THROMBOSIS RESEARCH 62; 93-96,199l 0049-3848/91 $3.00 + .OOPrinted in the USA. Copyright (c) 1991 Pergamon Press pk. All rights reserved.
MEGAKARYOCYTE PROGENITORS IN IMMUNE THROMBOCYTOPENIC PURPURA /ITP/ Maria
Podolak-Dawidziak
Department of Haematology , Medical Academy, Wrockaw , Poland
(Received 17.12.1990; accepted in revised form 14.1 .1991 by Editor H.A. Vinazzer)
ABSTRACT Megakaryocyte colony formation was increased in ?3 patients with ITP. Out of splenectomized ITP patients Colony Forming Unit-Megakaryocyte /CFU-Mk/ number normalized in four but was lower than normal in the remaining two. The proportion of immature megakaryocytes was higher in ITP than in normals or in ITP patients after splenectomy. In conclusion, in ITP accelerated platelet destruction leads to stimulatien of megakaryocytopoiesis , but some features of its ineffectiveness can be observed.
INTRODUCTION It is generally accepted that immune thrombocytopenic purpura /ITP/ is due to a plasma factor, which.is an IgG antiplatelet antibody directed towards a platelet and/or megakaryocyte assecia ted antigen /3,2/. The bone marrow responds with an elevatian of the platelet production. However, normal megakaryocyte maturation may be present besides immature cells /3,4/. In this study alterations of megakaryocytopaiesis in ITP were examined using a megakaryocyte colony assay. METIg)DS Megakaryocyte progenitors from 13 patients with ITP, nine females and four males, between 22 and 80 years of age /mean 41.4/ and from 15 haematologically healthy subjects were cultured in vitro. Key words
:
megakaryocyte progenitors , platelets, immune thrombocytopenia , splenectomy 93
MEGAKARYOCME
94
Vol. 62, Nos.
PROGENITORS...
1R
All patients were diagnosed and treated at the Dept.of Haematology Medical Academy, Wroclaw,Poland. Six of thirteen ITP patients resistant -- to pharmacological therapy had undergone splenectomy at the Dept,Of Surgery, Medical Academy, Wrockaw, Poland. They were examined additionally a year after surgery. Bone marrow,and blood samples were investigated at the Dept.of Haematology, University of Wales College of Medicine, Cardiff, U.K. Culture conditions were identical to those reported by Messner et a1./5/. Briefly, the cells were suspended at a final concentration od 2~10~ cells per m in a mixture of Iscove's Modified Dulbecco's Medium /IMDM/, 5x10-5M j3-mercapteaethanol 30% plasma obtained and pooled from a patient with severe aplastii anaemia, 5% phytohaem agglutinin-stimulated leukocyte-conditioned medium /PHA-LCM/ and 0.9% methylcellulose. The mixture /Iml/ was platgd into a 35- mm Petri dish and incubated for 12 to 14 days at 37 in a humified 9 atmosphere. The plates were examined for colonies under an inver ted microscope. Megakaryocytes from ITP and from normal bone marrow were counted per 1000 nucleated cells and classified by the crh teria of Levine et a1./6/ from Stage I /megakaryoblast/ to StageIV /mature megakaryocyte/. Two sets of data were compared using non parametric Mann-Whitney test. RESULTS As shown in Table I the number of CFU-Mk/ITP/ was significantly higher than normal and then diminished after splenectomy. Out of six splenectomized ITP patients in four the number of CFU-Mk nor malized , and in the remaining two was even lower than normal. The mean number of megakaryocytes and the percentage of immature megakaryocytes in ITP patients were markedly increased. After splenectomy the mean megakaryocyte number remained elevated, but the proportion of immature megakaryocytes was reduced. TABLE 1 Platelet Formation in Patients with ITP
