Webinaire sur l'introduction à l'hémostase

Today we're going to be talking about some hemostasis learning essentials and we're really going to be focusing in on introduction to hemostasis testing. My name is Doctor John Mezios and I'm your be presenter today. So before we talk about hemostasis and before going to the meat of the presentation, I always kind of want to start off with some learning objectives. So today we're going to be talking about the different stages of coagulation. We're going to describe some of the most commonly ordered assays in hemostasis and we're going to describe the clinical indications for a hemostasis assessment. So before we talk about the testing itself, it's kind of I always like to read baseline everybody before we go into that kind of the real, the points of the the particular presentation. So when we look at hemostasis, we really look at it. What does the word hemostasis actually mean? It's actually derived from 2 Greek words, one which is mean heme or blood and the other is status or stasis, which means standing or stoppage. So we look at hemostasis. Hemostasis really refers to the cessation of bleeding or the stopping of bleeding. Hemostasis also is synonymous of the word coagulation. And so throughout this presentation, I'm be kind of be using the words hemostasis and coagulation interchangeably. So before we kind of again talk about this, the testing specifically, there are really three normal stages of coagulation, right. So the first stage is primary hemostasis. In this particular case, we have the damage to the endothelial layer where we have the exposure of various sub endothelial matrix components like Von, Filibrand and Collagen which results in the adhesion and aggregation of plates to the area of injury. Secondary hemostasis is really the activation of our clotting factors in the subsequent formation of fibrin. And then finally we have tertiary hemostasis where we have fibrin cross linking in the subsequent fibrin analysis where we have the regulation let's say of that thrombin or the clot to to regulate its size. So this is not to go overboard. So the three stages of coagulation are primary hemostasis, secondary and tertiary hemostasis. So from an in, in vivo perspective, what we look as that first step is that adhesion right when we talk about primary hemostasis. So we have an injury to the endothelial layer, the exposure of various bio active compounds like Von, Filibrand and collagen all of which recruit platelets to the area of injury primarily mediated between the platelet glycoprotein 2B von filiband and collagen allowing that those plates to be recruited. And remember filiband it plays an important role as it's as it's exposed it kind of expands and grows larger in size and is able to recruit platelets in the area of injury. That second step is really the activation of our plates, right. So once they've been recruited to the area of injury, they undergo A conformational change. They go from the kind of a discoid shape to that of a kind of more of a philopodial expansion, which also allows the conformational change of glycoprotein 2B3A, which is the most common glycoprotein final platelets and it's also known as integrin alpha 2B. Beta three, as it goes from undergoes this conformational change from an inactive to an active state. It allows now the formation of fibrinogen bridges between adjacent plates. So you have your initial layer of platelets that are on the the the damaged vascular wall. Then you have recruitment of additional platelets that are kind of piling on to that by formation of these fibrinogen bridges and the subsequent formation of a thrombus. And then of course of lesser points. But of course still important is the association of glycoprotein 1B and its ability to bind to 1 villa brand. So now you have from an in vivo primary hemostatic response this initial interaction which we have that kind of the the working foundation of a thrombus that's essentially formed now. The importance of the secondary hemostasis now is really the coagulation cascade and we look at the coagulation cascade, many people are a little bit overwhelmed, this pretty good thing. You have the extrinsic pathway and the intrinsic pathway and I like to kind of first use this more simplified version. Both the extrinsic and intrinsic pathway feed into the common pathway, which is the activation of throbbing and the subsequent formation of a clot, right, and then that formation of these fiber and fibros. So this is done in tandem with your primary hemostasis, right, because the surface of the platelets are conducive or provide the appropriate surface in this particular case for these coagulation proteins to be activated. And for example, now we have kind of a regulation of that kind of that tertiary hemostasis where we have plasma engine that's converted to plasma and via TPA and this is where we have fibranalysis. And then we have fiber degradation products and probably one of the most well known markers of coagulation in D diamond. In addition, we have other markers like for example F 1 + 2 which is another marker of thrombus, but specifically not focusing on the forbidden olytics pathway from really focusing on the formation of prothrombin to thrombin. And we have natural anticoagulants like antithrombin and protein C and protein nests that regulate or balance the coagulation. And of course we have Pi one which regulates the activation of tissue plasminogen activator. So it inhibits the fibrinolytic process or high values of Pi one can result in fibrinolysis shutdown similar to what we saw in patients who had COVID-19. So from a kind of a molecular level, this is kind of the, the, the interplay of how everything kind of works together to really form a clock. Here you see the formation of A the recruitments of platelets, the plates undergoing shape change and the subsequent activation of the different coagulation proteins via different pathways. When the intrinsic extrinsic pathway we have it activation of tissues tissue factor to that of to factor 7, the formation of thrombin and then thrombin basically feeding back into and activating the other aspects of the intrinsic pathway via factors 8-9 and 