Webinaire sur les variables pré-analytiques en Hémostase

Welcome to the Latin American Educational Series. My name is Doctor John Mezios and I'm a Senior Clinical Consultant for Siemens Health and years. I've been with the company for about a little over 2 years and in my role as a Clinical consultant I basically discuss and and oversee hemostasis related issues as well as issues around plasma proteins, primarily focusing in on education. Prior to joining Siemens Health and years, I was an Assistant Professor of Pathology and Laboratory Medicine at Wild Cornell Medical College here in the United States as well as I was a former assistant Director of the Special Coagulation Laboratory at Bio Reference Laboratories, which is a reference lab laboratory here in the US So today we're going to be talking about pre analytical variables in hemostasis testing or pre analytical variables in coagulation testing. So I'm going to be using hemostasis and coagulation interchangeably throughout the presentation. So before we dive into the presentation itself, I just want to go over some learning objectives. So today we're going to identify proper coagulation sample collection techniques, identify proper coagulation sample preparation. We're going to explain the effects of pre analytical variables on coagulation results and we're also going to define some of the effects that let's say time and temperature have on coagulation results. So when we look at the samples journey from the patient to the results, it really goes through 4 distinct phases. We look at the pre analytical variables, we look at the analytical variables, the post analytical as well as the interpretation. So at any one of or throughout any one of these four stages, let's say of the journey, there could be issues that happens that arise. But the most common issue is those pre analytical variables that unfortunately the lab typically is not able to fully control. So when we look at in General Medical errors about 1,000,000 injuries in approximately 44,000 to 98,000 hospital deaths occur. And as a result of a medical error in the United States, medical errors are the 8th leading cause of death. Now interestingly about 2.4 million extra days of hospitalization are seen and a potential increase in hospital costs of approximately $17 billion have been associated with these medical errors. Now there was a really nice study that was published in 2013 which attempted to estimate the cost of these pre analytical variables or these errors. So this was done in a 650 bed hospital and what they know this is approximately $1.2 million per year were associated with these preanalytical variables. And in addition to the study, they also looked at let's say two specific continents. They looked at North America and Europe and when it broke down the cost by critical care for example, you can see again dollars and EUR slight differences due to the currency exchange. But you can see that overall in critical care it was roughly the same or average preanalytical cost that was per patient type. When you transition to the inpatient, again the costs are slightly higher but again between North America and Europe they're roughly similar. Interestingly when you transition to that outpatient clinical setting which includes the emergency department, you see those those pre analytical costs specifically in North America significantly increased. And again when you over, when you look at the overall cost of in a hospital setting pre analytical specimen error costs represent about 0.23 to about 1.2% of the total hospital operating costs. But if you look at the bottom, the overwhelming percentage of increasing cost is really associated with additional treatment that that patients are given as a result of a pre analytical variable. So I think that's very, very telling and the importance of pre analytical variables and how they could could affect laboratory data. So when you look in specifically at pre analytical variables or issues, you can see that it can happen in multiple aspects. It can happen outside the laboratory that is at the site of specimen collection and the specimen collection could happen within the hospital or it could happen to outside physicians offices. Then we have the transportation stability of that specimen. So going from the outside the laboratory to to the laboratory itself, that process of how the specimen is transported. And then finally, that specimen processing and storage stage within the laboratory itself. So those 3 distinct areas going from outside to the inside the laboratory and any one of those issues we can have a pre analytical variable or issue. When you look at the most common of these preanimal variables, it's really hemolyzed specimens and I think many laboratories. I know this is a a big problem in the United States that you have a a a fair amount of hemolyzed specimens that come to the laboratory approximately 10% that come in, they can see our our for example, insufficient sample. So either the sample was underfilled and therefore you don't have a lot of plasma to, for example, run all tests that are being requested or you can have an incorrect sample type, you know, unfortunately, the phlebotomist at one point wasn't really paying attention and put it into a potassium EDTA plasma tube and set that off to the Sam to the laboratory and you're going to get results that don't quite make sense. But in general, about 70% of the of the errors that you see in the laboratory are associated with pre analytical variables. So what is the real importance of getting an accurate result? And I just have, you know, 5 examples here. Well, for example, if you have a falsely normal result that you know for example, let's say patients going in for a surgical procedure and you get a value that's say for a factor 8, that's normal. But it's being masked by the fact that it's an acute phase response protein and it's elevated. In reality we have a low factor 8. So what that potentially does is it increases the the risk of the patient having a a bleeding during the surgical procedure. So it's important to ensure that we get accurate results because at the end of the day in any laboratory what we want to do is we want to ensure patient care. Another example is that they say you have a falsely abnormal result and this can range not only from hemostasis or coagulation testing but to a number of other testing that that the clinical lab does. For example, you can have an abnormal result that could result in unnecessary patient anxiety as well as additional testing that the patient may be required to do to to either confirm or to confirm the diagnosis and and so that results in additional costs as well. Well, what happens if we have let's say a falsely low or high result for anticoagulation monitoring? Well, that results in a that the patient then has a greater risk for either thrombosis or bleeding depending on those results. Well, what happens now for example if we have a patient that has a falsely normal DRBT screen when they're being screened for a lupus anticoagulant. Now that that results in the patient not being treated with an anticoagulant therapy because of that a falsely normal result and the patient perhaps subsequently developing a thrombotic event. And this last one is I've seen numerous times in my in, in, in practice where I was at the hospital where you have a false diagnosis of Von Villi brand disease. And yeah I think what all can attest to the fact how difficult it is sometimes to assess Von Villi brand disease. So for example we can have an inappropriate treatment for with DDADP and or affect the quality of life due to this lifelong label of disease. And again as I was, I've been signing out cases in the past. I would see clinical history and I would say the patient has been labeled with Von Willebrand's disease and then I would look at the results and the results are complete as normal. So that's why it makes a real important difference in giving patients the most accurate and correct results that we can give them. So we talked about pre analytical variables. There are some variables that again the lab is unable to control and these are specifically those patient