Heparin Assay and Protamine Titration
|
|
- Hubert Martin
- 7 years ago
- Views:
Transcription
1 Heparin Assay and Protamine Titration GUILLERMO ANIDO, M.D. AND DONNA JEAN FREEMAN, MT, H(ASCP) Anido, Guillermo and Freeman, Donna Jean: Heparin assay and protamine titration. Am J Clin Pathol 76: ,1981. By utilizing the fact that heparin and protamine sulfate directly neutralize each other, it is possible to quickly detect excess levels of protamine (anti-heparin activity) by back-titrating a plasma specimen with predetermined amounts of heparin. The approach suggested allows for the simultaneous deflnitiori of low levels of heparin, and incorporates an equally rapid and accurate measurement of high heparin concentrations. The methodology presented employs heparin assays using the "Protopath"* technic. The concept appears to be applicable to other test systems currently designed to monitor heparin activity. (Key words: Protopath; Heparin; Protamine; Thrombin; Synthetic substrates; Antithrombin III.) THE MAJORITY of requests received by this laboratory for heparin assays are generated by in-house surgical teams performing cardiovascular procedures which require the administration of high levels of heparin, followed post-surgically by neutralizing doses of protamine. Depending upon when the specimen is obtained, three distinct conditions can be encountered: 1) a "highly heparinized" specimen obtained during the course of surgery, 2) a "slightly heparinized" specimen obtained after partial neutralization with protamine, or 3) a "minus heparin" specimen that contains no residual heparin but, instead, excess levels of protamine. Our objective has been to develop an approach which would allow us to respond quickly and accurately to any of these three circumstances. The Protopath heparin assay is based upon the inhibition of thrombin by heparin as measured by using a synthetic fluorescent substrate. 2 The assay includes the following steps, and all reagents are supplied with the test kit: 1) patient plasma is added to diluted pooled normal plasma (Citrated Plasma Reagent = CPR) to eliminate effects of possible variations in levels of Antithrombin III (ATIII), 2) a standard amount of thrombin is added to the plasma dilution and the mixture is incubated for a specific length of time, 3) the mixture is added to a fibrinogen analogue, thrombin-sensitive, synthetic substrate (D-phenylalanine- Received January 16, 1981; received revised manuscript and accepted for publication March 2, Address reprint requests to Dr. Anido: Department of Laboratory Medicine, Broward General Medical Center, Fort Lauderdale, Florida * Dade Division, American Hospital Supply Corporation; Miami, Florida. Department of Laboratory Medicine, Broward General Medical Center, Fort Lauderdale, Florida proline-arginine-5-"aminoisophthalic acid dimethyl ester" = "AIE"), and 4) the fluorescence of AIE released by the action of thrombin is read in the Protopath. Equipment calibration establishes a reading of 100% thrombin activity when no detectable heparin is present. Dade recommends preparation of standard curves for each test kit lot number and each source of heparin (beef lung or porcine intestinal mucosa) as well as for each manufacturer. Heparin concentrations of 0, 0.1, 6.2, 0.4, and 0.8 U/ml heparin are assayed and results plotted on linear graph paper (See Fig. 1). Heparin concentrations in patient plasmas are read from the standard curve, relative to fluorescence. As set forth by Dade, the assay accurately measures therapeutic heparin levels commonly employed in the treatment of deep vein thrombosis, pulmonary emboli, etc., and the protocol includes brief suggestions for adapting the technic to measuring levels of heparin encountered during cardiovascular surgery. The following are the modifications and applications used in our laboratory. Materials and Methods Between June and December, 1980, 75 patient specimens were assayed for heparin activity. Of these, 50 specimens were "post-protamine" and were assayed for heparin and protamine. The other 25 were specimens from patients being heparinized for various conditions. Although we do use our technic to measure therapeutic levels of mucosal heparin, our most frequent assay is for beef lung heparin at either low or high concentrations. Beef lung heparin calls for the use of longer incubation times to stabilize assay results. Longer times, unfortunately, decrease the sensitivity of the curve at the low heparin concentrations. The first step of our investigation was to modify the protocol for constructing standard curves to suit our needs. Preparation of Standard Curve Heparin reference points of 0.2, 0.4, 0.6, and 0.8 U/ ml are assayed. The results are plotted onto three cycle /81/0010/0410 $00.80 American Society of Clinical Pathologists 410
2 o z z < oo o cc *» i O.k 0.5 HEPARIN, USP U/ml FIG. 1. Typical heparin standard curve obtained following the protocol established by the manufacturer of the Protopath. Plotting on regular graph paper fails to show straight linear relationship of remaining thrombin with heparin concentration.
