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Separation of hemoglobins and hemoglobin chains by high-performance liquid chromatography.

This paper evaluates various high-performance liquid chromatographic (HPLC) procedures useful for the detection and evaluation of numerous hemoglobin abnormalities in adults and in newborn infants. Two major approaches are discussed. The first concerns the analyses of hemoglobins in red cell lysates by anion- and cation-exchange chromatography; this type of HPLC has great potential in a routine laboratory, as quantitative data for numerous abnormalities can readily be obtained. The second concerns the separation of globin chains by reversed-phase HPLC that is most useful for the identification of hemoglobinopathies in adults, newborns, and fetuses.

Chromatography, High Pressure Liquid↗

Clinical characteristics of Japanese patients with primary myelodysplastic syndromes: a co-operative study based on 838 cases. Anemia Study Group of the Ministry of Health and Welfare.

The characteristics of Japanese (JPN) patients with myelodysplastic syndromes (MDS) were investigated in 838 retrospectively collected cases. The median age of the JPN patients was 60 years, about 10 years younger than that in most of the reports based on Western patients. Median survivals were 65 months for refractory anemia (RA), 58 months for RA with ring sideroblasts (RARS), 16 months for RA with an excess of blasts (RAEB), 10 months for RAEB in transformation (RAEBT), and 20 months for chronic myelomonocytic leukemia (CMML). Cumulative leukemia-free rates at final observation were 73% for RA, 79% for RARS, 24% for RAEB, 20% for RAEBT, and 53% for CMML. When low-risk (RA and RARS) patients were divided into two groups, those 40 years of age and older, and those under 40, the cumulative leukemia-free rate was 94% for the younger patients (n = 101), compared with 66% for the older patients (n = 318). The prognostic factors for survival were different from those in Western reports, i.e., variables representing quantitative abnormalities (hemoglobin levels, granulocyte, and platelet counts) were not major prognostic factors, while variables representing qualitative abnormalities (morphological abnormalities in granulocytic and megakaryocytic series cells) were highly significant. Two scoring systems for overall survival and for leukemic transformation were developed, based on multivariate prognostic factor analysis. Neither system included variables representing the degree of cytopenia. Whatever the reason for the different prognostic factors in JPN and Western MDS patients, the use of a scoring system based on Western patients for clinical decision-making in a JPN patient could be misleading, and vice versa.

Adolescent↗

Hemoglobin oxygen affinity is increased in erythropoietic protoporphyria.

Whole blood and hemolysates from seven normal and three erythropoietic protoporphyria patients were compared in terms of their hemoglobin function. The oxygen affinity (P50) of the erythropoietic protoporphyria hemolysates compared to normals (13.1 +/- 0.2 vs 17.5 +/- 0.3 mmHg; P < 0.001) and erythropoietic protoporphyria erythrocytes compared to normals (23.4 +/- 0.6 vs 27.1 +/- 0.5 mmHg; P < 0.001) were increased while oxygen-binding cooperativity (n-value of the Hill equation) were similar. We conclude that hemoglobin function in erythropoietic protoporphyria patients is altered, but without pathophysiologic consequences. Because hemoglobin in which protoporphyrin IX substitutes for heme has a low oxygen affinity, our findings of a higher than normal affinity in erythropoietic protoporphyria red cells and hemolysates may indirectly support the findings by others that protoporphyrin IX binds to hemoglobin at non-heme-binding sites. In addition, based on the effect of other abnormal hemoglobins, this shift in P50 will decrease any tendency for anemia in erythropoietic protoporphyria patients.

Erythrocytes↗

[Detection of combined forms of hemoglobino- and enzymopathies in newborns].

In order to develop a rational and early detection of combined forms of hemoglobin and enzymopathies, 1500 samples of neonatal cord blood were tested for alpha- and beta-thalassemia, of abnormal hemoglobins S and C, of methemoglobinemia and for hereditary persistence of fetal hemoglobin as well as 428 samples were examined for glucose-6-phosphate dehydrogenase (EC 1.1.1.49) and glutathione reductase (EC 1.6.4.2) deficiencies. For this purpose, isoelectrofocusing in Multiphor-2117 polyacrylamide-ampholine plates (LKB, Sweden) at pN 3.5-9.5 and pH 5.5-8.5 was carried out with subsequent laser densitometry of gels (Densitometer 2202, LKB). The data obtained were analyzed simultaneously in biochemical, hematological and genealogical studies. Hereditary impairments detected were evidenced by genealogical analysis.

Female↗

[Neonatal screening for hemoglobinopathies: a pilot study in Porto Alegre, Rio Grande do Sul, Brazil].

