Blood and blood products

Diana Walford

35

Blood and blood products

As refinements in the techniques of serology, blood banking and plasma fractionation have widened the scope and scale of therapy with blood and blood products, so attention has been focused increasingly on the problem of post-transfusion hepatitis and its possible progression to chronic liver disease. In the United States it has recently been estimated that more than 8 million units of blood and blood products are being used per year (1R), with 20--30,000 cases of posttransfusion hepatitis and a death rate from this cause alone of 3000 per a n n u m (2R). The first section of this chapter will therefore be devoted to a review of the most recent literature concerning the transmission of hepatitis from blood and its products. The remainder of the chapter will contain a review in depth of a newcomer to the blood products scene - haematin - and its use in the treatment of porphyria. This will be followed by a brief overview of other selected papers.

Hepatitis This year's literature has had two main themes: additional screening tests for hepatitis B virus infectivity (3 CR, 4 c, 5 C, 6CR), and the severity of non-A, non-B hepatitis and its progression to chronic liver disease (7 cr, 8Or). Anti-HBc and blood infectivity Antibody to the hepatitis B core antigen, anti-HBc, develops regularly during the course of Type B hepatitis and the highest titres are found in persons who become chronic carriers of the surface antigen, HBsAg(9 cR, 10CR). However, anti-HBc may persist for some time after HBsAg has fallen to undetectable levels and it has now been claimed that hepatitis B may be transmitted by units of blood containing high titre anti-HBc which are negative for HBsAg and anti-HBs(3 CR, 4c). This raises the question of whether additional tests are needed to screen out plasma containing hepatitis B virus. This additional

screening is likely to be of greatest importance in the preparation of coagulation factor concentrates where potentially undetectable levels of virus in the starting plasma may be selectively retained in the relevant Colin fractions. Hence Trepo e t al. (5 c ) have suggested that, besides HBsAg screening of blood units and plasma pools, the Cohn fractions used in the preparation of high-risk blood products should be screened for the presence of the soluble antigen (HBe) and for DNA polymerase, an enzyme which is closely associated with replication of the virus. Striking confirmation of the need for increased sensitivity of testing for the hepatitis B virus has come from a recent study of liver histology in a group of haemophiliacs in whom previous hepatitis B infection had resuited in persistent abnormalities of liver function (6CR). All 13 asymptomatic antiHBs-positive patients had histological evidence of liver disease, eight of them having the relatively benign chronic active hepatitis, five having severe liver disease. There was no apparent correlation between the titre of anti-HBs and the severity of the liver biopsy findings. Moreover, despite the presence of anti-HBs, the hepatocytes of eight out of 12 patients were found to contain hepatitis B virus, demonstrated by immunofluorescence with specific anti-HBs and anti-HBc antisera. The authors concluded that "throughout the world, a large number of asymptomatic haemophilic patients who have received numerous transfusions must have histological liver disease. In some, it must be severe. Adequate information is not yet available to evaluate fully haemophilic patients treated only with blood products negative by radioimmunoassay for HBsAg in both donors and final product. However, at least two patients cared for in Pittsburgh and treated only with such concentrates have had persistent biochemical abnormalities, and one has remained HBsAg positive for more than one year. In addition, the implications of non-A, non-B virus (or viruses) in haemophilia have

231

Blood and blood products yet to be evaluated. To return to less effective therapy for haemophilic bleeding would represent a step backward. It is apparent, then, that a major effort is necessary to develop a "clean" product rapidly for the treatment of the next generation of haemophilic patients.'

Epidemic hepatitis B caused by commercialhuman immunoglobulin Human immunoglobulin preparations correctly prepared by the cold ethanol fractionation method of Cohn are generally considered to be free from the risk of hepatitis transmission (llR). An epidemic of acute hepatitis B has been reported (12 Cr) which followed the administration of human immunoglobulin to members of staff of a mission hospital in India and their families. Jaundice developed in 123 (38%) of 325 persons inoculated. HBsAg was detected in three of the batches of immunoglobulin tested, but virus particles and e antigen were not detected. The batch material did contain anti-HBc, but not antiHBs, suggesting that anti-HBc was not protective. The authors considered that some failure of the fractionation method must have been responsible for the infectivity of the material and that the final quality control procedures cannot have been adequate. However, they stressed the importance of using only plasma units that are non-reactive for HBsAg by a sensitive method for the preparation of plasma protein fractions.

