Differentiation between Thalassemia Trait and Iron Deficiency Anemia Based on Low Hemoglobin Density and Microcytic Anemia Factor.

BACKGROUND: The most common causes of microcytic hypochromic anemia are thalassemia trait (TT) and iron deficiency anemia (IDA). Clinically, the differential diagnosis of TT and IDA is crucial, but it is typically challenging. Thus, in order to differentiate between TT and IDA, we seek to develop a new discriminative index on an automatic hematology analyzer utilizing the two new RBC characteristics of low hemoglobin density (LHD) and microcytic anemia factor (MAF). METHODS: We recruited a total of 323 subjects, including 115 healthy controls, 83 TT, and 125 IDA. An automated hematology analyzer (DxH800, Beckman Coulter) was used to determine peripheral blood parameters; LHD and MAF were calculated using the parameters of MCHC, Hb, and MCV. The receiver operating characteristic (ROC) curve was used to determine the cutoff values and evaluate the diagnostic value for TT and IDA. RESULTS: LHD was significantly lower in TT than IDA, whereas MAF was higher. To distinguish between TT and IDA, a new formula based on LHD and MAF was developed, with a cutoff value of 0.5, AUC of 0.9706 (95% CI: 0.9503 - 0.9909), and specificity, sensitivity, positive predictive value, and negative predictive values were 92.91%, 91.36%, 89.16%, and 94.40%, respectively. The new formula has proven advantages over conventional indices, such as RDW-SD, MCV, MCH, etc. Conclusions: The RBC parameters LHD and MAF detected by hematology analyzer could be useful for screening for TT and IDA. Our new formula outperforms other discriminant formulas in the literature with high sensitivity and specificity, is simple, rapid, and can aid in early detection and management.

Two Novel α-Thalassemia Mutations CD 39 -C [Thr > Pro] and CD 109 ACC > CCC [Thr > Pro] Identified in Two Chinese Families: A Case Report.

We reported the identification of two rare α-thalassemia silent carriers with novel HBA1 mutations of CD 39 -C [Thr > Pro] (HBA1: c.114del; p.Thr39Profs*11) and CD 109 ACC > CCC [Thr > Pro] (HBA1: c.325A > C; p. Thr109Pro), respectively. The two probands were pregnant women diagnosed with mild hypochromic anemia or microcytic hypochromic anemia by routine blood tests. They started iron therapy before taking differential diagnosis from iron deficiency anemia. After wait and watch approach, they both accepted thalassemia genetic screening, which identified CD 39 -C [Thr > Pro] and CD 109 ACC > CCC [Thr > Pro], respectively. Due to inappropriate iron therapy, worse anemia and iron overload were noticed in the first proband, but no obvious side effect was found in both probands. Functional analysis showed that, relative to the wild type, CD 39 -C [Thr > Pro] considerably reduced the expression of the HBA1 protein while CD 109 ACC > CCC [Thr > Pro] only had a minor impact. Our study highlighted the importance of gestational thalassemia screening based on next-generation sequencing for identifying novel rare thalassemia variants and increased our understanding about the relationship between genotype and phenotype of α-thalassemia.

TT@MHA: A machine learning-based webpage tool for discriminating thalassemia trait from microcytic hypochromic anemia patients.

BACKGROUND: Iron deficiency anemia (IDA) and thalassemia trait (TT) are the most common causes of microcytic hypochromic anemia (MHA) and are endemic in lower resource settings and rural areas with poor medical infrastructure. Accurate discrimination between IDA and TT is an essential issue for MHA patients. Although various discriminant formulas have been reported, distinguishing between IDA and TT is still a challenging problem due to the diversity of anemic populations. METHODS: We retrospectively collected laboratory data from 798 MHA patients. High proportions of α-TT (43.33 %) and TT concomitant with IDA (TT&IDA) patients (14.04 %) were found among TT patients. Five machine learning (ML) approaches, including Liner SVC (L-SVC), support vector machine learning (SVM), Extreme gradient boosting (XGB), Logistic Regression (LR), and Random Forest (RF), were applied to develop a discriminant model. Performance was assessed and compared with six existing discriminant formulas. RESULTS: The RF model was chosen as the discriminant algorithm, namely TT@MHA. TT@MHA was tested in an interlaboratory cohort with a sensitivity, specificity, accuracy, and AUC of 91.91 %, 91.00 %, 91.53 %, and 0.942, respectively. A webpage tool of TT@MHA (https://dxonline.deepwise.com/prediction/index.html?baseUrl=%2Fapi%2F&id=26408&topicName=undefined&from=share&platformType=wisdom) was developed to facilitate the healthcare providers in rural areas. CONCLUSION: The ML-based TT@MHA algorithm, with high sensitivity and specificity, could help discriminate TT patients from MHA patients, especially in populations with high proportions of α-TT patients and TT&IDA patients. Moreover, a user-friendly webpage tool for TT@MHA could facilitate healthcare providers in rural areas where advanced technologies are not accessible.

