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Infantile pyknocytosis: A cause of intrauterine haemolysis in two siblings
310
Pathology (1980), 12, April
HSA AND ASBT
groups of patients at this time. Early deaths, i.e. within the induction period, were by bleeding in 55 patients (357:J, infection in 69 patients (45%) and both in 12 patients (870. The trial was sponsored by the Australian Cancer Society and the National Health and Medical Research Council.
EOSINOPHIL COLONIES IN AGAR
J. MCCARTHY, N. NICHOLA,G. SZELARG, 0. M. CARSON & D. METCALF Cancer Research Unit, Walter und Elizu Hull Institute, Melbourne Using the agar culture system and human placental conditioned medium as a stimulus, we have examined some in vitro characteristics of eosinophil colony forming cells. Eosinophil colonies are compact and first appear after I0 d of culture. They continue t o proliferate while other colony types are dying and at the end of a 3 wk culture period they are the predominant colony type. The number ofeosinophil colonies are linear in relation to the number ofcells cultured and are dependent on CSF for in i d r o proliferation. In bone marrow cultures from 30 patients with nonhaemopoictic disease, the frequency of eosinophil colonies was 15% (range 340’73. Eosinophil progenitor levels were elevated in some cultures from 10 patients with Hodgkin’s disease (mean 25% and 3-67%) and 6 patients with chronic rnyeloid leukaemia 41% range 9-60%. In acute myeloid leukaemia there was no eosinophil colony formation although clusters were observed. In 3 cases of chronic myeloid leukaemia, eosinophil colonies were karyotyped and found to be Philadelphia chromosome positive.
CALCIUM PERMEABILITY IN THE HEREDITARY STOMATOCYTOSIS SYNDROME
J . S. WILm & C. C. SHALLER Department of Haematology, Austin Hospital, Melbourne cinti University O J Pennsylvania, Philadelphia, U . S . A . The syndrome of hereditary stomatocytosis includes a spectrum of mutations ranging from an overhydrated variety with typical stomatocytes and high red cell (Na K) to a dehydrated variety with target cells and low red cell (Na + K). Haemolysis is usually mild but can be severe a t the extremes of over- or under-hydration. Fluxes of Na and K are raised between 3 and 25 fold normal. In this study the specificity of the cation leak has been tested towards the divalent Ca2+cation by 2 types of experiments. First the influx of Ca2+was measured in erythrocytes depleted of ATP by preincubation for 90 min with inosine plus iodoacetate. This procedure inhibits the Ca pump and allows measurable uptake of Ca2+-ionswhen red cells are incubated in media containing 1.5 mM Ca2+.Calcium uptake into overhydrated stomatocytes was 12 nmol/ml cells/4 h and for dehydrated stomatocytes 20-50 nmol/ml cells/4 h. Control cells with comparable reticulocytosis (&lo%) allowed a Ca uptake of 20-30 nmol/ml cells/4 h while normal red cells took up 6 nmol/ml cells/4 h. The low Ca permeability of overhydrated stomatocytes was confirmed in a second type of experiment which involved measurement of K efflux from fresh, ATP-rich cells incubated in media of varying Ca concentrations. Raising the extracellular Ca’+ from 10 to 110 mM stimulated K efflux by 3-fold in normal cells and 5.5-fold in dehydrated stomatocytes. This effect is due to internal Ca stimulating a passive K + channel and has been termed the Gardos effect, Unexpectedly K C efflux from overhydrated stomatocytes was unaffected by extracellular Ca concentration. However, the Ca ionophore (A23187) induced a marked efflux of K + from overhydrated cells equal to that from normals or dehydrated stomatocytes. Thus overhydrated stomatocytes d o possess a Ca-stimulated K channel but these cells seem impermeable to extracellular C a ions. We conclude that overhydrated stomatocytes have a Ca permeability below that of control red cells despite Na and K fluxes which are 25-fold normal. This lack of correlation between Ca and Na permeabilities suggests that Ca enters red cells by a different pathway to the ‘leak’ for monovalent cations.