Severity of non-A, non-B hepatitis and progression to chronic liver disease Hitherto, direct evidence for the transmission of nonA, non-B hepatitis by blood products has been lacking. Recently, Wyke et al. (8 c r ) have demonstrated the transmission to chimpanzees of an agent responsible for two fatal cases and one non-fatal case of non-A, non-B hepatitis in patients with liver disease who received the same batch of commercial prothrombin-complex concentrate prior to liver biopsy. A further death apparently from non-A, non-B hepatitis occurred in their series of 17 patients with liver disease who received four different batches of concentrate. These well-documented cases underline the potential severity of this form of hepatitis in patients with pre-existing liver disease and call into question the wisdom of using prothrombin-complex concentrates to reverse coagulation abnormalities prior to liver biopsy. Since the publication of this report, all clinical trials of prothrombin-complex concentrates for indications other than haemophilia B in the United Kingdom have been suspended at the request of the Committee on Safety of Medicines. The need to limit

the non-essential exposure to coagulation factor concentrates is reinforced by reports that non-A, non-B hepatitis may progress to chronic liver disease(7 r 13c). In one of these reports(7C), histologically verified non-A, non-B hepatitis was shown to progress, both histologically and clinically, to chronic active hepatitis within a two-year period.

H a e m a t i n therapy in t h e p o r p h y r i a s

Haematin, a naturally occurring breakdown product o f haemoglobin, which can be crystallized from blood by simple chemical means, has recently been used as an experimental drug in the treatment o f porphyria and has thereby acquired the status o f a therapeutic blood product. A review o f the properties and adverse effects o f this compound has now become due following a series o f publications detailing its use in acute porphyria (14 CR, 15 Cr, 16 CR, 17 C, 18 CR, 19 r, 20C).

Biochemistry Naturally occurring haematin is one o f the early products o f haemoglobin catabolism, which takes place during the destruction o f effete red blood cells within the reticuloendothelial system. In the first step o f haemoglobin catabolism, the iron atom is oxidized to the trivalent state and methaemoglobin is formed. Next, the linkage between the haeme and the globin is severed, yielding haematin, which is the ferric complex o f protoporphyrin. The iron is released and the protoporphyrin ring is split with the production o f carbon dioxide and biliverdin, the latter being quickly reduced to bilirubin. Chemistry Defibrinated blood is added dropwise to a mixture o f boiling glacial acetic acid and sodium chloride. After cooling, the precipitated haematin is centrifuged, washed repeatedly and evaporated to dryness. The crude haematin is then dissolved in pyridine and recrystallized from a boiling glacial acetic acid mixture (21). Haematin for human use is prepared from HBsAg-negative blood. Preparation and administration of injectable solution Two methods have been employed: the crystals may be dissolved in 0.25~ sodium carbonate, filtered through a 0.22-micron Millipore filter, diluted in 500

