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Stem Cells, Vol. 14, No. 5, 558-565, September 1996
© 1996 AlphaMed Press

ORIGINAL PAPER

Serum Thrombopoietin Level in Various Hematological Diseases

Kensuke Usukia, Tomoyuki Taharab, Seiko Ikia, Mitsue Endoa, Mayumi Osawaa, Koichi Kitazumea, Takashi Katob, Hiroshi Miyazakib, Akio Urabea

a Division of Hematology, Kanto Teishin Hospital, Tokyo, Japan;
b Pharmaceutical Research Laboratory, Kirin Brewery Co., Ltd., Gunma, Japan

Key Words. Thrombopoietin • Serum level • Myelosuppressive state • Leukemia • Malignant lymphoma • Idiopathic thrombocytopenic purpura • Multiple myeloma • Myelodysplastic syndrome • Aplastic anemia • Myeloproliferative disorders • Liver cirrhosis

Correspondence: Dr. Akio Urabe, Division of Hematology, Kanto Teishin Hospital, 5-9-22 Higashi-Gotanda, Shinagawa-ku, Tokyo 141, Japan.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
To investigate the pathophysiological role of thrombopoietin (TPO) in thrombopoiesis, we measured its serum levels in 15 healthy individuals, 84 patients with various hematological diseases and 2 patients with liver cirrhosis using an enzyme immunoassay procedure. The TPO level was 0.84 ± 0.40 f mol/ml in normal individuals. TPO levels were considerably elevated in patients with myelosuppression after intensification chemotherapy of acute leukemia in complete remission (postchemotherapy group; n = 18; 18.46 ± 9.70 f mol/ml). When the data of normal individuals and the postchemotherapy group were combined, TPO levels were inversely correlated with the platelet count in this combined group. We compared these data of normal individuals and the postchemotherapy group with various hematological disease states. In aplastic anemia (n = 13; 16.03 ± 9.44 f mol/ml), acute lymphoblastic leukemia (n = 5; 10.36 ± 5.57 f mol/ml), malignant lymphoma (n = 6; 2.79 ± 2.27 f mol/ml), multiple myeloma (n = 3; 3.34 ± 0.20 f mol/ml) and chronic lymphocytic leukemia (n = 2; 1.71 ± 3.91 f mol/ml), the relationship of serum TPO levels and platelet counts was almost the same as in the combined group with normal individuals and the postchemotherapy group. However, the TPO levels were slightly higher in myeloproliferative disorders (n = 12; 1.99 ± 1.47 f mol/ml) and lower in acute myelogenous leukemia (n = 8; 2.27 ± 1.25 f mol/ml), hypoplastic leukemia (n = 3; 2.76 ± 2.23 f mol/ml), myelodysplastic syndrome (n = 2; 0.42 ± 0.60 f mol/ml), liver cirrhosis (n = 2; 1.50 ± 0.92 f mol/ml) and idiopathic thrombocytopenic purpura (n = 12; 2.08 ± 1.41 f mol/ml), when compared to the regression line for the combined group with normal individuals and postchemotherapy group. These findings suggest that TPO might play an important role in regulation of the platelet count in normal and pathological conditions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The regulation of platelet production is critical for maintenance of hemostasis. Inadequate megakaryopoiesis and/or thrombopoiesis can lead to serious bleeding. The humoral factors regulating these processes have been the subject of study for several decades [1]. Although several cytokines [1, 2] including interleukin (IL)-3 [3, 4], erythropoietin (EPO) [5-7], IL-6 [8, 9] and IL-11 [10-12] have been shown to influence megakaryocyte development and platelet production, none appeared to do so in a lineage-dominant fashion analogous to the situation of EPO in erythrocyte and G-CSF in neutrophil production. Thrombopoietin (TPO) was recently purified [13] and its DNA has been cloned [14, 15]. Several other groups independently cloned the cDNA of the ligand of c-Mpl product [16-19], and its cDNA sequence was found to be identical to that of TPO. This cytokine stimulates the growth of megakaryocyte colonies and the differentiation of megakaryocyte progenitor cells in vitro [20-22], and enhances platelet production in vivo [17, 23-25]. In thrombocytopenic animals treated with a myelosuppressive agent, the TPO was reported to increase inversely as the platelet mass declined [26]. However, the regulation of TPO production in hematological diseases is unknown.