variables. So for example, things that we can't control are obviously we know their gender differences, male and female press reference ranges, we know age differences as as we go from Pediatrics all the way to adults we have different age adjusted or age associated reference ranges. A really good example is D dimer where we can have different age adjusted D dimer values. Again, that's perhaps a topic for another day, but again you can see that age does play a critical role. Blood group also plays a significant role. For example, in patients who have a blood group O, their Von Rillibrand is actually going to be slightly lower compared to the other blood groups. And so therefore it's important to know when making that decision or when looking at these patients results that we know what the patient's blood group is on health status plays a really important flow and that kind of ties off with stress as well. So for example, if a patient has an inflammatory condition, we know that a number of coagulation proteins will be elevated such as factor 8 and Von Fillebrand as they are positive acute phase reactants and so they would be elevated. So if you're masking or looking for Von Fillebrand disease when the patient let's say has an hyperinflammatory response that deficiency could be masked by that increase in in value of of those acute phase reactants. And we have pregnancy, a really good example is protein S Protein S is decreased when in in pregnancy and there's trimester specific reference ranges for that Protein S transitioning a little bit of the phlebotomy again looking at a different type of tube collections, are we looking at whole blood versus tube collections, How do we collect, how do we collect the blood? Now the tourniquet technique really plays an important role tube size and and anticoagulant that are being used and I'll touch on these a little bit later as we go through the presentation. The order of blood draw that is really important as well because I've noticed many times that they could mix the blood draw and perhaps send the rung to the laboratory for coagulation testing and then finally fill volume and hematic adjustment. And again I'll touch on those in the in the in the coming slides. But again those also play an important role in in ensuring accurate or ensuring accurate results and proper specimen quality. Not only do we have to worry about, you know, patient variables as well as you know phlebotomy, but we also have, we have to worry about timing. I can't stress the importance of getting a proper clinical history on patients. You know for example if you're looking to assess the the thrombotic or thrombophilia of a patient and the patient just recently suffered suffered the thrombotic event, there's really no need to order any testing on this patient because for example the natural anticoagulants will be decreased such as protein S and protein C and fracture 8 May be elevated. So you're not going to get an accurate diagnosis. So knowing the clinical picture and knowing when and when to test is extremely important. Another example is of patients under heparin therapy, either unfraction heparin or low molecular heparin. You could have it could affect other assays, for example, it can affect your lupus workup or it can also affect your functional activated protein C resistant assays where it can give you falsely lower values or falsely elevated values mimicking a factor of five lighting mutation and then going in and doing additional testing in this case will be your DNA testing and getting discrepant results. Another good example is warfarin therapy we know there's certain vitamin K dependent factors, one of which you know two of which are protein C and protein S and they will be decreased in the context of warfarin therapy and your lupus anticoagon test may also be affected. So it's really critical to know when to order these tests. So for example, in many clinical laboratories it may be beneficial to develop clinical guidelines on ordering specific assays that allow and assist our our clinical colleagues on when to order these tests on these patients. Another really interesting point is that miscommunication or the order sets being confusing, for example factor 5 or factor 5 Lite in. So if a clinician is variant is not familiar, let's say it's not a hematologist who's not familiar with the differences between factor 5 Lite and Factor 5. They may suspect a factor 5 when they really wanted a factor 5 Lite in on other good examples factor aid or factor aid inhibitors. Of course these two are different assays and they're assessing two different things. But again, having clearly defined or assets allowing the clinicians, even those that are not proficient in let's say and are not hematologists or not laboratorians are able to go ahead and order these tests factor 10 and anti 10A heparin assay, that's a common problem. For example, in clinical laboratories, if someone's interested in getting anti 10A, they see a factor 10, then they'll go ahead and ordered it and what they're going to get is they're going to get a factor 10A activity and not the anti 10A which would be your head. So really defining and having orders, order sets there to the point and not confusing really helps in improving and and alleviating some of these pre analytical variables that we could be seeing. We also know there's a a number of pharmacological effects on coagulation. So here are just some examples that I'll go through. So for example, if a patient's on heparin either unfractionated heparin or low molecular heparin, we know that typical monitoring of these patients would be for unfraction heparin, we can use APDT. We know that APD is fraught with a lot of pre analytical issues itself because of the fact that when you're doing lot to lot changes those therapeutic ranges may in fact change. We can also transition to an anti 10A which can be used for unfraction heparin as well As for low molecular heparin. But what kind of impact do those anticoagulants have on coagulation testing? Well for example it could affect your lupus anticoagulant interpretation giving you falsely elevated or falsely low values and of course it's going to affect your PTT based single factor assays. So if you're reminding the measure let's say factor 8 or factor 9, you could potentially be getting lower values and and mimicking a normal response to a A deficiency For example, a good example is again vitamin K antagonist. We know that the most widely used one is warfarin or Coumadin and so again typically we would monitor that patient with a PTINR and I think Doctor Andreas Rechner is going to be talking about if that in his introduction for hemostasis section following my presentation. In addition to that, for example, if we look at how does that impact let's say coagulation testing, again it'll impact your lupus and as well as impact your PTT based single factor assays as well as your protein acid protein C A common issue that I've seen again in the clinical laboratory is the doacs or the direct oral anticoagulants and there's two types, there's the anti 10A, anti 10A such as riboroxaban apixaban and those will be monitored using some US drugs calibrated anti 10A assays. Or you have the direct thromp inhibitors such as the Bigotran and they could be used to, they could be monitored by say an E caring clotting time or an E caring chromogenic assay or even using a dilute thrombin time. But again, how does that impact your coagulation test? And I've seen this so many times in practice where you would get a sample and you would see, you know, an abnormal, let's say lupus profile. But then you look in the patient's history and you see the patient's underprox and we now know that these new oral anticoagulants of these doacs will affect your coagulation assessment. So therefore, it's absolutely important to get a proper clinical history. Now if the patient cannot be taken, let's say off anticoagulant medication, then you would try to mimic or get as close to the through value of that drug or get a sample right before the next dose to at least be able to somehow interpret those results. And again, that's also going to be applicable to the laboratory procedures. And