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Peripheral-blood mononuclear cells from an individual known to produce antibody to the rhesus D blood group were enriched for specific antibody-producing cells before transformation with Epstein-Barr virus. An anti-rhesus-D antibody-producing cell line (UCH D4) was obtained and cloned. Culture supernatants contained about 20 micrograms/ml of rhesus-D-specific IgG-class antibody. The antibody reactivity compared well with that of reagents used routinely for rhesus blood-grouping.

Plasma proteins glucosylated in vitro were injected repeatedly into 7 mice. A group of 4 was given multiple injections of non-glucosylated plasma proteins and another 10 were untreated. 5 of 7 mice given glucosylated proteins had glomerular-basement-membrane thickening on electron microscopical examination. This feature is characteristic of human and experimental diabetic renal disease. These results suggest that there may be a link between plasma-protein glucosylation and diabetic nephropathy.

THE irus macaque (Macaca irus) is polymorphic for a minor haemoglobin component which resembles human Hb-A2 in electrophoretic mobility and concentration but differs from it in having a common β-chain with the normal major component, Hb-Ami (ref. 1). Two major haemoglobin variants Hb-Pmi and Hb-Qmi are also frequent in this species and these also differ from Hb-Ami in their α-chains. Some animals carry three haemoglobins, for example Hb-Pmi (αPmi2βAmi2), Hb-Ami (αAmi2βAmi2) and the minor component (αXmi2βAmi2), and therefore have three types of α-chain (αAmi, αPmi and αXmi). Hb-Pmi and Hb-Ami are probably deteimined by allelic genes. It was therefore suggested1 that the minor component arose by duplication of the α-locus in a manner analogous to the proposed origin of Hb-A2 by a β-locus duplication2.

VARIANTS of human haemoglobin A with an electrophoretic mobility identical with Hb-S at pH 8.6, but not causing sickling, are designated HbD. This type of haemoglobin has been found in many widely separated geographical localities1. In six cases the haemoglobins D were structurally identical with the substitution glutamic acid→glutamine at position 121 of the β-chain2,3. These samples were obtained from a Punjabi, a Caucasian from North Carolina, an Italian family living in Chicago, a Portuguese, a Greek Cypriot and a white family living in Los Angeles in whom Hb-S was also present.

Summary. The effect of the concentration of haemoglobin S (Hb S) on its oxygen-dissociation properties was studied using either reconstituted Hb-S cells of different mean corpuscular haemoglobin concentrations (MCHCs) prepared by osmotic lysis, or cells in which Hb S is diluted by the presence of another haemoglobin. Only 4% (phosphate buffer) and 21%(bis Tris) of the low oxygen affinity of fresh Hb-S cells was found to be due to their slightly elevated intracellular 2, 3-DPG concentrations since when the cells were depleted of 2, 3-DPG most of the low affinity remained. The low affinity showed a marked dependence upon haemoglobin concentration which was absent for 2, 3-DPG-depleted Hb-A cells and, by extrapolation, the MCHC at which the oxygen affinities of the Hb-S cells became identical to that of the Hb-A cells was 14.5 g/dl in phosphate buffer and 13.1 g/dl in bis Tris. Both fresh and 2, 3-DPG-depleted cells containing another haemoglobin as well as Hb S (Hb-SA, Hb-SC and Hb-SF cells) were also found to have low oxygen affinities provided that the intracellular Hb-S concentration (MC(Hb-S)C) was above a certain level. These also showed a strong dependence upon the MC(Hb-S)C. The mean MC(Hb-S)C at which the low oxygen affinities of the DPG-depleted cells were abolished were 8.3 g/dl (phosphate) and 11.2 g/dl (bis Tris). Hb F in fresh Hb-SF cells brought about a much greater increase in oxygen affinity than the same amount of either Hb A or Hb C. In 2, 3-DPG depleted cells Hb A showed a greater ability to ‘dilute’ the Hb S than did Hb C. The conditions for the low oxygen affinity of Hb S were therefore found to be very similar to those required for the gelling of both pure Hb S, and Hb S in haemoglobin mixtures. It was concluded therefore that the low oxygen affinity of the Hb S was caused by the polymerization and that the difference between the oxygen affinities of Hb-S and Hb-A cells may be used as a measure of the polymerization process.