232 ml physiological saline and administered intravenously over about three hours (15 cr) or, prior to filtration, the p H o f the haematin-sodium carbonate solution may be brought to 8.0 with 1.5 N HCl and the filtered solution may be administered directly over a 5-minute period or after dilution in physiological saline as before. Both methods result in a sterile, non-pyrogenic material (16 CR ). Mechanism of action in porphyria In acute intermittent porphyria (ALP), as in the rarer forms o f hepatic porphyria, viz. hereditary coproporphyria (HC) and variegate porphyria (VP), there is a partial deficiency o f a single enzyme required for the synthesis o f haemoglobin (22R). The result is a reduced rate o f haeme production with a consequent increase in the activity o f the enzyme ~-aminolaevulinic acid synthetase (ALA synthetase), which is under negative feedback regulation by haeme. Since the enzyme defect in AIP is only partial, the compensatory increase in A L A synthetase activity is sufficient to maintain the normal rate o f haeme production. However, endogenous and exogenous factors (notably drugs such as barbiturates], may temporarily boost the demand for haeme production and at this stage the deficient enzyme becomes the rate-limiting step in haeme synthesis. The relative haeme deficit leads to a sustained de-repression o f A L A synthetase and a resultant build up o f hepatic A L A and porphobilinogen (PBG), which are synthesized above the enzymatic block. In the acute attack, large quantities o f PBG and, to a lesser extent, ALA, are excreted in the urine. Recent advances in the management o f acute porphyria have concentrated on trying to prevent the induction o f hepatic A L A synthetase activity, either by carbohydrate loading (23 R) or by infusion o f haematin (14 CR, 15 Cr, 17 C, 18 cR, 20c). Clinical use of haematin in porphyria In 1971, Bonkowsky et al. (14 c R ) administered intravenous haematin to an anuric patient in a severe attack o f AlP. On repeated administration, there was a striking effect on the serum A L A and PBG, the A L A decreasing from a range o f 7 5 - 1 2 5 lag/lO0 ml to 0 - 1 0 lig/100 ml and the PBG from 5 0 0 - 6 8 0 pg/lO0 ml to 1 2 0 - 3 0 0 fig~100 ml. Unfortunately, the patient died o f renal failure (unrelated to the haematin administra-

D. Walford tion) before it was possible to determine whether it was beneficial for the porphyric relapse. This study was followed by a report o f a rapid and complete remission o f an attack o f AIP in which clinical improvement occurred only after infusion o f haematin had produced a marked decline o f the serum and urinary A L A and PBG (15Cr). The haematin was still detectable in small amounts in the serum at 30 hours, though not at 36 hours after the infusion, and was well tolerated. Five cases o f hepatic porphyria, including three o f AIP, one o f VP and one o f porphyria cutanea tarda symptomatica (PCT) in remission were studied by Jeelani Dhar et al. (16 CR). In two o f the cases with AIP in relapse, infusion o f haematin had a marked lowering effect on serum and urinary PBG and A L A , with prompt improvement o f the clinical condition. The third patient with AIP was in chemical remission but suffering from long-standing psychoneurosis on which haematin administration had no effect. It was also ineffective in the patient with PCT. The patient with VP was almost moribund from the neurological effects o f the disease by the time it was decided to treat with haematin. Treatment was undertaken despite the fact that the urine A L A and PBG levels were negligible at the time, making it doubtful that haematin could be o f benefit in the absence o f biochemical evidence for hepatic A L A synthetase induction. Following the infusion, no change occurred in the high levels o f faecal proto- and coproporphyrin excretion and although the patient's condition improved concomitantly with the haematin, improvement could not be attributed with any degree o f certainty to the treatment. In all cases, the haematin infusions were generally well tolerated. Three o f the patients received a total o f 11 infusions o f unbuffered haematin-sodium carbonate solutions in 400 ml o f physiological saline over 3 - 4 hours. There were four episodes o f thrombophlebitis with or without a mild febrile reaction. In one case, all three infusions were followed by this type o f reaction. It was thought that the thrombophlebitis might have been the result o f the irritant effect of a n alkaline solution infused through a small vein. No thrombophlebitis occurred in those patients receiving solutions buffered to pH 8.0 (see above) through a large antecubital vein, although mild transient febrile reactions were observed, the source o f which might have been the de-ionized water prepared in the