3 412 semi-log paper which yields a linear relationship between the four points. Our subsequent assay approach is adjusted to utilize this linearity. A measured 1.0 ml specimen of pooled normal plasma is assayed using 5 /A to obtain a zero reference point (this point is not actually included in the curve). Then 5 /u.1 of a 40 U/ml heparin-saline preparation is added to the plasma specimen for a heparin concentration of 0.2 U/ml in exactly 1.0 ml volume; 5 /ul of the specimen are then used to assay that concentration. Another 5 IJL\ of the 40 U/ml heparin-saline preparation is then added to the specimen, resulting in a heparin concentration of 0.4 U/ml in the 1.0 ml volume. As before, 5 jxl are used to assay the 0.4 U/ml heparin concentration. This cycle of removing 5 p\ from the specimen for assay and then adding 5 fi\ of the heparinsaline preparation to it is repeated for the remaining two reference points of 0.6 and 0.8 U/ml heparin. The 40 U/ml heparin-saline preparation is stored at 2-8 Centigrade for later use. The resulting standard curve is illustrated in Fig. 2. Assay of "Highly Heparinized" Specimens Upon receipt of a highly heparinized specimen (obtained during surgery), a 1.0 ml volume of a 1:10 dilution of the specimen is prepared in normal pooled plasma (according to diluent requirements set forth by Dade) and assayed using 5 /ul of the dilution. If the original specimen contains between 2 and 6 U/ml heparin, the assay of the dilution reads between 0.2 and 0.6 U/ml on the standard curve prepared for that particular heparin. Multiplication by the dilution factor 10 yields the final result. A specimen dilution reading greater than 0.6 U/ml (indicating that the original specimen contained greater than 6 U/ml heparin concentration) would be further diluted 1:2 to obtain a reliable reading with which to calculate a final result. Usually the one step 1:10 dilution is sufficient. Should a specimen dilution read less than 0.2 U/ml, the final result would be obtained using the definitive assay described below. Assay of "Slightly Heparinized" Specimens For a specimen suspected of having a low heparin level, usually post-surgically, therapeutically, or "rebound," the assay is first run "as is" for confirmation using 5 /xl from a 1.0 ml volume. A definitive assay is then performed as follows: 5 (JL\ of the appropriate 40 U/ml heparin-saline preparation is added to the plasma - specimen, restoring exactly the 1.0 ml volume and increasing the specimen heparin concentration by 0.2 U/ml. 5 /u.1 of this specimen is then assayed for the cumulative heparin result. ANIDO AND FREEMAN A.J.C.P.. October 1981 If the original specimen contains, for instance, 0.06 U/ml heparin, the "as is" results falls below our lower curve limit of 0.2 U/ml. However, upon the addition of 0.2 U/ml heparin, the cumulative result is 0.26 U/ml, and that concentration can be read accurately from the curve. The final result issued for the patient is = 0.06 U/ml heparin. The addition of 0.2 U/ml heparin to the initial specimen shifts the readings into our standard curve where small changes in concentration can be reliably defined. Assay of "Minus Heparin" Specimens All post-protamine specimens are assayed as described in our protocol for "slightly heparinized" specimens. The "as is" result, under "minus heparin" conditions, will undoubtedly read below the 0.2 U/ml point on the standard curve. The definitive assay result, after the addition of 0.2 U/ml heparin, determines whether or not there is an excess of protamine, reported as "antiheparin activity." That is, if, after the addition of heparin, the specimen fails to read at a level of 0.2 U/ml or, in fact, stays near the zero level, the presence of excess protamine is confirmed. By adding incremental amounts of 0.2 U/ml heparin to the specimen and assaying it after each addition, it is possible to "back-titrate" the specimen to a reading at or above the 0.2 U/ml reference point. By deducting the accepted heparin reading from the amount of heparin added, a quantitative result can be issued in terms of "anti-heparin activity" demonstrated by the original specimen. For instance, if a total of 0.4 U/ml heparin is added to bring the specimen to a reading of 0.25 U/ml, the "anti-heparin activity" in the original specimen is = 0.15 U/ml. As in the procedure described for constructing a standard curve, an exact volume of 1.0 ml specimen volume is maintained throughout the process. Considerations 1. All results are confirmed by performing an assay after the addition of a known amount of heparin. Levels of less than 0.05 U/ml heparin or antiheparin activity are reported as "none detected." 2. Excess levels of protamine interfere with the coagulation process and thus act as an anticoagulant. It is our experience that protamine does not give an apparent (but false) heparin reading using this fluorescent technic. 3. The terminology "anti-heparin activity" is employed for reporting results of this nature because the assay is not a direct assay of protamine, but rather it is an assay of overall anti-heparin activity.
4 HEPARIN ASSAY AND PROTAMINE TITRATION 413 \ ^ \ \ Vw o.; 1 0. h HEPARIN, USP U/ml FIG. 2. Typical heparin standard curve obtained following our protocol. Linear relationship of remaining thrombin with heparin concentration is obtained by plotting on three cycle semi-log paper. Extrapolation to zero heparin concentration is eliminated.
5 414 ANIDO AND FREEMAN A.J.C.P. October 1981 In-vivo contribution of Platelet Factor 4 is reflected in the assay. 4. Heparin or anti-heparin activity is reported with the assay requisition which identifies the heparin given to the patient. In our laboratory, "backtitration" is routinely performed using the heparinsaline standard preparation. A result for either heparin concentration or anti-heparin activity corresponds to, for instance, beef lung heparin from a certain manufacturer and lot number. 5. The 0.2 U/ml lower limit for reliability was chosen because normal plasma (containing no detectable heparin) may give somewhat different results when incubated with thrombin for 90 sec (an assay specification for beef lung heparin). Random normal specimens yield similar results upon the addition of 0.2 U/ml heparin. Our graph is sufficiently accurate at 0.2 U/ml to allow the detection of small changes in heparin concentration above this point. Also, this point is far enough removed from the zero point to avoid erroneous interpretations of low level heparin; and, while the slope of the curve at this point allows for minor variations, the heparin concentration primarily determines the outcome. 6. The reference point of 0.8 U/ml on the standard curve is assayed and plotted; however, this point serves primarily to reaffirm the linearity of the previous points. 7. A statement of the exact time a specimen was obtained must accompany all assay reports. Delayed responses must take into consideration metabolic clearance of heparin or protamine. After an initial 10 min incubation of the substrate cuvettes, each assay is completed within 5 min. The possibility of specimen contamination at the time of collection should be considered by those interpreting results. 