More on PFA-100 Specimens

More on PFA-100 Specimens
Nov 12, 2015 7:07am

In follow-up to our 11/11/15 Collecting PFA-100 Specimens post, I've received responses from Donna Castellone and Drs. Ali-Sadeghi-Khomami and Emmanual Favaloro:


First from Ms. Castellone: Most important is that your reference range reflects how you draw the patients--21 gauge, butterfly, the patients should be the same.  Constancy is most important!


Next, this detailed information from Dr. Sadeghi-Khomami: I suggest under any circumstances laboratories need to follow recommendations provided by device manufacturers for “sample preparation and handling” in a particular intended use. I have referenced abstracts from two articles claiming that no difference observed between 23g and 21g needles in pediatrics, so it seems 21g needle sizes are good enough for PFA-100. Unfortunately, many studies published in scientific literature do not appreciate enough the limitations and industrial variability of assay system, including blood types and reagent lot-variability, in their conclusions.

I found the following relevant information that indicates 21g needle or larger (<21g) could be used. As you mentioned, patient discomfort using larger needles is another problem which needs to be considered. Obviously, laboratories need to adhere to their own method of sample preparation in order to use appropriately their own reference ranges [Geo adds--refer also to Ms. Castellone's comment above]:

1. Sample Collection:

  • System: either vacuum- or plunger-based
  • Collection medium: 3.2% (106 mM) or 3.8% (129mM) buffered sodium citrate
  • Technique : short cuff-time, needle-size ≥ 21g (g numbers <21)
  • Thorough mixing of sample by gently inverting tube several times

2. Sample Processing:

  • Transport and store the sample undisturbed at room temperature
  • Mix the sample carefully by inverting several times by hand
  • 800–1,000 μl blood inserted in cartridge, taking care that no air bubbles are introduced
  • Tubing must be validated by the site that wants to use it

3. Sample Stability: keep capped and test the sample within four hours of collection

4. Reference ranges(closure times, CT):

  • 3.8% sodium citrate (n=176 with confirmed normal platelet function); Collagen/epinephrine mean 132 s, range 94–193 s; Collagen/ADP mean 92 s, range 71–118 s
  • 3.2% sodium citrate (n=309 with confirmed normal platelet function): Collagen/epinephrine mean 110 s, range 82–150 s; Collagen/ADP mean 78 s, range 62–100 s

References:

Carcao MD, Blanchette VS, Dean JA. The Platelet Function Analyzer (PFA-100): a novel in-vitro system for evaluation of primary haemostasis in children. Br J Haematol. 1998;101:70–3. “We used the system to establish normal ranges for CTs in healthy children, adults and neonates. Mean CTs of healthy children were independent of the needle gauge used (21g or 23g) for blood sampling; they were very similar to the mean CTs of healthy adults, but longer than mean CTs of healthy neonates.”

Rand ML, Carcao MD, Blanchette VS. Use of the PFA-100 in the assessment of primary, platelet-related hemostasis in a pediatric setting. Semin Thromb Hemost. 1998;24:523–9. “CTs for healthy children are independent of the needle gauge (21g or 23g) used for blood sampling. They are similar to CTs for healthy adults, but neonates have significantly shorter CTs, likely due to increased levels of vWF .”


And a follow-up from Dr. Favaloro: I think the previous replies have said it all. We have been using 21g needles and butterfly-needle/cannulas for platelet function/PFA testing for at least a decade without problems. We stopped using 19g needles for platelet function testing also at that time. This is an unfortunate dogma that still exists among some die hard old school practitioners. It was what I was trained, but it is not really true. It is not so much the size of the needle, but the skill of the phlebotomist. A good phlebotomist will get a good sample for platelet function testing using a 21g needle. A bad phlebotomist will not--but they will also fail on the 19g. And 18g seems even more unnecessarily cruel! With some butterfly-needle/cannula sets you may need to use a discard tube because of the air in the cannula, which may thus underfill the first tube, but it's use will not hurt the platelet function/PFA.
 

1 Comment

In follow-up to our 11/11/15 Collecting PFA-100 Specimens post, I've received responses from Donna Castellone and Drs. Ali-Sadeghi-Khomami and Emmanual Favaloro:


First from Ms. Castellone: Most important is that your reference range reflects how you draw the patients--21 gauge, butterfly, the patients should be the same.  Constancy is most important!


