HETEROGENEITY OF OSTEOGENESIS IMPERFECTA CONGENITA

820 obtained during convalescences one month after the onset of typical illness with both hxmorrhagic and renal manifestations. The sera were reconstituted and tested by the indirect immunofluorescent test for the presence of antibody to the virus of KHF using three different KHF viral antigen systems-namely, KHF virus infected apodemus mouse lung tissue, Wistar rat lung tissue, and A-549 cells (a transformed continuous cell line derived from a human lung carcinoma). These three antigens were prepared on slides and fixed in acetone for several minutes. Serial four-fold dilutions of each serum, ranging from 1:16 to 1:8192, were reacted on each of these antigens for 30 mins. After washing, fluorescein isothiocyanate conjugated anti-human immunoglobulin was added to the slides and examined under the fluorescence microscope. Positive control serum consisted of a convalescent phase serum from a Korean patient taken 30 days after recovery from KHF virus infection; the titre of this serum was previously shown to be 1:8192. The normal control serum was from a healthy adult living in the United States. All three of the Chinese sera showed positive reactions with the three KHFV antigens. Antibody titres were high and fluorescence was very specific. In each instance the immunofluorescent antibody titres to KHF viral antigens in each of the three patients were 1:4096 with all three antigens. We conclude that HFRS in the People’s Republic of China is caused by a virus antigenically closely related or identical to the KHF virus of Korea, and the HFRS agents of the Soviet Union, Scandinavia, and Japan.

MEAN POTENCIES OF

were

Laboratory of Central Nervous System Studies, National Institute of Neurological and Communicative Disorders and Stroke, National Institutes of Health, Bethesda, Maryland 20205, U.S.A.

PYUNG-WOO LEE D. CARLETON GAJDUSEK

CLARENCE J. GIBBS

Department of Epidemiology,

Shanghai 1st Medical College, Shanghai, China

ZHI-YI XU

ALUMINIUM HYDROXIDE ADSORPTION AND FACTOR VIII CLOTTING ASSAYS

SIR,-Collaborative assays of factor VIII clotting activity have revealed

discrepancy

between results from different

laboratories, especially when plasma and

,

concentrate

are

assayed against each other. Differences between laboratories1 using the same method are largely due to the reagents used,’ and there is a basic 20% difference between the results of onestage and two-stage assays.2 Because of these problems, a working party has been formed by the U.K. hxmophilia centre directors with the object of improving agreement between laboratories. The investigations of the working party have been incorporated into the collaborative studies organised by the National Institute for Biological Standards and Control to calibrate British Standards for factor VIII. An obvious difference between one-stage and two-stage assays is the aluminium hydroxide (Al[OHh) adsorption of plasma, normally regarded as essential to the two-stage method but not done in one-stage assays. We report here the results of a collaborative study carried out by the working party to investigate the contribution of this step to the discrepancy between the two methods. Eight laboratories (four using one-stage methods and four two-stage) took part. Two freeze-dried plasmas were assayed against the International Standard forFactor VIII (concentrate), each laboratory performing two sets of six assays, one with and one without AI(OH)3 adsorption. 1. Kirkwood TBL, Rizza CR, Snape TJ, Rhymes IL, Austen DEG. Identification of sources of inter-laboratory variation in factor VIII assay. Br J Hæmatol 1977; 37: 559-68. 2. Kirkwood TBL, Barrowcliffe TW. Discrepancy between one-stage and twostage assays of factor VIII:C. Br J Hœmatol 1978; 40: 333-38.

8TH

B.S. PLASMA

(IU/AMPOULE)

I

I

All potencies are geometric means from four laboratories carrying out each method. Figures in brackets are geometric coefficients of variation of the meanResults for other plasma in the study were almost identical. * Normal techniques.

were used, including the same Al that the only variable was the presence or absence of the adsorption step. When adsorption was specified, both plasma and concentrate samples were adsorbed, the concentrate being first diluted in hamiophilic plasma. The results are shown in the table. The presence or absence of the Al(OH)3 adsorption step substantially influenced the potency of the plasma, assayed against the concentrate standard. Inclusion of adsorption in the one-stage assays reduced the potency by 8%, and omitting it from the two-stage method increased the potency by 13%. It seems clear that, in this study, the Al(OH)3 adsorption step accounted for rather more-than half of the observed 20% discrepancy between the two assay