8. The plasma diluent used in the assay (CPR) supplies sufficient ATIII to combine with heparin concentrations referenced on the standard curve. Thus, the results obtained from the assay represent the percent thrombin remaining when the heparin present functions at maximum reactivity. It is understood that, by artificially introducing a full complement of ATIII into the test system, a purely in-vitro measurement of heparin results. Patients on high doses of heparin demonstrate rapid saturation and resulting depletion of their own ATIII level. This must be considered when interpreting heparin assay results, or evaluating concurrent coagulation studies dependent upon clotting endpoints. The assay is of particular advantage if results are to be used for calculating neutralizing doses of protamine. 9. The assay uses 5 ul of plasma which is first diluted with 200 ul CPR, then further diluted with 2,000 ul substrate. This offers two advantages: 1) replicate determinations can be made from the 1.0 ml specimen volume without seriously disturbing the heparin concentration, and 2) hemolysis, often encountered during by-pass procedures, does not interfere with the accuracy or reproducibility of the assay. 10. The Protopath methodology for performing heparin assays requires meticulous technic. Incubation times, reagent specifications and other assay parameters are strictly standardized. A heparin control is provided with the test kit; and, for internal standardization, the heparin-saline preparation to be used in an assay is first assayed using known normal plasma to confirm the accuracy and reproducibility of the standard curve. Discussion Although heparin management during and after cardiovascular surgery is pertinent, the appearance of post-surgical bleeding can be due to a variety of causes. For appropriate patient follow-up, tests are provided to identify individual clotting factor deficiencies and inhibitors, ATIII levels, fibrin degradation products, plasminogen levels, etc. The methodology presented here introduces an approach to: distinguish the presence of protamine, define low levels of heparin; and rapidly determine high heparin concentrations. Results obtained using heparin as the titrating agent in this context are within 0.02 to 0.05 U/ml of the calculated heparin level. We have performed additional studies on the Protopath using protamine to titrate heparin of low, moderate, and high concentrations. Results correlate with calculated values within 0.05 to 0.10 U/ml heparin. Other methods for heparin assay include: 1) Activated Partial Thromboplastin Time (APTT), 4 2) Thrombin Time (TT), 5 3) Activated Coagulation Time (ACT), 1 and 4) Hepcon/System A-10* 3 an instrument utilizing protamine to measure heparin concentrations via an adaptation of the ACT. All four procedures are dependent upon clot production and prolonged times. In addition, the Hepcon/System A-10 is temperature dependent. The concept employed in our approach is simple and perhaps widely applicable. The addition of an internal standard of heparin to a specimen will either: 1) add to the effects of pre-existing heparin, 2) yield predictable results in patients having no detectable heparin, or 3) demonstrate an opposite or decreased effect from * HemoTec, Inc.
6 Vol. 76 No. 4 HEPARIN ASSAY AND PROTAMINE TITRATION 415 that normally found, due to the presence of protamine. It appears logical that, by using a heparin "back-titration" technic, laboratories now performing standard coagulation studies to monitor heparin activity could provide a test for excess protamine. Limited investigations using this concept with an APTT test system have been performed in this laboratory. The following is an example of the data obtained: Specimen Concentrations: Protamine Heparin mg/ml None mg/ml 0.2 U/ml mg/ml 0.4 U/ml None 0.2 U/ml None 0.4 U/ml APTT (Normal Range = 21 to 29 seconds) 36.5 seconds 35.2 seconds 31.9 seconds 58.9 seconds 86.3 seconds Although heparins of different types neutralize protamine to various degrees, a general ratio of 1 mg protamine to 100 units of heparin could be applied to the above data such that mg/ml protamine might be expected to neutralize 0.5 U/ml heparin. As shown, the addition of heparin to the specimen containing protamine yields APTT results correspondingly more normal. While the magnitude of the change is not great, the direction of the change is steady. It seems likely that, if 0.6 U/ml heparin were added to a specimen containing mg/ml protamine, a "pass through" phenomenon might occur whereby the protamine present would be neutralized and the residual heparin would yield a prolonged result. Yet, if compared with the effects of adding that same amount of heparin to a known normal plasma, the difference in results would be readily apparent. In fact, it appears possible to estimate the amount of protamine present by identifying the heparin concentrations necessary to achieve such a "pass through." Quantitative limitations would include those of the test system utilized. Specific plasma clotting factors, especially ATIII levels, hemolysis, etc., may affect the findings in studies of this type. Yet, the feasibility of determining whether an initially prolonged finding is due to one of the other medications is upheld. Conclusions Heparin assay using a synthetic substrate eliminates the dependency upon clot production as an end point. Coagulation factor abnormalities and inhibitor levels do not affect the assay results. The use of a heparin internal standard and a linear standard curve in all our determinations increase the accuracy and reproducibility of results. 3. This system allows for the simultaneous determination of either heparin or protamine. 4. The reverse titration technic represents a simple approach to determine the concentration of excess protamine. 5. The Protopath heparin assay in conjunction with the adaptations we suggest offer the advantages of speed, accuracy, reproducibility, and specificity. References I. Bull BS, et al: Heparin therapy during extracorporeal circulation. J Thorac Surg 69: , 1975 >. Dade Heparin Synthetic Substrate Assay Package Insert CO (Revised 6/80). I. HemoTec, Inc. Hepcon/System A-10. Heparin Analyzer Product Information Bulletin L Langdell RD: Partial thromboplastin time technics. Thrombosis and bleeding disorders. Edited by NV Bang, FK Beller, E Deutsch, et al., New York, Academic Press, Sirridge MS: Laboratory evaluation of hemostasis, Philadelphia, Lea and Febiger, 1967.
Contents. Abstract... i. Committee Membership... iii. Foreword... vii. 1 Scope... 1. 2 Introduction... 1. 3 Standard Precautions...
Vol. 28 No. 20 Replaces H47-A Vol. 16 No. 3 One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test; Approved Guideline Second Edition This document provides guidelines
More informationSession 3 Topics. Argatroban. Argatroban. Drug Use and Adverse Effects. Laboratory Monitoring of Anticoagulant Therapy
~~Marshfield Labs Presents~~ Laboratory Monitoring of Anticoagulant Therapy Session 3 of 4 Michael J. Sanfelippo, M.S. Technical Director, Coagulation Services Session 3 Topics Direct Thrombin Inhibitors:
More information75515-7 Lupus anticoagulant aptt & drvvt screening panel W Reflex
75515-7 file:///c:/users/cholck/appdata/local/temp/relma_2_49_user_75515-... Page 1 of 1 75515-7 Lupus anticoagulant aptt & drvvt screening panel W Reflex PANEL HIERARCHY LOINC# LOINC Name R/O/C CardinalityEx.
