Automation of LTA: Comparison of methods and challenges

Welcome to the Hemostasis Learning Institute, Master Class 2025. My name is Doctor John Mitzios and I'm a Medical Sciences partner at Siemens Health and Ears. I will be your host throughout this course. As a leader in hemostasis testing for more than 40 years, Siemens Health and Ears passionately supports hemostasis research and education, and we are thrilled to have you with us. For this expert LED course about laboratory diagnostics in thrombosis and hemostasis. Our first speaker is Professor Early, Sox. Professor Sox is a consultant in transfusion medicine, laboratory medicine, and thrombosis and hemostasis at the University Hospital in Geeson. His research focus is on primary hemostasis, especially immunoresponses against platelets. He has published more than 120 original papers and currently serves as chairman of the Plated Immunology Working Party of the International Society of Thrombosis and Hemostasis and is Chairman of the Standing Commission on Laboratory Testing of the German Society of Thrombosis and Hemostasis. His presentation today will be on Light Transmission Aggregometry, or LTA, which remains the gold standard method for evaluating plated function disorders. Recent efforts have tried to tackle the main issue affecting LTA, which is a lack of standardization. Professor Sachs will report on recent advances in automation and standardization of LTA, aiming to improve its reliability and comparability. Professor Sachs, the floor is yours. Welcome to this presentation on the automation of light transmission aggregometry. A comparison of methods and challenges. First of all, I would like to remind you what light transmission echogometry is about. This is where we put planted which plasma in a Corvette and this Corvette is usually incubated at 37° and a steel motor with a steel bar that takes care of the plate that's being in solution all the time of the measurement. Then light goes through that Corvette and behind the Corvette is being received and the data are being entered into a calculator so that in the end a recorder will provide us with a transmission and time diagram of which I will speak about in a minute or so. So agonists are then usually being added during this process and these agonists will lead to the clumping of platelets so that at the end of the process once The Agonist has been entered, all the platelets will stick together in one single come currently. Whereas light transmission as long as the platelets are in suspension is minimum. After that, the process of active clumping of the platelets, all the platelets will stick together and a maximum of light, 100% of light will pass through the Corvette. If you look at this curve in a bit more detail, and here we have the time on the X axis and the light transmission in percent on the Y axis, the diagram often looks like this. We have at the beginning quiescent platelets and the light transmission is so to say at zero and then after adding The Agonist light transmission will slightly go below 0 because platelets perform a shape change. And that transmission goes a bit beyond the transmission that is seen was quite simply it's UMM and then the reversible aggregation is being initiated. As a result of reversible aggregation, plated granules are being disgorged by the platelets and after these contents have been released from the granules, ADP will then stimulate irreversible aggregation of the platelets, which is the end of the classical light transmission acrogometry. So what types of agonists are being used to perform light transmission acrogometry? No worries, I'm not going to speak to you about all the details of the table. I just would like to point out that there are several agonists starting here, the first line with epinephrine and going down to in the last line in a 2. And all these agonists are being used to stimulate the platelets in order to make them form an aggregate through several biochemical pathways. And in the end, the response of the platelets to these different agonists will help us understand which pathway exactly in the plate that is being disturbed to come up with a preliminary diagnosis of a plated function disorder. This is an example of a patient that has been investigated by light transmission acrogometry. You can see in green and normal control and then the patient in double red and grey. You can see that out of The Agonist that I have just introduced to you, he did not respond to collagen and he did not respond to ADP and he also did not respond to acridonic acid, but he responded to risk to. And this is an interesting finding because all the other agonists act through receptors on the surface of the platelet and will lead to an active process of platelet clamping. Whereas Ristocetan has a special mode of binding, it binds to activate like a protein 1B alpha and induces a platelet, a plated aggregation process that is not active. It is more like sticking platelets together through this agonist. Then we will come back to this in a second. What types of plated function defects are known in