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IntroductionVariations in postnatal length of refrigerated, unfixed umbilical cords were studied over time to elucidate natural changes and times of stability.MethodsLength was measured in 132 cords following severance, repeated at varying timed intervals and studied by analysis of variance and regression analysis.ResultsData show immediate rapid initial phase shortening (mean 4.2+/-3.9 cm SD); an interval of lengthening; stable length at hours 3-4 following severance, a slower second phase shortening (mean 1.5+/-0.7 cm SD) beginning at 5 hours and peaking at 12 hours; and gradual lengthening to stable length after 23 hours. Overall, there was a significant net mean decrease of 3.49+/-2.29 cm SD. Shortening was greatest for intact long cord segments (p=0.0001), as much as 11 cm. Two highly significant models for predicting umbilical cord length at delivery (OL) were determined using the post-delivery lengths (Length) measured at different times following delivery (Hours), as follows: At ≤ 3 hours following delivery: OL=1.02xLength cm+1.11xHours At >3 hours following delivery: OL=1.07xLength+0.44xHours-0.01x(Hours)2ConclusionCord lengths stabilized between hours 3-4 and after 23 hours following severance. Phase one shortening resembles vasoconstriction; phase two resembles rigor mortis. The models allow prediction of the original umbilical cord length at delivery, regardless of the time of measurement.

More precise analysis of causes of death is needed to focus research efforts and improve morbidity and mortality in sickle cell disease. In this study, the morphological evidence of the cause of death was studied in 306 autopsies of sickle cell disease, which were accrued between 1929 and 1996. The most common cause of death for all sickle variants and for all age groups was infection (33-48%). The terminal infection was heralded by upper respiratory tract syndromes in 72.6% and by gastroenteritis in 13.7%. The most frequent portal of entry in children was the respiratory tract but, in adults, a site of severe chronic organ injury. Other causes of death included stroke 9.8%, therapy complications 7.0%, splenic sequestration 6.6%, pulmonary emboli/thrombi 4.9%, renal failure 4.1%, pulmonary hypertension 2.9%, hepatic failure 0.8%, massive haemolysis/red cell aplasia 0.4% and left ventricular failure 0.4%. Death was frequently sudden and unexpected (40.8%) or occurred within 24 h after presentation (28.4%), and was usually associated with acute events (63.3%). This study shows that the first 24 h after presentation for medical care is an especially perilous time for patients with sickle cell disease and an acute event. Close monitoring and prompt aggressive treatment are warranted.

Objective: To assess the effect of prenatal education about newborn sickle cell screening on parents’ compliance with the follow-up for infants with sickle cell trait. Subjects: Expectant mothers whose prenatal education included information about newborn sickle cell screening were the study group, and those whose prenatal education did not include such information were the control group. Methods: Mothers of infants with sickle cell trait were given the opportunity for in-person notification of screening results and follow-up counseling/education. Follow-up rates, anxiety and retention of information were assessed for the case-control study. Results: There were a total of 15 670 infants born in the region, and 647 infants were identified with sickle cell trait. The follow-up rate for parents of infants with sickle cell trait was significantly higher (76%) for study group than the control group (49%) ( P =0.0006). Parents whose prenatal education included sickle cell hemoglobinopathy information retained significantly more of the information given during the post-natal education than did controls. Conclusion: Our data suggest that prenatal education for expectant mothers which includes information about newborn sickle cell screening significantly increases the follow-up rate for infants with sickle cell trait and contributes to a greater retention of information.

The red blood cells (RBCs) derived from blood taken from homozygous sickle cell (SS) patients demonstrate densities that are inversely proportional to the intracellular reduced glutathione (GSH) content. Addition of 1 mM 1-chloro-2,4-dinitrobenzene (CDNB) to low-density sickle cells (LDSS), at 4 degrees C, results in a shift of LDSS erythrocytes to high-density sickle cells (HDSS), with corresponding decreases in GSH. We have previously demonstrated that this CDNB effect was due to increased K(+) leakage and that dense cell formation could be inhibited by clotrimazole (specific for the Gardos channel) but not DIOA (specific for the K(+)-Cl(-) co-transport system) at pH 7.4 (Shartava et al. Am. J. Hematol. 1999;62:19-24). Here we demonstrate that clotrimazole (10 microM) inhibits dense cell formation at pH 7.1 and 6.8, while DIOA (1 mM) has no effect. As pH 6.8 is the optimal pH for the K(+)-Cl(-) co-transport system, we can now reasonably conclude that damage to the Gardos channel is responsible for CDNB-induced dense cell formation.

