The clinical significance of immune-related marker detection in idiopathic thrombocytopenic purpura / 中华内科杂志

Objective To assess the clinical significance of detecting the immune markers in idiopathic thrombocytopenic purpura (ITP). Methods The frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombopoietin in 64 ITP patients and 31 healthy controls were measured with enzyme-linked immunospot assay (ELISPOT),modified monoclonal antibody immunobilization of platelet antigens assay (MAIPA), flow cytometry and sandwich enzyme-linked innnunosorbent assay respectively. Results Compared with the controls[1.3 ± 0. 5/105 peripheral blood mononuclear cell (PBMC), (0.33±0.06,0.41±0.03), (22.08±4.54)% and (8.19±2.46)%], the frequencies of circulating B cells secreting platelet-specific antibody (7.6±4.6/105 PBMC in acute ITP group, 5.3±3.0/105 PBMC in chronic ITP group), platelet-specific antibody (including the anti-GP Ⅱ b/Ⅲa antibody, anti-GP Ⅰ b/X antibody) (0.51 ±0.11, 0.48±0.06 in acute ITP group; 0.49±0.10,0.46±0.09 in chronic ITP group), the percentage of CD8+ T Lymphocyte (27.09±9.86 ) %, the percentage of reticulated platelet in ITP patients[the megakaryocyte cytosis group (24. 85 ± 19. 18)%, the normal megakaryocyte group (23.89±18.90)%]were significantly increased ( all P＜0.05).The frequencies of circulating B cells secreting platelet-specific antibody in acute ITP patients were notably increased (P＜0.05) compared to the chronic ITP patients. In T lymphocyte subsets, the percentage of CD3+T lymphocyte and CD4+ T lymphocyte and the ratio of CD4+/CD8+ in the patients with ITP[(60.88±14.59)%, (28.41±10.55)%, 1.18±0.59]were notably decreased than those in the healthy controls [(69.89±6.43)%, (35.38±5.05) %, 1.64±0.29, P＜0.05]. There was no apparent difference of the level of thrombopoietin between ITP patients with megakaryocyte cytosis (72. 09 ± 41.64 ) and health controls (75.37± 26. 32, P ＞ 0. 05 ), however, the level of thrombopoietin of ITP patients with normal megakaryocyte apparently increased (118.60±70.72, P＜0.05). Conclusion Detecting the frequencies of circulating B cells secreting platelet-specific antibody, platelet-specific antibody, the percentage of T lymphocyte subsets, the percentage of reticulated platelet and the level of thrombepoietin in the patients with ITP may improve the diagnosis and guide clinical therapy.

Clinical Usefulness of the Simple Technique to Diagnose Thrombocytopenia Using Immature Platelet Fraction / 대한진단검사의학회지

BACKGROUND: Immature platelet fraction (IPF) is the percentage of reticulated platelet (RP) of total platelet count. We measured an IPF reference range using XE-2100 blood cell counter with upgraded software (Sysmex, Japan) and evaluated the clinical utility of this parameter for the laboratory diagnosis of thrombocytopenia due to an increase in peripheral platelet destruction. METHODS: Peripheral blood samples collected into K2 EDTA (Beckton Dickinson, USA) were analyzed at Chonbuk National University Hospital. One hundred forty-two samples from apparently healthy adults (all routine full blood count parameters including platelets within the healthy reference range) were used to establish a normal reference range for IPF. The patients were classified into 3 groups including hypoplastic (consisted of 22 patients undergoing chemotherapy with falling platelet counts and 14 with aplastic anemia), cirrhotic (40 with cirrhosis of liver), and idiopathic thrombocytopenic purpura (ITP) (14 with ITP) groups. RESULTS: An IPF reference range in healthy individuals was established as 0.4-5.4%, with a mean of 1.7%. A significant increase in IPF values was found in the ITP patient group. The cut-off value of IPF was 6.1% and its sensitivity and specificity were 92.9%, and 82.9% respectively. Reproducibility was good. CONCLUSIONS: A rapid, inexpensive automated method for measuring IPF is feasible and should become a standard parameter in evaluating thrombocytopenic patients.

Comparison of an Immature Platelet Fraction and Reticulated Platelet in Liver Cirrhosis / 대한진단검사의학회지

BACKGROUND: The main causes of thrombocytopenia in cirrhosis are thought to be platelet destruction and the reduction of thrombopoietin (TPO) expression in the liver. Immature platelet fraction (IPF) has been measured by a fully automated analyzer (Sysmex XE-2100, Japan) as reticulated platelet (RP), which is reflected with thrombopoiesis in bone marrow. In this study, we tried to compare the percentage of IPF (IPF) with that of RP (RP) in patients with liver cirrhosis (LC) and controls. METHODS: We compared IPF to RP in 72 liver cirrhosis patients and 30 healthy normal controls. RP was stained with acridine orange, followed by FC500 (Beckman Coulter, USA) analysis and the IPF was identified by flow cytometry with the use of a nucleic acid specific dye in the reticulocyte channel on the Sysmex XE-2100 (TOA Medical Electronics Co., Ltd., Japan). RESULTS: IPF value in the healthy control was 2.2% (1.7-5.2). RP and IPF were significantly higher in the patients with liver cirrhosis (P<0.05). IPF appeared to be correlated with RP (y=0.19x+3.35, r=0.34, P<0.05). In ROC for diagnosis of LC, IPF was significantly more useful than RP. CONCLUSIONS: This results show that a rapid, inexpensive automated method for measuring the IPF is feasible and should become a standard parameter in evaluating reticulated platelets.