Blood and blood products research laboratory and used in making up the sodium carbonate solution. All subsequent solutions were prepared with 'sterile distilled water for injection, USP" No reaction o f any kind was encountered in two patients who received a total o f nine infusions o f undilu ted buffered haematin-sodium carbonate infusions over 4 - 5 minutes through a large arm vein. In addition, the authors briefly mentioned that haematin produced a transient renal injury in a patient with A l P in remission, o f whom further details are given below. Peterson et al. (17 C) have described the efficacy o f haematin in a patient with A l P in almost fatal relapse. Following progressive deterioration in his condition despite conservative treatment which included glucose infusions, haematin therapy produced marked improvement after a lag o f 42 hours. This was accompanied by a decrease in the levels o f serum PBG and A L A and an increase in their urinary excretion, followed by a sharp fall in the urinary levels. The lagphase preceding the clinical benefit was ascribed to an only partial effect o f the haematin in repressing A L A synthetase in the early part o f the treatment and to the activity o f the enzyme already formed and still available. There was also the possibility o f oscillation in the induction o f A L A synthetase by haematin, as demonstrated by Waxman et al. (24). The only adverse effect experienced by the patient was a mild superficial thrombophlebitis which did not recur when a larger vein was used for later infusions. Further confirmation o f the safety and efficacy o f haematin in AlP has come from McColl et al. (20C), who described a patient who developed a motor neuropathy during an acute attack whilst receiving a high oral glucose regime. The patient's general condition began to improve markedly the day after the last o f her three daily infusions o f haematin. The improvement in her condition paralleled a pronounced reduction in the activity o f leukocyte A L A synthetase and a reduction in the urinary excretion o f ALA. Since the excess A L A can be assumed to have originated from hepatic cells, the authors suggested that the level o f leukocyte A L A synthetase might reflect the activity o f hepatic A L A synthetase. The improvement in the patient's general condition was maintained for seven weeks, at which time she developed a sensory neuropathy. Once again, improvement in her condition followed on the

233 day after the last o f three daily haematin infusions and similar biochemical changes were observed. The haematin infusions produced mild temporary irritation when injected into small peripheral veins, a situation which was made necessary by the fact that most o f the patient's veins had been damaged by past infusions o f glucose. In contrast to the beneficial effect o f haematin in the acute neuropathy described here, haematin has been found o f little benefit in the chronic phase o f the neuropathy (25c). The reported experience o f haematin therapy in erythropoietic porphyria (EP) has been confined to a case o f congenital EP in whom haematin produced repression o f porphyrin biosynthesis in erythrocyte precursors (26C). The haematin evidently entered the precursors in the bone marrow. The time taken to achieve maximal repression o f porphyrin biosynthesis ( 5 - 7 days) was longer than that seen in hepatic porphyria (3 days) and corresponded with the maturation time from the proerythroblast to the circulating reticulocyte stage. With one exception, all the reported adverse effects o f haematin have related to mild febrile reactions and superficial thrombophlebitis. The one exception was an episode o f transient renal failure which followed rapid administration o f a relatively large amount o f haematin to a patient with AIP in clinical remission (18CR). The patient was a volunteer who was taking part in a study designed to provide information concerning the optimum dosage o f haematin required for the suppression o f hepatic A L A synthetase activity. Even in remission the patient's A L A and PBG were consistently elevated and an initial infusion o f 400 mg haematin in 50 ml solution, pH 8.0, over five minutes was well tolerated and produced a marked fall in the biochemical values. Subsequently, he was re-admitted to hospital for the second part o f the study and was given 1000 mg o f haematin in 135 ml o f solution over 15 minutes. The initial phase o f the infusion was uneventful, but after he had received two-thirds o f the dose, the patient complained o f dizziness and tingling and pain in the fingers and toes. His blood pressure remained steady but he developed a transient bradycarclia. The infusion was completed, by which time there was severe colicky abdominal pain and sweating. Thirty minutes later there was a transient rigor followed by a pyrexia o f 101~ which had returned to

