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A new approach to the antenatal diagnosis of beta-thalassaemia major (homozygous beta-thalassaemia) has been developed. Using rhodamine-labelled antibodies directed against gamma-globin, fetal erythrocytes can be identified in fetal blood samples contaminated with maternal cells, and using fluorescein-labelled antibodies directed against beta-globin, the presence of beta-chains (and thus Hb-A) may be determined in the same cells by double labelling immunofluorescence microscopy. The results obtained using this technique on blood samples from fetuses at risk for beta-thalassaemia major show that if fetal beta-chains are detected immunochemically the fetus is normal or heterozygous for beta-thalassaemia and over 85% of the fetuses unaffected by beta-thalassaemia major may be identified in this way. The technique is rapid, applicable to small numbers of fetal erythrocytes heavily contaminated with maternal cells and eliminates the necessity of determining chain synthesis ratios in those cases where beta-chains are detected immunochemically.

Restriction endonuclease analysis has been performed on the alpha and beta globin gene clusters of 57 Cypriots homozygous for beta thalassaemia, 30 with the transfusion dependent form of the condition (thalassaemia major) and 27 who are less severely affected (thalassaemia intermedia). There was a significant difference in the incidence of alpha thalassaemia between the two groups: 14/27 of the patients with thalassaemia intermedia also had deletion forms of alpha thalassaemia, while only 4/30 of the patients with thalassaemia major were similarly affected. Thus in Cypriot patients who are homozygous for beta thalassaemia the co-inheritance of alpha thalassaemia is an important factor in determining the clinical course.

We report an Asian Indian family in which two daughters have Hb Sun Prairie, a known unstable alpha 2-globin variant [codon 130, GCT--CCT; alpha 2 130(H13)Ala--Pro beta 2]. While the homozygous probands have chronic hemolysis-the same phenotype as previously reported, the heterozygous parents are asymptomatic with a thalassemia carrier phenotype, distinct from the chronic hemolytic state previously described in a heterozygote. Unlike the earlier cases in which family studies were not available, this family clearly exhibits autosomal recessive inheritance, unusual amongst variants within the same region of helix H. Globin chain biosynthesis ratios initially suggested a beta-thalassemic hemoglobinopathy-this was excluded by normal sequence analysis of both beta-globin genes. This case report further illustrates the complexity of phenotypes in the thalassemic hemoglobinopathies. It also demonstrates inversion of the alpha/beta-globin chain biosynthesis ratio, a phenomenon which had been noted in other alpha-globin variants and can be a confounding factor in the investigation of thalassemic hemoglobinopathies.

A fixation and permeabilization procedure using formaldehyde and acetone has been developed which allows immunostaining of intracellular haemoglobin for fluorescence activated cell sorter (FACS) analysis of erythrocytes. The treatment preserves antigenicity and light-scattering properties. Validation of the method was given by the correlation of F cell number in adults determined by FACS analysis with that assessed by microscopic examination of cell smears, and by the direct relationship between beta chain synthesis and intensity of beta chain/Hb A immunofluorescence within fetal erythrocyte samples known to vary in their beta chain/Hb A content. The procedure is rapid, non-subjective and sensitive, and makes analysis of haemoglobin content, type and distribution amongst red cell populations possible.

Liposomes containing the photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) were coupled to polyclonal sheep anti-mouse-Ig antibody and bound to cells coated with specific mouse monoclonal antibody. When illuminated with red light, the AlSPc in the liposomes was activated to produce singlet oxygen and the antibody and liposome targeted cells were destroyed. DW-BCL cells (an Epstein Barr virus immortalised B-cell line) were targeted with an anti-B-cell antibody (8A) and killed specifically, both alone and in the presence of bone marrow mononuclear cells (BM-cells), without phototoxic effects on the untargeted bone marrow CFU-GM progenitor cells. The presence of an excess of non-target cells did not interfere with antibody and liposome binding, or light access to target cells. Similar results were obtained with T-lymphocytes as target cells using anti-CD3 antibody. Specific targeting to the B-cells was demonstrated in the cell mixtures by use of fluorescent microscopy combined with a sensitive technique to detect low levels of AlSPc fluorescence, a cooled charge couple device (CCD) camera. This was also able to show low levels of non-specific background binding of AlSPc to BM-cells and a small population of cells that took up AlSPc in the absence of antibody. The latter were shown to be monocytes by flow cytometry. Images Figure 5