there's a really nice article I think that just came out and and that's talks to how to properly assess these patients and what protocols perhaps could be involved when you're assessing these patients on Dalek therapies. So let's transition a little bit to specimen collection and processing. Now everything really begins with the sample. I mean if you have a a sample that's of poor quality, then you're going to get poor quality results. So really it's important that we have the best sample that we can get for these patients. So that really comes from the phlebotomy. So you know, I again, this is just kind of a a fun little anecdote, but the phlebotomy, what is phlebotomy? Well, it's the act of venipuncture that initiates the hemisatic process. So if you think about it, you're playing, you know you're you're you're you're pointing or getting or you're causing damage to the skin and that's resulting in some type of hemostatic response. But not only does we have to worry about the phlebotomy procedure, but we also have to worry about the body position and how that could potentially affect the the results. So for example, your Hematica increases while lying and then sitting and standing. So getting these lying, sitting and standing positions could really affect your hematic hematocrit. It could cause some some level of hemo concentration and therefore they could result in this redistribution of blood. So that could ultimately affect your PTPTT which could be shortened as your fibrinogen increases. So if you think about it, as fibrinogen increases you have better clotting and you have about a shortened a shorter PT and PTT. So ideally you want to have these specific patients sit in a comfortable position for at least 20 to 30 minutes before any blood drawings done to allow the hematic to kind of readjust and be able to have a proper sample being drawn. So we talked again really briefly about the phlebotomy. So sample size of small turn needle size plays an important role. The small, the size of the needle, the more disruption that we'll have in the plates and perhaps have more hemolysis, 19 to 23 gauge needles are appropriate, 25 can be used in certain situations and for example if a butterfly is necessary, it can be used. But there's certain protocols that we would have to follow, not go over through some of those protocols in the coming slides. And of course, indwelling Catalyst, which is commonly seen in in patients, you know typically should be avoided. But if it can't be avoided, again there's a specific process that you would have to go through to ensure that you are able to draw a good sample tube type plays an important role. Obviously you would want to avoid silica siliconized glass or polypropylene plastic because that could effectively result in some type of activation. And again a smaller diameter tube could potentially increase your platelets. So therefore you have to be very wary of what how that sample and which tube type you're using. What's also important is a tourniquet. So I I've seen again in in many clinical laboratories where the tourniquet is placed in the arm and it's left for the duration of the blood draw and for coagulation testing specifically, That's not an ideal situation. Typically you don't want to leave the tourniquet on for longer than a one minute and the patient should not pump their fist because what that does is effectively causes some change in the coagulation properties and proteins, ultimately resulting in some small or minimal amount of activation which could potentially snowball into a larger hemostatic response. And as I can say it, as it says here in the slide, you can also slowly venous circulation. So if you're clamping onto your arm for an extended period of time, there's less blood getting to the area where you're drawing the blood from, which could potentially delay the process causing by the phlebotomist to draw more on the syringe, resulting in a little bit more platelet stressing and causing more plate activation of your platelets. So again, having these principles in place or having knowing these things is critical to ensuring that we get the best sample available for coagulation testing. What also plays an important role is the order of blood draw. And I think many people in the clinical lab have seen this type of picture before. So if you go through the how you're supposed to quote UN quote, how you're supposed to draw blood. You know, first of all, your blood culture tubes are the ones that are typically drawn first because you're all you're going to see is you're going to see whether or not there's specifics organisms and microorgans growing in that you would invert those and and put those aside. And then you transition to your sodium citrate tube which was for your coagulation testing. And then you would proceed to other tests if they're needed going from your chemistry all the way down to, let's say blood banking and and trace elements for example. But what's important for your coagulation testing result in, in your sodium citrate tube is really doing a gentle inversion about three to four times to ensure there's a proper mix of the anticoagulant of blood. So for in a situation where you would just need APT and APT, just a real quick PT and APDT, typically there's no discard tube that's required because if you think about what your PT and PDT, and again, Doctor Rechner is going to talk about this in the next lecture. But really there are global assays that are assessed the overall hemostatic function of of the patient's coagulation cascade. So globally you won't get a significant effect. But if you want to measure other analytes, for example, like your factor activities or looking at von Villar band disease or looking at plated aggregation, then you most likely would need a discard tube. However, if you are to use a butterfly you you are going to need a discard tube. And that's because you want to ensure that you maintain that nine parts blood to one part anticoagulant ratio in the overall sodium situated tube In dwelling catheter. Typically you would want to flush with about 5 mill MLS of sodium chloride and that discard about 6 Dead Space volumes of the tubing and that minimizes the deeper of the heparin contamination that you could potentially see. Again ensuring accurate and proper sample quality. Kind of already talked about this, but once soon as the blood is drawn and put into the sodium situated too, you want to immediate invert end over end about three to four times. But you want you want you want to avoid agitation that could potentially cause activation of your plate so it would be a gentle inversion. I've seen times where phlebotoms will just draw the blood, shake it vigorously and put it onto the thing and then send it back to the laboratory. And then you wonder why the results come back. The way that they come back, you also want to fill, you want to ensure that you have a proper fill. So we talked about there's a fill zone and I'll talk about that in in the coming slides. But you want to ensure that you're above that 90% fill zone to ensure accurate results and to ensure that the sodium amount, the amount of anticoagulant, the blood is maintained in that nine parts to one now 3.2% sodium Stitrade is more forgiving to the underfill. And I'll talk a little bit about the anticoagulants in the next slide. And then two manufacturers do in fact show and have a proper fill on their label to indicate at what point you have 100% fail and CSI guidelines do accept a 90% fill. But again I think it's good laboratory practice to see if there is let's say a 90 and 95 and a 100% does that affect your their laboratory results by doing an internal laboratory study as part of good laboratory practice. So when we talk about anticoagulants, we talk about there's two real two anticoagulants that are used for for coagulation testing and the main principle is that they're able to bind calcium therefore presenting preventing that initiation of the clotting cascade. One is 3.2% sodium citrate which is recommended and and used primarily in clinical practice. There's a second one which is a 3.8 sodium citrate that gives generally gives more prolonged results and