographic left ventricular ejection fraction, serum ferritin (mean of the last 5 years) and hemoglobin (mean of the last 2 years) have been evaluated in each patient. Hepatic iron content was determined in 5 patients with atomic absorption after liver biopsy. Six patients were on chelation therapy with subcutaneous desferrioxamine (mean 2 ± 1 grams/week). Mean ferritin was 637 ± 497 ng/ml and mean hemoglobin 8.0 ± 1.0 g/dl. Heart T2* was normal in all patients (mean 46 ± 11 msec, range 36 – 62 msec). The mean left ventricle ejection fraction was 61 ± 6 % (range 51 – 70 %). Echocardiogram showed in all the patients a mild enlargement of both ventricles. Three patients had pulmonary hypertension and two had an extrasystolic arrhythmia. To our knowledge this is the first study reporting the results of heart T2* in thalassemia intermedia. As a consequence of the mechanism and rate of accumulation patients with thalassemia intermedia do not have heart iron overload, while liver iron concentration is quite relevant. Cardiac complications in thalassemia intermedia are mainly due to the hyperdynamic circulation associated with chronic anemia and to pulmonary hypertension.

Description: The clinical phenotype of homozygous β thalassemia varies in severity from the mild thalassemia intermedia to the severe thalassemia major. This variability depends largely on the molecular heterogeneity of β thalassemia defects. We report the first case of a homozygous state for nondeletion Sardinian δ-β0 thalassemia, which resulted in a symptomless clinical phenotype with a peculiar hemoglobin (Hb) pattern (99.8% Hb F and 0.2% Hb A2). The molecular defect was characterized by the presence of 2 nucleotide substitutions: −196C〉T in the promoter of the Aγ-globin gene and β 39C〉T nonsense mutation. The absence of typical β thalassemia clinical findings was due to the high Hb F output, which compensated for the absence of β chains. The near absence of Hb A2 may have resulted from either alterations in the globin gene transcriptional complex with preferential activation of γ-globin genes and suppression of δ-globin genes or preferential survival of red blood cells with the highest Hb F content and low Hb A2 level.

Description: Abstract 5180 Alpha-thalassemia (α-thalassemia) has two clinically significant forms: hemoglobin Bart hydrops fetalis (Hb Bart) syndrome and hemoglobin H (HbH) disease. HbH disease is characterized by microcytic hypochromic hemolytic anemia, hepatosplenomegaly, mild jaundice, and sometimes thalassemia-like bone changes. Diagnostic of Alfa Thalassemia: Classic testing for α-thalassemia includes: hematologic testing of red blood cell indices, peripheral blood smear, supravital stain to detect RBC inclusion bodies, and qualitative and quantitative hemoglobin analysis. HBA1, the gene encoding α1-globin, and HBA2, the gene encoding α2-globin, are the two genes most commonly associated with α-thalassemia. Molecular genetic testing of HBA1 and HBA2 detects deletions in about 90% and point mutations in about 10% of affected individuals. 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 Alfa Thalassemia. Patient and Methods: We have collected 129 patients with Alfa Thalassemia Intermedia (HbH disease). 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). Results: Using ROC analysis, the best parameters differentiating the HbH Disease from the normals were: RDW (AUC 1. 000), @LHD(AUC 1. 000), @MAF(AUC 1. 000), @MCNRET (AUC 1. 000), MCV (AUC 0. 999), @MCRET (AUC 0. 999), @RSF (AUC 0. 998), HGB (AUC 0. 996), @MSCV (AUC 0. 995). Using ROC analysis, the best parameters differentiating the HbH Disease from other anemias (excluding normals) were: @LHD(AUC 0. 957), @MCNRET (AUC 0. 946), @MCRET (AUC 0. 902), @MAF(AUC 0. 873), MCV (AUC 0. 869). Using logistic regression we found a discrminant function that permits to differentiate/flag perfectly the patients with HbH disease from other anemias, and of course from normals: AUC 0. 996) Sensitivity: 91. 47% Specificity 94. 68% with a percent of cases correctly classified of: 93. 67 %. 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:Novartis: Research Funding, Speakers Bureau; Apopharma: Research Funding, Speakers Bureau; Ferrokin: Research Funding.

