Proficiency in the Detection and Titration of Blood Group Antibodies: Results of an Interlaboratory Trial

Twelve numbered samples of serum were distributed to 82 Canadian blood transfusion laboratories. The presence or absence of blood group antibodies in these samples was correctly reported by less than 50% of participants; inability to demonstrate the presence of weak anti-D or anti-c was responsible for most of the failures. On the other hand, about 85% of participants reported titres of anti-D and anti-Fy^a within an acceptable range. Two proposals are made that should help laboratories achieve a high and uniform level of proficiency in antibody detection and titration.     

Cell Response to Surface Stimulation

The injection of anti-D into Rh-negative subjects who have Rh-positive red cells in the circulation results in the inhibition of immunization against the D-antigen¹. On the other hand, subjects who have had a primary anti-D response to Rh-positive red cells frequently give a good secondary response to small doses of red cells despite the presence of anti-D in their plasma. The difference in action between the passively-administered and the actively-produced anti-D might lie in the fact that the injected IgG anti-D is derived from a pool of donors and therefore contains a number of IgG antigens which are foreign to the recipient, compared with the autologous nature of the anti-D present after a primary response.     

Lymphocyte function in patients with anti-D(Rh0) antibodies

The behaviour of humoral and cellular immunity as well as that of cells bearing Fc receptors was investigated in persons with anti-D antibodies. Peripheral lymphocytes could be identified in 6 of 26 examined test persons particularly sensitized against the stroma of Rh-positive erythrocytes. There was no relation to the anti-D titre. The parameters of humoral immunity showed no correlation to the anti-D titre. In 37 per cent of the persons with anti-D antibodies, which were produced "naturally" or artificially, an increased content of IgE could be proved in the serum. In each case, anti-D formation apparently leads to an increase of cells bearing Fc receptors, which can be recognized by the increased number of EA rosettes. A relation to the anti-D titre did not exist. None of the immunological test methods used is suitable for predicting the success of an artificial immunization for the purpose of gaining high titre anti-D antibodies.     

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.     

A design improvement in continuous blood sampling and analysis for glucose in rest and exercise.

In the past 15 yr attempts to sample blood continuously for glucose analysis with the AutoAnalyzer (Technicon Corp.) have been reasonably successful in resting subjects. However, they have sometimes required heparinization of the subjects so as to avoid clotting in the tubes of the system. To avoid the hazards of heparinization during exercise, a method is described for sampling and analyzing blood glucose continuously with the AutoAnalyzer, using a specially designed male Luer adaptor which fits into disposable Teflon catheters.     

High dose intravenous immunoglobulin repertoire versus anti-D. Disproportions in quantity and quality.

Here we consider certain therapeutic effects that intravenous administration of pooled high dose immunoglobulin and anti-D IgG share. Despite million-fold difference in doses such an effect occurs at least in idiopathic thrombocytopenic purpura (ITP). We postulate that spontaneous bleeding events may remit even when platelet numbers show refractoriness. We also mention the possible sparing of anti-D antibody-coated red blood cell (RBC) destruction and, finally, an acceleration of fibrotic involution. Fc receptors (FcRs) play a central role; beyond the well-established interactions with the immunoglobulin Fc fragment, FcRs are supposed to display special cognitive properties that enable them to pick out the therapeutic molecules from the recipient's IgG pool. Such subtle selection suggests some disarray in the host. On the other hand it may explain why the often-encouraging outcome of IVIG therapy remains unpredictable.     

An error in Rh testing in pregnancy.

Anti-D (anti-Rho) in the blood of two Rh-negative pregnant women was believed to be due to active immunization. In the first case, however, antibodies were no longer detectable 2 weeks later. In the second case they disappeared by the end of 31 weeks. It was discovered that both women had been given immune globulin (human) because of exposure to rubella. The globulin given to the first woman probably contained about 0.1 mug of anti-D per ml; that given to the second probably contained about 0.6 mug of anti-D per ml. Both babies were O Rh-positive. Both women were given Rh immune globulin after delivery. Both have completed a further pregnancy and no anti-D has been found on many tests. In tests carried out in 1971 all samples of immune globulin (human) examined contained anti-D, but usually in inconsequential trace amounts.     

