Screening Tests Selection of reagents & Mixing studies Dr Aboobacker Mohamed Rafi Assistant Professor Dept of IHBT JMMCRI Thrissur ,Kerala

Recap We understood the physiology of coagulation Importance of History taking Importance of Pre analyticals Bleeding Assessment Tool to be used & Scored So if the patient needs evaluation We start with certain Blood Tests The coagulation screening tests

Screening test Screening test is a preliminary procedure to detect the most characteristic sign of a disorder that may require further confirmation. The test should be :- - Done in a short time - Comparatively little effort - Cheap - Very sensitive - Serve as a selection procedure for further specific investigation Aim of the test/tests is not to miss any step that would put the patient at risk of bleeding

Screening Tests for Hemostasis Platelet count Bleeding time (BT) Prothrombin time (PT) Activated Partial thromboplastin time (aPTT) Thrombin Time (TT) Factor XIII screening Most important is - History

Value of history in Diagnosis An abnormal coagulation parameter has no diagnostic value, if the patient is not bleeding has not been a bleeder ( no past history of bleeding) not likely to be a bleeder – ( no family or medication history)

Tests for Secondary haemostatic Factor defects - All the clotting factors are present in the plasma Tests for Clotting factors - plasma based tests Important – care in preparing plasma Starts at Blood sampling itself ( Pre analyticals already discussed)

Preparing plasma Anticoagulate blood in Trisodium Citrate (3.2% ) (0.105 - 0.129mol/l) 1:9 citrate to blood ratio Carefully collected to prevent activation of factors Careful not to make bubbles

Preparing Plasma Centrifuge at high speed (1500 – 2000g for 10 min) - To separate Plasma as Platelet Poor Plasma (platelet count <10,000) Test immediately before labile factors are lost. If not keep in freezers Time sensitive - FV and FVIII labile (<4 hours) Storage - 4 hrs at 4°C - 1 month at -20°C - 6 months at -70 °C

Plasma Based Tests All clotting tests to be done in a 370C water bath. A circulating water bath is ideal. Keep test plasma and / or control plasma and reagents at 370C at least 5 min before doing the tests Always a PNP to be run as control

PROTHROMBIN TIME ( PT) The PT assess extrinsic and common pathways Detect deficiencies in factor VII,II,V & X as well as low fibrinogen levels PT reagent: Thromboplastin which contains Tissue factor Negatively charged phospholipids Calcium (recombiplastin) It is manufactured using recombinant human tissue factor produced in Escherichia coli and synthetic phospholipids TF in reagents are 25000 times than in our body

Prolongation of PT indicates Reduced concentration of one or more relavant clotting factors,factor VII deficiency warfarin usage (vitamin K antagonists) Hepatic impairement DIC Nutritional deficiency & malabsorption ( vitamin K)

PROCEDURE Add 0.1ml ( 100μl) plasma into duplicate small glass tubes placed in a 370C water bath, and leave 3 min to equilibrate to 370C. Add 0.2ml ( 200μl) pre warmed recombiplastin & simultaneously start stop watches. Mix and tilt tubes to nearly horizontal position at one second intervals, otherwise maintaining in 37oC water bath. Depress stop watch mechanism on first appearance of a clot. The time taken for the clot to form (i.e. between addition of Recombiplastin and the appearance of the clot) is the ‘Prothrombin Time' (PT).

Expression of the Results: The results are expressed as a Take the mean of the duplicate readings (providing that they don't differ by more than two seconds) otherwise repeat the procedure and check the reagent; replace if necessary Expression of the Results: The results are expressed as a mean of the duplicate reading in seconds Both mean of the patient time and mean of the PNP time Results are always interpreted with INR (International Normalized Ratio).

Normal Range Normal Range : 11 – 16 Secs Ideally, each laboratory should establish its own control Prothrombin Time Control PT: test at least 20 normal plasmas in that laboratory’s PT assay, and taking the mean PT & calculate the SD Each day the aliquot of the PNP to be run as control & plot in LJ chart

PT Result Variety of reagents (containing different thromboplastin) are available in the market some are good and some are bad giving different PR (Prothrombin Ratio) on the same sample. Prothrombin ratio (PR) = Patient prothrombin time Control prothrombin time Prolonged PR - >1.2 (3 Seconds more than Control) Ideally a good screening test reagent should be very sensitive so that all levels of low factors can be picked up when a PT is done.