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INFANTILE PYKNOCYTOSIS: A CAUSE OF INTRAUTERINE HAEMOLYSIS IN TWO SIBLINGS
R. TAMPI,D. J. MAXWELL, J. M. MILLER& R. SESHADRIDepartment of’ Haematology. Flinders Medical Centre, Adelaide Pyknocytosis is a rare cause of haemolytic anaemia in utero and in the neonatal period. Two unusually severe cases of infantile pyknocytosis in 2 siblings are described. The first sibling, a male infant, was delivered prematurely at 37 wk. At birth the child had moderately severe jaundice and hepatosplenomegaly. The cord blood film showed marked pyknocytosis with numerous nucleated red cells. During the first 6 h of life the child developed clinical and
ABSTRACTS OF ANNUAL MEETING
1979 3 1 I
laboratory evidence of disseminated intravascular coagulation. An attempt was made to d o exchange transfusion but the child died 6 h after birth. The autopsy showed evidence of marked extramedullary erythropoiesis consistent with intrauterine haemolytic anaemia. The second infant of the parents was closely monitored during pregnancy as the history in the first infant had suggested severe intrauterine haemolysis. Amniocentesis was performed at 33 wk and 35 wk of gestation and showed an increase in bilirubin suggestive of a moderately severely affected infant. The baby was delivered by caesarean section at 35 wk and at birth showed evidence of haemolytic anaemia. Cord b1ood:haemoglobin 1 I .2 g:dl; PCV 36%; MCV 104 pm3; MCHC 32.7::',; MCH 34 pg; reticulocyte count 21.3%; 126 nucleated red cells per 100 white cells; WCC 12,900 x lo3 and platelet count 273 x 10'. Peripheral blood showed marked pyknocytosis very similar to the blood film of the first infant. The investigations to exclude enzyme deficiencies such as G g P D , P-K. and glutathione reductase were normal. [Hipoproteins were also normal. No Heinz bodies were detected. To control hyperbilirubinaemia, the baby required 3 exchange transfusions. Despite exchange transfusions pyknocytosis persisted with evidence of recurrence of anaemia. The pyknocytosis and haemolytic anaemia persisted until 4 mth of age. A previous report suggested that the erythrocyte abnormality may have resulted from a plasma factor present in the affected infants. However, in our patient red cell morphology was not corrected by incubating the red cells with fresh plasma, nor were normal erythrocytes affected when incubated with the infant's plasma. It would appear therefore that the erythrocyte abnormality is a transient but intrinsic abnormality of red cell membrane.
THE DETECTION OF IRON DEFICIENCY IN CHRONIC DISEASES
H. P. ROESEK Department oj'Medicinr, Unrversify of Qurenslund. Royal Brisbune H o s p i t d , Brisbunr Inflammatory or neoplastic diseases affect the biochemical and haematological parameters generally used to detect iron deficiency (I.D.). Hence, much reliance has been placed on absent stainable iron in marrow fragments and on a positive response to iron therapy in the diagnosis of iron depletion in chronic disease (C.D.). Present evidence indicates that neither method reliably detects I.D. in C.D. In summary: (i) histochemical and chemical determinations of marrow iron content correlate poorly; (ii) absent stainable marrow iron may occur with substantial iron deposits elsewhere; (iii) response to parenterdl iron shows no correlation with other indices of iron stores. Three groups ofpatients with active rheumatoid arthritisand apparent I.D. were compared. Group A (7 patients) was selected purely o n the basis of low serum ferritin levels (6.0 f 2.3 pg/l, mean f I SD). Groups B and C ( 9 and 13 patients) taken from the literature, were selected o n the basis of absent stainable marrow iron. Their serum ferritin levels were 35 f 27 pg/l and 38 f 19 pg/l. Levels of haemoglobin, serum iron and iron saturation did not suggest the presence of I.D. in any group. However. in Group A values for MCV (66.4 f 5.7 fl) and TIBC (83 f 13.4 pmole/l) were characteristic of I.D., whereas in Groups B and C they were intermediate or normal (MCV: 71.8 f 7.4: 78.7 f 8.2 fl: TIBC: 60.5 f 9.1: 71.2 f I I .2 pmole/l).This study supports the view that absent stainable marrow iron in C.D. does not necessarily imply I.D. However, low serum ferritin values ( < 12 pgil) d o indicate I.D. and are usually accompanied by appropriate changes in MCV and TIBC.
