INTRODUCTION: Essential thrombocythemia (ET) is an entity of classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by thrombocytosis with megakaryocytic hyperplasia where in the thrombocytes are increased with abnormal function. Thrombotic events are seen frequently and represent the main cause of morbidity and mortality in patients with MPNs, mainly polycythemia vera and ET. This study has aimed to research the effects of clonally increased thrombocytes on plasma viscosity (PV) levels among patients with ET and the relationship between PV and thromboembolism history, according to the hypotheses about the effects of PV in thromboembolic events among patients with ET. METHODS: A total of 55 patients were enrolled in the study group, 18 of who had been newly diagnosed with ET according to 2016 World Health Organization criteria and had not previously been treated. 37 of them had already been diagnosed with ET and had been treated. There were 47 healthy volunteers in the control group. 5 cc blood samples were taken from the patients into tubes including an anticoagulant to measure their PV levels. RESULTS: PV of the control group was found to be lower than in the study group and both each patient groups (p < 0.05). No relationship was found between the patient groups in terms of PV (p = 0.404). The mean PV levels of the 16 patients with a history of thromboembolism and the 39 patients with no such history were 2.42±0.17 cP and 2.33±0.20 cP, respectively. The mean PV levels were found to be similar according to their history of thromboembolism in all patient groups and in treated patients (p = 0.572 vs p = 0.991). CONCLUSION: We have found that PV levels were increased in clonally increased thrombocytes in patients with ET when compared with the control group. This is the first study in this field according to our knowledge.
Keywords: Essential thrombocytosis; plasma viscosity; thrombosis
Essential thrombocythemia (ET) is an entity of classic Philadelphia chromosome-negative myeloproliferative neoplasms (MPNs), characterized by thrombocytosis with megakaryocytic hyperplasia where in the thrombocytes are increased with abnormal function [[
The mutation of the Janus Kinase 2 (JAK2) gene is commonly detected in MPNs. JAK2 is a tyrosine kinase and plays an important role in myelopoiesis. JAK2 V617F gene mutation has been found in almost every patient with polycythemia vera as well as in 50%of patients with ET and primary myelofibrosis. Studies investigating thromboembolism in these diseases have shown that leucocyte counts more than 15×10
There are two dimensions of blood viscosity determination including plasma viscosity and whole blood viscosity. Plasma viscosity (PV) is a major determinant of blood viscosity in circulation and consists of protein-based macromolecules of blood and water. The value of PV can be affected by inflammation and tissue injury because of the changes in plasma proteins; therefore C-reactive protein (CRP), fibrinogen and erythrocyte sedimentation rate (ESR) can increase parallel with PV [[
This study has aimed to find out the effects of clonally increased thrombocytes on PV levels among patients with ET and the relationship between PV and thromboembolism history, according to the hypotheses about the effects of PV in thromboembolic events among patients with ET.
The study was conducted at Ankara City Hospital Hematology Department. After the approval of the ethics committee, the patients who came to our hospital's hematology outpatient clinic between July 2020 and August 2020 were included in the study. A total of 55 ET patients were enrolled in the study group, 18 of whom had been newly diagnosed with ET according to 2016 World Health Organization criteria and had not previously been treated. 37 of them had already been diagnosed with ET. These patients had already treated with aspirin and/or hydroxyurea according to their risk classification. Patients using drugs that might affect viscosity, such as lipid-lowering agents; and or who has smoking history, recent surgeries, recently has an infectious condition were excluded. There were 47 healthy volunteers in the control group who had consulted internal medicine outpatient clinics for routine controls. Volunteers who had any systemic diseases, using drugs such as lipid-lowering agents, anti-platelet agents, anti-coagulants, and having smoking history, recent surgery or infectious disease were excluded. The patients diagnosed with ET were evaluated for comorbidities which could affect PV. Patients who had been diagnosed with chronic renal failure, hypertension, coronary artery disease, diabetes mellitus, hyperlipidemia, connective tissue disease, chronic anemia, iron deficiency anemia and infectious diseases were excluded from the study. Furthermore, we excluded anyone who had abnormal ESR, CRP and fibrinogen values from the study and control groups. The patients were divided into two groups, as newly diagnosed and previously treated patients.
5 cc blood samples were taken from the patients, who signed the informed consent form, into tubes including an anticoagulant to measure their PV levels. The samples were taken from newly diagnosed ET patients before treatment and from treated patients during their hospital visits. The samples were kept at –80°C after being centrifuged at 3000 rpm for at least 5 minutes for later examination so as to avoid any mistakes concerning the calibration of the test machine. All the samples were melted and re-centrifuged on the measurement day, then measured at 37°C in a Brookfield DV-II+ Cone Plate Viscometer (Brookfield, Stoughton, MA, USA) machine, which was calibrated with distilled water. Ethics committee approval was received from Ankara City Hospital with the number E1-20-857 and informed consent was taken from all patients.