More informationNew anticoagulants: Monitoring or not Monitoring? Not Monitoring
The 2 nd World Congress on CONTROVERSIES IN HEMATOLOGY (COHEM) Barcelona, Spain September 6 8, 2012 New anticoagulants: Monitoring or not Monitoring? Not Monitoring Anna Falanga, MD Immunohematology and
More informationHemostasis analyzer system
Hemostasis analyzer system Providing fast, actionable results to help you reduce risks, complications and costs Get the whole picture with TEG Hemostasis analyzer system For more than forty years, hospitals
More information510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY DEVICE AND INSTRUMENT TEMPLATE
510(k) SUBSTANTIAL EQUIVALENCE DETERMINATION DECISION SUMMARY DEVICE AND INSTRUMENT TEMPLATE A. 510(k) Number: K041502 B. Purpose for Submission: To change the name of a previously cleared analyzer, and
More informationNew Oral Anticoagulants
Laboratory Monitoring of New Oral Anticoagulants.....What you need to know Rita Selby MD Medical Director, Coagulation Laboratories Uniersity Health Network & Sunnybrook HSC Uniersity of Toronto The 15
More informationMCHENRY WESTERN LAKE COUNTY EMS SYSTEM OPTIONAL CE ADVANCED LEVEL (EMTP, PHRN, ECRN) August 2013. Anticoagulants
MCHENRY WESTERN LAKE COUNTY EMS SYSTEM OPTIONAL CE ADVANCED LEVEL (EMTP, PHRN, ECRN) August 2013 Anticoagulants Anticoagulants are agents that prevent the formation of blood clots. Before we can talk about
More informationConsultative Coagulation How to Effectively Answer Common Questions About Hemostasis Testing Session #5020
Consultative Coagulation How to Effectively Answer Common Questions About Hemostasis Testing Session #5020 Dorothy M. (Adcock) Funk, M.D. Colorado Coagulation Karen A. Moser, M.D. Saint Louis University
More informationDisclosure. New Agents for Treatment of DVT. Prevalence of DVT VTE. Normal Hemostasis 7/17/2015. Mark Oliver, MD, RVT, RPVI,FSVU
New Agents for Treatment of DVT Disclosure PI Adopt and Amplify trials Mark Oliver, MD, RVT, RPVI,FSVU BMS and Pfizer Speaker VTE Venous Thromboembolism Recognized DVT s New : 170,000 Recurrent : 90,000
More informationTo aid practitioners in prescribing unfractionated heparin and low-molecular-weight heparins to patients.
UNFRACTIONATED HEPARIN AND LOW-MOLECULAR-WEIGHT HEPARIN TARGET AUDIENCE: All Canadian health care professionals. OBJECTIVE: To aid practitioners in prescribing unfractionated heparin and low-molecular-weight
More informationUSE AND INTERPRETATION OF LABORATORY COAGULATION TESTS IN PATIENTS WHO ARE RECEIVING A NEW ORAL ANTICOAGULANT (DABIGATRAN, RIVAROXABAN, APIXABAN)
USE AND INTERPRETATION OF LABORATORY COAGULATION TESTS IN PATIENTS WHO ARE RECEIVING A NEW ORAL ANTICOAGULANT (DABIGATRAN, RIVAROXABAN, APIXABAN) TARGET AUDIENCE: All Canadian health care professionals:
More information3.4 TEST FOR BACTERIAL ENDOTOXINS. Final text for revision of The International Pharmacopoeia
Document QAS/11.5 FINAL July 01 3. TEST FOR BACTERIAL ENDOTOXINS Final text for revision of The International Pharmacopoeia This monograph was adopted at the Forty-sixth WHO Expert Committee on Specifications
More informationMouse IgM ELISA. Cat. No. KT-407 K-ASSAY. For the quantitative determination of IgM in mouse biological samples. For Research Use Only. 1 Rev.
K-ASSAY Mouse IgM ELISA For the quantitative determination of IgM in mouse biological samples Cat. No. KT-407 For Research Use Only. 1 Rev. 072309 K-ASSAY PRODUCT INFORMATION Mouse IgM ELISA Cat. No. KT-407
More informationLaboratory Detection of Newer Anticoagulant Drugs
Laboratory Detection of Newer Anticoagulant Drugs Dorothy M. (Adcock) Funk, M.D. Colorado Coagulation, Laboratory Corporation of America Holdings Outline Newer Oral Anticoagulant Therapies A brief introduction
More informationAnalytical Specifications RIVAROXABAN
Page 1 of 9 ANALYTE NAME AND STRUCTURE - RIVAROXABAN SYNONYMS Xarelto CATEGORY Anticoagulant TEST CODE PURPOSE Therapeutic Drug Monitoring GENERAL RELEVANCY BACKGROUND Xarelto (rivaroxaban) is an orally
More informationBleeding disorders or haemorrhagic diatheses are a group of disorders characterised by defective haemostasis with abnormal bleeding.
Bleeding disorders or haemorrhagic diatheses are a group of disorders characterised by defective haemostasis with abnormal bleeding. Bleeding may be spontaneous in the form of small haemorrhages into the
More informationLaboratory Testing in Patients on Novel Oral Anticoagulants (NOACs)
Laboratory Testing in Patients on Novel Oral Anticoagulants (NOACs) Dr. Art Szkotak artur.szkotak@albertahealthservices.ca University of Alberta Hospital Edmonton, AB NOACs Direct Thrombin Inhibitors (DTI):
More informationIgM ELISA. For the quantitative determination of IgM in human serum and plasma. For Research Use Only. Not For Use In Diagnostic Procedures.