11:00. So now can take we look at the simplified the complicated version and now let's get a schematic version of where everything essentially has to play. Here we have a kind of an again the coagulation cascade. In general, you'll see the extrinsic pathway that feeds into the common pathway where we have tissue factor plus factor 7. And of course we have the intrinsic pathway which will have fractures 8911 feeding back into 1010A and then subsequent down to the formation of fibrin into fibrin. And of course, once we have a stable fibrin clot, we have factor of 13 that kind of comes in and really forms that nice cohesive glue, let's put it this way, that forms that stable clot. And then we have subsequent fibrinolysis, right. So there's this regulation that maintains this balance between both bleeding and that of coagular thrombosis. So when we look at the intrinsic pathway, specifically focusing in on the intrinsic pathway, we'll see here there's a very complicated diagram that looks at kind of where I have all these coagulation proteins play an important role, right. So we have upstream of factors 11 and factors nine. We have high molecular kinetogen for example, we have precalicon, we have factor 12, all of which playing a role, but physiologically don't seem to add a higher risk. If they are deficient in bleeding for example, well, when we look at the coagulation cascade, we have the factors 11 feeds into nine. Factor 8 comes off and activates, all of which activating factors 10 to 10A and the subsequent formation of of thrombin, activation of Pro Thrombin to thrombin and the formation of fibrin into fiber fibrin. Now we also have another marker of coagulation, very similar to D dimer, but it's really what's called is referred to as this Pro Thrombin activator complex that cleaves the Pro Thrombin factor 2, the form thrombin or factor 2A and the Prothrombin fragment 1 + 2. Now the Prothrombin 1 fragment 1 + 2 as another marker of coagulation, right very similar to that of D dimer, but it's actually upstream of that final product and this is more towards in that common pathway where it can actually have an elevation of F 1 + 2. Remember the common path, the intrinsic factor, it feeds into the common pathway. When we look at the extrinsic pathway, now this is where we have any type of endothelial release, any type of damage, right. We have that endothelial damage. We have the exposure of those sub endothelial matrix proteins like Collagen and Volvo brand. But we also have the release of tissue factor that tissue pregnant subsequently activates or combines with factor 7 to kind of feed down and activate factor 10 to 10/8 and then the subsequent activation of your common pathway which leads to the fibrinogen going to fibrin. So but it's really important that that generation of throbbing actually feeds back into the intrinsic pathway to drive the intrinsic pathway again. So kind of you see how it feeds back into and kind of enhances the coagulation process. So when we look at specific that thrombosis, you know doctor Verchad developed this virtual triad which really focuses on three primary aspects, right look at endothelial dysfunction, hypercoagulability and the stasis of blood flow. Those these three components play an important role in thrombosis or thrombotic development. Specifically when we look at endothelial dysfunction, for example, if is there any trauma or surgery that happens, anything that would cause any damage to the vascular bad, venous puncture, atherosclerosis, all of which can cause some type of degree of damage. Hypercoagulability can be seen in patients who have cancer sepsis, patients who have a thrombophilia assessment or have an inflammatory condition and of course venous stasis, where we talk about atrial fibrillation as an example, immobility or paralysis and of course venous obstruction, something that results in a an obstruction such as a tumor or obesity. And we talk about how there's as as a nice balancing act right from when we look at an healthy individual, you have a balance between both thrombosis and fibanalysis. But what happens if the scales are tipped in favor of fibrino fibrinolysis? Therefore we have a higher bleeding risk. If the scales are tipped in the opposite direction where it enhances or supports that of thrombosis, there's a higher thrombotic risk. So can properly maintained that balance is extremely important to ensure that we don't have thrombosis or we don't have fibrinolysis or bleeding. Here are some just common disease states that we can see that causes a disruption or disbalance coagulation system. For example patients who have hemophiliate A or B, they're a higher risk for bleeding. This is a hereditary industries with a strong bleeding tendency and will require lifelong medication and care. And of course thankfully we've gotten better at treating these patients and I thought because it's excellent processive, it primarily effects males. Now females can be carriers and that will have depressed or low values of factor eight. However they're not won't you won't see as low values that you would see in males for example of less than 1%. Von filibrand disease is probably one of the most common bleeding disorders and it can range from mild that's that we see in type 1 to that of severe that we see in types two or type 3. Peri post surgical bleeding assessments when we're looking at either pre surgical we're looking at making sure that patient is has a normal coagulation profile to see whether or not they need to have either products available or are they at risk for thrombosis due to some type of hereditary deficiency. And of course, there's just a number of reasons why you would obviously want obviously posed surgery. You want to make sure that if the patient is on anticoagulant medication, the property anticoagulating from a thrombotic risk. We talked about patients who developed venous thromboembolisms or the most common are deep venous thrombosis or DVTS. And of course pulmonary embolisms for PE patients have stroke that is a Thromps that blocks the arterial blood flow to the brain can develop these thrombotic events. And of course patients who have a heart attack can be the result