Skeletal maturation of the human fetus assessed radiographically on the basis of ossification sequences in the hand and foot

In studies of postnatal human development the skeletal maturation of the hand has been found to be a better indicator of general physical maturation than attained body height. For assessment of prenatal human development the Crown-rump length (CRL) has so far been the most commonly used measure. The object of the present study is to examine the possibility of also using the skeletal maturation of the hand as a maturity indicator in fetal development. The study is based upon a radiographic and histochemical investigation of 169 human fetuses. On the basis of counting silver-impregnated diaphyses on radiographs of the hand and foot a maturity indicator (CNO = Composite Number of Ossified bones in hand and foot) was established. Owing to the marked regularity of the recorded ossification pattern, the CNO parameter can be used for evaluating fetal maturation during the early half of the prenatal period. To supplement the assessment of skeletal maturation during the later stages of development, a classification based on the shape of some bones was included in the study. In many cases fetuses of the same size (CRL) exhibited different stages of skeletal maturation (CNO). In accordance with findings from assessment of postnatal development, a more accurate evaluation of fetal development is obtained by combining the size parameter CRL with an assessment of fetal skeletal maturation, CNO.     

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.     

Enhanced efficacy of anti-D immunoglobulin for treating ITP is not explained by higher immunoglobulin polymer content.

Several reports have suggested that low dose anti-D immunoglobulin is superior to high dose immunoglobulin for treatment of idiopathic thrombocytopenia purpura (ITP). However, some findings suggest that it is not the anti-D activity per se that is responsible for efficacy for treatment of ITP with anti-D immunoglobulin. Amongst alternative explanations for the mechanism of action is a relatively higher immunoglobulin polymer content of anti-D compared to other immunoglobulin products, which is more efficient in causing reticuloendothelial Fc receptor blockade. In order to investigate this we have evaluated the polymeric IgG content of anti-D and other immunoglobulin products. Different products showed considerable variation in immunoglobulin polymer content. There was no clear correlation between aggregate or dimer content and product type and anti-D as a class of product did not contain higher amounts of either dimer or aggregate compared to other products. Some manufacturers' products increased in polymer content on storage, but others did not show this effect. Therefore, higher immunoglobulin dimer and/or aggregate content cannot explain the increased efficacy of anti-D immunoglobulin for treatment of ITP. The role of polymeric Ig in efficacy for treatment of ITP is unclear.     

Survival of human monoclonal anti-Rho (D) antibodies in the rhesus monkey.

In vivo half-life of a 125I-labeled human anti-D monoclonal antibody (mAb) and that of 131I-labeled Rho-GAM was assessed in a rhesus monkey injected simultaneously with both reagents. The half-life of the mAb was 7.9 days, compared to 17 days of Rho-GAM. Survival of the second dose of mAb, given 34 days after the first injection, was identical to that of the first dose, thus showing that the human mAb did not elicit an immune response. The in vitro produced human mAbs appear to be an alternative, unlimited source of anti-D antibodies for possible use in prevention of feto-maternal Rh immunization.     

The second to fourth digit ratio and variation in the androgen receptor gene

The second to fourth digit ratio (2D:4D) is sexually dimorphic, with lower mean values in males compared to females. It has been suggested that the sex difference in 2D:4D is determined prenatally, 2D:4D is negatively related to prenatal testosterone and positively to prenatal oestrogen, and that 2D:4D is a marker for levels of sex steroids during brain organisation. There is growing evidence that many sex-dependent behaviours are correlated with 2D:4D. However, there is no direct evidence for an effect of prenatal sex steroids on the digit ratio. The response to prenatal testosterone is dependent on the amount produced and the foetal sensitivity to the hormone. Variation in the X-linked androgen receptor gene (AR) determines sensitivity to testosterone. Alleles of AR with low numbers of CAG triplets respond to testosterone with high transactivational activity, while high numbers of CAG's are associated with increased insensitivity to testosterone. We show in a sample of 50 men (49 Caucasian subjects, 1 Caucasian/Chinese subject) that 2D:4D is a phenotypic correlate of AR structure. Right-hand 2D:4D was positively correlated with CAG number and individuals with low 2D:4D in their right hand compared to left hand had AR alleles with low CAG numbers. We discuss the implications of our findings for our understanding of the aetiology of 2D:4D, its relationships with sex-dependent behaviours, and the evolutionary implications of variation in 2D:4D and AR.     