Distributions of continuous variables were examined by Shapiro Wilk. Age and mean platelet volume (MPV) were given as mean±standard deviation (mean±sd) and other continuous variables were provided as median (min-max). Categorical variables were summarized by frequency (%).
Continuous characteristics were compared between groups by Independent samples t-test, Mann-Whitney U test or Kruskal-Wallis test with respect to the distribution of characteristics and the number of the groups. When necessary, Mann-Whitney U test with Bonferroni correction was applied as post-hoc test after Kruskal-Wallis test. Chi-square tests were performed to compared categorical characteristics between groups. Pearson (r) and Spearman (r
All statistical analyses were performed via IBM SPSS Statistics 21.0 (IBM Corp. Released 2012. IBM SPSS Statistics for Windows, Version 21.0. Armonk, NY: IBM Corp.).
The demographic features and the hematological measurements of the patients are given in Table 1. The mean age of the ET patients was higher than that of the control group (p < 0.001). The mean age of the newly diagnosed and the treated ET patients did not differ (p = 0.055), while the mean age of the treated patients was found to be higher than that of the control group (p < 0.05). The gender distribution across the groups was similar (p = 0.373).
Table 1 Comparisons of demographic features, hematological measurements, and PV across groups
ET patients Control ( Total ( Newly diagnosed ( Follow-up ( Mean±SD Mean±SD Mean±SD Mean±SD Median (min-max) Median (min-max) Median (min-max) Median (min-max) Age (year) 46.36±12.31¥ 58.51±15.17 52.11±16.13 61.62±13.86¥ 0.055 Gender (M/F) 16/31 15/40 3/15 12/25 0.600 0.373 0.335 Hemoglobin (g/dL) 14.1 (10.8 –16.3)¥ 13.0 (7.9 –15.6) 13.0 (11.5 –15.3) 13.0 (7.9 –15.6)¥ 0.501 WBC count (x109/L) 7.1 (4.6 –10.7) 8.2 (3.2 –23.6) 7.2 (3.2 –23.6) Platelet count (x109/L) 669.0 743.5 620.0 (175.0 –1838.0) (462.0 –1838.0) (175.0 –1154.0) MPV (fL) 10.74±0.79 10.48±0.82 10.28±0.70 10.58±0.87 0.105 0.170 0.243 MCH (pg) 28.7 (19.1 –31.5) 29.4 (20.7 –47.0) 27.7 (20.7 –37.0) Ferritin (μg/L) 41.0 (10.5 –406.0) 39.7 (8.0 –330.1) 34.0 (16.4 –254.0) 50.0 (8.0 –330.1) 0.783 0.450 0276 PV (cP) 2.36 (1.59 –3.43) 2.42 (1.83 –3.43) 2.29 (1.59 –3.35) 0.404
WBC: White blood cell count; MPV: Mean platelet volume; MCH: Mean corpuscular hemoglobin; PV: Plasma viscosity. Bold descriptive statistics are significantly different from other two groups as the result of post-hoc test.
The hemoglobin level in the control group was significantly higher than those in the ET patient group (p = 0.007). The hemoglobin levels of both ET patient groups were similar (p = 0.501, Table 1). The WBC counts of the ET patients were found to be higher than those of the control group (p = 0.028) and the WBC counts of the newly diagnosed patients were significantly higher than those of the treated patients and the control group (p < 0.05, Table 1). The groups were found to be similar in terms of MPV level and ferritin level (p > 0.05, Table 1).
The viscosity level in the control group was significantly lower than those in the ET group and in two patient groups (p < 0.05). In contrast, two patient groups were similar with respect to the viscosity (p = 0.404, Table 1).
The, JAK2, ESR, CRP measurements and thromboembolic-bleeding history of ET patient groups are shown in Table 2. There were 16 (29.1%) ET patients who had a history of thromboembolism and only one of them was a newly diagnosed patient. The history of thromboembolism was found significantly less frequent in the newly diagnosed patients than those in the treated patients (p = 0.018). JAK2 V617F mutation was similar in both ET patient groups (p = 0.055). There was not a significant difference between the groups in terms of their ESR and CRP levels (p > 0.05).