of thrombosis in the coronary arteries of the heart resulting in a thrombotic thrombosis. So some of the common risk factors that we see as it relates to the development of thrombosis of course these are critically I'll patients that are higher risk for developing clots. So for example a family history having some strong family history consistent with some type of A deficiency can resiled in thrombophilia and enhanced robotic risks pregnancy it's as it's known to be inflammatory condition because of the the change the body is undergoing. These patients may be at higher risk if they have a concomitant hereditary risk factor, prolonged immobility, diabetes, cardiovascular disease, severe inflammatory conditions like we saw in patients who have COVID-19, prolonged hospital stay and surgery all can enhance the risk of of thrombosis if there is a genetic, for example, predisposition to some type of thrombophilia. So what are the kind of the purposes of diagnosing or the laboratory evaluation of of hemostatic diagnosis or hemosthasis? Well, that's really threefold. We're really looking at the assessing the risk of the patient. So is the patient at risk for bleeding or thrombosis. So for example, prior to surgery, you would do a coagulation assessment to see if the patient has a normal hemostasis profile. And depending on the results, it leads either yes, we can go under go, we can safely have the patient undergo surgery or we need to do further evaluation because there is a suspected thrombophilia thrombophilia associated with it. It also is to monitor the progress of the patient. How is the hemostasis disorder progressing? You know, for example, if there's a a risk for thrombosis, right, you know, do we need to treat these patients with anticoagulation, Is it getting worse? Good example is lupus anticoagulant where you typically want to monitor your patients based on a therapy that's being given to see whether or not this inhibitor is being decreased. And so therefore there's less of effect on your coagulation, adjust and monitor patients that are on therapy, right? Are we using the correct dose? For example, patients that are on warfarin, we talk, we'll talk about the clinical utility of the PTI in our system, but you would use that to monitor these patients to make sure that they're within the therapeutic range. Deluxe for example. Again, if you're looking to monitor patients that are on anticoagulation, these direct oral anticoagulants or these new like Rubberox Van A pixaband A bigotran can be used to assess whether or not the patient has drug on board or it can be used more specific assays to quantify the anticoagulant. When we look kind of look at the four basic coagulation tests, we have four of them which are Pro Thrombin time which is a functional test of both of the extrinsic and common pathway and we like the activated partial problem plus and time or the APT. This is a functional test of the intrinsic and common pathway. I apologize for the the typo there. Thrombin time is a functional test of the common pathway and of course we have determination of fibrinogen concentration, which is a functional or quantitative measurement and depending on the methodology of fibrinogen. So if you're looking at the functional, you're looking at how is it able to do its function in this particular case acting kind of as the glue and of course looking at the quantitative, how much of the fibrinogen is present. So let's try it kind of take our first step into the prothrombin time or the PT and here really focusing in on tissue damage and the association with factor 7 and really feeding into the common pathway. In the conversion of fibrinogen to fibrin here typically you'd use some type of thromboplast or tissue factor and then the addition of calcium and that stimulates and activates that common or the proton of the extrinsic and into the common pathway. Now there's also different different type of clock detection systems. We have a mechanical call detection system as highlighted here in the cartoon representation, where we can have a little magnetic to a ball that goes back and forth and as the fibrance strands begin to form and they form a nice cohesive lattice work, that magnetic ball stops moving back and forth and therefore you get your clotting time at seconds most often. Most common is the photo optical, again using optical sensors to look at the decrease of light transmits or the increase of light transmits depending on the methodology, which basic results in the clock time in seconds. And of course more different types of assets would use this kind of viscoelastic type of testing where now you're looking at track tracking plus the numeric result. And here you would look at not only the formation of a clot, but you'd also look at for analysis specifically, for example, cold blood. The other two, mechanical and photo optical most often would be done in your your your patient's plasma. So what are the some of the clinical conditions that can result in your prolonged PT? For example, this is where you would have a decreased function or deficient amount of proteins. For example your vitamin K dependent factors like your factors 210 seven for example may be affected because either of as a result of medication. Most common would be warfarin or due to some type of liver disease. But this is what you would see your post common increase for prolonged PT. So some common causes, liver disease, for example decay and clotting fracture production or vitamin K you know, malabsorption for example could result in your prolonged PT patients who are in vitamin K antagonists like warfarin which blocks the carboxylation and the formation of some of these vitamin K dependent factors will result in a prolonged PT. Del X, in particular the vitamin, the direct factor 10A inhibitors such as rivaroxaman, apiximan and doximan will result in a prolonged PT. As well, patients who are undergoing fracture consumption of some type of Coagulopathy like the the seminary Intravascular coagulation or DIC will result in a prolonged PT. And again, if you have high fibrin or fibrin degradation products, which subsequently inhibits the coagulation which occurs in DIC, you'll see I