Correspondence Saxton, M.J. (1990) The membrane skeleton of erythtocytes; a percolation model. Biophysical lournal, 57, 1 1 67-1 177. Zachee. P.. Boogaerts, M.A., Hellemans, L. & Snauwaert, J. (1992) Adverse role of the spleen in hereditary spherocytosis: evidence by the use of the atomic force microscope. British Iournal o/Haematology, 80, 264-265.
Marques da Costa has made two comments, the first concerning the definition of the membrane irregularities found by the atomic force microscope (AFM) on the erythrocyte surface of unsplenectomized hereditary spherocytosis (HS) patients. In the second comment he made the speculation that our ‘pseudopodia’ could be the onset of bleb formation, which become vesicles and shed away from the cell as shown by Agre (1989). We accept Marques da Costa’sfirst comment and we will in future call our ‘pseudopodia’membrane protrusions. Our data could neither confirm nor reject his second comment. The protrusions with a base diameter of 120 nm by a length of 80 nm were the largest found by the AFM on HS red blood cells (RBCs), which is at least a factor of 5 smaller than the blebs seen by Agre (1989) in the scanning electron microscope. The high resolution of the AFM technique make it possible to detect such protrusions on RBCs of HS patients but only before splenectomy. This confirms the adverse effect
625
of the splenic environment on HS RBCs. Shedding of microvesicles, on the other hand, happens also in normal red blood cells (Bevers et al. 1992). Detailed analysis by the AFM was not able to detect small protrusions, would-be microvesicles, on the erythrocyte membrane of normal or other abnormal RBCs (target cells, echinocytes, sickle cells, acanthocytes).
Department of Haematology, University Hospital, B-3000 Leuven, Belgium Department of Chemistry, University of Leuven, B-3001 Leuven, Belgium
P. ZACHCE M. A. BOOCAERTS
L. HELLEMANS
J. SNAUWAERT
REFERENCES Agre, P. (1989) Partlal deficiencies of erythrocyte spectrin in hereditary spherocytosis. Red Blood Cell Membranes. Hematolog#, Vol. 11 (eds. Agre and Parker), pp. 707-727. Bevers. E.M., Wiedmer, T., Comfurius, P..Shattil, S.J.. Weiss, H.J.. Zwaal. R.F.A.& Sims, P J .( 1 992) Defective Ca2+-inducedmicrovesiculation and deficient expression of procoagulant activity in erythrocytesfrom a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome. Blood, 79, 380-388.
INFUSION OF CLOTTING FACTOR CONCENTRATES IN HAEMOPHILIA I read with interest Dr Evans et al’s research paper, ‘Consistently normal CD4 + , CD8 + levels in haemophilic boys only treated with virally safe factor VIII concentrate (BPL 8Y),’ British journal of Haematology, 1991. 79, 457-461. The paper is clearly of interest and topical at this juncture of evaluating the long-term health potential of individuals with haemophilia in the post-AIDS era. The key issue, of course, is the potential for the frequent infusion of clotting factor concentrates to induce pathophysiologic changes of which the immune and reticuloendothelial systems are prime targets. Research papers such as that presented by Evans and coworkers are of considerableimportance in contributing to the body of knowledge appertaining to this issue. It is of considerable concern when such an article is published which presents biased conclusions and partially presented data. The authors studied a small cohort of 15 boys and observed CD4 changes in one of them. Over time, in presumably a second child, the CD4 number fluctuations was related to the amount of treatment. Also, CD8 numbers were
related to frequency of treatment in two other cases and the ratio of CD4/CD8 changed with the cumulative amount of treatment in one further patient. The conclusion that I come to is that therapy is associated with T cell fluctuations in five of 15 patients studied. However, the authors do not consider these associations ‘significant’and conclude that this therapy shows a lack of effect on all subjects under study in the population evaluated. In addition, comparisons between the haemophilic group and the ‘normal’ control population were not reported in adequate detail, other than the observed age-effect. Also. while the authors mention that blood counts were carried out. they do not report on any of these laboratory observations.
Baxter Healthcare Corporation, Hyland Division, 5 5 0 North Brand Boulevard, Glendale, CA 91203, U.S.A.
