Serum Thrombopoietin (TPO) Levels in Patients with Amegakaryocytic Thrombocytopenia Are much Higher than those with Immune Thrombocytopenic Purpura

1996 ◽  
Vol 76 (05) ◽  
pp. 675-678 ◽  
Author(s):  
Harumi Y Mukai ◽  
Hiroshi Kojima ◽  
Kazuo Todokoro ◽  
Tomoyuki Tahara ◽  
Takashi Kato ◽  
...  
Keyword(s):  
Correlation Coefficient ◽  
Immunosorbent Assay ◽  
Immune Thrombocytopenic Purpura ◽  
Steroid Treatment ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombocytopenic Purpura ◽  
Platelet Counts ◽  
Healthy Donors

SummaryWe assayed serum thrombopoietin (TPO) levels in amegakaryocytic thrombocytopenia (AMT) and immune thrombocytopenic purpura (ITP) patients by using a newly established enzyme-linked immunosorbent assay (ELISA). TPO levels in AMT patients were quite high (mean ± SD = 13.7 ± 11.2 fmoles/ml, n = 4), whereas those in ITP patients were only slightly higher (1.25 ± 0.39, n = 12) than those of the healthy donors (0.55 ± 0.2, n = 20). Furthermore, in ITP patients no correlation was observed between platelet counts and serum TPO levels (correlation coefficient = 0.14). We further assayed serum TPO levels sequentially during steroid treatment in patients with AMT and ITP. In one AMT patient serum TPO levels started to decrease in accordance with the increase of megakaryocyte counts, which preceded the increase in platelet counts. However, in ITP patients serum TPO levels did not change significantly throughout the course of the treatment despite the recovery of platelet counts. Based on these findings, we conclude that serum TPO levels may be regulated at least in part by megakaryocyte counts.

scholarly journals Detection of platelet antibodies using a micro-enzyme-linked immunosorbent assay (ELISA)

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 311-317 ◽  
Author(s):  
CA Schiffer ◽  
V Young
Keyword(s):  
Immunosorbent Assay ◽  
Immune Thrombocytopenic Purpura ◽  
Hla Typing ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombocytopenic Purpura ◽  
Microtiter Plates ◽  
Platelet Antibodies ◽  
Liquid Suspension ◽  
Large Numbers ◽  
Specific Antigens

Abstract An enzyme-linked immunosorbent assay (ELISA) for the measurement of circulating platelet antibody and platelet-associated IgG (PAIgG) is described. The test is done in microtiter plates and rapidly provides quantitative and highly reproducible results. Alloantibodies from 28 of 30 multiple transfused patients and isoantibodies from 3 of 4 patients with immune thrombocytopenic purpura (ITP) were detected. PAIgG was elevated in all 4 patients with ITP, and HLA and platelet-specific antigens were reliably detected using HLA typing sera and anti-PIA1 antibody, respectively. Platelets preserved wither by dessication in the wells of the microtiter plates or in liquid suspension in saline at 4 degrees C gave results comparable to values using fresh platelets. Storage periods ranged from 30 days for dessicated platelets to more than 1 yr for platelets stored in suspension. The ability to utilize preserved platelets may allow relatively convenient screening of large numbers of potential platelet donors for alloimmunized patients.

Detection of platelet antibodies using a micro-enzyme-linked immunosorbent assay (ELISA)

Blood ◽  
1983 ◽  
Vol 61 (2) ◽  
pp. 311-317
Author(s):  
CA Schiffer ◽  
V Young
Keyword(s):  
Immunosorbent Assay ◽  
Immune Thrombocytopenic Purpura ◽  
Hla Typing ◽  
Enzyme Linked Immunosorbent Assay ◽  
Thrombocytopenic Purpura ◽  
Microtiter Plates ◽  
Platelet Antibodies ◽  
Liquid Suspension ◽  
Large Numbers ◽  
Specific Antigens

An enzyme-linked immunosorbent assay (ELISA) for the measurement of circulating platelet antibody and platelet-associated IgG (PAIgG) is described. The test is done in microtiter plates and rapidly provides quantitative and highly reproducible results. Alloantibodies from 28 of 30 multiple transfused patients and isoantibodies from 3 of 4 patients with immune thrombocytopenic purpura (ITP) were detected. PAIgG was elevated in all 4 patients with ITP, and HLA and platelet-specific antigens were reliably detected using HLA typing sera and anti-PIA1 antibody, respectively. Platelets preserved wither by dessication in the wells of the microtiter plates or in liquid suspension in saline at 4 degrees C gave results comparable to values using fresh platelets. Storage periods ranged from 30 days for dessicated platelets to more than 1 yr for platelets stored in suspension. The ability to utilize preserved platelets may allow relatively convenient screening of large numbers of potential platelet donors for alloimmunized patients.