think these are prolonged PT. Now patients who are on heparin, probably you won't get a prolonged PT. So those patients are much more sensitive, usually monitored using PTT and it's more sensitive to heparin. Patients who are on lupus who have a suspected lupus anticoagulant or lupus inhibitor of APPT is a better test for lupus and lupus anticoagulant. And we we can certainly talk about the diagnosis of lupus inhibitor I think in other presentations in an attempt to kind of standardize patients who are on orphan for example, because you can have different reagent variables from different vendors. What can happen is, is that it would make it challenging to have one patient monitor their PT at hospital A and then if they have to go to hospital B. So this kind of normalization of the PT was implemented by kind of using this formula it's indicated here in the slide where you take your patients per time divided by by the the mean geometric normal of a a normal pool plasma for PTS and they take into this ISI. So the ISI is really the international sensitivity index of the thromboplasm that's being used. And so it's a, it's compared to that of a who's standard and what you're essentially doing is you're kind of normalizing the data. So if patient has, if a patient has to go to a different hospital and they are work on warfarin, you can kind of get a same similar values between different hospitals by using this particular index or this particular formula. And the ISI is generally between 1:00 and two. And for example, the closer you are the one more likely that you're using some type of human tissue factor reagents and the higher it could be indicated you're using different type of reagents like for example rapid tissue most often the most often time the closer you are to one the better is for your per INR assistance. So how you know how is this PTI and are used? Again, it's really to assess whether or not the patients within the target or therapeutic range. So for example, if the target is 2.3 to 3.5, especially if a patient is out of mechanical heart valve being able to monitor that and assess that to either titrate down the warfarin dose or increase the dose depending on on the clinical situation and where the patient falls. Again it's really for standardization, normalization. So we can even actually have the ability to monitor patients globally by using this iron out the apartment for example. Now remember we talked about that is the intrinsic feeding into the common pathway we're here this plays an important role or as in PT we had you know tissue factor here we need phospholipids. So phospholipids are used, different type of activators are introduced, can hear elagic acid for example, KAOL and silicon can be used that drive the intrinsic into the common pathway again being able to give us a clot time in seconds. And this is just a nice little schematic that we can see here that we're utilizing those fractures 8911 that feed into the common pathway. Again where do we see a prolonged PPT. Clearly anything within the intrinsic pathway could result any prolongation. Clinically significant deficiencies or deficiency would be factors 8-9 and 1111. Also known as chemophilia C is autosomal recessive. Patients who have hemophilia B that is a deficiency in factor 9 which is an exit recessive. Also vitamin K dependent can cause a prolonged PTT and of course hemophilia A or deficiencies in factory. Again exit recessive would add severely affected males, but females can also be carriers. But remember that factor 8 is also bound to von Filiban factor because von Filiban is a carrier or a molecular bus or factor 8. So there's a lot of interplay here and being able to really truly assess these patients a different diagnostic tests that you would need but focusing on the PTT you would see either a deficiencies in one of these proteins. For example, factor 12 doesn't play a significant role in vivo, but if there is a deficiency, you can also get a prolonged PTT. Now if you go through your further work UPS, you'll see that if there's not deficiency 8911 but a factor 12, most likely this is not going to cause a risk for bleeding but again be doing a complete work. But we're just focusing here on the PTT itself. So some factors that can result in prolonged PTT Hepburn for example, we know that can prolong your PTT specific that are unfractionate Hepburn and could potentially be used to dose patients if you're using a heparin response curve. Again there are newer acids that are available at the anti Tana that's typically would be used. However again PPT can be used patients who are on doacs. I mentioned before that patients who are on your direct from anti Tana inhibitors, they can actually result in a prolongation in your PDT. On the other end here, patients who are on to big a trainer direct thrombin inhibitor can prolong your PDT as well. And of course if patients are undergoing some type of consumptive Coagulopathy like DAC can also get a prolonged PTT for patients who have a lupus and heparin, in this case a lupus anticoagulant. Again your PTT can be a better test to pick up a prolongation. Now there are some rare cases in which you get a prolonged PTT and a prolonged PT, significantly prolonged PT, and that would be for a hypoprothrombinemia which you have an antibody against. Though rather than having a risk for thrombosis, you're actually at risk for bleeding in those very, very rare synthesis. But again, understanding some of the basic principles of these assays work allows you to make more better assessments to what the next tests will be. And again, as we talked about patients who are on warfarin therapy, most left are not going to be used to monitor PTT. And again, we're going to be using the PTI INR method because it's a much more sensitive test to monitor patients on warfarin therapy, so kind of transition. So we talked about how we assess the extrinsic pathway, how we assess the intrinsic pathway, well, how do we assess the common pathway and that's typically done by a thrombin time. Again, this is where we add thrombin and calcium and it really stimulates and activates fiber into fiber into form a clot. Again, this is kind of a schematic