This study was conducted to establish the frequency of hemoglobinopathies among newborns undergoing screening tests for metabolic diseases at the University Hospital (Hospital de Clínicas) in Porto Alegre, Rio Grande do Sul, Brazil. Testing for abnormal hemoglobins was performed by isoelectric focusing electrophoresis on agarose gel with blood obtained by heel stick and applied to filter paper. For confirmatory testing of abnormal neonatal screening, a venopuncture blood sample was obtained from the infant and parents and then submitted to hemoglobin electrophoresis on cellulose acetate at pH 8.6 and citrate agar at pH 6.2. A total of 1,615 subjects were studied: 20 samples showed the Hb S pattern and six samples showed Hb C. Thus, frequency of the sickle cell gene was 1.2% and that of the Hb C gene was 0.4%, regardless of race or origin. These data suggest that the inclusion of universal neonatal screening for hemoglobinopathies in the ongoing projects for the detection of phenylketonuria and congenital hypothyroidism has many advantages and should be considered in health programs.

Anemia, Sickle Cell↗

Assessment of prognostic factors in alcoholic liver disease: toward a global quantitative expression of severity.

The prognostic significance of a battery of clinical, laboratory, and histological indicators was assessed in relation to mortality risk in a 1-year study of 253 patients with alcoholic liver disease, of whom 51 died within such time. The relative risk associated with each abnormality was calculated. A number of abnormalities was found to be statistically associated with a higher risk of death. Among the clinical abnormalities, these were: collateral circulation, edema, ascites, encephalopathy, spider nevi, anorexia, and weakness. Among the laboratory tests, these were: albumin, bilirubin, hemoglobin, abnormal prothrombin time, and alkaline phosphatase. Two hundred and sixteen of these patients had liver biopsies in which the quantifiable abnormalities were scored. Among the histological findings, the alterations significantly related to mortality were necrosis, Mallory, and inflammation, while the presence of cirrhosis per se did not influence the mortality risk. The relative risk factors for mortality associated with the histological alterations were lower than those derived from clinical or laboratory measurements. The advantage of using only clinical and laboratory items to derive a global, quantitative expression of severity is discussed. The relative mortality risks provided a means of calculating a "unit of severity" for each clinical and laboratory abnormality. A combined clinical and laboratory index (CCLI) results when these mortality-risk units are added. Such a combined index had a quasi-linear relationship with the risk of mortality for the complete population. This method compared well with severity scores derived from computerized, linear step-wise discriminant function (SDF) analysis and from a logistic regression (LR) analysis. The factors chosen to have independent prognostic significance by the SDF analysis were: encephalopathy, albumin, prothrombin time, and hemoglobin, while only encephalopathy, albumin, and hemoglobin were chosen by the LR analysis. Within a range of values, LR can provide a good discrimination in relation to mortality, similar to that observed for the CCLI in its complete range. However, there are some advantages to the CCLI method vs. the LR or SDF analyses. The CCLI is less susceptible to being unduly influenced by a nonspecific effect of treatment on the items chosen than the SDF and LR analyses, as the CCLI contains a large number of factors. Obtaining a single-severity score such as the CCLI is of value in: (a) assessing the effectiveness of treatment modalities; (b) analyzing the success of randomization; (c) separating cohorts of different severity, and (d) comparing new liver tests, histological abnormalities, or specific biological events with the severity of alcoholic liver disease.

Age Factors↗

Thalassemic erythrocytes inhibit in vitro growth of Plasmodium falciparum.

Blood specimens from 100 thalassemic patients were screened in vitro for inhibitory effects on growth and multiplication of Plasmodium falciparum. The culture medium mixture designated REM consisted of 9 volumes of minimum essential medium (GIBCO Laboratories, Grand Island, N.Y.) and 1 volume of RPMI 1640 (GIBCO) supplemented with 10% heat-inactivated human serum. Parasite multiplication in erythrocytes containing normal hemoglobin cultured in RPMI or REM was similar. Significant reduction in parasite multiplication rates was observed in erythrocytes containing abnormal hemoglobin when these were cultured in REM. The degree of reduction in five types of thalassemic erythrocytes was in the following descending order: hemoglobin H disease with Hb Constant Spring, classical hemoglobin H disease, beta(0)-thalassemia-hemoglobin E in which blood harbored a high percentage of hemoglobin F-containing cells, beta (0)-thalassemia-hemoglobin E in which blood harbored few hemoglobin F-containing cells, and beta-thalassemia heterozygous variant.

Animals↗

[Application of Helena SAS-3 full automatic hemoglobin agarose electrophoresis].

