Laboratory Variables That May Affect Test Results in Prothrombin ...

More documents

Recommendations

Info

seconds. These results may drastically affect the interpretation of the PT assay and the warfarin dosage in the individual patients. 3 The WHO in 1977 introduced a standardized thromboplastin that was touted as an international reference preparation (IRP). The hope was that each laboratory could develop a reference range for PT testing that was to be compared to the IRP range. Taking this initial step further the WHO in 1983 then put forth the idea for PT standardization based on a mathematical formula (INR) that uses the elements of the PT assay. The INR then would be the PT result that a laboratory would obtain if the test were performed using the standardized WHO reference thromboplastin reagent with an assigned ISI value of 1.0. Each assigned thromboplastin ISI could then compare the individual reagent sensitivity to an IRP that has been calibrated with a WHO reference plasma. The INR is calculated using the following formula 3 : INR = (patient PT) ISI Mean normal PT Each individual laboratory would supply the mean normal PT. The INR method would then allow the physician to compare the PT results with different laboratories reagent/instrument combinations. Manufacturers now produce a wide variety of thromboplastin with ISI values ranging from 0.9 to 3.0. This wide range of ISI will produce a huge difference of PT results. 3 The INR was designed to correct for this difference. Therefore, if a patient were traveling from one city to another the physician would have the advantage of expecting a standardized INR value to maintain a patient’s OAT range. As observed in the case report improper assignment of ISI values with different reagent/instrument combinations can result in clinically significant inaccurate values. This difference in ISI values resulted in improper management of a large group of patients on OAT with catastrophic consequences. Huge differences in INR values have been reported when there are significant differences between low versus high ISI sensitivities in thromboplastins. 4,5 Preanalytic Variables of INR Testing Citrate Concentration Prothrombin time assays are performed on citrated platelet-poor plasma. The concentration of the sodium citrate anticoagulant can affect the PT time thus affecting the INR value. 6-8 In the United States, until recently, approximately 80% of the PT assays were performed in plasma that was collected using vacutainer tubes that contained 3.8% (129 mol/L) sodium citrate. In 1998, NCCLS Subcomittee on Coagulation started to recommend that a 3.2% (109 mol/L) sodium citrate concentration be used for most coagulation studies. This concentration was selected because the WHO was calibrating thromboplastins using the 3.2% concentration. 9 Other international standardization organizations also recommend using the 3.2% citrated anticoagulant. The osmolality of the 3.2% concentration is apparently closer to plasma. Higher INR values have been found in some thromboplastin reagents in plasma collected with 3.8% citrated vacutainer tubes. In one direct comparison study between the 2 citrate concentrations using a low ISI thromboplastin, the INR difference in 18.0% of the specimens showed a INR difference of 0.7. In another study, the difference on all specimens gave an average difference of 19.0%. The lower the ISI value is the more difference the citrate concentration makes in the INR results. 9 In platelet studies and testing of blood bank products such as cryoprecipitate, the 3.8% citrate concentration is still recommended. Specimen Handling Improper specimen collection can also drastically affect the PT assay thus affecting the INR value. This area has become controversial due to a number of studies that discuss the length of time and how a specimen can be stored be performing a PT assay. One study showed that storage of specimens for up to 6 hours at room temperature did not affect the PT results. 10 In another study, it was found that specimens from patients who were on low dose warfarin could be shipped overnight at room temperature © laboratorymedicine> february 2003> number 2> volume 34 without significantly changing the INR values. 11 Another study looked at specimens stored as whole blood. It found that after 3 days stored at room temperature that there was no significant change in the INR. 12 NCCLS guidelines from 1998 stated that uncentrifuged specimens for the PT/INR test or centrifuged plasma left of top of the separated cells stored at various temperatures from 2 to 24°C should be run within 24 hours from the time the sample was obtained from the test subject. 13 The PT results may vary from the time of collection of the specimen and storage of the sample if the subject was receiving heparin and was on OAT. The laboratory must be sure that the thromboplastin has a heparin neutralizer to counteract the effects of heparin. Evidence has also been presented that if specimens are stored at approximately 4°C that the PT/INR results may be shortened due to cold activation of FVII. 9 Our recommendation is that all specimens should be treated as if they were the last specimens that could be obtained from the test subject without having to recollect the sample. If another assay is required, such as an APTT or factor assay, the prolonged storage times of >4 hours would possibly render the specimen compromised and required the patient to have a second venipuncture. This increases time and expense for all concerned. More recently it has been discovered that the coagulation tube has been found to be a clinically significant source of variability in coagulation testing. There has been controversy in using “partial-draw tubes” which required less vacuum to manufacture which resulted in 1.8 mL or 2.7 mL blood draws versus the conventional 4.5 mL draw with the blood to anticoagulant ratio of 9:1. Since the partial-draw tubes filled more slowly than the so-called full-draw tubes to platelets may have been activated due to lengthy exposure to shear forces arising from exposure to shear forces arising from drawing blood into the increased headspace. The activated platelets then may release platelet-factor 4 or phospholipids which could compromise coagulation testing. 14 125
