Chapter 41: Laboratory Evaluation of Hemostasis

MTY1215 Hematology 2 Laboratory [Midterm]

Mr. Sam Kevin Saclayan

OUTLINE § Add 500uL PRP to instrument-

I. Platelet Function Test compatible cuvette
A. Bleeding time § Drop 1 clean plasticized stir bar
B. Platelet Aggregometry § Incubate for 5 minutes at 37C
C. Von Willebrand Factor Activity Assay § Place sample in the reaction well and
D. Heparin-induced Thrombocytopenia start the stirring device and recording
Assay computer
II. Platelet Activation Markers • 800-1200rpm
III. Clot-Based Plasma Screening Tests for § After the recorder tracing stabilizes,
Coagulation Disorders add an agonist (platelet activator)
A. Prothrombin Time o Collagen, epinephrine, thrombin, and
B. Partial Thromboplastin Time track
C. PTT Mixing Studies
D. Thrombin Clotting Time
E. Venom Activated Assays

PLATELET FUNCTION TEST

- Designed to detect qualitative platelet
abnormalities
- Patient with symptoms if mucocutaneous
bleeding
- Qualitative platelet abnormalities
- Presence of bleeding symptoms: plt ct.
>50,000/uL

BLEEDING TIME
- Function test for platelet
- Obsolete
o But done with patient who undergo
surgery
- Original test of platelet function
- Reference interval = 2-9 minutes
- Affected by:
o Intracapillary pressure
o Skin thickness
o Size and depth of the wound
o Prolonged by MedTech: squeeze out the
blood

PLATELET AGGREGOMETRY
- Used to detect platelet adhesion, aggregation,
and secretion
- High-complexity test
- Requires a skilled, experienced operator
- Requested on cases that require specific
result for specific diagnosis
o For research purposes
- Platelet aggregometry using PRP
o Light – transmittance aggregometer
o Procedure:
§ Calibrate the instrument

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 Chapter 41: Laboratory Evaluation of Hemostasis
MTY1215 Hematology 2 Laboratory [Midterm]
Mr. Sam Kevin Saclayan

- Whole blood platelet aggregometry

o Measured by electric impedance
o Procedure:
§ Dilute (1:1) specimen using normal
saline solution
• 300 – 500 uL
§ Drop 1 stir bar
§ Incubate for 5 minutes at 37C
§ Transfer cuvette to a reaction well
§ Add agonist
§ Suspend a pair of low-voltage
cartridge-mounted disposable direct
current electrodes in the mixture
o Direct proportion to platelet activity
- Platelet lumiaggregometry
o Use for simultaneous measurement of
platelet aggregation and secretion of ATP
from activated platelet granules
o Uses PRP
o Procedure:
§ Prepare 2 aliquots of the sample
§ Add ATP to the first sample and
luciferin-luciferase
§ Test for full luminescence
§ Add luciferin-luciferase and an
agonist on second sample
o Shows full aggregation, eicosanoid
pathway

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 Chapter 41: Laboratory Evaluation of Hemostasis
MTY1215 Hematology 2 Laboratory [Midterm]
Mr. Sam Kevin Saclayan