general? You can see it's a very long list. And now, Boris, I'm not going to speak to you about all the different plated function defects. I'm just trying to explain to you how we can differentiate between these different types of disorders by using light transmission aggregometry. So if we just look on the left side of that extensive table and then give all the possible responses that can be seen in plated acrygometry with all the different agonists, so epinephrine, ADP, collagen, arachidonic acid and ristocetin. For each of these rows, a patient may have either and slightly impaired aggregation response which is given in yellow or a more stronger affected plated response which is given in red. And if we go through that, you can see that so after performing all these different tests on different patient samples, we end up with quite distinctive diagnostic patterns. So these patterns will as allow to come up with a preliminary diagnosis. It is not possible in all cases to come up with a diagnosis based on these testings. So in addition to Latin Mission acrogometry to come up with a final diagnosis or most likely diagnosis in these patients, we also need to assess the release of granules with two type alpha granules and delta granules. Not going to speak about that today. And we also and always have to look at the blood smear. The blood fill will tell us something about how the platelets do look like. If you look come in the first line where ARC is being introduced, you see that platelets are pale and that they are large, and sometimes we even observe changes to other blood cells. If you look at the third line, which is the Chidiaki Gashi syndrome, you see WBC, which means white blood cells because there are changes to white blood cells typical for this disorder. So in the end with late transmission acogometry, with the release of alpha and delta granules and with the interpretation of the blood smell, they're usually able to make a differential diagnosis between these different types of platelet function disorders. If you go back to the patient that I have introduced at the beginning with no response to collagen, ADP and arachidonic acid, but a response to Ristocetan, it is now a simple to make a diagnosis because this is a typical reaction pattern of Glandsman thrombosphemia. I'll go back just one slide and point it out to you. If you look at the second last line, which says Glandsman thombsteemia, you can see here that these patients do not respond to epinephrine, ADP, collagen and aerodonic assay. But they do respond to ristocetin, such as what we have seen in the patient that they gave to you as an example, such as what we can see here in these pictures. And, and in fact, then with deeper diagnosis, the patient was sequenced and this patient had a single amino acid substitution in the signal peptide, which prevented the protein that is responsible for the printing of fibrinogen to come up to the surface. So this is the second part of the table with all the different disorders, and I'm just going quickly over those because it's basically the same as in the first part of the table. You can see here different reaction types of all these disorders and light transmission equigometry. You can see that granular release, alpha granules and this case alpha and delta granules together will help making a differential diagnosis. And that again, a look at the size of the platelets and changes to other blood cells and the blood smear is helpful. Again, if you look in the second line from the bottom, which is we've got Eldridge syndrome, you can see that this is usually a disorder with very small platelets or S denoting small. These plant that's usually do not respond to epinephrine and collagen and granular release of both alpha and delta granules is usually affected. So uploaded functional defects relevant in daily routine. And this is a question which is difficult to answer. In this study here, which comes from the Vienna University Clinic and has been performed a couple of years ago, they looked at patients with a mild bleeding disorder. So on the ISTH bleeding assessment tool, the ISTHBAT, that range was between 3:00 and 7:00. You can see that if you assess these patients in up to 75%, you do not come up with a final diagnosis. However, 25% of patients do have a final diagnosis and if we look in the final diagnosis of those patients bleeding mildly plated function disorders are usually responsible for approximately 50% of patients with a diagnosis and they make up 13.9% of all patients being assessed with a mild bleeding disorder. However, in this study, only light transmission equigometry has been performed and you have seen there's a number of plated function defects that come up with a normal light transmission equigometry. So that's a little drawback of that study because granular release has not been tested and also differentiated between what we call here a possible and what we call a confirmed plated function disorder. And they said it was possible if the patient did not respond to 1 agonist at one visit and it was confirmed if the patient did not respond