Lab testing in Coagulation Flashcards
Coagulation testing temperature
always at 37 C
Coag specimen
plasma w/ sodium citrate (3.2%) at 9:1 ratio
requires Ca2+ to be added to the testing bc sodium citrate will bind all available calcium
Platelet-Poor plasma (PPP)
plasma containing less than 10x10^9/L platelets
Platelet-Rich Plasma (PRP)
plasma containing ~200-300x10^9/L
Testing of Primary Hemostasis
peripheral smear, platelet count, platelet aggregation, bleeding time & platelet function analyzer, platelet secretion studies, flow cytometry
Specimen problems w/ platelet counts & estimates
clumping- due to capillary puncture
satellitism - caused by EDTA & leads to falsely low counts
giant platelets- counted as WBC; falsely low count
fragmented RBCs- ocunt as plt; falsely elevated count
Platelet aggregation studies
addition of aggregating reagent to PRP - measure change in transmittance
2 waves: primary & secondary
* know charts on slide 21 in lab testing pdf
Bleeding time
measure of platelet function
3 types: duke, ivy, template (!)
prolonged w/ aspirin etc
Bleeding time: template
standardized back pressure & standardized depth
normal bleeding time 1-9 minutes
not very sensitive
should NOT be performed if plt count is less than 100x10^9/L
Platelet function analyzer
replaces the BT- eliminates many of the variables
pumps whole blood (in citrate) through each of 2 apertures containing either : collagen/epi or collagen/ADP
& measures time necessary for plt to occlude the aperture
Flow cytometry for primary hemostasis
useful for GPlb/IX deficiency (Bernard-Soulier) & GPIIB/IIIa deficiency (Glanzmann’s thrombasthenia)
Testing of Extrinsic pathway
prothrombin time (PT)
Prothrombin time (PT)
screen for inherited or acquired deficiencies in the extrinsic & common pathway
measures factors: I, II, V, VII & X
monitors oral anticoagulant therapy- coumadin /warfarin
range 10-13 seconds
PT procedure
specimen & reagents & 37C
plasma specimen + thromboplastin reagent (contains Ca2+)
PT & INR
INR is the international normalized ratio
used to correct for differences in coag instruments
INR formula
INR = (PT patient/ PT normal) ^ISI
0 to >6
theraputic is 2-3
PT sources of error
short draw - falsely shorten the PT
delay between collection & teting may lead to decreases in factor V
patient high hematocrit- can lead to prolonged PT
Testing of Intrinsic pathway
APTT or PTT-performed by adding platelet phospholipid substitute & contact activator (APTT reagent) & Ca2+ to activate factor XII
APTT
screen for intrinsic & common pathway
measures all factors except VII (extrinsic) & XIII
28-35 seconds
monitors heparin therapy
sources of error for APTT
blood collection error (short draw)
hematocrit
sample processing etc
Activated Clotting time (ACT)
used to monitor the effectiveness of high dose heparin therapy
not performed in the clinical lab - point of care test
Thrombin Clotting time
thrombin time TT, TCT
measures clotting time of the last step in the coag cascade- fibrinogen (!) to fibrin by thrombin (test of common pathway)
used to detect dysfibrinogenemia
TCT source of error
collection etc
can be falsely prolonged due to heparin
Reptilase Time
clotting time similar to thrombin time except that a snake venom is used instead of thrombin
reptilase is a thrombin like enzyme
NOT PROLONGED BY HEPARIN
Fibrinogen Assay
recommended test for estimating fibrinogen level
high [thrombin] is inversely proportional to [fibrinogen]
thrombin reagent is 25x more concentrated than in TCT, patient plasma needs to be diluted
APTT normal &
PT abnormal
possible causes:
- factor deficiency in extrinsic pathway probably VIII
- specific factor inhibitor
APTT abnormal &
PT normal
possible causes:
- Factor deficiency in intrinsic pathway (XII, XI, kallikrein, IX, or VIII)
- Specific factor inhibitor
- lupus anticoagulant
APTT abnormal
PT abnormal
TCT normal
possible causes:
- factor deficiency in common pathway
- vitamin K defect
- liver disease
- inhibitor present
APTT abnormal
PT abnormal
TCT abnormal
possible causes:
- factor deficiency (I)
- severe liver disease
- DIC
- inhibitor
Test to evaluate specific factor deficiency
when PT &/or APTT are prolonged
- mixing studies-differentiate factor def. from inhibitor
- specific factor assay
Mixing study
performed to differentiate between factor deficiency & inhibitor
this procedure will correct the prolonged PT or APTT if it is caused by a deficiency of one or more of the coag factors
Mixing study precedure
patient’s plasma mixed 1:1 w/ normal plasma
if normal then factor deficiency is suspected
if NOT normal then suspect an inhibitor
rerun after heating for 2 hours
if APTT is still normal = factor def.
then perform specific factor assay!
Mixing study results for Factor deficiency
immediate mixing study: corrected
after 2 hour incubation: corrected
Mixing study results for Lupus-like anticoagulant
immediate mixing study: no correction
after 2 hour incubation: no correction
Mixing study results for Specific inhibitor (factor VIII)
immediate mixing study: correction
after 2 hour incubation: no correction
Factor Assay
performed to confirm a specific factor deficiency & to determine the actual activity of that factor w/in the plasma
the degree of correction produced w/ the patient’s plasma is compared to a reference curve
most likely will be Factor VIII
Fibrinolytic System
2 major tests:
Fibrin degradation products & D-dimer
Fibrin Degradation Products (FDPs)
Plasmin cleaves fibrin & yields FDPs
normally D & E fragments do not reach plasma concentrations of 2 um/mL
assay test based on antigen-antibody reaction
Increased FDP is seen in:
DIC, deep vein thrombosis, pulmonary embolism, liver disease, alcoholic cirrhosis, kidney disease, etc etc etc
D-dimer Assay
more specific test for plasmin lysis of fibrin beginning to replace FDPs excellent marker for DIC (positive) 3 methodologies: semiquantitative assay quantitative assay latex agglutination
Testing of Thrombophilia
antithrombin assays
protein C assays
protein S assays
Mechanical clot detection
incorporates a change in electrical conductivity between 2 metal electrodes immersed in a solution
when clot formed, fibrin strands act as conductor between probes causing constant electrical current
Photo-optical detection of clot formation
measured by a change in optical density of a test sample
widely used today
Chromogenic detection of clot formation
color intensity is measured spectrophotometrically & is directly proportional to the concentration of chromophore tag
Immunologic detection methods of clot formation
antigen level is proportional to the amount of light absorbed
