Coagulation Testing Flashcards
Clotting End Point
Photo-optical
Electromechanical
Chromogenic
Immunologic
Photo-optical
Most Common
Measure change in optical density
Electromechanical
Clot completes the circuit
Clot impedes movement of steel ball
Chromogenic
Use of synthetic substrate
- Mimics natural counterpart
- Subtrate is linked to color indicator
- P-nitroanilide (yellow color)
Immunologic
Detection/measurement by antigen-antibody
- Electrophoresis
- Latex agglutination
- RID
- EIA
Prothrombin Time (PT)
Function of Extrinsic & Common Pathway
Factors: 7, 10, 5, 2, 1
PT Normal Time
12-15 seconds
PT Clinical Correlation
Prolonged
- Liver disease
- Vitamin K deficiency
- Factor deficiency of 1, 2, 5, 7, or 10
- Anticoagulatns
- DIC (Disseminated Intravascular Coagulation)
Activated Partial Thromboplastin Time (APTT)
Tests function of Intrinsic and Common Pathways
Factors: 12, 11, 9, 8, 10, 5, 2, 1
APTT Reagent
Phospholipid (PF3)
Contact activator (Silica, ellagic acid, kaolin)
CaCl2
APTT Normal Time
25-35 seconds
APTT Clinical Correlation
Prolonged
- Liver disease
- DIC
- Factor deficiencies: 12, 11, 9, 10, 5, 2, 1
- Anticoagulants
Thrombin Time (TT)
Thrombin added to plasma
Testing Factor 2
TT Normal Time
15-19 seconds
TT Clinical Correlation
Prolonged
- Hypofibrinogenemia
- Dysfibrinogenemia
- DIC
- Liver diease
- Increased FSP
- Heparin
Fibrinogen Assay
Determines amount of Factor 1 (fibrinogen)
Fibrinogen Assay Testing
Plasma diluted 1:10 with veronal buffer
Thrombin reagent added
Fibrinogen Normal Amount
200-400 mg/dl
Fibrinogen Clinical Correlation
Decreased amounts in:
- Liver disease
- DIC
- Fibrinogen deficiencies
FSP/D-Dimer Testing
Determines amount of FSP or D-Dimers present
Latex agglutination testing
- Visible agglutination above normal range
FSP (Fibrin Split Products) Normal Range
0-0.5 ug/mL or <400 ng/L
FSP Increased fibrinolysis Condition
DIC
Primary fibrinolysis
Pulmonary embolism
FSP Decreased or Incomplete Condition
Liver Disease
False Increase to Clotting Times
Hematocrit >55%
Unreliable Clot Times
Hematocrit <25%
Specimen Anticoagulant
Sodium Citrate
1:9 of citrate:blood
Chelates Calcium
Adjustment for Hematocrit Calculation
(0.5 x (100-patient hct))/60
Testing Correction with Normal Plasma
Mix equal volumes of Pooled Normal Plasma (NP) to patient plasma
Correction: within 2 sec PT within 5 sec APTT
Correction with NP
Consistent with Factor deficiencies
No/Partial Correction with NP
Consistent with presence of circulating anticoagulants
- Heparin, Coumadin, FSPs, Lupus Inhibitor, Factor Inhibitors
Testing Correction with Saline
Mix with equal volumes of Normal Saline and Patient Plasma
Correction: within 2 sec of PT or within 5 sec of APTT
Full/Partial Correction with Saline
Consistent with presence of circulating inhibitors
No Correction with Saline
Consistent with Factor deficiencies
Correction with NP, No with Saline
PT prolong, APTT normal
- Possible Factor 7
PT normal, APTT prolong
- Possible deficiency of PK, HMWK, 12, 11, 9, 8
PT prolong, APTT prolong
- Deficiencies of any factor(s)
Pre Kallekrein Screening
Repeat APTT with increased incubation time
- Allows for more contact activation
- If correction is seen, evidence of deficiency
Pre Kallekrein Followup Test
PK Assay
Factor VIII:c Inhibitor
Most Common specific inhibitor
Reasons for 8:c Inhibitor
Hemophiliacs
Drugs
Post partum women
Autoimmune disease
Lymphoproliferative disease
Unknown
Clinical Signs of 8:c Inhibitor
No response to therapy
Severe bleeding
Hemarthrosis (bleeding into joints)
Laboratory Signs of 8:c Inhibitor
Increased APTT, normal PT
Correction with NP at 0 hrs
No correction with NP at 2 hrs
* TIME AND TEMPERATURE DEPENDENT *
Bethesda Titer
Determine the strength of Factor 8:c inhibitor
Bethesda Test
Equal volumes patient plasma and NP
Incubate for 2 hrs at 37 C
Factor assay test to determine amount of residual 8:c
Other Specific inhibitors
