Evaluation of role of HPLC (Merits & Pitfalls), in the diagnosis of various hemoglobinopathies & thalassemic syndromes.

BACKGROUND: : HPLC is one of the most important tools for accurate diagnosis of hemoglobinopathies and thalassemias. The advantage of the HPLC system is the excellent resolution, reproducibility &quantification of several normal and abnormal hemoglobin. RESULTS: BIO RAD Variant II analyzer was used. Sickle cell syndromes including double heterozygous states accounted for 56.13% of total cases. HbSS, HbS/ß0-th, HbS/ß+-th ß-thal trait comprises 29%, 6.5%, 5.1%& 10% of total cases respectively with mean MCV (fl) = 84, 68,71,64 respectively. The Mean HbA2 for ß-thal trait, HbE trait &HbE-ß thal showed 5.1 ± 1.1, 19 ± 9 & 24 ± 8 respectively. HbF is increased in 8.6% case (excluding SC syndromes & ß-thal disorders), of these 5.5% were infants & 12 cases of Aplastic Anemias. Peak P2 >7% (2.4% cases) was seen in uncontrolled diabetes mellitus which on quantification showed HbA1C = 8 ± 2.1 mmol/L. DISCUSSION: : HPLC in correlation with CBC parameters & family studies can aid in the diagnosis of majority of Hemoglobinopathies and thalassemic syndrome. The CBC & HPLC parameters of the present study are in good correlation with the research conducted by Tejinder Sing, RiouJ & Alla Joutovsky. Present study showed HPLC comprehensively characterizing HbS, A, A2, F, S, C, D from each other & was also applicable for the quantification of HbA1c for the monitoring of Diabetes Mellitus. CONCLUSION: : The merits of HPLC are small quantity of sample required, economical, less TAT, accurate categorization of HbS, HbA2 & F. But one has to be aware of the limitations and problems associated with this method due to variant hemoglobin within the same retention windows. The present findings show HPLC as an excellent & powerful diagnostic tool for the direct identification of hemoglobin variants with a high degree of precision in the quantification of normal and abnormal hemoglobin fractions.

Study of red blood cell alloimmunization risk factors in multiply transfused thalassemia patients: role in improving thalassemia transfusion practice in Fayoum, Egypt.

BACKGROUND: ß-Thalassemia is considered the most common chronic hemolytic anemia in Egypt. Alloimmunization can lead to serious clinical complications in transfusion-dependent patients. The objective of this study was to determine the frequency and types of alloantibodies, and, in addition, to study the risk factors that might influence alloimmunization in multiply transfused thalassemia patients in Fayoum, Egypt, with the goal that this study could help minimize some of the transfusion-associated risks in those patients. STUDY DESIGN AND METHODS: A total of 188 multiply transfused thalassemia patients attending Fayoum University Hospital were analyzed. Alloantibody identification was performed by DiaMed-ID microtyping system. RESULTS: Alloimmunization prevalence was 7.98%. The most common alloantibody was D-related; anti-D was the most frequent alloantibody found in eight of the 188 patients (4.25 %), followed by anti-C in two patients (1.1%), anti- E in two (1.1 %), anti-c in two (1.1 %), anti-Fya in two (1.1%), anti-K in one (0.53 %), and an unknown antibody in one patient (0.53%). Higher rates of alloimmunization were found in female patients, in patients with ß-thalassemia intermedia, in splenectomized patients, in D- patients, and in patients who started blood transfusion after 3 years of age. CONCLUSION: The study reemphasizes the need for cost-effective strategy for thalassemia transfusion practice in developing countries. Red blood cell antigen typing before transfusion and issue of antigen-matched or antigen-negative blood can be made available to alloimmunized multiply transfused patients. Early institution of transfusion therapy after diagnosis is another means of decreasing alloimmunization.

Iron overload in thalassemia and related conditions: therapeutic goals and assessment of response to chelation therapies.