IgM ELISA For the quantitative determination of IgM in human serum and plasma For Research Use Only. Not For Use In Diagnostic Procedures. Please read carefully due to Critical Changes, e.g., Calibrator
More informationContent Sheet 7-1: Overview of Quality Control for Quantitative Tests
Content Sheet 7-1: Overview of Quality Control for Quantitative Tests Role in quality management system Quality Control (QC) is a component of process control, and is a major element of the quality management
More informationInc. Wuhan. Quantity Pre-coated, ready to use 96-well strip plate 1 Plate sealer for 96 wells 4 Standard (liquid) 2
Uscn Life Science Inc. Wuhan Website: www.uscnk.com Phone: +86 27 84259552 Fax: +86 27 84259551 E-mail: uscnk@uscnk.com ELISA Kit for Human Prostaglandin E1(PG-E1) Instruction manual Cat. No.: E0904Hu
More informationThrombin Generation Assay
Thrombin Generation Assay Kit insert Version: February 2013 Summary Thrombin is a key enzyme of the coagulation cascade. Its measurement gives direct information about the thrombogenicity of a biomaterial
More informationApplication Sheet for Rivaroxaban (Xarelto ) Standard Range with. BIOPHEN Heparin LRT (#221011/221013) RUO
Instrument Adaptation The attached instrument adaptation has been prepared and validated by the reagent manufacturer, Hyphen-Biomed. Instrument Siemens BCS-XP Product BIOPHEN Heparin LRT Analyte Rivaroxaban
More informationLupus anticoagulant Pocket card
Lupus anticoagulant Pocket card Issue number 5 2012 Antiphospholipid Syndrome 1 The antiphospholipid syndrome (APS) is diagnosed in patients with recurrent thromboembolic events and /or pregnancy loss
More informationAnticoagulation Essentials! Parenteral and Oral!
Anticoagulation Essentials! Parenteral and Oral! Anti-Xa and Anti-IIa! Parenteral Anticoagulants! Heparin family (indirect anti-xa and anti-iia):! UFH! LMWH (enoxaparin, fondaparinux)! Direct thrombin
More informationDVT/PE Management with Rivaroxaban (Xarelto)
DVT/PE Management with Rivaroxaban (Xarelto) Rivaroxaban is FDA approved for the acute treatment of DVT and PE and reduction in risk of recurrence of DVT and PE. FDA approved indications: Non valvular
More informationHuman Free Testosterone(F-TESTO) ELISA Kit
Human Free Testosterone(F-TESTO) ELISA Kit Catalog Number. MBS700040 For the quantitative determination of human free testosterone(f-testo) concentrations in serum, plasma. This package insert must be
More informationVALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY Q2(R1)
INTERNATIONAL CONFERENCE ON HARMONISATION OF TECHNICAL REQUIREMENTS FOR REGISTRATION OF PHARMACEUTICALS FOR HUMAN USE ICH HARMONISED TRIPARTITE GUIDELINE VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY
More informationChem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution.
Chem 405 Biochemistry Lab I Experiment 2 Quantitation of an unknown protein solution. Introduction: The determination of protein concentration is frequently required in biochemical work. Several methods
More informationMalondialdehyde (MDA) ELISA
Malondialdehyde (MDA) ELISA For the quantitative determination of MDA in serum, plasma and other biological fluids Cat. No. KT-21493 For Research Use Only. Not for use in diagnostic procedures. Page 1
More informationPHAR 7633 Chapter 19 Multi-Compartment Pharmacokinetic Models
Student Objectives for this Chapter PHAR 7633 Chapter 19 Multi-Compartment Pharmacokinetic Models To draw the scheme and write the differential equations appropriate to a multi-compartment pharmacokinetic
More informationGuidance for Industry
Guidance for Industry Q2B Validation of Analytical Procedures: Methodology November 1996 ICH Guidance for Industry Q2B Validation of Analytical Procedures: Methodology Additional copies are available from:
More informationDr Gordon Royle Haematologist, Middlemore Hospital
The New Oral Anticoagulants (NOACs) Dr Gordon Royle Haematologist, Middlemore Hospital Disclaimers Boehringer-Ingelheim Bayer Sanofi Douglas Pharmaceuticals Preventing disasters: lessons learned A cautionary
More informationINR = (patient PT/mean normal PT) ISI.
The Relationship of the International Normalized Ratio () to the Prothrombin Time (PT) By: William DePond MD, President and Chief Medical Officer MEDLAB In 1983, it was determined that patients receiving
More informationLAMC Reversal Agent Guideline for Anticoagulants 2013. Time to resolution of hemostasis (hrs) Therapeutic Options
LAMC Reversal Agent Guideline for Anticoagulants 2013 Medication resolution of hemostasis (hrs) Intervention Administration Instructions Heparin 3-4 Protamine 1mg IV for every 100 units of heparin Slow
More informationLaboratory 5: Properties of Enzymes
Laboratory 5: Properties of Enzymes Technical Objectives 1. Accurately measure and transfer solutions with pipettes 2. Use a Spectrophotometer to study enzyme action. 3. Properly graph a set of data. Knowledge
More informationAbnormal Basic Coagulation Testing Laboratory Testing Algorithms
Global Coagulation Testing Abnormal Basic Coagulation Testing Laboratory Testing Algorithms Jeffrey S. Jhang, M.D. No single global laboratory test Bleeding history is the strongest predictor of bleeding
More informationICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology. Step 5
European Medicines Agency June 1995 CPMP/ICH/381/95 ICH Topic Q 2 (R1) Validation of Analytical Procedures: Text and Methodology Step 5 NOTE FOR GUIDANCE ON VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND
More informationThe management of cerebral hemorrhagic complications during anticoagulant therapy
The management of cerebral hemorrhagic complications during anticoagulant therapy Maurizio Paciaroni Stroke Unit Division of Cardiovascular Medicine University of Perugia - Italy Perugia Stroke Registry
More informationBios 6648: Design & conduct of clinical research
Bios 6648: Design & conduct of clinical research Section 1 - Specifying the study setting and objectives 1. Specifying the study setting and objectives 1.0 Background Where will we end up?: (a) The treatment
More informationLABORATORY DIAGNOSIS OF BLEEDING DISORDERS
LABORATORY DIAGNOSIS OF BLEEDING DISORDERS Secondary Hemostasis CIRCULATORY SYSTEM Low volume, high pressure system Efficient for nutrient delivery to tissues Prone to leakage 2º 2 to endothelial surface
More informationCanine creatine kinase MB isoenzyme (CK-MB)ELISA Kit
Canine creatine kinase MB isoenzyme (CK-MB)ELISA Kit Catalog No. CSB-E15852c (96T) This immunoassay kit allows for the in vitro quantitative determination of canine CK-MB concentrations in serum and plasma.