SENSITIVE AND SPECIFIC ENZYME-LINKED IMMUNOSORBENT ASSAY FOR UROKINASE-TYPE PLASMINOGEN ACTIVATOR IN HUMAN PLASMA

1987 ◽  
Author(s):  
J Grøndahl-HANSEN ◽  
N Agerlin ◽  
L S Nielsen ◽  
K Danø
Keyword(s):  
Plasminogen Activator ◽  
Immunosorbent Assay ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mean Values ◽  
Amino Terminal ◽  
Type Plasminogen Activator ◽  
Healthy Donors ◽  
Urokinase Type Plasminogen Activator ◽  
The Mean

An enzyme-linked immunosorbent assay (ELISA) was developed for the measurement of human urokinase-type plasminogen activator (u-PA) in plasma and serum. Microtiter plates were coated with a monoclonal antibody and incubated with standard or sample. Bound u-PA was quantitated with polyclonal antibodies conjugated with biotin, followed by avidin-peroxidase. The assay was 10-fold as sensitive as other previously reported ELISAs, the detection limit being approximately 1 pg of u-PA in a volume of 100 μl with a linear dose-response up to 15 pg of u-PA. The assay detected active u-PA and its inactive proenzyme form equally well and the recovery of both forms was higher than 90% in plasma. A variety of structurally related proteins, including t-PA, were tested, but no reaction with proteins other than u-PA and its amino-terminal degradation product were observed. The intra-assay and inter-assay coefficients of variation for determination of u-PA in plasma were 7.6% and 8.4%, respectively. The assay was equally applicable to serum. The values obtained with plasma and serum were similar, and the results were not affected by small variations in the preparation of the samples. The ELISA was used to measure the concentration of u-PA in plasma from 34 healthy donors. The mean values for u-PA in plasma from healthy donors was 1.1 ng/ml ± 0.3 ng/ml (SD) (range 0.6 - 1.5 ng/ml). No significant differences were found between men and women and no correlation between u-PA concentration and age could be demonstrated.The mean u-PA concentration in plasma from healthy donors obtained in this study is substantially lower than that reported by others. This might be due to different methods of determination of the protein content of the standard preparations or to differences in the specificity of the assays.

scholarly journals Quantitation of platelet-associated IgG by radial immunodiffusion

Blood ◽  
1981 ◽  
Vol 57 (4) ◽  
pp. 809-811 ◽  
Author(s):  
BS Morse ◽  
D Giuliani ◽  
M Nussbaum
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenic Purpura ◽  
Radial Immunodiffusion ◽  
Thrombocytopenic Purpura ◽  
Platelet Counts ◽  
Normal Platelet ◽  
Serum Igg ◽  
Acute Itp ◽  
Igg Level

Abstract Platelet-associated IgG (PAIgG) was measured by a simple radial immunodiffusion technique using washed solubilized platelets and commercially available immunoplates. Subjects with normal platelet counts had PAIgG levels of 1.5--7.0 fg/platelet. Subjects with idiopathic immune thrombocytopenic purpura (ITP) had levels ranging from 5.7 to 70.5 fg/platelet. All patients with recurrent ITP and 85% of patients with acute ITP had elevated PAIgg. Elevated PAIgG was also found in 17% of patients with recovered ITP, 40% of patients with SLE and thrombocytopenia, 57% of patients with thrombocytopenia occurring during the course of septicemia, and 100% of patients with IgG myeloma in whom the serum IgG level was clearly elevated, regardless of the platelet count. The results are similar to reports that used more complex techniques.

scholarly journals Pneumococcal Type 22F Polysaccharide Absorption Improves the Specificity of a Pneumococcal-Polysaccharide Enzyme-Linked Immunosorbent Assay

2001 ◽  
Vol 8 (2) ◽  
pp. 266-272 ◽  
Author(s):  
Nelydia F. Concepcion ◽  
Carl E. Frasch
Keyword(s):  
Correlation Coefficient ◽  
Immunosorbent Assay ◽  
Pneumococcal Vaccination ◽  
Correlation Coefficients ◽  
Enzyme Linked Immunosorbent Assay ◽  
Igg Antibody ◽  
Functional Activities ◽  
Antibody Titers ◽  
The Common ◽  
Elisa Inhibition

