T C G Case Reports 127

A

T G

A C C

- G A

Fig 1A. DCGE analysis of fragment C of the /&globin gene amplified from an individual heteroqgous for P-globin gene framework 1 and 3 (lane 1 ) and an individual homozygous for framework 1 (lane 2). SamplesC (Pjy mutation) and P (patient) show a band faster than the normal heteroduplex. compatible with a destabilizing mutation.

KEFERENCES

Boehm. C.. Ilowling. C.E.. Waber. P.G., Giardina. J.V. & Kazazian. H.H.. ]r (1986) llse of oligonucleotide hybridization in the characterization of a p-thalassemia gene (/l”TGC+TCA) in a Saudi Arabian family. Blood. 67, 1185-1 188.

Goossens. M.. Fanen. P.. Attree. 0. & Vidaud. M. (1990) A new strategy for direct detection of b-thalassemia mutations. Annals the New York Academ!j of Science. 612, 74-80.

1 2 3 Fig 1B. DNA sequence autoradiograph of part of fragment C. Lane 1: patient: lane 2: control (p39 heterozygous): lane 3: normal control. A G to A substitution is shown in lane 1 at the third nucleotide of codon 37.

Gyllensten. U.B. & Erlich, H.A. ( 1 988) Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proceedings of the National AcademyofSciencesofthe UnitedStatesofAmericn. 85. 7652-7656.

TREATMENT OF SEVEKE APLASTIC ANAEMIA WITH TOTAL LYMPHOID IRRADIATION AND METHY LPKEDNISOLONE

The majority of patients with severe aplastic anaemia (SAA) have T-cell-mediated bone marrow suppression (Ascensao et al, 1976). Thus while bone marrow transplantation (BMT) remains the therapy of choice for SAA, various modalities of immunosuppression such as antithymocyte globulin and corticosteroids have been tried with varying success (Baciga- lupo et a / . 1988). Cyclosporine has been reported to induce a complete haematologic remission in SAA at full immunosup- pressive doses (Dundar & Damirkezik, 1990) or a t a low dose (Mehta et a/, 1992).

Total lymphoid irradiation (TLI) results in lasting immuno- suppression through a selective reduction in the number and function of helper T-cells and certain subsets of B-cells (Strober, 198 7). TIJ-induced immunosuppression has

resulted in long-term survival of allografts in experimental animals (Slavin et al. 1978). and marked improvement in autoimmune diseases such as rheumatoid arthritis (Trent- ham et al. 198 1) and lupus nephritis (Strober et al, 1985).

A 12-year-old female with known trisomy 2 1 and Down’s syndrome was admitted with a purpuric rash and vaginal bleeding of 2 d duration. She had fever 1 5 d before for which she had received empiric co-trimoxazole and chloroquine. Examination revealed fever, pallor, generalized purpuric eruption, and vaginal bleeding. There was no organomegaly or lymphadenopathy. Investigations revealed: haemoglobin 5.2 g/dl. leucocytes 1 * 1 x 1 09/1 (1 5% granulocytes), reticulo- cytes 0.1%. platelets 10 x 109/1, and a markedly hypocellular bone marrow on trephine biopsy. Ham’s acidified serum test

128 Case Reports was negative. Renal and hepatic function tests were within normal limits. No source of infection could be demonstrated on chest X-ray, and blood and urine cultures were negative. She was treated with transfusions of packed red cells and platelet concentrates, and broad-spectrum antibiotics. TWO days after admission, she had massive gastrointestinal haemorrhage as a result of which the haemoglobin decreased to 4 g/dl. Due to lack of availability of a histocompatible sibling for BMT, it was decided to try immunosuppression with total lymphoid irradiation (TLI) and intravenous meth- ylprednisolone.

The patient was given 800 cGy TLI in four fractions of 200 cGy each over 2 d followed by intravenous methylpredniso- lone at the following doses: 30 mg/kg/d x 3 d, 20 mg/kg/ d x 4 d, 10 mg/kg/d x 4 d, and 5 mg/kg/d x 4 d. On the fourth day of therapy the investigations showed: haemoglobin 7.3 g/dl. leucocytes 1.5 x 10y/l (50% granulocytes), platelets 90 x 10y/l, and reticulocytes 0.5%. At the end of 15 d of intravenous steroid administration the counts were: hae- moglobin 8 g/dl. leucocytes 2 . 7 x 10y/l (granulocytes 70%), platelets 180 x 10y/l, and reticulocytes 1%. The patient received 4 weeks of oral prednisolone 1 mg/kg after cessation of the intravenous methylprednisolone. At the end of all therapy the counts were: haemoglobin 11.5 g/dl, leucocytes 4.5 x 10y/l (granulocytes 71%). platelets 2 3 0 x 109/1, and reticulocytes 1 * 5%. The patient is asymptomatic and haema- tologically normal 6 months after therapy.

Long-term follow-up of patients of aplastic anaemia who achieved complete haematologic remission with immuno- suppression has shown late development of myelodysplasia and acute myeloid leukaemia (De Planque et al. 1988). Because aplastic anaemia is a stem cell disorder, the basic anomaly can be rectified only with BMT. However, BMT is not a suitable option in a number of patients due to lack of availability of suitable donors.