Hydroxyurea (HU) is a widely used cytotoxic agent that is known to induce fetal hemoglobin (HbF) production and is presently used to ameliorate the severity of pain episodes in patients with sickle cell anemia (HbSS). Previously we have shown that HU inhibits growth of burst forming unit-erythroid (BFU-E) colonies in a dose-dependent manner, while fetal hemoglobin levels were increased. In the present report, we extended our analysis demonstrating the number of S phase cells is significantly higher for HbSS patients that respond to HU therapy. Studies were completed in vitro using erythroid progenitors derived from umbilical cord samples or peripheral blood from patients with HbS-hereditary persistence of fetal hemoglobin (HbS-HPFH) or HbSS disease. The effect of HU on (a) S phase erythroid progenitors, (b) BFU-E colony growth, (c) HbF levels in BFU-E colonies, and (d) total cellular RNA synthesis was analyzed in vitro for the three groups. The level of S phase erythroid progenitors was similar for all three groups and BFU-E colony growth was inhibited 92-94% for all samples in a dose-dependent manner. The HbF levels were increased in BFU-E colonies from HbSS patients (control, 4.0% +/- 1.15% vs. +HU, 22.67% +/- 2.03%) whereas HbF levels were decreased in BFU-E colonies derived from umbilical cord samples (control, 80% +/- 9.07% vs. +HU, 35.7% +/- 4.81%) or HbS-HPFH patients (control, 49.67% +/- 3.84% vs. +HU, 23.3% +/- 0.88%). Total RNA synthesis measured by 3H-uridine incorporation increased with increasing concentrations of HU; however, actinomycin D inhibited HU-induced RNA synthesis. These results suggest that HU can inhibit an active globin gene without preference and that newly synthesized RNA is under transcriptional control mechanisms.

Clotrimazole, a specific inhibitor of the Ca2+ activated potassium (Gardos) channel, and the antioxidant N-acetylcysteine were found to inhibit the in vitro formation of high-density sickle cells induced by treatment with 1-chloro-2,4-dinitrobenzene (CDNB). The CDNB induced leakage of K+ can be inhibited by treatment of SS erythrocytes with 20 mM N-acetylcysteine. We conclude that the effect of N-acetylcysteine in preventing K+ efflux and formation of high-density sickle cells is related to its ability to protect the Gardos channel from oxidative damage caused by diminished levels of reduced glutathione. This effect is due to the ability of N-acetylcysteine to maintain an appropriate level of reduced glutathione and its direct antioxidant activity. Am. J. Hematol. 62:19–24, 1999. © 1999 Wiley-Liss, Inc.

In this paper we test the direct treatment effects in crossover designs under various assumptions on errors. We end up getting a battery of independent tests and discuss how to combine them. We illustrate our results using an example with actual data.

We show that N-acetylcysteine (NAC) has the ability to cause statistically significant diminishment in the in vitro formation of irreversibly sickled cells (ISCs) at concentrations greater than 250 μmol/L. Other antioxidants, approved for human use (cysteamine, succimer, dimercaprol), were not efficacious. NAC had the ability to cause statistically significant conversion of ISCs formed in vivo back to the biconcave shape. NAC was also shown to reduce the formation of dense cells and increase the available thiols in β-actin. We showed that diminishing reduced glutathione (GSH), by treatment with 1-chloro-2,4-dinitrobenzene, resulted in increased dense cells. We conclude the NAC blocks dense cell formation and ISC formation by targeting channels involved in cellular dehydration and β-actin, respectively. The efficacy of NAC is probably due to its combined antioxidant activity and ability to increase intracellular GSH.

Patients with sickle-cell anemia treated with hydroxyurea may have significant reduction in frequency and severity of pain episodes. However, previous clinical trials show a variable response to hydroxyurea. Criteria which can be used to select patients who are likely to respond to hydroxyurea treatment would be useful. Our laboratory has previously demonstrated an inverse linear relationship between the total number of burst-forming unit-erythroid (BFU-E) colonies and fetal hemoglobin levels in sickle-cell patients treated with hydroxyurea. In the present report, an in vitro cell culture system was established to evaluate the effects of hydroxyurea on BFU-E colony growth and induction of fetal hemoglobin production. Five Hb SS patients who were not previously treated with hydroxyurea and three Hb SS patients who failed to respond to hydroxyurea treatment were included in the study. The results show that the number of BFU-E colonies is decreased from 153.7 to 7.2 per 3 x 10(5) mononuclear cells, whereas fetal hemoglobin levels were increased from 5.1 to 19.4% in the presence of hydroxyurea in vitro in cultured erythroid progenitors, which were derived from 5 patients before treatment. The number of BFU-E colonies decreased from 153.7 to 2.0 per 3 x 10(5) mononuclear cells in the in vitro cultures obtained from serial peripheral blood samples over a 9- to 20-week period of oral hydroxyurea therapy. A simultaneous rise in fetal hemoglobin level from 10.2 to 28.6% in the peripheral blood over the same period of hydroxyurea therapy was also observed. Our results demonstrate that the increase in fetal hemoglobin levels in cells treated with hydroxyurea in vitro is comparable to the rise of fetal hemoglobin production following hydroxyurea therapy in these patients. On the contrary, these findings were not observed in three previously non-responsive sickle-cell patients. These results suggest that the changes in number of BFU-E colonies and fetal hemoglobin levels after in vitro exposure to hydroxyurea may be a useful approach to select sickle-cell patients who will respond to hydroxyurea therapy.