Diagnosis
.p
CFU-Mk /per 2 x JO5 cells/ z
SD Statistical significance 1TPvNomal
Megakaryocytes / % marrew cells/ j;
SD
Stages I+11 III+IV
Platelet count x log/L j;
SD
34.8 8.2
X
0.33
0.10
23
77
36.9
29.6
6
35.5
7.1
X
0.32
0.14
27
73
14.4
9.0
b/
6
18.8
3.8
NS
0.32
0.12
12
88
104.2
104.8
NoImal
15
18.3
3.1
0.19
0.06
93
278.0
13
ITP a/
7
79.5
x statisticalsignificance, p(O.001
; ITP, ImmuneThrumbocytopenic Purpura Abbreviations: CFWMLC, ColonyPenningUnit-Megakaryocyte a - before splenectomy, b - after splenectemy
Vol. 62, Nos. 112
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95
DISCUSS10 N
The results in the ITP patients studied has shown an increaued. number of marrow CFU-Mk. Similarly, Sugiyama et al./7/ observed an increased count of CFU-Mk in patients with IT?. However, it varied widely within the normal range in half of the patients in the study. The normal number of megakaryocyte progenitors showing increased cycling activity has been also found in ITP marrows /8/. Fur thermore, the previous study demonstrated in these patients a low plasma ability for stimulating the CFLJ-Mkdevelopment /9/. This s& milar to the reported by Hoffman et al./lO/, but not by Sugiyama et a1./7/. Marrow megakaryocytes were elevated in number and shifted towards immature forms /23%/ ; other authors indicated that After splenectomy, in they can amount to 40% /II/ or even 60% /7/. six IT? patients the number of CFU-Mk showed a tendency to norma lize, and though the megakaryocyte count remained increased they were more mature. Therefore, in ITP patients increased megakaryocyte colony for mation has been found , but in some of them the megakaryocytopoiesis was ineffective. The possible pathophysiologic role played by suppression of the development of megakaryocyte progenitors and/or maturation remains unclear. REFERENCES 1 BORNE, A.E.G.Kr.von dem, HdRMERHORYT, F.M., LEEUWEN , E.F., PE G&S q H.G., RIESZ , E. von and ENGELFRIET , C.P. Autoimmune thro::k bocytopenia : Detection of platelet autoantibodies with the sus pension immunofluorescence test. Br J Haematol,45, 319-327, 1980 2 MC KENNA , J.L. and PISCIOTTA , A.V. Fluorescence of megakaryocytes in idiopathic thrombocytopenic purpura /ITP/ stained with fluorescent antiglobulin serum. Blood , 19 , 664-675, 1962 C.A. Thrombokinetics in man, J Clin 4 MC MILLAN , R., LUIKEN , G.A., LEVY , R., YELENOSKY , R. and MNGMIRE , R.L. Antibody against megakaryocytes in idiopathic thr ombocytopenic purpura. J Am Med ASSOC., a , 2460-2462 , 1978 5 MESSNER , H.A., JAMAL , N. and IZAGUIRRE, C. The growth of large megakaryocyte colonies from human bone marrow. J Cell Physiol. 1, 45-51, 1982 6 LEVINE , R.F., HAZZARD , K.C. and LAMBERG, J.D. The significance of megakaryocyte size, -Blood,60,1122-1131 , 1982 7 SUGIYAMA , H., YAGITA , M., TAKAHASHI , T., NAKAMURA , K., IHG .T S., X)SHIND , T. and IMURA , H. Megakaryocytopoiesis in idiopa thic thrombocytopenic purpura. Acta Haematol JPN , 50, 119-128 , 1987 8 BALLEM, P.J., SEGAL , G.M., STRATTON , J.R., GERNSHEIMER , T., ADAMSON , J.W. and SLICHTER , S.J. Mechanisms of thrombocytope nia in chronic autoimmune thrombocytopenic purpura. J Clin Invest , 80, 33-40, 1987 9 PODOLAK-DAWIDZIAK , M. Ability of plasma from patients with immune thrombocytopenic purpura /ITP/ to promote the growth of mega karyocyte progenitors from normal marrow.Folia Haematol. Leipzig 117, 179-183, 1990
96
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PROGENITORS...
