Biomedical subjects
Publications and source records attributed to P F Milner.
Hematuria is a rather frequent complication in patients with sickling disorders. However, the occurrence, incidence and management of this complication during pregnancy have not been discussed in the medical literature. We treated three patients with sickle-cell-induced hematuria in pregnancy and developed an approach we find useful for such patients.
Between July 1977 and October 1981, nine patients with sickle cell anemia underwent tonsillectomy or tonsillectomy with adenoidectomy without operative or postoperative morbidity or mortality. Appropriate management is necessary to prevent vaso-occlusive events. The current protocol for elective surgery used at the Medical College of Georgia, Augusta, consists of the following: (1) preoperative transfusion and hydration, (2) intraoperative prevention of hypothermia and maintenance of blood volume, and (3) postoperative oxygen therapy and hydration. The critical requirement of this protocol is the preoperative transfusion of these patients with packed cells consisting of hemoglobin A erythrocytes to lower the percentage of Hb S to less than 45% as determined by quantitative hemoglobin electrophoresis.
We have reported the direct analysis of the allele for beta 2-globin by using restriction endonuclease Dde I coupled with blot-hybridization analysis. In that report we predicted that a major use of our analysis could be for the prenatal diagnosis of sickle cell anemia. Here we present such an analysis. In addition, this report also describes the use of a new enzyme Mst II, which also distinguish the beta s allele from the normal beta-globin allele. Blot-hybridization analysis with restriction endonuclease Mst II shows the 5' end of the normal beta-globin gene to reside on a fragment of approximately 1.14 kilobases, whereas the 5' end of the beta s-globin gene resides on a fragment of approximately 1.34 kilobases. Because the fragment sizes generated by Mst II are significantly larger than those generated by Dde I, one can easily perform a prenatal diagnosis for sickle cell by standard blot hybridizations onto nitrocellulose filters.
Bone marrow infarction was investigated by 99mTc-sulfur colloid imaging in 42 patients with sickle cell anemia (SS) over a period of 2 yr. Marrow defects were demonstrated in 28 patients (66.6%), and in 15 (aged 19--52 yr), they were matched by roentgenographic evidence of medullary bone infarction. Repeated images showed no change in the size or site of these defects. Among 13 patients (aged 6--32 yr), all in crisis when initially examined, marrow defects were not associated with roentgenographic changes, and in many cases, repeated images showed resolution or decrease in size of the defects in 3--6 mo, even if the limb had been swollen and the marrow defect large. Among 14 patients (aged 18--36 yr), all asymptomatic at the time of study, no defects were found. Comparison of hematologic variables revealed a higher mean hemoglobin and hematocrit level among those with marrow infarcts (p less than 0.0001). High levels of HbF, or the presence of alpha-thalassemia, did not protect against marrow infarction. Pulmonary fat embolism was not observed. 99mTc-sulfur colloid marrow imaging was considered to provide more useful information in the initial management of bone pain and swelling in sickle cell crisis than either roentgenographs or conventional 99mTc-methyldiphosphate bone images.
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Several reports have been published on the use of polymorphisms found in the human hemoglobin genes as a means for prenatal diagnosis of sickle cell anemia. The disadvantages of this approach reside in its limited application and the need for family analysis. Here we report that, by use of restriction endonuclease Dde I and diazobenzyloxymethyl-paper transfer procedures, a direct analysis can be made. Individuals with normal hemoglobin (AA) show two bands (175 and 201 base pairs) complementary to a 5'-specific beta-globin gene probe. Sickle cell trait individuals (AS) exhibit an additional band (376 base pairs). Individuals with sickle cell anemia (SS) show the band at 376 base pairs with a concomitant loss of the 175-base pair band. We interpret these changes in banding pattern to be the result of the elimination of a restriction site for Dde I in the altered codon associated with the sickle cell allele. Because an analysis can be performed on as little as 20 micrograms of cellular DNA, the application to prenatal diagnosis of sickle cell anemia should be possible.
The in-vitro synthesis of hemoglobin (Hb) chains was studied among 60 Hb S heterozygotes (AS) having different quantities of Hb S, including five with an associated alpha-chain heterozygosity (ASAG). Hematologic values and hemoglobin composition were studied in these cases and in 15 other ASAG heterozygotes. The percentages of Hb S (which fell between 27% and 42%) and the mean corpuscular volume values correlated directly with the alpha/non-alpha values, confirming previous suggestions (Huisman, Hemoglobin 1:349, 1977) that the concomitant occurrence of an alpha-thalassemia-2 heterozygosity (alpha alpha(0)/alpha alpha; beta/beta(S)) or homozygosity (alpha(0) alpha/alpha(0) alpha; beta/beta(S)) resulted in intermediate or lower levels of Hb S compared with Hb S heterozygotes having four active alpha-chain genes (alpha alpha/alpha alpha; beta/beta(S)). Among ASAG heterozygotes, the occurrence of low (about 25%), intermediate (about 33%), or high (about 45%) proportions of an alpha-chain variant resulting from a variability in the number of active alpha-chain genes due to alpha-thal-2 coincided with high (39%), intermediate (34%), or low (28%) levels of Hb S, respectively. However, the overlap of biosynthetic data between Hb S heterozygotes with four, three, or two active alpha-chain genes prevents a reliable diagnosis in individual cases.
