A Practical Guide to Laboratory Haemostasis


Pre-Analytical Variables


When making decisions about diagnosis, further investigation and management - clinicians rely, in many cases, on the results of blood tests. The conclusions and decisions that follow from those results are only valid if the results themselves are valid.
The purpose of quality control and laboratory participation in external quality assurance schemes is, of course, to ensure that the tests which produce those results are reliable, reproducible and accurate. The establishment and maintenance of secure, reliable and well-distributed information networks exist to ensure that the results, once available, can be accessed by the clinician or other authorised users in a timely fashion.
However, there is another element to laboratory testing which is often overlooked and which has only recently begun to be the focus of research and interest: pre-analytical variables. The processes mentioned above are only useful if we can make a basic assumption that the sample we are analysing, the sample that has reached the laboratory and been entered into the analytical process, actually reflects what is happening in the patient. If this simple requirement is not satisfied by the sample we analyse then the results that are produced will be meaningless, no matter how well quality controlled the testing process or how robust our means of transmitting the information to the end-user are, and the clinical decision making process will be, potentially disastrously, flawed.
How then do we ensure that the sample the laboratory analyses actually reflects the patient’s state? First we must consider what factors may influence the state of the sample and from that we may determine mechanisms to properly regulate these factor

Principles of Pre-Analytical Variables

These may be divided into three broad categories:

Category Variables
Sample Collection Patient identification and sample labeling
Phlebotomy technique
Sample volume
Sample collection tube
Sample Handling Storage
Agitation, centrifugation
Transport conditions
Delays in transport to the laboratory for processing
Patient Factors Physiological variables
Pathological states

The illustration below summarises the key variables which can affect the result of any assay:

Pre-Analytica Variables

Sample Collection

This begins with correct patient identification.

Variable Sources of Error
Patient Identification Incorrect patient identification is one of the major, and most dangerous, sources of pre-analytical error. Problems may arise if a patient is confused, lacks an identification bracelet or has a similar name to another patient. Samples may also be mislabeled, especially if samples are taken from multiple patients then batched for later labeling. Some institutions have introduced barcoded or RIF chip patient ID bands and handheld readers which generate sample labels at the bedside to reduce this problem although, obviously, these solutions are not suitable for out-patient settings
Venepuncture The venepuncture process itself may also contribute to pre-analytical error.
Common problems include:
Drawing blood downstream from an IV infusion
Applying the tourniquet for more than a minute (may cause haemoconcentration)
Slow venepuncture (encouraging clotting and an 'activated' sample)
An inappropriately narrow gauge needle (leading to clotting or haemolysis of the sample)
Failing to discard the initial sample from an indwelling catheter (otherwise can result in sample dilution and heparin contamination if the line contains heparin)
Incorrect tubes
Samples, if drawn through a needle into a syringe, should be drawn into plastic or siliconised glass to delay coagulation activation and should not be passed back through a needle into the sample tube for the same reason.
Inadequately filling the sample collection tube Inadequate filling results in an incorrect anticoagulant:plasma ratio and inaccurate results.
The ratio for blood taken into sodium citrate should be 1 part anticoagulant:9 parts blood. For patients with very low or high haematocrits (e.g. severe haemolysis or poorly controlled polycythaemia) specially prepared tubes with an appropriate volume of anticoagulant may be needed (or strictly measured volumes of blood, calculated from the haematocrit, taken into standard tubes) to ensure a correct anticoagulant:blood ratio. Most tubes are calibrated to collect 2.7ml of whole blood into 0.3ml of 3.2% (0.109M) sodium citrate to give a final volume of 3.0ml and a ratio of whole blood:anticoagulant of 9:1.

When the haematocrit exceeds 0.55 (55%) the reduced plasma volume requires a decrease in the volume of anticoagulant used to maintain the ratio of 9:1. The formula:

C (ml) = 1.85 x 10-3 x (100-Hct (%)) x V (ml)

where C = ml of 3.2% sodium citrate anticoagulant; Hct (%) = haematocrit of the patient in % and V = ml of whole blood in tube. So for a sample tube of 2.7ml and a patient haematocrit of 0.67 (67%) the correct ratio of anticoagulant is:
1.85 x 10-3 x (100-67) x 2.7 = 0.165ml.
For a collection tube of volume 10ml and a patient haematocrit of 0.67 (67%) the correct ratio of anticoagulant is: 1.85 x 10-3 x (100-67) x 10 = 0.61ml

The graph below shows an alternative method for calculating the concentration of 3.2% trisodium citrate to be used as an anticoagulant depending upon the haematocrit and which will ensure a ratio of 1 part anticoagulant:9 parts blood.