representation of what's essentially happening. But just imagine here now we're adding the throbbing, we're causing the activation, we're causing the activation of hibernation which results in the formation of a clot. So and what clinical situations would we see prolonged thrombin time of course in patients, fibroids and deficiency for example a dysfunction of a fibrointasin a low concentration that is a quantitative, we'll see a prolonged thromatine heparin contamination where we'd see a prolongation in in thromatine patients who are on a direct Robin inhibitor like the bigger trend clearly would see a prolongation if the patient develops thrombin antibodies that is the if at some point they were exposed let's say to bovine thrombin and they develop antibodies again very rare. And of course if patients are undergoing some type of disseminate intravascular coagulation or some type of Coagulopathy, you can also see an increase in your thrombin time. Now when we look at fibrinogen specifically, we talked about the importance of fibrinogen and here you'll see also a nice little schematic. But what essentially happens when thrombin activates fibrinogen, it causes a cleavage of these fibrino peptides resulting in those the the ability for fibrinogen to kind of form these fibrin fibrils and kind of act as that kind of a molecular glue for lack of a better word of all the coagulation cascade plus primary and secondary hemostasis, right. So as this black glycoprotein is circulating within the blood, once it's activated by thrombin, it results in kind of this cross linking that happens, right. So in addition to that, not only can it facilitate this cross linking, but it can also stop the thrombin enzymatic activity. So also kind of regulates the thrombin activity. So it kind of acts as kind of a negative feedback loop. So remember, it's all about this balance of the hemostasis system, right? So it's really important to balance the hemostasis system and this is what also Fibragen does. So how is Fibragen measured? Well, there are really 4 assays. The cost method, however, is probably considered one of the most widely used methods and it's a functional assay that's based upon the time for fibrin clot formation. You have APT derived Fibragen assay, you have an immunological acid which assesses the antigen concentration. So this is really more the quantitative versus Cos and the PT fibrin as a qualitative. And of course you have the gravimetric which is a method based upon clot weight not typically used in clot in routine clinical practice. Like I said before, the cost method of fibrinogen determination is considered the most common method. It is a functional assay. So therefore it's a qualitative assay and it really measures how well the fibrinogen is functioning and therefore you you're understanding whether or not you have a normal functioning fibrinogen. So for example, it's a thrombin kind of based assay. You add it and you base it on a calibration curve, which we look at your fibrinogen concentration and here depending on where your patient sample you read it off, it falls read it off your calibration curve to define how much the activity of your fibrinogen. In this particular case, the PT divided fibration curve is really using your routine PT that is performed on a patient's platelet poor plasma and then you you're using a calibration curve again to look at your where your patient's sample falls or patient concentration falls. Now this is a simple and inexpensive test and it is widely used. However, the test can give misleading results in some clinical disorders. It's typically not recommended for that routine laboratory use, whereas the class method is in fact represented or recommended. So what happens if we have an abnormal fibrinogen? Well, of course we can have congenital disorders like a fibrogen emia, hypofibrogenemia or disfibrogenemia for example. Now the importance of having these different types of fibrogene acids is important because not only we have to look at a qualitative assessment, we also have to look at a quantitative assessment, right. How much of the forbidden do we actually have? So in the previous slides I mentioned of an immunological, right, looking at the antigen. So for example, if we're worried about a disfibrogenemia, that is something's wrong with my furbogen molecule because it's not giving me it's not functioning correctly. Am I making enough? So that's where we have the difference in diagnosing of a hypo versus a dystrobogenemia, right? And that's pretty good case using not only our functional assay, our Klaus method, but also our quantitative method, you can just find whether or not am I, am I making enough fibrogen, but for whatever reason there's some molecular mutation or mutation within the fibrogen molecule that's not functioning. So I would expect any this fibrogenemia to have a low function but a normal quantity, right, because I'm making enough. But in hypo fibrogenemia, I would expect to have both decreased. So that's when it's important to know the different types of assays that are available for clinical use acquired disorders. You can have both hypo and disfibrogen that are acquired as a result of either liver disease consumption and so on. And of course we also know that fibrogen is a positive IQ phase reactant, right similar to that factory in fact in von Vilibram. So there would be elevated in inflammatory conditions. So it patients who have pregnancy, there is a pro coagulant state and of course there's a higher risk for thrombosis if they have a concomitant genetic mutation or a hereditary dysfunction abnormality like anti thrombin deficiency or something along those lines. Again understanding and putting everything together in proper clinical context is important. Again we kind of we talked about you know PTS, PTS fibrinogen, thrombin times but what happens is what how do we measure that fibrinolytic process, right. Really we're looking at the formation of D dimer. So we talked about the activation of thrombin, it cleaves the fibrino peptides allowing the the fibrin to form these monomers. Then we have factor of 13 that comes in, it facilitates the polymerization that is the formation of these