Transfusional iron loading inevitably results in hepatic iron accumulation, with variable extrahepatic distribution that is typically less pronounced in sickle cell disease than in thalassemia disorders. Iron chelation therapy has the goal of preventing iron-mediated tissue damage through controlling tissue iron levels, without incurring chelator-mediated toxicity. Historically, target levels for tissue iron control have been limited by the increased frequency of deferoxamine-mediated toxicity and low levels of iron loading. With newer chelation regimes, these limitations are less evident. The reporting of responses to chelation therapies has typically focused on average changes in serum ferritin in patient populations. This approach has three limitations. First, changes in serum ferritin may not reflect trends in iron balance equally in all patients or for all chelation regimens. Second, this provides no information about the proportion of patients likely respond. Third, this gives insufficient information about iron trends in tissues such as the heart. Monitoring of iron overload has advanced with the increasing use of MRI techniques to estimate iron balance (changes in liver iron concentration) and extrahepatic iron distribution (myocardial T2*). The term nonresponder has been increasingly used to describe individuals who fail to show a downward trend in one or more of these variables. Lack of a response of an individual may result from inadequate dosing, high transfusion requirement, poor treatment adherence, or unfavorable pharmacology of the chelation regime. This article scrutinizes evidence for response rates to deferoxamine, deferiprone (and combinations), and deferasirox.

International reproducibility of single breathhold T2* MR for cardiac and liver iron assessment among five thalassemia centers.

PURPOSE: To examine the reproducibility of the single breathhold T2* technique from different scanners, after installation of standard methodology in five international centers. MATERIALS AND METHODS: Up to 10 patients from each center were scanned twice locally for local interstudy reproducibility of heart and liver T2*, and then flown to a central MR facility to be rescanned on a reference scanner for intercenter reproducibility. Interobserver reproducibility for all scans was also assessed. RESULTS: Of the 49 patients scanned, the intercenter reproducibility for T2* was 5.9% for the heart and 5.8% for the liver. Local interstudy reproducibility for T2* was 7.4% for the heart and 4.6% for the liver. Interobserver reproducibility for T2* was 5.4% for the heart and 4.4% for the liver. CONCLUSION: These data indicate that T2* MR may be developed into a widespread test for tissue siderosis providing that well-defined and approved imaging and analysis techniques are used.

Outcome and management of pregnancy in women with thalassaemia in Cyprus.

We describe the management and clinical outcome of pregnancies among 100 Greek Cypriot women with thalassaemia: 88 with thalassaemia major and 12 with thalassaemia intermedia. A total of 152 successful pregnancies and 161 deliveries were included. All patients had endocrine assessment and frequent ferritin measurements. Multiple successful pregnancies included 7 twins and 1 triple pregnancy. Pregnant thalassaemics required significantly larger amount of total blood transfusion during pregnancy. There was a statistically significant increase in the ferritin levels during pregnancy, and levels remained significantly higher after pregnancy. Most pregnancies resulted in delivery of full-term healthy babies, and obstetric complications were rare, although some problems were encountered.

Ultrastructural assessment of Plasmodium falciparum in age-fractionated thalassaemic erythrocytes.

Culture of Plasmodium falciparum in age-fractionated thalassaemic red blood cells (RBC) has shown evidence of parasite damage on light microscopy in older cells during the third culture cycle (96-144 h). In this report, parasites growing in thalassaemic trait and normal RBC were examined ultrastructurally from 96 to 144 h. All parasite stages in old thalassaemic RBC showed evidence of damage worsening with culture duration. There were cytoplasmic alterations with ribosomal damage, and parasite cytoplasm became increasingly loose and grainy, with multiple fissures. Discontinuity of the nuclear membrane with an abnormal nucleolus was seen at l20 h. Cytosomes remained normal, but damage to the food vacuole and shrunken disintegrating parasites were observed at 144 h. These changes are compatible with cellular degeneration and developmental retardation and would account for the schizont maturation arrest and reduced reinvasion rates previously reported. Increased free radicals associated with thalassaemic erythrocytes would explain these changes, further supporting the role for oxidant stress in the protective mechanism.

Cost-effectiveness of prenatal screening for thalassaemia in Hong Kong.