Clinical Significance of Thrombopoietin Levels in Maintenance Hemodialysis Patients with Thrombocytopenia / 대한신장학회잡지

BACKGROUND: Thrombopoietin (TPO) is a major cytokine which plays a critical role in the regulation of thrombopoiesis and megakaryopoiesis. Since the kidney is one of the TPO-producing organs, it is hypothesized that TPO deficiency in end stage renal disease can give rise to thrombocytopenia. However, serum TPO levels and their clinical significance in maintenance hemodialysis patients with thrombocytopenia are not completely evaluated. The aim of the present study was to compare the percentage of reticulated platelets and serum TPO levels between non-thrombocytopenic group (platelet count > or =150x109/L, non-T group) and thrombocytopenic group (platelet count <150x109/L, T group) and to investigate the local and/or systemic effect of the TPO on the platelet count in hemodialysis patients. METHODS: We measured the percentage of reticulated platelets and serum TPO levels in samples obtained from venous returns of arteriovenous fistula (AVF) and contralateral peripheral veins in 44 hemodialysis patients. Serum reticulated platelets were measured by flow cytometry and serum TPO levels were determined with a commercially available ELISA kit. Patients with a history of HBV/HCV infection and hepatobiliary disease were excluded. RESULTS: Reticulated platelets of T group (4.57+/-2.32%) were significantly lower than non-T group (7.79+/-3.62%) (p<0.05). Serum TPO levels obtained from venous return of AVF in T group (78.37+/-15.48 pg/mL) were lower than non-T group (98.15+/-35.05 pg/mL) (p<0.05). Serum TPO levels obtained from contralateral peripheral veins in T group (77.20+/-17.28 pg/mL) were lower than non-T group (104.73+/-38.45 pg/mL) (p<0.01). There were no statistically significant difference of serum TPO levels between venous return of AVF and contralateral peripheral veins in T group. CONCLUSION: Decreased circulating reticulated platelets and serum TPO levels despite low platelet counts in comparison with normal platelet counts in hemodialysis patients, suggesting that the feedback mechanism, the TPO producing organ and bone marrow is not working with effect in the regulation of thrombopoiesis. An alteration in the production and regulation of TPO level is not influenced by local factor like an AVF endothelium.

Estimate of Thrombopoiesis by Flow Cytometric Analysis of Reticulated Platelets in Moderate Thrombocytopenia / 대한진단검사의학회지

BACKGROUND: Measurement of reticulated platelets (RPs) is useful in order to discriminate the causes of thrombocytopenia. In flow cytometric analysis for the percentage of RPs (RPs%), only thiazole orange (TO) fluorescence is considered, ignoring the significance of forward scatter (FSC). In this study, we intended to devise a new index reflecting the pattern of platelet clusters in the plot of FSC vs. TO fluorescence. METHODS: In the patients with moderate thrombocytopenia, 44 cases with decreased thrombopoiesis and 37 cases with increased thrombopoiesis were selected. In flow cytometry, several indices made with FSC and TO fluorescence were evaluated in discrimination between the two groups. RESULTS: Of the primary parameters, RPs% and FSC had relatively high efficiencies in discrimination. A new index incorporating these two parameters had higher efficiency than RPs% (P<0.05). New index=RPs%x(TO-positive platelet FSC-TO-negative platelet FSC)/TO-negative platelet FSC) CONCLUSIONS: In addition to the RPs%, more information about thrombopoiesis was obtained through the analysis of the plot of FSC vs. TO fluorescence and the index that quantifies the pattern of TOstained platelet clusters was devised.

Clinical Study of Idiopathic Thrombocytopenic Purpura: Focused on Reticulated Platelet / 대한소아혈액종양학회지

PURPOSE: Immune thrombocytopenic purpura (ITP) is divided into acute and chronic forms. Unfortunately, there have been no known specific laboratory or clinical predictors for the diagnosis of chronic ITP. This study was performed to elucidate the prognostic significance of various clinical and laboratory parameters, including reticulated platelet percentage. METHODS: We retrospectively analyzed 60 patients who were diagnosed as ITP at the Department of Pediatrics, Wonju Christian Hospital from January, 1989 to January, 2001. Various kind of clinical parameters such as age, sex, symptom duration, prior URI history, response to treatment, and laboratory parameters like platelet count at initial presentation, lowest platelet count, duration of thrombocytopenia, initial reticulated platelet percentage, antiplatelet antibody IgG and IgM, antinuclear antibody (ANA), direct and indirect Coombs' test were compared between acute and chronic ITP. RESULTS: Fifteen % of patients (9/60) was chronic ITP. The peak age incidence was from 1 to 3 year of age in both acute (29.4%) and chronic ITP (22.2%). The acute ITP was prevalent in spring season, May and June. There was no difference in the incidence of prior URI history between acute and chronic ITP. Higher proportion of chronic ITP patients (5/9; 55.5%) than acute ITP patients (7/51; 13%) had symptom duration longer than 1 month (P or =1 months duration of presenting symptoms and relapse after 2 months from initial presentation have the propensity of developing chronic ITP. Reticulated platelet percentage could not discriminate acute and chronic ITP. A more accurate detection method should be developed for reticulated platelets.