EXPERIENCE WITH D.D.A.V.P. IN THE CORRECTION OF BLEEDING TIME IN PATIENTS WITH VON WILLEBRAND'S DISEASE
N. C. MENON,E. W. BEKRY,P. A. OCKELFORD & J. G . BUCHANAN Depurtnzent o f H a e n i a / o l o g ~ ~ . Auckland Hospital, New Zeuland 1-Deamino-8-D-arginine vasopressin (D.D.A.V.P.), a synthetic analoguc of anti-diuretic hormone, when administered intravenously produces a dose related rise in Factor VIII coagulant activity (VIII:C.), Factor VIII related antigen (VIII R:AG) and VIII Ristocetin Cofdctor (V1II:Rcof) in patients with mild haemophilia and von Willebrand's disease. The published studies have shown that D.D.A.V.P. in the dose range .2-.5 pg:kg produces little or n o shortening in bleeding time. Eight patients with classical von Willebrand's disease were studied. Following intravenous administration of D.D.A.V.P. in a dose range .3-.6 pg/kg body weight, a shortening of bleeding time was observed in all cases. Six of these patients were studied in an attempt t o demonstrate a correlation between the shortening of the bleeding time and rise in Factor VIII:C, Factor VIII antigen, and VIII Rcof. This reduction in bleeding time, which lasted 3 to 5 h in the 6 patients who were serially studied, is related to increases in all Factors measured. although only Factor
Pathology (1980), 12, April
HSA AND ASBT
groups of patients at this time. Early deaths, i.e. within the induction period, were by bleeding in 55 patients (357:J, infection in 69 patients (45%) and both in 12 patients (870. The trial was sponsored by the Australian Cancer Society and the National Health and Medical Research Council.
EOSINOPHIL COLONIES IN AGAR
J. MCCARTHY, N. NICHOLA,G. SZELARG, 0. M. CARSON & D. METCALF Cancer Research Unit, Walter und Elizu Hull Institute, Melbourne Using the agar culture system and human placental conditioned medium as a stimulus, we have examined some in vitro characteristics of eosinophil colony forming cells. Eosinophil colonies are compact and first appear after I0 d of culture. They continue t o proliferate while other colony types are dying and at the end of a 3 wk culture period they are the predominant colony type. The number ofeosinophil colonies are linear in relation to the number ofcells cultured and are dependent on CSF for in i d r o proliferation. In bone marrow cultures from 30 patients with nonhaemopoictic disease, the frequency of eosinophil colonies was 15% (range 340’73. Eosinophil progenitor levels were elevated in some cultures from 10 patients with Hodgkin’s disease (mean 25% and 3-67%) and 6 patients with chronic rnyeloid leukaemia 41% range 9-60%. In acute myeloid leukaemia there was no eosinophil colony formation although clusters were observed. In 3 cases of chronic myeloid leukaemia, eosinophil colonies were karyotyped and found to be Philadelphia chromosome positive.
