HEMA LAB - Lab Evaluation of Hemostasis Part 2

Platelet function tests

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

50,000/uL

Quali ative platelet abnormalities are suspected only when bleeding symptoms are present and the platelet count exceeds

bleeding time test

now obsolete, was the original test of platelet function.

The phlebotomist used a lancet to make a small, controlled puncture wound and recorded the duration of bleeding, comparing the results with the reference interval of 2 to 9 minutes

2-9 minutes

reference interval of bleeding time test

intracapillary pressure, skin thickness at the puncture site, and size and depth of the wound

The bleeding time is affected by the nonplatelet variables of _______, all of which interfere with accurate interpretation of the test results

Platelet adhesion, aggregation, and secretion

are assessed using platelet aggregometry that can be detected by one of three different types of instruments

Platelet aggregometry

is a high-complexity laboratory test requiring a skilled, experienced operator

light-transmittance aggregometer

PRP aggregometry is performed using a specialized photome er called a ____________ distributed by Chrono-Log or BioData

Platelet Aggregometry Using Platelet-Rich Plasma

After calibrating the instrument in accordance with manufacturer instructions, the operator pipettes the PRP to instrument-compatible cuvettes, usually 500 mL; drops in one clean plasticized stir bar per sample; places the cuvettes in incubation wells; and allows the samples to warm to 37° C for 5 minutes. The operator then transfers the first cuvette, containing specimen and stir bar, to the instrument's reaction well and starts the stirring device and the recording computer. The stirring device turns the stir bar at 800 to 1200 rpm, a gentle speed that keeps the platelets in suspension. After a few seconds, the operator pipettes an agonist (platelet activator) (Table 41.4) directly into the sample to trigger aggregation. In a normal specimen, after the agonist is added, the shape of the suspended platelets changes from discoid to spherical followed by pseudopod extension, and the intensity of light transmittance initially (and briefly) decreases, then increases in proportion to the degree of shape change.

electrical impedance

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

Whole-Blood Platelet Aggregometry

Specimens are diluted 1:1 with normal saline and tested immediately. Suspen ion volume may be 300 to 500 mL. The operator drops in one stir bar per cuvette and places the cuvettes in 37° C incubation wells for 5 minutes. The operator transfers the first cuvette to a reaction well, pipettes an agonist directly into the specimen, and suspends a pair of low-voltage cartridge-mounted disposable direct current electrodes in the mixture.

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 platelet dense granules

Platelet Lumiaggregometry

the operator adds an ATP standard to the first sample and then adds luciferin-luciferase and tests for full luminescence. The operator then adds luciferin uciferase and an agonist to the second sample; the instrument monitors for aggregation and secretion simultaneously

Typical Platelet Impedance Lumiaggregometry Reference Intervals

inhibitor

If the immediate mix PTT corrects, a new 1:1 mixture is prepared and incubated 2 hours at 37° C. If the incubated mixture's PTT fails to correct to within 10% of the incubated PNP PTT, an ____ may be present

Von Willebrand Factor Activity Assays

requires a panel of quantitative and functional assays

VWF antigen (VWF:Ag) immunoassay

the clot-based coagulation factor VIII assay

the functional VWF ristocetin cofactor assay (VWF:RCo)

the dilute ristocetin-induced platelet aggregation assay (RIPA)

The VWF assays include the

dilute ristocetin-induced platelet aggregation assay (RIPA)

also called the ristocetin response curve, which is employed to diagnose VWD subtype 2B

VWF Activity Immunoassay (REAADS von Willebrand Factor Activity enzyme immunoassay)

employs a monoclonal antibody spe ific for an active VWF epitope

VWF Collagen Binding Assay (Technozyme VWF:CBA ELISA Collagen Type I)

mimics VWF's in vivo collagen adhesion property

VWF Activity Immunoassay (REAADS von Willebrand Factor Activity enzyme immunoassay) and VWF Collagen Binding Assay (Technozyme VWF:CBA ELISA Collagen Type I)

reflect VWF activity rather than concentration and offer improved precision when com

ared to the VWF:RCo

light-transmittance aggregometry, washed platelet light-transmittance aggregometry, washed platelet lumiaggregometry, and whole-blood lumiaggregometry

aggregation-based tests for HIT include

washed platelet carbon-14 (14C) serotonin release assay

is an aggrega ion assay that measures platelet activation and secretion in

uced by HIT antibodies in the presence of heparin

UFH

agonist in Heparin-Induced Thrombocytopenia Assays

C-SRA

is available from specialized reference laboratories and is regarded as both the reference and the confirmatory method for a diagnosis of HIT