We have tested the hypothesis that dense cell formation in sickle cell disease is associated with increased binding of calpromotin to the membrane, an event that occurs during the activation of calcium-dependent potassium transport. By SDS polyacrylamide gel electrophoresis, we found that sickle cell membranes contained more calpromotin than did normal membranes when stained with Coomassie brilliant blue or when transferred to nitrocellulose paper and immunostained with horseradish peroxidase. Also, the membranes from dense sickle cells contained significantly (P = 0.00055) higher levels of calpromotin, 2.62+/-1.59 microg/mg membrane protein, compared to light sickle cells, 1.40+/-0.70 microg/mg membrane protein, when measured by an enzyme-linked immunosorbent assay. The ratio of calpromotin associated with dense cell membranes to light cell membranes was significantly greater than 1.0 (P < 0.00005). Transmission electron micrographs of immunogold-labelled membranes supported the increase in calpromotin binding in dense sickle cell membranes. In addition, the immunogold probe demonstrated clustering, which was not observed in light sickle cell membranes nor in normal membranes. Finally, we incubated HbSS cells in vitro using a repetitive deoxygenation/ reoxygenation procedure to produce dense cells and then measured the levels of calpromotin associated with their membranes. As expected, the levels of calpromotin bound to the membrane doubled during the procedure relative to the basal levels at the beginning of the incubation. The correlation coefficient, calculated between the increase in dense cell formation and the increase in calpromotin associated with the membrane, was statistically significant (P = 0.038). The results demonstrate that an increase in calpromotin binding to the membrane is associated with dense cell formation presumably through the activation of the calcium-dependent potassium channel.

It has been demonstrated that cysteine modification in irreversibly sickled cell β-actin slows down the remodeling of membrane skeletons [Shartava et al.: J Cell Biol 128:805-812, 1995]. This slow remodeling can be due to alterations in spectrin-actin binding and/or actin-actin interactions in irreversibly sickled cell (ISC) membrane skeletons. In these studies we demonstrate that ISC actin binds spectrin normally. However, ISC β-actin polymerizes and depolymerizes more slowly than control β-actin, and forms unusual aggregates when placed under polymerizing conditions. Electron microscopic analysis of actin polymers indicated that ISC actin generates a large amount of aggregates which we conclude are due to the structural modification caused by the disulfide bridge between cysteine264 and cysteine373 in β-actin. Am. J. Hematol. 55:97-103, 1997. © 1997 Wiley-Liss, Inc.

This study compares the rate of mental disorders in children and adolescents with sickle cell disease with the rate in a corresponding control population. Using the clinical interview, a diagnosis based on DSM-III-R criteria for the presence of 13 mental disorders common to childhood was considered in a sequential sample of 39 subjects with sickle cell disease and a convenience sample of 26 same-race control subjects. Thirty-one percent of the sickle cell group and 42% of the control group screened positive for one or more of the selected mental disorders. The difference was not significant. When subgroups of mental disorders were considered (anxiety disorders, depressive disorders, disruptive behavior disorders, psychotic disorders, and other selected disorders), the differences were still not significant. This study suggests that children and adolescents with sickle cell disease do not have a greater risk for clinically significant mental disorders than same-race outpatient clinic controls. This study supports other reports in the medical literature that suggest that children attending outpatient medical clinics are at a higher risk for mental disorder than is seen in epidemiologic studies of nonmedical populations.

We have previously demonstrated that slow dissociation of HDSS membrane skeletons in high ionic strength Triton X-100 buffer was related to a posttranslational modification in beta-actin, in which a disulfide bridge was formed between cysteine 284 and cysteine 373[Shartava et al: J Cell Bio 128:805, 1995]. These previous dissociation assays were limited to two homozygous (SS) sickle cell patients and a single temperature (37 degrees C). In the current work, we have expanded the SS subjects to 9 and have carried out dissociation assays at 0, 24, 30, 34, and 37 degrees C. At 0 degrees C there was limited dissociation of spectrin and actin from normal(AA), low density sickle cell(LDSS), and high density sickle cell (HDSS) core skeleton up to 24 hr. The first order rate constants for dissociation of spectrin, at 0 degrees C, was 0.030-0.035 x 10-4 sec-1 for AA,LDSS, and HDSS core skeletons. However at 24, 30, 34, and 37 degrees C the rate of dissociation of spectrin from HDSS core skeletons was significantly slower than the rate of dissociation from AA core skeletons. Having determined the first order rate constants for spectrin dissociation at these specified temperatures, we then asked whether dithiothreitol (DTT) would hasten the dissociation of core skeletons. The presence of DTT caused the rate of dissociation of the HDSS membrane skeleton to become statistically indistinguishable from the rate of dissociation of AA membrane skeletons. This is consistent with the suggestion that reversible thiol oxidation is responsible for the slow dissociation of the HDSS membrane skeleton.

Glycogen in the placenta and its appendages is important for fetal well-being. The precise location of the glycogen stores, however, is unknown. This study was initiated to quantitate glycogen levels at well-defined sampling sites in more than 641 samples from 10 uncomplicated pregnancies and to correlate these glycogen levels with clinical and morphological variables. By biochemical assay, glycogen levels were greatest in the midumbilical cord section (29.08 ± 1.18 mg/g dry wt) and lowest in the amnionic membrane (2.31 ± 0.08 mg/g dry wt). Within the placental disk, parenchymal glycogen levels were greatest near the cord insertion (9.31 ± 2.68 mg/g dry wt) and lowest at the periphery (5.71 ± 1.14 mg/g dry wt). The midumbilical cord glycogen level showed strong direct correlations (P < . 001) with birth weight, umbilical cord weight, and total calculated umbilical cord glycogen and somewhat lower but significant (P < .037) direct correlations with the calculated mean umbilical cord glycogen level, total c...