EDWARDD. GOMPERTS
MEGADOSE METHYLPREDNISOLONE FOR PURE RED CELL APLASIA Dr Nakao et al’s (1991) patient with pure red cell aplasia responded toelevationofprednisolone from lOmg to 20mg/d in 2 weeks in the presence of cyclosporine (CYa) administration. despite an earlier failure to respond to high-dose methylprednisolone (dose not specified).
Megadose methylprednisolone (MDMP,daily 30 mg/kg for 3 d, 20 mg/kg for 4 d followed by 10, 5 , 2 and 1 mg/kg for 1 week each, each dose given 2-5 min before 9 a m . ) alone was used in a patient with pure red cell anaemia successfully, since this treatment has been used in the treatment of
62 6
Correspondence
Diamond-Blackfan syndrome (t)zsoylu, 1984, 1988). She was given conventional prednisone (2 mg/kg) for 6 months duration previously without any response (c)zsoylu, 1990). Normoblastaemia (2%) with reticulocytosis (3.2%) was observed on the 12th day of MDMP treatment. Haemoglobin increased to 11.34 g/dl (PCV 34%) on the 23rd day and reached 16.4 g/dl (PCV 50%)in 3 months with normoblastic erythroid hyperplasia of the bone marrow. Although spontaneous recovery could not be rejected as in Nakao et al's case, timing of the response may strongly suggest that MDMP was effective in this case. She has been off the treatment over 4 years and still enjoys a haematologically and clinically normal life. Although sustained improvement of the anaemia was not obtained with high-dose methylprednisolone administration in Dr Nakao et al's patient, some reticulocytosis was observed. It might be argued that a complete response would have been obtained if the methylprednisolone dose had been increased. I thought MDMP treatment in red cell aplasia should be emphasized since it is cheaper than other approaches and can be administered orally (c)zsoylu & Ertiirk. 1991). Department of Paediatrics, Hematology. Unit, Hacettepe University Faculty of Medicine and Hacettepe Children's Hospital, 06100 Ankara. Turkey
REFERENCES Nakao. S., Masaoka. H.. Shiobara, S.. Mori. T. & Matsuda T. ( 1 991 ) Dramatic improvement of pure red cell aplasia refractory to combined cyclosporine and prednisolone therapy induced by prednisolone dose escalation. British Iournal oJ Haematologg. 79. 520-521. ozsoylu. S. ( 1 984) High-dose intravenous corticosteroid for a patient with Diamond-Blackfan syndrome refractory to classical prednisone treatment. Acta Haematologica. 71, 207-210. ozsoylu. S. (1 988) High-dose intravenous corticosteroid treatment for patients with Diamond-Blackfan syndrome resistant or refractory to conventional treatment. American journal oJ Pediatric Hematology/Oncologg. 10, 21 7-22 3. h o y l u , S. (1990) High dose intravenous methylprednisolone for pure red cell aplasia. American journal oJHematologg. 34, 236. Ihsoylu, S. & Ertiirk G. ( 1 991) Oral megadose methylprednisolone for childhood acute thrombocytopenic purpura. Blood. 77, 18561857.