Is an expression system for producing therapeutic antibodies with immunosuppressive properties found at last? Comment to letter by Dr. Quagliaroli

The prophylaxis of the hemolytic disease of the newborn — a mandatory procedure in obstetrics — requires significant amounts of plasma-derived polyclonal anti-D immunoglobulin. Despite numerous attempts, the proper technology for mass production of effective monoclonal anti-D is still not available. LFB Biotechnologies is currently performing clinical trials with recombinant anti-D antibody that has low fucose content and is expressed in the cells of rat myeloma YB2/0. It was shown that this drug is well tolerated, accelerates fast clearance of D+ red blood cells, and can inhibit anti-D immune response in Rhesus-negative volunteers.     

Characterisation of a chromatographically produced anti-D immunoglobulin product

A chromatographic fractionation method has been developed for the production of a liquid-stable anti-D immunoglobulin product for intravenous and intramuscular use. An immunoglobulin fraction, highly enriched with anti-D immunoglobulins, was isolated by cation-exchange column chromatography and further polished, first by anion-exchange chromatography, followed by an aluminium hydroxide gel treatment. The process includes two specific steps for virus inactivation and removal, namely S/D treatment and nanofiltration. The overall anti-D process yield is about 56%. The final product is stabilised with human albumin and glycine and placed in ready-to-use syringes. The anti-D product was shown to be stable in liquid state for at least 30 months at 4°C.     

Prevention of Rh-haemolytic Disease: Results of the Liverpool “Low-risk” Clinical Trial

A clinical trial is reported in which Rh-negative primiparae, just delivered of an Rh-positive ABO-compatible infant and in whom fetal cell counts after delivery suggested less than 0·2 ml of circulating fetal blood, were treated with about 200 μg of anti-D gammaglobulin. Three (0·36%) out of 844 women thus treated developed anti-D in the subsequent six months; this is 10% of the incidence in untreated controls. Three (1·8%) out of 171 treated mothers had anti-D at the end of the second Rh-positive pregnancy, and this is 18% of the incidence in controls.Possible reasons for the occasional failure of the treatment are discussed and the results of this trial are compared with those of a previous trial in which 1,000 μg or more of anti-D was given to a different group of mothers. The combined results of the two trials lead to the conclusion that the passive administration of anti-D gammaglobulin after delivery affords in this population of Rh-negative women a 95% protection rate in the postdelivery period and an 89% protection rate by the end of the subsequent pregnancy.     

Indications of neutralising anti-idiotypic antibodies and selective proteolytic fragmentation in polyclonal anti-D IgG preparations

Proteolytic fragmentation is the only suggested cause of potency losses during storage of liquid human polyclonal anti-D Ig. Besides the effect of fragmentation, we have investigated the potential contribution of neutralising anti-idiotypic antibodies (anti-Ids). Potency changes during storage and/or upon pH reduction in anti-D IgG batches with or without addition of plasminogen and urokinase were quantitatively analysed by the autoanalyser (AA) method or by a special procedure of flow cytometry (FC). Moreover, simultaneous changes of the molecular size distribution pattern have been determined by size exclusion chromatography. In contrast to the AA procedure, the particular FC methodology was found to be almost insensitive to proteolysis comprising up to 30% of total IgG. Data interpretation was based on the assumption that both assays cannot detect Ids with neutralised paratopes. In the absence of detectable neutralisation (functional absence of anti-Ids), it could be demonstrated that the anti-D IgG subpopulation is more sensitive to fragmentation by endogenous protease as compared to the unrelated bulk. However, both methods detected batch- and assay-dependently variable potency losses during storage. Moreover, the increase of potency induced by pH reduction correlated with the increase of monomeric IgG, essentially on the expense of dimers. This finding was interpreted to indirectly indicate the neutralising action of anti-Ids known to be the major driving force of dimer formation in polyclonal IgG. A more or less pronounced pH-dependent potency increase was also detectable in three arbitrarily selected batches of two other manufacturers. The data allows to assume that anti-Id-mediated neutralisation can significantly contribute to losses of anti-D potency. In addition, it turned out that anti-D plasma itself can be the source of anti-Ids.     

