General Approach in Investigation of Haemostasis. Lecture 1: Introduction. Preanalytical Variables including. Sample Collection. Site Selection. Storage Requirements. Transportation of Specimen. Haemostasis.
Lecture 1: Introduction
Transportation of Specimen.
Hemostasis is a complex interaction between vessels, platelets and coagulation proteins that, when working properly, stops bleeding while maintaining blood flow in the vessel.
Specific tests are available to evaluate platelet function, coagulation proteins, natural occurring inhibitors and fibrinolysis.
Proper sample collection is of utmost importance for reliable test results to evaluate the bleeding patient, thrombosis or fibrinolysis (preanalytical phase)
All these tests are influenced by sample collection, sample processing and sample storage.
The laboratory will not evaluate samples that are hemolyzed, clotted, contain fibrin strands or improperly stored.
Reference Laboratory Services will immediately notify the client of any problems with the sample.
When blood is withdrawn from a vessel, changes begin to take place in the components of blood coagulation. Some occur almost immediately, such as platelet activation and the initiation of the clotting mechanism dependent on surface contact.
* CLSI : Clinical and Laboratory Standards Institute
Sample Collection : Samples with High hematocrits
* National Committee for Clinical Laboratory Standards
X= volume of sodium citrate
Vol =volume of whole blood drawn
PCV= patient’s hematocrit
Patients Hct= 60%, V= 5 mL
X=(100-60)*5 / (595-60)
= 40*5 / 535 = 0.34 ml
Patient Hct = 25%, V=5 ml
X=(100-25)*5 / (595-25)
= 75*5 / 570 = 0.65 ml
Send specimen on ice OR deliver to lab ASAP
Separate cells from plasma immediately via centrifugation
To prepare platelet-Poor plasma
Centrifuge the blue top evacuated tubes (CLSI, formerly NCCLS recommendation is 1500 rpm for 15 minutes).
Using a plastic pipette, immediately remove the top 2/3 of the plasma to a plastic aliquot tube.
Centrifuge this plasma sample and remove the top ¾ of the plasma to a second plastic aliquot tube with a fresh plastic pipette.
Freeze the specimen within one hour of collection.
The more detailed investigations of coagulation proteins also require caution in their interpretation depending on the type of assay performed. These can be divided into three principal categories, as described in the following sections.
Assays Using Chromogenic Peptide Substrates (Amidolytic Assays)
Include immuno-diffusion, immuno-electrophoresis, radioimmunometric assays, latex agglutination tests, and tests using enzyme-linked immunosorbent assays (ELISA).
Fundamentally, all these tests rely on the recognition of the protein in question by polyclonal or monoclonal antibodies. Polyclonal antibodies lack specificity but provide relatively high sensitivity, whereas monoclonal antibodies are highly specific but produce relatively low levels of antigen binding.
latex agglutination kit: Latex microparticles are coated with antibodies specific for the antigen to be determined. When the latex suspension is mixed with plasma an antigen–antibody reaction takes place, leading to the agglutination of the latex microparticles.
Agglutination leads to an increase in turbidity of the reaction medium, and this increase in turbidity is measured photometrically as an increase in absorbance.
Usually the wavelength used for latex assays is 405 nm, although for some assays a wavelength of 540 or 800 nm is used. This type of assay is referred to as immuno- turbidimetric.
Do not freeze latex particles because this will lead to irreversible clumping.
An occasional problem with latex agglutination assays is interference from rheumatoid factor or paraproteins. These may cause agglutination and overestimation of the protein under assay.
Chromogenic, or amidolytic, methodology is based on the use of a specific color-producing substance known as a chromophore.
the chromophore normally used in the coagulation laboratory is para-nitroaniline (pNA), which has an optical absorbance peak at 405 nm on a spectrophotometer.
Coagulation assays are functional bioassays and rely on comparison with a control or standard preparation with a known level of activity.
In the one-stage system optimal amounts of all the clotting factors are present except the one to be determined, which should be as near to nil as possible.
The best one-stage system is provided by a substrate plasma obtained either from a patient with severe congenital deficiency or artificially depleted by immuno-adsorption.
Coagulation techniques are also used in mixing tests to identify a missing factor in an emergency or to identify and estimate quantitatively an inhibitor or anticoagulant.
The advantage of this type of assay is that it most closely approximates the activity in vivo of the factor in question. However, they can be technically more difficult to perform than the other types described earlier.
Using snake venoms (The Taipan venom time employs a reagent isolated from the venom of the Taipan snake (Oxyuranus scutellatus) that directly activates prothrombin in the presence of phospholipid and calcium.)
Aassay of ristocetin cofactor (used to diagnose von Willebrand disease )
The clot solubility test for factor XIII.
DNA analysis is becoming more useful and more prevalent in coagulation. However, this requires entirely different equipment and techniques