ABSTRACT The specificity of the immune response to the 23-valent pneumococcal-polysaccharide (PS) vaccine in healthy adults and to a pneumococcal conjugate vaccine in infants was examined by measuring immunoglobulin G (IgG) antibody titers by enzyme-linked immunosorbent assay (ELISA) and the opsonophagocytosis assay. ELISA measures total antipneumococcal IgG titers including the titers of functional and nonfunctional antibodies, while the opsonophagocytosis assay measures only functional-antibody titers. Twenty-four pairs of pre- and post-pneumococcal vaccination sera from adults were evaluated (ELISA) for levels of IgG antibodies against serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F. Twelve of the pairs were also examined (opsonophagocytosis assay) for their functional activities. The correlation coefficients between assay results for most types ranged from 0.75 to 0.90, but the correlation coefficient was only about 0.6 for serotypes 4 and 19F. The specificities of these antibodies were further examined by the use of competitive ELISA inhibition. A number of heterologous polysaccharides (types 11A, 12F, 15B, 22F, and 33A) were used as inhibitors. Most of the sera tested showed cross-reacting antibodies, in addition to those removed by pneumococcal C PS absorption. Our data suggest the presence of a common epitope that is found on most pneumococcal PS but that is not absorbed by purified C PS. Use of a heterologous pneumococcal PS (22F) to adsorb the antibodies to the common epitope increased the correlation between the IgG ELISA results and the opsonophagocytosis assay results. The correlation coefficient improve from 0.66 to 0.92 for type 4 and from 0.63 to 0.80 for type 19F. These common-epitope antibodies were largely absent in infants at 7 months of age, suggesting the carbohydrate nature of the epitope.

Platelet Counts Following Eltrombopag Discontinuation in Patients with Chronic Immune Thrombocytopenic Purpura.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3517-3517
Author(s):  
Gregory Cheng ◽  
Michael Tarantino ◽  
Terry Gernsheimer ◽  
Oliver Meyer ◽  
Andres Brainsky ◽  
...  
Keyword(s):  
Platelet Count ◽  
Immune Thrombocytopenic Purpura ◽  
Research Funding ◽  
Rescue Medication ◽  
Study Medication ◽  
Bleeding Events ◽  
Thrombocytopenic Purpura ◽  
Baseline Platelet Count ◽  
Platelet Counts ◽  
Transient Decrease

Abstract Abstract 3517 Poster Board III-454 BACKGROUND Eltrombopag (PROMACTA®; GlaxoSmithKline, Collegeville, PA, USA) is an oral, small molecule (565 Da), thrombopoietin receptor agonist that has been approved in the United States for the treatment of patients with chronic immune thrombocytopenic purpura (ITP). It is also being studied in thrombocytopenic patients with chronic liver disease, hepatitis C, myelodysplastic syndromes, and cancer. Withdrawal of treatments that stimulate platelet production may theoretically result in recurrent thrombocytopenia below pretreatment levels (below baseline). OBJECTIVE: To determine whether worsening of thrombocytopenia (ie, platelet count decrease below baseline) occurs after discontinuation of eltrombopag in patients with chronic ITP. METHODS: The lowest median platelet counts during the first 4 weeks posttherapy were compared with median baseline platelet counts. Data from 369 patients treated in 3 randomized, double-blind, placebo-controlled studies were analyzed: TRA100773A and TRA100773B were 6-week studies, and RAISE was a 6-month study. For all 3 studies, a baseline platelet count <30,000/μL was required. Platelet counts, bleeding events, and the use of ITP medication were examined in the 4 weeks following the discontinuation of eltrombopag or placebo. A transient decrease in platelet counts (ie, worsening of thrombocytopenia) was defined as a platelet count below 10,000/μL and at least 10,000/μL below each patient's baseline platelet count (Bussel N Eng J Med 2006). RESULTS: Using pooled data from the 3 studies, no decreases below baseline median platelet counts (placebo, 16,300/μL; eltrombopag, 16,000/μL) were observed compared to the lowest median platelet counts within the first 4 weeks posttherapy (placebo, 14,000/μL; eltrombopag, 17,000/μL). Across the pooled studies, a total of 10/128 (8%) of placebo-treated patients and 20/241 (8%) of eltrombopag-treated patients had a transient decrease in platelet counts in the 4 weeks following discontinuation or interruption of treatment. None of the 10 placebo-treated patients had bleeding events associated with posttreatment platelet nadirs. Three of the 20 eltrombopag-treated patients had bleeding events and/or rescue treatment associated with the platelet nadir in the 4-week posttreatment period. One patient discontinued eltrombopag after achieving platelet counts >200,000/μL following on-therapy rescue medication (corticosteroid 0.5 mg/kg/day); 9 days after discontinuing study medication, the patient had grade 1 gum bleeding and resumed daily corticosteroids at an increased dose. The second patient had grade 3 menorrhagia and was administered vincristine (patient had a history of similar symptoms). The third patient had Henoch-Schoenlein purpura, interrupted eltrombopag due to platelet counts >400,000/μL, and 7 days after holding eltrombopag had a platelet count of 2000/μL, experienced grade 1 mouth hemorrhage and grade 2 petechiae, and did not require rescue medication. The patient continued in the study for the full 6 months and following permanent discontinuation of eltrombopag, this patient did not experience a transient decrease in platelet counts or any bleeding. CONCLUSION: Across 3 placebo-controlled studies, the incidence of transient decreases in platelet counts following discontinuation or interruption of study medication was similar in patients receiving eltrombopag or placebo. Therefore, these decreases may be unrelated to study medication and may represent normal fluctuations in platelet counts in patients with chronic ITP. Transient platelet count decreases were generally not associated with bleeding events. Disclosures: Cheng: GlaxoSmithKline: Research Funding. Tarantino:GlaxoSmithKline: Speakers Bureau; Lundbeck: Speakers Bureau; Baxter: Membership on an entity's Board of Directors or advisory committees. Gernsheimer:GlaxoSmithKline: Honoraria, Research Funding; Amgen: Honoraria, Research Funding. Meyer:GlaxoSmithKline: Consultancy, Honoraria. Brainsky:GlaxoSmithKline: Employment. Stone:GlaxoSmithKline: Employment.

