Reduced Mean Corpuscular Volume Research Articles

Sub-acute toxicity effects of <i>Garcinia kola</i> on serum electrolytes, haematological, visceral organ weights and histopathological profiles in Wistar rats

This study was undertaken to evaluate the toxicity effects of root extract of G. kola on serum electrolyte, haematological and histological parameters of rats. Rats of both sexes were randomized into groups and orally administered daily with determined doses of G. kola extract using distilled water as a control for 21 days. On the 22nd day, all the animals were sacrificed and dissected to collect blood and selected organs. The serum and whole blood were assayed for serum electrolytes and haematological parameters respectively while selected organs were examined for their weight and histopathological lesions. The extract of G. kola did not cause significant alteration in majority of the serum electrolytes and hematological indices. However, the extract significantly elevated the mean corpuscular heamoglobin concentration. On the other hand, the extract reduced mean corpuscular volume, haematocrit (150 and 600 mg/kg), mean platelet volume (150 and 600 mg/kg) and procalcitonin (150 mg/kg). In the vital organs, there were no significant lesions observed except at the highest dose. The root extract of G. kola is relatively safe in rats when repetitively administered orally in small doses for a prolonged period of time to ensure that its use is free of toxicity to humans.

Development of molecular diagnostic platform for α0 -thalassemia 44.6kb (Chiang Rai, --CR ) deletion in individuals with microcytic red blood cells across Thailand.

The α0 -thalassemia 44.6kb or Chiang Rai (--CR ) deletion has been reported in northern Thailand and is capable of causing hemoglobin (Hb) H disease and a lethal α-thalassemia genotype, Hb Bart's hydrops fetalis, in this region. However, there are no current data regarding the frequency of --CR nationwide due to a lack of effective diagnostic assay. Therefore, this study aimed to develop a reliable platform for simultaneous genotyping of--CR and two common α0 -thalassemias in Thailand (--SEA and --THAI ) and investigate the frequency of--CR across Thailand. Multiplex gap-PCR assay and five renewable plasmid DNA controls for--CR ,--SEA , --THAI , α2-globin (HBA2), and β-actin (ACTB) were newly developed and validated with reference methods. The developed assay was further tested on 1046 unrelated individuals with a reduced mean corpuscular volume (MCV) of less than 75 fl for investigating genotypic and allelic spectrum of --CR . Our developed assay showed 100% concordance with reference methods. The results were valid and reproducible throughout hundreds of reactions. Comparison of the genotypic and allelic spectra revealed that heterozygous --SEA (--SEA /αα) and --SEA alleles were dominant with the frequency of 22.85% (239/1046) and 13.34% (279/2092), respectively. Of these, --THAI and --CR were relatively rare in this population and comparable to each other with the allelic frequency of 0.14% (3/2092). This study successfully established a reliable molecular diagnostic platform for genotyping of --CR , --SEA , and --THAI in a single reaction. Additionally, we demonstrated the frequency of --CR in Thailand for the first time and provided knowledge basis for the planning of severe α-thalassemia prevention and control programs in Thailand, where thalassemia is endemic.

X-linked sideroblastic anaemia in a female fetus: a case report and a literature review

BackgroundX-linked sideroblastic anaemia (XLSA) is commonly due to mutations in the ALAS2 gene and predominantly affects hemizygous males. Heterozygous female carriers of the ALAS2 gene mutation are often asymptomatic or only mildly anaemic. XLSA is usually characterized by microcytic erythrocytes (reduced mean corpuscular volume (MCV)) and hypochromia, along with increased red cell distribution width. However, in females with XLSA the characteristic laboratory findings can be dimorphic and present with macrocytic (elevated MCV) in addition to microcytic red cells.Case presentationWe report a case of fetal anaemia, presenting in the early third trimester of pregnancy, in a female fetus. Ultrasound findings at 29 weeks were of cardiomegaly, prominent umbilical veins, a small rim of ascites, and mean cerebral artery peak systolic velocity (PSV) value above 1.5 Multiples of the Median (MoM). She underwent non-invasive prenatal testing that determined the rhesus genotype of the fetus to be rhesus B negative. No red blood cell antibodies were reported. Other investigations to determine the underlying cause of fetal anaemia included microarray comparative genomic hybridization, serology to exclude congenital infection and a peripheral blood film and fetal bilirubin to detect haemolysis. The maternal grandmother had a history of sideroblastic anaemia diagnosed at the age of 17 years. The mother had mild macrocytic anaemia with haemoglobin of 10.4 g/dl and MCV of 104 fl. The fetal anaemia was successfully treated with two in utero transfusions (IUTs), and delivery occurred via caesarean section at 37 weeks of gestation. The red cell gene sequencing in both the mother and fetus were heterozygous for an ALAS2 mutation causing in utero manifestations of XLSA. The haemoglobin on discharge to the local hospital at five days of age was 19.1 g/dl. Subsequently, the infant became anaemic, requiring regular 3–4 monthly blood transfusions and demonstrating overall normal development. Her anaemia was unresponsive to pyridoxine.ConclusionsThis is one of four cases reporting multiple female members presenting with discordant clinical features of XLSA from being entirely asymptomatic to hydropic in utero. Our report is novel in that there are no previous cases in the literature of anaemia in a female fetus heterozygous for ALAS2 mutation.

