The Possibility Of Serum TPO Level Serving As A Diagnostic Indicator Of ITP In Pregnancy

Immune thrombocytopenia (ITP) is one of the most common bleeding disorders in clinical practice. The main symptoms are mucocutaneous hemorrhage, gastrointestinal hemorrhage, genitourinary tract hemorrhage, and even intracranial hemorrhage, which might be life threatening. ITP is an acquired autoimmune disorder, including anti-platelet autoantibodies- and/or cytotoxic T lymphocyte (CTL)-mediated increased platelet destruction and decreased platelet production due to disorders of megakaryocyte maturation.Thrombocytopenia is a common complication in pregnancy, occurring in approximately 10% of pregnant women. It can be caused by many different factors; the most common cause is gestational thrombocytopenia (GT). ITP in pregnancy is one of the other common causes of pregnancy-associated thrombocytopenia, accounting for approximately 5% of cases. Gestational thrombocytopenia is a benign disease, which is not associated with adverse outcomes to the mother or the fetus and need no aggressive treatment. On the contrary, ITP in pregnancy may lead to severe complications of both the mother and the fetus, making the condition and treatment more complicated. The anti-platelet autoantibodies may travel across the placenta, reach the circulation of fetus and produce thrombocytopenia, leading to gastrointestinal and/or intracranial hemorrhage of fetus or infant. Therefore, early diagnosis of ITP in pregnancy is of great significance to close monitoring and timely treatment of the pregnant patients and their infants.Thrombopoietin (TPO) exerts its effect by binding to the specific c-Mpl receptor, stimulating the proliferation, differentiation and maturation of megakaryocytes and the production of functional platelets. TPO is produced mainly in the liver, and cleared from the circulation by binding to the c-Mpl receptors on platelets and megakaryocytes. As the hepatic production of TPO appears to be constant, the circulating level of TPO is determined mainly by the circulating platelet mass; the lower the platelet count, the higher the TPO level. However, several recent studies showed that the TPO levels of ITP patients were only slightly higher than healthy controls, or even within the normal range. On account of the platelet counts of ITP patients are significantly lower than normal, some hematologists propose that the insufficient endogenous TPO is one of the pathogenesis of ITP.The first purpose of our study was to evaluate whether or not serum TPO levels in pregnancy-associated ITP differ from those in GT, and to reveal the possibility of TPO serving as a marker for differential diagnosis. The second purpose of the study was to compare the TPO levels in pregnant versus nonpregnant ITP patients. The serum TPO levels of ITP in pregnancy patients, gestational thrombocytopenia patients, healthy pregnant women, age-matched nonpregnant ITP patients and nonpregnant healthy controls were determined. The serum TPO level of ITP in pregnancy was significantly higher than GT (P< 0.01). Twenty-nine of 35 patients with ITP in pregnancy had TPO values> 500 pg/mL, whereas none of the GT patients’TPO levels exceeded 500 pg/mL. These results indicate that measurement of the TPO level may give additional information in differentiating ITP from GT. Patients with TPO levels of 500 pg/mL or over need to be examined and observed carefully, since the thrombocytopenia is more likely to be caused by ITP.ITP in pregnancy patients presented significantly higher TPO levels than nonpregnat ITP patients (P< 0.01). Numerous studies over the past 2 decades have documented that TPO levels are high when thrombocytopenia is due to megakaryocyte deficiency and low when it is due to increased platelet destruction. We further investigated the bone marrow cytology of pregnant and nonpregnant ITP patients.The bone marrow megakaryocytes numbers of nonpregnant ITP patients were normal or increased; while more than half of the ITP in pregnancy patients had decreased megakaryocyte numbers. These data indicate that the pathogenesis of pregnant and nonpregnant ITP might be different.Part Ⅰ:The study on the serum thrombopoietin level of immune thrombocytopenia in pregnancyObjective:To detect the serum TPO level of ITP in pregnancy, gestational thrombocytopenia, and nonpregnant ITP patients. To analyze the difference of TPO levels between ITP in pregnancy and GT, evaluate the possibility of TPO level serving as a differential diagnostic indicator. And to analyze the difference of TPO levels between pregnant and nonpregnant ITP, explore the pathogenesis mechanism of ITP in pregnancy.Methods:1. Whole blood and serum were obtained from 35 ITP in pregnancy patients,31 gestational thrombocytopenia patients,32 healthy pregnant women,32 age-matched nonpregnant ITP patients and 35 nonpregnant healthy controls. Platelet count was determined by complete blood count (CBC)2. Serum TPO levels were measured by commercial enzyme-linked immunosorbent assay (ELISA) kits.3. Bone