Z score analysis: A novel approach to interpretation of an erythrogram.

Context: Z score defines the shift of an observed value from the mean. Aims: By determining the direction of this shift and its absolute value for mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC), one can quickly screen the hemogram for any spurious results in RBC parameters and also predict the type of anemia. This is because MCH and MCHC are derived parameters (from Hb, RBC, MCV) and thereby reflect the true as well as false changes in an erythrogram. Materials and Methods: A total of 975 hemograms were studied retrospectively. Basic statistical formulae using mean and standard deviation were applied to calculate z scores for MCH and MCHC. Results obtained were compared with the standard method and validated by an independent cohort of 100 random samples run on a different machine. Results and Statistical Analysis: Z score was found to be statistically significant (p <.001) in diagnosing iron deficiency anemias, megaloblastic anemias, hemolytic anemias, regenerative anemias, anemia of chronic disease and spurious findings. Z score was not significant (p = 0.9) in predicting beta thalassemia trait. The sensitivity was low for the differentials of microcytic hypochromic anemias. Conclusions: Despite this, Z score can be of immense help to the clinicians and pathologists in making quick interpretation of the underlying red cell abnormalities. Also, it can be used as a quality assessment tool in hematology laboratories taking pre analytical and analytical factors into account.

Iron in the General Population and Specificities in Older Adults: Metabolism, Causes and Consequences of Decrease or Overload, and Biological Assessment.

Iron is involved in many types of metabolism, including oxygen transport in hemoglobin. Iron deficiency (ID), ie a decrease in circulating iron, can have severe consequences. We provide an update on iron metabolism and ID, highlighting the particularities in older adults (OAs). There are three iron compartments in the human body: 1) the functional compartment, which consists of heme proteins including hemoglobin, myoglobin and respiratory enzymes; 2) iron reserves (IR), which consist mainly of liver stocks and are stored as ferritin; and 3) transferrin. There are two types of ID. Absolute ID is characterized by a decrease in IR. Its main pathophysiological mechanism is bleeding, which is often digestive and can be due to neoplasia, frequent in OAs. Biological assessment shows low serum ferritin and transferrin saturation (TS) levels. Furthermore, hypochromic microcytic anemia is frequent, and the serum-soluble transferrin receptor (sTfR) level is high. Functional ID, in which IR are high or normal, is due to inflammation, which is also frequent in OAs, particularly in its chronic form. Biological assessments show high serum ferritin, normal or low TS, and normal sTfR levels. Moreover, C-reactive protein is elevated, and there is moderate non-regenerative non-macrocytic anemia. The main characteristics of iron metabolism anomalies in the elderly are the high frequency of ID (20% of ID with anemia in adults ≥85 years) and the severity of its consequences, which include cognitive impairment in case of ID or iron overload and decrease of physical activity in case of ID. In conclusion, causes of ID are frequently intertwined in OAs as a result of the polymorbidity that characterizes them. ID can have dramatic consequences, especially in frail OAs. Thus, measuring the appropriate biological markers prevents errors in the positive diagnosis of ID type, clarifies etiology, and informs treatment-related decision-making.

Heterozygosity for the Novel HBA2: c.*91_*92delTA Polyadenylation Site Variant on the α2-Globin Gene Expanding the Genetic Spectrum of α-Thalassemia in Iran.

There are four copy numbers of α-globin genes (16p13.3) in the human genome and the number of defective α-globin genes dictates the severity of α-thalassemia (α-thal). Mutations that occur in the 3' untranslated region (3'UTR), and especially at the polyadenylation (polyA) sites, affect the translation, stability and export of mRNA. A patient with hypochromic microcytic anemia was referred to the Kariminejad-Najmabadi Pathology & Genetics Center, Tehran, Iran by the health network. Molecular analysis of genomic DNA for the evaluation of mutations on the α- and ß-globin genes was performed. Direct sequencing of the hemoglobin (Hb) subunit α2 (HBA2) gene revealed a two nucleotide deletion between +816 and +817 in the 3'UTR, located at the polyA site, which seems to be a novel pathogenic variant. This novel variant expands the genetic spectrum of α-thal in the 3'UTR of the HBA2 gene.

Analysis of rare thalassemia caused by HS-40 regulatory site deletion.

