Maternal anti-D concentrations and outcome in rhesus haemolytic disease of the newborn.

The relation between maternal anti-D concentrations, measured against the British working standard, and outcome of rhesus-sensitised pregnancies was studied. There is a clear relation between increasing anti-D concentrations and the chance of a severely affected baby. Of those pregnancies (78) where serial anti-D concentrations remained below 4 IU/ml, no baby had a cord haemoglobin below 10 g/dl and three had exchange transfusions. In contrast, of those mothers (106) with anti-D concentrations above 4 IU/ml, 23 had babies with a cord haemoglobin below 10 g/dl and 79 babies had exchange transfusions. It is suggested that those pregnancies where anti-D concentrations remain below 4 IU/ml represent a relatively safe group in which amniocentesis may be avoided.     

Turnover rate of anti-D IgG injected during pregnancy.

Anti-D IgG was injected into 15 Rh-negative women in the 28th week of gestation and into three non-pregnant women. The uptake of anti-D after the intramuscular injections was calculated by measuring the concentration of antibody in the plasma with an autoanalyser. The biological half life and the catabolic rate of anti-D IgG were calculated according to a compartmental model. The recovery in vivo of anti-D was an average 24% in the non-pregnant women and 21% in the pregnant women. The half life of anti-D were 24 and 21 days, respectively. With a dose of 125 micrograms the plasma anti-D concentration was less than 1 ng/ml at about 10 weeks after the injection. With double the dose the concentration at delivery was at least 1 ng/ml. Although 250 micrograms of anti-D IgG seems to be effective when given in the 28th weeks of gestation, the great individual variations in uptake and recovery rates will lead to occasional cases of Rh-immunisation during pregnancy despite all routine regimens.     

Haemolytic Disease of the Newborn Caused by Anti-Lan Antibody

The first recorded example of anti-Lan associated with haemolytic disease of the newborn is reported. This emphasizes the importance of screening for atypical antibodies early in pregnancy, even though prophylactic use of anti-D immunoglobulin will eventually reduce the incidence of haemolytic disease due to anti-D antibody.     

Vitamin D metabolism in human pregnancy. Concentrations of free and sulphated 25-hydroxyvitamin D3 in maternal and fetal plasma at term

The concentrations of free and sulphated 25-hydroxyvitamin D3 in 20 paired maternal-cord plasma samples obtained at delivery have been determined. The compounds were isolated by liquid-solid extraction at elevated temperature, and the sulphate was purified by anion exchange chromatography prior to hydrolysis and analysis by high-performance liquid chromatography. The study shows that unconjugated 25-hydroxyvitamin D3 is predominant in maternal plasma (mean 20 ng/ml) whereas the sulphate is the major form of vitamin D3 in fetal circulation (mean 21 ng/ml plasma). The total concentration of the two compounds in cord plasma (mean 35 ng/ml) was significantly higher than that in maternal plasma (mean 30 ng/ml). Positive correlations were obtained between maternal and cord plasma levels of free 25-hydroxyvitamin D3, between maternal and cord plasma levels of sulphated 25-hydroxyvitamin D3 and between plasma levels of the maternal free compound and the fetal sulphate. There was also a relationship between the levels of free and sulphated 25-hydroxyvitamin D3 in cord plasma. The results suggest that sulphation may be a physiologically important reaction for deactivating and/or trapping secosteroids in the fetus.     