Results and complications of surgical treatment for retinal detachment in thirteen patients with proliferative sickle cell retinopathy are reported. Eleven had maintained or improved visual acuity after treatment. Methods and complications of exchange transfusion in eight patients are described. The pathogenesis and methods of treatment of these retinal detachments and the benefits of exchange transfusion are discussed.

UCH D4 is a human monoclonal antibody produced by an Epstein-Barr (EB) virus-transformed lymphoblastoid cell line. The antibody is specific for the rhesus D antigen, and is a good laboratory reagent for red blood cell typing. UCH D4 can be purified from the culture supernatant medium with the removal of all detectable DNA and infectious EB virus particles. This purified antibody will be used for in vivo assessment of its ability to prevent Rhesus disease of the newborn.

The incidence and the clinical course of patients suffering from sickle-cell syndrome (Hb SS; Hb SC; Hb S thal) in England and Wales are not known. In 1979 an ad hoc committee was formed to investigate these problems. Initially, a questionnaire was sent to 227 haematologists in England and Wales to determine the number of cases in these countries. The replies have indicated that 1367 cases were seen in 1978 and 1979. Probably this may represent only half the total number of cases. From this survey it has been possible to draw up a composite map showing the location of patients, which has provided a basis to determine the clinical course of the disease, and for further studies into the complications and management of sickle-cell disease in England and Wales. From a second questionnaire preliminary data about the general management and mortality in England and Wales have been recorded.

The sequence of erythropoietic events occurring during normal development of an embryo to an adult is a useful system for demonstrating cellular differentiation in relation to function. In mice there are two generations of red cells. The first, the primitive erythroid cells, originate in the blood islands of the yolk sac from about 8 days gestation and enter the circulation at 9 – 10 days gestation. These are large nucleated cells which continue dividing after release, and synthesize the three embryonic haemoglobins, EI (x2y2), EII (α2y2), and EIII (α2z2). The second generation of red cells, the definitive erythroid cells, are produced in the liver and enter the circulation from about 12 days gestation. These are non-nucleated and contain only adult haemoglobin (α2β2) (Fantoni et al., 1967).

In three patients with march haemoglobinuria a well-defined protein abnormality has been revealed by high-resolution sodium-dodecyl-sulphate/polyacrylamide-gel electrophoresis.

Summary. In an attempt to predict the likelihood of successfully treating sickle cell disease by increasing haemoglobin S (Hb S) oxygen affinity, two liganded derivatives of Hb S have been studied in an in vitro system that measures deoxy-Hb S polymerization. The participation of these liganded forms in the polymers has been quantitated in terms of an exclusion factor that relates their behaviour to that of deoxy-Hb S. Carbonmonoxy-Hb S has an oxy-Hb-like conformation and did not participate significantly in the polymerization. It was calculated that 30% carbonmonoxy-Hb S would have to be maintained in vivo to prevent sickling. Met-Hb S has a conformational equilibrium intermediate between oxy- (or carbonmonoxy-) and deoxy-Hb S and behaved in a similarly intermediate manner with regard to deoxy-Hb S polymerization. 60% met-Hb S would be needed to prevent in vivo sickling. It is concluded that stabilizing the oxy(R)-conformation is a potentially useful way of preventing sickling, and that a level of 30% R-state Hb S would have to be maintained for this to be successful.

