SEED No 2 - Pre Analytical Factors That Influence Coagulation Test Results

8/2/2019 SEED No 2 - Pre Analytical Factors That Influence Coagulation Test Results

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Sysmex EducationalEnhancement & Development

SEED-Arica Newsletter | No 2 | December 2010

Preanalytical influences on coagulation test results

Routine coagulation testing is an integral part o laboratory

testing that inorms clinical decision making. There have

been major advances in technology with tight quality control

procedures in place or the analytical phase o testing.

The validity o results generated or patient sample results is

however highly dependent on several preanalytical variables

that will not be detected by conventional quality control

processes. This is a very important topic as preanalyticalactors inluencing the reliability o laboratory test results

are commonplace.

These variables can be broadly categorized as controllable

variables such as specimen collection and handling pre-

analysis and uncontrollable variables that relate to actors

that are endogenous and speciic to each individual

specimen (or example the presence o haemolysis

or jaundice).

Specimen CollectionThe process o phlebotomy is extremely common, yet it

is generally practiced with very little regard or the need

or standardized procedures, primarily because the impact

that a poorly collected specimen has on the accuracy o

laboratory test results is largely unrecognized by those

tasked with blood collection. Poorly collected blood samples

are a major cause o sample rejection and erroneous

coagulation test results as poor sample quality due to aulty

collection technique is not always overtly evident.

a) Site of venepuncture

n Wherever possible only antecubital veins should be used.

n As a general rule, the smaller and more distal the vein

the greater the chance that the sample collection will

be technically diicult and the specimen compromised.

Traumatic ex vivo haemolysis and clotted specimens

increase in requency when alternate sites are used.

n Blood should not be collected rom the same arm in

which a drip is inserted. This is important in order

to protect the integrity o the drip site as well as to

avoid any possible inadvertent sample dilution with

the inusate.

b) Blood collection technique

n Evacuated tube collection systems are superior to the

use o ordinary syringes.

nThe specimen is collected directly into the

appropriate anticoagulant thereby minimizing the

possibility o inadvertent sample clotting.

n The chance o tube under-illing is minimized.

n The gauge o the needle should not be too small

(ideal 9-G) or else there is a greater risk oex vivohaemolysis as red blood cells are sheared because o

the high vacuum orce applied during collection. A

signiicant increase in D-Dimer count is observed in

specimens collected with smaller diameter needles.

n Tourniquets are commonplace in venesection but should

not be used indiscriminately. The tourniquet should be

removed as soon as blood low into the collection tube

is established and should never exceed minute so as

to minimize venous stasis. Venous occlusion causes

haemoconcentration, an increase in ibrinolytic activity

and possibly activation o some clotting actors. A one

to three minute period o stasis will result in clinically

signiicant changes in the PT, APTT, ibrinogen and

D-Dimer values.

Introduction to coagulationThe purpose of this newsletter is to provide laboratory staff an overview of preanalytical factors that influence coagulation

test results to ensure that erroneous results are not released.


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n Whilst it used to be advocated that one should neveruse the irst draw tube or coagulation testing, the latest

CLSI guidelines have dropped this requirement as no

evidence exists to indicate that there is any meaningul

dierence in coagulation test results between irst

and second draw tubes. I more than one tube is

however being drawn, then it is recommended that the

coagulation tube should not be drawn irst.

n Blood should not be drawn rom indwelling catheters

as they are very likely to be diluted as well as

contaminated by heparin. Sometimes however, this

may be the only venous access possible in critically ill

patients, in which case, the irst ml should not be used

or coagulation tests.

c) Blood collection tubes

n Blood must be collected into a sodium citrate tube (light

blue top).

n Citrate is available as 3.% and 3.8%. Both are acceptable

and give the same results provided that the sample tube

is properly illed and analysed resh. The eect o under-

illing and aging is more pronounced with 3.8% citrate.

n Ensure that the tube has not expired.

d) Tube filling

n The tube must be adequately illed. Inadequate tube

illing is one o the commonest reasons or sample

rejection.

n The minimum ill volume required depends on the

actual parameter being tested but as a rule o thumb

tubes should have a minimum ill volume o 8% o thedemarcated level (in keeping with the vacuum volume).

A 5ml tube should thereore contain a minimum o 4ml

o blood.

n Proper illing is essential to ensure the proper blood to

anticoagulant ratio. The citrate removes calcium rom

the blood which prevents it rom clotting in the tube.

The ratio o anticoagulant to blood in the tube needs

to be correct otherwise the test results will be wrong.

All tests involving clotting as the end point o detection

require the addition o calcium chloride in the test

method. The amount o calcium chloride that is included

in the reagent has been determined based on the

expected quantity o citrate in plasma post collection.

Clearly i the tube is under-illed, there will be an excesso citrate in the plasma, and hence too little calcium

to compensate, so clotting times will be erroneously

prolonged.

n Samples with a very high haematocrit will have the

same outcome as i the tube were under-illed i.e.

there is a relative excess o anticoagulant to plasma.

Special tubes should be prepared or the patient by the

laboratory. A nomogram exists rom which one can see

how much citrate should be added to a tube with 5ml ill

volume speciic or haematocrit. This typically applies to

haematocrit values above 5%.

n Overilling will not occur i blood is collected using a

vacuum system. It is however possible to do so i blood

is collected using a standard needle and syringe and

tubes are decapped or illing.

n Both over and under-illed tubes will give erroneous

results.