Thalassaemia genes in Baghdad, Iraq

To estimate the prevalence of thalassaemia genes in Baghdad, a study was made of 502 randomly selected pregnant women attending a major maternity care clinic in the city. A reduced mean corpuscular volume [MCV] of < 80 fl was used as a screening test for thalassaemia in this population, followed by a battery of tests, including haemoglobin analysis and iron studies, to confirm the diagnosis. Using this approach, 22 cases were diagnosed of beta-thalassaemia trait, five cases of alpha-thalassaemia trait and one case of delta beta-thalassaemia trait. Based on these figures the estimated prevalence rates of beta-, alpha- and delta beta-thalassaemia traits in Baghdad, would be 4.4%, 1.0% and 0.2%, respectively. The study also includes a discussion of the above findings in the context of those reported in other neighbouring countries

Inherited microcytic anemias.

Inherited microcytic anemias can be broadly classified into 3 subgroups: (1) defects in globin chains (hemoglobinopathies or thalassemias), (2) defects in heme synthesis, and (3) defects in iron availability or iron acquisition by the erythroid precursors. These conditions are characterized by a decreased availability of hemoglobin (Hb) components (globins, iron, and heme) that in turn causes a reduced Hb content in red cell precursors with subsequent delayed erythroid differentiation. Iron metabolism alterations remain central to the diagnosis of microcytic anemia, and, in general, the iron status has to be evaluated in cases of microcytosis. Besides the very common microcytic anemia due to acquired iron deficiency, a range of hereditary abnormalities that result in actual or functional iron deficiency are now being recognized. Atransferrinemia, DMT1 deficiency, ferroportin disease, and iron-refractory iron deficiency anemia are hereditary disorders due to iron metabolism abnormalities, some of which are associated with iron overload. Because causes of microcytosis other than iron deficiency should be considered, it is important to evaluate several other red blood cell and iron parameters in patients with a reduced mean corpuscular volume (MCV), including mean corpuscular hemoglobin, red blood cell distribution width, reticulocyte hemoglobin content, serum iron and serum ferritin levels, total iron-binding capacity, transferrin saturation, hemoglobin electrophoresis, and sometimes reticulocyte count. From the epidemiological perspective, hemoglobinopathies/thalassemias are the most common forms of hereditary microcytic anemia, ranging from inconsequential changes in MCV to severe anemia syndromes.

Changes in Erythrocyte Morphology at Initiation of Cardiopulmonary Bypass Without Blood Transfusion Were Not Associated With Less Deformability During Cardiac Surgery