Maternal insulin and placental 3-O-methyl glucose transport

The effects of insulin in the maternal circulation on the placental clearance of 3-O-methyl glucose were investigated in 7 animals in the presence of a constant maternal glucose concentration. While maternal insulin concentration changed from 12 +/- 4 to 175 +/- 33 mu Units/ml, the placental clearance remained constant at 16.2 +/- 1.2 (control) and 15 +/- 1.3 ml/min per kg fetus under the influence of the insulin. To test the secondary hypothesis that in the control condition the hexose transport system was saturated, we performed a further series of experiments in 6 fasted animals. In these animals the control maternal plasma insulin concentration was 2 +/- 0.3 mu Units/ml and after the infusion of insulin it increased to 562 +/- 26 mu Units/ml. Under conditions of constant maternal and fetal plasma glucose concentrations, this massive elevation of plasma insulin did not change the placental clearance of 3MeG which was 15.2 +/- 1.6 in the control condition and 13.3 +/- ml/min per kg under the influence of high insulin. We conclude that maternal insulin ranging from 2 mu Units/ml to supraphysiologic doses does not effect a physiologically significant change in placental hexose transfer. Placental glucose transfer can probably therefore, be changed only be changing the concentration of glucose in the maternal and fetal plasma.     

In vitro development of rat embryos undergoing organogenesis in heparin-plasma.

This study examines the use of heparin-plasma as a culture medium for mammalian postimplantation whole-embryo culture. The growth and differentiation of head-fold rat embryo explants over 48 hours in a standard serum medium was compared with development of same stage explants over 48 hours in a plasma medium prepared using sodium heparin. Heparin disrupted the morphological differentiation of embryos, in a concentration-dependent manner, from 25 micrograms sodium heparin/ml media (i.e., 5 IU/ml media), with overall embryo growth being adversely affected from a concentration of 200 micrograms sodium heparin/ml media (i.e., 40 IU/ml media). Defects of cranial neural tube development were the first apparent structural anomalies resulting from culture in heparin media. Forebrain development was grossly abnormal and associated with failure of eye development. As the heparin concentration in media increased, the cephalic neural folds remained widely open and the edges became increasingly everted, although differentiation of the heart, otic primordia, and pharyngeal arch persisted. Similar concentration-dependent dysmorphogenic effects were seen when embryos were cultured in the standard serum media with added heparin. A minimum heparin concentration of 100 micrograms sodium heparin/ml media (i.e., 20 IU/ml media) was required to effectively inhibit coagulation of the plasma medium over the 48 hour culture period. Although embryonic growth was not adversely affected at this heparin concentration, morphological differentiation was severely disrupted. Therefore, heparin is not a suitable anticoagulant for the preparation of plasma for use in postimplantation whole-embryo culture.     

A highly sensitive fluorometric assay for determination of human coagulation factor XIII in plasma

Based on the iso-peptidase activity of human plasma FXIII, a novel fluorometric assay that determines FXIII concentrations in human plasma below 0.05 IU/ml is introduced. We considered a peptide sequence derived from alpha(2)-antiplasmin (n =12) to yield high sensitivity. Peptide Abz-NE(Cad-Dnp)EQVSPLTLLK exhibits a K(m) value of 19.8+/-2.8 microM and is used in a concentration of 50 microM. The assay design is suitable for measurements in cuvettes (1 ml volume) as well as for the microtiter plate (MTP) format (0.2 ml volume). It provides linear dose-response curves over a wide range of FXIII concentrations (0.05-8.8 IU/ml). The assay was validated with respect to precision, detection and quantitation limits, accuracy/specificity, linearity, and range. A comparison of the fluorometric assay with the photometric assay for FXIII determinations in plasma pools as well as single donor plasma revealed suitability of the fluorometric assay for FXIII determination in plasma of healthy individuals. FXIII concentrations in plasma samples of patients with severe FXIII deficiency are discussed in the context of FXIII antigen levels. These assays correlate well in the critical range below 0.1 IU/ml, whereas the photometric assay may overestimate residual FXIII activity in severe FXIII-deficient patients.     

Induction of sister-chromatid exchanges in cultured human cells by an organophosphorous insecticide: malathion.

Because malathion is a widely used organophosphorous insecticide, the effects of non-toxic concentrations (2.5--40 micrograms/ml) on sister-chromatid exchange (SCE) frequencies were determined. Human fetal fibroblasts were exposed once or twice to malathion, with 20 h between exposures. A single exposure to a concentration of 40 micrograms/ml resulted in a highly significant increase in the number of SCEs. After a double exposure, a concentration of 20 micrograms/ml induced an even greater increase in SCE frequencies. Comparison of Sce frequencies after single and double exposures indicated a cumulative effect; the number of exchanges at concentrations of 5 micrograms/ml or higher was significantly greater after the double exposure. An analysis of SCEs by chromosome group showed that exchanges were distributed approximately according to chromosome length.     