Standardised reagents

(OH)3 suspension,

so

methods. Two possible explanations for these results are removal of non-specific clotting activity from plasma by Al(OH)3, and direct adsorption of some factor VIII clotting molecules. Preliminary results with the factor VIII clotting antigen assay (I. R. Peake, unpublished) indicate the latter, and further a

studies of this aspect are in progress. Because of the possibility that some factor VIII molecules are being removed, it would clearly be preferable to omit the AI(OH)3 adsorption procedure from the two-stage method. We found that this could be done by making minor modifications to the assay procedure involving the use of a non-subsampling system, and high dilution of samples. With these simple technical modifications to the two-stage method, it should be possible in future to obtain substantially better agreement between onestage and two-stage assays of factor VIII standards. It remains to be seen, however, whether the two-stage assay without adsorption is applicable to measurement of factor VIII in hsemophilic plasma samples for control of transfusion therapy. We thank the directors and staff of the haemophilia centres in the following hospitals for participating in the collaborative study: Hallamshire Hospital, Sheffield; The London Hospital; Churchill Hospital, Oxford; Royal Free Hospital, London; Royal Infirmary, Manchester ; Royal Infirmary, Edinburgh; Addenbrooke’s Hospital, Cambridge. National Institute for Biological Standards and Control, London NW3 6RB

T. W. BARROWCLIFFE T. B. L. KIRKWOOD

Oxford Hæmophilia Centre

C. R. RIZZA

HETEROGENEITY OF OSTEOGENESIS IMPERFECTA CONGENITA

SIR,-Dr Young and Dr Harper (Feb. 23, p. 432) conclude that osteogenesis imperfecta congenita (OI[C]) is very rarely autosomal recessive. This conclusion may be prematurefirstly, because 01 is biochemically heterogeneous and secondly because decisions by parents of an affected child may influence computed segregation ratios. 01 which presents with spontaneous fractures in utero or within the first five weeks of life is called OI(C)L if there are 3. Kirkwood TBL. Geometric

1979; 35: 908-09.

means

and

measures

of

dispersion.

Biometrics

821 broadened misshapen bones (broad boned lethal) or OI(C)N (non-broad boned lethal) if there are not. Because bone and skin share similar collagens genetically the study of cultured skin fibroblasts and unfixed tissue can provide crucial information about diseased bone. OI(C)L is biochemically heterogeneous. Penttinen et al.l showed that cultured skin fibroblasts produce markedly reduced type I/III collagen ratios. Steinmann and his colleagues2 have proved that this same culture line produces much less collagen than normal but has normal degradation rates. Trelstad et al.3 observed increased amounts of hydroxylysine in skin and bones from a patient with OI(C) whose cultured fibroblasts produced

collagen ratios. We are studying a patient with consanguineous parents who differs from Penttinen and Trelstad’s patients. Biochemical heterogeneity of these parents’ collagens is distinctly possible. Other biochemical lesions await detection in such patients-e.g., procollagen extension peptide abnormalities are possible (D. J. Prokop, personal communication). OI(C)N may be even more variable.

in sporadic osteogenesis imreview. In classical OIC the recurrence rate is 25%. When the affected child is, on clinical grounds, a typical case of the dominantly inherited disorder a new mutation is the likely cause and the risk of recurrence is ’s negligible. In sporadic cases which cannot be classified into either group, it is probably reasonable to suggest an empirical recurrence rate of perhaps 3%.

Competent genetic counselling perfecta demands careful clinical

Department of Biochemical Medicine, Ninewells Hospital and Medical School, C. R. PATERSON

Dundee DD1 9SY

normal

We have

recently described’

a German child with conwho a total has sanguineous parents deficiency of the type I collagen (1.z chain. His parents are biochemically heterogeneous for this gene, producing about half as much a2 chains as nor-

mal. The biochemical study of collagen defects in 01 may evenidentification of heterozygotes and homozygotes in both fetal and adult life, and clinicians caring for such patients can help by providing samples for biochemical investigation. OI(C)L is such a severe disease-and OI(C)N only slightly less so-that families with affected children may be prejudiced against further pregnancies, whatever the quoted genetic risk. This would introduce bias into any ascertainment figures and tend to alter the segregation ratio from 0.25. A reduction in the genetic fitness of heterozygotes could also produce this effect. These possibilities may be relevant to Young and Harper’s survey.

tually permit

Division of Clinical Sciences, Clinical Research Centre, Harrow, Middlesex HA 3UJ