to two agonists and that was repeated during a second visit to the clinic. And this finally leads to flow chart such as this one. And this is taken from the recommendations of the International Society and on Thrombosis and Hemostasis Scientific Subcommittee on Plastic Function Disorders. You can see here that usually after the clinical evaluation in the top part of the diagram, a patient with abnormal history would receive a platelet count and routine coagulation tests. And from development factor screening and the platelet count, whether it be normal or low or it be low at all, it will lead to a potential platelet function disorder diagnosis. And these patients will then finally require initial testing of their platelet functions. So how about automated systems? All I've been speaking about so far was manual aggregometry and I will make a comparison in a minute or so. So how about automated LTA systems on the market? Basically there's three systems at the moment. There is this CS system series which comes from. These are integrated analyzers. They do plasmatic coagulation and they can perform ecrogometry if you put pleasure with plasma instead of citrated plasma on the analyzer and instead trombomate which is a system that only performs Placid aggregation, so no plasmatic coagulation on that analyzer. And the Italica COAG 360 system which will most likely be no longer available from next year on. So what is really the difference between automated and manual light transmission aggregometry systems? I have written down here the workflow which is sampling of the blood, then preparing the plated rich and plated poor plasma, dispensing it, adding The Agonist, detecting light transmission, reporting results and then interpreting results. And as you can see, the second part of the that manual method is only automated for detection of light transmission and and the reporting of the results. And an automated method is not fully automated. It is in addition automated for the dispensing of the platelet rich plasma and the adding of The Agonist. But preparing the platelet rich plasma and interpreting the results is still up to the laboratory and it's not integrated in these analyzers. Why is that so difficult to compare results from manual testing and from analyzers? And is it difficult to compare 1 analyzer to another analyzer? There's a number of aspects that need to be considered in order to answer this question. The first one is there's no gold standard. We do not know what the right response of a given platelet suspension from a patient with a given diagnosis is in general, if it's not a diagnosis that is one of the classical, such as Klansmann's thrombocytopenia, which I have shown to you a couple of minutes ago, where no agonists other than restoccitin can induce plated aggregation. There's no generally accepted protocol for the preparation of plated rich plasma. There's no generally accepted agonist concentration, There's no inter manufacturer control of the potency of agonists and I will speak about that in a minute. That is difficulty to access normal controls. That is healthy donors for a couple of laboratories. So they don't have a normal control and there's in fact very few potential in house positive controls because usually you do not have patients with Klansmann thrombocytopenia, Bruner Soyer syndrome, or those taking aspirin at hand every day when you run your essays. There's no established external quality assessment scheme. Extended internal laboratory, a testing or a comparison may help here. Maximum aggregation, of which we speak a lot if we speak about LTA, is not always the appropriate readout. Sometimes, and for some diagnosis, we also have to look at the exact way the curves look like. We have unclear inter SC variation, so we do not know whether the same patient tested today and tomorrow will lead to the same results for many of these measurements. And finally, we do not have any universal decision values or cut off. We cannot say that a regular plated response to a given agonist on a given machine should be 72120% and that this is a generally accepted goal for the responsible platelet suspension from a healthy person on all analysers with all agonists. Let's speak about imprecision first. Imprecision of manual and automated methods. So what happens if you repeat the same test with the same agonists on the same patient? As you can see here, there are 6 agonists studied here and in an expert laboratory. Let us testing manually on an aggregometer. The imprecision is below 5%, which is an excellent value for a functional essay, and you can see here that the thrombomate on the very right of the table more or less comes up with the same results. The instruments that do perform plasmatic regulation and are also performing automated equigometry are somewhat comparable. However, as you can see for the older instrument series, some of these imprecisions are above 5%, but still in the end they are all below 10%, and below 10% is an acceptable imprecision for a functional cellular essay. The more important is aspect. And please do not be worried