Deaths from rhesus haemolytic disease in England and Wales in 1977: accuracy of records and assessment of anti-D prophylaxis.

All the death certificates for deaths in 1977 where haemolytic disease of the newborn (HDN) was the principal, an antecedent, or a contributory cause were obtained from the Office of Population Censuses and Surveys (OPCS). The hospital notes of all 54 of the live-born cases and all of the 101 stillbirths were also obtained. The cause of the death indicated by the notes was compared with the cause and coding on the death certificate. In about a quarter of the cases death was not due to haemolytic disease of any type. The commonest errors arose because the International Classification of Diseases (8th edition) stipulates that hydrops without mention of cause should be coded as HDN and because stillbirths to rhesus-negative mothers tend to be attributed to rhesus HDN automatically. Though deaths from HDN may be overestimated in this way, they are also underestimated because rhesus disease, although mentioned on the certificate, is not selected as the underlying cause, which it may be. These cases were found through multiple coding of all the contributory causes of death, which OPCS performs on a 25% sample of all death certificates for research purposes. These two sources of inaccuracy tend to cancel each other out, but statistics from death certificates give a misleading picture of the efficacy of anti-D prophylaxis because anti-D can never prevent cases which are not in fact due to rhesus HDN. Most of the mothers studied had become immunised before anti-D became available, but in those who could have been treated 75% had not received prophylaxis. As this was a sample of deaths, however, it would not be accurate to extrapolate this high figure to the population at risk. Nevertheless, the organisation of prophylaxis is clearly deficient and should be remedied before providing antenatal anti-D to supplement postnatal treatment.     

Protein, cholesterol, acid phosphatase and aspartate aminotransaminase in the seminal plasma of turkeys (Meleagris gallopavo) producing normal white or abnormal yellow semen

Turkeys which produce yellow semen have abnormal ductuli efferentes' epithelial morphology, with blebbing of cytoplasmic material into the ductal lumen. This could possibly increase the activity or concentration of seminal plasma components. In the present study, seminal plasma from 270 Large White breeder turkeys was evaluated for protein and cholesterol concentrations and the activities of acid phosphatase and asparate aminotransaminase. In a separate experiment, protein concentrations of turkey seminal plasma were estimated by biuret or Bradford methods. Bradford estimates were 46.6% less than those obtained with the biuret assay, using bovine serum albumin as the standard. Estimates of seminal plasma protein concentration in the main study were obtained using the Bradford method, and should be adjusted accordingly when compared with other studies using the biuret technique. Abnormal yellow seminal plasma, compared to normal white seminal plasma, had elevated levels of total protein and cholesterol and increased activities of acid phosphatase and aspartate aminotransaminase. Overall means were: 14.3 mg/ml, 38.9 mg/dl, 232.6 IU/ml, 81.0 IU/ml, respectively. Correlation coefficients for cholesterol concentration, acid phosphatase and aminotransaminase activity with protein concentration were +0.65, 0.70 and 0.50 (P less than 0.0001), respectively. Specific activities of both enzymes showed a significant reduction as seminal plasma protein increased, indicating a disproportionate increase in proteins other than these enzymes in yellow seminal plasma.     

