From Kathleen Tobertga: Hello, We are having problems between sites with the results of our 2 hour normal pooled plasma (NPP) control (unmixed) for our mixing studies. Our site (the main lab) is getting an increased value for the PTT around 40 s, which we have researched and seems to be elevated due to heat labile factors being destroyed. The other site is getting normal results for their 2 hour incubated NPP control (~32 secs). We are trying everything to figure out what is going on short of actually going over there and performing the testing ourselves. We are both using the same company for the pooled plasma, they are thawing at 37°C, we at room temp. Tommorow we will be trying to replicate each other’s thawing techinque to see if that is the variable. What I’d like to know, because I could only find it in one resource, are we correct about the heat labile factors and that the 2 hour incubated control should be increased, and do you have any place I can go to find documentation? Would be much appreciated. Thank you.
Hi, Kathleen, and thank you. Are you in the same system as Louise Pleiss, whose question I posted about an hour ago, or is this just an amazing coincidence? My hunch is you’ll find that the difference in your approaches to thawing is the key, and I await the results of tomorrow’s experiment. I know it goes without saying, but you may want to confirm that everyone is adequately mixing the thawed control. I provided one (rather old) reference to Ms. Pleiss that addresses the effect of incubation on factor VIII, and am digging through old references for others. I’ve also sent messages to several experts around the world, one of whom, Dave McGlasson, had been unable to find a reference. Please watch Fritsma Factor, as I expect to get some comments and to collect additional references in the next few days.
Hello, Kathy T, and thank you for your comment. I’m still te
Hello, Kathy T, and thank you for your comment. I’m still teaching online, only now at Rutgers University. Margaret Giddens Fritsma, MS MT (ASCB) SBB is related to me by marriage and I mentioned your question to her this morning. She asked, “I wonder if their incubator is too warm?” She scares me, really she does!
Thanks all, we have done all our experiments and it seems th
Thanks all, we have done all our experiments and it seems that the dry incubator at our site was slightly on the high side. The company for the PNP recommends thawing in a circulating water bath at 37. GeorgeKing Biomedical has been very helpful with their suggestions and information. So, we have amended our policy to always thaw at 37 for the recommended time. And yes, Louise works at the alternate site, and you George are certainly a popular guy (I had you as a teacher on-line at UC a couple of years ago).
I would agree that thawing is a key step. CLSI guidelines ar
I would agree that thawing is a key step. CLSI guidelines are clear on this, and our personal experience also indicates that thawing at room temperature or slow thawing actually leads to gradations in plasma levels of FVIII. High levels at the bottom and low levels at the top. This is compounded by inadequate mixing. Also important is the actual level of FV & FVIII in the pool. If they are closer to 50U/dL than 100 U/dL then even small losses of labile factors in the pool may reduce the levels to those that will affect the APTT. So, suggest thawing at 37C for 5-10 min and then mixing well before use. Also, although some factor VIII inhibitors only acquire full inhibition at 2 hrs or even longer, a 1 hr incubation could provide a reasonable compromise for many cases tested. Excellent reviews: 1. Kershaw G, Jayakodi D, Dunkley S. Laboratory identification of factor
inhibitors: the perspective of a large tertiary hemophilia center. Semin Thromb Hemost. 2009 Nov;35(8):760-8.; 2. Kershaw G, Orellana D. Mixing tests: diagnostic aides in the investigation of prolonged prothrombin times and activated partial thromboplastin times. Semin Thromb Hemost. 2013 Apr;39(3):283-90.
Based on your statement: “We are both using th
Based on your statement: “We are both using the same company for the pooled plasma, they are thawing at 37°C, we at room temp”, I would say thawing at room temperature will increase cryoprecipitation of F8. That is why you observed 40s clotting time. I encourage you to run sensetive functional assay for F8, F5, F7 and Fibrinogen to see the difference between two thawing technique. Thawing frozen plasma at room temperature is not recommended.