Studies over the past few years have shown that it is possible to develop iron chelating agents that are active when given by mouth. Such compounds need to have a high binding constant for Fe(III) and an intermediate water and lipid solubility of both the unliganded compound and the iron complex with a Kpart of 0.2-1 for the free ligand. Hexadentate ligands would be preferable to bidentate compounds but no suitable compounds are available. In order to evaluate such compounds, simple cellular and animal screening models have been set up in a number of laboratories, and the potential of a new compound can be determined in a few weeks. Several groups have produced candidate compounds which are in various stages of development. DF is the established iron chelating drug, and the production of an orally active pro-drug must be high on the list of further developments, although this approach has so far not produced any useful compounds. It would appear that, at present, none of the other available hydroxamates will be sufficiently orally active or non-toxic to replace DF. Similarly, none of the available catecholates is promising enough to warrant further development at present. Among the amino carboxylates, although specificity may be a problem, the ester derivatives of HBED appear promising but have not yet been fully evaluated; this needs to be done before this group is discarded. Among the aryl hydrazones, PIH has reached the stage where it has been given to humans, but it may not be sufficiently active to be clinically useful. In this group there are several further compounds under development. PIB and a number of other derivatives need careful investigation before they are discarded, while pyridoxal-2-pyrimidyl-ethoxycarbonyl methbromide (PPEM) is in the early stages of animal testing and appears quite promising. Too little is known about the more recently synthesized hexadentate compounds based on the pyridoxal moieties, such as PLED, to judge whether this is a promising approach. Unfortunately, the naturally occurring siderophore, desferrithiocin, has proved too toxic for further development. Finally, a number of hydroxypyridin-4-ones have been synthesized and there are several that appear to be promising. CP20 (L1), the methyl derivative, has been given to humans, while at least two compounds with greater activity and relatively lower animal toxicity are close to being introduced. This group of compounds lends itself to the synthesis of a large number of derivatives with defined properties.(ABSTRACT TRUNCATED AT 400 WORDS)

Summary. Liposome-entrapped desferrioxamine was administered to iron-overloaded 59Fe labelled mice. When given orally or intraperitoneally entrapment did not enhance the effect of the chelator, but given intravenously liposomal desferrioxamine doubled the 59Fe excretion for a given dose of the drug, and excretion after a single dose continued for up to 3 d. In addition, liposomes containing ionophore A23187 administered concurrently with DTPA caused an excretion of 59Fe, whereas DTPA alone had no effect.

Using polyclonal human T cells and anti-CD3 monoclonal antibodies we have shown that small unilamellar liposomes covalently coupled with protein A become bound to T cells and not to B cells and that the binding was a specific liposome-antibody-receptor interaction. Intracellular delivery of liposome contents was demonstrated by the use of encapsulated carboxyfluorescein and flow cytometry and the transfer of membrane-bound liposomal carboxyfluorescein was virtually complete in 30 min. Liposomes containing methotrexate inhibited the growth of PHA-stimulated peripheral blood lymphocytes by 90%, after 48 h incubation. Potential applications are proposed in the study of the behaviour of surface membrane components and in T cell depletion and purging of bone marrow.

DF has a low general toxicity, perhaps because of its low lipid solubility, Kpart 0.01 (Porter et al, 1988b). This feature of the molecule may prevent it from penetrating most cells of the body. It appears that there may be a specific mechanism of uptake of the drug by hepatocytes (Porter et al, 1987), making the iron in these cells available for excretion via the bile, while the iron excreted in the urine may all come from extracellular chelation, particularly when iron leaves the reticuloendothelial cells (Hershko et al, 1978). On this hypothesis, cellular toxicity occurs only when DF penetrates sensitive cells in sufficient amounts so that some free DF remains after all the available iron in such cells has been chelated. Such a hypothesis accounts for the protection of cells by iron overload and therefore the greater sensitivity of unloaded patients. The retina and central nervous system are further protected by the blood-retinal or blood-brain barrier, and increased penetration of this barrier, mediated by high peak levels of DF, by drugs or other diseases would lead to the retinal or neurotoxic effects seen. In the ear, high levels of unliganded DF for a period of time may be necessary to cause deafness. Thus the very property that prevents its oral activity may be part of the reason for the low toxicity of DF. The severe toxic effects on vision, hearing and growth are all more likely at higher doses of DF and there appears to be partial protection against them by iron overload. These two conclusions have to be taken into account when deciding on the appropriate dosage for each patient. With care, the dosage can be adjusted to remove enough iron to prevent iron accumulation and therefore its toxic effects, whilst keeping doses low enough to prevent DF from being toxic itself. It appears that even in very iron-overloaded patients dosages higher than 125 mg kg-1 day-1 may cause visual disturbances and should be avoided. In patients on renal dialysis with aluminium toxicity great care is needed to avoid retinal toxicity even with dosages as low as 50 mg kg-1 day-1, although the drug should not be withheld if clinically indicated. The administration of DF to renal dialysis patients is described by Pogglitsch et al (1981, 1983), Pacitti et al (1983), Ihle et al (1986) and Molitoris et al (1987). DF should not be given to patients unless there is a clearly established clinical indication.(ABSTRACT TRUNCATED AT 400 WORDS)

