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Foremost among the beneficial effects of screening umbilical cord blood is the optimized quality of care that can follow the immediate involvement of an infant with sickle cell disease and his or her family in an appropriate health care system. This is exemplified by the reduction in the case fatality rate of pneumococcal septicemia that has been achieved. Appropriate follow-up of screening also includes transmission of information about the diagnosis of a hemoglobinopathy trait or alpha-thalassemia to affected families and their physicians, with ready availability of education and counseling.

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Glycosylated hemoglobin in red blood cell hemolysates of five patients homozygous for CC, 18 patients with SC condition, and 13 patients heterozygous for Hb C with or without insulin-dependent diabetes mellitus were separated by Bio-Rex 70 chromatography. The various glycosylated components were identified by analysis of the hemoglobin components for ketoamine and phosphate, in vitro glycosylation studies, and by the quantitative differences in the minor components between the participants with and without diabetes. The percentages of Hb A1a + b, Hb A1c, and Hb C1c were significantly increased in the Hb C heterozygote with diabetes. Similarly, the percentages of Hb S1a + b and Hb S1c were elevated in the SC patient with diabetes. It was noteworthy that the levels of these components became normal after adequate control of diabetes. Moreover, the levels of Hb C1c in the CC participants and Hb S1c (Hb S1c/total Hb S) in the SC patients were significantly higher than the Hb S1c levels previously reported in patients with sickle cell anemia. These findings might reflect the fact that CC and SC patients have less severe hemolytic anemia. Moreover, the relative proportions of Hb A1c and Hb C1c were nearly the same in Hb C heterozygotes, which indicated that Hb A and Hb C were glycosylated in vivo to approximately the same extent.

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Changes in Department of Defense regulations now permit persons with sickle cell trait to serve in all service branches. However, for purposes of the regulation, sickle cell trait is defined as 41% or less S hemoglobin. Our screening experience, based on 397 individuals with sickle cell trait, with quantitative scan of cellulose acetate electrophoretic sheets, indicates that 20-40% (depending on definition of terms) of individuals with sickle cell trait would be excluded by this criterion.

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Rabbits were made reticulocytotic by repeated bleeding or by injection of phenylhydrazine. Up to reticulocyte levels of 70%, the baseline activity of magnesium-dependent calcium-stimulated adenosine triphosphatase ( ( Ca + Mg)-ATPase, EC 3.6.1.3.) of the red cell plasma membrane was not significantly changed. Maximal activity in the presence of calmodulin was significantly reduced, the stimulation by the activator falling from about 350% to about 50%.

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For a severity classification of sickle cell disease to be accepted, it is necessary that clinicians agree upon relative disease severity between patients. This condition was shown to be satisfied for a randomly selected group of patients evaluated by four persons. All rank correlation coefficients between observer pairs were highly significant. Representative severity indices based on history and recent hospital events also correlated significantly with evaluator ranking. The results show that, in principle, a classification of sickle cell disease patients by severity is possible. Such a classification would be most useful to evaluate the prognostic significance of particular signs or symptoms, or the success of various treatments in affecting severity of disease.

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The sleep of two patients with sickle cell disease was punctuated by periods with notably reduced arterial O2 saturation despite simultaneous normal respiratory rates. The O2 levels returned to baseline during intermittent wakefulness. Gas exchange patterns during sleep may contribute to the morbidity of sickle cell disease.

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The sickle cell (Hb SS) membrane-bound Ca2+-ATPase was found to have a Vmax in a range of 30-100% of the Vmax of the normal enzyme. In all sickle cell preparations, the Ca2+-ATPase could be stimulated at least 4-fold by calmodulin, but the stimulation factor varied considerably (4-26 fold) in the different preparations. The affinity of the ghost sickle cell Ca2+-ATPase for Ca2+, ATP and calmodulin was comparable to that of the normal enzyme. The sickle cell Ca2+-ATPase was solubilized from the membrane with Triton-X-100, and purified through a calmodulin sepharose-4B column, a technique by which the Ca2+-ATPase from normal ghosts has been successfully isolated in a functionally active and pure form (see V. Niggli, E.S. Adunyah, J.T. Penniston and E. Carafoli, 1981, J. Biol, Chem. 256, 395 - 401). The specific activity of the isolated sickle cell enzyme was significantly decreased (up to 80%) with respect to that ot the normal enzyme, but the amount of protein isolated was comparable to normal. All other parameters of the ATPase (affinity for Ca2+, ATP and calmodulin) were comparable to those found for the normal enzyme. In SDS polyacrylamide gel electrophoresis, the purified enzyme appeared as a single band protein with a Mr comparable to that of the normal enzyme. In the absence of calmodulin the sickle cell enzyme could be activated by acidic phospholipids, as reported for the normal enzyme. After reconstitution into liposomes it transported Ca2+ with normal efficiency (about 1 Ca2+/ATP hydrolyzed). Therefore, the only difference between the purified normal and the sickle cell enzyme appears to be the lower specific activity of the latter.