OBJECTIVE: To assess the value of Halena SAS-3 full automatic hemoglobin agarose electrophoresis in the diagnosis of thalassemia. METHODS: Hemoglobin A and A2 was detected by agarose electrophoresis in 200 blood samples collected from 170 healthy subjects, 2 patients with beta-thalassemia, 12 with acute lymphoblastic leukemia, 12 acute myelocytic leukemia and with 4 non-Hodgkin lymphoma. RESULTS: The Mean+/-SD of hemoglobin A in the healthy subjects and patients with beta-thalassemia, acute lymphoblastic leukemia, acute myelocytic leukemia and non-Hodgkin lymphoma was 97.55+/-0.51, 97.01+/-0.329, 97.42+/-0.57, 97.44+/-0.55, and that of A2 was 2.44+/-0.51, 2.99+/-0.32, 2.84+/-0.28, 2.55+/-0.55, respectively. The values of electrophoresed hemoglobin F of the 2 patients with beta-thalassemia were higher than those of the normal adults. The within-run coefficient of variation (CV) and between-run CV was 0.17 and 7.76 for hemoglobin A, and 0.16 and 7.14 for hemoglobin A2, respectively. CONCLUSIONS: Halena SAS-3 full automatic agarose electrophoresis system possesses strong resolving power in detecting trace abnormal hemoglobin and accurately identifies unusual evaluation of hemoglobin A2 and F to facilitate the diagnosis of thalassemia.

Child, Preschool↗

Clinical expression of sickle cell anemia in the newborn.

The clinical records of 14 neonates with hemoglobin S homozygosity were reviewed. The infants were identified through a statewide cord blood testing program in Georgia for abnormal hemoglobins. Nine of the 14 patients had neonatal jaundice, most likely due to hemolysis. In one patient, significant anemia appeared as early as six weeks postnatally. It is suggested that neonatal jaundice may be an early sign of sickle cell anemia. A significant anemia can also develop before 3 months of age even when Hb F levels are usually still rather high.

Anemia, Sickle Cell↗

Hb A2-Canada or alpha 2 delta 2 99(G1) Asp replaced by Asn, a newly discovered delta chain variant with increased oxygen affinity occurring in cis to beta-thalassemia.

An Indian family is described in which the father has a delta chain abnormal hemoglobin which is the result of a mutation of the delta gene in cis to a beta-thalassemia heterozygosity. The abnormality concerns a substitution of the Asp residue in position 99 (G1) by an Asn residue. A similar substitution has been found in the beta chain of Hb Kempsey (alpha 2 beta 2 99 Asp replaced by Asn). The observed abnormality results in a greatly increased oxygen affinity of this newly discovered Hb A2 variant.

Adolescent↗

Hemoglobin Cowtown (beta 146 HC3 His-Leu): a mutant with high oxygen affinity and erythrocytosis.

A new mutant, hemoglobin Cowtown, has been found in a white man and his father, both of whom have erythrocytosis. The father had previously been treated with 32P for polycythemia vera. The abnormal hemoglobin is not detectable on electrophoresis in alkaline buffers, but it resolves distinctively on electrophoresis in citrate agar, pH 6.0; similarly, the abnormal beta-globin chain does not separate from beta-A in urea 2-mercaptoethanol buffers of pH 8.9, but it moves anodically to beta-A at pH 6.0. Peptide chromatography and amino acid analysis of the beta chain reveal that the C-terminal histidine residue (beta 146) has been replaced by leucine. Like several other hemoglobins substituted at this residue, Hb Cowtown has a high oxygen affinity and a diminished Bohr effect.

Adult↗

Measurement of glycosylated hemoglobins in black diabetic patients: a note of caution.

Inspection of chromatography columns used for measurement of glycosylated hemoglobins revealed that blood samples from certain black diabetic patients produced two residual hemoglobin bands after chromatography rather than one. The levels of glycosylated hemoglobin were significantly lower in these subjects than in other diabetics. Further investigation revealed that each of these subjects had the hemoglobin AS or AC phenotype. The presence of hemoglobin S or C appears to cause spuriously low levels of glycosylated hemoglobin as determined by ion exchange chromatography. Other means to assess diabetic control should be used for patients with these abnormal hemoglobins.

Black People↗

Investigation of microcytosis: a comprehensive approach.