Evidence of controlled corpus allatum activity in the adult Colorado potato beetle

The in vivo control of corpus allatum (CA) activity in females of Leptinotarsa decemlineata was investigated. Evidence was obtained that CA activity is adjusted by negative feedback when juvenile hormone (JH) titres are changed experimentally. Conclusions are based on determination of the rate of in vitro JH synthesis by the CA, on changes in CA volume, and on JH titres in the haemolymph. These assay methods are used alternatively in some of the experiments.After unilateral allatectomy, the remaining CA had doubled its activity 7 days later. On the other hand, the activity of CA in young adults was suppressed after the JH titre was elevated by the implantations of 2 CA taken from active females. Similarly, in beetles treated topically with exogenous JH the CA atrophied and showed a much reduced activity after 5 days. Denervation of CA in 0-day-old long-day and 7-day-old short-day females did not change CA activity when measured 1 day later.     

Trends in the BVDV serological response in the Upper Midwest.

Bovine viral diarrhoea continues to be an important disease affecting both beef and dairy animals of all ages. One of the quickest means of measuring bovine viral diarrhoea virus (BVDV) exposure and infection in the herd is a serum neutralization (SN) assay. Type 1 and type 2 BVDV SN results from the Animal Disease Research and Diagnostic Laboratory at South Dakota State University were collected over a seven-year period (1995-2001) to determine any trends. These results indicated that in 1996, 31% of the animals had titres > or =512 while in 2001, 74% of the titres were > or = 512. There has been a progressive increase over the seven-year period in the number of cases with titres > or = 512 with the exception of 1999 when there was a slight decrease. When analyzing the titres greater than 512, a further increase was seen. In 1995, 80% of the titres were < or = 1024 and 20% were 2048 and no titres were >2048. In 2001, 47% of the antibody levels were < or = 1024 while 53% were < or =2048 and 30% were >4096. The most dramatic increase in titres occurred in 1997 and the percentage of animals with titres from 512-8192 has remained fairly constant for the last five years (1997-2001). This increase is in part due to more extensive use of vaccination but probably also reflects a rise in field infections. In the future, standardizing existing BVDV SN serology along with developing new BVDV serology methods is necessary to provide continuity for any full-scale eradication programme.     

Prevention of Rh haemolytic disease.

Between 1970 and 1976 in the Yorkshire region the incidence of Rh antibodies in Rh-negative pregnant women fell by 70%. This decrease occurred in both old (long-standing) and new (first-affected) cases, which emphasised that the reduction in numbers was as much due to fewer pregnancies among Rh-negative mothers as to administration of anti-D immunoglobulin. Nevertheless, the incidence has begun to level out. The continued incidence of first-affected cases is caused by three main factors: failure of administration of anti-D immunoglobulin after normal deliveries and abortions; a steady incidence of antibodies in primigravidae; and cases in which administration of anti-D immunoglobulin had failed to protect. Administering anti-D antenatally might reduce the incidence of new cases among primigravidae who are sensitised before anti-D is normally given. Even without routine antenatal administration of anti-D, the incidence of severely affected Rh babies in the Yorkshire region could be reduced to one or two isolated cases a year in a population of three to four million by administering anti-D after all Rh-negative deliveries and after every abortion.     

A Stable Lyophilized Reagent for Use in a Potential Reference Assay for Quantitation of Anti-D in Immunoglobulin Products

Quality control of anti-D immunoglobulins intended for in vivo clinical use requires in vitro assay of potency. A lyophilized biotinylated monoclonal anti-D (biotinylated Brad-5; 99/698) has been evaluated for its suitability to serve as a working reagent in a competitive enzyme-linked immunoassay (EIA) for anti-D quantitation. The reagent demonstrated acceptable stability in accelerated degradation tests and following reconstitution. Twelve international laboratories obtained comparable potencies for each of nine anti-D samples using 99/698 in a standardized assay procedure using erythrocytes fixed to microtitre plates. We also describe the use of trehalose for stabilization of dried erythrocyte-coated microtitre plates.