typically like for example there's APT and PD that will be slightly more prolonged than that of 3.2% sodium citrate. But why is 3.2% recommended? Well, one is more forgiving to the improper blood to anticoagulant ratio. So that's why that greater than 90% of fill is is ideal because of that 3.2% sodium situation. And you found that it's when you do do analysis, the results do not deviate significantly. The 3.2% is close to that of plasma osmolality and in addition it's kind of more accurate for if APDT is going to be used for patients on heparin. Now it's really important to know. So here in the United States for example, there are vendors that will give you samples that are plasma samples for let's say a reference range study. And there's one specific company that I find out the hard way when I ordered that sample and it came to my lab that they actually did use 3.8% sodium citrate as an anticoagulant. So when I take those samples and I try to incorporate that into my reference range where the other samples were anticoagulant and 3.2%, you can see there's going to be a mismatch. So it's really important to know that the anticoagulant that was used for all your samples specifically let's say for a reference range study were used 3.2% sodium citrate. So how does the how does the how does different how do different anticoagulants affect your results And I really like this slide because it really just just drives on the point that even if someone comes back and says in jaws and gives you an EDTA plasma you're going to get a result but it's going to be an inaccurate result. So for example one of the things that I've I've I've seen again in in practice was that someone put in in a potassium EDTA tube send over plasma send to the cloud lab and you get these weird results that don't make sense. So fortunately you're able to check in many clinical laboratories you're able to check the clinical history to see does this make sense with the clinical picture because what you'll see here is that you'll you'll get APT and APTT result. Clearly they're they're going to be more elevated or significantly more prolonged. So that should raise suspicion that something is not right with the with the sample quality. For example, if you look at factor 8, your factor 8 is going to be very, very low. So you're you could potentially misdiagnose this particular patient having a factor 8 deficiency when in reality they don't. So it's really important to ensure that the proper anticoagulant is used, in this case sodium citrate, the 3.2%. If you are suspicious of let's say of an EDTA plasma, typically the most common one is potassium EDTA. What you could do is take that sample and run on the chemistry analyzer and you'll get a very, very high potassium indicating you know clearly this is not a a sodium situated sample. On the other hand, one of the things that I did when I used to teach medical students or residents, one of the things that I always would ask them and I would got I, I typically would get blank stairs was well what is the difference between plasma and serum. And you know for a medical student you would expect that should be a real quick response. But it it was one thing that typically many people don't think of well plasma, you have all your coagulation proteins plus fibrinogen. Well what happens in serum is that you actually activate it. So you're devoid of your fibrinogen, so you've consumed the the your fibrinogen. So therefore serum is absence of fibrinogen. So if your sample comes back and you get a routine PT and a PDT that looks there's no clot detected. Measuring fibrinogen C of very very low value can indicate that is most like it would indicate A serum sample. So there's ways of differentiating that the different type of sample types. But again, it's absolutely critical to ensure that all the, the, the clinical staff and the specific phlebotomy is using your propane anticoagulant with it and which is in this case 3.2% sodium citrate. So what happens when you get a clotted sample? Now there's different techniques in a lot of some of the new instrumentation from different vendors out there have the ability for the instrument to actually detect whether or not there's a clot on board. But for example, the traditional way is either taking like a wet applicator a stick and going through and seeing whether or not there's some type of clot present. But what you would typically see is here where you see a normal sample and you have your plasma which contains your fibrinogen. And if you do get a clot, there's a couple things that can happen. If you get a large clot as depicted here on the screen where you have your serum sample and your clot on the bottom that you can visibly see you're going to get a prolonged PTPT and thrombotime, which is what we would expect based on also on the previous slide because the fibrinogen is basically has been consumed. But if you have a micro clot, sometimes you can actually see these micro clots by using that wood applicator stick. You'll see a more of a shortened PTT that could indicate, let's say, an activated sample. So if you see a significantly activated sample that in my mind would trigger, let's say this patient needs to be redrawn and then retested. And you'd also perhaps see an elevated D dimer in these patients because you have the whole process of fibrin degradation as well happening in the sample. So we do see a large number of these of of these clotted samples coming through the clinical lab. Typically it's about 0.4 to 0.7% of the samples that are clotted. Now again, depending on the laboratory policy, typically clotted temples should be rejected and then ask for a redraw. So let's transition a little bit to the transportation and I think in the in the one of the learning objectives I mentioned is, is temperature. And temperature really in plays a really important role. So I I'm not sure how how things are done, let's say in Latin America, but I know here for example in the United States, there's different ways in which samples can be taken to the clinical laboratory. It can happen within the hospital system itself and then the samples are either transported via some type of transportation system to the lab or they can happen outside physicians offices where a physician is partnering with a specific laboratory and they'll draw the lab or they draw the blood, let's say at their office and then send it to the laboratory. So you can imagine that temperature plays a really important role because as the samples, let's say in in in winter months where the temperatures are cold or in the summer months where the temperatures are really warm, those very big fluctuations in temperature could wreak havoc on your specimen quality. In addition, what's also important is timing. CSI guidelines recommend that that coagulation testing should be performed on a sample that's drawn in about four hours. So less than four hours is the optimal time, greater than 4 hours. You could result in some level of activation that could be misleading for your results. And of course transportation. How are those samples transported? Are they racked and positioned upright? Or they're just thrown in the car, for example, or in this pneumatic tube system? And the samples are going all over the place because you do want to avoid the minimal amount of agitation. The more agitation there is, the more likely that you'll get some level of activation. Now the pneumatic transport systems happens typically in the larger hospital networks where the samples can go from one floor to the hospital laboratory via this pneumatic tube system within the hospital. So again, it's sometimes it's good to assess whether or not those specimens going from let's say the furthest floors away to the laboratory if for example, those have specific issues. So again looking at it and analyzing in greater detail I think is is extremely important. So now what happens when we get into the into the lab, we talked about that that samples journey, so three out of the four are listed here. We'll talk about the pre analytical phase. We look at centrugation sample preparation and aliquot freezing and thawing depending on how the samples are being transported. The