kind of complexes between the D domains and then you have this cross-linked fibrin. So in the event of fibrinolysis what do we have, what happens is we have that these formation of fibrin and degradation products. Now there could be many, many fibrin degradation products, but we talked about the importance of measuring D dimer right, which is one of those the, the, the components that we use to assess whether or not the patient has undergone our sulfurethral boses. In this particular case when we look at what our instruments and what are reagents analyzed, we were looking at this DDE fragment that's indicated by the green check mark in addition to the other fibrin degradation products that can happen. What we focusing on and it's specific is this YD or DDE fragment that we would see and that's what we are reagent antibodies target and are able to pick up and give us a D dimer concentration. So some of the clinical, sorry, when we look at where is D dimer elevated in normal conditions, right, because of DDM are having a higher half life around 8 hours, it saves much, much longer compared to the other ones that we can be is like a thrombin, anti thrombin complex or the Protrom and F 1 + 2 for example, if the patients have recently undergone let's say a heavy bout of exercise either weight lifting, running, you could also see slightly elevations in your D dimer. But of course the importance of D dimer and that is not only where it can be elevated normal conditions, but where is it pathologically elevated. And of course as you talked about D dimer is important as a marker of thrombosis. So in this particular case, what we're talking about when we have D dimer being elevated, it can be in a number of pathologic conditions such as patients who have DIIC, patients who are have DBTRP that's forming. So there's a number of clinical situations that can result. And basically this kind of slide highlights some of the clinical and more specific applications where you would see an elevation in D timer, right. So we talked about Afib, they're for artery disease, Cath, coronary or atherosclerosis, strokes, cancer, these are all can result in elevation Z dimer something that's causing that hypercoagulability that we talked about when we talked about in the context of purchase triad. But we also know the importance of D dimer when talked about DVT to PE exclusion right when we talked about excluding A venous non bibolic event. So for example, we know that D dimer has a very good and negative predictive value. That is, it's supposed to exclude the presence of a disease. So in this particular context, when we have a normal D dimer but we still have the patient experiencing chest pain, for example, that indicates most likely that that's not the result of a pulmonary embolism, but that could be the result of a cardiac dysfunction or in this case a heart attack that's not associated with a thrombosis or it's more associated with a stenosis of a coronary artery. So that's where D dimer plays an important role. So we covered a lot but really some of the take home points are as follows. You know we have hemostasis is is really balancing right. It's about it's a balancing app between both thrombosis and bleeding. Coagulation consists of three primary stages, primary, secondary and tertiary. Hemostasis and coagulation disorders can be inherited as well as acquired. So being able to distinguish those based on the temper testing that's available and we only talked about some basic tests today. Of course as we go through these, well we can we'll talk about more, more more specific testing. But again understanding some of the basic principles and what these tests actually have to do is really important. So some final thoughts, you know what is real purpose of hemostasis, right, the diagnosed the diagnostic testing process, right, it's to assess the risk of the patient, are they at higher risk for bleeding or thrombosis to monitor patients of how their, you know disease status is progressing to adjust and monitor that of of anticoagulant therapy. PTI you know is a good example for warfarin testing or and some of the Kia said the parameters that we kind of talked about today are PTPTD, fibrinogen, thrombotime, MF course. Briefly we touched on D dimer. But remember at the end of the day it's really important the laboratory has effective communication with that with the clinical staff because we're really in this line of business to enhance and improve patient care. And with that I'll be more than happy to take any questions. Thank you for your attention.

10 11 12 13 14 15 16 17 18 19 20 21 22 23 2-3 2.3-3.5 24 25 26 27 28 29 30 31 90 80 70 60 32 33 34 35 36 37 38 39 Hemostasis Learning Institute presents Introduction to Hemostasis John V. Mitsios, Ph.D. Powered by experts Mitsios, John Learning Objectives SIEMENS . Unrestricted O Siemens Healthineers, 2022 Healthineers What is Hemostasis Describe the different stages of coagulation Describe the most commonly ordered assays in hemostasis Describe the clinical indications for a hemostasis assessment Hemostasis - What Is It? Hemostasis is the mechanism that ensures that blood in an intact circulatory system remains fluid, but coagulates spontaneously in response to injury. Hemostasis = cessation of bleeding (Greek) Haem = blood Statis = standing, stoppage Synonym: Coagulation What Are The Normal Stages of Coagulation? Primary Hemostasis Adhesion and aggregation Modified from Bhat R et al. Hematol Oncol Clin North Am. 2017;31(6):1105-1122. Mitchell RN. Hemodynamic disorders, thromboembolic disease, and shock. In: Perkins JA, editor. Robbins and Cotran pathologic basis of disease. 