Variants of human tissue-type plasminogen activator. Fibrin binding, fibrinolytic, and fibrinogenolytic characterization of genetic variants lacking the fibronectin finger-like and/or the epidermal growth factor domains

Tissue-type plasminogen activator (t-pa) is a serine protease comprising four different putative structural domains with homologies to fibronectin finger-like structures (finger), epidermal growth factor, kringle structures, and the active site of serine proteases. Only the finger and epidermal growth factor domain are each entirely encoded by unique single exons. We assessed the functional contribution of these two structural domains by making mutants precisely deleted for one or both of the relevant exons. The three mutant genes were expressed in monkey cells, and the variant proteins, purified from the culture medium, were characterized for their fibrinolytic activity, fibrinogenolytic potential, and affinity for fibrin. No significant difference in any biochemical property was observed among the variants. All three variants retained a catalytic dependence on cyanogen bromide fragments of fibrinogen which could not be distinguished from the wild-type enzyme. The activities of the variants were also very similar to that of wild-type t-pa, showing no detectable fibrinogenolytic potential in human plasma at activator concentrations of 500 IU/ml, or when their fibrinolytic activity was tested in human plasma using the 125I-labeled fibrin clot lysis assay at activator concentrations of 150 IU/ml or greater. However, the variants were markedly defective in fibrinolysis at low activator concentrations such that essentially no fibrinolysis was detected at 15 IU/ml. Measurement of fibrin binding showed that the variants lacked the high fibrin binding characteristic of wild-type t-pa. These results demonstrate that the fibrin specificity and fibrin-dependent activity of t-pa are independent of the protein's high affinity for fibrin. The implication of these results is that the t-pa variants would be ineffective activators at a physiological concentration of approximately 2 IU/ml but would be expected to behave similarly to wild-type t-pa at the steady-state plasma concentrations of 0.75-1.25 micrograms/ml (approximately 500 IU/ml) currently required for coronary reperfusion in patients receiving t-pa for acute myocardial infarction (Garabedian, H.D., Gold, H.K., Leinbach, R.C., Yasuda, T., Johns, J.A., and Collen, D. (1986) Am. J. Cardiol. 58, 673-679).     

Dry Blood Spots a Reliable Method for Measurement of Hepatitis B Viral Load in Resource-Limited Settings

MethodsThe performance of DBS in HBV quantification was investigated using a modified commercial test (Abbott RealTime HBV assay). Paired DBS and plasma samples were collected from an HBV positive cohort in Addis Ababa, Ethiopia. DBS were stored at ambient temperature for 4–39 days before shipment to the laboratory.ResultsTwenty-six paired samples were selected covering the total range of quantification, from 2.14 log IU/ml to >7 log IU/ml. HBV was detected in 21 of 21 (100%) DBS from patients with a corresponding plasma viral load above 2.70 log IU/ml. The mean difference between plasma and DBS was 0.59 log IU/ml, and the correlation was strong (R2 = 0.92). In stability studies there was no significant change in DBS viral load after storage at room temperature for up to 12 weeks.ConclusionsThis study suggests that DBS can be a feasible and reliable alternative to plasma for quantification of HBV in resource-limited settings. DBS can expand access to antiviral treatment for patients in low- and middle-income countries.     

Interactions between model triacylglycerol-rich lipoproteins and high-density lipoproteins in rat, rabbit and man

There are inverse relationships between HDL cholesterol and plasma triacylglycerol concentrations in normal and in hypertriglyceridemic individuals. To investigate the interactions between triacylglycerol-rich lipid particles and HDL, a lipid emulsion model of the triacylglycerol-rich lipoproteins was prepared. When emulsion particles were incubated with rat high-density lipoproteins (HDL) in the presence of lipid transfer activity (d greater than 1.21 g/ml fractions) from rabbit or human plasma there was a rapid bi-directional exchange of cholesteryl oleate (CO) and phospholipid (PL) labels between lighter and heavier fractions of HDL and emulsion particles. The transfers of CO and PL labels between both light and heavy fractions of HDL and the emulsion particles were increased with increasing amounts of emulsion added to the incubations. Incubation with the d greater than 1.21 g/ml fraction from rat plasma resulted in only a small exchange of CO whereas PL exchange was similar to rabbit and human plasma. Retinyl palmitate label was not transferred from emulsion particles to the HDL fractions even in the presence of lipid transfer activity from rabbit or human plasma. The present study shows that the transfer protein-mediated exchanges of surface and core lipids between HDL and the triacylglycerol-rich lipoproteins are affected by the quantity of triacylglycerol-rich particles in the system. This mechanism may contribute to the inverse relationships between plasma triacylglycerol concentrations and HDL concentrations in normal and hypertriglyceridemic individuals.     

