Presentation on theme: "General Approach in Investigation of Haemostasis"— Presentation transcript:
1General Approach in Investigation of Haemostasis Lecture 1: Introduction
2Preanalytical Variables including Sample Collection.Site Selection.Storage Requirements.Transportation of Specimen.
3HaemostasisHemostasis 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.
4Sample CollectionProper 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stored.Reference Laboratory Services will immediately notify the client of any problems with the sample.When blood is withdrawn from a vessel, changes begin to take place in the components of blood coagulation.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.
5Sample 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 osmolalityAnticoagulant/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 anticoagulantCLSI guideline is +/- 10 % of fill linePurpose of the anticoagulant is to bind or chelate calcium to prevent clotting of specimen(CLSI ) Clinical and Laboratory Standards Institute.* CLSI : Clinical and Laboratory Standards Institute
6Sample CollectionOther 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.
7Sample Collection : Samples with High hematocrits 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 sampleFormula correction achieves a 40% hematocrit(NCCLS) National Committee for Clinical Laboratory Standards.* National Committee for Clinical Laboratory Standards
9Site Selection Untraumatic venipuncture is required Traumatic venipunctures release tissue factor and initiate coagulationFingersticks/Heelsticks are not allowedIndwelling IV line draws are discouragedContain heparin & diluted bloodFalsely increased resultsOrder of DrawEvacuated tube systemBlue top is 2ndIf 2nd tube drawn, 1st top must be anticoagulant free (i.e. red top)
10Storage Requirements Prothrombin Time: PT Uncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or oC must be tested within 24 hours of collectionActivated Partial Thrombin Time: APTTUncentrifuged or centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or oC must be tested within 4 hours of collectionOther AssaysFibrinogen, Thrombin Time, Factor AssaysCentrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 oC or oC must be tested within 4 hours of collection
11Storage Requirements TEST PLASMA STABILITY AT RT CENTRIFUGE TO PREPARE PLATELET-FREE PLASMAREFRIGERATION(Or transport on ice)FREEZEPLATELET-FREEPLASMAPT24 hoursDo not refrigerateIf >24 hour delay in testingPTRXPTT4 hoursIf >4 hour delay in testingPTTRX2 hoursWithin one hour of collectionIf >2 hour delay in testingTTOTHERASSAYS
12Storage Requirements Other general notes Perform coagulation tests ASAPSpecimen 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
13Transportation of Specimen Send specimen on ice OR deliver to lab ASAPSeparate cells from plasma immediately via centrifugation
14Platelet Poor Plasma 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 x gWhy is PPP essential?Contains platelet factor 4 (heparin neutralizer)Contains phospholipids (affects lupus anticoagulant and factor assay testing)Contains proteases (affect testing for vWF)
15Platelets Poor Plasma preparation: To prepare platelet-Poor plasmaCentrifuge 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.
16Platelets Rich Plasma (PRP) Platelet-Rich plasma (PRP)Used in platelet function studiesx /LSpecimen must be centrifuged for x g
17Common Collection Problems ErrorConsequenceCommentShort draw<2.7 mLPT/PTT falsely prolongedAnticoagulant to blood ratio exceeds 1:9Failure to mix specimen after collectionBlood clots form when anticoagulant & blood do not mixExcess vigorous mixingPT/PTT falsely shortenedHemolysis and platelet activation cause start of cascadeHemolysisReject specimenImproper storage: wrong temperature or held too longMust follow storage requirementsChilling in refrigerator or placing on icePT falsely shortenedChilling to 4 oC activates factor VII.
18Common Collection Problems ErrorConsequenceCommentInadequate centrifugationPTT loses sensitivity for lupus anticoagulants and heparin.Factor assays inaccurateProlonged tourniquet applicationFalsely elevates vWF, factor VIIITourniquet causes venous stasis,Drawing coagulation tube after to other anticoagulant tubesPT/PTT falsely affectedContaminationProbing the veinPT/PTT falsely shortenedTissue thromboplastin is released activating coagulationHeparin contamination from line drawPTT falsely prolongedHeparin keeps the blood from clottingLipemiaTest may not workPhoto-optical methods affected
19Principles 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.ImmunologicalAssays Using Chromogenic Peptide Substrates (Amidolytic Assays)Coagulation AssaysOther Assays
20ImmunologicalInclude 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.
22latex 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.
23Notes: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.
24Chromogenic AssayChromogenic, 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.
25Coagulation AssaysCoagulation 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.
26Coagulation AssaysCoagulation 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.Mixing studies are tests performed on blood plasma used to distinguish factor deficiencies from factor inhibitors, such as lupus anticoagulant, or specific factor inhibitors, such as antibodies directed against factor VIII. Mixing studies take advantage of the fact that factor levels that are 50 percent of normal should give a normal Prothrombin time (PT) or Partial thromboplastin time (PTT) result.If the problem is a simple factor deficiency, mixing the patient plasma 1:1 with plasma that contains 100% of the normal factor level results in a level ≥50% in the mixture (say the patient has an activity of 0%; the average of 100% + 0% = 50%). The PT or PTT will be normal (the mixing study shows correction). However, if there is an inhibitor that inactivates the added clotting factor, the resulting factor level will be low and the clotting test will be prolonged (fails to correct). Therefore, correction with mixing indicates factor deficiency; failure to correct indicates an inhibitor.
27Other AssaysUsing 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