Citrate Concentration
Age of tubes The age of the tubes used should be monitored as anticoagulant solutions begin to loose their effectiveness after 3 months depending on storage conditions.


Sample Handling

The conditions of transport and storage of samples may have a profound influence on their usefulness.

Variable Sources of Error
Temperature and agitation are key issues Exposure of samples to excess heat e.g. storage next to a radiator or exposure to strong sunlight through a car windscreen, can cause denaturation of plasma proteins resulting in the formation of variably sized protein microparticles the smaller of which may be misread by automated analysers as platelets giving spurious elevation of the platelet count.
Recent studies have shown that delays of >6 hours between collection and processing of INR or prothrombin time samples can cause significant (i.e. >10%) changes in measured values.
Mechanical agitation e.g. a long car or van journey can also contribute to this.
The temperature at which archived samples are stored affects their shelf-life. For most coagulation tests storage at -35°C or less gives a shelf life of several years but storage at -20°C is inadequate.
Repeated freeze-thaw cycles may affect factor level, for example, a reduction in vWF:CB activity and FXII levels. Freeze-thawing may also produce phospholipid rich membrane microvesicles from platelet damage which may then mask the presence of a lupus anti-coagulant.


Patient Specific Factors

A number of physiological variables can be associated with certain patient factors. These variables are largely outside the control of the laboratory but knowledge of their existence will permit adjustments to either eliminate their effect or permit proper interpretation such as, for the examples given above, adjusting reference ranges or changing sample handling techniques such as warming the sample or using a different anticoagulant.

Variable Sources of Error
Age At birth the levels of the vitamin K dependent clotting factors are reduced and only reach adult values at approximately six months of age.
Physiological variation Other physiological states such as pregnancy affect components of the haemostatic pathway, in this case a rise in the levels of factor VIII, Von Willebrand Factor, fibrinogen, a fall in protein S and often a mild fall in platelet count.
Cryoglobulins When blood specimens cool from body temperature to room temperature, cryoglobulins aggregate and may be falsely identified as platelets and/or WBCs by the haematology analyser.
Platelet Clumping due to EDTA Platelet clumping is a phenomenon occurring due to EDTA dependent antibodies against platelet surface glycoproteins and may result in a falsely low platelet count. Collection of samples for platelet counting into sodium citrate can sometimes eliminate this.
Elevated bilirubin or lipid concentration High levels of bilirubin or lipids may result in turbid plasma which can interfere the with optical density measurements used to determine end points of some tests.


What Test Next

Establishment of a quality manual addressing preanalytical variables is a prerequisite for implementing measures to recognize and control this crucial component of laboratory quality, which cannot be detected by traditional analytical quality control procedures. The preanalytical quality manual should address both patient and specimen variables. This manual would include details on:
 - Standard instructions to be given to patients for certain tests should be included, for example, fasting before homocysteine levels are tested.
 - If direct phlebotomy into vacuum tubes is not used the quality manual should address the minimum sample volume needed for a laboratory test so that, for frequently sampled patients (especially young children), there is minimal wastage of sampled blood to reduce the risk of iatrogenic anaemia.
 - Sample identification guidelines should be explicitly spelled out in the manual.
 - Sample processing guidelines, transportation and specimen storage conditions should be clearly stated.
 - The quality manual should have a comprehensive listing of analytes and notation on the effect of at least commonly encountered influence and interference factors.
 - Finally, since the quality manual will be a source book for the laboratory professional an updated bibliography of preanalytical standards should be included.

International standardization bodies such as the ISO 6710 have issued standards for type and concentrations of anticoagulants to be used for venous blood samples.

The Clinical and Laboratory Standards Institute [CLSI] constantly update their guidelines on several aspects of the preanalytical phase. An updated listing of the CLSI Standards can be found on their website [].

The checklist prepared by the College of American Pathologists for Laboratory Inspection and Accreditation addresses specimen related preanalytical variables.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.