Possible with any inherited factor deficiency
Caused by exposure to factor through treatment
Not time or temperature dependent
Heparin
Acts as co-factor with Antithrombin
Makes Antithrombin 280x more effective
Most common cause of prolonged APTT in hospitalized patients
Monitored by Heparin assay, Xa assay, or APTT
Heparin Laboratory Signs
Prolonged APTT, PT, TT
No Correction with NP
Correction/partial with Saline
Heparin Correction Testing
Protamine sulfate
Reptilase Time
Low Molecular Weight Heparin
Inactivates platelet bond with Xa and resists PF4 inhibition
No interference with platelets or endothelial cells
Less antigenic
Laboratory monitoring not required
Coumadin (Coumarin or Warfarin)
Vitamin K antagonist
Common cause of prolonged PT
Monitored with PT and INR
Coumadin Laboratory Signs
Prolonged PT, APTT
Correction with NP
No Correction with saline
Fibrin Split Products (FSP)
Acts by complexing with fibrin monomers and preventing polymerization
FSP Laboratory Signs
Prolonged PT, APTT, TT
No correction with NP
Correction/Partial with saline
Lupus Inhibitor (Anticoagulant)
Second most common cause of prolonged APTT
Antibody (IgM, IgG) directed against phospholipid
Inhibits reagent in APTT testing
Lupus Inhibitor Causes
SLE
Drugs
Infections
Lymphoproliferative Disease
Unknown
Lupus Inhibitor Clinical Signs
No Bleeding problems
Thrombosis
Possible increased platelet aggregation
Lupus Inhibitor Laboratory Signs
Prolonged APTT
No correction with NP
Correction/Partial with Saline
Correction with platelet Neutralization procedure
Tissue Thromboplastin Inhibition Test (TTIT)
Used for Lupus Anticoagulant (LA)
PT with dilute thromboplastin (contains phospholipids)
- LA will cause greater prolongation of PT
Results are compared with normal control
TTIT Calculation
Dilute PT/Orig PT
Ratio >1:3 is consistent with LA
Dilute Russell Viper Venom Time (RVVT)
Used in LA testing
Dilute RVV + phospholipid + CaCl2 + patient plasma
RVV directly activates X
if dRVV is higher than normal control = evidence of LA
Test Results Consistent with Heparin
FSP - normal
TT - corrected with protamine sulfate
PNP - NA
Immunological Assays
Laurel Rocket
PIVKA (Prothrombin Induced VItamin K Antagonist)
Laurel Rocket Assay
Antibody to factor in agar
Serial dilutions of NP, Control plasma, and patient are electrophoresed
Antigen/Antibody complex precipitate and are stained
Factors Tested Using Laurel Rocket
VWF:Ag
VIII Ag
IX Ag
PIVKA
Antibody to factor 2 in agar
Patient plasma is adsorbed with barium carbonate to remove carboxylated factor 2
Adsorbed sample is electrophoresed
Reptilase Time (RT)
Snake venom - Bothrox atrox
Clots fibrinogen in different way than thrombin
Increased RT Associated with
FSPs
Dysfibrinogenemia
Hypofibrinogenemia
RT Removes Interference
Heparin
Factor Assays
Reagents for PT or APTT
Factor deficient plasma - contains 100% factor of all factors except for specific one
Pooled Normal Plasma
Factor XIII (13) Assay
Not tested by PT or APTT
5M Urea or Clot Solubility Test
5M Urea or Cold Solubility Test
Patient Plasma + CaCl2 = clot
Add 5M urea
Observe clot for lysis
Clot lysis within 24 hrs indicates <5% XIII activity or inhibitor
Factor Antigenic Testing
Determines AMOUNT of protein present, not activity
Stypven Time (ST)
Also called Russell Viper Venom Time (RVVT)
Snake venom - Viper russellii
Activates coagulation pathway at X, bypassing 7
ST Interpretation
Prolonged PT and normal ST = 7 deficiency
Prolonged PT and ST = common pathway deficiency
Activated Clotting Time (ACT)
Screening test for coagulation system
Whole blood clotting time (performed at bedside)
ACT Testing
Venous sample
Tube contains silaceous earth
Normal Clot time with ACT
80-125 seconds
VIII: VWF:Rco
Determines activity of the VWF portion of factor 8 molecule
Serial dilutions of normal plasma, control, and patient
Dilution + ristocetin + reagent platelets
Observe for platelet aggregation
