HEMA: Lesson 13 Laboratory evaluation of the coagulation mechanism (MIDTERMS)

CLOT-BASED SCREENING TESTS

Performed using traditional clotting endpoint assays that detect the functional capacity of the coagulation system.

prothrombin time

It evaluates extrinsic and common coagulation pathways.

PROTHROMBIN TIME (PT)

It is an assay designed to screen for defects in fibrinogen, prothrombin, and factors V, VII, and X. It evaluates vitamin K status.

fibrinogen, prothrombin, and factors V, VII, and X

PT is an assay designed to screen defects in what factors

Prothrombin time

• It is used to monitor the efects of therapy with the oral anticoagulant warfarin (correct dosage) and for the presence of liver disease or damage

thromboplastin or tissue thromboplastin

PT reagents

thromboplastin or tissue thromboplastin

consist of recombinant or affinity-purifed tissue extract/factor (e.g. from brain, lungs, and placenta) suspended in phospholipids mixed with a bufered 0.025 M solution of calcium chloride.

citrated platelet-poor plasma (PPP)

is mixed with PT reagent, clotting starts by activating factor VII

optical or electromechanical methods

The time it takes to form a visible clot refects the activity of the extrinsic and common coagulation pathways and is measured using

false prolongation of results

Incubation beyond 10 minutes in Prothrombin time may lead to

factor degradation, evaporation, or pH shifts

Incubation beyond 10 minutes in Prothrombin time may lead to false prolongation of results due to

FXIII

prothrombin test will not determine what factor

quality controls for PT

commercially prepared normal and abnormal (prolonged) controls are used

frozen, lyophilized control

what form is in the Quality control of PT

frozen control

are thawed at 37° C and mixed well

lyophilized control

reconstituted with the supplied buffer, mixed well, and allowed to stand for 20 to 30 minutes following manufacturer specifcations

seconds, Patient’s percent of normal activity, INR

Reporting Prothrombin Time Results

12-14

reference range of reporting PT in seconds

Patient’s percent of normal activity

old reporting method of PT

INR

used for warfarin monitoring

International normalized ratio

It is the ratio of the patient’s PT to a normal control plasma (reference time mean) PT raised to the power of ISI

International Normalized Ratio (INR)

It is a standardized way of reporting Prothrombin Time (PT) results on patients who are receiving oral anticoagulant therapy

INR

allowing consistent results across different labs

reference thromboplastin

basis for ISI value in equivalent of 1.

international reference thromboplastin reagent

To standardize PT results, the WHO developed an

ISI

The manufacturers’ reagents are then assigned with

1.0

The reference thromboplastin is assigned an ISI of

International sensitivity Index (ISI)

Correction factor from WHO for each batch of lot numbers of thromboplastin reagents used.

1

ISI that is most sensitive

once

Each laboratory must establish its own interval for each new lot of reagents at least how many times a year

12-14 sec

a typical PT reference interval is

oral anticoagulant therapy patient

INR is not intended for normal or healthy adult it is best for

2-3

Warfarin (Prophylactic therapy for DVT) is usually targeted to an INR of

2.5-3.5

Artifcial heart valve: INR =

3.0

Pulmonary embolism Treatment: INR =

Factor VII

When the PT is prolonged, (but the aPTT and TT test results are normal) what factor is deficient

DIC, liver disease, Vitamin K deficiency

diagnostic assay for PT

liver disease

sensitive to PT because of the short half-life of Factor VII (6 hours)

vitamin K deficiency

seen in severe malnutrition, during use of broadspectrum antibiotics that destroy gut fora, and in malabsorption syndromes

factor 5 and 7

To distinguish between vitamin K deficiency and liver disease, determine what factor levels?

prolonged

If defcient with FV then APTT is also

5 and 7

what factors are reduced in liver disease

5,7,10, prothrombin, fibrinogen

Prolonged PT results are also seen in congenital single-factor defciencies

factor assay

Any suspected single-factor defciency is confrmed with a

7 or 9

what factor deficiency is PT NOT affected

greater than 55%

Anticoagulant volume must be adjusted when the hematocrit is___ to avoid FALSE PROLONGATION in results

plasma lipemia or icterus

may affect the results obtained with optical instrumentation.

Thrombin test

To detect unexpected heparin use what test?

Lupus Anticoagulants

are members of the antiphospholipid antibody family and may partially neutralize PT reagent phospholipids.