Recently, Tahara et al., developed an enzyme-linked immunosorbent assay (ELISA) specific for human TPO [27]. In order to investigate the regulation of TPO production, we determined serum levels of this cytokine in healthy volunteers and in patients with various hematological diseases and liver cirrhosis (LC).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Patients and Blood Samples
Blood samples were simultaneously collected to determine the hematological parameters, C-reactive protein (CRP) levels and TPO levels, after obtaining informed consent. The mean value and standard deviation of the hematological and CRP data for each group are listed in Table 1Go. Serum samples were separated by centrifugation. Blood was obtained from 101 subjects, including 15 healthy volunteers. Eighteen patients had a myelosuppression phase after intensified chemotherapy for acute leukemia in complete remission, and nine of them had received G-CSF. Seventeen patients received platelet transfusions within one week prior to the collection of blood samples. Twelve patients had idiopathic thrombocytopenic purpura (ITP): two of them were studied at initial diagnosis, one patient had undergone splenectomy and two were on steroid therapy. Thirteen patients had aplastic anemia (AA): two of them were studied at initial diagnosis, one patient had received G-CSF (Filgrastim, Kirin; Tokyo, Japan), three had received anabolic steroid therapy, six had received red blood cell transfusions and four had received antithymocyte globulin (Atgam, Upjohn Japan; Tokyo, Japan). Twelve patients had myeloproliferative disorders (MPD); two of them had polycythemia vera (PV) and had been treated with intermittent phlebotomy. Another five patients had chronic granulocytic leukemia (CGL); two of them were studied at initial diagnosis, while three others had received interferon {alpha} (Sumiferon, Sumitomo; Tokyo, Japan) and hydroxyuera (Hydrea, Bristol-Meyer-Squibb Japan; Tokyo, Japan). Five patients had essential thrombocythemia (ET), diagnosed according to the criteria of the PV study group [28]: two of them had received interferon {alpha} and three others had been given intermittent ranimustine (Cymerin, Tokyo-Tanabe; Tokyo, Japan) therapy. Eight patients were studied at the time of diagnosis of acute myelogenous leukemia (AML). According to the French-American-British classification [29-32], there was one patient with M0, three with M1, one with M3, one with M4 and two with overt leukemia developing from myelodysplastic syndrome (MDS). Six patients were studied at the initial diagnosis of malignant lymphoma: one had Hodgkin's disease, one had cutaneous T cell lymphoma (Sézary syndrome) and four had non-Hodgkin's lymphoma (diffuse large cell type, B cell). Two patients with MDS, three patients with multiple myeloma and two patients with B cell chronic lymphocytic leukemia (CLL) were also studied at initial diagnosis. The three patients with hypoplastic leukemia studied had received blood transfusions and intermittent chemotherapy, while the two patients with LC had not been on any treatment.


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  		Table 1. Hematological parameters, CRP concentration and serum TPO concentrations
 