CALCIUM PERMEABILITY IN THE HEREDITARY STOMATOCYTOSIS SYNDROME
J . S. WILm & C. C. SHALLER Department of Haematology, Austin Hospital, Melbourne cinti University O J Pennsylvania, Philadelphia, U . S . A . The syndrome of hereditary stomatocytosis includes a spectrum of mutations ranging from an overhydrated variety with typical stomatocytes and high red cell (Na K) to a dehydrated variety with target cells and low red cell (Na + K). Haemolysis is usually mild but can be severe a t the extremes of over- or under-hydration. Fluxes of Na and K are raised between 3 and 25 fold normal. In this study the specificity of the cation leak has been tested towards the divalent Ca2+cation by 2 types of experiments. First the influx of Ca2+was measured in erythrocytes depleted of ATP by preincubation for 90 min with inosine plus iodoacetate. This procedure inhibits the Ca pump and allows measurable uptake of Ca2+-ionswhen red cells are incubated in media containing 1.5 mM Ca2+.Calcium uptake into overhydrated stomatocytes was 12 nmol/ml cells/4 h and for dehydrated stomatocytes 20-50 nmol/ml cells/4 h. Control cells with comparable reticulocytosis (&lo%) allowed a Ca uptake of 20-30 nmol/ml cells/4 h while normal red cells took up 6 nmol/ml cells/4 h. The low Ca permeability of overhydrated stomatocytes was confirmed in a second type of experiment which involved measurement of K efflux from fresh, ATP-rich cells incubated in media of varying Ca concentrations. Raising the extracellular Ca’+ from 10 to 110 mM stimulated K efflux by 3-fold in normal cells and 5.5-fold in dehydrated stomatocytes. This effect is due to internal Ca stimulating a passive K + channel and has been termed the Gardos effect, Unexpectedly K C efflux from overhydrated stomatocytes was unaffected by extracellular Ca concentration. However, the Ca ionophore (A23187) induced a marked efflux of K + from overhydrated cells equal to that from normals or dehydrated stomatocytes. Thus overhydrated stomatocytes d o possess a Ca-stimulated K channel but these cells seem impermeable to extracellular C a ions. We conclude that overhydrated stomatocytes have a Ca permeability below that of control red cells despite Na and K fluxes which are 25-fold normal. This lack of correlation between Ca and Na permeabilities suggests that Ca enters red cells by a different pathway to the ‘leak’ for monovalent cations.
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INFANTILE PYKNOCYTOSIS: A CAUSE OF INTRAUTERINE HAEMOLYSIS IN TWO SIBLINGS
R. TAMPI,D. J. MAXWELL, J. M. MILLER& R. SESHADRIDepartment of’ Haematology. Flinders Medical Centre, Adelaide Pyknocytosis is a rare cause of haemolytic anaemia in utero and in the neonatal period. Two unusually severe cases of infantile pyknocytosis in 2 siblings are described. The first sibling, a male infant, was delivered prematurely at 37 wk. At birth the child had moderately severe jaundice and hepatosplenomegaly. The cord blood film showed marked pyknocytosis with numerous nucleated red cells. During the first 6 h of life the child developed clinical and
ABSTRACTS OF ANNUAL MEETING
1979 3 1 I
laboratory evidence of disseminated intravascular coagulation. An attempt was made to d o exchange transfusion but the child died 6 h after birth. The autopsy showed evidence of marked extramedullary erythropoiesis consistent with intrauterine haemolytic anaemia. The second infant of the parents was closely monitored during pregnancy as the history in the first infant had suggested severe intrauterine haemolysis. Amniocentesis was performed at 33 wk and 35 wk of gestation and showed an increase in bilirubin suggestive of a moderately severely affected infant. The baby was delivered by caesarean section at 35 wk and at birth showed evidence of haemolytic anaemia. Cord b1ood:haemoglobin 1 I .2 g:dl; PCV 36%; MCV 104 pm3; MCHC 32.7::',; MCH 34 pg; reticulocyte count 21.3%; 126 nucleated red cells per 100 white cells; WCC 12,900 x lo3 and platelet count 273 x 10'. Peripheral blood showed marked pyknocytosis very similar to the blood film of the first infant. The investigations to exclude enzyme deficiencies such as G g P D , P-K. and glutathione reductase were normal. [Hipoproteins were also normal. No Heinz bodies were detected. To control hyperbilirubinaemia, the baby required 3 exchange transfusions. Despite exchange transfusions pyknocytosis persisted with evidence of recurrence of anaemia. The pyknocytosis and haemolytic anaemia persisted until 4 mth of age. A previous report suggested that the erythrocyte abnormality may have resulted from a plasma factor present in the affected infants. However, in our patient red cell morphology was not corrected by incubating the red cells with fresh plasma, nor were normal erythrocytes affected when incubated with the infant's plasma. It would appear therefore that the erythrocyte abnormality is a transient but intrinsic abnormality of red cell membrane.