The authors report the association of erythroleukemia (FAB M6) and fetal hemoglobin (HbF) level of at least 65% after therapy for ovarian carcinoma. The patient's erythrocytes had many signs of reversion to fetal-like erythropoiesis including: elevated HbF with a fetal G gamma/A gamma (gly/ala) of 3/1, low hemoglobin A2 (HbA2), macrocytosis, and increased i antigen. These data and data from other case reports suggest that elevation of HbF to greater than 25% with reversion to fetal-like erythropoiesis is useful in differentiating erythroleukemia from other preleukemic disorders.
There are now some 80 known genetic abnormalities controlling the production of unstable hemoglobin variants, including one in the gamma chain. The clinical presentation of patients carrying these variants and the circumstances under which the variants have been detected vary considerably so that no one syndrome covers all cases, but perhaps the best general term is Unstable Hemoglobin Disease (UHD). It must be emphasized, however, that many variants which have been discovered by Hb electrophoresis, and subsequently shown to be heat unstable, do not cause hemolysis in vivo. Some examples of UHD are known as Congenital Heinz Body Hemolytic Anemia (CHBHA) because Heinz bodies are present in large numbers after splenectomy. In many clinical cases of UHD, however, Heinz bodies may not be found. This short review attempts to highlight the different clinical pictures associated with unstable hemoglobins and to discuss their diagnosis. The substitutions and deletions that are associated with heat instability of the hemoglobin molecule are listed with references to the literature.
Although there is as yet no definitive treatment for patients with sickle cell disease there has been considerable improvement in the overall management of these patients in recent years. This stems largely from a better understanding of the disease process. The risks from infection in infancy and childhood due to asplenia ae now well known, and children at risk ar being identified and treated. The role of platelets and the activation of clotting factors, while still controversial, are now acknowledged as appropriate areas for research. The judicious use of blood transfusions for cerebral infarction has improved the prognosis for this complication, as it has for pregnancy and the fetus. Blood transfusion, however, has its own hazards in sickle cell disease and the extent to which it can be used requires further investigation. Finally, the broad spectrum of hematological findings and individual clinical variations in sickle cell disease is now better understood.
The outcome of pregnancy has been analyzed in 72 women with sickle cell anemia (SS) and 26 women with sickle cell-hemoglobin C disease (SC), part of an unselected population of 148 women over 18 years of age with these hemoglobinopathies, who have been followed at a sickle cell disease clinic for 8 years. In SS women, 22% of first pregnancies were aborted spontaneously, and the overall early fetal loss was 19.2%. A similar figure was calculated from the literature since 1956. In SC women, 12% of first pregnancies were lost, but the overall early fetal loss was only 8.9%. The perinatal mortality, under quite variable conditions of prenatal care and delivery, was 10.2% in SS women and 2% in SC women. There were no stillbirths or midterm deaths in utero among SC women, but these accounted for most of the perinatal mortality in SS women, particularly in first and second pregnancies. There was one neonatal death in each group, but eight third pregnancies in SS women were completed without perinatal mortality. A remarkable finding among the SC women was the number of successful pregnancies, ten in one woman, and many of the pregnancies were completed without the supervision of a physician. Our findings were compared with those in the literature, and the conclusion that we drew is that termination of pregnancy and sterilization of young women are not generally indicated solely on the basis of these hemoglobinopathies.
A microchromatographic procedure (Isolab Fast Hb Test System) which was developed for the quantitation of Hb AI (10) has been found useful for the quantitation of Hb F in samples that contain Hb S and/or Hb C but no Hb A (% Fmicro). This method has been evaluated through analyses of known mixtures of Hb F and Hb S. The Hb Fmicro levels in patients with sickle cell anemia and related conditions were compared with results obtained by alkali denaturation (% FAD) and conventional DEAE-cellulose chromatography (% FDE). This microchromatographic technique is a fast, simple, and sensitive method for Hb F quantitation in patients with Sickle cell disease.
The thalassemias are a heterogeneous group of genetically determined disorders of hemoglobin synthesis and can be divided into alpha-thalassemias and beta-thalassemias. The genes for these disorders are carried as relatively harmless traits which can be detected in the laboratory by a series of tests. As there are several variant genes in each group, heterozygotes for two slightly different genes occur, and interaction of these thalassemia genes with the hemoglobinopathies is quite common. Severe clinical disease usually only occurs in homozygotes, as in Cooley's anemia. The problem for the laboratory is to distinguish thalassemia trait from other causes of microcytosis and hypochromia in an economical and efficient way. The various proposed schemes are discussed, and it is suggested that detection of these traits should be part of a comprehensive screening program for hemoglobinopathies and thalassemias.
