Dear George, I am a student and I would like to ask for your help regarding a platelet aggregometry question. If the graph shows no reaction to the agonists ADP, epinephrine and ristocetin what does that mean? Thank you. Maria Carmis.
Hello, Maria, and thank you for your question. If you happen to be using the textbook,Rodak BF, Fritsma GA, Doig K. Hematology Clinical Principles and Applications, 3rd Edition, the answer can be derived from pages 679 to 681.
ADP and epinephrine depend upon intact membrane receptor sites, P2Y1 and P2Y12 for ADP and an α-adrenergic site for epinephrine. They also depend upon functional platelet activation pathways, so a membrane receptor defect or a platelet activation pathway defect, which is also called a secretion defect or aspirin-like disorder can result in no aggregation. You’ll need to compare to arachidonic acid and collagen results to sort out the differences, and ensure the patient has not taken aspirin.
Ristocetin is the agonist used to detect von Willebrand disease, and a negative response is presumptive, but not conclusive for this common bleeding disorder. The ristocetin test is not very sensitive, and most laboratories choose to use the ristocetin cofactor assay or the collagen binding assay to screen for von Willebrand disorder. Von Willebrand disease is not actually a platelet defect, it is a deficiency of the plasma protein, von Willebrand factor, and platelets in von Willebrand disease respond normally to the other agonists. The combination of poor response to ADP, epinephrine, and ristocetin together can only lead to a multiple conclusion. I hope this is helpful. Geo
Sure George, here we go. While platelet aggregation studies
Sure George, here we go. While platelet aggregation studies are not as specific or sensitive to von Willebrand disease, they are very useful in pointing the physician in the right direction.
We use the following concentrations of Risto in our studies: 1.2, 1.0, 0.75 and 0.50 mg/mL. A normal patient will show platelet agglutination for 1.2 and 1.0 mg/mL, variable percent agglutination for 0.75 and minimal agglutination for 0.50 mg/mL.
Generally speaking, a type 2A will show minimal to no agglutination with the 1.2 and 1.0 mg/mL and should be tested using 1.5 mg/mL.
Generally speaking, a type 2B will show agglutination in all four concentrations (1.2, 1.0, 0.75 and 0.50) and should be tested using 0.25mg/mL and saline. Most likely these two additional studies will show agglutination.
Obviously, multimeric analysis is the confirmatory testing method.
Maria states she saw no platelet agglutination for Risto. If she was speaking of the 0.50 mg/mL, then she was seeing exactly what she was supposed to see. If she was speaking of 1.2 mg/mL, then possibly von Willebrand type 2A is in the differential diagnosis.
Herb Crown
[Thank you, Herb, this is extremely helpful, thoughtful and detailed]
Thank you, Herb, for your very helpful answer. It is particu
Thank you, Herb, for your very helpful answer. It is particularly helpful for Maria to be aware of all the potential preanalytical and analytical variables that can affect aggregometry. Of course, aspirin is the problem that comes up the most. Although I mentioned the negative response to ristocetin may indicate von Willebrand disease, I don’t see a clue in Maria’s question that indicates it is a type 2 VWD. Can you elaborate?
Thank you Maria for your question and George for your answer
Thank you Maria for your question and George for your answer. I have some additional insight that may be helpful.
My first reaction when I read this question is there must be some kind of preanalytical testing problem with either the specimen or the patient. Platelet aggregation testing is relatively complex, requiring a significant amount of practice, training and attention to detail in order to be performed correctly. Since you, Maria, indicate you are a student, can I infer that you are performing this test? May I also infer that this platelet aggregation study might be performed in a student lab, or during a clinical rotation. If so, do not be concerned that your results showed no aggregation on this, one of your first attempts. If the specimen was hemolyzed, sat around for an extended period of time or the testing was performed on platelet FREE plasma instead of platelet RICH plasma you could also end up with a lack of platelet aggregation response. The instrument should be “blanked” using platelet free plasma, if platelet rich plasma was used by accident, the instrument would not recognize any platelet aggregation. As well, the platelet rich plasma should have minimal contact with air, as this will cause a change in the pH of the plasma. If your patient represented him(her)self as fasting and was in fact not fasting, this could also result in the aggregometer not being able to detect platelet aggregates, as the lipids would obscure them and read as zero percent aggregation. You should also obtain a complete drug history of the “patient” including prescription, over the counter and herbal remedies.
In addition to the above preanalytic variables, I would want to see additional testing on this “patient” because we have been provided with limited information. I would want to see the results from ADP using 5uM, 3uM and 2uM concentrations. I would want to see tracings of Collagen using 2.5ug/ml, 1.25ug/ml and 0.833 ug/ml concentrations. Ristocentin concentrations should include 1.5 mg/ml, 1.2 mg/ml, 0.75 mg/ml and 0.50 mg/ml and Arachidonic Acid should also be included. In the event the Arachidonic Acid aggregation is absent, the patient platelets should be tested against U46619 in multiple concentrations. We rarely use Epinephrine as an agonist, only when the platelet count of the platelet rich plasma is greater than 600,000.
After its all said and done, I agree with George. This “patient” shows signs of vonWillebrand Type 2 and also potentially Glanzmans. It would be extraordinarily rare for a patient to exhibit both disorders. Therefore this “patient” should be properly prepared for retesting and the testing should be thorough and comprehensive with particular attention paid to the preanalytical process.
I hope this helps.
Herb Crown
St. Louis University Hospital Coagulation Reference Laboratory