We report two siblings with congenital spherocytosis, multiple phenotypic abnormalities and an inherited interstitial deletion of the short arm of chromosome 8 (8p). The propositus came to our attention with acute bone marrow hypoplasia secondary to B19 parvovirus infection. The bone marrow trephine biopsy appearances of intranuclear eosinophilic degeneration in the erythroblasts may be pathognomonic of B19 parvovirus induced acute bone marrow aplasia. The presence of B19 parvovirus DNA was demonstrated in erythroblasts by in situ hybridization. Chromosome analysis of peripheral blood lymphocytes from both siblings showed an interstitial deletion of the short arm of chromosome 8, del (8) (p11p21). This abnormal chromosome was inherited from their mother, who showed this deletion as well as a small fragment representing the deleted 8p chromosome portion, del (8) (p11p21), +f. Centromeric material from chromosome 8 was detected in this chromosome fragment by in situ hybridization using an alpha satellite probe (pJM 128), but not by C banding. Chromosome analysis of skin fibroblasts from the mother and a third sibling with a similar karyotype showed the deleted fragment in over 80% of cells. Cells in which the fragment was absent exhibited the deleted 8p, suggesting there was no mosaicism. The mother and the third sibling were phenotypically normal without spherocytosis. A fourth sibling and the father were normal. The chromosome abnormality was not observed in five of the mother's siblings, suggesting that it arose de novo in the mother. Our findings strongly support a locus for congenital spherocytosis on the short arm of chromosome 8. The frequency of defects at this locus is unknown.

To determine whether beta-thalassemia can be detected in the fetus, blood was obtained from abortuses of normal mothers and of mothers with beta-thalassemia trait. The red cells were incubated with radioactive leucine and the globin chains were analyzed by radiochromatography. Two independent methods were utilized to correct the results for contamination by maternal radioactive beta-chain, and the corrected beta/gamma ratios were compared to a previously established range of normal fetal beta/gamma synthetic ratios obtained by similar measurements in pure fetal cells. In the erythroid cells of three fetuses from mothers with beta-thalassemia trait, the beta/gamma synthetic ratio was normal in two. The third had a beta/gamma ratio of 0.04 at 10 1/2 weeks, a 50% reduction, consistent with fetal beta-thalassemia trait. Two other fetuses, derived from parents both of whom had beta-thalassemia trait, were also studied. One had a beta/gamma ratio of 0.029 at 8 weeks, a 65% reduction, also consistent with beta-thalassemia trait. The cells of the other had a ratio of essentially zero at 11 weeks, highly suggestive of homozygous beta-thalassemia. Although further experience will be needed to distinguish the homozygous and heterozygous states reliably, it now appears that the beta-thalassemia gene is expressed in the first trimester. Therefore these data suggest that the antenatal diagnosis of beta-thalassemia is becoming an attainable goal.

In a group of young patients with thalassaemia and iron overload treated by subcutaneous infusions of desferrioxamine we have found a number of minor alterations in retinal function. The incidence of such changes is not related to drug dosage or to ferritin level but to abnormality of the extended glucose tolerance test.