Standardization of U.S. Reference Rh0 (D) Immune Globulin by quantitative automated hemagglutination

An automated hemagglutination procedure was used to assess the relative potency of US Reference Rh0 (D) Immune Globulin, Lot 3, with respect to the International Reference Preparation, WHO Anti-D immunoglobulin, Lot 68/419. A value of 300 international units (IU) of anti-D per ampoule has been assigned to Lot 68/419. In 25 assays, the mean value for Lot 3 was 820 IU anti-D per milliliter when tested in parallel with Lot 68/419.     

International collaborative study to assess candidate reference preparations to control the level of anti-D in IVIG for use in Europe and the United States.

Regulatory requirements to control the level of anti-D in intravenous immunoglobulin (IVIG) products with European and United States (US) licences are to be introduced. A reference preparation of IVIG containing anti-D at 0.0475 IU/ml and having a nominal titre of 8 using the proposed direct haemagglutination reference method was deemed suitable to define the anti-D limit. This preparation, code 02/228, and a negative control IVIG preparation, code 02/226, were established by the World Health Organization as International Reference Reagents (IRRs). As stocks of the IRRs are limited, new larger fill stocks of positive and negative reference preparations, codes 04/132 and 04/140, respectively, were produced. The results from an international collaborative study involving 16 laboratories showed that preparations 04/132 and 04/140 are indistinguishable from the corresponding IRRs 02/228 and 02/226, respectively, using the proposed direct haemagglutination reference method. Stocks of 04/132 and 04/140 have been shared with the European Directorate for the Quality of Medicines (re-coded as 23613 and 23614, respectively) and with the Center for Biologics Evaluation and Research of the United States Food and Drug Administration (re-coded as CBER Lots 1B and 1N-b, respectively) for use as European and US Biological Reference Preparations, respectively.     

Identification and characterization of cyclo(His-Pro)-like immunoreactivity in amniotic fluid

Amniotic fluid (AF) from 25 term pregnancies was analyzed for cyclo(His-Pro)-like immunoreactivity (CHP-LI). CHP-LI was detected in all AF samples and was indistinguishable from synthetic CHP by immunoidentity, by gel chromatography on Sephadex G-25, and by high pressure liquid chromatography. The mean concentration of CHP-LI in AF was 13,622 +/- 1288 pg/ml (+/- SE) and concentrations were not altered by maternal labor. Plasma concentrations of CHP-LI were similar in 4 pregnant and 4 control subjects [2260 +/- 432 pg/ml vs. 2162 +/- 419 pg/ml (+/- SE), respectively]. We conclude that 1) CHP-LI is readily detected in AF from term pregnancies and is indistinguishable from synthetic CHP, and 2) concentrations of CHP-LI in human AF are significantly higher than concentrations of maternal plasma CHP-LI, suggesting CHP AF originates by mechanisms other than diffusion from maternal plasma.     

Secretion of unconjugated estrone during pregnancy and around parturition in goats

The secretion of unconjugated estrone and its production site in pregnant goats were investigated in vivo. The mean estrone concentration (n = 15) in the peripheral plasma increased gradually, being 83 pg/ml on Day 40 and 483 pg/ml on Day 140 after mating. The estrone concentration increased rapidly after Day 2 before partum, reaching a peak at parturition (2370 pg/ml), and falling to 171 pg/ml at Day 1 post partum. The concentrations of estrone from the umbilical vein and umbilical artery did not differ from that found in the maternal jugular vein, suggesting that the fetus does not take part in estrogen production. The estrone concentration from the uterine vein after the fetus was removed was higher than the concentrations found in the maternal jugular vein and umbilical artery. In the placental tissue, a high concentration of estrone (18157 pg per gram of wet tissue) was detected. These findings suggest that the main production site of unconjugated estrone is the placenta.     