Elevated plasma levels of PF4

may accompany thrombotic stroke or coronary thrombosis, reflecting raised platelet activation

PF4 immunoassay

is available from Hyphen BioMed and its measurement may be of diagnostic or prognostic significance

CTAD tubes

Blood collectors employ _____ to prevent in vitro platelet activation

Thromboxane A2

the active product of the eicosanoid path ay, has a half-life of 30 seconds, diffuses from the platelet, and spontaneously reduces to thromboxane B2, a stable, measurable plasma metabolite

in vitro platelet activation

Efforts to produce a clinical assay for plasma thromboxane B2 have been unsuccessful be ause specimens must be collected in CTAD tubes to prevent _________ and must undergo a cumbersome extraction step before the assay is performed

Liver enzymes

act on thromboxane B2 to produce an array of soluble urine metabolites, including 11-dehydrothromboxane B2, which is stable and measurable

Immunoassays of urine 11-dehydrothromboxane B2

, are employed to characterize in vivo platelet activation.64 These assays require no special specimen manage

ent and can be performed on random urine specimens

urinary 11-dehydrothromboxane B2 assay

may be used to monitor aspirin therapy and to identify cases of aspirin therapy failure

Lee-White whole-blood coagulation time test

was the first laboratory procedure designed to assess co gulation

is no longer used, but it was the first in vitro clot procedure that employed the principle that the time interval from the initiation of clotting to visible clot for

ation reflects the condition of the coagulation mechanism

activated clotting time (ACT) test

uses a particulate clot activator in the test tube, which speeds the clotting process

is still widely used as a point-of-care assay to monitor UFH therapy in high-dose applications, cardiac catheterization and coronary artery bypass graft surgery

PT reagents

often called thromboplastin or tissue thrombo lastin, consist of recombinant or affinity-purified tissue factor suspended in phospholipids mixed with a buffered 0.025 M so ution of calcium chloride

Prothrombin Time Procedure

The tissue factor-phospholipid-calcium chloride reagent is warmed to 37° C. An aliquot of test PPP, 50 or 100 mL, is trans erred to the reaction vessel, which also is maintained at 37° C. The PPP aliquot is incubated at 37° C for at least 3 and for no more than 10 minutes. Aliquots that are incubated longer than 10 minutes become prolonged as coagulation factors begin to deteriorate or are affected by evaporation and pH change. A premeasured volume of reagent, 100 or 200 mL, respectively, 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

Prothrombin Time Quality Control

The medical laboratory practitioner tests commercially pre

ared normal and prolonged control PPP specimens at the be inning of each 8-hour shift or with each change of reagent

Reporting Prothrombin Time Results and the International Normalized Ratio

The medical laboratory practitioner reports PT results to the nearest tenth of a second, along with the PT reference interval.

When the PT is used for Coumadin monitoring, the INR, detailed in Chapter 40, is employed to normalize results from laboratories around the world

12.6 to 14.6 seconds

Prothrombin Time Reference Interval

Prothrombin Time as a Diagnostic Assay

Acquired multiple deficiencies such as disseminated intravascular coagulation (DIC), liver disease, and vitamin K deficiency all affect factor VII activity and are detected through prolonged PT results

Factors That Interfere with the Validity of Clot-Based Test Results

prolonged

The PT is ______ in congenital single-factor deficien ies of factor X, VII, or V, profound prothrombin deficiency, and fibrinogen deficiency when the fibrinogen level is 100 mg/dL or less

Thrombin Clotting Time Reagent and Principle

Commercially prepared bovine thrombin reagent at 5 National Institutes of Health (NIH) units/mL cleaves fibrinopeptides A and B from plasma fibrinogen to form a detectable fibrin polymer

