The problem most people have with mixing studies is the interpretation of whether the original clotting time has actually "normalized" or not. For example, if the initial PT was 80 and the mixing PT clotting time (50/50 with pooled normal plasma) was 17 seconds --was this clotting time corrected ("normalized") or not? A 17 second PT is not "normal" in any laboratory reference range but the clotting time was significantly shortened by 63 seconds. Any presentation called "How to interpret a mixing study" that does not actually discuss how to decide (interpret) whether a clotting time has corrected or not is leaving out one of the most important parts of the discussion.
@@kv8585 The problem is that different studies/ articles use different cutoffs for their Rosner index and Chang % correction calculations. There is no agreement, from what I can see, on the correct cutoff for either of these methods. I did my own experiments with both of these methods and they just wasn't as conclusive as I would like. The Rosner index seemed to be a bit more accurate than the Chang % correction (in our laboratory) but neither method was correct in 100% of the test cases that I used in my study. I ended up using the Rosner index but adding a comment, to every result, in the form of a table with 4 different scenarios based on whether the result corrected back to the reference range and whether the Rosner index was below our established cutoff. Each scenario includes the words, "The results are most consistent with..." In other words, we are not reporting a definitive answer. Most clinicians are "OK" with this but there are some who are frustrated and would like a more conclusive interpretation. What are you doing in your lab and how did you validate it? By the way, we also add a comment that "mixing studies are not sensitive enough to detect all LA's. If clinically indicated, please order the lupus anticoagulant panel."
@@jeannecommer8042 for the aptt we have just validated the rosner index. I would also include the importance of clinical details ie thombotic or bleeding. Let us not forget the sensitivity of the aptt reagents to factors and lupus. Syntasil SS is very sensitive to even mild lupus whilst Actin FS is less sensitive.
@@jeannecommer8042 for the validation, we utilised residual plasma from previous cases. Reagent selection is very important and can be easily done. I once did an exercise in order to compare lupus sensitivity between two different PT reagents. So basically had excess stock of pos LA controls, did a dilution with NPP in order to obtain 100, 90, 80% etc up to 0% of normal controls, then analysed the PT. We plotted the data and the cutoff utilised was the upper limit of the PT reagent specific normal values. Despite both reagents are recombinant, they did have different sensitivities to LA, which might also pose a problem since LA strong patients are given warfarin. Personally, without proper clinical details I would not perform a mixing studies. Also important is the choice of NPP which without sounding sexist, I would not include females due to high fviii and fibrinogen. Controls and calibrators may also not be suitable for mixing studies. If you are interested, there is a very good e book from the wfh website by Dr. Steve Kitchen and is free to download.
@@kv8585 Fortunately, we do have access to all or most of the clinical patient data but sometimes, it is unclear--even to the clinician. We have had cases where both bleeding and clotting have been reported and recently had a case where the patient had both a strong LA and a strong factor VIII inhibitor. If a mixing study is ordered, we always also perform a lupus anticoagulant panel (we will call the doctor, if need be, to get this ordered). We use Hemosil DRVVT and SCT kits on the ACL TOP 750 and feel very confident about these results. With every lot number change we use 40 normal plasmas to determine normalized screening times and ratios. Our cutoffs are based on a 3 SD range-- following the recommendations of the manufacturer and ISTH. These procedure are clear and we have no problem identifying LA's. The problem lies with mixing studies. We use mixing studies to try to distinguish a factor inhibitor from a factor deficiency. We have strict guidelines on what plasmas we will test. We will not test patients on anticoagulant. We run heparin levels to rule out heparin from a line draw or any other source. We check the patient's medication history carefully. There are plenty of examples, in the literature, of ways to run mixing studies and how to interpret them--but no rules or recommendations from regulatory agencies, or the ISTH on the BEST method to use. Do we mix 1:1? or 1:4? Does "normalization mean correction back to the reference range? Should the cutoff for the Rosner index be 15? or 11? or somewhere in between? There are, however, plenty of admonitions that every lab should validate their own mixing study methods, cutoffs and interpretations. I spent almost a year doing just that. I collected a huge amount of data--from journal articles, web articles, web blogs (like this one) and designed my own validation study (ISTH does not have a detailed suggested mixing study validation procedure). I looked at plasmas from congenital factor deficient patients, patients with LA's, plasmas from patients that had factor VIII inhibitors and normal plasmas. We use FDA approved PNP from George King Biomedical for our mixing studies. This PNP contains citrated plasma from 30 or more carefully screened normal human donors and is platelet poor with no buffers or preservatives. They also supplied many of the frozen factor deficient plasmas and a plasma with a strong FVIII inhibitor. We also used frozen samples from patient samples. I determined, very quickly, that defining correction as "back to the normal reference range" was not acceptable at all in our laboratory. By the way--we establish our own reference ranges for adults and use the "Toulon study" pediatric reference ranges for children (2016). After months of experiments, I had narrowed my options down to the Rosner Index. But, it was not 100% accurate. I had to choose the cutoff that was the most accurate and supplement that with a "most consistent" comment. I feel that ISTH or regulatory agency should publish both a recommended mixing study validation method and a recommended method to establish a Rosner index cutoff if that is the chosen method. They have recommendations on every other kind of coagulation testing! I realize that I have gotten a little long-winded. Sorry! It just seems to me that there are too many articles on mixing studies that do not address the interpretation issue. They start with--If the mixing study normalizes--do this or if the mixing study does not normalize--do that. They tend to skip the part about how to determine if normalization has taken place. Thanks for sharing your thoughts and suggestions.