A dipeptide L-lysine-L-phenylalanine is shown to inhibit both cell sickling and the gelation of solutions of sickle-cell haemoglobin. The effect on deoxyhaemoglobin solutions and gels was followed by centrifugation; a progressive inhibition of gelation was observed up to 30 mM Lys-Phe. The haemoglobin concentration at the plateau (26 g/dl) suggests that an effect might be seen in vivo if suitable concentrations of Lys-Phe (about 20 mM) can be maintained in the blood stream. Additional studies of lag time before onset of gelation support this. Oxygen dissociation curves of sickle cells showed an effect of Lys-Phe only after incubation for 3 h before measurement, the P50 decreasing from 51 mmHg (6.8 MPa) to 41 mmHg (5.5 MPa) for cells depleted of 2,3-bisphosphoglycerate. The effect of Lys-Phe on intact sickle cells was more rapid. A marked increase in the number of unsickled cells in the presence of Lys-Phe was observed after only 15 min incubation. This result, together with measurements of uptake both into the cell and onto the cell membrane shows that the compound produces a membrane-mediated antisickling effect in addition to the effect on haemoglobin in solution within the cell. The membrane effect is not due to a change in cell volume. The properties of this dipeptide may be of value in the treatment of impending and early sickle crisis.

The polymerisation of deoxyhaemoglobin S at 37 °C has been characterised by the supernatant concentration remaining after ultracentrifugation of the gel. Other haemoglobins, that are often found to occur with haemoglobin S, influence the polymerisation process. The effects of such haemoglobins have been quantitated in terms of a parameter, which estimates the difference in tendency to polymerise between a particular haemoglobin and haemoglobin S. The interpretation also assesses the participation of hybrid molecules present in mixtures of haemoglobins. The results suggest that, while each of the haemoglobins can form mixed polymers with haemoglobin S, haemoglobin F shows a greater inhibition of polymer formation than haemoglobin A, and haemoglobin A 2 has the maximum inhibitory effect. Hybrids show the same gelling potential as an equimolar mixture of each of these haemoglobins (A, F or A 2 ) with haemoglobin S. Haemoglobin C shows a large inhibition of polymerisation, intermediate to haemoglobins F and A 2 . However, hybrids of haemoglobins C and S behave like haemoglobin S and contribute preferentially to polymer formation. This behaviour could explain the severity of SC-disease relative to haemoglobin AS trait.

Desferrioxamine was given intravenously, at higher doses than previously reported, to counter the effects of transfusion-induced iron overload in four patients with beta thalassaemia major. In two of them retinal abnormalities developed, presenting with night blindness and field defects, which improved on withdrawal of the drug.

A chromatographic procedure for the separation of human globin chains is described. This method uses CM-Sepharose CL-6B as ion-exchanger and NaCl gradients formed in sodium phosphate buffers containing urea and 0.05M mercaptoethanol to elute the chains. One advantage of this system is that a column, once packed, may be used repeatedly for several chain separations, thus reducing cost and practical work. The resolution in our hands was also better than that on CM-Cellulose using phosphate buffer gradients.

The effect of urea on the oxygen affinity of sickle cells and normal cells was studied up to a concentration of about 1.0 M. Besides the increase in oxygen affinity found in both normal and sickle cells there was a further increase found only in the sickle cells. This specific increase was caused by the direct inhibition of the polymerization of deoxygenated Hb-S by the urea and was used to measure the extent of this inhibition. Even at concentrations of 1.0 M the urea did not fully inhibit the polymerization. At the urea concentrations recommended for treatment of sickle cell crises and for the oral treatment of the disease there was only a very slight inhibition of polymerization. The small increase in oxygen affinity brought about by these concentrations of urea (equivalent to a log P50 change of 0.016) will cause some additional minimal inhibition of sickling at physiological partial pressures of oxygen. Oxygen dissociation measurements of Hb-A in dilute solution showed that 0.95 M urea had no effect on the interaction of 2,3-DPG and chloride with haemoglobin nor on the haem-haem interactions. This implies that the quaternary 'deoxy' form of the haemoglobin may still form and that the action of urea in inhibiting the polymerization is through interference with the intermolecular bonds rather than by inducing a conformational change which disrupts the bonding. Unless urea at the concentrations recommended for treatment inhibits the sickling of the cells by some mechanism other than its effect on the haemoglobin (such as by an effect on the membrane), these results suggest that it is of little use for the treatment of sickle cell disease.