Partial Thromboplastin Time

is employed to monitor the effects of UFH and to detect LAC and specific coagulation factor antibodies such as antifactor VIII antibody

prolonged

PTT is ______ in all congenital and acquired procoagulant deficiencies, except for deficiencies of factor VII or XIII

phospholipid and negatively charged particulate activator

The PTT reagent contains _____ (previously called partial thromboplastin or cephalin) and a ________ such as silica, kaolin, ellagic acid, or celite in suspension

Partial Thromboplastin Time Procedure

To initiate contact activation, 50 or 100 mL of warmed (37° C) reagent consisting of phospholipid and particulate activator is mixed with an equal volume of warmed PPP. The mixture is allowed to incubate for the exact manufacturer-specified time, usually 3 minutes. Next, 50 or 100 mL of warmed 0.025 M calcium chloride is forcibly added to the mixture, and a timer is started. When a fibrin clot forms, the timer stops, and the interval is recorded

Partial Thromboplastin Time Quality Control

The medical laboratory practitioner tests normal and pro onged control plasma specimens at the beginning of each 8-hour shift or with each new batch of reagent

26 to 38 seconds

Partial Thromboplastin Time Reference Interval

PTT

has been the standard method for monitoring UFH, which is used to treat patients with ve

ous thrombosis, pulmonary embolism, myocardial infarction, and a variety of thrombotic events

60 to 100 seconds

A typical therapeu ic range of PTT is ______; however, the range varies widely and must be established locally

prolonged

The PTT result is _____ when there is a deficiency of one or more of the following coagulation factors: prothrombin; factors V, VIII, IX, X, XI, or XII; or fibrinogen when the fibrinogen level is 100 mg/dL or less

deficiencies of fac ors VIII, IX, or XI; hemophilia A, hemophilia B, and Rosenthal syndrome, respectively

The most common deficiencies that are reflected in an isolated prolonged PTT are

Lupus Anticoagulants

are IgG immunoglobulins directed against a number of phospholipid-protein complexes

are called nonspecific inhibitors because they have a variety of target antigens

potential thrombotic risk

Every acute care laboratory provides a means for their initial detection as their presence signals a

Specific factor inhibitors

are IgG immunoglobulins directed against coagulation factors

Anti-factor VIII

the most common of the specific inhibitors, is detected in 10% to 20% of patients with severe hemophilia and anti-factor IX is detected in 1% to 3% of factor IX-deficient patients

10%

If the mix ure PTT corrects to within ___ of the PNP PTT (or to within the reference interval) and the patient is experiencing bleeding, the operator presumes there is a coagulation factor deficiency (coagulopathy

Thrombin Clotting Time Procedure

Reagent thrombin is warmed to 37° C for a minimum of 3 and a maximum of 10 minutes. Thrombin deteriorates during incu

ation and must be used within 10 minutes of the time incuba ion is begun. An aliquot of patient plasma, usually 100 mL, is also incubated at 37° C for a minimum of 3 and a maximum of 10 minutes. The operator pipettes 200 mL of thrombin into the PPP aliquot, starts a timer, and records the interval to clot forma ion

15 to 20 seconds

Thrombin Clotting Time Reference Intervals

prolonged

The TCT is ______ when the fibrinogen level is less than 100 mg/dL (hypofibrinogenemia) or in the presence of antithrombotic materials such as FDPs, paraproteins, or UFH

TCT

may also assess the presence of the oral direct thrombin inhibitor dabigatran

Reptilase

is a thrombin-like enzyme, batroxobin, isolated from the venom of Bothrops atrox that catalyzes the conversion of fibrinogen to fibrin (Pefakit Reptilase Time; Pentapharm)

this enzyme cleaves only fibrinopeptide A from the ends of the fibrinogen molecule, whereas thrombin cleaves both fibrinopeptides A and B.

reptilase time test

useful for detecting hypofibrinogenemia or dysfibrinogenemia in patients receiving UFH therapy, and it may be used to work up a bleeding patient whose factors VIII, IX, and XI are normal. The reptilase time is prolonged in the presence of FDPs and paraproteins

Russell viper venom (RVV) from the Daboia russelii viper

which triggers coagulation at the level of factor X, was once used as an alternative to the PT. The assay was named the Stypven time, but is now obsolete

buffer-diluted RVV

is prolonged by the LAC

dilute RVV time

is rou inely employed to detect LAC