Thrombopoietin Levels May Predict Responsiveness to Therapy with Thrombopoietin Agonists Among Patients with Immune Thrombocytopenic Purpura,

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3288-3288 ◽  
Author(s):  
Robert Makar ◽  
Olga S. Zhukov ◽  
Mervyn A. Sahud ◽  
David J. Kuter
Keyword(s):  
Platelet Count ◽  
Clinical Response ◽  
Immune Thrombocytopenic Purpura ◽  
Myeloproliferative Disorders ◽  
Interquartile Range ◽  
Wilcoxon Test ◽  
Response To Treatment ◽  
Thrombocytopenic Purpura ◽  
Platelet Production ◽  
Platelet Counts

Abstract Abstract 3288 INTRODUCTION: Thrombopoietin (TPO) is the major regulator of platelet production. In prior clinical studies, thrombopoietin levels have been shown to vary inversely with circulating platelet mass and with the rate of platelet production. Thus, TPO levels may help distinguish between the various disorders of thrombocytopenia. In addition, the introduction of TPO agonists has created an interest in predicting the response of patients to these agents. Determining TPO levels may help predict such treatment responses. METHODS: Sera from 121 patients with a history of abnormal platelet counts were tested using a novel, commercially available ELISA assay that measures TPO levels. The TPO assay detected TPO levels as low as 7 pg/mL and was linear for levels up to 2000 pg/mL. The coefficient of variation ranged from 27% near the lower limit of detection to 9% at a TPO concentration of 669 pg/mL. The reference range for TPO was established in serum samples from 118 apparently healthy individuals (58 males and 60 females) and was 7–99 pg/mL. The Wilcoxon test was used to compare continuous variables and the Fisher's exact test was used to compare categorical variables. RESULTS: The patient population included 40 patients with a consumptive thrombocytopenia (38 with primary or secondary immune thrombocytopenic purpura (ITP), 2 with thrombotic thrombocytopenic purpura), 34 patients with myeloproliferative disorders (23 with essential thrombocytosis, 9 with polycythemia vera, 2 with an ill-defined myeloproliferative disorder), and 47 patients with hypoproliferative thrombocytopenia (29 with chemotherapy-related thrombocytopenia, 19 with primary or secondary bone marrow failure syndromes). Among the 38 patients with ITP, 11 were taking TPO agonists (9 on romiplostim, 2 on eltrombopag), 19 were taking immunomodulatory agents (16 on steroids alone or in combination with other therapies, 2 on azathioprine, 1 on danazol), and 12 were off ITP-specific therapy when the TPO level was measured. 9 out of 38 (24%) patients with ITP had undergone splenectomy and/or been previously treated with rituximab. The median serum TPO level in patients with consumptive thrombocytopenia was 64.5 pg/mL (interquartile range, 48.5–97.5 pg/mL) and the corresponding median platelet count was 68,000/μL (interquartile range, 27,000–144,500) (Figure). While patients with myeloproliferative disorders had similar TPO levels [median 87.0 pg/mL (38.0–125.5)], their platelet counts were significantly higher than those of patients with consumptive thrombocytopenia [median 549,500/mL (431,250–693,000] (P <0.0001). Contrastingly, comparable platelet counts [median 61,000/μL (31,000–118,000)] were observed among patients with hypoproliferative thrombocytopenia, but serum TPO levels were significantly higher than those of patients with consumptive thrombocytopenia [844 pg/mL (409.5–1551.5), P <0.0001]. Among 22 evaluable patients meeting diagnostic criteria for primary or secondary