During cardiac surgery, circulating red blood cells (RBCs) are at risk of exposure to environmental factors during extracorporeal circulation and transfusion of stored RBCs. For this study, the authors observed morphological differences, deformability, density distribution, and erythrocyte indices of RBCs during cardiac surgery with cardiopulmonary bypass (CPB). Prospective study. Tertiary care center affiliated with a university hospital. Adults who underwent elective cardiac surgery requiring CPB. Blood samples were obtained from 13 patients before incision (baseline), at initiation of CPB, after separation from CPB, and at completion of surgery. The morphological index (MI) in RBCs using light microscopy and the maximum deformability index (DImax) using an ektacytometer were evaluated. In addition, the fractionation of RBCs and erythrocyte indices were measured. The MI at initiation of CPB was significantly higher without blood transfusion compared with baseline, although the DImax did not significantly decrease simultaneously. The DImax after separation from CPB and at completion of surgery were significantly lower than that at baseline. This lowered DImax was accompanied by a significantly reduced mean corpuscular volume and elevated mean corpuscular hemoglobin concentration compared with baseline. Dense RBC subpopulations increased after initiating CPB. The MI after separation from CPB and at completion of surgery partially recovered. Administered stored RBCs showed a high MI and the lowest DImax. Morphological changes at initiation of CPB are considered potentially reversible transformations without loss of the membrane surface area and do not have a significant effect on the DImax. A decrease in deformability likely is due to transfusion of stored RBCs.

Comparative study of haematology of two teleost fish (Mugil cephalus and Carassius auratus) from different environments and feeding habits

Haematological parameters are valuable indicators of fish health status. This study is aimed to provide baseline data of the blood profile of two teleost fish species living in different environments and with divergent feeding behaviour, namely the flathead grey mullet Mugil cephalus Linnaeus, 1758, a marine herbivorous fish, and the goldfish Carassius auratus (Linnaeus, 1758), a freshwater omnivorous fish. Using an automated system coupled with flow cytometry and light microscopy, significant variations were found between M. cephalus and C. auratus blood parameters, except for haemoglobin concentration (Hgb). A significant increase in red blood cell count (RBC) and haematocrit (Hct) levels, associated with reduced mean corpuscular volume (MCV), was revealed in mullets in respect to goldfish. These data may be attributable to differences in fish species, or to their divergent physiological activeness as high RBC values are associated with fast movement and high activity with streamlined bodies, or to environmental factors such as water salinity, an increase in which may lead to erythropoiesis as an adaptive process in seawater fish. Additionally, lower values of white blood cell count (WBC) and thrombocyte count (TC) were recorded in mullets with respect to goldfish, and these changes may be due to divergent feeding habits of the two fish species, or to their different environments since increased salinity may inversely affect WBC. Overall, findings from this study provide a better understanding of the influences of divergent environmental conditions and feeding habits on fish blood parameters. The combined use of an automatic haematological count with flow cytometry was demonstrated to be effective for an early assessment of blood parameters in different fish species.

Prenatal Diagnosis and Molecular Analysis of a Large Novel Deletion (– –JS) Causing α0-Thalassemia

α-Thalassemia (α-thal) is a very common single gene hereditary disease caused by large deletions or point mutations of the α-globin gene cluster in tropical and subtropical regions of the world. Here, we report for the first time, a novel large α-thal deletion in a Chinese family from Jiangsu Province, People’s Republic of China (PRC), which removes almost the entire α2 and α1 genes from the α-globin gene cluster. Thus, it was named the Jiangsu deletion (– –JS) on the α-globin gene cluster causing α0-thal. Heterozygotes for this deletion showed an α-thal trait phenotype with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (Hb) (MCH) levels. The sequencing results showed that a 2538 bp deletion (NG_000006.1: g.35801_38338) existed in this novel genotype on the basis of –α4.2 (leftward), indicating a deletion of about 6.8 kb from the α-globin cluster. In addition, a 29 bp sequence was inserted into the deletion during the recombination events that led to this deletion. Through pedigree analysis, we knew that the proband inherited the novel allele from his mother.

Hematological and serum biochemical findings in clinical cases of cattle naturally infected with lumpy skin disease