Description: The increasing number of laboratory parameters and diagnostic information makes every day more difficult to remember the entire differential Diagnosis list for all of them. The high prevalence or rare anemias in the island of Sardinia (Italy) makes mandatory to screen the general population for the possible presence of hemoglobinopathies. In fact, the prevalence of Beta Thalassemia goes from 9.1 % of the population in the City of Nuoro to 11.7% in the City of Muravera. The prevalence of Alpha Thalassemia genetic mutations is much more frequent, going from 9.1% in the City of Tempio to 39% in the City of Alghero. DAO-2 is a software tool made using C## (C Sharp) and SQL relational databases, that permit to do Multiple Differential Diagnoses that consist in the Differential Diagnosis of more than one symptom at the same time. After, using the probabilistic theory called Bayes Theorem or Rule of Bayes, gives the probability of every possible disease or medical condition. We have checked the utility of DAO-2 as a software tool for the screening of the rare anemias in Sardinia. The Ospedale Pediatrico Microcitemico is a Hospital a Specialized in thalassemia and other rare anemias. We have cheked the utility of DAO-2 in 394 consecutive patients with a known anemia that came to our hospital and also in 195 persons in that we have excluded the presence of anemia or genetic conditions that predispose to a rare anemia (controls). TableDiagnosticnNON BETA (control)195HbH (alfa thalasemia intermedia)129Thalassemia intermedia Splenectomized33Alfa Thalassemia Trait diag.suspected32Beta Thalassemia Trait30Iron Deficiency Anaemia22Thalassemia intermedia (Beta-Thal Interm)18Alfa Thalassemia Heterozygous14Thrombopenia11microdrepanocitosi (Beta-S)8Thalassemia homozygous (young children Intermedia/Major)7Beta Thal Thalassemia major transfused7HbH (Alfa Thalassemia intermedia) transfused6Hereditary Spherocytosis Splenectomized5Blackfan-Diamond Anemia3Dyserithropoietic Anaemia type I (CDA I)3Sickle cell anaemia3Iron Deficiency Anaemia treated3Piruvate-Kinase deficiency3IRIDA iron-refractory iron deficiency anemia3Beta Thalassemia major3Fanconi's Anaemia2Dyserithropoietic Anaemia type II (CDA II)2Hyporegenerative anemia2Anemia of Newborn2Megaloblastic Anemia1Piruvate-Kinase deficiency Splenectomized1Hemoglobinopaty (Taybe) + α- thalassemia1Hemoglobinopaty hyperunstable (Hb Cagliari)1Hemoglobinopaty Koln1Hemoglobinopaty E (Hb E)1Hemoglobinopaty G (Hb G-Copenaghen)1Hemoglobinopaty J (Hb J-SARDEGNA)1HbH (alfa thalasemia intermedia) Splenectomized1Leucopenia & Neutropenia1Metahemoglobinemia (MET-Hb)1Pyropoikilocytosis1Evans's Syndrome1Hereditary Spherocytosis28Delta-Beta Thalassemia (Sardegna)1HbH (alfa thalasemia intermedia) + Beta Thalassemia Trait1Total394 The use of this new software, called DAO-2 makes possible to predict in the majority of the cases the presence of a rare anemia or a heterozygous non clinical carrier of genetic changes that can provoke a rare anemia. It is necessary to do prospective studies to check the efficiency of this software before to use it in the clinical practice. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 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). 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.

Description: Abstract 5163 Introduction: Hereditary Spherocytosis (HS) is one of the most common inherited hemolytic anemias. Many of them are autosomal dominant, being about 25% of the cases transmitted recessively. Diagnostic of HS: Classic testing for HS includes: Hematologic testing of red blood cell indices (RDW, MCHC, Reticulocyte count), peripheral blood smear (presence of spherocytes), osmotic fragility and Eosin-5-maleimide binding to band 3 and Rh-related proteins forms that may be used as screening tests for hereditary spherocytosis. Objective: Recently have been developed new parameters and information in the new automated hematology analyzer called DxH8008™ from Beckman Coulter as @MSCV (@Mean Sphered Cell Volume), @RSF, @MAF, @ LHD%. 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 Hereditary Spherocytosis. There are previous studies showing the possible benefit of using MCV minus @MSCV for the detection/flagging of cases with spherocytes. Patient and Methods: We have collected 28 patients with Hereditary Spherocytosis. All of them were confirmed by red cell morphology, osmotic fragility and Eosin-5-maleimide binding to band 3 and Rh-related proteins forms. Results: Using ROC analysis, the best parameters differentiating the Hereditary Spherocytosis from the normals were: RET% (AUC 0. 996), MCV - @MSCV (AUC 0. 996), @MSCV (AUC 0. 969), RDW(AUC 0. 892), MCHC (AUC 0. 860), HGB (AUC 0. 787). Using ROC analysis, the best parameters differentiating the Hereditary Spherocytosis from other anemias (excluding normals)were: MCV - @MSCV (AUC 0. 991), MCHC (AUC 0. 987), RET% (AUC 0. 857). 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:Novartis: Research Funding, Speakers Bureau; Apopharma: Research Funding, Speakers Bureau; Ferrokin: Research Funding.

