A Practical Guide to Haemostasis

Pre-Analytical Variables


A large number of variables can potentially affect the results of a laboratory test and these can be divided into:
  - Pre-analytical variables
  - Analytical variables
  - Post-analytical variables

There is increasing evidence that an awareness of the significance of the pre-analytical variables that can potentially affect a lab test, will ensure the reliability of the final result.

Pre-Analytical Variables

These may be divided into three broad categories:

Category Variables
Sample Collection Correct patient identification and sample labelling
Correct phlebotomy technique
Correct sample volume which may involve taking into account the patient's Haematocrit
Correct s ample collection tube
Sample Handling Storage
Mixing & centrifugation
Transport conditions and delays in transport
Patient-specific Factors Physiological variables
Pathological states

Samples for haemostasis tests are frequently collected into trisodium citrate in a ratio of 1 part Anticoagulant:9 parts blood. Under-filling of the tube results in an increased ratio of citrate and therefore when the sample is re-calcified, only partial re-calcification takes place and the clotting times are prolonged.

Similar results may be seen if the patient has an elevated Haematocrit [Hct]. 

When the haematocrit exceeds 0.55, the reduced plasma volume requires a decrease in the volume of anticoagulant used to maintain the ratio of 9:1. The formula shown below:

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

where C = ml of 3.2% Trisodium citrate; Hct (%) = haematocrit of the patient in % and V = ml of whole blood in tube - can be used to calculate the volume of 3.2% Trisodium citrate required to maintain the correct ratio of anticoagulant to whole blood [1:9].

A sample tube of 5ml and a patient with a haematocrit of 0.65 (65%) the correct ratio of anticoagulant is: 1.85 x 10-3 x (100-65) x 5.0 = 0.324ml.

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.

In the case of the neonate, 1ml sample tubes should be used and ideally, the volume of anticoagulant in the sample tube should be based on the volume of plasma and not on the total volume of blood taken and it should be reduced in proportion to the increased neonatal haematocrit in order to avoid dilution of coagulation factors and particularly if the neonate has a very high haematocrit.  However, most laboratories and neonatal units take a more pragmatic approach to this problem and accept a degree of artefactual prolongation of the coagulation times and most neonatal reference ranges are based on this.

Transport and storage of samples for laboratory testing can have a profound influence on the results of specific tests.  Recent studies have shown that delays of >4 hours between collection and processing of samples for routine laboratory testing can affect the results.
The temperature at which samples are stored affects their shelf-life..
Freeze-thawing should be avoided if samples are collected for Lupus Anticoagulant [LA] screening.  This can lead to lysis of any remaining platelets with the release of Phospholipids and which may neutralise weak LA is present. 

Patient Specific Factors

A number of physiological variables can be associated with certain patient factors and whilst these are largely outside the control of the laboratory their knowledge will allows adjustments to either eliminate their effect or a proper interpretation.

Variable Sources of Error
Age Neonatal reference ranges should be used for all new born infants.  The vitamin K dependent clotting factors are reduced at birth and only reach adult values at approximately six months of age.
Physiological variation Physiological states such as pregnancy may affect components of the haemostatic pathway e.g. Protein S levels.  Similar effects may be seen in the acute phase situation e.g. following surgery.
Platelet Clumping due to EDTA Platelet clumping due to EDTA-dependent antibodies against platelet membrane glycoproteins has been reported in 0.07–0.20% of cases and may result in a falsely low platelet count [Pseudo-thrombocytopaenia].
Elevated Bilirubin or Lipid levels High levels of Bilirubin or Lipids may lead to turbid plasma which can interfere with optical density measurements used to determine the end points in some tests.
Gender Higher levels of some clotting factors have been reported in females compared with males.  In addition there are decreased levels of Protein S and increased levels of Antithrombin activity in females.

What Test Next

Education of all staff is fundamental to ensure they are aware of the pre-analytical variables that may affect the results of any test and which may, indirectly therefore, influence, patient care.  See References for further information.