Summary Young red cells (YRBC) prepared on a cell washer were compared to whole blood in a randomized prospective trial in 48 transfusion dependent patients with thalassaemia major. A minor but statistically significant decrease in blood consumption was observed in the group receiving YRBC. However, no difference was seen in transfusion interval, mean haemoglobin and rate of haemoglobin fall between the two groups. Filtered blood was shown to be as effective as frozen blood in eliminating non-haemolytic febrile transfusion reactions in all the trial patients. We conclude that any reduction in the rate of iron loading brought about by the use of YRBC is clinically insignificant and does not justify the expense, time and work required to produce young red cells for use in a large transfusion dependent thalassaemic population.

Eight subjects with sickle-cell disease in the symptom-free steady-state received a single one-hour infusion of the new anti-sickling agent BW12C on a total of eleven occasions. A dose-dependent increase in wholeblood oxygen affinity was observed, resulting from the action of BW12C in stabilising the oxy-conformation of haemoglobin and causing a left shift of the oxygen saturation curve. At the highest dose given (20 mg/kg bodyweight), up to 23% of haemoglobin was modified to a BW12C-reacted high-affinity form without evidence of tissue hypoxia. There was biochemical and rheological evidence for a transient decrease in haemolytic rate.

A symptomless Iranian patient homozygous for beta thalassaemia has haematological changes similar to the beta thalassaemia trait. This remarkably mild phenotype is probably the result of coexistent alpha thalassaemia and increased gamma chain synthesis. Restriction endonuclease mapping analysis of the beta globin genes indicates that the patient is homozygous for a single nucleotide substitution at the 5' donor splice junction in the second intervening sequence of the beta globin gene. No other changes were observed in the non-alpha globin gene cluster. It seems unlikely that the augmented gamma chain synthesis in this patient is related to the molecular defect responsible for this beta o thalassaemia.

Two abnormal high oxygen affinity haemoglobins have been studied for their ability to inhibit the polymerization of deoxy-Hb S. They were used as models to predict the effect of chemically modifying haemoglobin to increase oxygen affinity since the oxy(R)-conformation is the most potent inhibitor known of cell sickling. The participation of these variants in deoxy-Hb S polymers has been described quantitatively in terms of an exclusion coefficient, f, that relates their behaviour to that of deoxy-Hb S, and qualitatively in terms of an exponent of hybridization. Hb Bethesda was a potent inhibitor of polymerization, its behaviour being similar to that of Hb F studied previously. Hb Radcliffe demonstrated atypical behaviour, with hybrid molecules of the formula alpha 2 beta S beta Rad participating in the polymerization as effectively as Hb S, as has been shown for Hb C. These data have implications for the development of anti-sickling agents designed to increase oxygen affinity by covalent binding to Hb S.

The potential value of sulphonated aluminium phthalocyanine (AISPc) as a purging agent for bone marrow autografts in acute myeloblastic leukaemia (AML) has been studied using in vitro clonogenic assays for normal (GM-CFC) and leukaemic (AML-CFC) progenitor cells. In nine out of 13 cases, the leukaemic blasts were found to be highly sensitive to AISPc. In six of the sensitive cases clonogenic assays revealed that only 2 +/- 1% of AML progenitor cells survived AISPc treatment under conditions which permitted a GM-CFC recovery of 60 +/- 11%. AISPc photosensitization was also shown to selectively eliminate the leukaemic cell line K562 from an in vitro model of minimal residual disease. Thus photosensitization using AISPc may be an effective method of purging marrow autografts in some cases of AML. Evaluation of the sensitivity of AML clonogenic cells at diagnosis may identify those patients in whom AISPc photo-purging may be of benefit at the time of an autologous bone marrow transplant.

