(LAB) CHAPTER 41: Laboratory Evaluation of Hemostasis

Blood specimen collection and management

________ and _______ are the most vulnerable stages of the hemostasis blood testing process because every stage is manual and thus error-fraught

platelet- poor plasma

Most hemostasis procedures are performed on _______ prepared from venous whole blood collected by venipuncture

10,000/uL

A platelet poor plasma (PPP) is a plasma with a platelet count of less than _________

23-gauge

In the case of small, scarred, or fragile veins, the collec- tor may fit a ______ Luer-adapter needle or _____ "butterfly" needle infusion tube to a syringe, which enables the collector to reduce and control the negative collection pressure.

20 or 21 gauge ; 1.0 or 1.25 inches long

preferred needle gauge and length for adult with good veins

23-gauge, winged-needle set

preferred needle gauge and length for child or adult with small, fragile, or hardened veins

19-gauge

needle gauge for transfer of blood from syringe to tube

blue top ; sodium citrate

Most hemostasis specimens are collected in plastic _________ sterile evacuated blood collection tubes containing a measured volume of buffered _____ anticoagulant.

Siliconized

_______ (plastic-coated) glass tubes are available, but their use is declining because of concern for potential breakage, with consequent risk of exposure to bloodborne pathogens

calcium

Sodium citrate binds ______ ions to prevent coagulation, and the buffer stabilizes specimen pH as long as the tube closure remains in place.

9 parts blood : 1 part anticoagulant

Upon collection, the anticoagulant solution mixes with blood to produce a _____ ratio

55%

The 9:1 blood-to-anticoagulant ratio is effective, provided the patient's hematocrit is ____% or below

prolonged

In polycythemia the decrease in plasma volume relative to whole blood unacceptably raises the anticoagulant-to-plasma ratio, which causes falsely ______ results for clot-based coagulation tests.

C = (1.85 x 10^-3) (100 - HCT) V

Where:

C = volume of sodium citrate in mL,

V = volume of whole blood-sodium citrate solution in mL

HCT = the hematocrit in %

Formula used to compute for amount of anticoagulant

ethylenediaminetetraacetic acid (EDTA, purple/lavender top)

_______ is not used for anticoagulated specimens for clot-based coagulation testing because it irreversibly chelates calcium ions.

calcium chloride

Calcium ion chelation with citrate is reversed with the addition of ________.

heparinized (green top)

__________ specimens have never been validated for plasma coagulation testing but may be necessary to produce accurate platelet counts in cases of platelet satellitosis (satellitism) as a substitute for specimens collected in EDTA.

short draw ; falsely prolonged

Specimen rejection: _______ is the error when the whole-blood volume is less than 90% of required volume or less than manufacturer specified minimum.

> PT and PTT are falsely ______

rejected ❌❌❌

Each specimen must be inspected visually before centrifugation or during analysis. When a specimen clot occurs, even if just small, the specimen must be _____ because it causes interference.

unpredictable hemostasis

Specimen Rejection: Pink or red plasma indicates in vitro activation of platelets and coagulation; causing ____________ test interference. Further, hemolysis interferes with optical endpoint coagulometer results.

lipemic or icteric

Specimen rejection: The specimen is _____ or _____ when optical instruments may fail to measure clots in cloudy or highly colored specimens. Interferes with chromogenic substrate methods. The practitioner must employ an electro-mechanical detection method instrument.

Tourniquet application > 1 minute ; falsely shortening

Specimen rejection: _____ is the error that causes blood stasis (slows venous flow), resulting to activated endothelial cells and hemoconcentration (elevated factors V, VII, VIII, XII, VWF and fibrinogen)

> Falsely _______ clot-based test results.