Next, this detailed information from Dr. Sadeghi-Khomami: I suggest under any circumstances laboratories need to follow recommendations provided by device manufacturers for “sample preparation and handling” in a particular intended use. I have referenced abstracts from two articles claiming that no difference observed between 23g and 21g needles in pediatrics, so it seems 21g needle sizes are good enough for PFA-100. Unfortunately, many studies published in scientific literature do not appreciate enough the limitations and industrial variability of assay system, including blood types and reagent lot-variability, in their conclusions.

I found the following relevant information that indicates 21g needle or larger (<21g) could be used. As you mentioned, patient discomfort using larger needles is another problem which needs to be considered. Obviously, laboratories need to adhere to their own method of sample preparation in order to use appropriately their own reference ranges [Geo adds--refer also to Ms. Castellone's comment above]:

1. Sample Collection:

  • System: either vacuum- or plunger-based
  • Collection medium: 3.2% (106 mM) or 3.8% (129mM) buffered sodium citrate
  • Technique : short cuff-time, needle-size ≥ 21g (g numbers <21)
  • Thorough mixing of sample by gently inverting tube several times

2. Sample Processing:

  • Transport and store the sample undisturbed at room temperature
  • Mix the sample carefully by inverting several times by hand
  • 800–1,000 μl blood inserted in cartridge, taking care that no air bubbles are introduced
  • Tubing must be validated by the site that wants to use it

3. Sample Stability: keep capped and test the sample within four hours of collection

4. Reference ranges(closure times, CT):

  • 3.8% sodium citrate (n=176 with confirmed normal platelet function); Collagen/epinephrine mean 132 s, range 94–193 s; Collagen/ADP mean 92 s, range 71–118 s
  • 3.2% sodium citrate (n=309 with confirmed normal platelet function): Collagen/epinephrine mean 110 s, range 82–150 s; Collagen/ADP mean 78 s, range 62–100 s

References:

Carcao MD, Blanchette VS, Dean JA. The Platelet Function Analyzer (PFA-100): a novel in-vitro system for evaluation of primary haemostasis in children. Br J Haematol. 1998;101:70–3. “We used the system to establish normal ranges for CTs in healthy children, adults and neonates. Mean CTs of healthy children were independent of the needle gauge used (21g or 23g) for blood sampling; they were very similar to the mean CTs of healthy adults, but longer than mean CTs of healthy neonates.”

Rand ML, Carcao MD, Blanchette VS. Use of the PFA-100 in the assessment of primary, platelet-related hemostasis in a pediatric setting. Semin Thromb Hemost. 1998;24:523–9. “CTs for healthy children are independent of the needle gauge (21g or 23g) used for blood sampling. They are similar to CTs for healthy adults, but neonates have significantly shorter CTs, likely due to increased levels of vWF .”


And a follow-up from Dr. Favaloro: I think the previous replies have said it all. We have been using 21g needles and butterfly-needle/cannulas for platelet function/PFA testing for at least a decade without problems. We stopped using 19g needles for platelet function testing also at that time. This is an unfortunate dogma that still exists among some die hard old school practitioners. It was what I was trained, but it is not really true. It is not so much the size of the needle, but the skill of the phlebotomist. A good phlebotomist will get a good sample for platelet function testing using a 21g needle. A bad phlebotomist will not--but they will also fail on the 19g. And 18g seems even more unnecessarily cruel! With some butterfly-needle/cannula sets you may need to use a discard tube because of the air in the cannula, which may thus underfill the first tube, but it's use will not hurt the platelet function/PFA.
 

By George Fritsma
Nov 12, 2015 7:29am
In follow-up to Dr. Sadegi-Khomami's 3.8% sodium citrate reference intervals, in McGlasson DL, Shah AD, Fritsma GA. Ability of the Innovance PFA P2Y system to detect clopidogrel-induced ADP receptor blockade in preangiocath individuals. Blood Coag Fibrinolysis 2011;22, we showed that PFA-100 CTs on specimens collected in 3.8% sodium citrate more closely predicted reference whole blood lumiaggregometry clopidogrel responses than those collected in 3.2%. Back when 3.2% sodium citrate collection was originally validated, the findings were generalized to platelet function testing specimens without direct comparisons, though 3.2% sodium citrate collection is now standard for all hemostasis specimens.

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