Collecting PFA-100 Specimens

Collecting PFA-100 Specimens
Nov 11, 2015 7:51am

Here is a stimulating question sent to Dennis Ernst, Center for Phlebotomy Education, from a participant in his Phlebotomy Supervisors' Boot Camp:

One of our physicians asks that we collect specimens for PFA-100 assays using an 18g needle. Our laboratory scientists support this requirement, indicating that lab protocol requires that we not use a needle smaller than 21g and that we may not use a 21g infusion ("butterfly") set, as the tubing air "rips" the RBCs apart. Our department distributed a message that we must use evacuated tube specimen collection devices for every patient. We have 75–85% Medicare patients for whom a 21g Vacutainer will not work. What is your opinion?


Hi, Dennis, thanks for the question. No matter how many guidelines and standards we write, we just can’t predict the ideas professionals develop with the best of intentions. Fortunately, a literature search provides some straight answers, references listed below. I've also attached a full-print article; Wollowitz A, Bijur PE , Esses D, Gallagher JE. Use of butterfly needles to draw blood is independently associated with marked reduction in hemolysis compared to intravenous catheter. Acad Emerg Med. 2013 ;20:1151–5.

There is nothing to prevent us from using 18g needles to collect specimens for the PFA-100, but all data support  21g needles as the standard of care. The 18g needle introduces concerns for patient comfort that could raise interest at the institution’s administrative level. The physician and lab scientist are probably motivated by concerns for hemolysis, however the references all support using a 21g needle.

Here is a helpful abstract: Lippi G, Fontana R, Avanzini P, Aloe R, Ippolito L, Sandei F, Favaloro EJ. Influence of mechanical trauma of blood and hemolysis on PFA-100 testing. Blood Coagul Fibrinolysis. 2012;23:82–6: For investigations of platelet function it is recommended that venipuncture should be performed using ordinary needle systems instead of butterfly cannulae systems. Platelets might be activated in the long plastic tubes of butterfly systems. The aim of this study was to investigate the dependency of platelet function results on blood sampling using different collection systems. Therefore, blood of 25 healthy volunteers was collected from both arms using at the same time on one side a 21-gauge needle and on the other side a 21-gauge butterfly cannula system. Both samples of each volunteer were analyzed on the PFA-100. Platelet aggregation was performed on the Behring Coagulation Timer (BCT) and the optical aggregometer PAP-4 using ADP , collagen and arachidonic acid to induce platelet aggregation in platelet-rich plasma. No significant prolongation of the closure times on the PFA-100 with the COL/EPI cartridge and the COL/ADP cartridge was observed when using butterfly cannulae. The results of optical aggregometry were not significantly different. The maximum aggregation response did not differ significantly for both collection systems. Aggregometry and the PFA-100 system are not affected by different blood collection systems. Therefore butterfly cannulae can be used for sample collection to investigate platelet function.

The abstract indicates that “butterfly” infusion set-collected specimens are equivalent to a 21g needle for platelet aggregometry and the PFA-100. I assert the apparent lack of platelet activation in the tube may be generalized to say that hemolysis is not an issue.

Though the idea that the air in the infusion set tube “rips” the RBCs apart is incorrect, the physician and lab scientist are correct in thinking that collection with a butterfly may be a source of hemolysis, not because of the air in the tube, but because of the needle gauge, as butterfly sets come equipped with 21g, 23g, and 25g needles. The Lippi article referenced below asserts there is no effect from using 23g needles. This is disputed in the attached Wollowitz paper, however, which documents a 1.9% hemolysis rate for 21g versus 3.2% for 23g needles. Taken together, the papers imply that specimens collected using a 23g needle should be collected slowly to prevent the small needle from damaging RBCs.

A key issue when using infusion sets is to ensure the displaced air from the tube doesn’t result in a short draw when the hemostasis tube (blue stopper, 3.3% citrate) is the first tube collected. To prevent this, the phlebotomist should use an initial discard tube and retain the specimen collected in the second tube.

A bigger concern, raised in several papers, is the hemolysis rate generated when collecting through a vascular access device; a whopping 14.6% in the Wolfowitz paper. Catheters, PICC tubes, and hep locks really do “rip” the RBCs. You can't ask ED personnel not to collect through the access device while performing the phlebotomy, a humane practice, but since the apparatus is not meant for blood collection, they must collect the blood "gently."

Another paper by Lippi cited below indicates syringe collection gives shorter PFA-100 closure times than venipuncture. I suspect in general lab conditions syringe collection would make little difference, but there are lab directors who require syringe collection for all platelet testing.

Here are the additional citations referenced above:

  • Lippi G, Salvagno GL, Montagnana M, Brocco G, Cesare Guidi G. Influence of the needle bore size used for collecting venous blood samples on routine clinical chemistry testing. Clin Chem Lab Med. 2006;44:1009–14.
  • Kennedy C, Angermuller S, King R, et al.  A comparison of hemolysis rates using intravenous catheters versus venipuncture tubes for obtaining blood samples. J Emerg Nurs. 1996;22:566–9.
  • Lippi G, Ippolito L, Zobbi V, Sandei F, Favaloro EJ. Sample collection and platelet function testing: influence of vacuum or aspiration principle on PFA-100 test results. Blood Coagul Fibrinolysis. 2013;24:666–9.
  • Mani H, Kirchmayr K, Kläffling C, et al. Influence of blood collection techniques on platelet function. Platelets. 2004;15:315–8.

