Coagulation testing
- tests performed at 37C
- PLASMA is specimen of choice
- Sodium citrate is anticoagulant (9:1)
- most tests require addition of Calcium
Platelet Poor Plasma (PPP)
- plasma containing < 10 x 10^9/L platelets
- prepared by centrifuging plasma for 15 mins at high speed
- need because platelets can neutralize heparin and vWF
Platelet Rich Plasma (PRP)
- plasma containing 200-300 x 10^9/L platelets
- prepared by centrifuging for 10 mins at low speed
Specimens for Coagulation testing
- sodium citrate (3.2%) binds Ca to prevent clotting
- requires plasma (contains coagulation factors)
- blue tops should be collected first
- proper ratio (9:1) is important - no short draw tubes
Platelet tests
Peripheral smear Platelet count Platelet aggregation Bleeding time and platelet function analyzer Platelet secretion studies Flow cytometry
Platelet counts and estimates (specimen problems)
- Clumping due to antibodies
- Satellitism as a artifact of EDTA
- Giant platelets not counted as platelets
- Fragment RBC’s counted as platelets
Platelet aggregation studies
- determines whether plts will clump with other plts when stimulated by agonists
- activating agents: ADP, collagen, ristocetin, epinephrine, arachidonic acid
- addition of activators results in shape change
- causes an increase in % light transmission
- aggregation occurs in two waves
- Specimen = platelet rich plasma
- aggregation inhibited by aspirin
Primary wave (platelet aggregation)
- direct response of platelets to aggregating reagent
- platelet shape change and formation of small aggregates
Secondary wave (platelet aggregation)
- represents complete aggregation
- result of endogenous ADP being released from activated platelet dense bodies
Bleeding Time (BT)
- measure of platelet function
- 3 Types: Ivy, Duke and Template
- Prolonged by aspirin
- NORMAL: 1 - 9 minutes
Duke Bleeding Time
one incision, usually earlobe
Ivy Bleeding Time
- standardized back pressure (blood pressure cuff)
- below inner aspect of elbow
- 1-3 incisions
- depth not standardized
Template Bleeding Time
- standardized back pressure
- standardized depth
- below inner aspect of elbow
- blot filter paper every 30 seconds until bleeding stops
BT Controversies
- prolonged if low plt count, decreased fibrinogen, vascular disease
- not very sensitive
- platelet function can be impacted by drugs, food, etc.
- SHOULD NOT BE PERFORMED IF PLT < 100,000
BT Compromises
- poor circulation at puncture site
- disturbance of platelet plug
- excessive movement by patients (kids)
- scaring, bruising
Platelet Function Analyzer (PFA)
- replaced BT test
- specimen at room temp
- blood pumped through 2 apertures (collagen/epi (CEPI) and collagen/ADP)
- measures time needed to occlude aperture
- time need is function of plt count, plt activity, vWF activity and hematocrit
abnormal CEPI, normal CADP
probably due to aspirin
abnormal CEPI, abnormal CADP
probably a platelet function abnormality
Platelet secretion studies
- not done in most labs
- secretion of granules monitored by chemiluminescence
Flow Cytometry
- antibodies reactive with platelet surface available
- useful for GP1b/IX and GP1b/IIIa deficiency
Prothrombin time (PT)
- tests extrinsic pathway
- patient plasma + tissue thromboplastin (with Ca)
- screen for inherited or acquired deficiencies in extrinsic and common pathways
- measures factors I, II, V, VII, X
- monitors oral anticoagulants (coumadin, warfarin)
- INR used to standardize results
INR (International normalized ratio)
- used to correct for differences in coagulation instruments, reagents, etc.
- calculation is reported with all PT results
- 0 < INR < 6
PT Sources of error
- traumatic venipuncture can introduce tissue factor and premature activation of factor VII (falsely shorten PT)
- delay between collection and testing decreases factor V
- increased Hct = decreased plasma, improper ratio of anticoagulant
Activated Partial Thromboplastin time (APTT)
- add plt PL substitute and activator (APTT reagent) and Ca to activate factor XII
- tests intrinsic pathway
- measures all factors except VII and XIII
- monitors IV anticoagulant therapy (heparin)
- same sources of error as PT