Vol. 62, Nos. l/2
10 HOFFMAN , R., MAZUR , E., BRUNT , E. and FL&YD , V. Assay of an activity in the serum of patients with disorders of thrombopoiesis that stimulates formation of megakaryocytic colonies. N En&l J Med., 305 , 533-538 , 1981 11 BIZANMG-I KUTTI J. and WEINFELD A. Platelet survival and platelet'pr&ction in idiopathic throibocytopenic purpura / ITP/. Br J Haematol , 27 , 127-143 , 1974
MEGAKARYOCYTE PROGENITORS IN IMMUNE THROMBOCYTOPENIC PURPURA /ITP/ Maria
Podolak-Dawidziak
Department of Haematology , Medical Academy, Wrockaw , Poland
(Received 17.12.1990; accepted in revised form 14.1 .1991 by Editor H.A. Vinazzer)
ABSTRACT Megakaryocyte colony formation was increased in ?3 patients with ITP. Out of splenectomized ITP patients Colony Forming Unit-Megakaryocyte /CFU-Mk/ number normalized in four but was lower than normal in the remaining two. The proportion of immature megakaryocytes was higher in ITP than in normals or in ITP patients after splenectomy. In conclusion, in ITP accelerated platelet destruction leads to stimulatien of megakaryocytopoiesis , but some features of its ineffectiveness can be observed.
INTRODUCTION It is generally accepted that immune thrombocytopenic purpura /ITP/ is due to a plasma factor, which.is an IgG antiplatelet antibody directed towards a platelet and/or megakaryocyte assecia ted antigen /3,2/. The bone marrow responds with an elevatian of the platelet production. However, normal megakaryocyte maturation may be present besides immature cells /3,4/. In this study alterations of megakaryocytopaiesis in ITP were examined using a megakaryocyte colony assay. METIg)DS Megakaryocyte progenitors from 13 patients with ITP, nine females and four males, between 22 and 80 years of age /mean 41.4/ and from 15 haematologically healthy subjects were cultured in vitro. Key words
:
megakaryocyte progenitors , platelets, immune thrombocytopenia , splenectomy 93
MEGAKARYOCME
94
Vol. 62, Nos.
PROGENITORS...
1R
All patients were diagnosed and treated at the Dept.of Haematology Medical Academy, Wroclaw,Poland. Six of thirteen ITP patients resistant -- to pharmacological therapy had undergone splenectomy at the Dept,Of Surgery, Medical Academy, Wrockaw, Poland. They were examined additionally a year after surgery. Bone marrow,and blood samples were investigated at the Dept.of Haematology, University of Wales College of Medicine, Cardiff, U.K. Culture conditions were identical to those reported by Messner et a1./5/. Briefly, the cells were suspended at a final concentration od 2~10~ cells per m in a mixture of Iscove's Modified Dulbecco's Medium /IMDM/, 5x10-5M j3-mercapteaethanol 30% plasma obtained and pooled from a patient with severe aplastii anaemia, 5% phytohaem agglutinin-stimulated leukocyte-conditioned medium /PHA-LCM/ and 0.9% methylcellulose. The mixture /Iml/ was platgd into a 35- mm Petri dish and incubated for 12 to 14 days at 37 in a humified 9 atmosphere. The plates were examined for colonies under an inver ted microscope. Megakaryocytes from ITP and from normal bone marrow were counted per 1000 nucleated cells and classified by the crh teria of Levine et a1./6/ from Stage I /megakaryoblast/ to StageIV /mature megakaryocyte/. Two sets of data were compared using non parametric Mann-Whitney test. RESULTS As shown in Table I the number of CFU-Mk/ITP/ was significantly higher than normal and then diminished after splenectomy. Out of six splenectomized ITP patients in four the number of CFU-Mk nor malized , and in the remaining two was even lower than normal. The mean number of megakaryocytes and the percentage of immature megakaryocytes in ITP patients were markedly increased. After splenectomy the mean megakaryocyte number remained elevated, but the proportion of immature megakaryocytes was reduced. TABLE 1 Platelet Formation in Patients with ITP
Diagnosis
.p
CFU-Mk /per 2 x JO5 cells/ z
SD Statistical significance 1TPvNomal
Megakaryocytes / % marrew cells/ j;
SD
Stages I+11 III+IV
Platelet count x log/L j;
SD
34.8 8.2
X
0.33
0.10
23
77
36.9
29.6
6
35.5
7.1
X
0.32
0.14
27
73
14.4
9.0
b/
6
18.8
3.8
NS
0.32
0.12
12
88
104.2
104.8
NoImal
15
18.3
3.1
0.19
0.06
93
278.0
13
ITP a/
7
79.5
x statisticalsignificance, p(O.001
; ITP, ImmuneThrumbocytopenic Purpura Abbreviations: CFWMLC, ColonyPenningUnit-Megakaryocyte a - before splenectomy, b - after splenectemy
Vol. 62, Nos. 112
MEGAKARYOCYTE
PROGENITORS...