marrow was aspirated from the posterior superior iliac spines of 23 ITP in pregnancy patients and 27 age-matched nonpregnant ITP patients, and stained according to Wright-Giemsa. Bone marrow megakaryocyte numbers were determined in double blind.4. Anti-platelet autoantibody was analyzed by MAIPA assay in 25 ITP in pregnancy patients and 32 age-matched nonpregnant ITP patients.Results:1. TPO levels of ITP in pregnancy patients were significantly higher than gestational thrombocytopenia patients. The TPO level of ITP in pregnancy was significantly higher than GT (mean±SD,1283±646 pg/mL vs.187±64 pg/mL, P< 0.01). Twenty-nine of 35 patients with ITP in pregnancy had TPO values> 500 pg/mL, whereas none of the GT patients’TPO levels exceeded 500 pg/mL. These results indicate that measurement of the TPO level may give additional information in differentiating ITP from GT. Patients with TPO levels of 500 pg/mL or over are more likely to be caused by ITP.2. TPO levels of ITP in pregnancy patients were significantly higher than age-matched nonpregnant ITP patients. The TPO level of ITP in pregnancy was significantly higher than nonpregnant ITP (mean±SD,1283±646 pg/mL vs.88±41 pg/mL, P<0.01).3.Bone marrow megakaryocyte numbers decreased in most of the ITP in pregnancy patients. The number of megakaryocytes was rated as normal (1 megakaryocyte per 1 to 3 low power fields), increased (>2 megakaryocytes per low power field), or decreased (1 megakaryocyte per 5 to 10 low power fields). Most of the nonpregnant ITP patients (17/27) had increased numbers of megakaryocytes, while the rest (10/27) had normal numbers of megakaryocytes. Conversely, among 23 patients with ITP in pregnancy,14 had decreased numbers of megakaryocytes,5 had normal numbers and 4 had increased numbers of megakaryocytes. These results indicated that most ITP in pregnancy patients had decreased megakaryocyte numbers, while most nonpregnant ITP patients had increased megakaryocyte numbers, the pathogenesis of pregnant and nonpregnant ITP might be different.4. There was no significant difference of anti-platelet autoantibodies between pregnant and nonpregnant ITP patients. Platelet glycoprotein-specific autoantibodies were determined by MAIPA assay. An absorbance higher than the mean absorbance+3 SD recorded for the controls was considered as positive. The positive rate of pregnant and nonpregant ITP were 44.0% and 62.5%, respectively. There was no significant difference (P> 0.05).Conclusions:1. ITP in pregnancy patients displayed significantly higher serum TPO levels than GT patients. Serum TPO level might act as a differential diagnostic indicator for these two diseases.2. Serum TPO levels in ITP in pregnancy patients were significantly higher than age-matched nonpregnant ITP patients.3. Most of the ITP in pregnancy patients had decreased megakaryocyte numbers. The pathogenesis of pregnant and nonpregnant ITP might be different.Part II:The study on the regulation of platelet desialylation by recombinant human thrombopoietin in ITPObjective:To determine the platelet desialylation level of ITP patients and healthy controls, and analyze whether there is significant difference. And to detect the alteration of platelet desialylation level of ITP patients before and after recombinant human thrombopoietin (rhTPO) treatment, explore the mechanisms of rhTPO in treating ITP patients.Methods:1. Twenty-two chronic ITP patients who failed to response to first-line treatment or relapsed were enrolled in this study, peripheral blood samples were collected before treatment. Twenty-four healthy volunteers were enrolled as controls, their blood samples were also collected, centrifuged to get platelets.2. The enrolled chronic ITP patients received rhTPO treatment (300 U/kg/d), until complete response within 14 days, or 14 days later with or without response. Blood samples were collected again after treatment, centrifuged to get platelet.3. Platelets were stained with PE-Cy5-CD41 a monoclonal antibodies, and the β-Gal residues exposed by platelet desialylation were stained with FITC-RCA-I monoclonal antibodies. Detect the mean fluorescence intensity of RCA-I by flow cytometry, to represent for the platelet desialylation level.Results:1. Of the chronic ITP patients treated with rhTPO therapy,40.9% achieved complete response (CR) within 14 days,31.8% achieved response (R) after 14 days of treatment, and 27.3% had no response (NR) after 14 days. No obvious adverse events were observed during rhTPO treatment.2. The platelet desialylation levels of ITP patients were significantly higher than healthy controls (P< 0.01).3. The platelet desialylation levels of ITP patients were significantly decreased after rhTPO treatment (P< 0.01).Conclusions:1. RhTPO therapy is effective and safe in treating ITP patients.2. The increased level of platelet desialylation may contribute to the pathogenesis of ITP.3. RhTPO therapy reduces the platelet desialylation level of ITP patients, which may partially explain the therapeutic mechanisms of rhTPO in treating ITP patient.