Measurement of TPO Levels
Serum samples were stored at –70°C until analysis (range: four months to three years). TPO levels were determined by ELISA as described elsewhere [27]. Briefly, each well of 96-well flat-bottomed microtiter plates (Maxisorp, Nunc; Roskilde, Denmark) was coated at 4°C overnight with 100 µl of monoclonal antibody against recombinant human (rHu) TPO, termed TN1 [27], at a concentration of 10 µg/ml in 50 mM carbonate buffer, pH 9.4. After washing with 20 mM Tris-HCl containing 0.5 M NaCl and 1.0% NaN3, pH 7.5 (TBS), 200 µl of a blocking reagent (Super block in TBS PIERCE; Rockville, IL) were added to each well and incubated for 30 min at room temperature. After the blocking reagent was aspirated, 100 µl of rHuTPO standards or serum sample were added to each well and allowed to react with the coated TN1 overnight at room temperature. After washing each well with TBS containing 0.1% Tween 20 (T-TBS), 100 µl of the biotinylated anti-rHuTPO F(ab')2 (prepared by pepsin digestion and biotinylation of polyclonal rabbit antibody against rHuTPO) [27] at a concentration of 500 ng/ml in dilution buffer (20 mM Tris-HCl containing 0.5 M NaCl, 1% bovine serum albumin, 2% PEG 6000, 0.05% Tween 20, and 0.1% NaN3, pH 7.5) were added to each well and incubated for three h at room temperature. After washing with T-TBS, 100 µl of strepravidin alkaline phosphatase conjugate (1 mU/ml in dilution buffer, Boehringer Mannheim; Tokyo, Japan) were added to each well, then incubated for one hour at room temperature. The color development was carried out using an amplification system (GIBCO BRL; Grand Island, NY). The color intensity was measured using the A630 nm subtracted from A492 mm on a plate reader. The quantitative range of rHuTPO was from 0.05 to 3.28 f mol/ml [27]. The within-assay and between-assay coefficients of variation were 3.0% to 4.9% and 5.9% to 6.1%, respectively, for the range of 0.24 to 2.95 f mol/ml. There was no significant cross-reactivity or interference by other cytokines including rHuIL-3, rHuIL-6, rHuIL-7, rHuG-CSF, rHuEPO and rHu stem cell factor. Furthermore, the addition of the soluble mpl, within the concentrations of 0.16 to 50 µg/ml, had only a minimal effect on the assay, and the recoveries of rHuTPO at 10 and 50 µg/ml of soluble mpl were 97.1% and 92.5%, respectively [27]. Curves obtained from the serial dilutions of positive serum samples always paralleled the standard curve, and TPO values of the test samples could be reproducibly determined by superimposing them on the standard curve.

Calculations and Statistical Analysis
The value for each measured variable is given as mean values ± standard deviation (SD). Mann-Whitney's nonparametric test was used for the calculation of the differences between groups. Spearman's rank correlation test was used in the calculation correlations between TPO levels and platelet counts.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Serum TPO Levels
The TPO levels determined by ELISA are shown with the hematological parameters in Table 1Go. In all 101 subjects investigated, there was weak but significant negative correlation between the TPO level and the platelet count (r = –0.493, p < 0.0001; Spearman's rank correlation coefficient), while there was no relationship between the TPO and the leukocyte count, hemoglobin concentration or CRP level.

All 15 healthy individuals had platelet counts within normal range (153 x 109 to 319 x 109/l) and TPO levels between 0.300 and 1.718 f mol/ml (Fig. 1AGo). In the 18 acute leukemia patients with myelosuppression after intensification chemotherapy in complete remission, the platelet count ranged between 6 x 109 and 182 x 109/l, while the TPO levels ranged from 3.679 to 43.205 f mol/ml and were considerably higher than normal individuals (Fig. 1AGo). Although there was no relationship between the TPO level and the platelet count in each group, the TPO levels were negatively correlated with the platelet counts when combining the normal individuals and the postchemotherapy group. We used these data of normal individuals and patients with acute leukemia in complete remission (postchemotherapy group) as control for comparison to various hematological disease states.



