From Diane Thornton, Section Supervisor of Hematology St. John’s Medical Center, Joplin, MO: When performing factor activity assays, are you determining the concentration or the function? Thanks.
George Fritsma: When you perform a clot-based coagulation factor assay using factor-depleted plasma and a partial thromboplastin time (PTT)-based, or prothrombin time (PT)-based assay, you are assaying coagulation factor activity or function. Likewise, a chromogenic factor assay measures function. In contrast, when you perform an immunoassay, such as an enzyme-linked immunoassay (ELISA), you are measuring factor concentration, just the amount of protein present. George continues with examples using factor VIII deficiency.
Freezing Specimens (March 6)
George Fritsma referenced a discussion of control plasmas in Nougier C, Sobas F, Nguyen TK: Analytic variability due to change of deficient plasma vials: application to one-stage clotting factor VIII assay. Blood Coagul Fibrinolysis 2011;22:151-4, and asked for guidelines on specimen freezing and storage.
Stephen Duff, Precision BioLogic Co-CEO commented there is, regrettably, no standard or guideline for specimen freezing, but made the following recommendations:
- A household freezer holds a temperature of -17°C, providing a “slow freeze,” which is acceptable when the analytes to be preserved are not temperature labile.
- A household freezer must have no defrost cycle, or the defrost cycle must be disabled.
- Standard laboratory -70°C or -80°C freezers provide a “fast freeze” speedy enough to preserve most labile proteins. Samples held in -70°C or -80°C freezers maintain their integrity for extended periods.
- If an analyte is labile, “snap-freezing” may be necessary. Snap-freezing employs liquid nitrogen at -196°C. Snap-freezing is seldom necessary for managing routine specimens.
- Plasmas shipped to coagulation reference laboratories must be shipped with at least 5 lbs of dry ice to maintain integrity.
Does HCT Affect Plavix Monitoring? (March 10)
A participant is currently using the VerifyNow P2Y12 for identifying those patients sensitive to Plavix or Prasugrel. There is a push to bring platelet mapping into the lab because there is a perceived problem with the measurement system of the cartridges, specifically at hematocrits less than 30%.
From George: According to Accumetrics, the VerifyNow P2Y12 assay is not indicated when the HCT is below 30%. However, you may wish to look into the effect of hematocrit on any other method you may choose. For instance, PFA-100 manufacturers set a HCT limit of 33%, and instruments like the TEG may be affected as well.
Dave McGlasson sent the following hemodilution information from Elaine Haney at Haemoscope:
We tested normal donor blood that was diluted ex-vivo with 0.9% saline to 0% (blank), 10%, 20%, 30%, 40%, 50%. The concentration at which hemodilution first became an interferent (showed a statistical and clinically significant difference from control in TEG parameters) was 40% dilution.
INR: Report to One Decimal (March 10)
From Diane Thornton, Section Supervisor Hematology, St. John’s MC, Joplin, MO: We have been reporting INRs as only one decimal as 1.2. We did this from the suggestions from CAP and their surveys. Also we have always rounded PTTs to a whole number. Should we be reporting 2 decimals on the INR and one on the PTTs? Thanks.
George responded: There is no clinical need to report INR to two decimals, and according to the rules of significant numbers, the math supports reporting only to tenths. For the PTT, whole numbers are correct, though I know of some labs that report halves, for example 35.5. PTs are typically reported to tenths.
More on Venoms (March 14)
From Vilas Hiremath at United Labs: I have spiked afibrinogenemia plasma with Russell viper (RV) and saw-scaled viper (Echis, SSV) crude venom. Prothrombin time (PT) and partial thromboplastin time (PTT) both failed to correct with RVV. The PT, but not the PTT, corrected to within the normal range with SSV. This appears to mean that SSV does not need fibrinogen to clot but RVV does need fibrinogen to clot.
George suspects your SVV preparation is contaminated with fibrinogen. SVV activates coagulation at the level of prothrombin, but requires fibrinogen to form a clot. You may wish to analyze both venoms for contamination in order to accurately assess their activity.
Effect of ACD on Platelet Satellitosis (March 14)
Donna Kaiser asks, “Is the use of acid-citrate-dextrose (ACD) tubes acceptable for patients who present with platelet satellitosis? We have several patients that continue to clump in sodium citrate tubes, mostly oncology patients. If ACD is acceptable what would the conversion factor be?
