LAB EVAL. OF HEMOSTASIS

• Blood collectors, including medical laboratory practitioners, phlebotomists, patient care technicians, nurses, and physicians who collect and manage hemostasis blood specimens must adhere closely to published protocols to ensure assay validity
• Blood specimen collection and management are the most vulnerable stages of the hemostasis blood testing process because every stage is manual and thus error-fraught
• Most hemostasis procedures are performed on platelet-poor plasma (PPP, plasma whose platelet count is less than 10,000/µL) prepared from venous whole blood collected by venipuncture
• Platelet-rich plasma (PRP)


  Plasma whose platelet count is approximately 200,000/µL
• Patient Management


  • Medical laboratory practitioner, pharmacist, nurse, and physician shoulder substantial responsibility for preparing a patient for hemostasis testing
  • Diligent bleeding and thrombosis risk assessment
  • Ask about family clotting and bleeding history, blood group, pregnancy, liver or kidney disease, leukemia, anemia, and malnutrition
  • Ask about bleeding and clotting characteristics including location, frequency, volume, and whether events are spontaneous or follow an injury or a surgical or dental procedure
  • Ask about 2-week drug history, including anticoagulants, antiplatelet drugs, contraceptives, and hormone replacement therapy
  • Ask about over-the-counter remedies and dietary supplements
  • Instruct patient to discontinue nonprescription drugs at least a week prior to blood collection
  • Anticoagulants and antiplatelet drugs are continued when the purpose is to assess their efficacy
  • Examine the patient for evidence of bleeding; bruises and swelling
  • Standardized bleeding assessment tools (BAT) may be employed to enhance bleeding history accuracy
  • Patients need only fast under arranged circumstances but are advised to avoid caffeine and exercise for 2 hours and smoking for 30 minutes before collection
  • Patients should be inactive for 5 minutes before collection
• Blood collectors


  • Manage patients using standard protocols for identification, cleansing, tourniquet use, and venipuncture
  • If there is a reason to anticipate excessive bleeding, the phlebotomist should extend the time for observing the venipuncture site from the usual 1 minute to 5 minutes and should apply a pressure bandage before dismissing the patient
• Needle Selection


  • 20 or 21 gauge, thin-walled, 1.0 or 1.25 inches long for adults with good veins
  • 23-gauge, winged-needle set for children or adults with small, fragile, or hardened veins
  • 19-gauge safety transfer unit for slowly injecting blood through tube closure
• Hemostasis Specimen Collection Tubes

  • Plastic blue-closure (blue-top, blue-stopper) sterile evacuated blood collection tubes containing a measured volume of buffered sodium citrate anticoagulant
  • Siliconized (plastic-coated) glass tubes are available, but their use is declining
  • Uncoated soda-lime glass tubes are unsuitable
• Sodium citrate


  • The anticoagulant used for most hemostasis testing is buffered 0.105 to 0.109 M (3.2%) sodium citrate, Na3C6H5O7.2H2O, molecular weight 294.1 Daltons
  • Sodium citrate binds calcium ions to prevent coagulation, and the buffer stabilizes specimen pH as long as the tube closure remains in place
  • Upon collection, the anticoagulant solution mixes with blood to produce a 9:1 ratio-9 parts whole blood to 1 part anticoagulant
• Sodium citrate volume adjustment for elevated hematocrits


  1. The 9:1 blood-to-anticoagulant ratio is effective, provided the patient's hematocrit is 55% or below

  2. In polycythemia the decrease in plasma volume relative to whole blood unacceptably raises the anticoagulant-to-plasma ratio, which causes falsely prolonged results for clot-based coagulation tests

  3. The phlebotomist must prepare specially marked tubes with relatively reduced anticoagulant volumes for collection of blood from a patient whose hematocrit is known to be 55% or higher

  4. The amount of anticoagulant may be computed using a formula
• There exists no evidence suggesting a need for increasing the volume of anticoagulant for specimens from patients with anemia, even when the hematocrit is less than 20%
• Other anticoagulants for hemostasis specimens