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  		Figure 1. Relationship between the platelet count and the serum TPO level in the following subgroups: A, in healthy volunteers ({circ}) and patients with myelosuppression due to intensification therapy for acute leukemia in complete remission (postchemotherapy group) ({square}); B, in AA ({blacktriangleup}); C, in ALL ({diamond}), malignant lymphoma (+), multiple myeloma ({bigtriangleup}) and CLL ({diamondsuit}); D, in AML (x), hypoplastic leukemia ({diamond}–), MDS (*) and LC (); E, in CGL (•), PV ({blacksquare}) and ET ({triangledown}); F, in ITP ({blacktriangledown}). In A, the regression line for the combined group with normal individuals and postchemotherapy group is shown. In B-F, each plot is overlaid onto the regression line for the combined group with normal individuals and postchemotherapy group for comparison.

 
In AA, ALL, malignant lymphoma, multiple myeloma and CLL, the relationship of serum TPO levels and platelet counts was almost the same as in the combined group with normal individuals and postchemotherapy group, as shown in Figures 1B and 1CGo. However, in AML, hypoplastic leukemia, MDS, LC and ITP, serum TPO levels were lower compared to the regression line for the combined group with normal individuals and the postchemotherapy group, as shown in Figures 1D and 1FGo. There was no statistical significance in relationship between the TPO level and platelet count in each disease. However, when combined, in the lymphoid malignancies (ALL, malignant lymphoma, multiple myeloma and CLL), there was a significant negative correlation between platelet count and the TPO level (r = –0.724, p = 0.0051) similar to that in the combined group with normal individuals and the postchemotherapy group (Fig. 1CGo). In contrast, there was no relationship between the TPO levels and the platelet counts in myeloid malignancies including AML, MDS and hypoplastic leukemia (r = 0.269, p = 0.3510).

In the 12 MPD patients, the TPO level (mean: 1.99 f mol/ml) was slightly higher when compared to the regression line for the combined group with normal individuals and the postchemotherapy group (Fig. 1EGo). In these patients, there was no relationship between the TPO level and the platelet count. All five ET patients (platelet count: 475 x 109 to 752 x 109/l) had slightly raised TPO levels (1.94 to 4.82 f mol/ml). The two patients with PV showed TPO levels (1.01 and 0.90 f mol/ml) similar to those in the normal individuals despite the presence of thrombocytosis (523 x 109 and 1,000 x 109/l, respectively), and TPO levels varied irrespective of the platelet counts in CGL. There was no relationship between the TPO levels and the platelet counts in each disease.

Effect of Cytoreductive Therapy on the Serum TPO Level
Serial changes in the serum TPO level were examined during the course of cytoreductive therapy in one patient who had acute promyelocytic leukemia in complete remission (Fig. 2Go). The TPO level began to increase with a decrease in the platelet count, and peaked at 22.31 f mol/ml on day 10. Then a plateau was noted until the level dropped in response to an increase of the platelet count following platelet transfusion. The TPO level subsequently fell rapidly with the recovery of the platelet count.



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  		Figure 2. Serial changes in serum TPO level and the platelet count in a 20-year-old woman with AML (M3) in complete remission who received BHAC-AMP intensification therapy, comprising behenoyl cytosine arabinoside (200 mg/m2/day for 7 days), aclarubicin (15 mg/m2/day for 7 days), 6-mercaptopurine (70 mg/m2/day for 7 days). Arrows indicate the transfusion of five units of platelet concentrate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we measured the serum levels of TPO in 101 subjects, including healthy volunteers and patients with various hematological diseases or LC. We found a considerable elevation of TPO in AA and postchemotherapy patients. In addition, a transient elevation of TPO levels was observed during the myelosupression period after cytoreductive therapy. These findings are consistent with the results obtained in thrombocytopenic animal models [26]. In the normal individuals, AA or postchemotherapy groups, there was no significant relationship between the platelet count and the TPO level, respectively. This may have been due to too few patients and the narrow range of platelet counts in these patients, since only one patient in the AA group and three in the postchemotherapy group had a count over 100 x 109/l. Since the postchemotherapy patients had been in complete remission of acute leukemia prior to chemotherapy, one can neglect the influence of leukemia on their TPO level and blood count. Therefore, we combined the data of normal individuals and postchemotherapy patients, and there was a negative correlation between the TPO level and the platelet count in this combined group.