Testing the equality of several binomial proportions was first proposed by Pearson (1900) and the discussion of this test can be found in standard statistics books. In this article we consider testing the equality of several binomial proportions to a known standard. For testing such a hypothesis against a two sided alternative, an intuitive test statistic and its null distribution have been mentioned in Rohatgi (1976). Here we study the non-null distribution of that statistic and provide a simple way of calculating the approximate power via a moment approximation method (MAM). A procedure of constructing simultaneous confidence intervals is also given here. Additionally, we propose test statistics for testing the above null hypothesis against one sided alternative hypotheses. We give simple methods of obtaining the p-value and the power for these test statistics. Finally, two examples are given to illustrate the techniques.

The relationship between free radicals and scavenger enzymes, and the disorders called the neuronal ceroid-lipofuscinoses, has long been an argumentative one. Recent evidence would seem to support the fact that such a relationship might exist but that it is indirect. The relationship does not seem due to an inborn error of free radical scavenger enzyme metabolism. Anticonvulsants play a role, as they influence free radical generating systems. At this juncture, no one has studied the relationship of anticonvulsant therapy, neuronal ceroid-lipofuscinosis, and the free radical-scavenging enzyme system, and their interplay. We have studied a large number of patients with epilepsy who are on either monotherapeutic or polytherapeutic regimens of most of the common anticonvulsants. We have found excessive free radical production in many of these patients, ranging from minor effects in the simpler anticonvulsants when used monotherapeutically, to more complex changes in polytherapeutic combinations. Likewise, we have found subtle and inconsistent findings in the free radical-scavenging enzyme system in a variety of examples of neuronal ceroid-lipofuscinosis. When refractory seizure disorders stimulate the vigorous use of polytherapy with a variety of free radical-facilitating anticonvulsants, free radical production becomes deleterious. Likewise, in certain types of neuronal ceroid-lipofuscinosis, polypharmacy with anticonvulsants, by enhancing the production of free radicals or suppressing scavenging enzymes, tends to be deleterious and induces a worsening in the disease process.

The problem of testing equality of several normal means to a specified value when the population variances are unknown is considered. Two test procedures, one obtained by using Fisher's method of combining independent tests and the other based on the ma..

Previously, we reported that a 70 kDa nuclear protein may regulate fetal haemoglobin gene expression in haemin treated K562 cells. To obtain further evidence of the specific role of this 70 kDa nuclear protein, we compared the nuclear fractions isolated from phenylacetate, hydroxyurea and haemin treated K562 cells. Both phenylacetate and hydroxyurea have been used to induce fetal haemoglobin synthesis in K562 cells. Cell growth was measured by biochemical events including DNA, RNA and protein synthesis. Differentiation of K562 cells was determined by both [3H]-leucine incorporation into fetal haemoglobin and scoring benzidine-stained positive cells. Unlike the haemin treated cells, phenylacetate and hydroxyurea induced growth arrest and increased fetal haemoglobin gene expression in K562 cells. After four days of treatment with phenylacetate and hydroxyurea more than 50% of the cells stained positive with benzidine. The SDS-Polyacrylamide gel electrophoretic analysis of nuclear proteins isolated from phenylacetate and hydroxyurea treated K562 cells showed that the 70 kDa protein was reduced in nuclear protein extract in both groups similar to haemin treated cells. These results suggest that the loss of the 70 kDa protein from a nuclear protein extract is not restricted to only haemin treated cells but also occurs in hydroxyurea and phenylacetate treated cells. Our results provide further evidence that the 70 kDa nuclear protein may be involved in regulating fetal haemoglobin expression through a negative control mechanism.

DNA-synthesis stage and total number of circulating burst-forming-units-erythroid (BFU-E) have been inversely correlated with hemoglobin F levels in the peripheral blood, as well as in the cells from the BFU-E-derived colonies obtained from homozygous sickle cell anemia (SS) patients during steady state. Similar studies in SS patients treated with cytotoxic agents have not been reported. However, regeneration of the erythroid marrow that follows the cytoreduction phase of chemotherapy has been suggested as one of the mechanisms of stimulation of fetal hemoglobin synthesis. Therefore, a longitudinal study of hemopoiesis in hydroxyurea-treated SS patients was conducted. Thirty-two sets of hemopoietic studies, including total circulating BFU-E and S-phase BFU-E, were obtained from three patients treated with hydroxyurea. A dose-dependent decrease in total BFU-E colonies occurred in peripheral blood of all three patients (r = -0.58, -0.85, and -0.97, respectively, with each P < 0.05). There was a strong positive correlation between hydroxyurea dose and fetal hemoglobin levels in two of the three patients who responded clinically (r = 0.89618 and 0.88632, respectively, with each P < 0.01). When data from all patients were combined (n = 32), there was a strong, inverse, linear relationship between total number of BFU-E and percentage S-phase BFU-E with fetal hemoglobin levels (r = -0.6649 and -0.7404, respectively, with each P < 0.0001). A stronger, curvilinear, multiple relationship was detected between total BFU-E and percentage S-phase BFU-E with fetal hemoglobin levels (R = 0.8351 and 0.8602 with each P < 0.0001).

