General Approach in Investigation of Haemostasis Lecture 1: Introduction

Preanalytical Variables including Sample Collection. Site Selection. Storage Requirements. 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.

Sample Collection 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 When blood is withdrawn from a vessel, changes begin to take place in the components of blood coagulation.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. * 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. APTT tests are more sensitive to the presence of heparin. Additional benefits of trisodium citrate are that the calcium ion is neutralized more rapidly in citrate.

Sample 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% or lower than 25% High hematocrits may cause falsely prolonged test results due to an over- anticoagulated sample Formula correction achieves a 40% hematocrit (NCCLS) National Committee for Clinical Laboratory Standards. * National Committee for Clinical Laboratory Standards

HCT Citrate (ml) 0.20 0.70 0.25 0.65 0.30 0.61 0.55 0.39 0.60 0.36 0.31 0.27

Site Selection 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 The needle should not be more than 21 gauge (for infants a 22 or 23 gauge needle may be necessary). Order of Draw Evacuated tube system Blue top is 2nd If 2nd tube drawn, 1st top must be anticoagulant free (i.e. red top)

Storage Requirements 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 TEST PLASMA STABILITY AT RT CENTRIFUGE TO PREPARE PLATELET-FREE PLASMA REFRIGERATION (Or transport on ice) FREEZE PLATELET- FREE PLASMA PT 24 hours Do not refrigerate If >24 hour delay in testing PTRX PTT 4 hours If >4 hour delay in testing PTTRX 2 hours Within one hour of collection If >2 hour delay in testing TT OTHER ASSAYS

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

Transportation of Specimen Separate cells from plasma immediately via centrifugation Send specimen on ice OR deliver to lab ASAP Prior to analysis, frozen samples must be thawed rapidly at 37°C for 3 –5 min. Thawing at lower temperatures is not acceptable because some cryo-precipitation is possible.

Platelet 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 15 minutes @ 2500 x g Why is PPP essential? Platelets Contains platelet factor 4 (heparin neutralizer) Platelets Contains phospholipids (affects lupus anticoagulant and factor assay testing) Platelets Contains proteases (affect testing for vWF) Platelet factor 4 (PF4) is a small cytokine belonging to the CXC chemokine family that is also known as chemokine (C-X-C motif) ligand 4 (CXCL4) . This chemokine is released from alpha-granules of activated platelets during platelet aggregation, and promotes blood coagulation by moderating the effects of heparin-like molecules. Due to these roles, it is predicted to play a role in wound repair and inflammation.[1] It is usually found in a complex with proteoglycan. PF4 paly role in Thrombogensis by neutralizing anticoagulantly active vacscular HSPGs (Heparin Sulfate Proteoglycans) releaed by Endothelial Cells

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.

Platelets Rich Plasma (PRP) 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

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 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 Reject specimen Improper storage: wrong temperature or held too long Must follow storage requirements Chilling in refrigerator or placing on ice PT falsely shortened Chilling to 4 oC activates factor VII.

Common Collection Problems 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 venous stasis causes local release of fibrinolytic components into the vein.

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. Coagulation Assays Immunological Assays Using Chromogenic Peptide Substrates (Amidolytic Assays) Other Assays

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. 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.

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.

A chromogenic method for the determination of factor VIII activity A chromogenic method for the determination of factor VIII activity. Test plasma is incubated with calcium, phospholipid, and excess amounts of purified factors IX and X. The activated factor X generated by the reaction hydrolyzes a chromogenic substrate, generating a colored reaction product that is measured by a spectrophotometer. The amount of generated factor Xa is directly proportional to the concentration of factor VIII activity.

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 A diversity of new diagnostic assays resulting from these discoveries is expected in the near future, but at present the major clinical role for molecular analysis is in the diagnosis of inherited thrombophilia. Reptilase time (RT) is a blood test used to detect deficiency or abnormalities in fibrinogen,[1][2] especially in cases of heparin contamination. Reptilase, an enzyme found in the venom of Bothrops snakes, has activity similar to thrombin. Unlike thrombin, reptilase is resistant to inhibition by antithrombin III. Thus, the reptilase time is not prolonged in blood samples containing heparin, hirudin, or direct thrombin inhibitors, whereas the thrombin time will be prolonged in these samples. Reptilase also differs from thrombin by releasing fibrinopeptide A, but not fibrinopeptide B, in its cleavage of fibrin. Other causes of prolonged reptilase time include the presence of fibrin degradation products, which interfere with fibrin polymerization. Ristocetin is an antibiotic, obtained from Amycolatopsis lurida, previously used to treat staphylococcal infections. It is no longer used clinically because it caused thrombocytopenia and platelet agglutination. It is now used solely to assay those functions in vitro in the diagnosis of conditions such as von Willebrand disease (vWD) and Bernard-Soulier syndrome. Platelet agglutination caused by ristocetin can occur only in the presence of von Willebrand factor multimers, so if ristocetin is added to blood lacking the factor (or its receptor -- see below), it will not coagulate. In an unknown fashion, the antibiotic ristocetin causes von Willebrand factor to bind the platelet receptor glycoprotein Ib (GpIb), so when ristocetin is added to normal blood, it causes agglutination Clot solubility test Urea Method The plasma sample [patient and control] are clotted by the addition of an excess of calcium and incubated at 37°C for 30 minutes. An alternative approach involves clotting the plasma with thrombin and saline. The clot is removed and placed in 5M urea and incubated for 24hours at either room temperature or 37°C. If the clot has dissolved this suggests FXIII deficiency and a formal FXIII assay should be undertaken. Acetic Acid [Monochloroacetic acid [TCA] Method/Trichloroacetic [TCA] acid] The clot is removed and placed in either 2% acetic acid or 1% MCA and incubated for 24hours at either room temperature or 37°C . If the clot has dissolved this suggests FXIII deficiency.

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