VON WILLEBRAND FACTOR ACTIVITY ASSAY PLATELET ACTIVATION MARKERS

- ADAMPS – cleaves into smaller VWF to blood - PF4
clotting o Elevated plasma levels = thrombotic
- Quantitative and qualitative assay stroke or coronary thrombosis
- Requires panel of quantitative and functional § Reflecting raised platelet activation
assays o PF4 immunoassay (Hyphen BioMed)
o VWF antigen (VWF:Ag) immunoassay § Uses CTAD tubes
o Clot based coagulation factor VIII assay • Prevents in vitro platelet
o VWF ristocetin cofactor assay activation
(VWF:RCo) - Thromboxane A2
o Ristocetin-induced platelet aggregation o Half-life of 30 seconds
assay (RIPA) o Active product of eicosanoid pathway
§ Ristocetin response curve o Spontaneously reduces to thromboxane
§ For diagnosis of VWD subtype 2B B2
- If madami ung nasa test baka mag cause ng § No immunoassay available
pathologic clotting o 11-dehydrothromboxane B2
- Fewer can be detrimental § Stable and measurable
- Manual ELISA § Used to characterize In vivo platelet
o Reflect VWF activity rater that activation
concentration and offer improved § Random urine sample
precision when compared to the § May be used to monitor aspirin
VWF:Rco therapy and to identify cases of
o VWF activity immunoassay aspirin therapy failure
§ Uses monoclonal antibody specific for • Aspirin = painkiller, side effect =
an active VWF epitope platelet activation
§ Produced by natural rapid test for
animals CLOT BASED SCREENING TEST FOR
• 1 rabbit = 1 antigen COAGULATION DISORDERS
o VWF collagen binding assay - Lee-White whole-blood coagulation time test
§ Mimics VWFs in vivo collagen o First in vitro clot procedure
adhesion property o The time interval from the initiation of
clotting to visible clot formation reflects
HEPARIN-INDUCED THROMBOCYTOPENIA the condition of the coagulation
ASSAY mechanism
- Aggregation-based tests - Activated clotting time (ACT) test
o Assess the functional platelet response o Uses a particulate clot activator in the
to the HIT antibodies test tube that speed the clotting process
o Includes o Used as a POCT assay to monitor UFH
§ Light transmittance aggregometry therapy in high-dose application, cardiac
§ Washed platelet light transmittance catheterization
aggregometry
§ Whole blood aggregometry PROTHROMBIN TIME
§ Washed platelet carbon-14 serotonin - PT reagents (thromboplastin or tissue
assay (SRA) thromboplastin)
• Aggregometry assay that - Components:
measures platelet activation and o Recombinant or affinity purified tissue
secretion induced by HIT factor
antibodies in the presence of § Para hindi magdeteriorate ang tissue
heparin factor
• Reference and confirmatory § Para mastablized ang tissue factor
method for Hit § Tissue factor = rabbit ring or lungs of
o Use UFH as agonist rabbit

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 Chapter 41: Laboratory Evaluation of Hemostasis
MTY1215 Hematology 2 Laboratory [Midterm]
Mr. Sam Kevin Saclayan

o Phospholipids
o 0.025M calcium chloride
- PT assay
o Specimen: PPP
o Procedure
§ Incubate PT reagent at 37C
§ An aliquot of test PPP (50 or 100 ml)
Is transferred to the reaction vessel
and incubated at 37C for at least 3-
10mins
§ A pre-measured volume of reagent
(100 or 200 ml) is speedily and
directly added to the PPP aliquot, and
a timer is started
§ As the clot forms, the timer stops, and
the elapsed time is recorded
o QC reagent
§ Commercially prepared normal and
prolonged control PPP specimens
(either frozen or lyophilized)
o Frequency
§ 8-hour shift or with each change of
reagent
§ For calibration
o PT ref range
§ 12.6 to 14.6 seconds
o Reporting
§ Nearest tenth of a second
o Use INR if PT is used for Coumadin
monitoring

PARTIAL THROMBOPLASTIN TIME

- To monitors the effects of UFH and to detect
- PT as a diagnostic assay LAC and specific coagulation factor
o Conditions associated with prolonged PT antibodies
§ Liver disease o Antibody VIII antibody
§ Vit k deficiency - Prolonged in all congenital and acquired
§ Congenital single-factor deficiencies procoagulant deficiencies
of factor X, VII, or V, profound o Except for deficiencies of factor VII or XIII
prothrombin deficiency and fibrinogen - PTT reagent
deficiency o Phospholipid
§ Previously called partial
thromboplastin or cephalin
o Negatively charged particulate activator
§ Silica, kaolin, ellagic acid, or celite

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 Chapter 41: Laboratory Evaluation of Hemostasis
MTY1215 Hematology 2 Laboratory [Midterm]
Mr. Sam Kevin Saclayan