D. Walford

234 normal by the next morning. Although the patient remained asymptomatic from that time, it was noticed that he had become oliguric, with a urinary output o f only 200 ml in the 24 hours after the haematin administration and he was found to be uraemic. Initially the urine was almost black but cleared rapidly over the next 36 hours. A bicarbonate infusion was given without any improvement and this was followed by i.v. mannitol and ethacrynic acid, but the urinary output continued to fall. Over the following 12 hours he received repeated injections o f ethacrynic acid and, two days after the haematin infusion, urinary function began to improve with a gradual return o f the blood urea and creatinine levels to normal values. Six days after the haematin administration, a plain abdominal X-ray showed bilaterally enlarged kidneys. The initial dark urine was found to contain 13.8 g/l o f protein, 1 - 2 5 RBC/HPF, 2 - 5 WBC/HPF and granular casts. Haematin was demonstrated in the urine and was present in association with albumin. The infusion o f 1000 mg o f haematin was accompanied by a paradoxical increase in the serum PBG which the authors attributed to the reduced renal clearance resulting from the episode o f acute tubular necrosis. The renal damage was thought to have been caused by unbound haematin, the normal protein binding sites on haemopexin and albumin having been exhausted by the large quantity o f haematin infused and the rapidity o f the infusion in relation to the dose. Certainly, large doses o f haematin have been observed to produce albuminuria and renal casts in dogs (27, 28) and marked toxic reactions, particularly alterations in vascular resistance, haemorrhage and thrombosis and death, in animals given injections too rapidly (28, 29). The patient received haematin at a rate o f 0.8 mg/kg/min. The authors considered that the transitory abdominal distress was caused by renal vasoconstriction, similar to that which has been noted above a certain dosage in dogs (29). They attributed the haematin-containing urine to the e~ccretion o f haematin not bound to plasma haemopexin or albumin. The capacity o f free haematin to produce renal tubular impairment has been clearly demonstrated in vitro by Braun et al. (30) who found that free haematin depressed hippurate transport by rabbit kidney slices whereas methaemalbumin did not. In a preliminary report o f the above case (16 Cj the authors concluded that there was a need

for limitation o f the amount and rate o f haematin infusion. Their policy was to give not more than 4 mg/kg per infusion at a rate not exceeding 40 mg/min, for 6 - 1 0 minutes, with at least a 12-hour interval before a second infusion, the total in a 24-hour period not exceeding 6 mg/kg.

MISCELLANEOUS

Systemic anaphylaxis secondary to the use of 5% plasma protein fraction A severe anaphylactic reaction developed in a 26-year-old woman after administration of only 40 ml of 5% plasma protein fraction (Plasmanate) (31). The reaction was characterized by generalized tingling, chest pain, sudden severe hypotension and urticaria and responded to intravenous fluids, adrenalin, antihistamine and steroids. Plasma protein fractions have been known to produce hypotension, urticaria, chills and fever (see SED 9, Ch. 35), but this is the first documented case of frank anaphylaxis.

Post-transfusion purpura treated by plasma exchange Laursen et al. (32 c R ) have described a case of post-transfusion purpura in a 61year-old multiparous woman whose serum contained the platelet alloantibody antiZWa. Following the first plasma exchange and concomitant treatment with prednisolone, there was a reduction in the titre of antibody from an initial value of 1:64 to 1 : 16 and after the second exchange the titre fell to 1:2. The platelet count began to increase about two days after the second exchange and normal values were reached within 6 days and remained at normal levels. Neuromuscular hyperexcitability due to citrate-induced hypercalcaemia developed during one session on the Haemonetics 30 cell separator and was treated with calcium. The authors concluded that it was not possible to evaluate the role of plasma exchange in their patient's recovery but suggested that repeated plasma exchanges should be tried in patients with post-transfusion purpura, both in an effort to shorten the duration of the thrombocytopenia and to allow studies of the quantity and composition of circulating immune complexes in this condition.

Blood and blood products Risks from multiple infusions of factor VIII concentrates in haemophiliacs Allain(33 R) has reported on a recent symposium which dealt with the risks attendant on the repeated infusion of factor VIII concentrates in haemophiliacs. The incidence of antibodies to factor VIII was given as about 12%, with 20% of patients with antibodies being low responders who could be treated normally. Patients with high titre antibody could be treated by multiple plasma exchanges and porcine factor VIII. The porcine material regularly caused profound thrombocytopenia, but this could be avoided if high-purity concentrate, prepared by the newly developed solid-phase poly-electrolyte method of fractionation, were used. It was reported that the incidence of factor VIII inhibitors was greater in blacks and in subjects of blood group AB, than in other individuals. A multicentre study in America had revealed an age-related increased incidence of hypertension amongst haemophiliacs. Those subjects who had developed hypertension seemed to have received a greater number of infusions than those who did not. 11% of the hypertensives had disturbed renal function, whilst 34% had persistent abnormalities of liver function. Multi-transfused haemophiliacs were known to develop numerous allo-antibodies to serum proteins, the most common being anti-Gm. It was reported that in such individuals, transfusion could be followed by the formation of immune complexes and the activation of complement. The high incidence of hepatitis B and non-A, non-B hepatitis in haemophiliacs was discussed. The demonstration of persistent abnormalities in liver function tests had led several investigators to perform diagnostic liver biopsies but concern was expressed as to the justification of such procedures in haemophiliacs in view of the considerable risks and the lack of direct benefit to the patient.