95
DISCUSS10 N
The results in the ITP patients studied has shown an increaued. number of marrow CFU-Mk. Similarly, Sugiyama et al./7/ observed an increased count of CFU-Mk in patients with IT?. However, it varied widely within the normal range in half of the patients in the study. The normal number of megakaryocyte progenitors showing increased cycling activity has been also found in ITP marrows /8/. Fur thermore, the previous study demonstrated in these patients a low plasma ability for stimulating the CFLJ-Mkdevelopment /9/. This s& milar to the reported by Hoffman et al./lO/, but not by Sugiyama et a1./7/. Marrow megakaryocytes were elevated in number and shifted towards immature forms /23%/ ; other authors indicated that After splenectomy, in they can amount to 40% /II/ or even 60% /7/. six IT? patients the number of CFU-Mk showed a tendency to norma lize, and though the megakaryocyte count remained increased they were more mature. Therefore, in ITP patients increased megakaryocyte colony for mation has been found , but in some of them the megakaryocytopoiesis was ineffective. The possible pathophysiologic role played by suppression of the development of megakaryocyte progenitors and/or maturation remains unclear. REFERENCES 1 BORNE, A.E.G.Kr.von dem, HdRMERHORYT, F.M., LEEUWEN , E.F., PE G&S q H.G., RIESZ , E. von and ENGELFRIET , C.P. Autoimmune thro::k bocytopenia : Detection of platelet autoantibodies with the sus pension immunofluorescence test. Br J Haematol,45, 319-327, 1980 2 MC KENNA , J.L. and PISCIOTTA , A.V. Fluorescence of megakaryocytes in idiopathic thrombocytopenic purpura /ITP/ stained with fluorescent antiglobulin serum. Blood , 19 , 664-675, 1962 C.A. Thrombokinetics in man, J Clin 4 MC MILLAN , R., LUIKEN , G.A., LEVY , R., YELENOSKY , R. and MNGMIRE , R.L. Antibody against megakaryocytes in idiopathic thr ombocytopenic purpura. J Am Med ASSOC., a , 2460-2462 , 1978 5 MESSNER , H.A., JAMAL , N. and IZAGUIRRE, C. The growth of large megakaryocyte colonies from human bone marrow. J Cell Physiol. 1, 45-51, 1982 6 LEVINE , R.F., HAZZARD , K.C. and LAMBERG, J.D. The significance of megakaryocyte size, -Blood,60,1122-1131 , 1982 7 SUGIYAMA , H., YAGITA , M., TAKAHASHI , T., NAKAMURA , K., IHG .T S., X)SHIND , T. and IMURA , H. Megakaryocytopoiesis in idiopa thic thrombocytopenic purpura. Acta Haematol JPN , 50, 119-128 , 1987 8 BALLEM, P.J., SEGAL , G.M., STRATTON , J.R., GERNSHEIMER , T., ADAMSON , J.W. and SLICHTER , S.J. Mechanisms of thrombocytope nia in chronic autoimmune thrombocytopenic purpura. J Clin Invest , 80, 33-40, 1987 9 PODOLAK-DAWIDZIAK , M. Ability of plasma from patients with immune thrombocytopenic purpura /ITP/ to promote the growth of mega karyocyte progenitors from normal marrow.Folia Haematol. Leipzig 117, 179-183, 1990
96
MEGAKARYOCYTE
PROGENITORS...
Vol. 62, Nos. l/2
10 HOFFMAN , R., MAZUR , E., BRUNT , E. and FL&YD , V. Assay of an activity in the serum of patients with disorders of thrombopoiesis that stimulates formation of megakaryocytic colonies. N En&l J Med., 305 , 533-538 , 1981 11 BIZANMG-I KUTTI J. and WEINFELD A. Platelet survival and platelet'pr&ction in idiopathic throibocytopenic purpura / ITP/. Br J Haematol , 27 , 127-143 , 1974