More informationThe new oral anticoagulants & the future of haemostasis laboratory testing. Alcohol: the good, the bad and the ugly
The new oral anticoagulants & the future of haemostasis laboratory testing Emmanuel J Favaloro Diagnostic Haemostasis Laboratory, Institute of Clinical Pathology & Medical Research, ICPMR, Pathology West,
More informationDabigatran (Pradaxa) Guidelines
Dabigatran (Pradaxa) Guidelines Dabigatran is a new anticoagulant for reducing the risk of stroke in patients with atrial fibrillation. Dabigatran is a direct thrombin inhibitor, similar to warfarin, without
More informationAnalytical Test Method Validation Report Template
Analytical Test Method Validation Report Template 1. Purpose The purpose of this Validation Summary Report is to summarize the finding of the validation of test method Determination of, following Validation
More informationHow to Biotinylate with Reproducible Results
How to Biotinylate with Reproducible Results Introduction The Biotin Streptavidin system continues to be used in many protein based biological research applications including; ELISAs, immunoprecipitation,
More informationMouse Creatine Kinase MB isoenzyme (CKMB) ELISA
KAMIYA BIOMEDICAL COMPANY Mouse Creatine Kinase MB isoenzyme (CKMB) ELISA For the quantitative determination of mouse CKMB in serum, plasma, cell culture fluid and other biological fluids Cat. No. KT-57681
More informationUSING CLSI GUIDELINES TO PERFORM METHOD EVALUATION STUDIES IN YOUR LABORATORY
USING CLSI GUIDELINES TO PERFORM METHOD EVALUATION STUDIES IN YOUR LABORATORY Breakout Session 3B Tuesday, May 1 8:30 10 am James Blackwood, MS, CLSI David D. Koch, PhD, FACB, DABCC, Pathology & Laboratory
More informationRat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit
Rat Creatine Kinase MB isoenzyme,ck-mb ELISA Kit Catalog No: E0479r 96 Tests Operating instructions www.eiaab.com FOR RESEARCH USE ONLY; NOT FOR THERAPEUTIC OR DIAGNOSTIC APPLICATIONS! PLEASE READ THROUGH
More informationPlatelet Review July 2012. Thomas S. Kickler M.D. Johns Hopkins University School of Medicine
Platelet Review July 2012 Thomas S. Kickler M.D. Johns Hopkins University School of Medicine Hemostasis Hemostasis is the process that leads to the stopping of bleeding Hemostasis involves blood vessels,
More informationValidation and Calibration. Definitions and Terminology
Validation and Calibration Definitions and Terminology ACCEPTANCE CRITERIA: The specifications and acceptance/rejection criteria, such as acceptable quality level and unacceptable quality level, with an
More informationClinical Study Synopsis
Clinical Study Synopsis This Clinical Study Synopsis is provided for patients and healthcare professionals to increase the transparency of Bayer's clinical research. This document is not intended to replace
More informationClinical Study Synopsis
Clinical Study Synopsis This Clinical Study Synopsis is provided for patients and healthcare professionals to increase the transparency of Bayer's clinical research. This document is not intended to replace
More informationReversal of Antiplatelet and Anticoagulant Therapy: What You Need To Know. Ronald Walsh, MD Chief Medical Officer Community Blood Services
Reversal of Antiplatelet and Anticoagulant Therapy: What You Need To Know Ronald Walsh, MD Chief Medical Officer Community Blood Services HEMOSTATIC PROCESS Initiation and formation of the platelet plug
More informationDr Gordon Royle Haematologist, Middlemore Hospital
The New Oral Anticoagulants (NOACs) Dr Gordon Royle Haematologist, Middlemore Hospital Disclaimers Boehringer-Ingelheim Bayer Sanofi Douglas Pharmaceuticals Preventing disasters: lessons learned A cautionary
More informationENZYME KINETICS ENZYME-SUBSTRATE PRODUCTS
ENZYME KINETICS INTRODUCTION The study of reaction rates catalyzed by enzymes and the factors affecting them is generally referred to as enzyme kinetics. The basic components of an enzyme catalyzed reaction
More informationAnticoagulant therapy
Anticoagulation: The risks Anticoagulant therapy 1990 2002: 600 incidents reported 120 resulted in death of patient 92 deaths related to warfarin usage 28 reports related to heparin usage Incidents in
More informationHiPer Ion Exchange Chromatography Teaching Kit
HiPer Ion Exchange Chromatography Teaching Kit Product Code: HTC001 Number of experiments that can be performed: 5 Duration of Experiment: Protocol: 5-6 hours Storage Instructions: The kit is stable for
More informationStep-by-Step Analytical Methods Validation and Protocol in the Quality System Compliance Industry
Step-by-Step Analytical Methods Validation and Protocol in the Quality System Compliance Industry BY GHULAM A. SHABIR Introduction Methods Validation: Establishing documented evidence that provides a high
More informationEXERCISE 5: ERYTHROCYTES SEDIMENTATION RATE - ESR, SED RATE
EXERCISE 5: ERYTHROCYTES SEDIMENTATION RATE - ESR, SED RATE Textbook: Skills: None 10 points Objectives: 1. State the principle of the Erythrocytes Sedimentation Rate - ESR. 2. List two factors which may
More informationReversal of Anticoagulants at UCDMC
Reversal of Anticoagulants at UCDMC Introduction: Bleeding complications are a common concern with the use of anticoagulant agents. In selected situations, reversing or neutralizing the effects of an anticoagulant
More informationInpatient Anticoagulation Safety. To provide safe and effective anticoagulation therapy through a collaborative approach.