The human erythroleukaemic cell line K562, in response to various chemical agents, undergoes differentiation and exhibits exclusive production of fetal and embryonic haemoglobins. In this study we have compared the efficiency of natural growth factors interleukin-3 and erythropoietin and three chemical inducers such as dimethyl sulfoxide (DMSO, 1.9%), phorbol-12-myristate-13-acetate (PMA, 50 ng/ml) and hemin (25 microM) on growth and differentiation of these cells. Erythropoietin significantly stimulated the growth of K562 cells (P

Ischemia-reperfusion injury has been implicated as playing a major role in the development of necrotizing enterocolitis, a major cause of morbidity and mortality in the newborn. A tungsten-supplemented molybdenum-free diet can reduce xanthine oxidase (XO) enzyme activity in the intestine, which in turn reduces the generation of oxygen radicals after an ischemia-reperfusion insult. This study evaluated the ability of this diet to be effective by indirect means, ie, transplacental and breast-feeding routes. XO activity of the intestine was measured in three groups of CD-1 white rats: I, weanlings fed the tungsten diet or standard chow for 1 week; II, 1-day-old rat pups whose mothers were maintained on the tungsten or standard chow for 7 to 10 days prior to term; and III, rat pups at 1 and 3 weeks after birth whose lactating mothers were maintained on the tungsten or standard chow. Some animals from group III also underwent either a 30- or 60-minute episode of occlusion of the superior mesenteric artery (SMA) to evaluate the protective effects of the diet. XO activity was significantly reduced in all groups receiving the tungsten diet (P less than .0001). Blinded histopathologic studies of the entire small bowel showed significantly less villar necrosis (P less than .05) and fibrosis (P less than .0001) in the tungsten-treated group than in the controls. In the 60-minute occlusion study all tungsten-group animals survived, whereas 7 of 12 in the control group died of intestinal infarction within 24 hours (P less than .001).(ABSTRACT TRUNCATED AT 250 WORDS)

A longitudinal, prospective, controlled evaluation of magnetic resonance images (MRI) of long bones in sickle cell patients was undertaken simultaneously with assessment of clinical status and hematologic parameters, including dense erythrocytes. MRI of bone marrow in sickle cell patients during steady states appeared patchy and were markedly different from those in matched controls (P approximately 0). Patients with severe patchiness were older than those with mild or moderate patchiness (P less than .03). Sixty-nine MRI were performed during 28 painful episodes occurring in 14 subjects with sickle cell disease (SCD). Increased signals on intermediate and T2-weighted images were detected in 35.7% of painful episodes. These abnormalities were distinct and not observed to occur spontaneously during the steady- state examinations (P approximately 0). Bone marrow infarcts were confirmed by biopsy in two instances and autopsy in one instance. Dense red cells decreased by 40.81% of baseline during pain crises (P = .00005), more remarkably in those who had pain in the lower extremities (P = .0145). Patients with change in MRI during pain crises had a greater percentage change in the dense cells than those without the change in MRI (69.7% v 31.3%, P = .0120).

Context.—Magnetic resonance imaging of bone marrow in homozygous sickle cell disease (hemoglobin [Hb] SS) shows nonhomogeneous, mottled signals that increase with age and number of crises. The pattern of these signals is reminiscent of the underlying vascular architecture, but histopathology of this tissue has not been adequately studied.Objective.—To elucidate the histopathology of blood vessels in the bone marrow in sickle cell disease.Design.—Retrospective histochemical morphometric study of bone marrow arteries by point counting in HbSS (13 cases) and sickle cell Hb C (HbSC) (8 cases) compared to nonanemic normal controls (HbAA) (10 cases). All patients were nondiabetic, normotensive, younger than 37 years, and matched for age group.Results.—The mean point count for perivascular fibrous tissue was significantly greater in the HbSS group (P < .001) in both small (P < .001) and medium-sized (P = .002) vessels, and in both age groups (pediatric, P < .001; adult, P = .005) compared with the HbAA group. Additional analysis showed the difference was significant in HbSS pediatric small vessels (P < .001) and in pediatric and adult medium vessels (P = .045 and P = .03, respectively). Ratios of fibrous tissue to muscle showed proportionately greater fibrous tissue in HbSS pediatric small (P < .001) and medium-sized vessels (P = .02), and in adult large vessels (P = .03). Mean point counts for muscle were significantly decreased in HbSS small vessels when all ages were compared as a group (P = .02), but when compared by age groups, counts were significantly increased in adult HbSS medium-sized vessels (P = .01). Overall mean point counts for muscle and fibrous tissue in the HbSC group were intermediate between those of the HbSS and HbAA groups, but were not significantly different from counts in the HbAA group (P = .78 and P = .35, respectively).Conclusion.—In sickle cell disease, arterial vessels in the bone marrow show significantly increased fibrous connective tissue and changes in muscle that vary with age and vessel size.