235 Some further quantitative data on the latter point are provided by a recent retrospective study from the Netherlands (34C) relating to haemophiliacs treated with clotting factor preparations of varying origin. Of 32 haemophilia A patients treated for a total of 128 patient years, 12 showed SGPT elevation, seven of these during a period of more than six months. Of six patients treated with imported factor VIII concentrate prepared from large plasma pools, four showed SGPT elevation within six months. In a series of haemophilia B cases treated with prothrombin-complex concentrates for a total of 38 patient years, two showed a brief and 10 a long period of increased SGPT. The data showed that four-donor cryoprecipitate preparations entailed a relatively smaller risk of hepatitis than prothrombin-complex concentrates and that by far the most dangerous were the commercial high pool factor VIII concentrates.

Immediate reactions with the use of factor VIII and factor IX concentrates

The frequency and character of all immediate reactions to factor VIII and factor IX concentrates in Pennsylvania state have been reviewed(35R). Immediate reactions were of two broad types: allergic and non-specific. The allergic type reaction comprised one or more of the following: excessive lachrymation, periorbital oedema, sensation of warmth, rhinitis and urticaria. The nonspecific symptoms included dizziness, headache and shortness of breath. All reactions occurred either during the infusion or within 5 minutes of the infusion. There were no anaphylactic reactions and no deaths. The factor VIII had been supplied by six different pharmaceutical companies and the factor IX by two different companies. The frequency of reactions was unrelated to any particular commercial source of concentrate. There were nine reactions per 15,948,930 units of factor VIII and two reactions per 3,161,033 units of factor IX. The authors concluded that the risk of an immediate reaction to factor VIII and IX concentrates is remote.

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D. Walford

236 Type B hepatitis after transfusion with blood containing antibody to hepatitis B core antigen. New Engl. J. Med., 298, 1379. 4. Katchaki, J.N., Brouwer, R. and Siem, T. H. (1978): Anti-HBc and blood infectivity (Letter). New Engl. J. Med., 298, 1421. 5. Trepo, C., Hantz, O., Jacquier, M. F., Nemoz, G., Cappel, R. and Trepo, D. (1978): Different fates of hepatitis B virus markers during plasma fractionation. A clue to the infectivity of blood derivatives. Vox Sang., 35,143. 6. Spero, J. A., Lewis, J.H., Van Thiel, D.H., Hasiba, U. and Rabin, B. S. (1978). Asymptomatic structural liver disease in hemophilia. New Engl. J. Med., 298, 1373. 7. Iwarson, S., Lindberg, J. and Lundin, P. (1979): Progression of hepatitis non-A, non-B to chronic active hepatitis. A histological follow-up of two cases. J. clin. Path., 32,351. 8. Wyke, R.J., Tsiquaye, K.N., Thornton, A., White, Y., Portmann, B., Das, P. K., Zuckerman, A.J. and Williams, R. (1979): Transmission of non-A, non-B hepatitis to chimpanzees by factorIX concentrates after fatal complications in patients with chronic liver disease. Lancet, I, 520. 9. Hoofnagle, J.H., Gerety, R.J. and Barker, L.F. (1973): Antibody to hepatitis-B-virus core in man. Lancet, H, 869. 10. Hoofnagle, J. H., Gerety, R.J., Ni, L. Y. and Barker, L.F. (1974): Antibody to hepatitis B core antigen: a sensitive indicator of hepatitis B virus replication. New Engl. J. Med., 290, 1336. 11. World Health Organization (1977): Advances in viral hepatitis. Report of the WHO Expert Committee on Viral Hepatitis. Technical Report Series 602. Geneva. 12. John, T. J., Ninan, G.T., Rajagopalan, M. S., John, F., Flewett, T. H., Francis, D. P. and Zuckerman, A.J. (1979): Epidemic hepatitis B caused by commercial human immunoglobulin. Lancet, 1, 1074. 13. Knodell, R.G., Conrad, M.E. and Ishak, K.G. (1977): Development of chronic liver disease after acute non-A, non-B post-transfusion hepatitis. Role of "r-globulin prophylaxis in its prevention. Gastroenterology, 72,902. 14. Bonkowsky, H. L., Tschudy, D.P., Collins, A., Doherty, J., Bossenmaier, I., Cardinal, R. and Watson, C. J. (1971): Repression of the overproduction of porphyrin precursors in acute intermittent porphyria by intravenous infusions of hematin. Proc. nat. Acad. Sei. USA, 68, 2725. 15. Watson, C. J., Jeelani Dhar, G., Bossenmaier, I., Cardinal, R. and Petryka, Z.J. (1973): Effect of hematin in acute porphyric relapse. Ann. intern. Med., 79, 80. 16. Jeelani Dhar, G., Bossenmaier, I., Petryka, Z.J., Cardinal, R. and Watson, C. J. (1975): Effects of hematin in hepatic porphyria. Further studies. Ann. ir~tern.Med., 79, 80. 17. Peterson, A., Bossenmaier, I., Cardinal R. and Watson, C.J. (1976): Hematin treatment of acute porphyria. Early remission of an almost fatal relapse. J. Amer. reed. Ass., 235, 520.