Inpatient Anticoagulation Safety Purpose: Policy: To provide safe and effective anticoagulation therapy through a collaborative approach. Upon the written order of a physician, Heparin, Low Molecular Weight
More informationINTRODUCTION Thrombophilia deep vein thrombosis DVT pulmonary embolism PE inherited thrombophilia
INTRODUCTION Thrombophilia (Hypercoagulability) is a condition in which a person forms blood clots more than normal. Blood clots may occur in the arms or legs (e.g., deep vein thrombosis DVT), the lungs
More informationSYBR Green Realtime PCR Master Mix -Plus-
Instruction manual SYBR Green Realtime PCR Master Mix -Plus- 0810 F0925K SYBR Green Realtime PCR Master Mix -Plus- Contents QPK-212T 1mLx1 QPK-212 1mLx5 Store at -20 C, protected from light [1] Introduction
More informationTitle of Guideline. Thrombosis Pharmacist)
Title of Guideline Contact Name and Job Title (author) Guideline for patients receiving Rivaroxaban (Xarelto ) requiring Emergency Surgery or treatment for Haemorrhage Julian Holmes (Haemostasis and Thrombosis
More informationNHS FORTH VALLEY RIVAROXABAN AS TREATMENT FOR DEEP VEIN THROMBOSIS AND PULMONARY EMBOLISM IN ADULTS
NHS FORTH VALLEY RIVAROXABAN AS TREATMENT FOR DEEP VEIN THROMBOSIS AND PULMONARY EMBOLISM IN ADULTS Date of First Issue 01/12/ 2012 Approved 15/11/2012 Current Issue Date 29/10/2014 Review Date 29/10/2016
More informationDetermination of the Rate Law for Food Dye Bleaching with Hypochlorite
This is an example report of an investigation performed in General Chemistry lab. Pay attention to format and content, not on the results or the experiment itself. The report is best explored on screen
More informationNew Anticoagulants: When and Why Should I Use Them? Disclosures
Winship Cancer Institute of Emory University New Anticoagulants: When and Why Should I Use Them? Christine L. Kempton, MD, MSc Associate Professor of Pediatrics and Hematology and Medical Oncology Hemophilia
More informationEnvironmental Water Testing: Surface Water, Groundwater, Hard Water, Wastewater, & Seawater
Document: AND Sol Env 08 2013 Environmental Water Testing: Surface Water, Groundwater, Hard Water, Wastewater, & Seawater Matrix specific sample preparation and testing methods for environmental waters
More informationIV solutions may be given either as a bolus dose or infused slowly through a vein into the plasma at a constant or zero-order rate.
د.شيماء Biopharmaceutics INTRAVENOUS INFUSION: IV solutions may be given either as a bolus dose or infused slowly through a vein into the plasma at a constant or zero-order rate. The main advantage for
More informationMETHODS OF VITAMIN ANALYSIS
METHODS OF VITAMIN ANALYSIS Specimen requirements; Fasting plasma or serum Lithium Heparin is the anticoagulant of choice for vitamins such as; thiamine, riboflavin, retinol, tocopherols & cholecalciferol
More informationReversed Phase High Presssure Liquid Chromatograhphic Technique for Determination of Sodium Alginate from Oral Suspension
International Journal of PharmTech Research CODEN (USA): IJPRIF ISSN : 0974-4304 Vol.2, No.2, pp 1634-1638, April-June 2010 Reversed Phase High Presssure Liquid Chromatograhphic Technique for Determination
More informationDesign of Experiments for Analytical Method Development and Validation
Design of Experiments for Analytical Method Development and Validation Thomas A. Little Ph.D. 2/12/2014 President Thomas A. Little Consulting 12401 N Wildflower Lane Highland, UT 84003 1-925-285-1847 drlittle@dr-tom.com
More informationRat creatine kinase MM isoenzyme (CK-MM) ELISA Kit
Rat creatine kinase MM isoenzyme (CK-MM) ELISA Kit Catalog Number. CSB-E14405r For the quantitative determination of rat creatine kinase MM isoenzyme (CK-MM) concentrations in serum, plasma and tissue
More informationph Alkalinity of Water
ph Alkalinity of Water DOC316.52.93085 Based on ISO standard 9963-1:1994 ph-metric Titration 0.4 to 20 mmol/l of Total Alkalinity 1. Introduction Alkalinity of water is its acid-neutralizing capacity.
More informationJOINT COMMISSION INTERNATIONAL ACCREDITATION STANDARDS FOR. 2nd Edition
JOINT COMMISSION INTERNATIONAL ACCREDITATION STANDARDS FOR CliniCAl laboratories 2nd Edition Effective 1 April 2010 International Patient Safety Goals (IPSG) Goals The following is a list of all goals.