Specimen storage at 1-6 degree Celsius

Specimen rejection: ______ causes precipitation of large von Willebrand factor multimers, activation of coagulation factor VII, activation of platelets, and destruction of platelet integrity

Specimen storage at > 25 degree Celsius

Specimen rejection: ______ causes coagulation factors V and VIII to deteriorate

cocktail shaking

Excessive specimen agitation, called "________," causes hemolysis (RBC rupture), procoagulant activation, and platelet activation.

5 mL of saline

Before blood is collected for hemostasis testing during specimen collection from vascular access devices [i.e. heparin or saline locks, ports in intravenous (IV) lines, peripherally inserted central catheters (PICC tubes), central venous catheters, or dialysis catheters]:

The line must be flushed with ________, and the first 5 mL of blood, or six times the volume of the blood collection tube, must be collected and discarded (do not flush with heparin)

Once flushed, and before the IV drip is started, collect specimen via syringe, exerting minimal negative pressure.

24

Specimens collected for PTs may be held uncentrifuged or centrifuged at 15° C to 25° C and tested within ___ hours of the time of collection.

4

Specimens collected for PTTs and most other clot-based assays are also held uncentrifuged or centrifuged at 15° C to 25° C, but they must be tested within ___ hours of time of collection.

1

If a patient is receiving unfractionated heparin (UFH) therapy, PTT specimens must be centrifuged within ___ hour of the time of collection to avoid depletion of the heparin.

1

If any plasma-based hemostasis assay cannot be completed within the prescribed interval, the practitioner must centrifuge the specimen within ___ hour of collection.

plastic

Plasma is transferred by plastic pipette to a ____ freezer tube, taking care to avoid stirring up the buffy coat.

intrinsic

Never use glass containers because glass stimulates the ______ coagulation pathway.

-20° C

Plasma may be stored at _____ for up to 2 weeks

-70° C

Plasma may be stored at _____ for long-term

VWF

To avoid cryoprecipitation of ____, specimens may not be frozen and thawed more than once.

1

At the time the test is performed, the frozen specimen is thawed rapidly at 37° C, mixed, and tested within ____ hour of the time it is removed from the freezer.

1° C to 6° C ; 2 hours

If specimen cannot be tested immediately, it may be stored at _____ for up to ___ hours after thawing.

si julia

sino ang baho lubot

Platelet function tests

designed to detect qualitative (functional) platelet abnormalities in patients who are experiencing the symptoms of mucocutaneous bleeding

50,000/mL

Qualitative platelet abnormalities are suspected only when bleeding symptoms are present and the platelet count exceeds ______

platelet count ; blood film

Before platelet function tests begin, ____ is performed and _____ is reviewed.

Bleeding time test

Obsolete test, was the original test of platelet function

2-9 minutes

Reference interval for bleeding time test

Duke (1912)

____ first described the bleeding time test in ____

Ivy (1941)

____ modified the bleeding time test in ____

Mielke

1969, ____ attempted to standardize the bleeding time by specifying a lan.cet that used a template to establish incision depth and applying a blood pressure cuff inflated to 40 mm Hg to the upper arm.

prolonged

A ______ bleeding time could theoretically signal von Willebrand disease (VWD), Glanzmann thrombasthenia functional platelet disorder); or scurvy or vasculitis (vascular disorder), and was thought to have a predictable result in therapy using aspirin and other NSAIDs.

(1) nonplatelet variables of intracapillary pressure,

(2) skin thickness at the puncture site,

(3) size and depth of the wound

These affect the bleeding time and interfere with accurate interpretation of test results

platelet aggregation

In ________, functional platelets adhere to subendothelial collagen, aggregate with one another, and secrete the contents of their a-granules and dense granules.

adhesion, aggregation, secretion

Platelet ______, _______, and _______ are assessed using platelet aggregometry.