Here is the promised article: /sites/default/files/wollowitz_et_al-2013-academic_emergency_medicine.pdf

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Here is a stimulating question sent to Dennis Ernst, Center for Phlebotomy Education, from a participant in his Phlebotomy Supervisors' Boot Camp:

One of our physicians asks that we collect specimens for PFA-100 assays using an 18g needle. Our laboratory scientists support this requirement, indicating that lab protocol requires that we not use a needle smaller than 21g and that we may not use a 21g infusion ("butterfly") set, as the tubing air "rips" the RBCs apart. Our department distributed a message that we must use evacuated tube specimen collection devices for every patient. We have 75–85% Medicare patients for whom a 21g Vacutainer will not work. What is your opinion?


Hi, Dennis, thanks for the question. No matter how many guidelines and standards we write, we just can’t predict the ideas professionals develop with the best of intentions. Fortunately, a literature search provides some straight answers, references listed below. I've also attached a full-print article; Wollowitz A, Bijur PE , Esses D, Gallagher JE. Use of butterfly needles to draw blood is independently associated with marked reduction in hemolysis compared to intravenous catheter. Acad Emerg Med. 2013 ;20:1151–5.

There is nothing to prevent us from using 18g needles to collect specimens for the PFA-100, but all data support  21g needles as the standard of care. The 18g needle introduces concerns for patient comfort that could raise interest at the institution’s administrative level. The physician and lab scientist are probably motivated by concerns for hemolysis, however the references all support using a 21g needle.

Here is a helpful abstract: Lippi G, Fontana R, Avanzini P, Aloe R, Ippolito L, Sandei F, Favaloro EJ. Influence of mechanical trauma of blood and hemolysis on PFA-100 testing. Blood Coagul Fibrinolysis. 2012;23:82–6: For investigations of platelet function it is recommended that venipuncture should be performed using ordinary needle systems instead of butterfly cannulae systems. Platelets might be activated in the long plastic tubes of butterfly systems. The aim of this study was to investigate the dependency of platelet function results on blood sampling using different collection systems. Therefore, blood of 25 healthy volunteers was collected from both arms using at the same time on one side a 21-gauge needle and on the other side a 21-gauge butterfly cannula system. Both samples of each volunteer were analyzed on the PFA-100. Platelet aggregation was performed on the Behring Coagulation Timer (BCT) and the optical aggregometer PAP-4 using ADP , collagen and arachidonic acid to induce platelet aggregation in platelet-rich plasma. No significant prolongation of the closure times on the PFA-100 with the COL/EPI cartridge and the COL/ADP cartridge was observed when using butterfly cannulae. The results of optical aggregometry were not significantly different. The maximum aggregation response did not differ significantly for both collection systems. Aggregometry and the PFA-100 system are not affected by different blood collection systems. Therefore butterfly cannulae can be used for sample collection to investigate platelet function.

The abstract indicates that “butterfly” infusion set-collected specimens are equivalent to a 21g needle for platelet aggregometry and the PFA-100. I assert the apparent lack of platelet activation in the tube may be generalized to say that hemolysis is not an issue.

Though the idea that the air in the infusion set tube “rips” the RBCs apart is incorrect, the physician and lab scientist are correct in thinking that collection with a butterfly may be a source of hemolysis, not because of the air in the tube, but because of the needle gauge, as butterfly sets come equipped with 21g, 23g, and 25g needles. The Lippi article referenced below asserts there is no effect from using 23g needles. This is disputed in the attached Wollowitz paper, however, which documents a 1.9% hemolysis rate for 21g versus 3.2% for 23g needles. Taken together, the papers imply that specimens collected using a 23g needle should be collected slowly to prevent the small needle from damaging RBCs.

A key issue when using infusion sets is to ensure the displaced air from the tube doesn’t result in a short draw when the hemostasis tube (blue stopper, 3.3% citrate) is the first tube collected. To prevent this, the phlebotomist should use an initial discard tube and retain the specimen collected in the second tube.

A bigger concern, raised in several papers, is the hemolysis rate generated when collecting through a vascular access device; a whopping 14.6% in the Wolfowitz paper. Catheters, PICC tubes, and hep locks really do “rip” the RBCs. You can't ask ED personnel not to collect through the access device while performing the phlebotomy, a humane practice, but since the apparatus is not meant for blood collection, they must collect the blood "gently."

Another paper by Lippi cited below indicates syringe collection gives shorter PFA-100 closure times than venipuncture. I suspect in general lab conditions syringe collection would make little difference, but there are lab directors who require syringe collection for all platelet testing.

Here are the additional citations referenced above:

  • Lippi G, Salvagno GL, Montagnana M, Brocco G, Cesare Guidi G. Influence of the needle bore size used for collecting venous blood samples on routine clinical chemistry testing. Clin Chem Lab Med. 2006;44:1009–14.
  • Kennedy C, Angermuller S, King R, et al.  A comparison of hemolysis rates using intravenous catheters versus venipuncture tubes for obtaining blood samples. J Emerg Nurs. 1996;22:566–9.
  • Lippi G, Ippolito L, Zobbi V, Sandei F, Favaloro EJ. Sample collection and platelet function testing: influence of vacuum or aspiration principle on PFA-100 test results. Blood Coagul Fibrinolysis. 2013;24:666–9.
  • Mani H, Kirchmayr K, Kläffling C, et al. Influence of blood collection techniques on platelet function. Platelets. 2004;15:315–8.

Here is the promised article: /sites/default/files/wollowitz_et_al-2013-academic_emergency_medicine.pdf

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