General Approach in Investigation of Hemostasis Ms. Ibtisam H. AlAswad Mr. Mohammed A. Jabar

Preanalytical Variables including: 1.Sample Collection. 2.Site Selection. 3.Storage Requirements. 4.Transportation of Specimen.

Many misleading results in blood coagulation arise not from errors in testing but from carelessness in the preanalytical phase. Ideally the results of blood tests should accurately reflect the values in vivo. When blood is withdrawn from a vessel, changes begin to take place in the components of blood coagulation.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 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 neutralised more rapidly in citrate, and APTT tests are more sensitive to the presence of heparin. Sample Collection

Side note: Samples with High hematocrits 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 Sample Collection

(1.85 x )* (100-Hct)*V= C Where: C= volume of sodium citrate V=volume of whole blood drawn Hct= patient’s hematocrit Example: Patients Hct= 63% V= 5 mL (1.85 x )* (100-63)*5= C (1.85 x )* (37)*5= 0.34 mL Correction Formula: High Hematocrits HaematocritCitrate (ml)

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 Draw Order of Draw Evacuated tube system Blue top is 1 st or 2 nd tube. If 2 nd tube drawn, 1 st 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 o C or o C 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 o C or o C must be tested within 4 hours of collection Storage Requirements

Other Assays Fibrinogen, Thrombin Time, Factor Assays Centrifuged with plasma remaining on top of cells in unopened tube kept at 2-4 o C or o C must be tested within 4 hours 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 o C for two weeks -70 o C 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) <10 x 10 9 /L Specimen has been centrifuged for x g Why is PPP essential? 1.Contains platelet factor 4(heparin neutralizer) 2.Contains phospholipid (affects lupus anticoagulant and factor assay testing) 3.Contains proteases (affect testing for vWF) Terms

Platelet-Rich plasma(PRP) Used in platelet function studies x 10 9 /L Specimen has been centrifuged for x g Terms

Common Collection Problems ErrorConsequenceComment Short draw <2.7 mL PT/PTT falsely prolongedAnticoagulant to blood ratio exceeds 1:9 Failure to mix specimen after collection PT/PTT falsely prolongedBlood clots form when anticoagulant & blood do not mix Excess vigorous mixingPT/PTT falsely shortenedHemolysis and platelet activation cause start of cascade HemolysisPT/PTT falsely shortenedReject specimen Improper storage: wrong temperature or held too long PT/PTT falsely prolongedMust follow storage requirements Chilling in refrigerator or placing on ice PT falsely shortenedChilling to 4 o C activates factor VII. Inadequate centrifugationPTT loses sensitivity for lupus anticoagulants and heparin. Factor assays inaccurate Desire platelet poor plasma Prolonged tourniquet application Falsely elevates vWF, factor VIIITourniquet causes venous stasis,

Common Collection Problems ErrorConsequenceComment Drawing coagulation tube PRIOR to other anticoagulant tubes PT/PTT falsely affectedContamination Probing the veinPT/PTT falsely shortenedTissue thromboplastin is released activating coagulation Heparin contamination from line draw PTT falsely prolongedHeparin keeps the blood from clotting LipemiaTest may not workPhoto-optical methods affected

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: D o n ot f reeze l atex 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. Applications of latex particles include the following: Calibration standards Filter challenges Agglutination assays Phagocytosis studies Flow cytometry standards Light scattering studies

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.

Chromogenic Assay Enzymatic activity of coagulation proteins and other substances can be measured by the chromogenic method in two ways: Direct measurement- the greater the change in optical density, the higher the level of the analyte. In essence, the change in optical density is pro­portional to the amount of the substance being measured (e.g., protein C activity assays). Primary assays, in which a substrate specific for the enzyme to be measured is used. Secondary assays, in which the enzyme or proenzyme measured is used to activate a second protease for which a specific substrate is available.

Chromogenic Assay Indirect measurement- the substance being measured has an inhibitory effect on another target enzyme that has activity directed toward the syn­thetic substrate. In this case, the change in optical density is inversely proportional to the amount of the substance being measured (e.g., heparin anti­factor Xa assays). Specific substrates are available for many coagulation enzymes. However, the substrate specificity is not absolute and most kits include inhibitors of other enzymes capable of cleaving the substrate to improve specificity.

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 Other assays include measurement of coagulation factors using snake venoms, assay of ristocetin cofactor, and 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