The problem most people have with mixing studies is the interpretation of whether the original clotting time has actually "normalized" or not. For example, if the initial PT was 80 and the mixing PT clotting time (50/50 with pooled normal plasma) was 17 seconds --was this clotting time corrected ("normalized") or not? A 17 second PT is not "normal" in any laboratory reference range but the clotting time was significantly shortened by 63 seconds. Any presentation called "How to interpret a mixing study" that does not actually discuss how to decide (interpret) whether a clotting time has corrected or not is leaving out one of the most important parts of the discussion.
One can utilise the Rosner index for the aptt or percentage correction for both pt and aptt
@@kv8585 The problem is that different studies/ articles use different cutoffs for their Rosner index and Chang % correction calculations. There is no agreement, from what I can see, on the correct cutoff for either of these methods. I did my own experiments with both of these methods and they just wasn't as conclusive as I would like. The Rosner index seemed to be a bit more accurate than the Chang % correction (in our laboratory) but neither method was correct in 100% of the test cases that I used in my study. I ended up using the Rosner index but adding a comment, to every result, in the form of a table with 4 different scenarios based on whether the result corrected back to the reference range and whether the Rosner index was below our established cutoff. Each scenario includes the words, "The results are most consistent with..." In other words, we are not reporting a definitive answer. Most clinicians are "OK" with this but there are some who are frustrated and would like a more conclusive interpretation.
What are you doing in your lab and how did you validate it? By the way, we also add a comment that "mixing studies are not sensitive enough to detect all LA's. If clinically indicated, please order the lupus anticoagulant panel."
@@jeannecommer8042 for the aptt we have just validated the rosner index. I would also include the importance of clinical details ie thombotic or bleeding. Let us not forget the sensitivity of the aptt reagents to factors and lupus. Syntasil SS is very sensitive to even mild lupus whilst Actin FS is less sensitive.
@@jeannecommer8042 for the validation, we utilised residual plasma from previous cases. Reagent selection is very important and can be easily done. I once did an exercise in order to compare lupus sensitivity between two different PT reagents. So basically had excess stock of pos LA controls, did a dilution with NPP in order to obtain 100, 90, 80% etc up to 0% of normal controls, then analysed the PT. We plotted the data and the cutoff utilised was the upper limit of the PT reagent specific normal values. Despite both reagents are recombinant, they did have different sensitivities to LA, which might also pose a problem since LA strong patients are given warfarin.
Personally, without proper clinical details I would not perform a mixing studies. Also important is the choice of NPP which without sounding sexist, I would not include females due to high fviii and fibrinogen. Controls and calibrators may also not be suitable for mixing studies.
If you are interested, there is a very good e book from the wfh website by Dr. Steve Kitchen and is free to download.
@@kv8585 Fortunately, we do have access to all or most of the clinical patient data but sometimes, it is unclear--even to the clinician. We have had cases where both bleeding and clotting have been reported and recently had a case where the patient had both a strong LA and a strong factor VIII inhibitor.
If a mixing study is ordered, we always also perform a lupus anticoagulant panel (we will call the doctor, if need be, to get this ordered). We use Hemosil DRVVT and SCT kits on the ACL TOP 750 and feel very confident about these results. With every lot number change we use 40 normal plasmas to determine normalized screening times and ratios. Our cutoffs are based on a 3 SD range-- following the recommendations of the manufacturer and ISTH. These procedure are clear and we have no problem identifying LA's.
The problem lies with mixing studies. We use mixing studies to try to distinguish a factor inhibitor from a factor deficiency. We have strict guidelines on what plasmas we will test. We will not test patients on anticoagulant. We run heparin levels to rule out heparin from a line draw or any other source. We check the patient's medication history carefully. There are plenty of examples, in the literature, of ways to run mixing studies and how to interpret them--but no rules or recommendations from regulatory agencies, or the ISTH on the BEST method to use. Do we mix 1:1? or 1:4? Does "normalization mean correction back to the reference range? Should the cutoff for the Rosner index be 15? or 11? or somewhere in between?
There are, however, plenty of admonitions that every lab should validate their own mixing study methods, cutoffs and interpretations. I spent almost a year doing just that. I collected a huge amount of data--from journal articles, web articles, web blogs (like this one) and designed my own validation study (ISTH does not have a detailed suggested mixing study validation procedure). I looked at plasmas from congenital factor deficient patients, patients with LA's, plasmas from patients that had factor VIII inhibitors and normal plasmas. We use FDA approved PNP from George King Biomedical for our mixing studies. This PNP contains citrated plasma from 30 or more carefully screened normal human donors and is platelet poor with no buffers or preservatives. They also supplied many of the frozen factor deficient plasmas and a plasma with a strong FVIII inhibitor. We also used frozen samples from patient samples.
I determined, very quickly, that defining correction as "back to the normal reference range" was not acceptable at all in our laboratory. By the way--we establish our own reference ranges for adults and use the "Toulon study" pediatric reference ranges for children (2016). After months of experiments, I had narrowed my options down to the Rosner Index. But, it was not 100% accurate. I had to choose the cutoff that was the most accurate and supplement that with a "most consistent" comment. I feel that ISTH or regulatory agency should publish both a recommended mixing study validation method and a recommended method to establish a Rosner index cutoff if that is the chosen method. They have recommendations on every other kind of coagulation testing!
I realize that I have gotten a little long-winded. Sorry! It just seems to me that there are too many articles on mixing studies that do not address the interpretation issue. They start with--If the mixing study normalizes--do this or if the mixing study does not normalize--do that. They tend to skip the part about how to determine if normalization has taken place. Thanks for sharing your thoughts and suggestions.
If the pt does not correct it can also be lupus anticoagulant
if the aptt is corrected why can't we have deficiency of factors 1, 2 or 10 (they are included in both pathways)?
Very good explanation of mixing studies!
Again great lesson
Thank you so much for this video!