18. Jeelani Dhar, G., Bossenmaier, I., Cardinal, R., Petryka, Z. J. and Watson, C. J. (1978): Transitory renal failure following rapid administration of a relatively large amount of bematin in a patient with acute intermittent porphyria in clinical remission. Acta. reed. scand., 203,437. 19. Pierach, C.A. and Watson, C.J. (1978): Treatment of acute hepatic porphyria. Lancet, 1, 1361. 20. McCoU, K. E.L., Thompson, G.T., Moore, M. R. and Goldberg, A. (1979): Haematin therapy and leukocyte ~-aminolaevulinic acid-synthetase activity in prolonged attack of acute porphyria. Lancet, 1, 133. 21. Fischer, H. (1955): Heroin. Org. Synth., 3, 442. 22. Meyer, U. A. and Schmid, R. (1978): The porphyrias. In: The Metabolic Basis of Inherited Disease, 4 th Edition, Ch. 50, p. 1166. Editors: J. B. Stanbury, J. B. Wyngaarden, and D. S. Fredrickson. McGraw Hill, New York. 23. Editorial (1978): Treatment of acute hepatic porphyria. Lancet, 1, 1024. 24. Waxman, A.D., Collins, A. and Tschudy, D.P. (1966): Oscillations of hepatic 8-aminolevulinic acid synthetase produced in vivo by heme. Biochem. Biophys. Res. Commun., 24, 675. 25. Bosch, E. P., Pierach, C.A., Bossenmaier, I., Cardinal, R. and Thorson, M. (1977): Effect of hematin in porphyric neuropathy. Neurology, 27, 1053. 26. Watson, C.J., Bossenmaier, I., Cardinal, R. and Petryka, Z. J. (1974): Repression by hematin of porphyrin biosynthesis in erythrocyte precursors in congenital erythropoietic porphyria. Proc.

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Blood and blood products 34. Kunst,V. A. J.M. and Geerdink, P. (1979): SGPT verhogingen bij hemofilie-pati6nten tijdens behandeling met stollingsfactorpreparaten. Ned. T. Geneesk., 123, 1513. 35. Prager, D;, Djerassi, I., Eyster, M.E., Gill,

23 7 F.M., Kajani, N.K., Lewis, J.H., Lusch, C., Rice, S. and Shapiro, S. S. (1979): Pennsylvania state-wide hemophilia programme: summaxy of immediate reactions with the use of factor VIII and factor IX concentrate. Blood, 53, 1012.