We studied both serum-free colony-forming unit-megakaryocyte (CFU-meg) numbers and serum thrombopoietin (TPO) levels in 14 patients with aplastic anemia (AA), 37 patients with myelodysplastic syndromes (MDS) and 23 patients with idiopathic thrombocytopenic purpura (ITP) to assess thrombopoiesis in these thrombocytopenic disorders. The mean CFU-meg numbers were lower in AA and MDS patients (10.7 ± 11.4 and 42.3 ± 58.5/105 BMLD cells) than in healthy controls (103.1 ± 57.3/105 BMLD cells) (P < 0.0001 and P = 0.0053, respectively), although they were distributed variably in MDS. ITP patients showed higher CFU-meg numbers (223.2 ± 143.5/105 BMLD cells) (P = 0.017). The mean TPO concentrations were higher in both AA (986.8 ± 500.8 pg/ml) and MDS patients (838.2 ± 639.1 pg/ml) than in healthy controls (80.7 ± 38.8 pg/ml) (P < 0.0001), although they were distributed from high to low in MDS. ITP patients showed a slight elevation of TPO (123.1 ± 55.3 pg/ml) P = 0.0106). The TPO levels was inversely correlated to both platelet counts and CFU-meg numbers (correlative coefficient (CC): −0.719 and −0.682, P < 0.0001) in AA, but not in ITP. In MDS, the inverse correlation to TPO was stronger in CFU-meg (CC: −0.678, P < 0.0001) than in platelet counts (CC: −0.538, P = 0.0014), suggesting that CFU-meg plays an important role in regulating TPO production in this heterogenous disorder. CFU-meg and TPO may provide useful information for understanding thrombopoiesis of MDS, especially for application of TPO.
In vitro colony-forming unit-megakaryocyte (CFU-meg) assay can provide useful information on thrombocytopenic disorders, although CFU-meg data obtained by a standardized method for these disorders is lacking. CFU-meg values obtained by methods such as fibrin clot culture, vary depending on the serum used as the stimulator for megakaryopoiesis.123 The lack of sufficient CFU-meg data might also be due to the inconvenience of identifying whether cells in semisolid cultures such as agar or methylcellulose are of megakaryocyte lineage by immunocytochemical methods.
Thrombopoietin (TPO), c-Mpl ligand, is considered to be a central humoral regulator of platelet production and its receptor, c-Mpl, is expressed not only on platelets but on CD34 positive cells and megakaryocyte progenitors.4 It is known that TPO is synthesized in liver, kidney, spleen, and lung.5 An inverse correlation between the platelet count and blood TPO level was shown in thrombocytopenic animals and the existence of a feedback mechanism dependent on platelet mass was proposed.6789 Then, it was considered that the concentration of circulating TPO is directly determined by platelets which bind and remove it from serum, thus the platelet mass plays a direct role in the regulation of serum TPO. This hypothesis, the so-called sponge theory, was supported by the observation that a semiquantitative reverse transcription-polymerase chain reaction assay did not demonstrate upregulation of TPO mRNA in the tissue of thrombocytopenic animals and the increase of TPO activity during thrombocytopenia was not caused by regulation at the level of TPO mRNA.10 These results give the impression that TPO protein was regulated at a post-transcriptional level and/or directly through absorption and metabolism by platelets.
However, it has been reported that TPO levels are regulated, at least in part, by modulating mRNA levels in bone marrow.11 In addition, it was recently shown that idiopathic thrombocytopenic purpura (ITP) patients had lower TPO concentrations than was to be expected from their platelet counts and showed no obvious correlation between TPO level and platelet count, suggesting a regulatory role for megakaryocyte mass.12131415
A serum-free collagen-based system for CFU-meg, Megacult-C, has been developed and is expected to become the standard assay for CFU-meg. We employed this system for the quantification of megakaryocyte progenitors of patients with aplastic anemia (AA), myelodysplastic syndromes (MDS) and ITP, and assessed serum TPO levels, in order to clarify the relationship between CFU-meg and TPO levels in these thrombocytopenic states, and the clinical impact especially in MDS.
Materials and methods
Bone marrow samples were obtained from 14 patients with AA (13–67 years old; eight males and six females), 37 patients with MDS (18–72 years old; 20 males and 17 females), 23 patients with ITP (13–81 years old; 16 males and seven females) and 11 healthy volunteers (18–42 years old; seven males and four females). MDS patients were diagnosed with refractory anemia (RA) in 31 cases, refractory anemia with an excess of blasts (RAEB) in two cases, RAEB in transformation (RAEB-t) in two cases and chronic myelomonocytic leukemia (CMML) in two cases by FAB subtype.16 Sera were obtained from 14 patients with AA, 31 patients with MDS, 17 patients with ITP and 15 volunteers. Informed consent was obtained from all subjects.