about the number of data that are shared here on this file. I will lead you to these data in a second or so. Then do not so often address question. Is the question about the relative potency of the agonists that you use? Most of you will most likely think that if you order arachadonic acid from one company or the other, because it's a chemically defined substance, this arachadonic acid will lead to the same responses of platelets. But this is not the case. And in this paper by Marie Christine Alessi that is now printed in the Journal of Thrombosis and Hemostasis, a number of laboratories and who were part of that group were allowed to test a number of different agonists in comparison to what was the standard agonist, which was given a relative potency of 1.0. If you look at the left hand panel that says ADP and then just at the red Asterix, you can see here that in comparison to the standard ADP, the chrono log ADP and the bio data ADP had roughly 1.2 fold increased potency. And you can see that also on the graphs on the left hand side that say chrono log and bio data with a red Asterix where you see the comparison between the responses to the standard and to the chrono log of bio data ADP. This means that if you do measure the same patient with different ADP solutions, they may give up to 20% higher equigomity responses. And please keep that in mind because it's responsible when we speak about checking the the resistance to drugs that can be used to block the specific receptance. And also please have a look on the right side of that table. This is arachadonic acid I was speaking about. This is even more interesting in that study. If you just look at the red acid rexes again, they appear in the table and they also appear on the graphs which are related to the table. And we have a relative potency of one for our standard preparation of arachadonic acid. Then you will realize that at the bottom part of the table, the high for the arachadonic acid had less potency, 10% left and the Sigma arachidonic acid had more potency, 40% more. So that means that the same patient compared with-, and Sigma would have a difference of 50%, which for testing, of course, is a lot. And this has to be kept in mind. What are the effects of agonist concentrations and aggregometer brands? In the same paper that came from Marie Christina Lesi and her group, they compared the effect of agonists that are not going to speak about all these comparisons, but just look at that one which popped up in panel D with a little red background. It was found out that 19% of healthy donors used in these essays do not fully respond to ADP 2.5 micromolar. This is interesting because in a minute or so you will see that ADP2 micromolar is a recommended concentration by by some international societies. Which is strange because apparently a high amount of healthy controls will not respond to that dose. And also if you look in in panel C for collagen again it appeared red on that graph. 3% do not fully respond to collagen 2.5 micrograms per milliliter. Interestingly, when all these differences in agonist concentration. And agonist potencies were compared to Agrogamata brands in multifactorial analysis. There was no effect that could be shown for the aggregator brand that would have affected inter individual variability. That means things we were speaking about previously that we thought if you use aggregometer A and I use aggregometer B, does apparently not so much have effects on the test results. But it is the concentration of The Agonist, it's the potency of The Agonist that have most effects on the results that we obtain when we compare healthy donors on different instruments, not the instrument physics itself. So this is a table with recommended agonist concentrations. And again, you can see here The Agonist I have been speaking about. And if you just concentrate maybe on the first line, which is epinephrine given the micro mole per liters. And you can see here International Society of Thrombosis and Hemostasis recommends to start with five, the British Society of Hematology with five and CLSI with Five Below. If you look at ADPISTH recommends to start with two, BSH with 2.5, 19% of healthy people will not respond to 2.5 and most likely they would not respond to 2. So it is to be questions whether this is a clever suggestion. And CLSI starts with five. And then the pluses and minuses indicate that these societies recommend to increase the concentration as a result is abnormal or to decrease a concentration as a result is normal. The studies that I have cited previously in the SALT, the Platinum study and the LSC study have a number of different concentrations. I'm just going to share those with you. Please realise that in the LSS study, we came up with a recommendation for epinephrine of 25 micromyals per litre and for ADP with five micromyals per litre. So whenever a patient has been assessed in the one or the other laboratory, it strictly depends on The Agonist concentrations that we use in order to answer the question whether this patient did receive a normal result or an abnormal result, whether