8th Edition. Philadelphia: Elsevier; 2010; Secondary Hemostasis Activation of clotting factors and fibrin formation Tertiary Hemostasis Fibrin cross linking & fibrinolysis In-vivo hemostasis Platelet adhesion: high shear stress: requires vWF Platelet Platelet (monolayer) vWF Endothelial injury Collagen Platelet activation: shape change Granule secretion ADP, TXA2 fibrinogen Thrombus development Of lesser importance in platelet aggregation: GPIb - binds vWF Secondary Hemostasis simplified F1+2: Prothrombin fragments 1 and 2 TF: tissue factor TFPI: tissue factor pathway inhibitor FXII: coagulation factor XII PAI: plasminogen activator inhibitor tPA: tissue plasminogen activator Extrinsic pathway Initiator: tissue factor (TF) surface + FXII Thrombin Clot formation Fibrin fibrils tPA Plasmin Plasminogen Fibrinolysis Fibrin degradation products (FDP) D-dimer TFPI Antithrombin Protein C/Protein S F1+2 PAI Molecular level Fibrinogen Fibrin Prothrombin Xa Va Xa IX uPA XI Fibrin Clot FDP VII TF Coagulation cascade Surface Contact IXa Tissue damage Extrinsic Common Intrinsic Inactive factor Active factor PL Platelet phospholipid Vit. K-dependent factors Stable Fibrin Clot Ca2+ Ca2 Fibrin Monomer Secondary Hemostasis (Plasma Coagulation System) - Intrinsic Pathway Collagen activates the surface-sensitive factors XII and XI (contact activation). Factor Xlla activates prekallikrein to kallikrein, which, together with HMWK Activating surfaces (high molecular weight kininogen) as a cofactor in an amplification loop, When HMWK is present, FXlla activates additional FXI to FXIa, . which activates FIX to FIXa in the presence of calcium ions. · FIXa activates FX to FXa. This activation is accelerated 200,000-fold by FVIlla (cofactor). FXa forms a complex (prothrombin activator complex) together with FVa (cofactor), calcium ions and phospholipids. Like FVIlla, FVa has no enzyme complex activity, but instead serves to speed up reactions by a factor of 100,000. This prothrombin activator complex then cleaves prothrombin (factor II) to form thrombin (Flla) and the prothrombin fragment 1+2 (F1+2) activator · Common pathway of intrinsic and extrinsic pathways Activation within minutes Subendothelial collagen Tenase FXII is activated in-vitro by glass, kaolin, celite or ellagic acid. Ca Tissue factor (TF) is released from injured tissue. Together with phospholipids, tissue lactor is formed, which, as a cofactor together with FVIla, then activates FX to FXa > Common pathway of intrinsic and extrinsic pathways What is important here is that a smail amount of FVIla must already be present in order to initrate the coagulation cascade (release of TF). However, as soon as FXa is formed, the pathway itself Is amplifled to convert FXa together with the tissue factor FVII to FVIla. Activation within seconds Endothelial damage Endothelium Virchow's triad Hypercoagulability Endothelial dysfunction Stasis of blood flow Endothelial Dysfunction Trauma or surgery Malignancy Venipuncture Sepsis Atherosclerosis Thrombophilia Inflammation Venous Stasis Atrial Fibrillation Venous Obstruction (e.g ., tumors, obesity) Immobility or paralysis Hemostasis is a Balancing Act Healthy Thrombosis Bleeding Risk Thrombosis Risk Diseases related to disbalanced coagulation Bleeding Hemophilia A or B Hereditary disease with a strong bleeding tendency Requires life-long medication and care Von Willebrand Disease (VWD) Most common bleeding disorder ranging from mild (type 1) to severe (type 2s, type 3) Peri- or post-surgery bleeding Due to medication such as anticoagulants or anti- platelet drugs Numerous other reasons, hereditary or acquired Venous Thromboenbolism Deep Vein Thrombosis (DVT) Pulmonary Embolism (PE) Stroke Thrombus blocking arterial blood flow to the brain Heart attack Thrombus in the coronary arteries of the heart Common risk factors for the development of thrombosis Due to a number of factors, critically ill patients are at high risk of developing clots. Several other comorbidities and triggers increase a patient's risk, including: Diabetes Immobility Cardiovascular disease Estrogen-based Severe therapy Inflammatory conditions Pregnancy Hospital stay Family history Surgery Purpose of Hemostasis Diagnostics Assess Risk Is there a risk of hemorrhaging or thrombosis, for example prior to surgery? Monitor Progress How is the hemostasis disorder progressing? Adjust and Monitor the Therapy Correct dosage of medications? Examples: phenprocoumon (Marcumar®, Falithrom®), heparin, acetylsalicylic acid (e.g. aspirin), DOACs (rivaroxaban, apixaban, dabigatran, etc.) The four Basic Coagulation Tests Test Abbreviation Comment Prothrombin time PT Functional test of extrinsic and common pathway Activated partial thromboplastin time APTT Thrombin time TT Functional test of the common pathway Fibrinogen Concentration Fib Functional or quantitative measurement (depending on the methodology) Prothrombin Time (PT) Fibrinogen -> Fibrin Tissue factor (thromboplastin) Ca2+ TF + VIIA Xa + VA Clot Clot Detection Systems Mechanical Magnet Clotting time (secs) Photo-optical Optical Sensor Viscoelastic Tracking + numeric result Increased PT: deficient function or concentration or inhibition of: vit K dependent Increased PT Liver disease Decreased clotting factor production/potential vit K malabsorption (if severe: aPTT prolonged) Vit. K antagonists Warfarin (Coumadin, blocks carboxylation) DOACs In particular direct Fxa-inhibitors such as Rivaroxaban, apixaban, edoxaban Disseminated Intravascular Coagulation (DIC) Factor consumption Fibrin/fibrinogen degradation products Inhibits coagulation (occursw/ DIC) Note RE: Heparin: aPTT is more sensitive to heparin RE: Lupus anticoagulant (LA): aPTT is a better test for LA Standardization of PT -> International Normalized Ratio (INR) ISI Ptpatient INR = PT (pooled normal) ISI = International Sensitivity Index of the thromboplastin (thromboplastin = TF + PL) ISI - generally between 1 (human tissue factor reagents) and 2 (rabbit tissue factor reagents) Ptp Ptpa How is the PT/INR used? Titrate warfarin dose to achieve a "target" INR Normal PT/INR Target for anticoagulation Mechanical heart valve Reason for anticoagulation Tx of DVT or PE Activated Partial Thromboplastin (aPTT) XII PL. Phospholipids (cephalin) contact activator (e.g. Kaolin, micronized silica or ellagic acid) Ca2+ Xia + IXa Villa + Xa + VA Increased aPTT: deficient function or concentration or inhibition of: def: hemophilia C (AR) ›def: hemophilia B (XLR) vit K dependent VIII def: hemophilia A (XLR) bound by VWF (fibrinogen) Other factors that can increase aPTT Heparin Inhibits Xa and lla (thrombin). Overdose prolonged PT) In particular direct thrombin inhibitors such as Dabigatran Inhibits coagulation (occurs w/ DIC) DIC Lupus anticoagulant (LA) Anti-phospholipid autoantibodies; aPTT is a better test for LA than PT RE: Vit K antagonists: warfarin; PT is a more sensitive test; warfarin overdose: prolonged aPTT Thrombin Time > functional measurement of fibrin generation Thrombin, Activation Monomer Soluble Polymerization Fibrinopeptides A&B E domain D domain Clot Formation Thrombin Time can be increased by: Fibrinogen deficiency Low concentration, dysfunction or inhibition Heparin is administered for anti-coagulation - inactivates lla (thrombin), etc. Dabigatran Direct oral anticoagulant for anticoagulant therapy directly inhibiting active thrombin (Factor lla) Thrombin antibodies May develop following operating room exposure to bovine thrombin Function of Fibrinogen Glycoprotein that circulates in the blood Occlude blood vessels to stop excessive bleeding It crosslinks platelets at the site of injury Is subsequently converted fibrin fibrils forming a three-dimensional mesh occluding the injury Can bind thrombin and abolish its enzymatic activity This regulatory function is important for the balance of the coagulation system Cleavage D Domain Y Chain E Domain Fibrinogeninogen measured? How is fibrinogen measured? Assays Clauss A functional assay based upon the time for fibrin clot formation PT-derived Fibrinogen Assays A derived fibrinogen based upon the prothrombin time An immunological method which measures fibrinogen antigen rather than functional fibrinogen Gravimetric A method based upon clot weight How is fibrinogen measured? letermination (most common method) Clauss method of fibrinogen determination (most common Clauss Technique Functional assay Fibrinogen concentration is inversely proportional to the thrombin time of diluted plasma A reference (standard) curve is prepared using known fibrinogen concentrations (diluted 1:5-1:40) vs the respective thrombin time Fibri Log 50 Sec 40 25 50 75 100 125 150 175 200 Log Fibrinogen, mg/dL Patient Sample PT-derived fibrinogen curve A routine PT is performed on the patient's platelet poor plasma and the Fibrinogen derived from the change in optical density compared to the calibration curve. The derived Fibrinogen is a simple and inexpensive test and is widely used. However, the test can give misleading results in some disorders and is not recommended for routine laboratory use. Vessel Damage [Contact Phase] Extrinsic Pathway Vascular Injury Tissue Factor [TF] Factor X Thrombin [lla] FIBRIN Monomer FIBRIN Polymer Cross-linked FIBRIN Abnormal fibrinogen .... Low functional fibrinogen concentration Congenital disorders Afibrinogenemia Homozygous Def Hypofibrinogenemia Heterozygous Dysfunctional fibrinogen Acquired disorders Liver disease, Consumption (DIC), thombolytic therapy Liver disease, Hepatic malignancy Increase functional fibrinogen concentration Pregnancy, estrogen, Acute phase response Procoagulant state Cross-linked fibrin is built from fibrinogen Fibrinopeptide A Congenital Fibrin monomer Cross-linked fibrin D-dimer is the final product of the plasmin-mediated cleavage of cross-linked fibrin molecules - Fibrinolysis is initiated to degrade the clot: Plasmin cleaves a) fibrinogen, b) b) non cross-linked fibrin, c) soluble fibrin and d) d) cross-linked, soluble or non-soluble fibrin YD YY XY XX XXD Only fibrin polymers that have undergone factor XIII-mediated cross-linking will produce fragments containing covalent bonds between two adjacent D domains (i.e. D-dimer). Therefore, D-dimer (DD/E fragment) should be considered as a specific FDP that is recognized by most of the reagent antibodies used in the laboratory assessment of thrombosis. Favresse J, et al. Crit Rev Clin Lab Sci 2018;55:548-577 c) Clinical samples - D-dimer antigen Normal conditions Pathological conditions - Half-life ca. 8 hours i.e. much longer than - The longer the fibrin degradation is progressing, the the thrombin antithrombin complex (TAT) smaller are the fragments (10-15 min) or F1+2 (1.5 h) Conditions with an interrupted blood flow (e.g. VTE), Clearance by the renal metabolism and tend to produce shorter fragments the reticulo-endothelial system Conditions with a maintained flow (e.g. DIC) are - D-dimer in the patient comprises multiple considered to produce longer fragments: Clinical plasma fragments of different molecular weight from DIC patients contain large amounts of high - Concentration correlates with the total molecular weight crosslinked fibrin derivatives, whereas thrombolytic activity in the body fragment D-dimer represents only a minor fraction of total D-dimer antigen - Approx. 2-3% of fibrinogen is converted into fibrin which instantaneously is entering the fibrinolytic pathway SIEMENS Clinical applications proven or considered for D-dimer testing Disseminated intravascular coagulopathy (DIC) Peripheral artery disease (PAD) Severe infections, e.g. COVID-19 Acute myocardial infarction (AMI) Cerebro-vascular Accidents (CVA) Progressing ischemic stroke Cancer Anticoagulation (AC) therapy Wells PS. J Thromb Haemost 2007;5:41-50 Approved Guideline. CLSI document H59-A. Wayne, PA: Clinical and Laboratory Standards Institute; 2011 Soomro AY, et al. Eur Heart J 2016; 2:175-184 Suzuki K, JTH 2018; 16:1442-4 Cul J, et al. Medicine 2015; 94:4 Abou-Ismail MY, et al. Thromb Res 2020;194:101-115 Atrial fibrillation (AF) VTE Acute aortic dissection (AAD) Coronary atherosclerosis (CAD) Aortic aneurysm (AA) DVT/PE exclusion CLSI. Quantitative D-dimer for the Exclusion of Venous Thromboembolic Disease; Golledge J, et al. Eur Heart J 2011; 32:354-364 Take Home Points Testing Hemostasis is a balance between clotting (thrombosis) and bleeding Coagulation consists of three stages (primary, secondary and Tertiary) A Coagulation disorder can be inherited as well as acquired Final thoughts · Purpose of Hemostasis Diagnostic testing . Assess the risk for bleeding or thrombosis? Monitor: How is the hemostasis disorder progressing? . Adjust and monitor the Therapy Key parameters to assess: . - PT, APTT, Fibrinogen, TT AND D-dimer D-Dimer Q&A Contact information: John V. Mitsios, PhD Email: johnmitsios@siemens-healthineers.com Siemens Healthcare Diagnostics Inc ., 2024 The products and features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed. SLS-23-3102-76