The TPO level was also slightly elevated in ITP patients, but was not as high as in the AA and postchemotherapy patients. The mechanism of thrombocytopenia in ITP is different from that in those other conditions, since the destruction of platelets is increased in ITP while platelet production is decreased in AA and after chemotherapy [33]. In contrast, increased thrombopoiesis is well-known to occur in ITP [33]. In an animal model, the TPO level was reported to increase inversely as the platelet mass declined [26], and platelets were shown to have TPO receptors [34] and to remove TPO from thrombocytopenic plasma in vitro [35]. Thus, it is possible that increased platelet turnover kept the serum TPO level from being elevated despite an increase of its production in ITP. Alternatively, another regulatory factor, in addition to TPO, may play a role in part, if at all, in the regulation of thrombopoiesis in ITP.

In our ALL patients, TPO levels raised inversely as the platelet counts decreased, as seen in the combined group with normal individuals and the postchemotherapy group, but there was no statistical significance between TPO levels and platelet counts (r = –0.700, p = 0.1615). It seems to be due to too few patients. ALL is one of the lymphoid malignancies that includes lymphoma, myeloma and CLL, and the thrombocytopenic mechanism is mainly a decrease of platelet production due to marrow replacement by malignant cells in all of these diseases. When combining the data of these lymphoid malignancies, we found a significant negative correlation between platelet count and the TPO level in these lymphoid malignancies. Thus, in these disorders that are associated with thrombocytopenia due to a decrease of platelet production, the TPO level increased in accordance with the decrease of the platelet count.

In AML, MDS and hypoplastic leukemia, although the mechanism of thrombocytopenia is also decreased platelet production, the TPO levels were lower compared with AA, postchemotherapy and ALL patients in relation to the degree of thrombocytopenia. AML cells are reported to express c-mpl (the TPO receptor) [36] and to grow in response to TPO [37]. Thus, it is possible that TPO was removed from the serum by binding to the receptors on AML cells, similarly to the mechanism that may in ITP. However, TPO levels also were not elevated in MDS and hypoplastic leukemia despite the low blast cell mass in contrast to AML.

On the other hand, all five ET patients showed slightly higher TPO levels than the normal individuals despite having thrombocytosis. This suggests that the mechanism of thrombocytosis in ET is different from that of erythrocytosis in PV [38], and is possibly mediated by an autocrine mechanism involving TPO rather than by the autonomous proliferation of megakaryocytes. In contrast, the two patients with PV showed TPO levels similar to those in the normal individuals despite the presence of thrombocytosis, and TPO levels varied irrespective of the platelet counts in CGL. Further analysis of TPO expression at the mRNA level will be necessary to ascertain whether there is aberrant production of TPO by these malignant cells.

TPO levels in LC were similar to those in the normal individuals and lower than the regression line of the combined group with normal individuals and the postchemotherapy group despite the presence of thrombocytopenia. TPO is reported to be produced mainly in the liver [15-17, 39]. It is therefore possible that thrombocytopenia in LC is mediated by failure of TPO production, as occurs with Epo in anemia secondary to renal insufficiency [40].

In summary, there was heterogeneity with respect to TPO levels in various clinical conditions, as well as their relationship to platelet counts. The circulating platelet count may modulate the TPO level or may be partly regulated by the TPO level in normal individuals and patients after chemotherapy, or with AA or lymphoid malignancy. In ITP, MPD or myeloid malignancy (AML, MDS and hypoplastic leukemia), the TPO level may be regulated in part by another unknown mechanism, if any. Since our study included a few patients in each diagnostic class, a further investigation with more patients is necessary in each disease.


   Footnotes
 
Provisionally accepted May 1, 1996.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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Received March 4, 1996; accepted for publication June 10, 1996.




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