INR -- INTERNATIONAL NORMALIZED RATIO

The current method for monitoring vitamin K antagonist (AVK) anticoagulant therapy is the international normalized ratio (INR) that provides consistency and standardization for the prothrombin time (PT) assay value. Even after the standardization of the INR, inaccuracies of this value have still been reported. To make the INR even more accurate, better local assessments of INR parameters are becoming available. These new methods use plasmas with certified INR values to locally verify and, if necessary, recalculate the international sensitivity index (ISI) for the local laboratory's reagent and instrument system. This CE Update will discuss the concepts of local verification and calibration to better define the manufacturer's assigned ISI value, thus reporting more accurate INR results.
Introduction

Our understanding of the basic science and clinical issues surrounding hemostasis has skyrocketed within the last 10 to 15 years. These advances have: 1) established better treatment for patients at risk for hemorrhage or thrombosis; 2) identified new hereditary coagulation disorders; and 3) provided mechanism(s) for clinical hemostatic diseases. With this in mind, clinicians have begun to put more demand on the coagulation laboratory. The laboratory has had to keep pace by developing new coagulation tests and better standardizing those tests already in use. The cornerstones of clinical coagulation testing are the prothrombin time and/or international normalized ratio (PT/INR) and the activated partial thromboplastin time (aPTT) for identifying and monitoring clinical coagulation disorders and therapeutics. Manufacturers have varied the sensitivities of these reagents to more easily assess this variety of clinical conditions. It now has become important for each laboratory to evaluate the commercial reagents to determine the most appropriate one for their clinical needs. The criteria for how reagents should be evaluated include: sensitivity for intended use, compatibility with instrumentation, number of assays performed each day, and cost.

In this CE Update, we discuss: 1) how to determine the INR; 2) how to locally verify and calibrate the PT reagent for more accurate INR values; and 3) the clinical use of the INR for monitoring anti-vitamin K anticoagulant therapy.

Oral Anticoagulant Therapy Monitoring Using the PT/INR

Anticoagulant therapy is used to treat and/or prevent both arterial and venous thrombosis.[1] Currently in the United States, IV anticoagulants (heparin and direct thrombin inhibitors [DTI]) are administered to inhibit further clot formation. The oral drug Coumadin (or warfarin) is given for long-term prevention of new thrombus formation; however, it is an indirect acting drug requiring 3 to 5 days to reach therapeutic effectiveness.[1] Warfarin derivatives are the standard therapy worldwide.[3] Annually, there are more than 21 million prescriptions written in the United States.[3] Warfarin derivatives (generally known as vitamin K antagonists [AVK]) act by inhibiting the vitamin K-associated post-translational modifications of the vitamin K-dependent clotting factors.[1] Unfortunately, warfarin has a very narrow therapeutic window and dosing responses vary between individuals of up to 10-fold and 2–3 fold within an individual caused by changes in medications, diet, or health status.[4] Warfarin is the second most common mismanaged therapeutic drug, requiring more than 40,000 emergency room visits per year and about $40 million to $60 million in additional medical costs per year.[4] The therapeutic window for oral anticoagulant drugs is very narrow; therefore the accuracy for monitoring these drugs' INR is essential since inadequate dosing increases thrombotic risk and excessive doses significantly increase the bleeding risk.

Monitoring AVK anticoagulant therapy using the INR is in its final phases of a slow transition in the United States compared with most European countries routinely using the INR value. The INR is a mathematically transformed or calculated value converting the PT in seconds to a standard ratio value.[1,2] The INR theoretically eliminates the differences in sensitivity of various PT reagents. However, within individual coagulation laboratories, the INR may not eliminate all of the variables of the PT assay, requiring local adjustment to make the reagent's INR more accurate with a specific instrument within the local environment.

Sensitivity to AVK anticoagulant therapy using the INR is the most important consideration when choosing a PT reagent.[2] A single plasma sample from an anticoagulated patient may give clinically significant different PT clotting times when tested against a variety of PT reagents; theoretically the INR value should be the same. It is important to pick a reagent with clotting times based on a scientific rationale rather than one with clotting times "familiar" to your clinicians.

Concept of the INR

The differences in PT results are dependent upon the composition of the PT reagent.[2] The reagent is composed of thromboplastin (tissue factor and phospholipid), extracted from a variety of sources (human and rabbit are the most common). Historically, each laboratory extracted thromboplastin from the human brain, but as commercial reagents were made available the major tissue source became rabbit brain. This is still used in a number of commercial reagents today. Now manufacturers are again starting to make reagents using human thromboplastin (placenta and recombinant).[5] The therapeutic responsiveness of the PT reagent is different depending on the source and composition of the thromboplastin.[1]

When the PT assay was first used to monitor AVK therapy, most providers based their therapeutic decisions on PT values in the range of 1.5 to 2.5 times the control value. However, now with the production of different reagent sensitivities with new sources of thromboplastin, the 1.5 to 2.5 range is not an accurate reflection of therapeutic anticoagulation.