Lumpy skin disease (LSD) is an acute viral disease of cattle that is currently emerging in the Middle East region and poses a serious threat to Europe and the rest of the world. The objective of this study was to describe hematological and serum biochemical findings associated with natural clinical infection of LSD in cattle. A total of 129 animals clinically infected with LSD were enrolled in the study. Venous blood sample were collected from study animals, and hematological and serum biochemical parameters were measured. Leukocytopenia was found in 8.7%, while leucocytosis was found in 18.2% of affected cattle. Decreased hematocrit concentration was seen in 18.3%. Most affected cattle had reduced mean corpuscular volume (43.7%), mean corpuscular hemoglobin (14.3%), and mean corpuscular hemoglobin concentration (11.5%). All cattle with abnormal platelets count had thrombocytopenia. Hyperfibrinogenemia, hyperproteinemia, and hyperalbuminemia were found in 69%, 59.6%, and 37.2% of affected cattle, respectively. Decreased creatinine concentration was seen in 65.8%. Hyperkalemia and hyperchloremia was found in 9.6% and 10.4% of the affected cattle, respectively. LSD appears to be associated with inflammatory leukogram, anemia, thrombocytopenia, hyperfibrinogenemia, hyperproteinemia, decreased creatinine concentration, hyperchloramia, and hyperkalemia. These are likely due to the associated severe inflammatory process and disease complications such as anorexia and reduced muscle mass. This is the first study that documents hematological and serum biochemical findings associated with LSD infection. Understanding the blood profile picture may give further insight to the pathogenesis of the disease and help in treatment of individual cattle.

Consumption of transgenic milk containing the antimicrobials lactoferrin and lysozyme separately and in conjunction by 6-week-old pigs improves intestinal and systemic health

Lactoferrin and lysozyme are antimicrobial and immunomodulatory proteins produced in high quantities in human milk that aid in gastrointestinal (GI) health and have beneficial effects when supplemented separately and in conjunction in human and animal diets. Ruminants produce low levels of lactoferrin and lysozyme; however, there are genetically engineered cattle and goats that respectively secrete recombinant human lactoferrin (rhLF-milk), and human lysozyme (hLZ-milk) in their milk. Effects of consumption of rhLF-milk, hLZ-milk and a combination of rhLF-and hLZ-milk were tested on young pigs as an animal model for the GI tract of children. Compared with control milk-fed pigs, pigs fed a combination of rhLF and hLZ (rhLF+hLZ) milk had a significantly deeper intestinal crypts and a thinner lamina propria layer. Pigs fed hLZ-milk, rhLF-milk and rhLF+hLZ had significantly reduced mean corpuscular volume (MCV) and red blood cells (RBCs) were significantly increased in pigs fed hLZ-milk and rhLF-milk and tended to be increased in rhLF+hLZ-fed pigs, indicating more mature RBCs. These results support previous research demonstrating that pigs fed milk containing rhLF or hLZ had decreased intestinal inflammation, and suggest that in some parameters the combination of lactoferrin and lysozyme have additive effects, in contrast to the synergistic effects reported when utilising in-vitro models.

HbA2: at the borderline of the KLF

In this issue of Blood, Perseu et al provide new insights into our understanding of the genetic basis of elevated hemoglobin A2.1 This is a major step forward for physicians interpreting hemoglobin electrophoreses of patients with borderline hemoglobin A2, normal or slightly reduced mean corpuscular volume (MCV), and normal mean corpuscular hemoglobin (MCH).

MTOR inhibition and erythropoiesis: microcytosis or anaemia?

Anaemia and microcytosis are common post kidney transplantation. The aim of this study was to evaluate the potential role of mammalian target of rapamycin (mTOR) inhibition in the development of anaemia and microcytosis in healthy animals and in human erythroid cultures in vitro. Rats with normal kidney function were treated with sirolimus (n = 7) or vehicle (n = 8) for 15 weeks. Hemograms were determined thereafter. In the sirolimus withdrawal part of the study, rats received sirolimus (SRL) for 67 days (n = 4) 1 mg/kg three times per week or for 30 days (n = 4) and were observed until Day 120. Hemograms were performed regularly. Peripheral blood mononuclear cells from healthy controls (HC; n = 8), kidney transplant patients with sirolimus treatment with (SRL + MC; n = 8) or without microcytosis (SRL - MC; n = 8) were isolated and cultured in the absence or presence of SRL (5 ng/mL). SRL-treated animals had a reduced mean corpuscular volume (MCV) and elevated erythrocyte count compared with control animals after 15 weeks of treatment. This effect was evident as early as 4 weeks (MCV: 61.5 ± 1.8 versus 57 ± 1.7 fL; P = 0.0156; Red blood count 7.4 ± 0.3 × 10(9)/L versus 8.6 ± 0.5 × 10(9)/L; P = 0.0156) and was reversible 90 days after SRL withdrawal. SRL in the culture medium of erythroid cultures led to fewer colonies in cultures from HC as well as from kidney transplant patients (without SRL: 34.2 ± 11.4 versus with SRL: 27.5 ± 9.9 BFU-E-derived colonies P = 0.03), regardless if the cultures were derived from recipients with normocytic or with microcytic erythrocytes. The presence of tacrolimus in the culture medium had no influence on the number and size of colonies. mTOR inhibition induces microcytosis and polyglobulia, but not anaemia in healthy rats. This might be caused by growth inhibition of erythroid precursor cells.