SINASIOZSOYLU
CLINICAL STUDY ON DETERMINATION OF SERUM OESTRADIOL IN CHRONIC IDIOPATHIC THROMBOCYTOPENIC PURPURA Chronic idiopathic thrombocytopenia purpura (ch-ITP) generally occurs in young adults, particularly women in the third and fourth decade, with an overall ratio of female to male of 3-4 to 1 which suggests that sex hormones modify and indicate the susceptibility to ch-ITP. Recently 50 patients in our hospital were studied. Their ages ranged from 13 to 79 years with a duration of ch-ITP from 6 months to 20 years. There were no clinical manifestations of effemination in the males and no excision of the ovaries or other organs in the female patients. The control group was composed of 33 healthy volunteers. Serum oestradiol (E2)concentrations were measured by radioimmunoassay. 20 of the patients were analysed by T lymphocyte subgroup, showing a higher proportion of OKT 8-positive cells and a lower proportion of OKT 4-positive cells. The results of the serum E2 concentrations are shown in Table I. Statistically there was a significant difference between the serum E2 concentration of the patients and the controls ( t = 3.12, P < O - O l ) . Among the female patients. there was also a significant difference between climacteric (I) and unclimacteric (11)(t'= 1.196, P
Marques da Costa has made two comments, the first concerning the definition of the membrane irregularities found by the atomic force microscope (AFM) on the erythrocyte surface of unsplenectomized hereditary spherocytosis (HS) patients. In the second comment he made the speculation that our ‘pseudopodia’ could be the onset of bleb formation, which become vesicles and shed away from the cell as shown by Agre (1989). We accept Marques da Costa’sfirst comment and we will in future call our ‘pseudopodia’membrane protrusions. Our data could neither confirm nor reject his second comment. The protrusions with a base diameter of 120 nm by a length of 80 nm were the largest found by the AFM on HS red blood cells (RBCs), which is at least a factor of 5 smaller than the blebs seen by Agre (1989) in the scanning electron microscope. The high resolution of the AFM technique make it possible to detect such protrusions on RBCs of HS patients but only before splenectomy. This confirms the adverse effect
625
of the splenic environment on HS RBCs. Shedding of microvesicles, on the other hand, happens also in normal red blood cells (Bevers et al. 1992). Detailed analysis by the AFM was not able to detect small protrusions, would-be microvesicles, on the erythrocyte membrane of normal or other abnormal RBCs (target cells, echinocytes, sickle cells, acanthocytes).
Department of Haematology, University Hospital, B-3000 Leuven, Belgium Department of Chemistry, University of Leuven, B-3001 Leuven, Belgium
P. ZACHCE M. A. BOOCAERTS
L. HELLEMANS
J. SNAUWAERT
REFERENCES Agre, P. (1989) Partlal deficiencies of erythrocyte spectrin in hereditary spherocytosis. Red Blood Cell Membranes. Hematolog#, Vol. 11 (eds. Agre and Parker), pp. 707-727. Bevers. E.M., Wiedmer, T., Comfurius, P..Shattil, S.J.. Weiss, H.J.. Zwaal. R.F.A.& Sims, P J .( 1 992) Defective Ca2+-inducedmicrovesiculation and deficient expression of procoagulant activity in erythrocytesfrom a patient with a bleeding disorder: a study of the red blood cells of Scott syndrome. Blood, 79, 380-388.
INFUSION OF CLOTTING FACTOR CONCENTRATES IN HAEMOPHILIA I read with interest Dr Evans et al’s research paper, ‘Consistently normal CD4 + , CD8 + levels in haemophilic boys only treated with virally safe factor VIII concentrate (BPL 8Y),’ British journal of Haematology, 1991. 79, 457-461. The paper is clearly of interest and topical at this juncture of evaluating the long-term health potential of individuals with haemophilia in the post-AIDS era. The key issue, of course, is the potential for the frequent infusion of clotting factor concentrates to induce pathophysiologic changes of which the immune and reticuloendothelial systems are prime targets. Research papers such as that presented by Evans and coworkers are of considerableimportance in contributing to the body of knowledge appertaining to this issue. It is of considerable concern when such an article is published which presents biased conclusions and partially presented data. The authors studied a small cohort of 15 boys and observed CD4 changes in one of them. Over time, in presumably a second child, the CD4 number fluctuations was related to the amount of treatment. Also, CD8 numbers were
related to frequency of treatment in two other cases and the ratio of CD4/CD8 changed with the cumulative amount of treatment in one further patient. The conclusion that I come to is that therapy is associated with T cell fluctuations in five of 15 patients studied. However, the authors do not consider these associations ‘significant’and conclude that this therapy shows a lack of effect on all subjects under study in the population evaluated. In addition, comparisons between the haemophilic group and the ‘normal’ control population were not reported in adequate detail, other than the observed age-effect. Also. while the authors mention that blood counts were carried out. they do not report on any of these laboratory observations.
Baxter Healthcare Corporation, Hyland Division, 5 5 0 North Brand Boulevard, Glendale, CA 91203, U.S.A.