(1) platelet membranes and platelet activation pathways are intact

(2) plasma fibrinogen concentration is normal

(3) normal secretions are released from platelet granules

Normal aggregation requires:

light-transmittance aggregometer

Platelet-rich plasma (PRP) aggregometry is performed using a specialized photometer called a ______________ distributed by Chrono-Log or BioData

500

[In PRP aggregometry]

Pipette (usually) ____ uL of PRP ➡️ drop 1 clean plasticized stir bar ➡️ place cuvette into incubation well ➡️ warm to 37 degree celsius for 5 minutes ➡️ transfer to reaction well

800 to 1200

[In PRP aggregometry]

After transferring to reaction well, PRP is stirred at _______ rpm. As this occurs, the recorder tracing stabilizes to generate a baseline, near 0% light transmittance.

agonist

[In PRP aggregometry]

After a few seconds, the operator pipettes an ______ (platelet activator) directly into the sample to trigger aggregation

(1) spherical ; pseudopod extension

(2) degree of shape change

[In PRP aggregometry]

In a normal specimen, after addition of agonist:

(1) shape of suspended platelets change from discoid to _____ followed by _____________,

(2) intensity of light transmittance initially (and briefly) decreases, THEN increases in proportion to the _________

100

[In PRP aggregometry]

As platelet aggregates form, more light passes through the PRP (platelets form large clumps that allow more light transmission) and the tracing begins to move toward ____% light transmittance.

40

[In PRP aggregometry]

Platelet function deficiencies are reflected in diminished or absent aggregation; many laboratory directors choose ____% aggregation as the lower limit of normal.

electrical impedance

In whole-blood platelet aggregometry, platelet aggregation is measured by __________

normal saline

[In whole-blood platelet aggregometry]

Specimens are diluted 1:1 with _______ and tested immediately.

300-500

[In whole-blood platelet aggregometry]

Pipette aliquots of properly mixed whole blood suspension + add equal volume saline (suspension volume may be _____ uL) ➡️ drop stir bar ➡️ place cuvette into incubation well ➡️ warm to 37 degree celsius for 5 minutes ➡️ transfer to reaction well

cartridge-mounted

[In whole-blood platelet aggregometry]

After transferring to reaction well, pipette an agonist directly into the specimen, and suspend a pair of low voltage ____________ disposable direct current electrodes in the mixture

electrodes

[In whole-blood platelet aggregometry]

As aggregation occurs, platelets adhere to the _______ and one another, impeding the current.

directly proportional

[In whole-blood platelet aggregometry]

The rise in impedance is __________ to platelet aggregation (amplified and recorded by instrument circuitry)

*impedance is measured in Ohms

True

True or False: whole-blood aggregometry tracing closely resembles a PRP-based light-transmittance aggregometry tracing

Resting platelet; stable baseline

*baseline at 0% aggregation

What is the 1st phase of platelet aggregation?

Shape change

*occurs after addition of agonist

What is the 2nd phase of platelet aggregation?

Primary aggregation

What is the 3rd phase of platelet aggregation?

Secretion

*release of adenosine diphosphate (ADP) & adenosine triphosphate (ATP) from platelet granules

What is the 4th phase of platelet aggregation?

Secondary aggregation

*formation of large clumps

What is the 5th phase of platelet aggregation?

light transmittance

The percentage of aggregation is measured by intensity of _______________________ through the specimen.

Platelet lumiaggregometry

differs little from light-transmittance or whole blood aggregometry, but because of its detection properties, it simplifies the diagnosis of platelet dysfunction

platelet dense granules

[In platelet lumiaggregometry]

The Chrono-Log Model 700 Whole Blood/Optical Lumi- Aggregometer is used for the simultaneous measurement of platelet aggregation and the secretion of adenosine triphosphate (ATP) from activated _________.

luciferin-luciferase

[In platelet lumiaggregometry]

As ATP is released, it oxidizes a firefly derived ______________ reagent (Chrono-Lume from Chrono-Log) to generate cold chemiluminescence PROPORTIONAL to the ATP concentration

luminescence

[In platelet lumiaggregometry]

The photodetector amplifies the _________, which is recorded as a second tracing on the aggregation report

second

[In platelet lumiaggregometry]