Description: Neutrophil hypergranulation, Dohle Bodies and Neutrophil vacuolation (Neutrophil Toxic Changes) are morphological cell changes seen in a number of clinically significant situations e.g. Bacterial Infection, Myelodysplastic Syndromes, etc. With increased automation, auto validation and reduced blood film review rates within Haematology, clinically important Neutrophil morphological changes may be missed, with potential delay in patient management / treatment. The detection/flagging of Neutrophil degranulation using Cell Population Data has been previously published (Laboratory Haematology 13:98-102, 2007), but there are no published DxH references for the detection / flagging of Neutrophil Toxic Changes, especially using the new CPD data from DxH 800. Cell Population Data (CPD) - numerical data developed through VCS technology (Volume, Conductivity, Scatter Laser (Light scattering) 3D cube on DxH800 & LH700 series instruments, gives additional information about the leukocytes analysed. This data is available as part of the Research cell population Data, around 8200 leukocyte events are anlaysed and the information reported as the Mean and SD of each leukocyte population. Additionally the DxH 800 uses five angles of Laser Light Scatter called: AL2, MALS, LMALS, UMALS and LALS. Study: We have identified 89 consecutive routine cases with Neutrophil Toxic Changes (NTC) and compared our data with normal cases (31). This information was analysed to identify the Neutrophil CPD that permit the detection of Neutrophil toxic changes. This discriminant CPD function was subsequently used to re-evaluate the same 89 samples with NTC, from a pool of samples from a routine lab (149 normal samples and 555 pooled abnormal samples) to determine the efficiency of the parameters found. Analysis of this data identified that the best parameter was the Neutrophil Standard Deviation of Axial Light Loss (SD-AL2-NE ): Abstract 4966. Table ROC analysis of 89 cases with NTC compared with 31 normal casesMean NormalMean NE Toxic ChangesStudent T-test pAUCSENSSPECIFCUT-OFF Sign.ROCn3189SD-AL2-NE8.874213.9263p 〈 0.00010.98692.1100〉10.31 p 〈 0.0001MN-LMALS-NE139.2903122.5843p 〈 0.00010.90983.190.312.79 p 〈 0.0001SD-MALS-NE9.19911.3715p 〈 0.00010.88580.993.5〉9.75 p 〈 0.0001SD-LALS-NE27.627738.85p = 0.12090.86580.996.8〉28.57 p 〈 0.0001 Table ROC analysis of 89 cases with NTC compared with 704 pooled samples (149 normal 555 pooled abnormal) AUC SENS SPECIF CUT-OFF Sign.ROC SD-AL2-NE 0.94 95.5 86.4 〉10.75 p 〈 0.0001 MN-LMALS-NE 0.89 85.4 85.2 13.1 p 〈 0.0001 SD-MALS-NE 0.835 82 76.3 〉10.51 p 〈 0.0001 SD-LALS-NE 0.83 79.8 80.3 〉28.27 p 〈 0.0001 We believe that adding this decision rule to the DxH800 instrument workstation - IF SD-AL2-NE 〉 〉10.75 then “Suspect flag of Neutrophil Cytoplasmic Toxic Changes (Dohle, Hypergran, Vacuoles)” may help to detect cases with these neutrophil abnormalities. We suggest that First time presentations with this suspect flag should result in the examination of the blood smear review the presence of these cell abnormalities. Prospective studies are necessary to validate these preliminary results. Disclosures No relevant conflicts of interest to declare.