ABSTRACT Objectives: To investigate the effect of HS-40 regulatory site deletion on α-globin gene expression and its clinical significance. Methods: Venous blood samples of subjects were analyzed using a hematology analyzer and high- performance liquid chromatography; fetal cord blood was analyzed by a capillary electrophoresis analyzer. Gap-polymerase chain reaction (PCR), reverse dot blot (RDB), and multiple-link-dependent probe amplification (MLPA) were used for genotyping of thalassemia. Results: The proband was POLR3 K, HS-40 heterozygous deletion; the proband's wife was -SEA/αα; the fetus was POLR3 K, HS-40 heterozygous deletion combined with -SEA deletion; all of them had microcytic hypochromic anemia. Fetal umbilical cord blood electrophoresis revealed a suspected Hb Bart's band to be 88.4%, and the fetus was, thus, diagnosed as Hb Bart's fetus. The red blood cell parameters of the sporadic case showed that he had microcytic hypochromic anemia. Hemoglobin (Hb) electrophoresis analysis showed Hb H to be 5.3%, leading to a diagnosis of Hb H disease. Gap-PCR and RDB identified the genotype to be -α3.7/αα, ßA/ßA. MLPA detected heterozygous deletion or -α3.7 deletion on one allele and deletion of the HS-40 regulatory site on the other allele. Conclusion: The deletion of HS-40 regulatory site reduced expression of α-globin. HS-40 heterozygous deletion manifested as mild anemia, which was of microcytic hypochromic type. When compounded with -α3.7/αα, it manifested as Hb H disease; and when compounded with -SEA/αɑ, it manifested as Hb Bart's fetus.

Application of HbA2 levels and red cell indices-based new model in the differentiation of thalassemia traits from iron deficiency in hypochromic microcytic anemia Cases.

INTRODUCTION: Thalassemia traits and iron deficiency anemia are the most common types of hypochromic microcytic anemia with similar clinical and laboratory features. It is vital to establish a new screening model based on HbA2 levels and red cell indices for the differentiation of TT from IDA in hypochromic microcytic anemia cases. METHOD: The data comprised of the red blood cell indices and HbA2 prenatal diagnostic test results of 810 individuals who were identified to conform to the following criteria: MCV < 80 fl or MCH < 26 pg. We launched a new model consisting mainly of significative red cell indices and HbA2 levels, as well as proposing cutoff values by using decision trees and logistic regression analyses. Next, we evaluated our new method by comparing the sensitivity, specificity, positive, and negative predictive values with those of the previous formulas. RESULTS: We put forward a new model and compared it with 5 efficient formulas. The new model exhibited the highest accuracy (0.918), with its sensitivity and specificity calculated as 0.917 and 0.921, respectively. Our new model's Youden index was 0.838, which is higher than the other formulas' Youden indices. CONCLUSIONS: The new screening model, based on HbA2 levels and red cell indices, is suitable for the screening of thalassemia patients in the hypochromic microcytic anemia group and has the best efficiency in distinguishing TT and IDA.

Nondeletional α-Thalassemia: Two New Mutations on the α2 Gene.

About 10.0% of α-thalassemia (α-thal) cases are due to point mutations, small deletions, or insertions of one or more bases on the α genes that can alter mRNA processing at the transcription, translation, or post-translation level; these cases are called nondeletional α-thalassemias (α-thal). Most occur within the domain of the α2 gene without changes in the expression of the α1 gene. We present two new frameshift mutations on the HBA2 gene, associated with a nondeletional α-thal phenotype. The probands were referred to our clinic because of persistent microcytosis and hypochromia. The molecular characterization was performed by automatic sequencing of the α-globin genes. Two new mutations were detected on the HBA2 gene; HBA2: c.85delG, p.(Ala29fs*21), and HBA2: c.268_280delCACAAGCTTCGGG, p.(His90Trpfs*9). These new mutations cause a change of the reading frame, the first on codon 28 and the second from codons 89 to 93. In the first mutation, the result is an altered amino acid sequence and a premature termination codon at position 87, while the elimination of 13 bp generates a protein of 95 residues and in this case, the premature termination codon is at position 96. These types of mutation are among the most damaging changes to the coding of a protein. Not only do they lead to changes in the length of the polypeptide, but they also vary the chemical composition, which would result in a nonfunctional protein. The importance of identifying these new mutations lies in their possible association with α0-thal, which could lead to a severe thalassemia.

α-Globin Genotypes Associated with Hb H Disease: A Report from Oman and a Review of the Literature from the Eastern Mediterranean Region.