- PTT specimen: PPP § Patients with severe hemophilia

- PTT assay - Detection of Lupus Anticoagulants, other
o Procedure Inhibitors, and Factor Deficiencies
§ Initiate contact activation o Procedure:
• 50 or 100 uL of warmed (37C) § Check PTT
reagent containing phospholipid, § Prolonged PTT -> TCT -> check UFH
particulate activator, and equal • TCT exceeds the upper limit of
volume of warmed PPP the TCT reference interval is
§ Incubate for the exact manufacturer- evidence for UFH
specified time: 3 minutes § UFH often prolongs the TCT to 30-40
§ Add 50 or 100 uL of warmed 0.025M seconds
calcium chloride, and a timer is • UFH may be neutralized using
started polybrene or heparinase
§ Fibrin clot forms, timer stops, record (Hepzyme, Siemens)
the interval § PTT mixing studies
o Reference interval § Mixed 1:1 UFH with reagent PPP
§ 26 – 38 seconds (normal)
o Quality control o If the mixture PTT corrects to within 10%
§ Same with PT of the PNP PTT and the patient is
- Monitoring Heparin Therapy with PTT experiencing bleeding, there is a
o Pampalabnaw ng dugo coagulation factor deficiency
o PPT is the standard method for (coagulopathy)
monitoring UFH o PPT corrects
o Used to treat patients with venous § new 1:1 mixture is prepared and
thrombosis, pulmonary embolism, incubated 2 hours at 37C
myocardial infarction, and a variety of o If the incubated mixture’s PTT fails to
thrombotic events correct to within 10% of the PNP PTT
o Typical therapeutic range and the patient is experiencing bleeding,
§ 60 – 100 seconds an inhibitor may be present
- PTT as diagnostic assay
o PTT result is prolonged: THROMBIN CLOTTING TIME
§ Deficiency of one or more of the - Activates factor XIII
following coagulation factors - Reagent
• Prothrombin o Commercially prepared bovine thrombin
• Factors V, VIII, IX, X, XI, or XII reagent at 5 NIH units/mL
§ Fibrinogen when fibrinogen level is - Principle
<100mg/dL o Cleaves fibrinopeptides A and B from
§ Hemophilia A, hemophilia B, and plasma fibrinogen to form a detectable
Rosenthal syndrome fibrin polymer
o Fibrin to polymer
PTT MIXING STUDIES - Procedure
- Lupus anticoagulants o Reagent is warmed at 37C for a
o Directed against a number of minimum of 3 and a maximum of 10
phospholipid-protein complexes minutes
o LAC = nonspecific inhibitors o 100 uL aliquot of patient plasma is
o Signals a potential thrombotic risk incubated at 37C for a minimum of 3 and
- Specific Factor inhibitors a maximum of 10 minutes
o IgG immunoglobulins directed against o 200 uL of thrombin into the PPP aliquot
coagulation factors o Start a timer and record the interval to
o Anti-factor VIII clot formation
§ Most common - Reference Interval
§ Hemophilia A o 15 – 20 seconds

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 Chapter 41: Laboratory Evaluation of Hemostasis
MTY1215 Hematology 2 Laboratory [Midterm]
Mr. Sam Kevin Saclayan

- Reporting
o TCT is prolonged when the fibrinogen
level is less than 100mg/dL
(hypofibrinogenemia)
o Afibrinogenemia and dysfibrinogenemia
o Presence of antithrombotic substances
§ UFH, FDPs, paraproteins: must be
ruled out
o Presence of the oral direct thrombin
inhibitor dabigatran

VENOM ACTIVATED ASSAYS

- Reptilase Time
o Reptilase
§ Isolated from the venom of Bothrops
artox (snake)
§ Catalyzes the conversion of
fibrinogen to fibrin
§ Cleaves only fibrinopeptide A from
the ends of the fibrinogen molecule
o Insensitive to UFH and FXIII deficiency
o Useful for detecting hypofibrinogenemia
or dysfibrinogenemia
o Reptilase time
§ Prolonged in the presence of FDPs
and paraproteins
- Russell Viper Venom Test
o Russell viper venom
§ Daboia russelii viper
§ Triggers coagulation at the level of
factor X
o Assay: Stypven time
o Dilute RVV time is routinely employed to
detect LAC

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