THE DETECTION OF IRON DEFICIENCY IN CHRONIC DISEASES
H. P. ROESEK Department oj'Medicinr, Unrversify of Qurenslund. Royal Brisbune H o s p i t d , Brisbunr Inflammatory or neoplastic diseases affect the biochemical and haematological parameters generally used to detect iron deficiency (I.D.). Hence, much reliance has been placed on absent stainable iron in marrow fragments and on a positive response to iron therapy in the diagnosis of iron depletion in chronic disease (C.D.). Present evidence indicates that neither method reliably detects I.D. in C.D. In summary: (i) histochemical and chemical determinations of marrow iron content correlate poorly; (ii) absent stainable marrow iron may occur with substantial iron deposits elsewhere; (iii) response to parenterdl iron shows no correlation with other indices of iron stores. Three groups ofpatients with active rheumatoid arthritisand apparent I.D. were compared. Group A (7 patients) was selected purely o n the basis of low serum ferritin levels (6.0 f 2.3 pg/l, mean f I SD). Groups B and C ( 9 and 13 patients) taken from the literature, were selected o n the basis of absent stainable marrow iron. Their serum ferritin levels were 35 f 27 pg/l and 38 f 19 pg/l. Levels of haemoglobin, serum iron and iron saturation did not suggest the presence of I.D. in any group. However. in Group A values for MCV (66.4 f 5.7 fl) and TIBC (83 f 13.4 pmole/l) were characteristic of I.D., whereas in Groups B and C they were intermediate or normal (MCV: 71.8 f 7.4: 78.7 f 8.2 fl: TIBC: 60.5 f 9.1: 71.2 f I I .2 pmole/l).This study supports the view that absent stainable marrow iron in C.D. does not necessarily imply I.D. However, low serum ferritin values ( < 12 pgil) d o indicate I.D. and are usually accompanied by appropriate changes in MCV and TIBC.
EXPERIENCE WITH D.D.A.V.P. IN THE CORRECTION OF BLEEDING TIME IN PATIENTS WITH VON WILLEBRAND'S DISEASE
N. C. MENON,E. W. BEKRY,P. A. OCKELFORD & J. G . BUCHANAN Depurtnzent o f H a e n i a / o l o g ~ ~ . Auckland Hospital, New Zeuland 1-Deamino-8-D-arginine vasopressin (D.D.A.V.P.), a synthetic analoguc of anti-diuretic hormone, when administered intravenously produces a dose related rise in Factor VIII coagulant activity (VIII:C.), Factor VIII related antigen (VIII R:AG) and VIII Ristocetin Cofdctor (V1II:Rcof) in patients with mild haemophilia and von Willebrand's disease. The published studies have shown that D.D.A.V.P. in the dose range .2-.5 pg:kg produces little or n o shortening in bleeding time. Eight patients with classical von Willebrand's disease were studied. Following intravenous administration of D.D.A.V.P. in a dose range .3-.6 pg/kg body weight, a shortening of bleeding time was observed in all cases. Six of these patients were studied in an attempt t o demonstrate a correlation between the shortening of the bleeding time and rise in Factor VIII:C, Factor VIII antigen, and VIII Rcof. This reduction in bleeding time, which lasted 3 to 5 h in the 6 patients who were serially studied, is related to increases in all Factors measured. although only Factor