α-thalassaemia

Alpha-thalassaemia is inherited as an autosomal recessive disorder characterised by a microcytic hypochromic anaemia, and a clinical phenotype varying from almost asymptomatic to a lethal haemolytic anaemia.It is probably the most common monogenic gene disorder in the world and is especially frequent in Mediterranean countries, South-East Asia, Africa, the Middle East and in the Indian subcontinent. During the last few decades the incidence of alpha thalassaemia in North-European countries and Northern America has increased because of demographic changes. Compound heterozygotes and some homozygotes have a moderate to severe form of alpha thalassaemia called HbH disease. Hb Bart's hydrops foetalis is a lethal form in which no alpha-globin is synthesized. Alpha thalassaemia most frequently results from deletion of one or both alpha genes from the chromosome and can be classified according to its genotype/phenotype correlation. The normal complement of four functional alpha-globin genes may be decreased by 1, 2, 3 or all 4 copies of the genes, explaining the clinical variation and increasing severity of the disease. All affected individuals have a variable degree of anaemia (low Hb), reduced mean corpuscular haemoglobin (MCH/pg), reduced mean corpuscular volume (MCV/fl) and a normal/slightly reduced level of HbA2. Molecular analysis is usually required to confirm the haematological observations (especially in silent alpha-thalassaemia and alpha-thalassaemia trait). The predominant features in HbH disease are anaemia with variable amounts of HbH (0.8-40%). The type of mutation influences the clinical severity of HbH disease. The distinguishing features of the haemoglobin Bart's hydrops foetalis syndrome are the presence of Hb Bart's and the total absence of HbF. The mode of transmission of alpha thalassaemia is autosomal recessive. Genetic counselling is offered to couples at risk for HbH disease or haemoglobin Bart's Hydrops Foetalis Syndrome. Carriers of alpha+- or alpha0-thalassaemia alleles generally do not need treatment. HbH patients may require intermittent transfusion therapy especially during intercurrent illness. Most pregnancies in which the foetus is known to have the haemoglobin Bart's hydrops foetalis syndrome are terminated due to the increased risk of both maternal and foetal morbidity.

Perfil hematólogico e características das fezes de equinos consumindo dietas hiperlipidêmicas

Objetivou-se avaliar os parâmetros sanguíneos, o consumo de matéria seca e as características fecais de equinos alimentados com dietas hiperlipidêmicas. Foram utilizados 15 equinos em delineamento experimental inteiramente casualizado, com três tratamentos e cinco repetições. As dietas utilizadas foram: dieta sem inclusão de óleo de soja (controle); dieta com inclusão de 8,5% de óleo de soja; dieta com inclusão de 19,5% de óleo de soja. O ensaio teve duração de 34 dias, sendo 30 dias de adaptação dos equinos às dietas e quatro dias de coleta de amostras. As amostras sanguíneas para avaliação hematológica e bioquímica foram coletadas no 34° dia, as características das fezes foram avaliadas no 33° e no 34° dia do ensaio e, a partir do 30° dia, o consumo de matéria seca (CMS) foi avaliado. Os equinos alimentados com as dietas hiperlipidêmicas apresentaram aumento no número de eritrócitos e redução no volume corpuscular médio (P<0,05). O nível de hemoglobina dos equinos avaliados diferiu (P<0,05) somente entre os animais que consumiram as dietas de controle e com inclusão de 8,5% de óleo de soja, com menor valor na dieta de controle de 9,5d dL-1. A inclusão de 19,5% de óleo de soja na dieta elevou os níveis séricos de triglicerídios nos equinos. Houve redução no CMS diário de 1,9 e 2,8kg, nos equinos alimentados com as dietas com 8,5 e 19,5% de óleo de soja em relação à dieta de controle de 9,0kg MS. A produção fecal diária reduziu em 0,7 e 1,1kg MS, nos equinos alimentados com as dietas com 8,5 e 19,5% de óleo de soja, respectivamente, em relação à dieta de controle de 3,4kg MS. A utilização de dietas hiperlipidêmicas para equinos com inclusão de óleo de soja possibilita a redução do consumo dietético e o aumento da disponibilidade plasmática de triglicerídeos sem a ocorrência de diarreias ou alteração das características das fezes.