OBJECTIVES: To determine the cost effectiveness of a universal prenatal screening program for alpha- and beta-thalassaemia. METHODS: We retrospectively reviewed our program from 1998 to 2002, and calculated the direct and indirect costs of various components. RESULTS: 18,936 women were screened at our prenatal clinic and 153 couples were subsequently referred to our Prenatal Diagnostic Centre for counselling and further investigations. In addition, there were 238 tertiary referrals and 157 self-referrals. After investigations, 84 fetuses were at risk of beta-thalassaemia major/beta-E thalassaemia, 19 of them were affected and 18 were aborted. The total expenditure on our program (HK 10.0 million dollars) would be less than the postnatal service costs (HK 40.4 million dollars) for 18beta-thalassaemia major fetuses if they were born. Of 361 women at risk of carrying a homozygous alpha0-thalassaemia fetus, 311 (86.2%) opted for the indirect approach (using serial ultrasound examinations to exclude Hb Bart's disease), and 76 (24.5%) subsequently underwent an invasive test for a definitive diagnosis. The sensitivity and false positive rate of this indirect approach was 100.0% and 2.9% respectively. CONCLUSION: It is cost effective to run a universal prenatal screening program in an area where both beta-thalassaemia and alpha-thalassaemia are prevalent. The indirect approach can effectively avoid an invasive test in unaffected pregnancies.

Comparative study on serum iron determination by different methods.

Iron is an important trace element in humans. Depletion or overload iron status can result in a number of aberrant physiological functions. Determination of serum iron is one of the valuable test panels for iron status assessment. A comparative study was performed on 3 different methods of serum iron determination, nonprecipitating ferrozine colorimetric method, precipitating ferrozine colorimetric method, and iron liquicolor cleaning factor colorimetric agar-based (CAB) method (Human, Taunusstein, Germany). The correlation coefficients between nonprecipitating ferrozine colorimetric method and precipitating ferrozine colorimetric method, between nonprecipitating ferrozine colorimetric method and iron liquicolor cleaning factor CAB method, and between precipitating ferrozine colorimetric method and iron liquicolor cleaning factor CAB method were found to be 0.998 (P = .067), 0.999 (P = .067), and 0.951 (P = .934), respectively. For a setting with limited resources such as Thailand, use of the first analytical technique, nonprecipitating ferrozine colorimetric method, is recommended because of its low cost and ease of performance.

HPLC--how necessary is it for haemoglobinopathy diagnosis in India?

Cation exchange high performance liquid chromatography (HPLC) is emerging as the method of choice for the initial screening of thalassemias and haemoglobinopathies and quantification of Haemoglobins (Hbs) like HbA, HbA2 and HbF. Since it is expensive, the present study was conducted to evaluate the need for HPLC in Indian laboratories and identify situations where it would be imperative. Eighty three patients suspected to have thalassemia and haemoglobinopathies were analysed. Both HPLC and alkaline gel electrophoresis detected 14 cases of HbE syndrome and 14 cases of HbS syndrome. However of the 14 cases diagnosed as HbD syndrome by alkaline electrophoresis, eight cases were diagnosed as Hb Q India, 1 case as HbD Iran and 5 cases of HbD Punjab on HPLC. Thirty-one cases were detected to have beta heterozygous thalassemia based on the high HbA2 levels (>3.9%) and eight cases were diagnosed as beta homozygous thalassemia by both HPLC and gel electrophoresis. One of them had an unknown Hb migrating in F-A region. Her mother also had same unknown Hb variant. In view of electrophoretic migration and retention time (RT) on HPLC, possibility of HbG-San Jose was considered. HPLC being an automated instrument is highly sensitive and specific, has high resolution and helps in quantification of various haemoglobins. However in a developing country like India where economical factors play a major role in planning for management of patients, the role of HPLC is limited.

Assessment of erythrocyte deformability with the laser-assisted optical rotational cell analyzer (LORCA).

The erythrocyte deformability of 28 patients with anemia was evaluated with the laser-assisted optical rotational cell analyzer (LORCA), an image analyzer that converts into numerical form the degree of refraction of a laser beam induced by red cells subjected to a range of torsional stresses. The patients were 10 thalassemics, including three with intermediate forms (1 HbC/beta degree, 1 homozygote beta for Orkin's haplotype VI, 1 beta degree/beta delta Sicilian type) and seven heteroygotes for beta Th; six with hereditary spherocytosis (including 2 with structural alteration of the spectrin beta chain); three with type II congenital dyserythropoietic anemia (HEMPAS), two hemizygotes and one heterozygote for G-6PD deficiency, and six with severe hypochromic hyposideremic anemia. Red cell deformability was reduced in intermediate thalassemia, hereditary spherocytosis and HEMPAS, normal in heterozygous beta thalassemia and G-6PD deficiency, and increased in hypochromic hyposideremic anemia. These results show that erythrocyte deformability can be impaired by an Hb chain imbalance, membrane and cyto skeleton structure anomalies and changes in the red cell area/volume ratio.