F.M. POPE A. C. NICHOLLS

Dr Pope and Dr Nicholls I was concerned about the letter from Dr Young and Dr Harper. While osteogenesis imperfecta is clearly heterogeneous5 there is little doubt that most cases of classical osteogenesis imperfecta congenita (OIC) are inherited in an autosomal recessive manner, as suggested by Sillence and his colleagues from Australia.1 I am reviewing the genetic counselling given to 272 families with osteogenesis imperfecta, including 66 cases of OIC. In 28 of these the parents were told that further children would not be affected. In 13 of these families further pregnancies occurred ; of a total of 18 pregnancies, 11 normal children, 4 similarly affected children, and 3 spontaneous abortions resulted. This recurrence rate is uncomfortably close to the 25% expected with autosomal recessive inheritance. In none of these cases was there evidence of parental consanguinity, but in 4 additional families OIC resulted from consanguineous mar-

SIR,-Like

riages. 1. Penttinen RP, Lichtenstein JR, Martin GR, McKusick VA. Abnormal collagen metabolism in cultured cells with osteogenesis imperfecta. Proc Natl Acad Sci USA 1975; 72: 586-89. 2. Steinmann BU, Martin GR, Baum BI, Crystal G. Synthesis and degradation of collagen by skin fibroblasts from controls and from patients with osteo-

genesis imperfecta. FEBS Lett 1979; 101: 269-72. 3. Trelstad RL, Rubin D, Gross J. Osteogenesis imperfecta congenita: Evidence for a generalized molecular disorder of collagen. Lab Invest 1977; 36: 501-08. 4. Nicholls AC, Pope FM. Biochemical heterogeneity of osteogenesis imperfecta: a new variant. Lancet 1979; i: 1193. 5. Sillence DO, Senn A, Danks DM. Genetic

perfecta. J Med Genet 1979; 16: 101-16.

heterogeneity

in

osteogenesis

im-

SPLENOMEGALY IN CHILDHOOD LEUKÆMIA

SIR,-We should like to endorse the conclusion of Dr Manoharan and others (March 1, p. 449) that splenomegaly in children with acute lymphoblastic leukxmia (ALL) in hxmatological remission does not usually indicate a relapse. We have seen persistent splenomegaly in at least six children in remission on chemotherapy; this was associated with chronic cytomegalovirus infection in two cases and with hepatic fibrosis, presumably due to drug toxicity, in two others; the cause in the remaining two is still unresolved, but both children remain in haematological remission 7 and 4 years after the splenomegaly was first detected. None has had a splenectomy. One of the patients with cytomegalovirus infection died of cor pulmonale in his first remission, 2 years after the splenomegaly was first noted; the other one’s spleen receded behind the costal margin soon after chemotherapy was stopped 12 years ago, and she remains in complete remission. One boy with hepatic fibrosis had splenomegaly 15 months after the original diagnosis of leukaemia, while in his first remission; he had a bone-marrow relapse over 2 years later, 5 months after stopping chemotherapy, and died of septicaemia during his second remission, the splenomegaly having persisted throughout. The other boy with hepatic fibrosis is still in his first bone-marrow remission, having had splenomegaly since May, 1976. Thus only one of these six children has had a bone-marrow relapse, and this was unrelated to the splenomegaly: in at least four patients the splenomegaly was associated with complications of chemotherapy, which also accounted for both deaths. Department of Hæmatology, Hospital for Sick Children

R. M. HARDISTY JUDITH CHESSELLS

London WC1N 3JH

SIR,-Dr Manoharan and colleagues conclude that splenomegaly in a child in complete remission (CR) from acute lymphoblastic leukxmia (ALL) need not indicate relapse-indeed persistence of an enlarged spleen, they suggest, might represent immunosurveillance against the leukxmia so that splenectomy could be harmful. Other causes of isolated splenomegaly in ALL in CR may be equally important. Case 1.-ALL was diagnosed in this 20-year-old man in 1978. CR was achieved with vincristine and prednisone, and intrathecal methotrexate was given, followed by oral 6-thioguanine maintenance. Biochemical signs of impaired liver function regressed after interrupting, and reappeared after restarting, the 6-thioguanine. The reinduction cycle of June, 1979, could not be given because of severe thrombocytopenia (20 x 109/1) and leucopenia (2.1 x 109/1), not corrected by interrupting maintenance therapy. Bone marrow examination disclosed normal myelopoietic and megakaryopoietic series. A spleen tip was felt just below the costal margin. Biochemical signs of toxic hepatitis were still present, and a scintigram disclosed an enlarged spleen and reduced take-up of tracer by the liver. Platelet studies showed a shortened half-life (8 h) and significant pooling in the spleen. The low cell-counts were thought to be due to hypersplenism and the spleen was removed. It weighed 477 g, but showed no signs of leukxmic infiltration. The liver had

a

nodular

macroscopic

appearance,