More informationHow to Verify Performance Specifications
How to Verify Performance Specifications VERIFICATION OF PERFORMANCE SPECIFICATIONS In 2003, the Centers for Medicare and Medicaid Services (CMS) updated the CLIA 88 regulations. As a result of the updated
More informationStatistical estimation using confidence intervals
0894PP_ch06 15/3/02 11:02 am Page 135 6 Statistical estimation using confidence intervals In Chapter 2, the concept of the central nature and variability of data and the methods by which these two phenomena
More informationValidating Microarray Data Using RT 2 Real-Time PCR Products
Validating Microarray Data Using RT 2 Real-Time PCR Products Introduction: Real-time PCR monitors the amount of amplicon as the reaction occurs. Usually, the amount of product is directly related to the
More informationBlood, Plasma, and Cellular Blood Components INTRODUCTION
Blood, Plasma, and Cellular Blood Components INTRODUCTION This chapter of the Guideline provides recommendations to Sponsors of Requests for Revision for new monographs for blood, plasma, and cellular
More informationChemistry 111 Laboratory Experiment 7: Determination of Reaction Stoichiometry and Chemical Equilibrium
Chemistry 111 Laboratory Experiment 7: Determination of Reaction Stoichiometry and Chemical Equilibrium Introduction The word equilibrium suggests balance or stability. The fact that a chemical reaction
More informationThe laboratory and new anticoagulant drugs
The laboratory and new anticoagulant drugs Andreas Hillarp Department of Clinical Chemistry and Transfusion Medicine Halland County Hospital, Sweden andreas.hillarp@regionhalland.se Disclosures for Andreas
More informationPoint-of-care (POC) versus central laboratory instrumentation for monitoring oral anticoagulation
Point-of-care (POC) versus central laboratory instrumentation for monitoring oral anticoagulation David M Dorfman a, Ellen M Goonan a, M Kay Boutilier a, Petr Jarolim a, Milenko Tanasijevic a and Samuel
More informationThis value, called the ionic product of water, Kw, is related to the equilibrium constant of water
HYDROGEN ION CONCENTRATION - ph VALUES AND BUFFER SOLUTIONS 1. INTRODUCTION Water has a small but definite tendency to ionise. H 2 0 H + + OH - If there is nothing but water (pure water) then the concentration
More informationANALYTICAL METHODS INTERNATIONAL QUALITY SYSTEMS
VALIDATION OF ANALYTICAL METHODS 1 GERT BEUVING INTERNATIONAL PHARMACEUTICAL OPERATIONS TASKS: - Internal auditing - Auditing of suppliers and contract manufacturers - Preparing for and guiding of external
More informationAssay Qualification Template for Host Cell Protein ELISA
Assay Qualification Template for Host Cell Protein ELISA Introduction: With ever increasing importance being placed on host cell protein (HCP) removal during the purification process, it is critical to
More informationOverview of ISO 17511 for 'Biologicals'
JCTLM Symposium on Reference Measurement Systems for Biologicals International Bureau of Weights and Measures Pavillon de Breteuil, Sèvres, FR 2004-12-15 Overview of ISO 17511 for 'Biologicals' R. Dybkaer
More informationWhat to do in case of hemorragia. L Camoin Jau Service d Hématologie APHM Marseille
What to do in case of hemorragia with NOAC? L Camoin Jau Service d Hématologie APHM Marseille Disclosure Boehringer Bayer Daishi Sanofi BMS Pharmacodynamic and kinetic properties of new oral anticoagulants.
More informationDevelopment and Validation of In Vitro Diagnostic Tests. YC Lee, Ph.D. CEO
Development and Validation of In Vitro Diagnostic Tests YC Lee, Ph.D. CEO 1 Validation of In Vitro Diagnostic Tests Validated d Diagnostic Test should: Provides test results that identify if positive i
More informationTOTAL PROTEIN FIBRINOGEN
UNIT: Proteins 16tproteins.wpd Task Determination of Total Protein, Albumin and Globulins Objectives Upon completion of this exercise, the student will be able to: 1. Explain the ratio of albumin and globulin
More informationLab 2. Spectrophotometric Measurement of Glucose
Lab 2 Spectrophotometric Measurement of Glucose Objectives 1. Learn how to use a spectrophotometer. 2. Produce a glucose standard curve. 3. Perform a glucose assay. Safety Precautions Glucose Color Reagent
More informationLecture 11 Enzymes: Kinetics
Lecture 11 Enzymes: Kinetics Reading: Berg, Tymoczko & Stryer, 6th ed., Chapter 8, pp. 216-225 Key Concepts Kinetics is the study of reaction rates (velocities). Study of enzyme kinetics is useful for
More informationNew Anticoagulants: What to Use What to Avoid
New Anticoagulants: What to Use What to Avoid Bruce Davidson, MD, MPH Clinical Professor of Medicine Pulmonary and Critical Care Medicine Division University of Washington School of Medicine Seattle USA
More informationASSURING THE QUALITY OF TEST RESULTS
Page 1 of 12 Sections Included in this Document and Change History 1. Purpose 2. Scope 3. Responsibilities 4. Background 5. References 6. Procedure/(6. B changed Division of Field Science and DFS to Office
More informationGUIDELINES FOR THE VALIDATION OF ANALYTICAL METHODS FOR ACTIVE CONSTITUENT, AGRICULTURAL AND VETERINARY CHEMICAL PRODUCTS.
GUIDELINES FOR THE VALIDATION OF ANALYTICAL METHODS FOR ACTIVE CONSTITUENT, AGRICULTURAL AND VETERINARY CHEMICAL PRODUCTS October 2004 APVMA PO Box E240 KINGSTON 2604 AUSTRALIA http://www.apvma.gov.au
More informationImplementing New USP Chapters for Analytical Method Validation
Implementing New USP Chapters for Analytical Method Validation March 2010 Ludwig Huber Fax.: +49 7243 602 501 E-mail: Ludwig_Huber@labcompliance.com Today s Agenda Handling Method Changes vs. Adjustments
More informationI know my value. CoaguChek XS Plus system Smart INR monitoring at your practice. 1 of 6
I know my value CoaguChek XS Plus system Smart INR monitoring at your practice 1 of 6 CoaguChek XS Plus system A better choice for your patients and your practice There has been an extraordinary increase
More informationCollection Guidelines for Routine & Special Coagulation Testing
Revised May 2010 Collection Guidelines for Routine & Special Coagulation Testing Best samples come from peripheral stick with evacuated tube system 19 to 22 gauge needle (smaller or bigger could cause
More informationBiomarkers for new anticoagulants vice and virtue
Biomarkers for new anticoagulants vice and virtue Dagmar Kubitza, MD AGAH, München 2014 Page 1 Definition of Biomarkers?. Surrogate markers are primary measures the effectiveness of investigational drugs.
More informationGene Expression Assays
APPLICATION NOTE TaqMan Gene Expression Assays A mpl i fic ationef ficienc yof TaqMan Gene Expression Assays Assays tested extensively for qpcr efficiency Key factors that affect efficiency Efficiency
More information