The CFU-meg number was determined with a commercially available CFU-meg assay kit (Megacult-C, Stem Cell Technologies, Vancouver, BC, Canada). Briefly, bone marrow (BM) cells were obtained with heparinized syringes and light- density (LD) cells were separated by Ficoll-Conray gradient centrifugation (1.077 g/μl). BMLD cells (1 × 105 cells/ml) were incubated with 5% CO2 and more than 95% humidity for 12 days, in double chamber slides in a 100 mm petri dish along with an open 35-mm petri dish containing 3 ml of sterile water. The culture contained 1.1 mg/ml of collagen, 50 ng/ml of recombinant thrombopoietin, 10 ng/ml of recombinant IL-6 and 10 ng/ml of recombinant IL-3 at final concentrations. The slides were fixed with methanol-acetone solution after removing the double chamber and stored at −20°C. Immunocytochemical stainings were performed with anti-human GPIIb/IIIa (CD41) antibody, biotin conjugated goat anti-mouse IgG, avidin-alkaline phosphatase conjugate and alkaline phosphatase substrate. CFU-meg was recognized as a group of more than three CD41 positive cells, subdivided into three groups by size as: small (three to 20 cells per colony), medium (21 to 49 cells per colony) and large (more than 50 cells per colony).
The serum TPO concentration was determined with a commercially available ELISA kit (Quantikine Human TPO Immunoassay, R&D Systems, Minneapolis, MN, USA). Briefly, human TPO standard and sera were added in duplicate to the wells of a microtiter plate precoated with an anti-TPO monoclonal antibody and incubated for 3 h at 4°C. After washing, horseradish peroxidase conjugated anti-TPO antibody was added and incubated for 1 h at 4°C. The color was developed by using tetramethylbenzidine as substrate. The absorbance was recorded at 450 nm. The sample TPO concentration was calculated from the corresponding standard curve.
Data are presented as the mean ± s.d. unless otherwise noted. Differences between two groups of data were analyzed by Student's t-test. Correlation analyses between two groups of data were performed with Spearman's rank correlation test. All of the above analyses were conducted using StatView Software (Abacus Concepts Inc., Calabasas, CA, USA.
The mean CFU-meg of AA patients was significantly low (10.7 ± 11.4/105 BMLD cells), compared to healthy controls (103.1 ± 57.3/105 BMLD cells), as shown in Figure 1 (P < 0.0001). The mean CFU-meg of MDS patients was significantly low, too (42.3 ± 58.5/105 BMLD cells) (P = 0.0053), but values varied from very low to high. There was no significant difference between AA and MDS patients. In the patients with ITP, the mean CFU-meg was 223.2 ± 143.5/105 BMLD cells, significantly higher than in healthy controls (P = 0.017), AA patients (P < 0.0001) and MDS patients (P < 0.0001).
Serum TPO concentrations
The mean concentration of serum TPO in AA patients (986.8 ± 500.8 pg/ml) was remarkably high, compared to healthy controls (80.7 ± 38.8 pg/ml) (P < 0.0001). The mean TPO level was significantly higher in MDS patients (838.2 ± 639.1 pg/ml) (P < 0.0001), too. However, serum TPO levels were distributed from very low to high among MDS patients as shown in Figure 2. There was no significant difference between AA and MDS patients. ITP patients showed a slightly elevated but significant mean concentration of serum TPO (123.1 ± 55.3 pg/ml) (P = 0.0106). There were significant differences of serum TPO level between ITP patients and AA patients or MDS patients (P < 0.0001).
Relationship between platelet counts, TPO concentrations and CFU-meg
A comparison of CFU-meg and TPO concentrations by diagnosis is shown in Table 1. There was no difference of platelet counts among diagnoses (65.2 ± 83.3 × 109/l, 55.2 ± 46.5 × 109/l and 35.3 ± 26.1 × 109/l in AA, MDS and ITP patients, respectively). Small colonies were mainly observed in AA and MDS patients, whereas almost equal numbers of small, medium and large colonies were observed in healthy controls. Large colonies were frequently observed in ITP patients. The TPO levels in ITP patients were lower than to be expected from the thrombocytopenia, similar to the levels in AA and MDS patients.
MDS patients were subdivided into three groups based on CFU-meg numbers/105 BMLD cells as: group I (more than 150), group II (50–149) and group III (less than 49), as shown in Table 2. Some medium and large colonies were observed in group I and group II, whereas most were small colonies in group III. In addition, very high TPO levels similar to those in AA patients were observed in group III, suggesting that small numbers of remaining progenitors were strongly stimulated to maintain peripheral platelet levels. On the other hand, some patients with sufficient numbers of CFU-meg and megakaryocytes in group I and II still showed thrombocytopenia, suggesting a defect in the final step of thrombopoiesis. These patients showed elevated but not very high TPO levels. Only patient 1 in group I with low TPO had liver cirrhosis. There was no difference in TPO levels by FAB subtype, or karyotype. Instead, an inverse correlation between TPO levels and CFU-meg numbers was found in MDS (correlative coefficient (CC): −0.678, P < 0.0001), whereas there was a weak inverse correlation between TPO levels and platelet counts (CC −0.538, P = 0.0014), as shown in Figure 3. On the other hand, in AA, a similar strong inverse correlation was found both between TPO levels and CFU-meg numbers (CC: −0.719, P < 0.0001), and between TPO levels and platelet counts (CC: −0.682, P < 0.0001). No obvious inverse correlation between TPO levels and CFU-meg numbers, and between TPO levels and platelet counts was found in ITP.