he is indeed having a planted function disorder or not having a planted function disorder. There's only very few data that do a comparison between the Sysmax's and the Siemens systems and the manual method. And one of these comparisons was presented in 2020 by a previous group from Graz. As you can see here in green, if you go for collagen and you do a comparison for collagen 2.2 milligrams per milliliter on the Siemens and Seismics analyzers, there's usually a very good correlation, which is above 88%. But you see that there are differences. So the relations, for example the Siemens Italica analyzer with the CS system from Sysmax was much better than the correlation between Siemens Italica and the correlation with a manual method. A chronolog 700 was used in these studies and again comparable data you can see between the seismic series and the chronolog. Please see that for a number of comparisons, they will say less than 10%. There was more than 10% deviation in the correlation, but with the two analyzers. So what? What can we say at that point of the presentation? What can we say at that point of light transmission, acrogometry in general, we can say that we now have better evidence for appropriate agonist concentrations. And I hope they will soon find their way into the recommendations of international societies. We have good evidence for a low imprecision on all instrument types and we have some evidence for acceptable inter essay variability. But for automated methods we can only say that we have advantages of a walk away system. We have advantages of smaller amounts of planet rich plasma which may these systems may be made more usable for pediatric samples. And we have advantages of shorter turn around times. What we do not have is large multi centre studies for automated methods on healthy individuals, multi centre studies for automated methods on patients with inherited plate function disorders, studies for drug monitoring with appropriate number of individuals and adequate controls. Which makes it difficult to draw any final conclusions on what I have presented so far. So let's turn our view to one more interesting and important question other than detecting plated function disorders and this is can light transmission eclogometry support pharmacote therapeutic decision making? And why would that be of interest? Well, one example is clopidogrel, which is the P2Y12 receptor antagonist. It's been used in patients with etholo thrombotic complications. And as you can see here in this selection of studies published between 2000 and 2006, the rate of resistant patients is between 5% and 44%, meaning that patients that are put on this drug do not respond adequately in relation to the plated function. And that meaning while you put the patient on the drug and you think he's safe now for a new event may not be safe because the drug is not working. And it's a question whether checking the planted function for patients that do receive the drugs that interact with planted function can help in decision making such as finding out that the patient is resistant and should better be put on a different drug. One important trial, and I would keep it very short to discuss this trial, but one important trial that has been done in that field is the sieving trial that was published in Lancet in 2017 and it came out of Munich University Hospital. And what they did in the study is they either put patients with acute coronary syndrome on prazigrel, which is more potent than clopidogrel but has a higher bleeding rate, or they put the patient first on prazigrel for seven days and then on clopidogrel for seven days. And then they did a standard test which was a multiplied essay which has a whole blood essay of plated function and at the decision value of 46 units. That was the cut off. When the patient was well responding to clopidogrel, he was kept on clopidogrel and otherwise if he was not well responding, it was switched back to prazogrel. And what you can see here on the left diagram is the hazard ratio and there's no difference in the hazard ratio with regard to ischemia. So this strategy with detecting patients who do well respond to clopidogrel, they were not inferior to those kept on praziquel, which was an interesting finding at the first finding to study that in a large patient population. Unfortunately, as you can see on the right hand side, it didn't affect bleeding. The bleeding range seem to be different. Statistically they would not. So patients on clopidogrel had bleeding things as often as you'd have patients to learn browser well, so can that be used in an automated system? So we use the platted acridometers that use plasma to do such a comparison essay. Problem is, if you look here at at that curve, and I'm sorry for the red line, the red line should be at ADP5 micromolars below, so not above. But if you look there, you can see that the correlation rates are rather poor. If you look into Sysmax CS 2500 correlation with Kronalox 700, you'll see that the correlation is O .83 7 and this is in comparison to a sharp cut off at A specific where you may be