In 1983, the World Health Organization (WHO) adopted a method to establish consistency of the PT value for patients on AVK.[6] This mathematical expression of the PT value is termed INR.[2] The INR calculation is based on the international sensitivity index (ISI) value specific to each PT reagent[1] and is valid only for stable AVK anticoagulant therapy and only up to an INR of 4.5.[1,2] An ISI value is determined for each thromboplastin reagent by comparing the responsiveness of the PT reagent with a WHO international reference preparation (IRP).[6,7]

Theoretically, the INR of a patient receiving AVK therapy would be the same regardless of the reagent and instrument used or in which the laboratory the sample was tested.[1,2,6] The specific PT reagent ISI value is determined using a WHO standard protocol comparing both PT reagent and IRP values from 20 normal individuals and 60 stable AVK individuals.[6,7] The slope of the regression calculation (orthogonal) of the 2 values is the ISI value of the unknown reagent.[2,6] Variation in the assigned ISI calculated by the manufacturer can vary +5%. This type of error can contribute to clinically different INR results.[6]

The INR is calculated using the assigned ISI value and the mean normal PT (MNPT) value (Figure 1).[2] The MNPT is determined by the laboratory using 20 to 40 (40 being optimal) healthy individuals reflecting the laboratory's patient base.[2] The mean is calculated using the geometric mean rather than the arithmetic mean.[2,8] The geometric mean assumes a non-normally distributed set of values. Geometric mean calculation transforms the data to log values before determining the mean. The arithmetic mean assumes a normally distributed normal population. The use of the arithmetic mean instead of the geometric mean can lead to a clinically significant difference in the MNPT and INR value.

Accuracy of the Manufacturer-assigned ISI

The ISI value reported in the PT reagent package insert has been determined by the manufacturer using the WHO standard method.[6] However, there are potential sources of error associated with this assigned ISI value (Table 1).[2,9] When these errors are compounded, a significant difference in the reported INR value and the true INR value can be found. These types of errors must be reduced by each laboratory. A manufacturer usually reports 2 ISI values for any PT reagent.[9,10] The first is the "generic ISI" and is determined for instruments using the same end-point detection method but not a specific instrument. This ISI must be used when the reagent of 1 manufacturer is used on an instrument made by a second manufacturer. However, the accuracy of this ISI value for the reagent-instrument system may be of clinical concern. The second reported ISI value is specific to the PT reagent and instrument combination ("instrument-specific ISI"). In several studies, the accuracy of INR results was enhanced when instrument-specific ISI was used instead of the generic ISI.[10,11] The range of discrepancy between the manufacturer-derived ISI and the local validated ISI can vary between +15% and 30%, thus making clinically significant differences in the INR value. Therefore the ISI value should be verified, especially in laboratories using generic ISI values. If the local validated ISI value is significantly different from the reported ISI, then the ISI must be determined locally.
i Initially, the ISI is assigned by the manufacturer based on the WHO ISI IRP. The yellow-shaded area shows the validation of the ISI at the local laboratory level. Every new reagent lot must at least be verified by the local laboratory. For proper local verification, the locally determined ISI must agree within 15% to be acceptable. If greater than 15%, then the ISI must be locally calibrated and re-verified.
Initially, the ISI is assigned by the manufacturer based on the WHO ISI IRP. The yellow-shaded area shows the validation of the ISI at the local laboratory level. Every new reagent lot must at least be verified by the local laboratory. For proper local verification, the locally determined ISI must agree within 15% to be acceptable. If greater than 15%, then the ISI must be locally calibrated and re-verified.

The definition of verification is "the confirmation through the provision of objective evidence that the specified requirements have been fulfilled" (within a pre-established set of criteria).[2,9] Verification is usually a 1-time process completed to confirm test performance before the INR system is used for patient testing. If significant differences in the INR test system are present, then calibration is performed followed by repeating the verification process. Calibration is "a set of operations establishing, under specified conditions, the relationship between true quantitative values indicated by the measuring test system."[9] For the INR test system, if the verification is not different from the reported ISI value, then calibration does not need to be performed. If significant differences (>15%) are found between the ISI values, then calibration of the ISI is required, followed by re-verification to ensure INR accuracy.[9,10]

The local verification and calibration (if necessary) of the ISI is performed with FDA-approved kits that provide all of the certified plasmas, the procedure, and data calculations. This ISI verification can be performed by any technologist or supervisor in all laboratories performing patient PT/INR results. The procedure usually takes 3 days of performing PT/INR testing on the certified plasmas and about 20 to 30 minutes of calculations that can be performed either by the manufacturer of the kit or through the manufacturer's Web-based program. This local verification and calibration procedure should be included in all new reagent and/or instrument contracts, cost per test, and cost per reportable result contracts. International sensitivity index verification and calibration (if necessary) should be performed (as part of the contract) at installation of the instrument, when starting a new reagent, for lot changes, and after instrument repair or internal or external QC issues. The procedure itself is relatively straightforward and has safeguards built in to provide a more accurate local ISI with clinically correct INR values.