analytical phase, HIL plays a really important role, as does fill volume and clotted samples. And then finally the post analytical phase we talk about how the samples are stored and if additional testing is then requested, how those samples are then thawed and if there's a proper protocol in place. So we talked about temperature. So in different situations temperature can affect your results. For example, if there's an increased temperature, it could potentially degrade factor 5 and and and eight, giving falsely lower values a decreased temperature. Let's say for example, if the sample is exposed to very, very cold air in the winter months, it could potentially activate factor 7, therefore causing a more of a shortened PT. Refrigeration of the samples or refrigeration of your whole blood again could have wreak havoc on factor 7-8 and bundle aban. And you want to avoid, let's say, storing samples on ice or in Colpac prior to any assay being performed. And of course avoiding refrigerated centrifuges if possible. Not recommended for coagulation testing. So this this Donna Castle alone she's a laboratory supervisor in in one of our in in the hospital system here in the in in New York in in Columbia and she just kind of coined this phrase. And I I kind of like this I kind of I took it from her is good coagulation happens in four hours where great coagulation happens in two hours. So obviously the faster you get the the the the sample to the laboratory the better it is for the laboratory and better is for the for the results. So this just looks at this list. Figure here just looks at let's just take PTS and PDTS. Obviously here we have PTS for unfraction, heparin for von filiband working, and for others. But let's just look at your PT and PT when your most routinely ordered tests in the clinical laboratory. If your sample is stored as whole blood, you can store the sample for room temperature for up to 24 hours and then spin it and get an accurate PT. But if you refrigerate it or freeze that whole blood, it's unacceptable specimen quality. We'll contrast that to the APDT where you can see if you store that room temperature, it's really good for up to 24, up to 4 hours. So that difference between PT and PT is really significant. And most labs, if you look at plasma, that's a you process the whole blood into plasma, you can store the sample for up to 24 hours and still get a normal or good PT. Same thing applies for room PTT where again it's only good for four hours, but if you refrigerate that plasma, excuse me, you refrigerate that past plasma, it's going to be unacceptable to get APT result, but you still will be able to get APTT result. So looking at how the specimens react under different extreme conditions is also really important. In the clinical laboratory you then freeze the samples for freezing. These samples are good for about 12 months for PTS and PTS for routine testing. But I also would recommend that in a clinical lab when you're doing these kind of internal studies to assess and just put samples into the freezer and then periodically pull them out foam and then retesting them and comparing that to the baseline value to ensure that it is indeed accurate for up to 12 months or however long you the lab is required to store samples, Let's kind of transition a little bit to centrifugation. So the goal is really to have platelet for plasma and and I'll touch a little bit about that in in the next slide. But really you want to be able to have a a platelet count that's less than 10,000 and that's critical to ensure proper coagulation results. You want to use about 1500 Genius for 15 minutes at room temperature. Typically double centrifugation is recommended especially if you want to do lupus into coagulant testing and you want to want you want to use let's say a swing out bucket rotor for the for your centrifuge which is you know essentially recommended. And it's ideal to validate the centrifuge and platelet count every six months to ensure that you are getting platelet poor plasma and then you can store that sample into -20 or -70 freezer. But you want to make sure that it's a non frost free freezer. So why plated poor plasma, Why does it really matter? Well, we know that plates are a rich source of phospholipids. Now in addition we also know that the reagents that are used for coagulation testing are also rich source of phospholipids. So you can imagine that you have excess amount of plates plus your reagent phospholipids. It could in fact shorten new results and therefore given inaccurate test results. We also know that platelet factor 4 is housed within the plate of the granules. So when they are excreted or secreted into the into the plat into the sample, it could effectively neutralize happening, therefore shortening the APDT and giving you a misrepresentation of the anticoagulated status. It's also for example, when we freeze and thaw these plates, platelets, valves, those plates can then just dissolve or disintegrate or break apart releasing more platelet factor 4 and therefore again affecting those results in which you get lower more of a problem shortened PPT and inaccurate PPT for patients on heparin. So just again heparinized PPT. If you have these platelets, it could decrease it giving you a misrepresentation of the anticoagulant status lupus anticoagulant. Again it could result in decreasing and therefore giving you a misrepresentation as well as protein As for example. These are three of of examples that we can that plates would significantly affect the results on. So when you do take these samples, and let's say you are a reference lab for a a larger group of of practices, you the samples come in, they're frozen, and then you have to have, let's say, a proper thawing procedure. Typically you'd want to thaw these samples about in a 37° water bath, no longer than 10 minutes. Ideally, it's around about that 5 minute mark You want to monitor and make sure as soon as they turn liquid that they're properly mixed. You want to mix thoroughly prior to any testing and you want to observe if there's any particular matter in the in the bottom. So often times there are these cryoprecipitates that form. So once the samples are brought out of of the freezer, they're put into this water bath, they thaw, you can get a cryoprecipitates forming. This has happened to me numerous times, especially in the context of von Villebrand testing where I would, you know, the technicians would well go ahead and run the test. It would get these results that don't quite make sense and they would come to me, and most likely you'd see a small particulate matter in the bottom because they just wouldn't spun, they didn't spin the sample correctly. And once the samples are thawed, you want to be able to keep them, you know, kind of at room temperature, ideally for about 10 to 15 minutes, allowing them to equilibrate prior to putting them on the analyzer for testing. If you have to, you can spend store those samples at 4° for no more than two hours. Again, you'd want to verify that, for example, if your PT will be affected in that in that context, but again, something that the clinical lab should verify their own on their own. So kind of looking at from the pitfalls, if your assays are greater than or if your sample is greater than four years old, four hours old, sorry, old, your factor 8 could be decreased and then your plate of factor 4 could inactivate happen, therefore lowering your PDT. If you're using let's say a glass tube or pipette, it could activate or shorten your PT and PDT if you have an ad on this again problem. In many, many clinical laboratories, a physician realizes that they need to order additional tests in this patient. They send out an order and the sample is you know, more than four hours old. You can say it's just not a good idea to order it if a sample has been stored properly and you should just don't do it and and really just order a new sample. If it's not platelet for plasma, you could potentially shorten your results. Uncapped tube for example can alter the pH within the tube itself and and and prolonging your PT and PPT. If you're using frost freeze, frost free freezers, it could happen those because