EDWARDD. GOMPERTS
MEGADOSE METHYLPREDNISOLONE FOR PURE RED CELL APLASIA Dr Nakao et al’s (1991) patient with pure red cell aplasia responded toelevationofprednisolone from lOmg to 20mg/d in 2 weeks in the presence of cyclosporine (CYa) administration. despite an earlier failure to respond to high-dose methylprednisolone (dose not specified).
Megadose methylprednisolone (MDMP,daily 30 mg/kg for 3 d, 20 mg/kg for 4 d followed by 10, 5 , 2 and 1 mg/kg for 1 week each, each dose given 2-5 min before 9 a m . ) alone was used in a patient with pure red cell anaemia successfully, since this treatment has been used in the treatment of
62 6
Correspondence
Diamond-Blackfan syndrome (t)zsoylu, 1984, 1988). She was given conventional prednisone (2 mg/kg) for 6 months duration previously without any response (c)zsoylu, 1990). Normoblastaemia (2%) with reticulocytosis (3.2%) was observed on the 12th day of MDMP treatment. Haemoglobin increased to 11.34 g/dl (PCV 34%) on the 23rd day and reached 16.4 g/dl (PCV 50%)in 3 months with normoblastic erythroid hyperplasia of the bone marrow. Although spontaneous recovery could not be rejected as in Nakao et al's case, timing of the response may strongly suggest that MDMP was effective in this case. She has been off the treatment over 4 years and still enjoys a haematologically and clinically normal life. Although sustained improvement of the anaemia was not obtained with high-dose methylprednisolone administration in Dr Nakao et al's patient, some reticulocytosis was observed. It might be argued that a complete response would have been obtained if the methylprednisolone dose had been increased. I thought MDMP treatment in red cell aplasia should be emphasized since it is cheaper than other approaches and can be administered orally (c)zsoylu & Ertiirk. 1991). Department of Paediatrics, Hematology. Unit, Hacettepe University Faculty of Medicine and Hacettepe Children's Hospital, 06100 Ankara. Turkey
REFERENCES Nakao. S., Masaoka. H.. Shiobara, S.. Mori. T. & Matsuda T. ( 1 991 ) Dramatic improvement of pure red cell aplasia refractory to combined cyclosporine and prednisolone therapy induced by prednisolone dose escalation. British Iournal oJ Haematologg. 79. 520-521. ozsoylu. S. ( 1 984) High-dose intravenous corticosteroid for a patient with Diamond-Blackfan syndrome refractory to classical prednisone treatment. Acta Haematologica. 71, 207-210. ozsoylu. S. (1 988) High-dose intravenous corticosteroid treatment for patients with Diamond-Blackfan syndrome resistant or refractory to conventional treatment. American journal oJ Pediatric Hematology/Oncologg. 10, 21 7-22 3. h o y l u , S. (1990) High dose intravenous methylprednisolone for pure red cell aplasia. American journal oJHematologg. 34, 236. Ihsoylu, S. & Ertiirk G. ( 1 991) Oral megadose methylprednisolone for childhood acute thrombocytopenic purpura. Blood. 77, 18561857.
SINASIOZSOYLU
CLINICAL STUDY ON DETERMINATION OF SERUM OESTRADIOL IN CHRONIC IDIOPATHIC THROMBOCYTOPENIC PURPURA Chronic idiopathic thrombocytopenia purpura (ch-ITP) generally occurs in young adults, particularly women in the third and fourth decade, with an overall ratio of female to male of 3-4 to 1 which suggests that sex hormones modify and indicate the susceptibility to ch-ITP. Recently 50 patients in our hospital were studied. Their ages ranged from 13 to 79 years with a duration of ch-ITP from 6 months to 20 years. There were no clinical manifestations of effemination in the males and no excision of the ovaries or other organs in the female patients. The control group was composed of 33 healthy volunteers. Serum oestradiol (E2)concentrations were measured by radioimmunoassay. 20 of the patients were analysed by T lymphocyte subgroup, showing a higher proportion of OKT 8-positive cells and a lower proportion of OKT 4-positive cells. The results of the serum E2 concentrations are shown in Table I. Statistically there was a significant difference between the serum E2 concentration of the patients and the controls ( t = 3.12, P < O - O l ) . Among the female patients. there was also a significant difference between climacteric (I) and unclimacteric (11)(t'= 1.196, P