Five patients with heterozygous beta-thalassaemia with an unusually severe clinical picture, low haemoglobin levels occasionally requiring blood transfusion, splenomegaly and unusually prominent basophilic stippling were found to have co-inherited a triple alpha-globin gene arrangement on one chromosome (alpha alpha alpha/alpha alpha). It seems probable that the expression of a single extra alpha-globin gene is sufficient in some patients with heterozygous beta-thalassaemia to give rise to a clinically significant degree of dyserythropoietic anaemia.

In all of six cases of congenital dyserythropoietic anaemia, type II (HEMPAS), gel electrophoresis in the presence of SDS revealed abnormally rapid migration of the preponderant integral membrane protein, band 3. After proteolysis of intact cells, the remaining part of the band 3, comprising the intramembrane segment and the cytoplasmic domain, migrated electrophoretically as a single band, identical in mobility to that from normal cells treated in the same manner. The anomaly thus resides in the extracellular domain of the protein, which is the glycosylated part of the chain. Peptide digests of the band 3 showed no evidence of a missing protein segment in the abnormal cells and the amino acid composition of the peptides derived from proteolysis of the extracellular protein of intact cells was also normal. We infer that the anomaly is one of glycosylation. The major glycoproteins, detected by carbohydrate-specific (PAS) stain appear normal in SDS gels. However, when the more sensitive procedure of reacting after electrophoresis with radioiodinated lentil lectin is employed, some additional minor protein components are revealed. In particular one species of apparent subunit molecular weight about 150 000 appeared in all cases of HEMPAS examined and in no normals. This component is not accessible to proteolysis by chymotrypsin or Streptomyces griseus protease, and may be associated with the inner membrane patches, characteristic of the HEMPAS condition. Overall cell shape and microviscosity of the membrane bilayer, as measured by fluorescence polarization of a lipid-soluble fluorophore, were substantially normal in HEMPAS cells.

Summary. Oxygen dissociation studies were carried out on red cells and lysates from a patient heterozygous for Hb Hammersmith. The oxygen affinity of the cells at pH 7.1 was decreased, partly by an increased cellular concentration of 2, 3-DPG and more importantly by an intrinsic low affinity of Hb Hammersmith. Haemhaem interactions were reduced and an abnormal Bohr effect (pH 7.1–7.4) was found in the cells which would bring about an additional decrease in oxygen affinity at physiological pH. Oxygen dissociation studies on the lysates showed a low oxygen affinity, a normal Bohr effect at 50% saturation, slightly decreased haem-haem interactions and a normal interaction with 2, 3-DPG. Estimation of the percentage of Hb Hammersmith in the lysates of the patient's cells by selective precipitation of the abnormal β chain with p-chloromercuribenzoate (PCMB) showed the presence of 30–33% abnormal haemoglobin. Assuming no interaction between the Hb A and the Hb Hammersmith, oxygen dissociation curves were calculated for Hb Hammersmith alone and these indicated that it has greatly decreased oxygen affinity, a normal Bohr effect, normal 2, 3-DPG interaction and (somewhat) decreased haem-haem interactions.

High levels of committed erythroid and granulocytic/monocytic progenitor cells have been demonstrated in fresh blood obtained at fetoscopy. The fetal progenitor cells were more sensitive to appropriate stimuli (erythropoietin and colony-stimulating factor) than adult progenitor cells grown under the same conditions, and this was shown to be due to intrinsic differences in the progenitor cells at the different developmental stages.