Lowered plasma erythropoietin in hypoxic rats with kidney tubule lesions

The role of the kidney tubules in the renal formation of erythropoietin is incompletely understood. Therefore, the capability to produce erythropoietin in response to hypoxia was studied in rats with tubular lesions. Nephron damage was induced in two different ways. First, rats were treated with the nephrotoxic aminoglycoside gentamicin (67.5 mg/kg and day) for 14 days. The animals were then subjected to simulated altitude (6,800 m) for 6 h. The resulting plasma erythropoietin concentration was significantly lower (0.5 IU/ml) than in saline treated control rats exposed to hypoxia (1.0 IU/ml). Second, unilateral hydronephrosis was induced by ureteral ligation. The contralateral kidney was removed immediately before the animals were exposed to simulated altitude for 6 h. The plasma erythropoietin concentration in the ureter-ligated rats did not increase above the value (0.3 IU/ml) in hypoxia exposed anephric rats. These results indicate that the production of erythropoietin is reduced following tubular injury. Tubule cells may directly produce the hormone or interfere with the O2-sensing mechanisms controlling its synthesis. The latter hypothesis would seem to be supported by our failure to demonstrate in vitro erythropoietin production by the two established kidney tubule cell lines, LLC-PK1 and PK-15.     

Fetomaternal adrenomedullin levels in diabetic pregnancy.

We investigated whether maternal and fetoplacental adrenomedullin, a newly discovered hypotensive peptide involved in the insulin regulatory system, is modified in diabetic pregnancy. We studied its correlation with pregnancy complications associated with this disease. Thirty-six pregnant women with diabetes (13 with type I and 23 with gestational diabetes mellitus) and in 40 uncomplicated pregnancies were included. 10 out of 36 diabetic pregnancies were complicated by gestational hypertension. In each woman, adrenomedullin concentration in maternal and fetal plasma and in amniotic fluid was assessed by specific radioimmunoassay. We found that overall mean amniotic fluid adrenomedullin concentration was higher (p < 0.05) in diabetic (14.7 +/- 1.6 fmol/ml) than in uncomplicated pregnancies (10.8 +/- 0.9 fmol/ml), whereas no differences were present in maternal and fetal plasma adrenomedullin levels between diabetic and uncomplicated pregnant women. High levels of amniotic fluid adrenomedullin were found in both type I and gestational diabetes mellitus pregnancies (13.7 +/- 1.4 and 15.6 +/- 2.2 fmol/ml, respectively). Diabetic pregnancies complicated by gestational hypertension showed lower (p < 0.05) amniotic fluid adrenomedullin concentrations than normotensive diabetic patients. These findings suggest that placental adrenomedullin production is upregulated in diabetic pregnancy, and it may be important to prevent excessive vasoconstriction of placental vessels.     

Aluminium bone disease in patients receiving plasma exchange with contaminated albumin.

Aluminium balance studies were carried out on eight patients with various immunological disorders who were receiving plasma exchange with albumin solutions known to be contaminated with aluminium. Four patients with impaired renal function (creatinine clearance less than 50 ml/min) retained between 60% and 74% of the aluminium infused during a single plasma exchange. Transiliac bone biopsy specimens from three patients in this group had a high content of aluminium and showed histological evidence of current or previous bone disease related to aluminium. Two of these patients suffered intermittent bone pain. The main route of excretion of injected aluminium was in urine, only a small proportion of the total input being removed in the "plasma bag" during plasma exchange. The extent of aluminium retention and bone deposition was not reflected by the plasma aluminium concentration before or after plasma exchange. Treatment of five patients with intravenous desferrioxamine increased the plasma aluminium concentration and urinary output of aluminium in those with evidence of aluminium retention. These studies show that patients with poor renal function receiving treatment with albumin contaminated with aluminium retain the metal and deposit it in bone, where it may eventually cause aluminium bone disease. Plasma exchange should be used with caution in patients with renal impairment.