From George Fritsma: Platelet satellitosis most often appears in EDTA tubes, however like you, I have seen it in citrate tubes. In those instances, I have gone to heparin tubes, which introduce no correction factor and provide for an accurate platelet count, though they cannot be used to make Wright stain films. In a discussion with Dan Southern of Western Carolina University, we speculate that since citrate is the anticoagulant, the same problem may appear in ACD. The ACD tubes that are distributed by Becton-Dickinson appear to be glass only, I find no plastic ACD tubes in their catalogue.
TCoag KC1 Delta Coagulation Instrument (March 16)
From Rob Lair, MLT Program Instructor, Medvance Institute, Nashville, TN: I was reading an article from Dan Southern about the TCoag KC1, and would very much like to receive a copy of the procedure he created for it. Our school has purchased the KC1 but none of the current instructors have been in-serviced and the manual is confusing. Could someone please email Prof. Southern’s procedure to me?
George emailed this directly to Prof. Southern, who responded off-line. This request arrived March 25: Hi, I would also appreciate a copy of the procedure. Mona Gleysteen, Lake Area Tech
PT Mixing Studies (March 19)
From Dr. Manohar Pravin: I have a patient with a history of painless gross hematuria for 8 months. He had severe anemia and hepatosplenomegaly. His USG abdomen revealed only hepatosplenomegaly. CT abdomen and pelvis is normal. LFT, KFT, sickling test, CBC is normal. His PT and PTT are prolonged. How should I approach?
George Fritsma: From the hemostasis perspective, follow-up mixing studies of the prolonged PT and PTT is the first step. Mixing studies will point you to a possible single or multiple factor deficiency, the result may be followed by individual factor assays. Once the factor deficiency is established, appropriate procoagulant therapy using single or multiple-factor preparations may be used. One additional laboratory assay that is useful when identifying factor deficiencies is the fibrinogen assay.
More on Specimen Freezing (March 21)
On March 6 George posted comments from the Precision BioLogic, Inc. technical staff on specimen freezing. Co-CEO Steve Duff and I both commented on the dearth of data that may support freezing temperature choices. Here is another reference on the subject, McGlasson D. Standardization of temperature in handling and storage of plasma for coagulation testing. Texas J Med Technol 1985; 2: 10-11. The article indicates that factor VIII levels become significantly reduced with stored at -20°C when compared to fresh specimens and those stored at -70°.
Overfilled Tubes (March 22)
I want to hear from you regarding the effects of overfilled coagulation tubes on PT, PTT, D-dimer, and fibrinogen. Thank you, Elvin Tiamzon.
George responds that, while there are a number of definitive articles on underfilling, there are none on overfilled tubes. In his experience, overfilled citrate tubes are regrettably commonplace. In most instances, overfilling occurs when a phlebotomist first collects blood in a syringe, then applies pressure to the syringe plunger while transferring to the citrate tube. The proper way to transfer is to passively allow tube vacuum to draw the blood from the syringe. Applying pressure overfills the tube. Removing the closure and needle and transferring through the syringe tip is also likely to cause overfilling.
Overfilling theoretically risks clotting, as the ratio of additive anticoagulant to whole blood may be insufficient. In practice, however, we seldom detect clots in overfilled tubes, though we nonetheless reject overfilled tubes on general quality assurance principles. Any clotted tube must be rejected, of course, as the results of all parameters cannot be predicted. I would guess that if you performed a PT, PTT, D-dimer, or fibrinogen on a non-clotted but overfilled tube, the results would parallel those from a properly collected tube, not that I advocate running tests on specimens from overfilled tubes.
Heparin in Pneumonia (March 29)
From Betty Ciesla, MS, MT (ASCP) SH, Morgan State University, Baltimore, MD:
Hi George. My mom age 85 was recently hospitalized for pneumonia. Although she was on IVs she was mobile for 3 days. They gave her a shot of heparin in the belly and when I questioned this, they called it standard protocol and DVT preventive. Needless to say I was pretty shocked. Is this really SOP, with no thought to heparin sensitization and HIT?
George responds, First, what she really got was probably low molecular weight heparin (LMWH), or Lovenox. Many experienced physicians and nurses just refer to Lovenox as heparin, though it is not standard unfractionated heparin (UFH). Lovenox is standard therapy for prevention, is relatively safe as it takes a large overdose to cause bleeding, and it causes HIT at a rate about 1% of UFH, probably lower at prophylactic doses.
We most often associate Lovenox with followup to surgery, especially orthopedic surgery, but it is also recommended for serious medical conditions. The reference is Geerts WH, Bergqvist D, Pineo GF, et al. Prevention of venous thromboembolism. American College of Chest Physicians Evidence-Based Clinical Practice Guideline (8th Edition). Chest 2008; 133:381-453S.