a bit poor to make a proper decision on whether the patient does respond to clopidogrel or not. Unfortunately, in this study here the multiplate essay as a validated reference has not been applied. And also we do not know from that study how high the number of patients that were still highly reactive with their platelets although they were put on has been included in the study. So we're missing these data and we currently cannot say that we can use that transmission echogometry for guiding pharmacal therapeutic decisions. I would like to also mention that if you consider the impact of the reagent and we have talks about the potency of different reagents, this should also be kept in mind. If you look here, this is a different a relative potency difference of one point for the chronal lock reagent on ADP where the Gray box gives you the in house reagent and the white box gives you the comparator. So be aware that if you use a different ADP reagent in the same concentration, it will maybe lead to higher reactivity 30 to ADP and that makes it difficult to allow for comparison. But between different systems also in this comparison of automated method on on on the Sysmics analyzer and the manual method. I just want to point out this aspect here because these authors conclude that in general that such an approach is comparable. But if you see here down here at ADP10 micro bolars, the difference is over the range between the manual method and the automated method. There's a number of dropouts. So these people do respond much better to one method than to the other that that means if they were in such a study of clopidogrel resistance, they would definitely not be picked up as being resistant. And you can see that here from the correlation, which is only 0.706 in this study. And again also this study here that came up in clinical Plaid thrombosis and hemostasis where they were looking on whether clopidogrel treatment could be well picked up by comparing a manual method with an automated method. You can see here that it was quite a significant difference. So quite a number of patients did have significantly different values on the one method in comparison to the other. And this is, as we all know now, most likely not so much an effect of the manual method, but it's may be more likely an effect of the different agonists that are used in these two instruments. So what can we conclude from what I have spoken to you about today? We can say that the automation of light transmission echogometry has the advantage of lower blood volumes and shorter turn around times, which is a clear benefit. We can say that when you use the Sysmex or Siemens Health in the application, you do not need an additional instrument. So it does do the plasmatic testing and the light transmission echogometry and maybe less training is required for medical scientists who run light transmission echogometry because it's partially automated. But however, do not forget that a tricky step, the preparation of platelet bridge plasma is still a manual method. Blood transmission echogometry could be offered more frequently maybe if it's being automated. But however, do not forget that pre analytics remain demanding, so nobody wants to do a platelet function analysis on a blood sample that has been put in a chute in case of suspected plated function defects. The interpretation of findings remains challenging, however, and a better standardization of agonist concentration and of potency is urgently required because otherwise we will never come up with something that is usable between hospitals, between institutions and between laboratories. And with this, I would like to finish my conclusion with saying that in case of drug effects, well justified decision values need to be implemented. Because we do have them for whole blood echogometry, but we don't have them for classical light transmission echogometry, because we want to know whether the effect is adequate. And sometimes we want to know whether such a substance is on board at all. And with this, I would like to thank you very much for your attention.

100 10 1.2 0.5-0.7 50 0.5% 1.9% 6.9% 7.7% 73.3% 2.1 3.7 4.6/4.2 4.8 1.6 3.0 2.2 1.4 3.3 2.3 4.6 2.8 2.6 1.7 4.4 2. 1.20 1.04 1.13 1.15 1.18 1.07 1.11 0.89 1.43 40 150 200 0.25 0.125 1.25 25 0.625 3.125 6.25 100- 2.5 0.5 12. 0.12 1.5 12.5 (2013) (2011) (2008) 1.2-1.5 Hemostasis Learning Institute presents Automation of LTA: Comparison of methods and challenges Prof. Dr. Ulrich Sachs Powered by hemostasis experts V. Mitsios PhD, Medical Sciences Partner Siemens Healthineers John V. Mitsios Light transmission aggregometry (LTA) JLU NEUE WEGE. SEIT 1607 cuvette platelet-rich TUKGM® plasma UNIVERSITATSKUNKUM GIESSEN UND MARBURG and Hemotherapy SIEMENS Healthineers incubator stir motor (600-1,500 rpm) stirrer light source receiver calculator recorder transmission time light transmission (%) quiescent platelets shape change irreversible aggregation ATP ADP Agonist Short form Start dose* Dimension epinephrine EPI umol/l adenosine diphosphate collagen COL ug/ml arachidonic acid ARA ristocetin RIS (high) g/l RIS (low) thrombin receptor TRAP activating peptide thromboxane A2 U46619 receptor agonist *Cattaneo M et al, J Thromb Haemost 2013;11:1183-1189. N control Collagen 100 ug/ml patient Arachidonic acid 2 mmol/l Ristocetin 1,5 g/l What types of PFDs are known? ARC, arthrogryposis renal dysfunction and cholestasis syndrome Medich platelet syndrome BSS, Bernard-Soulier syndrome MYH9-RD, MYH9-related disorders CHS, Chediak-Higashi syndrome P2Y12 defect COX-1, cyclooxygenase-1 deficiency PSD, primary secretion defect cPLA2, cytoplasmic phospholipase A2 deficiency PTS, Paris Trousseau syndrome Filaminopathy PT-VWD, platelet type von Willebrand disease FPD/AML/MDS, familial platelet disorder with propensity to acute myeloid leukemia and QPD, Quebec platelet disorder myelodysplastic syndrome Scott syndrome GATA1, macrothrombocytopenia with SPD, storage pool disease dyserythropoiesis/anemia/beta-thalassemia Stormorken syndrome GPS, grey platelet syndrome VCF, giant platelets and velo-cardio-facial syndrome GT, Glanzmann thrombasthenia WAS, Wiskott-Aldrich syndrome HPS, Hermansky Pudlak syndrome White platelet syndrome EPI ADP COL ARA RIS RIS EP COL ARA ADP COL ARA RIS COL ARA RIS ADP COL ARA EPI ADP ADP COL EPI ADP COL GRA-a ADP 20 umol/l Phylogenetic tree W11R HUMAN MRARPRPRPLWATVLALGALAGVGVGGPNICTTRGVSSCQQCLAVSPMCA Pig -- MRARRLWAAVLVVGALAGVGVGGPNICATRGVSSCQQCLAVSPTCA Bovine Mouse Rat Waxmann Y et al, Thromb Haemost 2022;122:1951-1953. EPI ADP COL ARA GRA What types of PFDs are known? vant in daily routine? Gebhart J et al, Haemophilia 2018;24:405-413. Are platelet function defects relevant in daily routine? n=418 prospective patients with mild bleeding ISBT-BAT: 5 [3-7] 2.6% 0.7% 0.2% Possible PFD Confirmed PFD Low VWF FIX deficiency FXI deficiency Possible PFD+ FXI deficiency Dysfibrinogenaemia BUC platelet function disorder (PFD)* 13.9% (= 52% with a diagnosis) *LTA only 1 agonist / 1 visit = possible 2 agonists, confirmed = confirmed PROBAND NORMAL ABNORMAL Potential platelet VWD DIAGNOSTIC ALGORITHM Flowchart Personal and family history and bleeding score: futher Physical examination: bleeding manitestations studies typical of IPFD renal function; cardiac function; mental redation; facial dysmorphism; eyes; bone; skin Preliminary laboratory investigation DIAGNOSIS LOW Platelet count function disorder Routine coagulation tests Blood clotting defect VWF screening PLATELET FUNCTION GENERATION SEQUENCING Gresele P et al, J Thromb Haemost 2015;13:314-322. No NEXT Automated LTA systems XRA CS-system series (Sysmex)1 Thrombomate (Behnk) Atellica® COAG 360 System2 (Siemens Healthineers) CS2x00i series, Sysmex (sysmex-europe.com); Atellica COAG 360 (siemens-healthineers.com); Thrombomate (behnk.de) 1 Product not available for sale in the U.S. 2 The Atellica COAG 360 System is no longer commercially available as it has been discontinued. The product is no longer supported by the manufacturer, distributor, or any affiliated parties. Automated versus "manual" LTA systems workflow manual method automated method blood sampling manual prepare PRP and PPP dispense PRP and PPP automated add agonist detect light transmission report results interpret results PRP, platelet rich plasma PPP, platelet poor plasma Why is it so difficult to compare? · no gold standard · no generally accepted PRP preparation · no generally accepted agonist concentration(s) · no inter-manufacturer control of agonist potency · difficult access to "N controls" for some laboratories · few potential in-house "P controls" (e.g ., GT, BSS or COX-1) · no external quality assessment established (ILC may help) · maximum aggregation not always appropriate read-out · unclear inter-assay variation · no universal decision values ("cut-off") Imprecision (CV%) of manual and automated methods manual1 CS-51002 ** Thrombomate1 3.4 / 7.6 1Sachs UJ et al, TH Open 2023;22:e56-e64; 2Patel I et al 2015, poster presentation: „Precision and throughput of light transmission aggregometery on a routine coagulation analyser"; 3Ling L-Q, Platelets 2017;28:712-719. 'Product not available for sale in the US. "Product availability varies by country. 6.8 /4.0 5.4 /6.4 4.6 /4.2 3.4 /7.6 6.5 /4.8 5.4 / 6.4 Supplier IH relative potency Biodata Hart Chronolog Helena Sigma Hyphen Alessi M-C et al, J Thromb Haemost, under revision. (C=1.0) Effects of agonist conc. and aggregometer brands Ristocetin Arachidonic acid Maximal intensity (%) mM Collagen Epinephrine TRAP6 19% do not fully respond to ADP 2.5 uM. 3% do not fully respond to collagen 2.5 ug/ml ADI No effect of aggregometer brand on interindividual variability! Agonist concentrations ISTH BSH CLSI mmol/l + 5 +/- 5 + 2.5 + 1.25 +/- 2 + 0.5-1.6 RIS (high) g/l + 1.2-1.5 ≤0.6 + 1 + increase concentration if abnormal; - decrease concentration if normal Cattaneo M et al, J Thromb Haemost 2013;11:1183-1189; Harrison P et al, Br J Haematol 2011;155:30-44; Christie D et al, Clin Lab Standards Inst 2008,38:32; Platton S et al, RPTH 2018;2:778-789; Alessi M-C et al, J Thromb Haemost, under revision. RIS (low) g/l Platton Alessi et al. 1.25, 2.5 +/- 1.25, 2.5 +/- 10 / 5 / 2.5 10/5/2.5 5 10 / 5 / 2.5