Recent development of hematology analysis technique and its clinical application / 中华检验医学杂志

The paper introduces the development of hematology analysis technique in recent 10 years,and mainly concentrates in WBC differential technique,the extended functions and automation.The clinical application of red cell volume distribution width,reticulated platelets and reticulocyte subpopulation are also described in the paper.The common problems in the usage of hematology analyzer and correction methods are pointed out.

The Diagnostic Significance of the Reticulated Platelet in Thrombocytopenia of Newborn, Prematurity and Perinatal Asphyxia / 대한소아혈액종양학회지

BACKGROUND: Reticulated platelets(RPs) are young platelets with high RNA content. The RP percentage(RPP) reflects bone marrow thrombopoietic activity. The aim of this study is to determine the normal value of RPPs in fullterm and premature neonates at birth and at 7 days of age, and to find out the diagnostic significance of RPP in thrombocytopenic neonates of various causes. METHODS: Flow cytometric measurements of RPP were performed in 38 healthy fullterm(Group 1), 30 premature(Group 2), 15 healthy, spontaneously recovered thrombocytopenic neonates(Group 3), 21 neonates with thrombocytopenia due to sepsis, perinatal asphyxia or hyaline membrane disease(HMD)(Group 4), and 25 healthy adults(Group 5). RESULTS: The RPPs of groups 1 and 2 were 3.10+/-2.00%(mean+/-SD) and 3.80+/-2.96% at birth, 2.66+/-1.44% and 2.85+/-1.69% at 7 days of age respectively. That of group 5 was 7.17+/-2.01%. Normal RPPs of healthy term and preterm neonates were significantly lower than that of healthy adults. The RPPs of term groups 3 and 4 were 2.05+/-2.43% and 6.94+/-3.66% respectively. That of preterm groups 3 and 4 were 2.00+/-1.04% and 7.29+/-4.87% respectively. The RPP of group 4 was significantly higher than groups 1, 2, or 3. There was no significant difference of Mean Platelet Volume(MPV) or Platelet Distribution Width(PDW) between group 3 and group 4. Reasonable cut-off value of RPP for the diagnosis of Group 4 was 8%, by which we could confirm 9 cases among 21 cases of neonatal thrombocytopenia due to sepsis, perinatal asphyxia, or hyaline membrane disease. CONCLUSIONS: The bone marrow thrombopoietic activity of neonates seems to be lower than that of adults. Decreased thrombopietic activity of bone marrow seems to be a cause of spontaneously recovering idiopathic neonatal thrombocytopenia. Increased platelet destruction seems to be a cause of neonatal thrombocytopenia due to sepsis, perinatal asphyxia, or hyaline membrane disease. The RPP higher than 8% could be a good supportive diagnostic criterion for neonatal thrombocytopenia due to sepsis, perinatal asphyxia, or hyaline membrane disease.

Diagnostic Value of Reticulated Platelet in Thrombocytopenia

PURPOSE: We performed this study to determine the diagnostic significance and useful cut-off value of reticulated platelet, which can be used for discriminating the destructive thrombocytopenia from underproductive thrombocytopenia. METHODS: We evaluated 37 patients with thrombocytopenia who were admitted to the Wonju christian hospital from March to July, 1995. All patients were evaluated with bone marrow megakaryocyte count. We divided them into two groups, group 1 was consisted of underproductive thrombocytopenic patients and group 2 was consisted of destructive thrombocytopenic patients. We measured the peripheral blood reticulated platelet count by FACScan flowcytometry after thiazole orange staining. RESULTS: 1) Among 37 patients, underproductive group consisted of 21 patients and destructive group consisted of 16 patients. 2) There was no significant difference in platelet count between two groups (41.7 31.8x109/L vs. 41.0 29.1x109/L). 3) Destructive group showed higher reticulated platelet count than underproductive group (7.7 3.7% vs. 19.1 11.1% : p<0.01). 4) Ideal cut-off value of reticulated platelet count for the discrimination of two groups was 11%. Its sensitivity and specificity were 88% and 95%, respectively. CONCLUSIONS: We suggest that reticulated platelet count is closely correlated with thrombopoiesis of bone marrow. The measurement of reticulated platelet can be a simple and useful diagnostic tool to discriminate destructive thrombocytopenia from underproductive thrombocytopenia.