Back to the subject, "should all coagulation specimens be PPP?" Dave McGlasson referred me to an interesting poster comparing PT, PTT, and fibrinogen assays from PPP and whole blood. This was presented at the 2001 meeting of the American Society for Clinical Laboratory Science, and I've provided a paraphrase here...

At the Mayo Medical Laboratories Coagulation Testing Quality conference in Minneapolis, April 14–17, Dr. Dorothy Adcock Funk of Esoterix reported on the 2008 revision of Clinical and Laboratory Standards Institute (CLSI) Standard H21-A5, Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays. One interesting comment she made is that it is not necessary to prepare platelet-poor plasma (PPP; plasma with a platelet count less than 10,000/uL) for routine coagulation testing on fresh plasma. She reconfirmed, however, that plasmas for freezing and lupus anticoagulant (LA) testing must be PPP. Bioactive platelet granule materials are released upon freezing, including platelet factor 4 (PF4), which neutralizes heparin. In LA testing, platelets provide high concentrations of membrane phospholipid, which may neutralize low-titer LAs.

An interesting question from Mary Coleman at the University of North Dakota:

A female student reported to student health with a severe headache. She had a complete blood count performed using a standard EDTA tube and she had a low platelet and elevated lymphocyte count. The laboratory scientist also prepared a direct blood film from the needle. On the film the patient's platelet estimate was normal and her platelets and lymphocytes were distributed normally.

For a follow-up collection the scientist used a syringe and transferred the blood to both an EDTA and a sodium citrate tube. She immediately ran a sample from the EDTA tube and the platelet count was normal. Several minutes later a repeat count on the EDTA and citrate tube showed that the platelet count had dropped and the lymphocyte count went from 29 to 46%. I would guess the patient has a cold platelet antibody, and the platelet clumps were being counted as lymphs.

But my question is, this student had been providing plateletpheresis donations. Should she be advised to discontinue pheresis, since the blood goes from body temperature to room temperature, and also, could something in the pheresis process stimulated her immune system to produce platelet antibodies?

Thanks, Mary Coleman

Another good one from Kim Kinney at Clarian:

Hi George,

I have a question regarding cold agglutinins and clotting coag samples.  I have heard from one of our sites that specimens from someone with a cold agglutinin will clot in citrate tubes. In order to get a good sample the tubes and the blood must be heated to 37 degrees...I have not heard of this.  Any opinions out there??

(Kim, I assume this means the blood is collected and maintained at 37 degrees until separation, right?) Geo.

 

From Mary B. Paoli at Palmetto Health:

What is the best collection method for coagulation specimens from tiny babies?  Our NICU nurses draw these sometimes with butterfly adapters and sometimes with the Gelco IV needles.  They are often clotted or hemolyzed and the nurses are naturally upset with the need to re-draw specimens.  We must limit the amount of blood taken from these premies so large discard amounts are discouraged.  I'm interested in how others handle getting good specimens from these smallest patients.

A question from Renee Briggs:

George,
My laboratory recently switched to the Greiner Bio-One plastic collection tubes for coagulation.  Greiner has various fill size tubes (2 mL, 3 mL, and 3.5 mL) for their Vacuette sandwich tubes for coagulation.  These tubes have specific fill lines in the tube label and even give a recommendation that the tubes be filled to the line +/- 10%.
My question is my hospital has lots of nurse/physician collected tubes and many time these tubes are sent to the lab incorrectly filled (i.e. outside this +/- 10% of the fill line identified by the manufacturer).  I searched the literature and there are various articles and CLSI documents that discuss the importance of maintaining the correct blood-to-anticoagulant ratio in order to obtain correct results.  However all the articles I found focus mainly on how "underfilled" coagulation tubes can affect patient test results and adjusting the amount of sodium citrate in collection tubes for patients with hematocrits >55%, but I have found none that address the coagulation collection tubes that are "overfilled".  Obviously many of the grossly overfilled collection tubes come into the laboratory clotted (probably due to inadequate sodium citrate), but what do the experts recommend for those tubes that are slightly overfilled and not clotted?  Should our laboratory reject these solely based on the incorrect blood-to-anticoagulant ratio? If so, how does this overfill issue possibly affect the patient's coagulation test results?

Another great question from Kim Kinney at Clarian:

Hi again George.  I have an observation that I am wondering if anyone else has noticed.  We use full-draw, BD glass tubes for all of coagulation, but we have had the 1.8 mL pediatric tube come in from Riley Children's Hospital for PT/PTT.  We always check for a clot before spinning.  We often get a warning on a normal PT (say, 11.0 s) on these 1.8 mL tubes indicating the fib level may be low.  When we re-check the tube for clots, we then pull out a visible clot. 

Could these tubes be activated due to, for example, a difficult stick and the process of spinning causes the visible clots to form?  It has only happened as far as I know in the 1.8 mL tubes--just curious!

Here is another great question from Kim Kinney at Clarian in Indianapolis:

Hi, George. Thank you for getting Dr. Olson's D-dimer response, it was very helpful.  But here is another dilemma of ours!  I was reviewing the slides from Dr. Adcock's ASCP audio conference today on coagulation pre-analytical variables and came across the requirements for sample storage.  The CLSI guideline, H21 A5 refers to whole blood or processed and aliquoted samples.  What about centrifuged, on cells, and capped?  Would the same requirements apply as whole blood??  I know APTTs suspected of containing heparin must be centrifuged within one hour, but what about PT samples that have been centrifuged, still 24 hrs at room temp?  Thanks for the help.

Here is a question on platelet clumping or satellitosis in citrate tubes from Georgina Gibbons at Summit Health.

Do you have any information on patients that have platelet clumping in both the EDTA and sodium citrate tubes? We have found on occasion that these people may have no clumping in sodium heparin green tops. Have you read or heard anything similar to this?

Here are two new questions from Kim Kinney at Clarian in Indianapolis:

We are validating VWF antigen and VWF activity (automated ristocetin cofactor) on our new TOP analyzer using the IL kits.  One issue we see is that the kits are not very forgiving when it comes to hemolysis. Now, I know, hemolyzed samples are not the best, but, we see a high number of kids from Riley Children's Hospital that have had DDAVP challenges with severe hemolysis post dose.  It is almost a given that the post samples will be hemolyzed. Could it be the DDAVP or is it just the drawing from a catheter?

Also, we were establishing our reference ranges and the new ranges were quite a bit lower than the old.  So low that they did not correlate well with our old ristocetin cofactor and antigen assays.  Most of these samples were frozen Did I read somewhere that freezing can decrease your VWF levels?