ITP who had taken a TPO agonist for at least 1 month, serum TPO levels appeared to predict responsiveness to the drug. A clinical response to a TPO agonist was defined as achieving a platelet count ≥50,000/μL after starting the drug and maintaining it at or above that count in ≥50% of subsequent complete blood counts from initiation until discontinuation of the drug, loss to follow-up, or 6 months had passed, whichever was longest, without the need for recurrent rescue therapy. Whereas 14 out of 16 (88%) ITP patients with a TPO level <99 pg/mL met our definition for a clinical response to treatment with a TPO agonist, only 1 out of 6 patients (17%) with a TPO level >99 pg/mL responded (P <0.005 for the difference in clinical response to TPO agents.) CONCLUSIONS: TPO levels may have diagnostic utility in discriminating between patients with hypoproliferative and consumptive thrombocytopenia. High TPO levels among patients with ITP may predict a poor clinical response to treatment with TPO agonists. Further studies are required to confirm these data. Disclosures: Zhukov: Quest Diagnostics: Employment. Sahud:Quest Diagnostics: Employment. Kuter:Quest Diagnostics: Consultancy, Research Funding.

scholarly journals An enzyme-linked immunosorbent assay for erythropoietin using monoclonal antibodies, tetrameric immune complexes, and substrate amplification

Blood ◽  
1989 ◽  
Vol 74 (2) ◽  
pp. 622-628 ◽  
Author(s):  
AW Wognum ◽  
PM Lansdorp ◽  
AC Eaves ◽  
G Krystal
Keyword(s):  
Monoclonal Antibodies ◽  
Immunosorbent Assay ◽  
Biological Fluids ◽  
Normal Serum ◽  
Detection Sensitivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Response Curves ◽  
Human Erythropoietin ◽  
Healthy Donors ◽  
Noncovalent Labeling

We recently reported the development of several monoclonal antibodies (MoAbs) to native human erythropoietin (Ep). In the present study we have used the two antibodies with highest affinity to develop a two- sided or sandwich enzyme-linked immunosorbent assay (ELISA) to measure Ep in human serum. In this assay Ep is incubated in microtiter wells precoated with the first (IgE) anti-Ep antibody. Assay wells are then incubated with the second (IgG1) anti-Ep antibody, which is labeled noncovalently with the enzyme alkaline phosphatase (AP) by means of bispecific tetrameric antibody complexes consisting of IgG1 anti-Ep cross-linked to IgG1 anti-AP using rat MoAbs specific for mouse IgG1. Application of this noncovalent labeling procedure, in combination with substrate amplification, results in a detection sensitivity of 0.5 to 1.0 mU/sample (5 to 10 mU/mL), which makes this assay suitable for measuring normal serum Ep levels. The validity of this ELISA for quantitating Ep in biological fluids was demonstrated by the parallelism obtained between pure recombinant Ep dose-response curves and those obtained with plasma and serum from healthy donors and patients with various hematologic disorders. Normal plasma Ep levels detected with this ELISA ranged from 9 to 101 mU/mL with a mean of 32 +/- 23 (SD) mU/mL. Ep levels in sera from patients with polycythemia vera were in the low to normal range, whereas Ep levels in sera from patients with secondary polycythemia and patients with aplastic anemia were moderately to strongly elevated. These results demonstrate that the Ep-ELISA is a sensitive, reliable, and nonradioactive immunologic method for quantitating Ep levels and should prove useful in a variety of clinical and laboratory settings.

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