More precise analysis of causes of death is needed to focus research efforts and improve morbidity and mortality in sickle cell disease. In this study, the morphological evidence of the cause of death was studied in 306 autopsies of sickle cell disease, which were accrued between 1929 and 1996. The most common cause of death for all sickle variants and for all age groups was infection (33-48%). The terminal infection was heralded by upper respiratory tract syndromes in 72.6% and by gastroenteritis in 13.7%. The most frequent portal of entry in children was the respiratory tract but, in adults, a site of severe chronic organ injury. Other causes of death included stroke 9.8%, therapy complications 7.0%, splenic sequestration 6.6%, pulmonary emboli/thrombi 4.9%, renal failure 4.1%, pulmonary hypertension 2.9%, hepatic failure 0.8%, massive haemolysis/red cell aplasia 0.4% and left ventricular failure 0.4%. Death was frequently sudden and unexpected (40.8%) or occurred within 24 h after presentation (28.4%), and was usually associated with acute events (63.3%). This study shows that the first 24 h after presentation for medical care is an especially perilous time for patients with sickle cell disease and an acute event. Close monitoring and prompt aggressive treatment are warranted.

Existing data regarding time between stroke and presentation for treatment are largely derived from hospital-based or multicenter databases and may not accurately reflect presentation patterns for most hospitalized stroke patients.We evaluated a consecutive series of all hospitalized patients in Mobile County, Alabama.We identified 1,010 hospitalized stroke patients. Of all patients with out-of-hospital stroke, 42% came to a hospital within 3 hours of symptom onset. There were no statistically significant interhospital differences. Being asleep at the time of stroke or being transported by family or friends significantly increased the likelihood of late arrival.A minority of stroke patients arrive at a hospital early enough to qualify for acute intervention. Until development of acute therapies with longer therapeutic windows or more robust therapeutic benefit than tissue plasminogen activator (t-PA), effective stroke prevention strategies will exert a greater influence on stroke incidence and morbidity.

Some of the commonly used univariate and multivariate tests on means are studied where the usual hypothesis-independent variance estimates are replaced with the corresponding hypothesis-dependent variance estimates. This approach leads to tests that are equivalent to the corresponding traditional tests in the cases considered here except in one case. Understanding this can be beneficial to practitioners and students of statistics.

Objective. Intravenous recombinant tissue plasminogen activator (rt-PA) is the only therapy of proven value for patients with acute ischemic stroke (AIS). Controversy exists with regard to the prognostic significance of early computed tomography (CT) changes in patients receiving rt-PA for AIS. The authors retrospectively reviewed all cases of AIS who received intravenous rt-PA for AIS in University of South Alabama hospitals between January 1996 and May 1999. A neuroradiologist, blinded to clinical outcomes, reviewed all baseline CT scans for the presence of the following signs: hyperdense middle cerebral artery (HMCA), loss of gray-white differentiation (LGWD), insular ribbon sign (IRS), parenchymal hypodensity (PH), and sulcal effacement (SE). Modified Rankin Scale (mRS) score was recorded 90 days after thrombolysis, and clinical outcome was dichotomized as favorable (0–1) or unfavorable (2–6). The authors performed both univariate and multivariate analyses to investigate the relationship between early CT signs, baseline clinical variables, and functional outcome as measured by the 90-day mRS scores. Any one early CT finding was detected in 23 (64%) patients. The frequency of specific findings were as follows: SE in 13 patients (36%), LGWD in 12 patients (33%), PH in 9 patients (25%), HMCA in 4 patients (11%), and IRS in 3 patients (8%) patients. There was no statistically significant association between the occurrence of these imaging findings and subsequent functional outcome after thrombolysis. The data suggest that the presence of subtle acute CT changes in AIS patients is not predictive of clinical outcome following administration of rt-PA as per National Institute of Neurological Disorders and Stroke protocol.

The synthesis and turnover of alpha-erythroid, beta-erythroid, alpha-nonerythroid and beta-nonerythroid spectrins was investigated in cultured rat hippocampal neurons. [35S]methionine and subunit specific antibodies were used to label and immunoprecipitate newly synthesized spectrins in 12- to 14-day-old cultures. Synthesis experiments, performed under normal resting conditions, showed that the ratio of newly synthesized alpha-erythroid/beta-erythroid and alpha-nonerythroid/beta-nonerythroid spectrins is 1/1 (mol/mol) both in the soluble and insoluble fractions. Soluble and insoluble alpha and beta erythroid spectrin turn over rapidly (half-life=16-24 min). Soluble nonerythroid alpha-spectrin (half-life=80 min) and beta spectrin (half-life=53 min) turn over more slowly than their insoluble counterparts (30-34 min). The nonerythroid alpha spectrin turnover was significantly different (p0.05) from the other measurements except for nonerythroid beta spectrin, indicating that these subunits are protected from rapid proteolytic degradation until they are assembled in the membrane skeleton.