α-Thalassemia (α-thal) is the most common autosomal recessive hemoglobinopathy. There is a vast diversity and geographical variability in underlying genotypes in Hb H (ß4) patients. Herein, we describe the genotypes found in the largest report of Omani Hb H patients. Moreover, we reviewed and summarized the literature published from the Eastern Mediterranean region. A retrospective review of all genetically confirmed Hb H disease patients diagnosed between 2007 and 2017 at Sultan Qaboos University Hospital, Muscat, Oman, was performed. Hematological parameters and clinical presentations were assessed. Both α-globin genes were screened for deletional and nondeletional mutations using a stepwise diagnostic strategy as described before. A total of 52 patients (27 females and 25 males) with a mean age of 20.6 years (range 0.23-80.0) were molecularly confirmed to carry Hb H disease. The patients had a hemoglobin (Hb) level of 9.3 g/dL (range 5.7-13.0) and mean corpuscular volume (MCV) of 58.4 fL (range 48.2-82.1). A total of eight genotype combinations were identified, with α2 polyadenylation signal mutation (polyA1) (AATAAA>AATAAG (αPA1α/αPA1α), often cited as αT-Saudiα/αT-Saudiα, being the most common (53.8%) followed by -α3.7/- -MED I (28.8%). Our cohort also included patients with combinations of αPA1 with other Hb variants: αPA1α/αPA1α with Hb S (HBB: c.20A>T) trait (n = 2), -α3.7/αPA1α (n = 2) and αcodon 19α (HBA2: c.56delG)/αPA1α (n = 1). Nondeletional Hb H disease due to the αPA1 mutation is the most common in Omanis. Molecular diagnosis is necessary for accurate confirmation of the diagnosis of α-thal, determination of underlying genotypes, follow-up and counseling.

An Unusual Compound Heterozygosity for Hb O-Arab (HBB: c.364G>A) and Hb D-Los Angeles (HBB: c.364G>C).

We report a newborn with a compound heterozygosity for Hb O-Arab (HBB: 364G>A) and Hb D-Los Angeles (HBB: 364G>C). To the best of our knowledge, the combination of these two hemoglobin (Hb) variants has not been identified and reported before. The variants of the proband and parents were identified by high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE). DNA analysis was performed to confirm the variants. The levels of Hb variants of the proband were determined post-partum, at 3 months and 1 year after birth. Blood count analysis after 1 year revealed that the proband had a mild microcytic anemia. Furthermore, HPLC and CE analysis revealed an equal distribution of Hb D-Los Angeles compared to Hb O-Arab at the age of 1 year. The follow-up of the patient, suggested that the Hb combination is clinically silent or mild.

Association between Different Polymorphic Markers and ß-Thalassemia Intermedia in Central Iran.

ß-Thalassemia intermedia (ß-TI) is a clinical condition characterized by moderate, non transfusional anemia and hepatosplenomegaly. The main objective of this study was to determine the molecular basis of the clinical phenotype of ß-TI in Iran. To elucidate the mild phenotype of many patients with ß-TI, we screened for three prevalent ß-globin gene mutations [IVS-II-1 (G>A) HBB: c.315+1G>A, IVS-I-110 (G>A) HBB: c.93-21G>A and IVS-I-5 (G>C) [HBB: c.92+5G>C], deletions on the α-globin genes, XmnI polymorphisms and restriction fragment length polymorphism (RFLP) haplotypes on the ß-globin gene cluster in 50 ß-TI patients. Fifty-eight percent of the patients (29 cases) were associated with the mentioned mutations. We showed that the HBB: c.315+1G>A mutation is linked to haplotype [+ - + +] (57.69%). This haplotype is in linkage disequilibrium with the XmnI polymorphism (NG_000007.3: g.42677C>T) and has been associated with increased expression of Hb F in ß-TI patients. The XmnI polymorphism is defined in association with this prevalent mutation. Two patients had a single α-globin gene deletion [-α3.7 (rightward) deletion]. The main genetic factor in mild phenotype ß-TI patients is the linkage of an XmnI polymorphism (NG_000007.3: g.42677C>T) with the HBB: c.315+1G>A (80.76%), which is associated with increased production of Hb F and coinheritance of haplotype [+ - + +] with ß-TI, especially with the homozygous HBB: c.315+1G>A mutation. Molecular basis of ß-TI could be explained by the involvement of different factors that tend to develop the disease phenotype.

A Novel ß-Thalassemia Mutation [IVS-I-6 (T>G), HBB: c.92+6T>G] in a Chinese Family.