Verification of the ISI in the Local Laboratory

The laboratory should not accept the initial ISI value assigned by the manufacturer as the local laboratory's working parameters, and conditions may significantly affect the patient's calculated INR results. With this in mind, it becomes the laboratory's responsibility to verify the validity of the ISI (Table 2).[9]

Two basic methods for local verification of ISI are available.[9] The first is the impractical method of using the WHO standard protocol. It is not feasible since it requires fresh plasma samples, an IRP, and the ability to perform the labor-intensive tilt tube assays. The second, more practical method uses certified plasmas to determine the ISI. These plasmas are purchased from the manufacturer of the reagent system or from an independent vendor especially for the reagent system. The verification kit should be part of the validation process for new installations, new reagents, and changes in reagents lots (Table 2). The procedure for verification per the provided instructions must be followed. The verification kit should be FDA approved following the established guidelines of Clinical and Laboratory Standards Institute (CLSI).[2,9] In brief, the procedure requires a minimum of 3 certified plasmas in the range of 1.5 INR to 4.5 INR. These plasmas can be lyophilized or frozen. The certified plasmas are evaluated exactly as patient samples. The PT and INR values are determined for each plasma tested in duplicate once per day for 3 days. The INR values obtained are compared to the assigned INR of the certified plasma. If these values compare within 15%,[2,9] then the ISI has been verified, and the PT reagent can be used with the assigned ISI value. If the verification procedure fails (INR values >15%), then a local calibration must be performed (see below).[2,9] Table 3 and Table 4 show examples of a passing verification and a failing verification, respectively. In Table 3, the mean INR values are within 15% of the assigned value for all 3 plasmas, the ISI is verified, and the assigned ISI value is correct. In Table 4, the highest INR certified plasma failed verification (>15%), so the assigned ISI cannot be used, and local calibration must be performed to determine a local valid ISI.

Local System Calibration of the ISI for the Local Laboratory

If verification fails, then the laboratory must not report patient results until the correct ISI is determined.[9] The laboratory must establish that: 1) instrument(s) was(were) working properly; 2) reagents were reconstituted correctly; 3) MNPT was determined accurately (geometric mean); and 4) no clerical or mathematical errors were made. If these are correct, then proceed to local calibration of the ISI.

Local calibration can be accurately determined using 2 methods: 1) calculating a local ISI, or 2) generating a PT/INR calibration line on which PT values are read as the INR value.[9] Whichever method is used, the calibration kit must be FDA approved. The certified plasmas must be compatible for the reagents and instruments used in the laboratory.[9] As an example, if the laboratory is using recombinant human thromboplastin, the calibration kit should be certified for use with human thromboplastin. The local ISI calibration procedure is a modification of the WHO protocol.[8,9] The PT values for each of the certified plasmas are determined using the local PT reagent and instrument. The local PT values are plotted against the assigned PT values of the certified plasma and an orthogonal regression line is calculated. The ISI calibration is considered valid if the slope has a CV of <3%.[9] The resulting slope of the line is the correct local ISI.[8,9] The procedure is similar to the verification method. The certified plasmas must be run in duplicate for at least 3 days to account for variation due to random error.

The necessary number of certified plasmas depends on a variety of factors, such as the source of the plasma (immuno-depleted or treated individual), type of plasma (frozen or lyophilized), single donor or multiple donors, and the IRP used for the certification of the plasma.[9] The manufacturer of the calibration kit in consultation with and approval by the FDA has determined the minimum number of certified plasmas needed. Most manufacturers will help the local laboratory determine the ISI value through either the Internet or as a "send-in" service. The manufacturer must provide detailed documentation of these ISI determination calculations for accreditation documentation. Of important note, after local calibration, the ISI must again be re-validated to confirm that the calibration and changed ISI are truly correct.

The second method to determine the local ISI is the direct INR calibration line, which is independent of the ISI value and the MNPT.[9] A disadvantage of this method is that many laboratory information systems and/or instrument systems are not programmed for calculating the INR by this method. Using this protocol, the PT values of certified plasmas are determined using the laboratory's system, also testing for 3 days in duplicate. The PT values determined locally are plotted on the y axis against the assigned certified plasma INR values (x axis). Using orthogonal regression, the best fit line is plotted. For a valid calibration curve, the r2 value of the regression line must be >0.95. The patient's INR is mathematically or graphically determined from this calibration line. Significant changes in the reagent-instrument system, such as lot changes, instrumentation repair, QC validity changes, and proficiency testing problems will all require establishing the INR calibration line and recertification

The INR is an important clinical tool for monitoring AVK therapy. The introduction of the INR has added several orders of magnitude of accuracy to anticoagulant monitoring. Still, more accuracy is needed to further reduce clinical problems associated with anticoagulation. To this end, the development of local reagent accuracy through the use of local ISI verification and ISI calibration adds even greater confidence to the correct reporting of INR values. The methods for ISI verification and the local ISI calibrations are just beginning to be used in the United States. The College of American Pathologists (CAP) Laboratory Accreditation program checklist (HEM.23220) requires documentation that the ISI is validated. The local ISI verification and calibration method fulfills that requirement. The methods may appear complex and the mathematics difficult, but kit manufacturers should assist with these calculations. Local determination of the ISI will significantly increase clinical confidence in oral anticoagulant monitoring.