Most frequently, placental glycogen has been studied as an index of fetal nutrition. There are no published studies of placental glycogen as an index of fetal stress. In this study of 1573 samples from 71 placentae, glycogen levels in the placental disk, fetal membranes and umbilical cord of normal uncomplicated pregnancies were compared with those in complicated pregnancies. The complicated pregnancies included preterm delivery, hypertensive disorders, inadequate prenatal care, substance abuse, maternal fever or infection, obesity, diabetes mellitus, premature rupture of membranes, intrauterine growth retardation, sickle cell trait, and acute meconium staining of amniotic fluid at delivery. The data showed that the only significant differences were in the subgroup complicated by meconium-stained amniotic fluid in which the placental disks and umbilical cords had significantly lower (P=0.0006) glycogen levels. This finding suggests a relatively specific association. It is interesting to speculate that the passage of meconium with its vasoconstrictive effect increases utilization of local glycogen stores, decreases local glycogen reserves needed for the work of further vasoconstriction, and, in the event of subsequent acute stress, impairs vascular perfusion of tissues. In this way, meconium could predispose the infant to asphyxia.

Irreversibly sickled cells (ISCs) remain sickled even under conditions where they are well oxygenated and hemoglobin is depolymerized. In our studies we demonstrate that triton extracted ISC core skeletons containing only spectrin, protein 4.1, and actin also retain their sickled shape; while reversibly sickled cell (RSC) skeletons remodel to a round or biconcave shape. We also demonstrate that these triton extracted ISC core skeletons dissociate more slowly upon incubation at 37 degrees C than do RSC or control (AA) core skeletons. This observation may supply the basis for the inability of the ISC core skeleton to remodel its shape. Using an in vitro ternary complex dissociation assay, we demonstrate that a modification in beta-actin is the major determinant of the slow dissociation of the spectrin-protein 4.1-actin complex isolated from the ISC core skeleton. We demonstrate that the difference between ISC and control beta-actin is the inaccessibility of two cysteine residues in ISC beta-actin to labeling by thiol reactive reagents; due to the formation of a disulfide bridge between cysteine284 and cysteine373 in ISC beta-actin, or alternatively another modification of cysteine284 and cysteine373 which is reversible with DTT and adds less than 100 D to the molecular weight of beta-actin.

Patients with sickle cell anemia often express myriad clinical signs and symptoms that affect their life-style and academic performance. Certain psychoeducational and psychosocial factors have been shown to influence the academic achievement of older patients with sickle cell anemia. However, studies evaluating the school readiness skills of younger children have not been published. To determine whether sickle cell anemia delays preschool development in children aged 4 to 6 years, we studied 10 affected children and 10 normal subjects matched for age, sex, and race. School readiness was evaluated by the Pediatric Examination of Educational Readiness (PEER), which assess a child's performance in areas of developmental attainment such as visual input, verbal output, and short-term memory. The presence of associated movements (minor neurologic signs) and other areas of behavior such as selective attention, activity level, adaptive behavior, and processing efficiency are also observed. The children with sickle cell anemia scored significantly lower than their normal counterparts in several parameters of the PEER. The McCarthy Scales of Children's Abilities, a standardized psychometric test, showed that these children with sickle cell anemia were within the normal range of intelligence. Magnetic resonance imaging done on three children with sickle cell anemia who scored lowest on the PEER revealed no cerebrovascular infarcts. These preliminary studies demonstrate significant differences in school readiness skills between children with sickle cell anemia and normal subjects.

Erythroid progenitors circulating in peripheral blood and their response to erythropoietin (EPO), interleukin-3 (IL3), and phytohemagglutinin-stimulated, lymphocyte-conditioned medium (PHALCM) were assessed in sickle cell anemia (SCA) patients and controls. SCA patients have significantly higher numbers of circulating burst-forming unit-erythroid (BFU-E) compared with controls (mean +/- SEM, 940.27 +/- 129.11 per ml and 86.56 +/- 19.74 per ml, respectively; P less than 0.0001). At low doses of EPO, BFU-E-derived colonies were significantly increased in SCA patients compared with controls (each P less than 0.05). The EPO dose required to produce 50% of maximum colony numbers was 47 times greater in control subjects than in SCA patients. Moreover, in 11 of 17 patients with SCA, spontaneous BFU-E-derived colonies were formed without added erythropoietin. This phenomenon was not observed in control subjects (P = 0.035). PHALCM developed from mononuclear cells of SCA patients had significantly greater stimulatory effect than did that derived from controls regardless of the source of target cells (each P less than 0.05). A two-step study of IL3 sensitivity of erythroid progenitors was conducted. First, in a liquid culture system, circulating erythroid progenitors of SCA patients and controls were incubated in the presence of varying doses of IL3. During a second step, CFU-E-like colonies were observed in methylcellulose cultures of these cells. The mean numbers of colony-forming unit-erythroid (CFU-E)-like colonies was significantly higher in SCA patients compared with control subjects at low doses of IL3 (each P less than 0.02). The increased response of erythroid progenitors to IL3 and the increased production of hemopoietic growth factors (IL3 or non-IL3) contribute to the hemopoietic response in SCA patients. These mechanisms and increased sensitivity of the BFU-E to EPO may explain lower than expected EPO levels in SCA patients.

DNA-synthesis state of circulating burst forming unit-erythroid (BFU-E) was evaluated in patients with sickle cell anemia and correlated with percent of fetal hemoglobin synthesized in the BFU-E-derived cells. Percentage of S-phase BFU-E inversely correlated with percent fetal hemoglobin synthesized in the BFU-E-derived cells (simple linear correlation coefficient, r = -0.8, P = 0.0302; polynomial regression, R = -0.99, P = 0.0002). This observation is of relevance to our understanding of the relationship between the developmental stage of the erythroid progenitors and expression of globin genes.