A partially purified fraction of the water soluble photosensitive dye sulphonated aluminium phthalocyanine (AlSPc) was encapsulated in liposomes which were then linked to a targeting monoclonal antibody 791T/36 using a heterobifunctional linking agent. The photocytotoxic effects of the liposomes were determined on two cell lines bearing an antigen with which the targeting antibody binds: 791T, an osteosarcoma and C170, a colorectal carcinoma; and a control cell line not bearing the antigen; DW-BCL, an Epstein-Barr virus immortalised B-cell line. Antibody dependent cytotoxicity was observed in 791T and C170 cells and was proportional to the number of antigens on the cells, the AlSPc concentration and the time of exposure to activating red light. No significant toxicity was seen using untargeted liposomes, control cells or free AlSPc fraction under similar conditions. Targeted cells and controls kept in the dark also showed no significant toxicity. A possible mechanism of action is postulated and simple adaptations which demonstrate the versatility of the model are discussed. Some suggestions as to the clinical situations to which this system might be applied in the form of photodynamic therapy (PDT) are made.

The reaction between p-chloromercuribenzoate (PCMB) and haemoglobin has been investigated to define the optimum conditions for dissociation to the subunits. The production of various molecular species has been followed by electrophoresis, gel filtration and ultracentrifugation. The reagent was applied to human haemoglobins A, S, C, Ja (Glasgow), A2, H and F, and also to the haemoglobins of dog, rabbit, horse and pig. Evidence is presented for the occurrence of haemoglobins without α-chains containing dissimilar β-chains, i.e. β2Yβ2X The behaviour of haemoglobins in the presence of PCMB was found to correlate qualitatively and quantitatively with the nature and number of their sulphydryl groups. It is shown that attachment of reagent at position β-93 causes initial dissociation of the tetramer to the dimer: α2β2 ag 2αβ. From the location of these reactive sulphydryls it is concluded that the initial dissociation is along the bc plane of the haemoglobin molecule with the formation of two α1β1 dimers. Further dissociation by PCMB at pH 6.0 only occurred with haemoglobins containing a second sulphydryl group on the β-chain (β-112). From the data it is concluded that substitution at both unreactive thiols β-112 and α-104 causes final dissociation to the monomeric species. The contribution of the various sulphydryl groups to dissociation was also indicated by the behaviour of alkylated haemoglobins when treated with PCMB. Evidence is presented that dissociation of haemoglobin to half-molecules in several media occurs across the same plane as that initially affected by PCMB. The connection between the sigmoid oxygen tension curve and dissociation is discussed.

Diseases of protein synthesis can be divided into two main groups: in the first group the rate of synthesis of the protein or one of its constituent poly­ peptide chains is reduced, while in the second an abnormal protein is pro­ duced. For hemoglobin, the first group is the thalassaemias, and these have already been reviewed in this series (1). This article will deal with the other group, those caused by the structurally abnormal hemoglobins. These dis­ eases are important for two reasons. First, some of them, such as sickle cell disease, are very common in certain parts of the world and present an im­ portant clinical problem. The second reason is the relative ease, compared to other human proteins, with which the abnormal hemoglobins can be isolated and their structural abnormality determined. In this way, the abnormal hemoglobins have provided a model indicating the type of protein abnor­ malities which can cause disease and also a natural "testbed" for the rules thought to govern the assembly of protein molecules. Recent work on the structure and function of hemoglobin has not only increased our understanding of the mechanism of sickle cell disease but has led to the discovery of two further groups of diseases (Table 1). The first consists of hemoglobins which cause disease because of abnormal oxygen dissociation properties. The second group is the "unstable hemoglobins" which lead to hemolytic anemia. Some of these hemoglobins also have al­ tered oxygen dissociation. In this short review only the more recent advances in the diseases caused by abnormalities of structure of hemoglobin will be discussed. Previous work has been reviewed elsewhere (2-6), and a compre­ hensive list of the variations in structure of the abnormal hemoglobins is given by Lehmann & Carrell (7).

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.

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.

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)

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.

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 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.