Hemostasis Specimen Collection

• The general rule for blood collection using sodium citrate is to analyze the sample within 2 hours.

Hemostasis Specimen Collection and Handling

• Hemostasis laboratory procedures use venous whole blood.
• 3.2% buffered sodium citrate is an anticoagulant with a 9:1 ratio to blood.
• Venous whole blood is the most common sample.
• Sodium citrate acts as a buffering agent and is preferred for patients undergoing Heparin Therapy.
• Sodium citrate preserves factor V and Factor VIII and is satisfactory in platelet aggregation.
• The sodium citrate to blood ratio is 1:9.
• Light blue top tubes usually hold 2.7 mL of blood with 0.3 mL being anticoagulant.

Specimens Used in Testing

• Whole blood is used for platelet aggregometry and platelet count.
• Platelet rich plasma (PRP) is used for platelet aggregometry and platelet function tests.
• Platelet-poor plasma (PPP) is used for coagulation testing/assays
• PPP is the most common specimen used for Hemostasis.

Whole Blood Aggregometry

• Uses whole blood diluted 1:1 with saline without centrifugation
• Dilution applies if platelet count is >100,000; no dilution if <100,000
• Electrical Impedance/ Electrical Resistance used as principle (RBC, WBC, & PLT, resists electricity).
• Light Transmittance Aggregometry uses PRP and is more common, along with WBA, for platelet aggregometry.

Platelet Lumiaggregometry

• Detects aggregation activity and ability to secrete granules using whole blood or PRP.

• Luminescence is the principle.

• Whole blood is used for Platelet counting as well as EDTA (Lavender) for CBC or Sodium Citrate (Light blue) for Coagulation testing

• EDTA problem: causes RBC shrinkage theoretically.

• Platelet Function Tests use PRP to determine platelet quality, not using PPP

• Tests that use PRP include Platelet Adhesion/Retention Testing or Glass Bead Retention Test along with EDTA or Heparin.

• Platelet Aggregation Test is used and is tested using Platelet Factor 3 Availability

• Platelet Factor 4 & Beta-Thromboglobulin requires centrifugation at ref. temp. and uses CTAD as anticoagulant

• Latex D-dimer Assay uses EDTA, heparin, or sodium citrate as an anticoagulant.

• Platelets are most responsive 30 minutes to 3 hours post-collection.

• If not processed within 3 hours, refrigeration is possible, but can activate Factor VII and XI, causing platelet aggregation or vWF precipitation.

• Freezing is an alternative (-20C for two weeks, -70C for over two weeks) when sample cannot be processed within those hours.

• PPP specimens are centrifuged and separated and is used to detect activity within secondary Hemostasis.

• PPP is platelet-poor to avoid bias when assessing secondary hemostasis with false shortened results.

• Clot-Based Tests like PT, APTT, Thrombin-clotting time, and Reptilase time use PPP to assess fibrin clot formation speed.

• Light blue top specimens should not be stirred or disturbed post-centrifugation, potentially causing false shortened results from lysed RBCs.

Summary of Tests for Each Specimen:

• Whole Blood:
  • Whole Blood Aggregometry
  • Platelet Lumiaggregometry
  • Platelet Count
• PRP:
  • Light Transmittance Aggregometry
  • Platelet Lumiaggregometry
  • Platelet Function Test
  • Platelet Adhesion/ Retention Testing or Glass Bead Retention Test
  • Platelet Aggregation Test
  • Platelet Factor 3 Availability Test
  • Platelet Factor 4 & Beta-Thromboglobulin
• PPP:
  • Clot-Based Tests:
    • PT
    • APTT
    • Thrombin-clotting time
    • Reptilase time
    • CT/BT

Patient Management During Hemostasis Specimen Collection

• Patients need not fast, but should avoid vigorous activities and rest quietly for 30 minutes before hemostasis testing.
• Patient must not be tired or out-of breath; it is necessary to avoiding nicotine and exercise
• Patient avoids caffeine, garlic, ginger, Vit. K supplements and St. John's wort (Antidepressant).
• Avoid Aspirin, NSAIDS, Oral Contraceptive pills for collection.
• Nicotine raises blood pressure, avoid substances that increase blood pressure.
• Aspirin and NSAIDs affect platelet aggregation, impacting primary hemostasis.
• Long term Mefenamic acid usage leads to ulcer and chronic blood loss causing low MCV.
• Patients with anxiety patients that take St. John's Wort(Antidepressant) leads to increased activity of Warfarin as well as high fibrinogen groups in patients taking contraceptive pills.
• Scurvy (Vit. C Deficiency) leads to Petechiae (purpura) after tourniquet application
• Vitamin C is crucial for hemostasis via blood vessel integrity.