BFU-E COLONY GROWTH IN RESPONSE TO HYDROXYUREA: CORRELATION BETWEEN IN VITRO AND IN VIVO FETAL HEMOGLOBIN INDUCTION 158

Objective: To look at the role of a standardized screening test for assessing depression in children and adolescents with and without sickle-cell disease and to compare findings with this instrument with clinical evaluations by child psychiatrists. In addition, to suggest the prevalence of clinical depression in children and adolescents with sickle-cell disease. Design: Nonrandomized, sequential sample of subjects, convenience control sample, prevalence study using the Children's Depression Rating Scale—Revised and an interview assessment by a child psychiatrist based on criteria from the Diagnostic and Statistical Manual of Mental Disorders, Revised Third Edition . Setting: Comprehensive Sickle Cell Center outpatient clinic and ambulatory Resident Practice Clinic at the University of South Alabama Children's Medical Center, Mobile. Study Participants: Patients aged 6 through 18 years with homozygous sickle-cell disease (hemoglobin SS) served as study subjects. Subjects of the same age and race who did not have sickle-cell disease served as controls. Measurements and Results: The mean Children's Depression Rating Scale–Revised total scores were 27.1 and 22.1 in study and control groups, respectively ( P =.0073). Eleven (29%) of 38 children in the study group had scores indicating a high risk for clinical depression. Four (12%) of 34 children in the control group were in this category. The four items accounting for most of this significant difference were excessive fatigue, physical complaints, selfesteem, and morbid ideation. This contrasted with the clinical evaluation by a child psychiatrist in a clinical interview in which 13% of study subjects and 15% of control subjects had evidence of clinical depression ( P =.85). Conclusion: Excessive fatigue and physical complaint factors contributed to a high false-positive rate when the Children's Depression Rating Scale–Revised was used to screen for depression among patients with sickle-cell disease. Based on the clinical interview by a child psychiatrist, the actual prevalence of clinical depression was not increased in children with sickle-cell disease compared with those without this chronic illness. (Arch Pediatr Adolesc Med. 1994;148:457-460)

Suppose one is trying to model the relationship between the dependent variable, Y, and the p independent variables, X1, X2, . . ., Xp, using the method of least squares in multiple regression. Let one of the independent variables, say X1 (without loss of generality), and Y be the measurement on the same characteristic under two different conditions. For example, one could think of X1 as volume of the left hemisphere of the brain and Y as volume of the right hemisphere of the brain, or X1 as height of the experimental group of children at the initial stage and Y as height of the same group of children at a later stage, or X1 as the weight of a fish-catch on shipboard and Y as the weight of the same fish-catch in a laboratory. Note that, in the first example a dependent variable could have been y = Y + X1 = total volume of the brain, while in the last two examples, a dependent variable could have been y = Y = growth in height of children or error in fish-catch weight measurement on shipboard, respectively. Notice that, in each case discussed, y incorporates X1 in it. In this note, a general case of y = Y + dXI is considered, where d is any constant. Now suppose the two fitted models based on n observations (to predict Yi and yi) are respectively

Both one-sided and two-sided alternative hypotheses are considered. For each case, an overall test procedure is initially discussed and then followed by a multiple comparison procedure. The critical values required for the multiple comparison procedures..

The two group t test is generalized here to produce a hypothesis testing procedure on the overlapping coefficient of two normally distributed populations with common variance, assuming that the researcher knows the direction of the population means. The confi¬dence intervals are constructed on the overlapping coefficient. An illustrative example is given using the proposed procedures

(1985). C237. On the coefficient of determination. Journal of Statistical Computation and Simulation: Vol. 22, No. 2, pp. 181-184.

Two simple approximation formulas for approximating the areas under the t distribution are presented here which are easily programmable on pocket calculators. Their accuracy should be sufficient for most of the practical applications.

Reciprocal control of platelet function in the circulation has been proposed for the platelet-produced platelet proaggregatory prostanoid thromboxane A2 (TxA2) and the vascular endothelium-produced antiaggregatory prostanoid prostacyclin (PGI2). Forty drug-free healthy subjects were given a single dose of ibuprofen (0, 8, 10, 12, or 14 mg/kg) in a randomized, double-blind study. Blood samples were drawn 0, 2, 4, and 6 hours and 7 days after dosing for determination in serum (from untreated or in vitro indomethacin-treated portions of the blood) of TxA2 and PGI2 by radioimmunoassay of their stable metabolites (TxB2 and 6-keto-PGF1 alpha). Maximal platelet release of TxA2 (untreated serum) was lower in all drug groups 2, 4, and 6 hours after dosing. There was no significant decrease in PGI2 release. All doses of ibuprofen (except 0 mg/kg) induced essentially identical plasma levels at the times of measurement (postpeak decline), and effects could not be distinguished by dose for 8, 10, 12, or 14 mg/kg at these times. It is concluded that ibuprofen induces antiplatelet effects for at least 6 hours while preserving normal antiplatelet mechanisms.