General Approach in General Approach in Investigation of HaemostasisInvestigation of Haemostasis

Lecture 1: Introduction

Preanalytical Variables Preanalytical Variables includingincluding

1. Sample Collection.2. Site Selection.3. Storage Requirements.4. Transportation of Specimen.

Haemostasis 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When blood is withdrawn from a vessel, changes begin to take place in the components of blood coagulation.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.

Sample Collection

Anticoagulant of choice 3.8% or 3.2% Sodium Citrate

3.2 % Preferred as the standard measure due to stability and closeness to the plasma osmolality

Anticoagulant/blood ratio is critical (1:9) Exact amount of blood must be drawn. No short draws

are acceptable, this will falsely increase results due to presence of too much anticoagulant

CLSI guideline is +/- 10 % of fill line

Purpose of the anticoagulant is to bind or chelate calcium to prevent clotting of specimen

* CLSI : Clinical and Laboratory Standards Institute

Sample Collection

Other anticoagulants, including oxalate, heparin, and EDTA, are unacceptable.

The labile factors (factors V and VIII) are unstable in oxalate, whereas heparin and EDTA directly inhibit the coagulation process and interfere with end-point determinations.

Additional benefits of trisodium citrate are that the calcium ion is neutralized more rapidly in citrate, and APTT tests are more sensitive to the presence of heparin.

Sample Collection

According to the latest CLSI (formerly NCCLS) guideline on coagulation testing, it is important to adjust the sodium citrate volume when a patient’s hematocrit is greater than 55%.

Examples of patients who may have elevated hematocrit values are newborns or people with polycythemia vera.

NCCLS* recommends adjusting anticoagulant ratio for patients with hematocrits exceeding 55%

High hematocrits may cause falsely prolonged test results due to an over- anticoagulated sample

Formula correction achieves a 40% hematocrit

* National Committee for Clinical Laboratory Standards

Sample Collection : Samples with High hematocrits

X = (100–PCV)*vol./(595–PCV)Where: X= volume of sodium citrateVol =volume of whole blood

drawnPCV= patient’s hematocrit

Examples: Patients Hct= 60%, V= 5 mLX=(100-60)*5 / (595-60) = 40*5 / 535 = 0.34 ml

Patient Hct = 25%, V=5 mlX=(100-25)*5 / (595-25) = 75*5 / 570 = 0.65 ml

HCTCitrate

(ml)

0.20 0.70

0.25 0.65

0.30 0.61

0.55 0.39

0.60 0.36

0.65 0.31

0.70 0.27

Untraumatic venipuncture is required Traumatic venipunctures release tissue factor and initiate

coagulation Fingersticks/Heelsticks are not allowed Indwelling IV line draws are discouraged

Contain heparin & diluted blood Falsely increased results

Order of DrawOrder of Draw Evacuated tube system

Blue top is 2nd

If 2nd tube drawn, 1st top must be anticoagulant free (i.e. red top)

Site Selection

Prothrombin Time: PT Uncentrifuged or centrifuged with plasma remaining on top of cells in

unopened tube kept at 2-4 oC or 18-24 oC must be tested within 24 hours of collection

Activated Partial Thrombin Time: APTT◦ Uncentrifuged or centrifuged with plasma remaining on top of cells in

unopened tube kept at 2-4 oC or 18-24 oC must be tested within 4 hours of collection

Other Assays Fibrinogen, Thrombin Time, Factor Assays Centrifuged with plasma remaining on top of cells in unopened tube

kept at 2-4 oC or 18-24 oC must be tested within 4 hours of collection

Storage Requirements

TESTPLASMA

STABILITY AT RT

CENTRIFUGE TO PREPARE

PLATELET-FREE PLASMA

REFRIGERATION(Or transport on

ice)

FREEZEPLATELET-

FREEPLASMA

PT 24 hours Do not refrigerateIf >24 hour delay

in testing

PTRX 24 hours Do not refrigerateIf >24 hour delay

in testing

PTT 4 hoursIf >4 hour delay

in testing

PTTRX 2 hoursWithin one hour of

collectionIf >2 hour delay

in testing

TT 4 hoursIf >4 hour delay

in testing

OTHERASSAYS

4 hours Within one hour of

collectionWithin one hour

of collection

Storage Requirements

Other general notes Perform coagulation tests ASAP

Specimen may deteriorate rapidly (especially factors V and VIII)

If the testing is not completed within specified times, plasma should be removed from the cells and placed in a frost free freezer - 20 oC for two weeks -70 oC for six months

Storage Requirements

Send specimen on ice OR deliver to lab ASAP

Separate cells from plasma immediately via centrifugation

Transportation of Specimen

Platelet –Poor plasma (PPP) Platelet-Poor plasma is necessary for coagulation testing to

prevent activation of platelets and release of PF4, a heparin inhibitor.

The plasma platelet count must be < 10,000 /mm3. Specimen has been centrifuged for 15 minutes @ 2500 x g Why is PPP essential?

1. Contains platelet factor 4 (heparin neutralizer)

2. Contains phospholipids (affects lupus anticoagulant and factor assay testing)

3. Contains proteases (affect testing for vWF)

Platelet Poor Plasma

Platelets Poor Plasma preparation:

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.

Platelet-Rich plasma (PRP) Used in platelet function studies 200-300 x 10 9 /L Specimen must be centrifuged for 10 minutes @ 200 x

g

Platelets Rich Plasma (PRP)

Common Collection Problems Error Consequence Comment

Short draw<2.7 mL

PT/PTT falsely prolonged

Anticoagulant to blood ratio exceeds 1:9

Failure to mix specimen after collection

PT/PTT falsely prolonged

Blood clots form when anticoagulant & blood do not mix

Excess vigorous mixing

PT/PTT falsely shortened

Hemolysis and platelet activation cause start of cascade

Hemolysis PT/PTT falsely shortened

Reject specimen

Improper storage: wrong temperature or held too long

PT/PTT falsely prolonged

Must follow storage requirements

Chilling in refrigerator or placing on ice

PT falsely shortened Chilling to 4 oC activates factor VII.

Error Consequence Comment

Inadequate centrifugation PTT loses sensitivity for lupus anticoagulants and heparin.

Factor assays inaccurate

Prolonged tourniquet application

Falsely elevates vWF, factor VIII

Tourniquet causes venous stasis,

Drawing coagulation tube after to other anticoagulant tubes

PT/PTT falsely affected Contamination

Probing the vein PT/PTT falsely shortened Tissue thromboplastin is released activating coagulation

Heparin contamination from line draw

PTT falsely prolonged Heparin keeps the blood from clotting

Lipemia Test may not work Photo-optical methods affected

Common Collection Problems

Principles of Laboratory Analysis 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.

1. Immunological

2. Assays Using Chromogenic Peptide Substrates (Amidolytic Assays)

3. Coagulation Assays

4. Other Assays

Immunological 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.

Notes: 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 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 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 Assays 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.

Other Assays 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