Chest pain and small red cells: size does matter

A 63-year-old woman was admitted to our hospital in June, 2008, with a 24-h history of central chest pain radiating to her neck. In the 7 days before admission, she had experienced intermittent chest tightness without any obvious precipitating cause. Her medical history was unremarkable. She did not smoke and was on no regular medication. There were no abnormalities on clinical examination. ECG showed inversion of T waves in leads III, V2, and V3. Serum troponin T concentration after 12 h was 1·22 μg/L (normal range [NR] 0–0·01 μg/L) indicating an inferior non-STelevation myocardial infarction. We started our patient on aspirin, clopidogrel, and dalteparin. Serum cholesterol was slightly high (5·3 mmol/L; normal up to 5 mmol/L) but no additional risk factors for coronary artery disease were evident. Blood tests showed haemoglobin 137 g/L (NR 120–160 g/L), leuco cytes 14·5×109/L (NR 4–11×109/L), neutrophils 12×109/L (NR 2·0–7·5×109/L), and platelets 614×109/L (NR 150–400×109/L), with normal eosinophil (0·08×109/L; NR 0–0·4×109/L) and basophil counts (0×109/L; NR 0–1×109/L). C-reactive protein concentration was 4 mg/L (normal up to 5 mg/L). After 24 h, leucocyte count had normalised, and the platelets decreased to 528×109/L. Coronary angiography showed normal left coronary arteries. The right coronary artery was dominant with a long layer of fresh thrombus in the proximal/mid vessel (fi gure A), clearly the cause of her symptoms. Because of the risk of distal embolisation, angioplasty was deferred and the patient was started on an infusion of the glycoprotein IIb/IIIa inhibitor, tirofi ban. Repeat angiography after 72 h of tirofi ban showed almost complete resolution of the thrombus (fi gure B) and we managed our patient conservatively thereafter, with the initiation of dual antiplatelet therapy. On closer scrutiny of the presenting blood count, despite normal haemoglobin and packed-cell volume, there was micro cytosis with reduced mean corpuscular volume (74·5 fL; NR 80–96 fL) and hypochromia (mean corpuscular haemoglobin 23 pg; NR 27–32 pg). The serum ferritin concentration (12 μg/L; NR 13–150 μg/L) confi rmed iron defi ciency. This conclusion was supported by the microscopic appearance of the blood, which showed anisopoikilocytosis with microcytic hypochromic cells, elliptocytes, and pencil cells (fi gure C). In addition, platelet anisocytosis and giant platelets were seen (fi gure D). Paradoxically, despite iron defi ciency, there was erythrocytosis (red cell count 5·96×1012/L; NR 3·8–4·8×1012/L) indicating loss of homoeostatic control of erythropoiesis. The suspicion of an iron-defi cient myelo proliferative disorder underlying the myocardial infarction was confi rmed by the presence of a heterozygous mutation (V617F) in the JAK2 gene, detected in 90–95% of patients with myeloproliferative polycythaemia. We started our patient on hydroxy carbamide, resulting in satisfactory control of the platelet count. When last seen in May, 2009, our patient was asymptomatic with no further angina. Myeloproliferative disorders are associated with thrombotic complications with predominance of arterial over venous events. The recognition of the somatic mutation JAK2V617F in most patients with myeloproliferative polycythaemia has improved the diagnostic accuracy and bone-marrow examination is not mandatory. Diagnosis of myeloproliferative polycyth ae mia is therefore straight forward in the setting of an elevated haemoglobin and packed-cell volume. Some patients may present with a normal haemoglobin and only subtle changes in red cell indices, as a result of iron-defi ciency; identifi cation of these patients is important because of the potential for life-threatening thrombosis through mechanisms independent of haemoglobin and packedcell volume. Despite iron defi ciency, these patients should not be given iron-replacement therapy unless closely supervised, because it could stimulate a rapid and potentially catastrophic increase in haemoglobin.