  • EDTA (lavender closure) is not used for clot-based coagulation testing because it irreversibly chelates calcium ions
  • Heparinized (green closure) specimens have never been validated for plasma coagulation testing but may be necessary to produce accurate platelet counts in cases of platelet satellitosis
  • EDTA, acid citrate dextrose (ACD, yellow closure), and dipotassium EDTA (K2EDTA, white closure) gel tubes may be used for molecular diagnosis
  • Citrate theophylline adenosine dipyridamole (CTAD, blue closure) tubes suppress in vitro platelet or coagulation activation for specialty assays
• Venipuncture


  • The blood collector adheres strictly to standard venipuncture procedures for evacuated tube or syringe collection
  • The hemostasis specimen must be collected first or immediately after a nonadditive tube
  • The hemostasis tube may not immediately follow a tube that contains heparin, EDTA, sodium fluoride, or clot-promoting silica particles
  • Evacuated tubes are designed so that the negative internal pressure draws the blood into the tube
• Table 41.2 lists hemostasis specimen collection and management errors that require specimen rejection
• Electro-mechanical detection method instrument

  Used to employ for blood stasis detection
• Blood stasis


  Activates endothelial cells and elevates the concentration of von Willebrand factor and fibrinogen, falsely shortening clot-based test results
• Storage at refrigerator temperatures


  Causes precipitation of large von Willebrand factor multimers, activation of coagulation factor VII, activation of platelets, and destruction of platelet integrity
• Storage at above standard room temperature

  Causes coagulation factors V and VIII to deteriorate
• PT


  Prothrombin time
• PTT


  Partial thromboplastin time
• Red-closure or serum separator (gold or red-gray closure with gel, SST) tubes

  Additives may transfer to the hemostasis specimen and invalidate hemostasis test results
• Nonadditive tubes


  Red-closure glass (but not plastic) tubes and clear-closure plastic tubes
• When following an additive tube


  Phlebotomist may use and discard a preliminary blue-closure tube
• Evacuated tubes


  Designed so that the negative internal pressure collects the correct volume of blood
• Collection tube manufacturers indicate the allowable range of collection volume error in package inserts and provide a minimum volume indication on each tube
• In most cases the volume of blood collected must be within 90% of the calibrated volume
• Short draw - a specimen with a smaller volume than the minimum specified by the manufacturer


  Generates erroneously prolonged clot-based coagulation test results because the excess anticoagulant relative to blood volume adversely neutralizes test reagent calcium
• When collecting a specimen using a butterfly set

  Blood collector compensates for the internal volume of the tubing by collecting and discarding a nonadditive tube or a blue-closure tube
• When collecting by syringe

  Collector replaces the Luer adaptor needle or butterfly needle with a Luer adaptor-equipped large-bore safe transfer device and gently transfers the specimen from the syringe into evacuated tubes, using the same order of tube selection as for standard evacuated tube collection
• Clotted specimens are useless for hemostasis testing, even if the clot is small
• After collection


  Blood collector must gently invert the specimen end over end at least three times to mix the blood with the anticoagulant and prevent clot formation
• Excessive specimen agitation, called "cocktail shaking", causes hemolysis (RBC rupture), procoagulant activation, and platelet activation
• Before centrifugation and testing


  Medical laboratory practitioner visually examines the specimen for clots
• Many coagulometers are equipped to detect the presence of clots
• Clotted specimens are discarded, and a new specimen is collected
• Excess needle manipulation


  May promote the release of connective tissue procoagulants that contaminate the specimen by activating clotting factors
• Time to clot test results from specimens collected during a traumatic venipuncture


  May be falsely shortened and unreliable
• During blood collection


  Phlebotomist must remove the tourniquet within 1 minute of its application to avoid stasis, which slows venous flow
• Stasis


  Results in endothelial cell activation and hemoconcentration with the local accumulation of coagulation factors V, VII, VIII, XII, and von Willebrand factor (VWF), which may falsely shorten clot-based coagulation test results
• CLSI Standard GP41 discourages the practice of collecting specimens from vascular access devices