ß-Thalassemia (ß-thal) is one of the most common inherited hemoglobin (Hb) disorders in southern China. Up to now, the mutation spectrum of ß-thal has been increasingly broadened through various molecular methods. In this study, a 34-year-old female displaying microcytic, hypochromic anemia was first detected with a novel IVS-I-6 (T>G) (HBB: c.92+6T>G) mutation by Sanger sequencing. Pedigree analysis performed on her family showed that her mother and her daughter, who had abnormal hematological indices, also carried this mutation, while her other family members with normal hematological phenotypes, were not detected to carry any mutation. Based on the observed symptoms in this Chinese family, we concluded that this novel mutation was associated with a mild ß-thal phenotype.

ThalPred: a web-based prediction tool for discriminating thalassemia trait and iron deficiency anemia.

BACKGROUND: The hypochromic microcytic anemia (HMA) commonly found in Thailand are iron deficiency anemia (IDA) and thalassemia trait (TT). Accurate discrimination between IDA and TT is an important issue and better methods are urgently needed. Although considerable RBC formulas and indices with various optimal cut-off values have been developed, distinguishing between IDA and TT is still a challenging problem due to the diversity of various anemic populations. To address this problem, it is desirable to develop an improved and automated prediction model for discriminating IDA from TT. METHODS: We retrospectively collected laboratory data of HMA found in Thai adults. Five machine learnings, including k-nearest neighbor (k-NN), decision tree, random forest (RF), artificial neural network (ANN) and support vector machine (SVM), were applied to construct a discriminant model. Performance was assessed and compared with thirteen existing discriminant formulas and indices. RESULTS: The data of 186 patients (146 patients with TT and 40 with IDA) were enrolled. The interpretable rules derived from the RF model were proposed to demonstrate the combination of RBC indices for discriminating IDA from TT. A web-based tool 'ThalPred' was implemented using an SVM model based on seven RBC parameters. ThalPred achieved prediction results with an external accuracy, MCC and AUC of 95.59, 0.87 and 0.98, respectively. CONCLUSION: ThalPred and an interpretable rule were provided for distinguishing IDA from TT. For the convenience of health care team experimental scientists, a web-based tool has been established at http://codes.bio/thalpred/ by which users can easily get their desired screening test result without the need to go through the underlying mathematical and computational details.

Carcinoma of the colon in a 40 year old female: a case report.

We present a histologically proven mucinous adenocarcinoma of the colon in a 40 year old female from Gulu, Northern Uganda. Her elder sister died at 25 years with advanced adenocarcinoma of colon similarly with her mother who died of the same illness 10 years apart. Using the Amsterdam criteria for the diagnosis of the carcinoma of the colon, this is descriptive of Hereditary Non Polyposis Colorectal Carcinoma (HNPCC). Blood examinations revealed microcytic hypochromic anaemia. The Renal and Liver function parameters were essentially normal. The abdominal ultrasonography showed an ill-defined mass in the right hypochondrial region which was heterogeneous with central echogenicity approximately 7.2cm wide and with no intra-abdominal lymphadenopathy or ascitis. At laparotomy, the sonographic findings were confirmed with a demonstrable mass in the hepatic flexure of the colon with hyperemic areas on its serosa. Macroscopically, there was an annular fungating mass with a central necrosis in the hepatic flexure measuring over 7.0cm. Histology of the colonic tumour showed a mucinous adenocarcinoma of the colon (Duke's B). This finding highlights the occurrence of colonic adenocarcinoma in the young person in Northern Uganda, a finding which draws the attention of the medical community towards having a higher index of suspicion for carcinoma of the colon in patients with similar presentation.

The association between microcytic anemias and spirometric parameters.

INTRODUCTION: Microcytic anemia is a type of anemia with smaller than normal red blood cells. Iron deficiency anemia and thalassemia are some of the major causes. The aim of the study was to compare the pulmonary function of the subjects with microcytic anemia to the respective results of the normal population. MATERIAL AND METHODS: This was a cross-sectional study in Bandar Abbas, Iran, conducted on the patients attending yearly occupational health checkups. Complete blood cell count and a standard spirogram were attained from each consenting participant and occupational histories of exposure to dust, fumes, solvents, and noxious gases were obtained. RESULTS: At last, 2,199 subjects were included in the analysis, of which 335 cases had microcytic anemia. There was a significant association between having microcytic anemia and forced vital capacity (FVC) reduction, and to a lesser degree, the reduction of forced expiratory volume in the first second (FEV1). These parameters were also significantly increased together with the rise of mean corpuscular volume (MCV) in the sample population. CONCLUSION: It can be concluded that having microcytic anemia may reduce some spirometric parameters. Even though these changes are small, adjusting for the reduced values can help prevent an overestimation of lung disorders, mostly in borderline cases.