Autologous bone marrow transplantation (ABMT) in first remission of acute non-lympho-blastic leukaemia (ANLL) has increased the proportion of patients eligible for intensive myeloablative therapy and has produced encouraging early results. However, more patients relapse after ABMT than after allogeneic BMT and this may be due in part to reinfusion of leukaemia with the graft. We have examined the phototoxicity of sulphonated chloro-aluminium phthalocyanine (SPC) on leukaemic blasts from patients with ANLL and normal myeloid progenitors (GM-CFC) to determine whether in vitro phototherapy might be used to eliminate residual ANLL from bone marrow grafts. ANLL blasts were shown to take up SPC readily and the phototoxicity was shown to be dependent on the concentration of SPC, duration and temperature of incubation, the interval from SPC incubation to light exposure and the duration of light exposure using 675 nm light delivered by an optical fibre at 50 mV from an Ar pumped dye laser. After a 2 hour incubation at 25 μ.g SPC m−1 recovery of ANLL blasts at 24 h was 5 ± 2% whilst GM-CFC recovery from normal marrow was 47 ± 9%. This study suggests that ANLL blasts may be more sensitive to SPC phototoxicity than normal marrow GM-CFC, and further studies to examine the potential of this compound for the elimination of residual ANLL from marrow autografts are in progress.

The 59Fe excretion caused by a range of bidentate N-substituted [R group = methyl (CP20), ethyl (CP21), propyl (CP22), isopropyl (CP23), butyl (CP24) or hexyl (CP25)] 3-hydroxypyrid-4-one chelators in iron-overloaded mice is presented. All the compounds cause significant iron excretion when given intraperito-neally, but that the most hydrophobic compounds, CP24 and CP25, were toxic except at low doses. The excretion caused by CP21, CP22 and CP23 were significantly greater than that caused by CP20 and slightly larger than that caused by an equivalent dose of desferrioxamine. These compounds (CP20 through CP24) also caused significant excretion of 59Fe when administered orally. Compounds CP21, CP22 and CP24 were significantly more active than compounds CP20 and CP23. It is concluded that the N-ethyl or N-propyl 3-hydroxy-pyrid-4-ones are the most promising compounds for clinical application. Preliminary experiments using a hexadentate pyrid-2-one, CP130, show that this causes significant 59Fe excretion both when given intraperitoneally or orally

Three members of an Oxfordshire family have polycythaemia. In each case their whole-blood oxygen affinity is increased. This is due to a previously undescribed haemoglobin variant which has been named haemoglobin Radcliffe (alpha2beta299(Gl)Ala). In addition to having a high oxygen affinity haemoglobin Radcliffe shows virtually no haem-haem interaction and a diminished Bohr effect. It is synthesized at the same rate and is as stable as haemoglobin A. X-ray analysis indicates that crystals of deoxyhaemoglobin Radcliffe are isomorphous with those of deoxyhaemoglobin A. Solutions of haemoglobin Radcliffe were also studied by high-resolution proton nuclear magnetic resonance spectroscopy. The structure/function relationships of haemoglobin Radcliffe are discussed in the light of these studies.

The aim of this study was to make sequential measurements on a series of premature infants with and without respiratory distress syndrome to assess the effect of acidosis and hypoxaemia on oxygen transport. 3 infants with an uneventful postnatal course (33 weeks gestation, birth weights 1325 gms, 1455 gms and 1484 gms) were compared with 6 infants who needed assisted ventilation for respiratory problems after birth (mean gestation 31–5 weeks, range 29 to 34 weeks; mean birth weight 1132 gms, range 800 to 1905 gms).

A series of bidentate hydroxypyridinone iron chelators that have therapeutic potential as oral iron chelators, have been studied systematically to determine which properties are the most critical for the mobilization of hepatocyte iron. The relationship between lipid solubility of the free and complexed forms of each chelator and hepatocyte iron release has been investigated as well as the contribution of the binding constant for iron (III). Hydroxypyridin-4-ones that were approximately equally soluble in lipid and aqueous phases were the most active compounds, the partition coefficient of the free chelator appearing to be more critical in determining iron release than that of the iron-complexed form. Highly hydrophilic chelators did not mobilize intracellular iron pools, whereas highly lipophilic compounds were toxic to hepatocytes. The contribution of the binding constant for iron (III) to cellular iron release was assessed by comparing hydroxypyridin-4-ones (log beta 3 = 36) and hydroxypyridin-2-ones (log beta 3 = 32), which possess similar partition coefficients. The results show that the binding for iron (III) is particularly important at low concentrations of chelator (less than 100 mumol/L) and that at higher concentrations (greater than 500 mumol/L) iron mobilization is limited by the available chelatable pool. Measurement of iron release with other chelators confirms the importance of both the lipid solubilities and iron (III)-binding constants to iron mobilization. The most active hydroxypyridin-4-ones released more hepatocyte iron than did deferoxamine when compared at equimolar concentrations. The results suggest that the ability of an iron chelator to enter the cell is crucial for effective iron mobilization and that once within the cell the binding constant of the chelator for iron (III) becomes a dominant factor.

