From Joanna Carroll, NCSU College of Veterinary Medicine. We just purchased a new Stago STA Satellite. We have not been using Stago reagents, but we will probably switch. If our comparisons are close, can we adjust our current ranges or should we reestablish new?
From George: Where normal ranges exist, use a confirmatory approach, which typically specifies 20 aliquots from normal subjects. Assay the specimens, compute mean and 95% confidence interval or ±2 SD, compare to the existing normal range, and adjust if necessary. In instances where normal aliquots are difficult to collect, published reference intervals are a reasonable substitute, though slightly less accurate. This is the first question we’ve had on the topic of hemostasis in veterinary medicine, and I would be interested to learn how other veterinary labs establish normals, and also to learn are the most common applications for PTs, PTTs, and other hemostasis tests.
Dave McGlasson contributes: I would suggest to purchase Schalm’s Veterinary Hematology for reference intervals for different species of animals. To perform coagulation testing on a variety of animal species one should have an instrument that can measure short and prolonged times in PT, APTT and specialty coagulation assays. Many species have vastly different coagulation protein levels than humans. Make sure your instrumentation and reagents have maximum flexibility.
Joanna responds: Typically we try for 30 normal samples from each species that we deal with (dogs, cats, horses). Getting 30 normal dogs is fairly easy, cats and horses however not so easy. We do the best we can and our ranges seem comparable with other veterinary teaching hospitals.
“PMorer” at Stago adds: One of the issues in veterinary medicine is the breed of the animal. For example Beagles may have slightly different results compare to Spaniels. In humans the age is of equivalent importance. The Start4 is used in a French veterinary schools to manage PTs under 10 seconds and PTTs under 30. These numbers are not seen in human patients.
From DF Moreno: Hi, I’m from Argentina and I work in a hemostasis lab. An anticoagulated woman had a prolonged prothrombin time (PT). The coagulometer read +++, meaning more than 300 seconds and an INR more than 20. However she wasn’t bleeding. What could be the reason?
George responds: Assuming your coagulometer is working properly and the reagents are valid, the most likely possibility is that the specimen was partially clotted and the coagulation system proteins had been consumed. This occurs, for instance, when the specimen is not properly mixed immediately after collection, and the clot may go undetected after centrifugation.
DF Moreno responds: The patient told me she takes 4 mg Acenocumerol per day. I took the sample and centrifuged immediately. The patient didn’t return to control.
From George: from your description it seems the specimen was managed correctly, however this still appears to be an analytical error, as there is no information that leads towards a patient concern.
From DF Moreno: Hi again. In my hospital we store fractionated samples of calibrator and plasma depleted of factors V, VII, VIII, II, and thrombin for fibrinogen in a freezer to -70°. How much time can it last? Inserts don’t mention or recommend anything about it.
George replies, yes, you are wise to carefully store these reagents. Most distributors stamp an expiration date on their packaging material that indicates its shelf life. The date is not usually provided on the product’s package insert. Most products are stable for at least two years when properly stored. Once you have thawed them for use, most are stable for eight hours if refrigerated. Of course, once thawed and used, the remainder is discarded; you cannot refreeze for later use.
“pmorer” at Stago adds, “Just to say that it is the same for freeze-dried reagents because they have been frozen one time before drying. So you cannot refreeze them. BR
From Tiffany Whittle: Is there a difference between factor V Leiden mutation and factor V deficiency? The reason I ask is that when I donate blood and tell them I am FV Leiden positive they “call” to see if I can donate, since only FV deficiency is in their book. They have told me to always say I am FV deficient so they do not have to call because the blood center will discard plasma for both disorders.
George responded: You are absolutely right, and your blood center should know the difference. We laboratory scientists shoot ourselves in the foot by the way we name our assays. Every day someone gets confused about factor V deficiency and factor V Leiden.
George goes on about confusion affecting other coagulation assay names and suggests the next time Tiffany donates, she should them she is activated protein C resistant (APCR positive) instead of factor V Leiden positive. They mean almost the same thing, and maybe APCR is in the donor service database. She should not say she is factor V deficient, that would cause you to be deferred and identified as hemophilic.
From Pearl Gabrielle: Our ED wants to add PT/INR to their i-STAT. I am looking for information or studies done comparing the i-STAT to coagulation analyzers, specifically Stago analyzers.
George Fritsma: There is a large volume of literature that correlates POC instruments to central laboratory plasma-based PT/INR testing. I’ll start with Abbott’s own package insert for their Prothrombin Time PC/INR cartridge. Their data are encouraging, with CVs of less than 5%. They performed correlations with an STA Compact®, but using Dade Innovin® reagent, the r value was 0.943, acceptable.
Most literature compares plasma-based coagulation to whole blood PT/INR results from the Roche Coag-U-Chek®, which probably occupies a larger market share than the i-STAT. One example is Toulon P, Ozier Y, Ankri A, et al. Point-of-care versus central laboratory coagulation testing during haemorrhagic surgery. A multicenter study. Thromb Haemost 2009;101:394-401. These studies reflect CVs and correlations equivalent to the data published in the package insert.
Anticoagulation clinics prefer POC testing because the patient need not wait the usual 45 minutes for a conventional specimen to be delivered to the lab, centrifuged, analyzed, and reported. EDs may be less inclined to use POC for this purpose, as they may not require a rapid turn-around time. Many EDs, however, already have i-STATs for other purposes such as electrolytes, glucose, or cardiac markers. The down side of POC testing is that the CVs are usually a little larger than plasma-based PT/INR, and there is an automatic multiplier factor added in POC circuitry that converts a whole blood PT to an equivalent plasma PT. However, studies that compare the percentage of time the patient spends within the INR therapeutic range consistently show more desirable data when using self-administered PT testing with a portable POC device compared to using a central laboratory.
Finally, you may wish to obtain a copy of Van Cott EM. Point-of-care testing in coagulation. Clin Lab Med 2009 ;29:543-53. This article appears in an entire issue devoted to point-of-care testing and could give you the answers you are looking for.
From Michele Drejka, Newark Beth Israel Medical Center: I have a newly installed PAP-8E, and want to validate its performance. I am planning to run several comparisons with my old PAP-4, but I don’t know what is required or recommended in terms of number and types of comparisons or any other studies that will prove performance.
From George: congratulations on your new PAP-8E and thank you for your question and compliment. The document you need for validation is the CLSI Approved Guideline H58A: Platelet Function Testing by Aggregometry, 2008. This is a fairly recent publication, chaired by Doug Christie, PhD, and provides a validation section.