Table 2 Comparisons of JAK2, ESR, CRP measurements and thromboembolic and bleeding history across patient groups
Total Newly diagnosed Follow-up Median (min-max) Median (min-max) Median (min-max) Thromboembolic history 16 (29.1) 1 (5.6) 15 (40.5) Bleeding 5 (9.1) 1 (5.6) 4 (10.8) 1.000 JAK2 V617F 27 (49.1) 5 (27.8) 22 (59.5) 0.055 ESR (mm/h) 20.0 (1.0–89.0) 22.0 (3.0–89.0) 20.0 (1.0–75.0) 0.114 CRP (mg/L) 0.30 (0.10–4.40) 0.40 (0.10–4.40) 0.30 (0.10–1.50) 0.212
ESR: Erythrocyte sedimentation rate; CRP: C-reactive protein.
The mean PV levels of the 16 patients with a history of thromboembolism and the 39 patients with no history were 2.42±0.17 cP and 2.33±0.20 cP, respectively (Fig. 1). The mean PV levels of treated patients with a history of thromboembolism (n = 15) and without history (n = 22) were 2.35±0.14 cP and 2.35±0.21 cP, respectively (Fig. 1). The mean PV levels were found to be similar according to their history of thromboembolism in all patient groups and in treated patients (p = 0.466 and p = 0.991, respectively).
Graph: Fig. 1 Mean plasma viscosity level with respect to the history of thromboembolism in all patients (right) and in the treated patients (left).
The PV was positively, weakly, and linearly correlated with the age (r
Table 3 Correlations between PV and age, hemoglobin, and platelet counts
PV Whole sample ( ET patients ( rS rS Age 0.227 0.213* 0.118 Hemoglobin –0.241 –0.168 0.221 Platelet count 0.303 –0.062 0.656
r
The effect of PV on the thromboembolism history in ET group was examined by both a univariate model and a multivariate model considering age and gender of the patients. Neither univariate analysis nor multivariate analysis reveal a significant effect of PV (Table 4).
Table 4 Effect of PV on thromboembolism history
b±SE OR 95%CI Univariate model Age1 1.258±0.632 3.520 1.020–12.145 Gender2 1.103±.0.641 3.014 0.858–10.592 0.085 PV (cP) –0.553±0.747 0.575 0.133–2.485 0.459 Multivariate model Age1 1.156±0.676 3.176 0.844–11.955 0.087 Gender2 0.792±0.680 2.207 0.582–8.375 0.245 PV (cP) –0.807±0.810 0.446 0.091–2.183 0.319
b: Regression coefficient, SE: Standard error, OR: Odds Ratio, CI: Confidence interval.
The ability of PV levels in terms of separating the newly diagnosed patients from the control group was found to be lower than the platelet count but it was statistically significant (AUC±SE = 0.801±0.059, p < 0.001, Figure 2). The optimal cut-off for PV was determined as≥2.145 cP with 77.8%(95%CI: 54.8%–91.0%) of sensitivity and 74.5%(95%CI: 60.5%–84.8%) specificity.
Graph: Fig. 2 ROC curve of platelet count and plasma viscosity.
Thromboembolic events are the main cause of mortality and morbidity in MPNs [[
It was shown that elevated viscosity caused by high hemoglobin levels was the reason for thromboembolic events in polycythemia vera [[
The effects of thrombocyte count on PV levels had been searched in different studies. Toprak et al. had searched the effects of thrombocyte counts on PV levels. Twenty patients who had thrombocytosis caused by iron deficiency anemia were included in their study. There was no relationship between thrombocyte counts and PV levels. In fact, fibrinogen, hyperlipidemia, and inflammation that could affect PV levels had not been evaluated clinically or laboratory in the study [[
Another study which was conducted among 113 patients with iron deficiency anemia, pancytopenia, polycythemia vera, essential thrombocythemia, idiopathic thrombocytopenic purpura, myelodysplastic syndrome, aplastic anemia, and thalassemia, showed that WBC and platelet counts could affect complete blood viscosity. In contrast they could not show the effect on PV levels [[
Our results were similar to these studies, which were conducted with different ET patient groups. We have found that there was no relationship between thrombocyte count and PV levels in ET patients.
A study which was aimed to investigate the relationship between thromboembolism and PV levels had showed that patients with pulmonary thromboembolism had higher PV levels in than healthy controls [[
JAK2 V617F mutation positivity in our ET patients was 49.1%, thromboembolic event history rate was 29.1%and bleeding history rate in was 5.1%. These results showed a good correlation with literature.
The most important limitation of this study was the number of participants. Another limitation was the difference of age between ET patients and control group, even if we have tried to control the effects of such confusing factors, age remained as a problem.
In conclusion, PV levels were found higher in patients with ET. The relationship between increased plasma viscosity and thromboembolism which plays such an important role in terms of mortality and morbidity among these patients should be searched among larger patient populations.
We would like to thank Prof. Dr. Osman İlhan for giving us permission to use the apheresis unit of Ankara University to measure viscosity.
By Tekin Güney; Ferda Can; Afra Alkan; Sema Akıncı and İmdat Dilek
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