Relationships between oxidative stress markers and red blood cell characteristics in renal azotemic dogs

Oxidative stress parameters and erythrocyte characteristics were studied in 15 normal healthy dogs and 33 renal azotaemic dogs from Small Animal Hospital, Faculty of Veterinary Science, Chulalongkorn University. Dogs with renal azotaemia had reduced mean corpuscular volume (MCV) ( P < 0.01), packed cell volume (PCV) ( P < 0.001) and increased mean corpuscular hemoglobin concentration (MCHC) ( P < 0.001). The relationship was found between degree of azotaemia and MCV, PCV and MCHC. Dogs with severe renal azotaemia had higher intraerythrocytic sodium contents (RBC-Na) ( P < 0.05). The red blood cell catalase activity and glutathione and plasma malondialdehyde were unaltered while urinary malondialdehyde-creatinine ratio (U-MDA/Cr) increased significantly ( P < 0.001). The U-MDA/Cr was correlated significantly with plasma creatinine concentration ( P < 0.05), urinary protein-creatinine ratio ( P < 0.05) and fractional excretion of sodium ( P < 0.001). The results suggest some changes in RBC characteristics and urine oxidative stress marker in renal azotaemic dogs. Moreover, the U-MDA/Cr is a sensitive biochemical parameter which increased along with degree of renal dysfunction.

A Novel C to A Substitution in the CCAAT Box of beta Globin Gene.

The promoter of the human beta-globin gene contains three positive cis-acting elements required for maximal transcription: the CACCC box located between −86 and −90, the CCAAT box located between −72 and − 76 and the TATA box located between −28 and −31 relative to the start site of transcription. Naturally occurring mutations within the TATA and the CACCC box regions have been recorded in patients with beta+ thalassemia. Mutations within the TATA box disrupt assembly of the basal transcription complex, while mutations at the CACCC box prevent binding of an erythroid-specific transcription factor EKLF. Surprisingly, no mutations have so far been identified in the highly conserved element CCAAT box and the transcription factors responsible for the regulatory activity of the CCAAT site in vivo have been less intensively studied. We report a novel mutation −76 C>A (HBB c. −126) detected by sequencing analysis of beta globin gene in a Italian beta+ thalassemic patient. The transversion C>A hits the first nucleotide in the CCAAT box of the beta globin gene. The carrier, a male 44 years old, shows a mild hypochromic and microcytic anaemia with reduced mean corpuscular volume and mean corpuscular haemoglobin (MCV 75 fl, MCH 25 pg) and Hb A2 level slightly increased (3.9%). Recently, studies in vitro in gel-shift and reporter assays, investigating the transcriptional activity of human beta globin CCAAT box, have identified five factors: NF-Y (CP1) a ubiquitous CCAAT box binding complex, GATA-1 an erythroid-specific transcription factor, C/EBPbeta, C/EBPgamma and C/EBPdelta members of CCAAT/enhancer-binding protein family involved in hemapoietic regulation. This represents the first report of a natural mutation of the human beta-globin CCAAT box and confirms its functional significance for in vivo transcription.

Identification of α-thalassemia mutations in subjects from Eastern Sicily (Italy) with abnormal hematological indices and normal Hb A2

We analyzed the prevalence of alpha-thalassemia mutations in 298 subjects from Eastern Sicily (Italy) with reduced mean corpuscular volume (MCV) and mean corpuscular hemoglobin (MCH), normal HbA2 and HbF, and normal serum iron. In 131 subjects (43.9%) we found six different genotypes of alpha-thalassemia: -alpha3.7/alphaalpha (36.6%), -alpha3.7/-alpha3.7 (27.5%), -(MED)/alphaalpha (10.0%), -alpha20.5/alphaalpha (9.1%), alphaHphIalpha/alphaalpha (8.4%), alphaHphIalpha/alphaHphIalpha (6.1%), and -alpha3.7/alphaHphIalpha (2.3%). Our data underline that in Eastern Sicily populations, the molecular screening of alpha-thalassemia mutations and/or deletions may be useful to better characterize the clinically asymptomatic subjects with a slightly reduced MCV and MCH and normal iron status.

High prevalence of alpha-thalassemia among individuals with microcytosis and hypochromia without anemia.