those types of freezers actually have different types of freeze, thaw cycles that cooling and warming of the temperature of the sample could affect the specimen quality. So that's where you'd want to use non frost free freezers. And of course the presence of a clot could prolong or shorten the results. So if you can look at the summary of general pre analytical issues, we talked briefly about the collection principles. So we talked about patient selection, knowing the patient's clinical history, the the medication the patient is on, age, gender and so on and so forth. Phlebotomy plays an important role in ensuring that you have a good specimen that's sent to the clinical laboratory. And I'll touch about fill volume and hematic adjustment in the next section where I talk about the different types of interferences. We talked about the specimen transportation stability, the importance of transportation conditions. We want to make sure that we have optimal temperatures. We want to make sure we minimize agitation. We're going to analyze the distance that the sample has to travel to ensure that there's the minimal amount of disturbance within the sample that could potentially affect your specimen quality. Of course we're looking at sample stability. For example, if there's any type of influence, that presence of excess amount of heparin, all those factors that could potentially affect the quality of your specimen and then transition to that inside the laboratory phase, we look at centrifugation, double centrifugation to make sure you have a plate of poor plasma. How are you preparing that sample? If the samples need to be aliquot or the frozen then thawed, having proper procedures in place and then finally specimen condition about hemolysis, lipena and icreas and I'll touch about in the next session when I talk about interferences. But those in general are the preanalytical issues that we have to take into consideration. And there's just a lot that you have to think about to ensure that the sample that gets from the patient to the laboratory is of the best quality to ensure accurate results for the patient. So now let's transition to these common interference substances that we see in the clinical laboratory, hemolysis, icteris and leukemia. So we know that hemolysis can can wreak havoc on your coagulation test and the main reason is as soon as you draw the blood or as soon as that that vena puncture is initiates that hemostatic response you draw, you know the the plunger is pulled you you cause lysis. That lysis results in releasing of ADP and other pro coagulant factors resulting in plate activation and the activation of the coagulation escape. So therefore resulting in results that will not be able to be accurately interpreted. So we look at many. So there's let me start by clarifying there's two different types of analyzers and I think Doctor Rechner will talk about mechanical versus photo optical clock detection systems. But in brief on mechanical clock detection system is where you have a mechanical ball that goes back and forth that really doesn't look at the, let's say the photo optical outside of things, but really is reliant on the the presence of fibrinogen whereas the photo optical looks at changes in absorbance values to assess the clock time in seconds. But typically you'll see here is a hemolysis. Leukemia and Ichterus have different effects at different absorbance, they'll have values. So for example, hemoglobin, the presence of you know excess amount of hemoglobin as we would see in a hemolyzed specimen has a profound effect around the 400 and five 410 nanometer range. Whereas let's say Ichterus has a more very broader distribution around the 400 to 500 nanometer range. And the newer instruments that require photo optical clot detection systems have the ability to use multiple wavelengths. For example, if there's hemolyzed specimen, it'll transition from the 405 to let's say to the 660 nanometer range or the 800 where the level of the OR the degree of interference is is significantly less. So again I I I showed this slide before where we know about the majority of the samples that come into the clinical laboratory are typically hemolyzed in nature and that's where we have the bulk of our our issues. So if you look at the next few slides, we'll touch base on underfilling. We'll touch base on hemolysis echterst and and Latinia and we know that hemolysis which is the most relevant parameter for interviewing substances that we see in the clinical liberty and it's really independent from the measuring technology. And again not to go into a greater detail but there are the the, the one system that uses the mechanical clot detection system even though it uses a a metal ball it is subject to interferences as seen in in from hemolysis or like hemic and ectors ectors as well. So we know really what hemolysis is, is really you have an excess amount of excessive amount of hemoglobin and or erythrosis salt destruction that's in the sample and typically many clinical laboratories result result, you know result on vision visual inspection of the sample to see what degree of hemolysis there is. And obviously that's not the most accurate way but you know comparing it to a chart that's typically you know in the in the laboratory you'll see that this patient is grossly hemolyzed and you go ahead and reject it. Many analyzers now have pre analytical sample integrity checks on board which allow the instruments to assess the degree of hemolysis seen and provide different levels for the laboratory to either approve, reject or approve with a comment. So what are some of the general causes of hemolysis? Well it's a hemolytic process that can happen in vivo. Severe infections for example DIC are also known as disseminate intravascular coagulation which is again a widespread coagulation that happens it can see in patients who have sepsis for example or transfusion reaction. So those are some of the in vivo events and what we really see is in vitro. What could happen is you can have a difficult venopuncture or also known as a difficult stick, you know, using a small born needle freezing the whole blood sample for example was then following that simple results in hemolysis. And this is what really accounts to about 70% of the injected samples that you see in the clinical laboratory from the in vitro, In vitro causes of hemolysis. So if you specifically look at the effect that's and it has on a number of analytes, for example for the PDT it can either cause a falsely increased or falsely decreased result. For PTD dimer factor 5/8 and 10, it can arose in falsely increased results, whereas Fibonagen anti throb and throbotamin and protein C for example, it can cause a falsely decreased results in the context of Humalog specimens transitioning now to a the lesser of the less less least common one of the three. In this case it's it's Icterus. Again it goes about again by visual inspection to detect if it's at what degree you have a a significantly inter example. Again this is a very laborious process and it's prone to significant errors. Again many analyzers now that are available to do this automatically, but let's just focus some of the in vivo causes. You can have elevated bilirubin as a result of severe liver disease for example. So that excessive amount of bilirubin that's present causes and could potentially influence and affect some of your hemostatic assays. So if you look at those hemostatic acids that are affected, for example falsely increased results you would see in the context of PT and fibrinogen unaffected results, your PDT and factors most likely won't be affected and of course falsely decreased results. You know, you may see an antithrombin bipedia as is another regularly occurring HIL interference and that is common as hemolysis. But again, this is really the result of an excessive amount of lipids that are present and again you rely on the text, typically rely on visual inspection to detect lipaemia, again very laborious and very prone to errors. But what are some of the causes of lipaemia? For example, there could be genetic reasons that that prediction has a genetic predisposition to a hyperlipidemia or there's a