Specimen handling, transport and storage

a) Specimen mixing

n The blood must be thoroughly mixed with the

anticoagulant in the tube by inverting the tube

several times.

n This is essential to prevent micro clot ormation in the

tube. The process o clot activation consumes clotting

actors and can result in alse short or long clot-based

coagulation test results.

b) Time delays between collection and analysis

n The time between sample collection and processing

is critical. The maximum time permitted depends on

speciic test to be perormed. As a rule o thumb,

samples should be processed within 6 hours o

collection, ailing which samples should be spun

down, the plasma portion removed, and snap rozen

to a minimum o -C. Only one reeze thaw cycle

is permissible.

n Old samples become activated. Just like in poorly

mixed specimens, mini clots orm in the tube thereby

consuming clotting actors which can result in alseshort or long clotting times. Such micro clots are

invisible to the operator.

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c) Storage conditionsn The eect o time also depends on the temperature at

which the specimen is stored.

n PT results are stable or up to 4 hours on condition

that the specimen is stored at 4-6C or room

temperature and was not exposed to any excessive

mechanical agitation or high temperatures that may

be experienced during road transportation or non-air-

conditioned laboratories. The sample may be stored as

whole blood or plasma.

n The APTT and most other coagulation tests are probably

stable up to 8 hours unless the patient is being treated

with the anticoagulant heparin. The sample may be

stored as whole blood or plasma.

nA maximum o hours delay prior to testing is

recommended or samples rom heparinised patients.

d) Specimen centrifugation

n Samples must be centriuged adequately in order to

ensure that the plasma is ree o platelets. As platelets

are a source o phospholipid, residual platelets,especially i the plasma is going to be rozen and

thawed, are a source o phospholipid that may give alse

short clotting times.

n It is not important to have a temperature controlled

centriuge.

n For routine coagulation tests centriugation at 5 x g

or 5 minutes at room temperature is recommended.

n

Ideally specimens should be centriuged within hour ocollection.

n As a general rule, it is best to centriuge specimens and

remove plasma rom above the red cell layer as soon

as possible i any delay in coagulation tests is likely.

Contact with red cells is a source o activation.

n It is essential to ensure that the buy layer and platelet

rich plasma is avoided when plasma is separated or

storage. As platelets are a rich source o phospholipid,

clotting times may be erroneously short i plasma is

contaminated with platelets. Some tests are more

aected than others.

Other specimen variables that may give erroneouscoagulation test results

a) Haemolysis

n Haemolysis is a leading source o intererence in

coagulation testing.

n The source o haemolysis, whether it occurred in vivo or

occurred as a result o traumatic venepuncture or poor

sample handling is irrelevant.

n Causes o traumatic haemolysis include:-

n the use o small bore needles

n diicult phlebotomy

n excessive shaking or mixing o the specimen

n exposure to excessively hot or cold temperatures

n centriugation at a too high speed or or too long

n Haemolysis results in the release o haemoglobin

into the plasma. This may interere with the results

obtained on coagulation analysers using optical clot

detection systems.

n Chromogenic assays work on the principle o measuringcolour in the plasma. These assays exploit the act

that proteins involved in maintaining haemostatic

balance are principally enzymes that unction by

cleaving a speciic substrate. In the assay system

the natural substrate is replaced with a chromogenic

substrate which when acted upon by the active enzyme,

causes the plasma to change colour in proportion to the

enzymatic activity. As such, any colour that is observed

is interpreted as being a consequence o the activity

o the analyte in question. Free plasma haemoglobin

will be identiied as colour and be assumed as havingbeen elicited by enzyme activity which would be

erroneous in this case. Assay results may be either

too high, i the parameter in question (e.g. Protein C)

is directly proportional to colour change, or too low

i activity is inversely proportional to colour change

(e.g. Antithrombin).

n The degree o intererence depends on the test principle

or the coagulation parameter under analysis.

n It is important to be aware that haemolysis may

interere with results but most assays have some inbuilt

sae guards and are only prone to wrong results above a




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certain threshold level. In this regard it is very importantto read package inserts very careully as dierent

reagents or the same test have dierent tolerances.

n I in doubt about whether or not the degree o

haemolysis within a sample will interere with test

results, one could measure the plasma haemoglobin level

on a haematology or chemistry analyser.

n I samples are visually grossly haemolysed it would be

best to request a repeat specimen to be collected. I

the patient has a haemolytic condition the doctor must

be inormed to treat results with reserve, as well as to

give an indication o whether results are likely to be

erroneously too high or too low. Some interpretation

may still be possible.

b) Jaundice

n The bilirubin present in jaundiced samples has the same

eect as haemolysed samples although this is always

inherent to the patients pathology and not induced in

the collection process.

c) Lipaemian High lipid levels in plasma result in very turbid

specimens which may interere with clotting tests using

optical detection systems as this relies on a measure o

change in light transmission through a sample.

Take home messagesn The quality o coagulation test results is highly

dependent on several preanalytical variables and may be

erroneous under certain conditions.

n Correct tube ill volume is critical as the incorrect plasma

to anticoagulant ratio will give wrong results.

n Sample collection should be undertaken with adequate

precautions to prevent excessive stasis and traumatic

haemolysis.

n Excessive delay between collection and sample analysis

will aect test results.

n Permissible sample storage times beore results are

aected, only apply i the tubes are also adequately

illed and the sample not exposed to excessively warm

temperatures and excessive mechanical agitation.

n The combination o tube under-illing, high

temperatures and agitation result in accelerated

compromise and hence recommended maximum storage

times no longer apply.

n Samples should be centriuged and plasma separated

rom red cells as soon as possible.

n As a rule o thumb, analyse samples within 6 hours o

collection and i this is not possible, reeze the plasma

or later analysis.

n The presence o haemolysis, jaundice and lipaemia may

aect the accuracy o results.

The next edition will ocus on the prothrombin time test, the

concept o the INR and oral anticoagulation.



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SEED No 2 - Pre Analytical Factors That Influence Coagulation Test Results