In order to determine the contribution of alpha-thalassemia to microcytosis and hypochromia, 339 adult outpatients seen at Unicamp University Hospital (with the exception of the Clinical Hematology outpatient clinics), who showed normal hemoglobin (Hb) levels and reduced mean corpuscular volume and mean corpuscular hemoglobin, were analyzed. Ninety-eight were Blacks (28.9%) and 241 were Caucasians (71.1%). In all cases, Hb A2 and F levels were either normal or low. The most common deletional and nondeletional forms of alpha-thalassemia [-alpha3.7, -alpha4.2, --MED, -(alpha)20.5, alphaHphIalpha, alphaNcoIalpha, alphaalphaNcoI and alphaTSAUDI] were investigated by PCR and restriction enzyme analyses. A total of 169 individuals (49.9%) presented alpha-thalassemia: 145 (42.8%) were heterozygous for the -alpha3.7 deletion (-alpha3.7/alphaalpha) and 18 (5.3%) homozygous (-alpha3.7/-alpha3.7), 5 (1.5%) were heterozygous for the nondeletional form alphaHphIalpha (alphaHphIalpha/alphaalpha), and 1 (0.3%) was a --MED carrier (--MED/alphaalpha). Among the Blacks, 56 (57.1%) showed the -alpha3.7/alphaalpha genotype, whereas 12 (12.2%) were -alpha3.7/-alpha3.7 and 1 (1.0%) was an alphaHphIalpha carrier; among the Caucasians, 89 (36.9%) were -alpha3.7/alphaalpha, 6 (2.5%) had the -alpha3.7/-alpha3.7 genotype, 4 (1.7%) presented the nondeletional form (alphaHphIalpha/alphaalpha), and 1 (0.4%) was a --MED carrier. These results demonstrate that alpha-thalassemia, mainly through the -alpha3.7 deletion, is an important cause of microcytosis and hypochromia in individuals without anemia. These data are of clinical relevance since these hematological alterations are often interpreted as indicators of iron deficiency.

Evidence that microdeletions in the alpha globin gene protect against the development of sickle cell glomerulopathy in humans.

There is a large variability in the severity of the clinical manifestations of sickle cell anemia (SSA), including renal involvement. Haplotypes in the beta-globin gene cluster associated with the geographical origin of the sickle mutation, as well as microdeletions in the alpha-globin genes, could provide an epigenetic influence on the heterogeneous outcome in SSA. It has been determined that the cause of progressive renal insufficiency in SSA is a glomerulopathy, clinically detected by the presence of macroalbuminuria (albumin excretion rate >300 mg/g creatinine). To investigate the role of the alpha-globin gene microdeletion and beta-globin gene cluster haplotypes on the degree of glomerular involvement, 76 adult SSA patients (hemoglobin SS) were studied to determine the relationship between these genetic markers and the development of sickle cell glomerulopathy. Macroalbuminuria was present in 22 (29%) of 76 adult SSA patients. The coinheritance of microdeletions in one or two of the four alpha-globin genes (alpha-thalassemia) was associated with a lower prevalence of macroalbuminuria (13%) versus patients with intact alpha-globin genes (40%, P = 0.01). By contrast, there was no association between albuminuria and beta-globin gene haplotypes (Central African Republic [CAR] versus non-CAR haplotypes). Patients with alpha-globin gene microdeletions had lower mean corpuscular volumes and mean corpuscular hemoglobin concentration than patients with all four alpha genes (86+/-2 versus 99+/-3 fl, and 33.9+/-0.2 versus 34.9+/-0.2%, respectively, P<0.05). There were no such hematologic differences between CAR and non-CAR beta-globin haplotypes. There were no differences in duration of disease (age), hemoglobin levels, reticulocyte index, and lactate dehydrogenase levels between those with and without glomerulopathy, but the mean arterial pressure was higher (87+/-1 mm Hg) in patients with intact alpha gene locus versus those with microdeletions (80+/-2 mm Hg, P<0.05). It is concluded that the coinheritance of microdeletions in the alpha-globin gene locus in SSA patients confers "renoprotection" by mechanisms not related to the degree of anemia or the severity of hemolysis, but could be related to a reduced mean corpuscular volume or to a lower erythrocyte hemoglobin concentration.