While the duration of the follow-up is still short, the preliminary result in this patient seems to be highly encour- aging. We did not encounter any serious adverse reaction to the irradiation and the high-dose methylprednisolone. In our opinion, TLI and methylprednisolone should be considered a therapeutic option in patients with SAA who are not candidates for BMT or antithymocyte globulin, or those who

have failed one course of therapy with antithymocyte globulin.

'Blood Research Centre, Vivina Bldg 3A, S V Road, Bombay 400 058, and Departments of

4Haematology, Nanavati Hospital and Research Centre, Bombay 400 056, India

JAYFSH MEHTA' SEFMA SINCHAL'* NACRAJ HUILGOL~

B. c . MEHTA'.4 2Radiotherapy, 'Pediatrics and RASHID MERCHANT'

REFERENCES

Ascensao, J., Pahwa. R.. Kagan, W., Hansen. J.. Moore, M. &Good. R. ( 1976) Aplastic anaemia: evidence for an immuno\ogica\mechan- ism. Lancet, i, 669-671.

Bacigalupo, A.. Hows. J.. Gluckman. E., Nissen, C.. Marsh. J.. Van Lint, M.T.. Congiu, M.. De Planque. M.M., Ernst. P.. McCann. S.. Raghavashar. A.. Frickhofen, N., Wursch. A., Marmont. A.M. & Gordon-Smith, E.C. ( 1988) Bone marrow transplantation (BMT) versus immunosuppression for the treatment of severe aplastic anaemia (SAA): a report of the EBMT SAA Working Party. British Iournal ol Haematolog& 70. 177-182.

De Planque. M.M., Kluin-Nelemans, H.C.. Van Krieken. H.J.M., Kluin. P.M., Brand, A., Beverstock. G.C.. Willemze. R. & Van Rood, J J . (1988) Evolution of acquired severe aplastic anaemia to myelodysplasia and subsequent leukaemia in adults. British journal OJ Haematology. 70. 5 5-62.

Dundar. S. & Damirkezik, A. (1990) Cyclosporin for refractory aplastic anaemia. Acta Haematologica, 83. 112.

Mehta. J., Singhal. S. & Mehta, B.C. ( 1 992) Low-dose cyclosporine in severe aplastic anemia. American journal of Hematology (in press).

Slavin. S.. Reitz. B., Bieber. C.P.. Kaplan, H.S. & Strober. S. (1978) Transplantation tolerance in adult rats using total lymphoid irradiation (TLI): permanent survival of skin. heart, and marrow allografts. journal OJ Experimental Medicine, 147, 700-707.

Strober. S. (1987) Total lymphoid irradiation in alloimmunity and autoimmunity. journal ~ J h d i a t r i c s . 111, 1051-1055.

Strober, S., Field, E., Hoppe. R.T., Kotzin, B.L.. Shemesh, 0.. Engelman. E.. Ross. J.C. & Myers, B.D. (1985) Treatment of intractable lupus nephritis with total lymphoid irradiation. Annals oJInternal Medicine. 102, 450-458.

Trentham, D.E.. Belli, J.A.. Anderson, R.J.. Buckley, J.A.. Goetzl. E.J.. David, I.R. & Austen. K.F. ( 1 981) Clinical and immunologic effects of fractionated total lymphoid irradiation in refractory rheumatoid arthritis. New England journal oJ Medicine. 305 , 976-982.

USE OF RECOMBINANT HUMAN GRANULOCYTE COLONY STIMULATING FACTOR IN RETICULAR DYSGENESIS

Reticular dysgenesis (RD) is an extremely rare disease classified within the severe combined immunodeficiencies. Since only lymphoid and myeloid cell development is impaired, it has been advocated that this disorder might be due to a failure of differentiation and proliferation of a precursor cell distinct from the totipotent haematopoietic stem cell (Moore, 1991: Rosen et al, 1985). In idiopathic congenital neutropenia recombinant granulocyte-colony sti- mulating factor (rG-CSF) has been shown to increase the number of myeloid cells (Bonilla et al, 1989: Welte et al, 1990). Such treatment was unsuccessful when applied to our patient suffering from RD.

A female newborn from consanguinous parents was treated for a suspected septicaemia diagnosed 2 4 h after delivery. At 1 1 d of age the haemoglobin was 12.9 g/dl. the platelet count was 4 9 7 x 10y/l, the white cells count was 0 . 7 x 109/1 with 0% neutrophils, 80% lymphocytes and 20% monocytes. A granulocytic alloimmunization test was nega- tive. A bone marrow examination showed a large number of undifferentiated cells with a blockage in maturation at the promyelocytic stage. However. cells of erythroid and mega- karyocytic origin were present at all stages of maturation. Bone marrow immunophenotype was: &A-DR. 50.3%: CD10. 13.8%; and a large panel of T (CD2.3.4, 5.6. 7.8) . B