We employed a new collagen-based CFU-meg assay which has the advantage that incubation, fixation and staining of the entire culture can be performed on the same slide and stable data can be expected, independent of sera. The mean CFU-meg number of healthy controls by this method was three or four times the values obtained previously with the fibrin clot culture system.17181920 Although the new system seems better than previous methods of culturing CFU-meg, it only provides the number of CFU-meg per BMLD cell for different types of bone marrow cells in different diseases. We need to be cautious when interpreting the data as it does not provide the absolute number of CFU-meg per constant number of BM cells. Still, the data obtained were very informative as to the extent of megakaryopoiesis in each disease. The CFU-meg numbers in ITP were significantly elevated in the present study, although they varied from low to high in previous reports.171921 This discordance of CFU-meg data among studies might be due to the low efficacy of colony formations dependent on sera. The present study disclosed that CFU-meg numbers in AA are very low in most patients and relatively low even in the patients with normal platelet counts. In addition, most CFU-meg of AA patients were small.
Corresponding to a decrease in CFU-meg, serum TPO levels were extremely high in AA patients, as reported pre- viously.121314152223242526 One AA patient who had a complete hematological response to immunosuppressive therapy still showed a relatively low CFU-meg number and a high TPO concentration. Elevated levels of TPO may be required to maintain normal or near normal platelet counts in remission of AA, because recovery of stem cells or progenitors is incomplete, as suggested by a previous study.27
Previous studies have demonstrated that CFU-meg numbers in MDS were normal in some patients, but decreased in most.1828 In addition, the number of megakaryocytes per colony was also reported to be reduced.29 Consistent with these studies, two-thirds of the patients, group III in our study, showed reduced numbers of CFU-meg; most of them were small colonies. However, there were some patients with normal or increased numbers of CFU-meg (group II and I) and thrombocytopenia simultaneously, suggesting poor platelet production in spite of sufficient megakaryocytic progenitors. The administration of TPO to these patients may be effective in improving thrombocytopenia because their TPO levels were not increased. In the past, studies on TPO levels in MDS showed significantly high levels in RA patients and generally low levels in RAEB and RAEB-t patients.153031 It was also reported that a significant inverse relationship between the TPO concentration and platelet count exists in RA patients, but not in RAEB or RAEB-t patients. However, in this study, both RAEB and CMML patients showed high TPO levels when their CFU-meg were reduced, suggesting no difference among subtypes. The MDS patients with reduced CFU-meg showed high TPO levels, and some patients with normal or increased CFU-meg showed no significant elevation of TPO even with low platelet counts. These results suggest that the simple administration of TPO to MDS patients might not be effective enough to increase the platelet count although we do not know the effect of a super high concentration of TPO on the megakaryopoiesis in these patients.
We found a strong inverse correlation between serum TPO concentrations and CFU-meg numbers in MDS patients, while there was a weak inverse correlation between serum TPO levels and platelet counts in the same patients. This suggests that there is an important relationship between megakaryocyte progenitors in bone marrow and TPO regulation, at least in MDS. In the present study, TPO levels in ITP patients showed only slight elevation and were lower than expected from the platelet counts, as reported previously.121322 There was no significant inverse relationship between either TPO and platelet count, or TPO and CFU-meg, different from MDS. Therefore, we could not tell whether the serum TPO level is directly determined by the platelet mass, or whether megakaryocyte progenitors play an important role in TPO regulation in physiological and pathological situations.
A recent study demonstrated that TPO mRNA levels in bone marrow and spleen of mice vary in response to thrombocytopenia while those in liver and kidney do not change, and suggested the possibility that the TPO regulatory mechanism responds to thrombocytopenia in bone marrow and spleen.11 In addition, it was also reported that mice that lacked the transcription factor NF-E2 showed an absence of platelets, a normal megakaryocyte development and normal TPO levels, suggesting that the TPO regulation is not based only on platelet mass but on megakaryocyte mass in mice.32 More recently, it was disclosed that TPO was synthesized by bone marrow stromal cells,33 and TPO levels in bone marrow were higher than those in peripheral blood and correlated with TPO mRNA expression in bone marrow stromal cells.26 These studies suggest that TPO in a microenvironment such as bone marrow stromal cells also plays an important role in thrombocytopenia. It will be important to clarify the role of marrow stromal cells in TPO regulation and thrombopoiesis in a future study.
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The authors are grateful to Mr Shigehiro Nakashima for his technical assistance.
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Wang, W., Matsuo, T., Yoshida, S. et al. Colony-forming unit-megakaryocyte (CFU-meg) numbers and serum thrombopoietin concentrations in thrombocytopenic disorders: an inverse correlation in myelodysplastic syndromes. Leukemia 14, 1751–1756 (2000) doi:10.1038/sj.leu.2401898
- colony-forming unit-megakaryocyte (CFU-meg)
- myelodysplastic syndromes
- idiopathic thrombocytopenic purpura
- aplastic anemia
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