We have examined the effect of 2, 3-diphosphoglycerate (DPG) on the solubility of deoxy-sickle hemoglobin (deoxy-Hb S) under conditions such that concentration, pH, and osmolarity of deoxy-Hb S solutions approached physiological. The range of DPG/Hb molar ratios encompassed the extremes found for this ratio in erythrocytes from individuals with sickle cell anemia. After monomer-polyer equilibrium had been established, the phases were separated by centrifugation and assayed for concentrations of Hb and DPG. DPG had no effect on the solubility of deoxy-Hb S. Furthermore, at DPG/Hb molar ratios less than one, there was no preferential incorporation of deoxy-Hb S containing bound DPG into polymers. At DPG/Hb molar ratios greater than one, concentrations of free DPG in monomer and polymer phases were virtually identical. Thus, under the specified equilibrium conditions, DPG is not a determining factor in the polymerization of deoxy-Hb S.
The syndrome thalassemia intermedia can be the clinical expression of heterozygosity for different tyes of thalassemia, beta-thalassemia and hereditary persistence of fetal hemoglobin, beta-thalassemia and Hb-Lepore, and in blacks it may even represent a true beta-thalassemia homozygote. This report describes thalassemia intermedia in a white male due to beta-thalassemia and an unstable hemoglobin. Chain-synthesis studies showed an excess of alpha-chain production over beta-chain production in the propositus and his mother but balanced chain synthesis in the clinically normal father, who is heterozygous for the unstable hemoglobin. The unstable hemoglobin was found to be beta14 (A11) Leu leads to Pro, which has previously been described in a clinically normal African woman, and named Hb-Saki. This hemoglobin is not distinguishable from Hb-A on routine electrophoresis at alkaline or acid pH and tests for unstable hemoglobins are necessary for its detection. The increasing list of such hemoglobin variants and previous cases of heterozygosity for beta-thalassemia and unstable hemoglobins are reviewed.
The proportion of Hb G Philadelphia (alpha68-Asn leads to Lys) in heterozygotes has been found to have a well-defined bimodal distribution around means of 33% and 46% Hb G. microcytosis and hypochromia are consistently associated with the latter group, who also have a decreased ratio of alpha/beta-chain synthesis in the peripheral blood, but these characters are not linked to the Hb-Galpha gene, because a parent with microcytosis and 46% Hb Galpha may have offspring with 33% Hb G without significant microcytosis. In one family a subject with Hb G and Hb G2 but no Hb A or Hb A2 is presumably a homozygote for alphaG. This subject has microcytosis and a decreased ratio of alpha/beta chain synthesis. In another family a subject with Hbs H, G and G2 but without Hbs A or A2 is heterozygous for both Hb G and alpha thalassaemia I. These findings are compatible with the hypothesis that the alphaG mutation occurs on a chromosome with only a single alpha-chain locus and that the expression in heterozygotes as 46% or 33% Hb G is determined by the homologous chromosome in trans having either one or two normal alphaA genes respectively. The significance of this polymorphism for chromosomes carrying alpha-chain genes is discussed.
Five families are described in which there have been matings between individuals doubly heterozygous for beta thalassaemia and the delta-chain variant haemoglobin A2' to normal persons. In all there were 24 informative offspring. There were no crossovers between the beta-thalassaemia and delta-chain loci; in three of the families the genes were linked in cis and in two families the genes were found in trans. Together with previously reported families there have now been 58 opportunities for crossing over between the beta-thalassaemia and delta-chain loci and there have been two possible and one highly probable crossovers. Of the total of 9 families reported to date 4 have had the genes for beta thalassaemia and Hb A2' in cis and 5 in trans. These findings are contrasted with the findings in families where a beta-chain structural variant and Hb A2' have been observed together and these genes have always been found in trans and never in cis. The reasons for linkage disequilibrium of this type are discussed. It is concluded tentatively that the distance between the delta-structural and beta-thalassaemia loci is greater than that between the delta-structural and beta-structural loci. To date this conclusion can only be applied to the beta+ -thalassaemia and beta-thalassaemia genes as found in the African population, since this is the only population with a high incidence of delta-chain mutants which allow linkage analysis of this type to be carried out.
A system for rapid hemoglobin electrophoresis on both cellulose acetate and citrate-agar combined with a simple solubility test for sickling variants is described. By use of a saponin hemolysate of once-washed erythrocytes, all three tests can be performed at one time so that an accurate result of the hemoglobin phenotype can be provided expeditiously. A technic for rapid hemoglobin electrophoresis on citrate-agar, using standard equipment is described in detail.