Drugs That Affect Testing:

• Aspirin: impairs platelet function and stops cyclooxygenase to stop thromboxane A2
• Coumadin (warfarin): inhibits Factors IX, X, VII, II, Protein S, C, Z (prolongs PT)
• Heparin: Clotting factors prolonged causes prolonged APTT
• Penicillin: causes changes of blood vessel walls and induces hemolysis
• Warfarin inhibits Vit. K dependent group and Factor VII is affected, so PT determines amount of Warfarin intake.
• Warfarin is converted to a metabolite post digestive tract.
• Heparin given intravenously or subcutaneously and is preferred.
• Penicillin causes platelet dysfunction (Ex. Beta-lactams antibiotics, Broad-spectrum antibiotics)

Collection Tubes:

• Light blue top, plastic-coated tubes are preferred.
• Most hemostasis specimens are collected in plastic blue-stopper (blue-top, blue-closure) sterile evacuated blood collection tubes containing a measured volume of 0.105 to 0.109 M (3.2%) buffered sodium citrate anticoagulant
• Tubes of uncoated soda-lime glass are unsuitable they activate platelets and the plasma
• Siliconized (plastic-coated) glass tubes are available, but their use is waning because of concern for potential breakage, with consequent risk of exposure to blood borne pathogens

Important Note:

• Plastic, polystyrene, silicone coated glass syringes used for hemostasis testing.
• Evacuated tube system (ETS) is the preferred collection method.

Specimen Collection Protocol

• The evacuated blood collection tube system (ETS) is the preferred method
• Syringe collection with initial "discard tubes" needed when necessary
• In venipuncture, the order of draw is collection of discard first, followed in the second draw
• Hemostasis testing discards 3mL blood because of Tissue thromboplastin (Factor III). In ETS, a 3 mL discard tube is used before a light blue top.

1. Non-additive tube (cleansing of tissue thromboplastin; can be used for serum tests)
2. Light blue top tube
3. EDTA
4. Plastic syringe (additional platelet tests)

Important Note:

• The phlebotomist may use and discard a preliminary blue-topped tube if non-additive tubes are unavailable,.

Blood to Anticoagulant Ratio (9:1)

• Short draw generates prolonged clot-based coagulation due to excess anticoagulant neutralizes the calcium in test reagents
• Excess anticoagulant prolongs PT and APTT
• Reject and recollect short draws because saturation does not occur
• Overfilled samples cause false shortened results.

Important Note:

• A sample with a Ratio ay ↑9 (ex. 10:1) gives false shortened results
• A sample with a Ratio ay ↓9 (ex. 8:1) gives false prolonged results

Clotted Specimens:

Are useless for hemostasis testing and require inversion to avoid clot formation

• Always reject clotted specimens when the tube inversion step is skipped after donation
• Excessive Specimen Agitation causes hemolysis; requires a phlebotomist to shake the tube
• Hemolysis releases procoagulants ADP to create clot formations
• Excess Needle Manipulation causes procoagulant substances from outside the skin to activate coagulation
• Traumatic venipuncture causes falsely shortened and unreliable clotting time tests

Important Note:

Traumatic Venipuncture releases tissue thromboplastin to the extrinsic pathways of coagulation; the tourniquet should be released within 1 minute

• A tourniquet may cause Stasis, slowing down venous flow and accumulating coagulation factor VIII and von Willebrand factor (VWF), which may result in false shortening of clot-based coagulation test results
• Stasis occurs as a result of prolonged the tourniquet for blood flow reasons and causes Factor VII, Factor VIII, Factor XII and vWF concentration
• Tourniquet application prevents stasis and requires observation of blood flow for vein selection
• It's not possible to do Catch methods as IV. lines can be caught. If the IV line causes catching, discard 6mL.

Table 42-1 Hemostasis Specimen Collection Errors

• Errors requiring new collection includes: - Short Draw: volume of blood used is 90% less. -Clot in Specimen: centrifuge; the presence of even a small clot requires that the specimen be recollected, so use for visuals - Causes Hemolysis: creates activations that indicate platelets and coagulation results Visible Hemolysis generates pink or red plasma in vitro - Generates Lipemia or Icterus: practitioners must employ a mechanical instrument. If tools are not able to measure clots due to highly colored specimens.

  • Causes Prolonged Tourniquet application: the concentration of von Willebrand factor and factor VIII elevate to decease parameters. Stasis occurs
  • Generates Specimen temperature causing destroyed integrity V and VII factors and triggers Willybrand multimers at 1-6 degrees C.
  • Storage at above generates storage: deteriorates coagulation Standards

• Needle bores prevents hemolysis and activation of platelets

• Large Specimens (25mL) require 19 g Needle usage

• Children require 23g needles to minimize vein pressure

• High hematocrit creates adjustments due to decreased volume of plasma with high anticoagulant

• adjustment is used after hematocrit exceeds 55%

Important Notes:

Ethylenediaminetetraacetic Acid (EDTA) is irreversible from binding so coagulation must never used. Inhibits thrombin from reacting and Is useful to collect blood count as well as testing for Molecular.

• Use for the platelet to bind and test
• CTAD stops coagulation and activates specialized assays
• Specimens with prothrombin require 20-21 Gauge needle/ standard
• Gauge requires small samples for tests and Gauge 19 to retrieve clots -
  • 23 G Is for patients that are very hard Other Anticoagulants: Test for specialized factors that has problems clotting.

SpecimenStorage Timeand Temperatures must stored in

• 18 - C to 24 C

Important Notes:

Must be in sealed sealed tube for longer periods in low temperature

• Cannot be heated; always in temperature
• Cold temperature increase V and XI

Important Notes Table:

• 4 h or 24 h after PT and PTT without fractions added to specimens.
• 18 - or 3h after

Preparation for Specimen Assay:

• Use for 3.2% Sodium citrated
• Whole Bloods must have clots
• Samples 200 uL in the centrifuge
• 150 gx for 11 to 15 mins to produce