Serological profile assessment of the infection with hepatitis C virus (HCV) in haemophiliacs and thalassemic patients.

Polytransfused patients represent a major risk group for hepatitis C (HCV) acquirement. Haemophiliacs and thalassemic patients treated with virus contaminated blood or blood derivatives frequently exhibit anti-HCV antibodies and signs of chronic hepatitis. The serological profile for the HCV infection was investigated in 13 haemophiliacs, 18 cases of thalassemia and in 14 polytransfused patients affected by other diseases. The presence of anti-HCV antibodies was detected by means of the ORTHO HCV 3.0 ELISA kit and confirmed by Western-blot Murex. The serotyping used synthetic peptides mimicking the immunodominant epitopes in the NS4 region, characteristic of each of the six HCV genotypes in an ELISA blocking reaction (Murex). Serotype 1 was prevalent (51.1%), while serotype 2 was detected in 13.3% of patients, with a higher frequency in thalassemia cases. The remaining samples were multireactive, and serotype 3 alone was not detected. The profile of Western-blot bands was distinct for the monoreactive samples belonging to serotype 1 or 2. The analysis of the multireactive samples in young (thalassemic, age mean 15.17 +/- 6.5) and aged patients (haemophiliacs, age mean 32.64 +/- 13.5) allows us to suggest a different succession of reinfection acquirements. The infection with one of the subtypes does not confer protection against the reinfection with others. However, a certain attenuation of the symptomatology is obvious in the case of reinfections, indicating the existence of crossantigenic reactivities which contribute to protection. This protection is more evident in the case of primary infection with type 2 and is partially due to antigens coded by the NS4 genomic segment.

Utilization of red-cell FAD by methaemoglobin reductases at the expense of glutathione reductase in heterozygous beta-thalassaemia.

FAD-dependent methaemoglobin reductases (MHR) were studied in red cells in heterozygous beta-thalassaemia to investigate how they related to low FAD-dependent glutathione reductase (GR). In contrast to GR, MHR activities were usually normal or increased. In particular, whether expressed in relation to haemoglobin or number of red cells, NADPH-MHR activity was markedly increased in most subjects, probably being a response to increased oxidative stress. Oral riboflavin had no effect on MHR activities, indicating saturation with FAD even though GR was deficient. A strong correlation between percent stimulation of GR by FAD and NADPH-MHR activity indicates that FAD is utilized by MHR at the expense of GR. This could be an important influence on GR in heterozygous beta-thalassaemia. Thus, the low activity resulting from an inherited deficiency of FAD is decreased further.

[Evaluation of erythrocyte deformability in carriers of thalassemic trait with the Hanss' hemorheometer]. / Valoración de la deformabilidad eritrocitaria en portadores del rasgo talasémico con el hemorreómetro de Hanss.

Red-cell deformability was assessed with the Hanss' haemorheometer in 63 carriers of the thalassaemic trait (20 beta and 43 delta beta). Impaired deformability (rigidity index 10.5 +/- 1.4) was present in 80% of the carriers of both beta- and delta beta-traits, as compared with a control group (rigidity index 8.7 +/- 0.6). No correlation was found between such indices and several parameters capable of influencing upon red-cell deformability, namely, MCV, MCH, RDW and MDA. The possibility of any impairment of lipid compounds in red cell membrane is suggested as a cause of decreased deformability in thalassaemia carriers.

Hemoglobin distribution width: a rapid assessment of dense red cells in the steady state and during painful crisis in sickle cell anemia.