Warfarin

is often prescribed to prevent thrombosis in patients with LACs, but the PT maybe an unreliable monitor of warfarin therapy. Instead, monitoring shall be done using chromogenic endpoint factor X assay

chromogenic endpoint factor X assay

Warfarin is often prescribed to prevent thrombosis in patients with LACs, but the PT maybe an unreliable monitor of warfarin therapy. Instead, monitoring shall be done using

chromogenic factor X assay

useful for monitoring pediatric patients and patients with mitral valve defects who are on warfarin therapy

clotting time

It is an insensitive test and has poor reproducibility

Capillary Method or Slide or Drop method, Lee & White Whole Blood Coagulation Time

methods for clotting time

clotting time

measures period required for blood to clot after it was removed from the body.

heparin

what can alter results for clotting time

2-4 min

reference value for clotting time

LEE-WHITE WHOLE BLOOD COAGULATION TIME TEST

the first laboratory (in-vitro) procedure to assess coagulation

lee-white whole blood coagulation time test

the time interval from the initiation of clotting to visible clot formation refects the condition of the coagulation mechanism

coagulopathy

A prolonged clotting time indicates a

7-15 min

reference value for lee-white whole blood coagulation time test

nature of collecting surface, diameter of tube, temperature, admixture of blood with tissue juice

Factors affecting clotting time

faster the coagulation time

the rougher the surface, the

faster the coagulation time

the narrower the tube, the

increases

coagulation time ___ with temperature

37C

most ideal temperature for coagulation test

faster

coagulation time is ___ when blood is with tissue juice

1953

activated clotting time was described in

ACTIVATED CLOTTING TIME (ACT)

modifcation of Lee-White whole blood clotting time

ACTIVATED CLOTTING TIME (ACT)

Measures the intrinsic pathway

activated clotting time

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

ACTIVATED CLOTTING TIME

Still widely used as a near-patient assay (Bedside test or Point- of- care testing) to monitor UFH (unfractionated Heparin) therapy in hypertherapeutic applications

Activated clotting time

It is more sensitive to factor defciencies and heparin therapy than the LWCT

platelets, coagulation factors and inhibitors

Activated clotting time is affected by

Partial Thromboplastin Time (PTT)

aPTT is also known as

Partial Thromboplastin

the reagent ONLY contain phospholipid (PL), which is a platelet substitute and NO tissue factor as in the prothrombin time assay

ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT)

It is used to screen for abnormalities of the intrinsic and common clotting system

7 and 13

APTT is used to detect all congenital and acquired procoagulant deficiencies EXCEPT for deficiencies of factors

Factor 13

what factor cannot be detected with PT and aPTT

ACTIVATED PARTIAL THROMBOPLASTIN TIME (aPTT

Used to monitor the effects of UFH (heparin therapy) and to detect LAC and specific coagulation factor antibodies such as the antifactor VIII antibody

phospholipid, activator, 0.025M calcium chloride

reagents of PTT

partial thromboplastin or cephalin

phospholipid reagent is previously called

activator

provides the negatively charged particle that activates factor XII (12)

silica, kaolin, ellagic acid, or celite

examples of activator in PTT reagents

7,9,11,12

Most PTT reagents are made to show a prolonged result if the blood has less than approximately 30% of factors —

Citrated platelet-poor plasma (PPP)

sample pf 0.025M calcium chloride in PTT

nearest tenth of a second

In PTT, results are reported to the

2-3

therapeutic range for warfarin

23-25 seconds

reference of APTT

ex-vivo Brill edward curve

Monitoring Heparin Therapy, Establish a therapeutic range using the

1.5 to 2 times normal (60-100 sec)

Patients receiving therapeutic heparin should have an APTT between

APTT

is requested/ordered when a hemorrhagic disorder is suspected or when recurrent thrombosis or the presence of an autoimmune disorder points to the possibility of LAC

factor 7 and 13

deficiencies have NO effect on the APTT

APTT

is LESS sensitive to vitamin K deficiency or warfarin therapy than the PT

prothrombin; factors V, X, VIII (Hemophilia A), IX (Hemophilia B), XI (Hemophilia C or Rosenthal Syndrome), XII or fibrinogen

prolonged APTT results in the deficiency of one or more of which coagulation factors

factor XII, prekallirein or HMWK

prolonged PTT results in deficiencies of factor

anti-factor VIII or anti-factor IX

prolonged PTT results in presence of a SPECIFIC INHIBITOR

LAC

prolonged PTT results in the presence of a NON SPECIFIC INHIBITOR

LAC

an immunoglobulin with affinity for phospholipid-bound proteins