postprandic collection after some type of a fatty meal that requires that it has this significant amount of lipids present analytically. It could cause, for example, having higher baseline readings, let's say at a 405 nanometers or so transition to. Having this multi wavelength system allows you to go let's say to a higher wavelength where there's a less of a pronounced effect. You also have a biological effect. It can cause an elevation in your factor 7 or it can have an acute effect on plated function, or it can cause decrease in some of the clotting factors that are available, for example like factors 29 and 10. But really your best approach in a sample that has a higher degree of lipids is really recollecting the patient's fasting to ensure proper specimen quality. But how does that affect your coagulation results? Again, you can get falsely increased results in antithrombin unaffected results and PPPT and factors. And of course falsely decreased results in fibrinogen, thromatine, lupus and plasminogen. So we touched in the initial part of the the discussion talked about filling and how the proper filling and the proper filling tubes. So here are four tubes and you'll see the fill zone that's between that 90 and 100% mark. And you can see that two out of the four are not properly filled, one is underfilled and one is overfilled And how does that really affect your coagulation testing? So if we just focus in on the two tests, you'll see the two ones in the middle, which are the ones that fall within that fill zone, greater than 90% but lower than 100%. Those are the ones that would be acceptable specimen quality. So let's just take two examples, one that's filled correctly and one that's under filled and what would affect and how does that under filling affect your coagulation tests. If you look at the sample that is filled correctly, your anticoagon, the blood ratio is appropriate. So that means when you do the centrugation process and you have plasma ready and you recalcify, there's a proper amount of calcium, calcium in there that's not affected by the sodium citrate allowing you to get accurate testing. Where in the sample that has underfilled that means there there's more anticoagulant, there is blood and the ability to sequester that additional calcium that's added in that recalcification step when doing the test will affect your clot times and typically will prolong your clot times giving you inaccurate results. So that that's why it's absolutely critical to ensure that we have that proper anti anticoagulant to blood ratio, 99 parts blood to one part anticoagulant. I mentioned briefly about hematocrit. You know again I'm not going to go through these formulas that are indicated on the slide here. But what you'll see in patients who have a hematocrit of greater than 55% typically requires you to adjust the amount of anticoagulant on board in order to ensure a proper anticoagulant to blood ratio. Many laboratories have this kind of a quick reference. For example, this is a reference to using that 2.7 ML 2 that's that overall 3ML blue top that has about .3 ML of anticoagulant and takes about 2.7 ML of blood. So in this particular case for example, if your patient has a hematocrit of 60, you would want to remove 0.12 ML of anticoagulant and allowing to having along the proper anticoagulant to blow ratio. Again, I'm not going to go into details because many labs already have this in place in the clinical laboratories. So if we look at improper tube filling, we talked about this already, you would get falsely increased results for example in your PT and PDT and you could get falsely decreased results in in in D dimer. So we can see that the effect of HIL and improper tube filling have on the different types of coagulation testing that that we see in clinical practice. So here's just again just a summary. You'll see that hemolysis ictosymopenia as well as improper tube filling can either falsely increase or falsely decrease your results depending on which interfering substance we're looking at. Again focusing a little bit of HL interferences, you know HL may affect the ability of optical reading instruments to accurately assess plated poor plasma. But again with newer technology and how the instruments have essentially evolved. The instruments on board typically have these multi wavelength parameters that allow to switch wavelengths and get not allowed to give you accurate results. If for example there is suspected X vivo or in vitro hemolysis ideally as to reject the sample independent of the measuring technology out there. Because again irrespective of if it's photo optical or mechanical cloud detection systems there still will be an effect of hemolysis. Latimia samples can be processed using ultra centrifugation techniques for example. This can be done in in many clinical laboratories if the sample is of critical importance. You know you would take one aliquot ultrasentifugic to remove the sediment, let's say the the the the the lipids remove the superning and then basically do a comparison before and after to see if there's an improvement or you can actually see if you can get results. But again, before this is implemented in any laboratory this should be then verified in the clinical lab to ensure that this is an accurate technique to be performed. Icarus again may interfere with the accurate assessment of various chromogenic methods. And the last point here is the infusion of these hemoglobin based oxygen carrier or this quote UN quote fake blood that's available in certain medical institutions and it could create a kind of a pseudo hemolysis appearance and may affect some of your cloud based assays as well as your chromogenic methods. So some of the take home messages is that coagulation samples are subject to a number of free analytical variables and the time from blood draw to assays critical for coagulation results. Remember, 4 hours is good coagulation, 2 hours is great coagulation. Hemolysis may have adverse effects on coagulations. Also of a severely hemolyzed specimen is given to the laboratory. It's better to eject that sample and then order a new one. Sample handling can also result values. Again we talked about the processing and this and how the the samples are transported. So minimizing amputation and shrinking samples are upright position. They're in a cool, an appropriate environment to minimize these, you know, changes in in, in temperatures. Patient variables contribute to test results. We now talked about age, gender, different types of stressors, you know, inflammatory conditions, sitting, standing. There's a number of variables that you have to take into consideration when you're looking at these results that sometimes don't make sense and when results do not make sense, consider pre analytical variables. I know this is kind of kind of a touchy subject in many clinical laboratories and I've had this experience where I've had people come and and call me and says well these results are wrong and then you end up figuring out that this patient was on some type of medication which affected your your results. And you know often times clinicians that are not familiar with laboratory medicine when they see the results and and and they're doing their job in into ensuring patient care, they question the results and rightfully so. But it's also good to educate the the the non laboratories or the non pathologists or laboratory directors that there are a number of clinical pre analytical variables that could potentially affect your results. And it's good to and it's really important for the laboratory staff as well as the clinical staff to always be on the same page. And by that I'd like to thank everyone for your attention and I would be more than happy to take any questions if there are any. Thank you again for your attention.