Microcytosis is a highly prevalent finding during blood examination. This study investigates the causes of microcytosis (defined as mean corpuscular volume (MCV) < 82 fl) in 466 patients referred to our laboratory for suspected hemoglobinopathy. The following data were obtained: Hb, MCV, serum iron, transferrin, ferritin, HbA2, HbF, isoelectric focusing of the Hb, gene mapping of chromosome 16 with Xba I and Bgl II and hybridization with an alpha- and a zeta-probe, inflammatory status. Results show that iron deficiency remains the first cause of microcytosis (35.2% of our patients), even in a selected population such as ours. Deletional alpha-thalassemia, probably the most frequent hemoglobinopathy throughout the world, represents the second most frequent cause of microcytosis (31.1%), followed by beta-thalassemia heterozygous state (18.9%). Of our patients, 1.3% had microcytosis due to the presence of an abnormal hemoglobin (HbC, Hb S/C, HbE). Three cases (0.6%) had other possible causes of microcytosis. Of the remaining 60 cases, 28 had an inflammatory state. Finally, 32 cases (6.9%) remain unexplained; taking into consideration the origin of these cases, their hematological parameters and their family history, we postulate that these cases are at high risk for non-deletional alpha-thalassemia.

Chromosome Mapping↗

Anemia in patients with neoplastic diseases.

A specific and characteristic type of anemia is not a feature of all malignant disease. To the contrary, the nature of the anemia will depend upon the causative mechanism, of which blood loss and accelerated erythrocyte removal appear to be the most frequently seen and the most clearly defined. Recognition of anemia due to loss of blood is relatively simple if the subtlety of blood loss in the stool is borne in mind and persistent testing to demonstrate it is carried out. Indeed, anemia characterized by chronic loss of blood in men can only mean chronic gastrointestinal bleeding if certain rare hemoglobin abnormalities can be ruled out. Anemia due to accelerated erythrocyte removal may also be recognized by simple measures. After transfusions raise hemoglobin values to near normal levels, the disappearance of the transfused blood and the rapid return of the pretransfusion severity of anemia are good evidence of the presence of such a mechanism, if blood loss can be ruled out.Adequate management of the anemia of malignant disease depends upon a clear understanding of the various mechanisms involved. It is highly probable that attention to this feature will, in many instances, significantly prolong the productive life of persons with malignant disease.

Anemia↗

[Hemoglobinopathies in West-African immigrant workers in France (author's transl)].

In this paper are brought the results obtained in two Parisian hospitals during a survey of abnormal hemoglobins in 540 immigrant workers coming from Africa, mostly from Mali, Mauritania and Senegal. All the subjects investigated were male and between 20 and 40 years old. The studies were performed following internationally standardized technics. The most frequent abnormalities were: Hb S found in 16.3%, Hb C (6.6%), alpha-thalassemia trait (3.1%) and beta-thalassemia trait (3.1%). Some rare abnormalities were also found: delta-chain variants, hereditary persistance of foetal hemoglobin, Hb Hope and Hb Grady. This work emphasizes the high frequency of the different hemoglobin disorders in this population. The easy diagnostic of electrophoretically detectable variants is compared to the more complex situation of thalassemia leading probably to an under estimation of the percentage.

Adult↗

[School screening of hemoglobinopathies in the Marseilles area. An analytic study of 35,289 tests].

A systematic screening for thalassemias and hemoglobinopathies was performed in 35,289 children with a double test associating blood cell counts and hemoglobin electrophoresis. Four hundred thirty-seven (1.23%) hemoglobin abnormalities were found, consisting of 274 cases with thalassemia (0.77%) and 159 hemoglobinopathies (0.45%) among which there were 80 cases with sickle-cell trait (0.22%), 30 with hemoglobin C (Hb C) (0.08%), 19 with Hb D (0.05%), 12 with HbJ (0.03%), 12 with Hb E (0.03%). There was an obvious ethnic influence as, among 17,180 autochtonal French children, only 15 abnormalities were found (0.08%) while 392 were found in 16,850 non autochtonal children (2.32%) and 30 in 1,259 French children from Corsica (2.38%). The highest incidence was found in Italians (173 of 6,455-2.68%) and Maugrabins (105 of 3,014-3.48%).

Adolescent↗

Post-translational modification of hemoglobin in alcoholism.

Hemolysates from a majority of alcoholic individuals contain a hemoglobin fraction having chromatographic properties resembling those of hemoglobin AIc, a normally occurring glycosylated hemoglobin, but differing from the latter in not being bound by boronated agarose. A hemoglobin fraction having similar properties was also isolated from human erythrocytes exposed to acetaldehyde. These observations and other evidence suggest that the abnormal hemoglobin fraction present in men and women addicted to alcohol is produced by addition to hemoglobin Ao of an aldehyde or ketone formed metabolically from ethanol and thus may serve as a diagnostic indicator of alcoholism.

Alcoholism↗