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The enthalpy of activation of entry of 45Ca into erythrocytes under conditions of zero net transport (19 Kcal/mol), inhibition of entry by N-ethylmaleimide, and transport pH optimum of 7 - 7.5 are consistent with a protein carrier for inward calcium movement. Initial rate analysis shows saturation kinetics and transstimulation, consistent with a carrier-mediated exchange-diffusion mechanism.

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The effect of the addition of radiographic contrast material (Renografin) to blood on the oxyhemoglobin dissociation curve and P50 was measured by a metabolic deoxygenation technique in a strongly buffered red cell suspension. With incubation time constant, increasing doses produced progressive decreases in P50. With incubation time varied at a constant dose, a decrease in P50 was seen after only one minute. In addition, in vivo studies were performed on 11 patients undergoing cardiac catheterization. Simultaneous proximal coronary sinus and aortic samples were drawn as controls, and then at one minute and five minutes after injection of the left coronary artery. In eight patients studies were performed after, and in three prior to left ventriculography. At one minute after left coronary injection there was a significant decrease of coronary sinus as compared to aortic P50 (p less than .10) (only when left ventriculography was performed prior to coronary arteriography). The magnitude of these effects in vivo is unknown, but they would be expected to be more severe in areas distal to a critical coronary lesion due to stasis of blood flow and ischemic metabolic changes.

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There is an exchangeable calcium pool in both normal and sickle cell erythrocytes, comprising about 10-15% of the total cellular calcium. Sickle cells show increased calcium as compared to normal cells in the oxygenated state. Specific differences between sickle and normal cells which may be associated with this fact are an increased rate of calcium exchange in sickle cells at low external calcium, an increased "leak" of calcium into sickle cells (i.e., phosphate independent exchange), and a pattern of magnesium loss in sickle cells which is consistent with a Mg-Ca exchange diffusion resulting in the increased intracellular calcium in these cells. The exchangeable calcium in sickle cells is more labile, almost all of it being available for re-exchange out of the cell over a short-time-course experiment. Analyses of flexibility and osmotic fragility of sickle cells are consistent with expected effects of increased intracellular calcium.

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The dissociation of cervical cell suspensions after various chemical and enzymatic treatments was monitored by using the Centrifugal Cytology rotor to produce glutaraldehyde-fixed dispersions on conventional microscope slides and subsequent Pap staining. A special program was written in RPG II to record and analyze the results of the dissociation experiments in terms of white blood cells and the true cervical cells ("other cells"), and the degree of dissociation and recovery of both classes of cells. Since accurate differential counts on the untreated Ayre scrapes were difficult, the samples were syringed gently to break up the large or adventitious clumps. Cumulated results from control preparations indicate that the white blood cells and "other cells" are composed respectively of 92 and 63% single cells. The cells were further dissociated by: dissolving the cervical mucin sequentially with dithiothreitol and iodoacetic acid; depolymerizing the nucleohistone gel with ribonuclease; solubilizing the desmosomes with EDTA; removing the remaining cellular agglutinins with Varidase; and finally mechanical dispersion by hypertonic shock. The optimum procedure for dissociation involves the use of ribonuclease, dithiothreitol, iodoacetic acid EDTA, Varidase and sucrose shock. The white blood cells are now monodisperse and 81% of the "other cells" are found as single cells. If nuclear separation by two diameters is considered sufficient 98% of the "other cells" are single. The slide preparations are now sufficiently good that a scanning system is feasible.

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Under exchange conditions (no net increase in calcium), erythrocytes incubated in isoosmotic phosphate-buffered saline have an exchangeable calcium pool comprising about 10% of the total erythrocyte calcium. This pool reaches exchange equilibrium, for either inward-directed or outward-directed transfer of the 45Ca-exchange label, with a half-time of about 20 min. The uptake of Ca2+ requires phosphate, even under hypo-osmotic conditions, where the calcium loading expected as the cells swell is obtained only when phosphate is present. The phosphate requirement is not due to Ca2+ transport as a phosphate salt. This exchangeable-calcium pool is also present in sickle-cell-anemia erythrocytes, and comprises a similar proportion of total cellular calcium.

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