44,000-98,000 162.18 177.98 337.15 245.37 337.02 107.62 80 60 40 20 64% 10% 13% 70% 10 11 12 13 19 14 15 16 20-30 17 18 29 21 22 11.6-12.6 18.9-21.7 27.7-35.3 10.9-11.4 12.7-13.2 258-372 <20 95-110 100-128 73-106 63-122 150-237 91-139 <1-17 4-19 84-132 128-192 206-401 75-108 75-115 112-182 65-122 77-142 274-451 66-111 33-68 80-106 57-72 26-70 88-111 90-128 31-67 280-444 78-108 59-95 23 45-92 14-64 59-9 31-6 24 0.4-0 25 Hemostasis Learning Institute presents Post-analytical Variables in Hemostasis Testing John V. Mitsios, Ph.D. Powered by hemostasis experts Objectives SIEMENS . Identify proper Explain effect of Define effect of coagulation sample time and collection preparation temperature on coagulation results Objectives ources of Spurious Results Variables Analytical Calibrators Transport Reagents Result Processing Technologist Clinical Storage Procedure Interval Calculation Statistics Controls Instrument & Patient Comparison Reference Green SF Clin Biochem 2013;46:1175-1179 Medical Errors Zhan C ct al. J Am Med Assoc 2003:290:1868-74. 1 million injuries and approximately hospitals deaths annually 8th leading cause of death in the United States 2.4 million extra days of hospitalization and possibly increase of hospital costs by $17 billion Estimated Cost of Pre-analytical Errors Average preanalytical error cost per patient type North America ($) Europe (€) Critical Care Inpatient (other) 650 bed hospital Outpatient (including ED) approximately Preanalytical specimen error costs represent between 0.23% and 1.2% of total hospital $1,199,122/year operating costs. in preanalytical costs % Cost Blood Collection Redraw and Lab Instrument Downtime Patient Treament Consumables Investigation Costs Outside the laboratory In the laboratory General Pre-analytical Issues Specimen collection Healthineers Specimen Transportation and stability Specimen processing and storage Pre-analytical Sample Integrity Challenges in Hemostasis Lab Pre-analytical errors and unsuitable samples account for ... Hemolyzed Samples Insufficient Samples Incorrect Samples Others ... up to 70% of lab errors. b errors Accurate Results Make a Difference False Normal could False Abnormal False low or high False normal False diagnosis of prevent further could lead to costly result for DRVVT assay von Willebrand diagnostic testing and time anticoagulation Disease consuming tests monitoring Factor deficiency Unnecessary Patient at risk for Patient not treated Inappropriate bleeds during patient anxiety and thrombosis or with anticoagulant treatment with surgery expense bleeding therapy and DDAVP and/or develops affect the quality of thrombosis life due to lifelong label of disease Specimen Collection Pre-analytical Variables Specimen Collection Patient Conditions Gender Age Blood Group Health Status Stress Pregnancy Phlebotomy procedure Whole blood vs tube collection. Systems and blood collection Tourniquet technique Tube size and anticoagulant Order of Draw Fill Volume and Hematocrit Adjustment VS Se Aware of Proper Time to lytical Variables Investigate Root Cause of Thrombophilia Following a thrombotic event · natural anticoagulants are decreased Heparin Therapy · AT decreased Guidelines for Ordering can · LA workup effected assist physicians · APCR effected Warfarin Therapy · PC and PS decreased · LA effected Be Aware of Proper Time to Development of Clinical Doctor's Order Factor V or Factor V Leiden? Lupus Anticoagulant or Lupus Prep? PT ør Prothrombin Gene Mutation? Inhibitor Workup or Immediate Mixing Study?R effected · FX or anti-Xa Heparin assay? Even the orders are confusing Study? Pharmacological Effects on Coagulation - Some Examples Drug Class Heparin Vitamin K Anti-thrombotics DOAC Factor Replacement antagonist Platelet function Anti-lla, ECT, ECA, Factor level (e.g. Hem A or B) Anti-Xa (UFH & assays (e.g. Light VWF, FVIII (DDAVP/ transmittance Drug calibrated vasopressin) aggregometry, anti-Xa PFA-100) PT/INR, APTT FFP, plasma based products Lupus Anticoagulant, No consensus on Lupus, PT-based Sample collection just Newer replacement Impact on method for before next dose! therapies might require other factor assays Protein S, Protein C measuring or Impact on other special testing methods coagulation Methods include coagulation tests - (long acting factor PFA, Ultegra, and concentrates, Emicizumab)! traditional platelet e.g. Lupus, APTT, methods PT/INR, single factor aggregation assays APTT (UFH) PT/INR Monitoring LMWH) Watch-out APTT-based single single factor assays, & Processing Everything begins with the Sample! 1st Things 1st Phlebotomy ... in vivo becomes in vitro "The act of Venipuncture initiates Hemostatic Response" CLSI Guidelines = 17 Steps for Phlebotomy Procedure Standing PT & a Body Position @ Draw Effects Results Hct increases Lying - Sitting - Standing reversible hemoconcentration · redistribution of the blood PT & aPTT can be shortened and Fibrinogen increased 20-30 minutes before Hct adjusts Other Factors .... Draw Effects Results Small size = platelet disruption & Tube Type Standing Tourniquet hemolysis versible hemoconcen tEvacuated blood collection tube . No longer than 1 minute Size based on patient and blood the preferred · Patient should not pump fist volume · Siliconized Glass or Polypropylene (change coagulation proteins) 19 - 23 gauge for adults, 25 can Plastic Can slow venous circulation & be used PTT can be shortened.a Small diameter tubes increase d increase FVIII, vWF, Tissue Syringe/needle draw increases platelet activation Plasminogen hemolysis risk Syringe to tube via cap puncturet adjusts Can cause platelet activation for correct fill Butterfly ... may be necessary for certain patients Indwelling Catheter should be avoided Other Factors .... Tube Type hemolysis · Evacuated blood collection tube be used · Small diameter tubes increase RED LAVENDER Blood culture GOLD PINK YELLOW Start here Blood Bank GREEN GRAY Collection Tube Draw Order Single Coagulation Order PT or aPTT .. ... no discard tube required Other analytes. .... discard tube required Butterfly · Discard tube required to fill tubing dead volume: assures 9:1 ratio Indwelling Catheter · Flush with 5 ml NaCl and discard 6 dead-space volumes of the tubing: minimizes heparin contamination CLSI (NCCLS) Guidelines Fourth Edition December 2003; H21 A4 vol. 23 No. 35 . PT or aPTT. Mix Tube Single Coagulation OrdeRT end over end 3 -4 times Tube Fill Butterflyder fill prolongs PT / aPTT due to excess sodium citrate Tube manufacturer shows proper fill on label > 90% ... possible clot activation Sample Tube · Immediately INVERT end over end 3 -4 times Excessive shaking or inversion can activate platelets 3.2% sodium citrate more forgiving of under fill · CLSI Guidelines accept 90% fill < 90% ... normal results can become abnormal Anticoagulant for Coagulation Sample 3.2% trisodium citrate ... RECOMMENDED ivate platelets 3.8% gives prolonged PT/aPTT · More accurate aPTT for heparinized patients · More forgiving of improper blood:citrate ratios sodium citrate Closer to plasma osmolality re forgiving of under fill · Do not mix concentrations for reference range, etc. · Binds calcium preventing clotting · Closer to plasma osmolality Assay Sodium Citrate EDTA Plasma Serum PT (s) NCD Effect of incorrect anticoagulant may be "obvious" or aPTT (s) "subtle" and lead to an erroneous result. Thrombin Time Fibrinogen (mg/dL) Factor VIII (%) Factor V (%) 78 108 some factors Factor level loss vs. activation events. Heparin contamination prolongs aPTT, TT and decreases Factor levels & Fib Protein C (%) Chromogenic AT activity (%) Effect of Incorrect Anticoagulant EDTA sample gives abnormal vWF:RCo results = Dx Type 2 VWD EDTA increases PT, aPTT and decreases FV and FVIII, false weak LA . Serum sample can give "normal" results for vWF.Ag and Partial clot in tube: increase or decrease based on Fib / Clotted Samples Clotted samples must be rejected Plasma (contains fibrinogen) White blood cells and Buffy coat platelets Red blood cells Prolonged PT/aPTT/TT ... If fibrinogen consumed by clot Shortened aPTT ... If sample activated but fibrinogen still present Elevated D-dimer Frequency in large laboratory lab si lab size: 80-100 per month 0.4-0.7% of samples clotted (Minus fibrinogen) Clot (Blood cells in fibrin clot) Outside the Lab: Transportation conditions of whole blood samples can have an impact on the final result Laboratory Temperature: Time Transportation System: Racked and positioned upright 4 hrs according to CLSI Guideline Minimal agitation Pneumatic transport systems should not be used for samples requiring platelet-function testing