Hemoglobin distribution width (HDW) is a measurement of the heterogeneity of the red cell hemoglobin concentration as determined by the Technicon H1 automatic counter. The utility of this measurement for patients with sickle cell disease was assessed in 275 samples from 61 patients. We found that patients with sickle cell disease in steady state had an elevated HDW (3.89 +/- 0.82 gm/dl) compared with the normal range of 2.2 to 3.2 gm/dl, and that this value correlated strongly with the number of dense red cells (r = 0.72, p less than 1 x 10(-10]. When HDW was determined daily for patients with SS in painful crisis, the decline in dense cells observed during this event was paralleled by a decrease in HDW. The percent of dense cells fell from steady state levels of 10.0% +/- 9.5% to 5.7% +/- 4.9% at the beginning (days 1 through 3) to 3.1% +/- 2.0% at the end of painful crisis (days 6 through 9). HDW decreased from the steady state value of 3.89 +/- 0.82 gm/dl to 3.44 +/- 0.73 gm/dl at the beginning of crisis and fell further to 3.14 +/- 0.40 gm/dl at the end. We conclude that HDW is a rapid and reliable assessment of the percent of dense cells in sickle cell disease and is a useful objective parameter to follow in sickle cell crisis.

Assessment of splenic and RES function of patients with thalassemia major long after partial splenic embolization: in vivo clearance study.

The activity of the remaining reticuloendothelial system (RES) and the function of the splenic remnants was estimated in 5 thalassemic patients who had undergone successful partial splenic embolization (PSE) 6 yr previously. The kinetics of 125I-heat-denatured human albumin as well as that of 51Cr-heat-damaged homologous red blood cells were applied for this purpose and the parameters derived were compared to those of nonsplenectomized as well as splenectomized thalassemics with the following results: (a) The parameters of splenic function in embolized thalassemics were found to be within the limits observed in nonsplenectomized patients. (b) Their maximum phagocytic capacity was significantly lower, not only than that found in nonsplenectomized, but also than in thalassemic patients splenectomized at about the same time. It is concluded that, 6 yr after PSE has been performed, a reorientation of the altered circulatory dynamics has taken place in the splenic remnants allowing previously blockaded areas to gain normal function. It therefore seems that, despite the continuing hemolytic stimulus, RES hyperplasia is prevented, resulting in the stable, low-level transfusion requirements that have been observed in embolized thalassemics.

Haemoglobinopathies, malaria, and other interferences with HBA1 assessment.

Total glycosylated haemoglobin (HbA1) was determined by a rapid minicolumn chromatography technique in 438 diabetic patients and correlated with the mean of fasting and post-prandial blood glucose values for the preceding six weeks. In 360 of them, free of congenital haemoglobinopathies and other detected causes of HbA1 mis-interpretation (reference group), a significant correlation was established between the HbA1 and glucose values: y = 0.54 X + 4.91; r = 791; (p less than 0.01). In 28 of the 29 patients with heterozygous haemoglobinopathies (Hb S = 17; Hb C = 8; Hb D Pundjab = 1; Hb E = 2) the apparent HbA1 values were inappropriately low. The apparent HbA1 value was above the 95% confidence limits in the 29th patient, with beta thalassaemia. In 10 out of 14 diabetics with recurrent hypoglycemic attacks, the HbA1 value was lower than the 95% confidence limits of expected values. Out of 21 diabetics with a shortened red cell lifespan (occult blood losses: 10; haemolysis: 11) 15 displayed a lower than expected HbA1 value. Among these was a diabetic patient with malaria and severe anaemia. Out of 14 diabetics with severe chronic renal failure only 3 presented with apparent HbA1 values above the 95% confidence limits.

[Heterozygote thalassemias screening. Contribution of data analysis methods (author's transl)]. / Le dépistage des thalassémies hétérozygotes. Apport de méthodes d'analyse de données.

In order to reduce the cost of a systematic heterozygote thalassemias screening, and using a discriminant analysis, the authors propose a pre-selection performed on the erythrocytometric data. In the present conditions of applicability, the methodology we used appears to be very efficient in the pre-screening process. An electrophoresis with the determination of hemoglobin A2 is then carried out on the pre-selected individuals as a confirmation of the diagnosis.

Assessment of iron stores in subjects heterozygous for beta-thalassaemia based on serum ferritin levels.

Serum ferritin concentration was assessed in male and female pregnant and non-pregnant thalassaemia carriers and in normal subjects of both sexes. Low ferritin levels were found in 61% of non-pregnant and in 32% of pregnant female beta-thalassaemia heterozygotes whereas male thalassaemia carriers had normal iron stores. Increased ferritin levels were not observed in any of the subjects examined. These findings show that iron deficiency is a common finding in female thalassaemia carriers of reproductive age who are not receiving iron supplementation.