Add ATP standard to first sample ➡️ add luciferin-luciferase & test for full luminescence ➡️ add luciferin- luciferase + agonist to ______ sample (instrument monitors for aggregation and secretion simultaneously)

Thrombin (thrombin receptor-activating peptide, TRAP)

[In platelet lumiaggregometry]

________ (or _______________, _______) is typically the first agonist used because it induces full secretion.

1 ; 1-2

[In platelet lumiaggregometry]

___ unit/mL of thrombin will be used to induce the release of _____ nM of ATP, detected by luminescence of firefly luciferin-luciferase. Luminescence will be measured, recorded, and used for comparison with the luminescence produced by additional agonists.

50

Normal secretion induced by agonists (other than thrombin) produces luminescence at a level of about ___% of that resulting from thrombin.

True

T or F: Thrombin-induced secretion may be diminished to less than 1 nM in storage pool deficiencies, but it is relatively unaffected by membrane disorders or pathway enzyme deficiencies.

True

T or F: The agonists used to activate platelets are thrombin or synthetic TRAP, adenosine diphosphate (ADP), epinephrine, collagen, arachidonic acid, and ristocetin.

True

T or F: Laboratory practitioners use platelet agonists to test for abnormalities of specific membrane binding sites.

Arachidonic acid

_________________ is the agonist used to check for deficiencies in the eicosanoid synthesis pathway, including deficient or aspirin-suppressed cyclooxygenase which result in reduced aggregation and secretion

Ristocetin

____________ is used to check for abnormalities of plasma VWF in VWD

Lumiaggregometry

__________________ provides more definitive information for conditions where recording platelet secretion, in addition to platelet aggregation, is an important diagnostic factor.

PAR-1 and PAR-2

Thrombin (or TRAP) cleaves the ______ and ______, both members of the seven-transmembrane repeat receptor family.

saline

Reagent thrombin:

> stored dry at -20° C or -70° C

> reconstituted with physiologic ______

> leftover reconstituted thrombin may be aliquoted and frozen for later use

True

T or F: Thrombin has the disadvantage that it often triggers coagulation (fibrin formation) simultaneously with aggregation, abolishing the value of the aggregation tracing.

ADP (adenosine diphosphate)

_______ is the most commonly used agonist, particularly in aggregometry systems that measure only aggregation and not luminescence.

-20° C or -70° C

Reagent ADP:

> stored at ______ or ______

> reconstituted with physiologic saline

> leftover reconstituted ADP may be aliquoted and frozen for later use

thienopyridine antiplatelet drugs:

clopidogrel & prasugrel , and NSAIDs

Platelet aggregometry is employed to monitor response to which antiplatelet drugs

Primary aggregation

__________ involves shape change with formation of microaggregates; both are reversible processes

Secondary aggregation

____________ is full platelet aggregation after release of platelet dense granule ADP

Secretion

__________ is absent in storage pool deficiency when thrombin or TRAP is used as the agonist.

Epinephrine

__________ binds platelet a-adrenergic receptors, identical to muscle receptors, and activates the platelets through the same metabolic pathways as reagent ADP

1° C to 6° C

Epinephrine:

> stored at ____ to ____

> reconstituted with distilled water immediately before use

> aliquots of leftover reconstituted epinephrine may be frozen for later use

aggregation

The results of epinephrine-induced aggregation match those of ADP, except that epinephrine cannot induce ___________ in storage pool disorder or eicosanoid synthesis pathway defects no matter how high its concentration. Hence, epinephrine does not work in whole-blood aggregometry.

collagen

_______ binds GPIa/IIa and GPVI, but it does not induce primary aggregation.

may not

Collagen:

> stored at 1° C to 6° C

> no further dilution

> (may or may not?) be frozen

aspirin

Loss of collagen-induced aggregation may indicate a membrane abnormality, storage pool disorder, release defect, or the presence of __________