THE human embryonic haemoglobins (Hbs) Gower 1 and Gower 2 (ref. 1) are present in fetuses up to 60 mm crown–rump measurement2. However, relatively little is known about the structure and function of these haemoglobins because of the difficulty in obtaining suitable material, particularly since the introduction of the suction method for terminating pregnancy in the first trimester. Here we present the amino acid composition and sequence data for the human e-chain, prepared by purifying Hb-Gower 2 (α2e2) from early abortion samples. In addition, separation of embryonic red cells from the majority of the maternal red cells has led to the isolation of Hb-Gower 1 and the demonstration that it has the composition ζ2e2.

Monoclonal antibodies for the beta-globin chain of human HbA (UCH beta) and the gamma-globin chain of human HbF (UCH gamma) have been made. UCH beta indirectly labelled with rhodamine-labelled goat anti-mouse Ig and directly flouresceinated UCH gamma have been used, via double labelling immunofluorescence microscopy, to assay for the presence of beta-chains in fetal erythrocytes obtained at fetoscopy from fetuses at risk for beta-thalassaemia major. The results from 111 cases demonstrate that beta-chain synthesis of as low as 1.8% can be detected in fetal erythrocytes. The immunochemical labelling can be rendered more sensitive by the use of biotinylated UCH beta and avidin-FITC conjugates. This method is rapid and can be used for a sample that is highly contaminated with maternal erythrocytes.

Summary. Oxygen dissociation studies were carried out on haemoglobin E (Hb E) at both high and low haemoglobin concentrations. Oxygen affinities of fresh red cells from three people homozygous for Hb E and from one with Hb E-β thalassaemia (Hb-E trait/β-thai trait) were low in three out of four patients studied, while the oxygen affinity of red cells from an individual with Hb-E trait was normal. 2, 3-DPG concentration in the fresh cells from the people with homozygous Hb E or Hb-E trait/β-thal trait which showed low oxygen affinities were elevated sufficiently to account for the shifts observed. When the cells from two of these people with homozygous Hb E were depleted of 2, 3-DPG, their oxygen affinities became the same as that of similarly treated normal cells. Pure ‘stripped’ Hb E in dilute solution behaved identically to Hb A in respect of P50, Bohr shift, haem-haem interaction, and interaction with inorganic phosphate or 2, 3-DPG. Hb E, therefore, has the same oxygen dissociation properties as Hb A both in dilute solution and in the red cell. The low oxygen affinities found in the fresh cells and in whole blood are caused by high 2, 3-DPG concentrations within the cell.

Erythropoietin was estimated by radioimmunoassay in serum from 78 cord blood samples, collected in the second and third trimesters in 72 pregnancies. In 43 samples obtained during or after normal pregnancy (from 19 to 42 weeks gestation) erythropoietin levels increased with gestation. Cord blood haemoglobin also increased with gestation, but the rate of increase was less during the last weeks of pregnancy. Erythropoietin levels were similar in the cord blood of infants of the same gestation, whether born vaginally or by caesarean section. The fetus can respond to severe anaemia or hypoxia with increased erythropoietin levels as early as 24 weeks gestation. Elevated erythropoietin levels were found in two out of eight infants born after labour in which there was 'acute' fetal distress, suggesting the presence of unrecognized chronic fetal hypoxia in these pregnancies.

The oxyhaemoglobin dissociation curve (ODC) was investigated in 15 patients with Eales's disease and 11 controls to test the hypothesis that Eales's disease is caused by retinal hypoxia secondary to impaired oxygen release from the blood. Patients with the peripheral form of Eales's disease did not have any significant difference in the P50 values when compared with the controls. However, the P50 values were significantly lower (P < 0.005) for the patients with mixed retinopathy. This degree of increased oxygen affinity is not physiologically significant but may reflect an intrinsic abnormality of the red cells in these cases.