126 Instrumentation There are over 300 different methods of performing a PT assay in the United States alone. Because of this wide variety of testing it is almost impossible for manufacturers to provide accurate reagent/instrument specific ISI values for each reagent/instrument combination. The WHO protocol for assigning ISI values to thromboplastin reagents is determined by performing a manual tilt-tube method in quadruplicate. 15 Laboratories then have to depend on manufacturers to accurately assign the proper ISI of their reagents based on some adaptation of the WHO protocol. 16 Therefore, the instrumentation can vastly affect the PT test results. If a coagulation laboratory is using a reagent from one company and an instrument from another supplier they have to rely on the reagent companies generic assignment of mechanical or photo-optic ISI values. Laboratories may not be aware that different automated coagulation systems can directly affect the ISI and the INR result. There may be distinct differences between mechanical and photo-optical coagulation systems with different reagents. 16,17 In our institution, we performed a protocol with 7 different reagent/instrument combinations on the same specimens. 16 In many specimens, we saw clinically significant differences of INR values between the different reagent/ combinations. T1 dis- plays some of these INR result differences. Observe that the specimen #1180 has an INR value of 3.95 with one reagent/instrument combination and 7.16 with another system. This is an 81.3% difference. The 3.95 value might be considered a high therapeutic range value that would probably require no intervention. The 7.16 INR might cause some institutions to consider this result a panic value. Many other variables can affect the coagulation instrument ISI sensitivity used in the PT analysis. These are temperatures of the system and reagents, accuracy of volume of the reagents being dispensed, and the citrate concentration of the specimen collected. 15,16 Looking for a simpler method to locally calibrate ISI values we performed a protocol at our institution that was sponsored by Dade Behring M (Deerfield, IL) using a calibration system supplied by Precision-Biologic M (Dartmouth, NS, Canada). Precision Biologic has proposed a simplified method of locally calibrating the reagent ISI using a 5-day ISI calibration protocol. The method used a set of 5 OAT frozen plasmas and 1 normal frozen plasma that are assayed against a standard (WHO IRP) thromboplastin. The frozen OAT calibrators and the normal plasma encompassed the 4 therapeutic categories used in OAT (4.5). Two recent papers discussed in detail how any laboratory can use the frozen calibrator plasma Comparison of Selected INR Differences of Human Subject’s Plasma laboratorymedicine> february 2003> number 2> volume 34 © method to locally calibrate their reagent/instrument combinations and how they would affect subject and manufacturers suggestions for ISI values with many different thromboplastins and instruments. 15,17 Laboratories that run PT/INR assays can then set up there own ISI calibration of the locally used thromboplastin. This method may be the most practical for improving inter-laboratory INR agreement. See the results in T2 and T3 on differences in local and manufacturer calibrations of ISIs that were used in our study. 14 Heparin Effects on PT/INR Testing Unfractionated heparin contamination can adversely affect the results of a PT/INR assay. Many subjects starting on OAT are coming off heparin therapy, which could give physicians problems with falsely elevated INR values when trying to establish baseline INR ranges for patients. Each manufacturer’s thromboplastin has an individual sensitivity to the effect of heparin. Some reagents have a heparin neutralizer present such as polybrene or protamine sulfate that can counteract the effects of heparin in the therapeutic range so the PT/INR results are not compromised. However, reagents without a heparin neutralizer present have been found to have PT results prolonged in some subjects by as long as 10 seconds. This could lead to Human MLA 900C CI+ MLA 900C T-D MLA 900C T-R MLA 1600C T-D STA CI+ STA T-D STA T-R Sample 1167 2.77 3.41 3.72 3.62 2.68 3.24 3.41 1181 2.53 3.2 3.65 3.36 2.64 2.85 2.91 1180 3.95 5.98 5.83 7.16 4.02 4.93 5.05 1398 2.94 3.94 3.44 4.14 3.06 3.61 3.35 1393 3.57 4.98 4.48 5.0 3.57 4.39 4.18 1391 3.03 4.14 3.41 4.43 3.13 3.82 3.14 1684 3.72 5.82 4.99 6.29 3.99 5.57 4.47 1688 2.95 3.94 3.93 4.44 3.12 3.7 3.79 1814 3.15 1.97 4.0 4.85 3.24 4.23 3.58 1669 3.25 2.41 3.66 3.93 3.24 4.29 3.28 T1 Done on the STA and MLA 900 C with thromboplastins: Neoplastine CI+, Diagnostica Stago (CI+); Thromboplastin D, Pacific-Hemostasis (T-D); Recombiplastin, Hemolance (T-R) and the MLA 1600C with T-D only.
Page 1: 124 science [coagulation and hemato
Page 5 and 6: 128 Reporting of PT/INR results The

Laboratory Variables That May Affect Test Results in Prothrombin ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?