Sensitivity of a PT reagent Most sensitive PT reagent – picks up the mildest of deficiency and exaggerates the difference between ranges of deficiency. PT is abnormal even if FVII is 40% and there is a significant difference in PT time when levels are 13% and 17% - GOOD Reagent Least sensitive PT reagent- misses deficiency and will not show the difference between ranges of deficiency . PT is normal even if FVII is 21% and there is no significant difference in PT time when levels are 11% and 17% - BAD Reagent

Sensitivity & ISI and Importance of INR International sensitivity index (ISI)- is a value that is assigned indicating the sensitivity of the reagent. Most sensitive reagent has ISI close to a value of 1 Less sensitive reagents will be more than 1.4 onwards International normalized ratio (INR) is normalizing a PT expressed as PR by incorporating the allocated sensitivity (ISI) of the PT reagent used = PRISI

INR (International Normalized Ratio) INR= ( Test PT /Control PT)ISI ISI = International Sensitivity Index. There has been more success in standardizing the PT than the APTT INR is used as a standardized system for oral anticoagulant monitoring Keep the patient’s INR between 2-3,If the patient is on warfarin <2:thrombosis >3:bleeding This overcame reagent variabilities found when using international sensitivity index (ISI)

Activated partial thromboplastin time (aPTT) It is the time required for the clotting of citrated plasma after the addition of activated partial thromboplastin reagent and calcium chloride Partial thromboplastin reagent –a reagent lacking the apoprotein component of the complete thromboplastin reagent APTT should detect a deficiency of clotting factors II,V,VIII,IX,X,XI,XII and fibrinogen ( Intrinsic Pathway) Great variability between available reagents in the composition of phospholipids

aPTT Reagents Various reagents are available even in developing countries for both PT and aPTT 17 aPTT reagents and 20 PT reagents are used in India among the laboratories participating in the National EQAS program (Mammen J, et al. Semin Thromb Hemost. 2007; 33: 265-72) . Select reagents Sensitive to factor deficiency. Reflect good responsiveness It is very important for each laboratory to carefully identify its upper limit of normal Barna L, Triplett DA. Ric Clin Lab 1989; 19: 345-54

PROCEDURE Add 0.1ml ( 100μl) of plasma and 0.1ml ( 100μl) APTT reagent in a glass tube (12 x 75) and place in a 37oCwater bath Mix, and leave for 5 minutes to equilibrate to 370 C and to provide suitable activation of plasma with contact factor Add 0.1ml ( 100μl) of warmed 0.025M cacl2, and simultaneously start stopwatch Mix and tilt tubes to nearly horizontal position at one-second intervals, otherwise maintaining in 370C water bath Depress stopwatch mechanism on first appearance of a clo The time taken for the clot to form (i.e., between addition of CaCl2 and the appearance of the clot) is the APTT Always do in duplicates Take the mean of the duplicate reading (provided that they don’t differ by more than 2 seconds otherwise repeat procedure and check reagents.

RESULTS Report APTT results in seconds. REFERENCE RANGE: Each lab has to establish their own range As a rough guide the aPTT of normal plasma should be between 25 to 35 sec. A difference of more than 6 sec between the control and patient is considered as abnormal and needs further evaluation

The common causes of a prolonged APTT are as follows: Deficiency of a coagulation factor other than factor VII Disseminated intravascular coagulation Liver disease Massive transfusion with packed cells only Administration of or contamination with heparin or other anticoagulants A circulating anticoagulant (inhibitor) Moderately prolonged in patients taking oral anticoagulant drugs and in the presence of vitamin K deficiency.

aPTT reagent selection Concentration of phospholipid varies markedly between reagents. If a lupus-sensitive reagent is used for the initial APTT, it is useful to perform a second APTT using a reagent which has a very high phospholipid concentration (Actin FS) If the prolongation with the first reagent is caused by lupus anticoagulant, then the second APTT is almost always normal So it is always better to use Lupus insensitive aPTT reagents for screening

REAGENTS FOR SCREENING TESTS Many different reagents are available throughout the world. The following sources of information in relation to the likely performance of a particular reagent can be considered: Comparative data in relation to other reagents from EQA schemes Published data Local testing of plasma from patients with known defects Manufacturers data sheets

Reagent selection To have at least 2 types of APTT reagents in the lab For Routine factor assays , inhibitor assays & patients on Heparin , a lupus insensitive reagent should preferentially be used. In patients clinically suspected of lupus inhibitors, LA-sensitive APTT reagents should be used.In such patients the clinical history of patients also should be integrated with the report APTT Reagents for Different Coagulation Tests: One Size Does Not Fit All

THROMBIN TIME Thrombin time assesses the final step of coagulation i.e conversion of fibrinogen to fibrin by thrombin.

Principle Thrombin is added to patients plasma and time required for clot formation is noted. Reagent :thrombin solution Specimen :citrated platelet poor plasma .

Procedure Pre warm thrombin reagent at 37 deg C Pipette out 0.2 ml(200μL) of test and control plasma into glass tubes and incubate at 37c for 2 mins. Add 0.2 ml (200μL) of thrombin reagent to each tube ,and immediately start the stopwatch and mix tube contents Tilt tubes to near horizontal position at one second intervals maintain solution at 370C. Depress stopwatch mechanism upon formation of a clot . This is the thrombin time. Always perform the tests in duplicates . Take the average reading and report time in secs. Normal range: ±3 seconds of control.

Result interpretation Report TT results in seconds . Each lab has to establish their own range. A patient’s TT should be within 2 s of the control (i.e. 15–19 s). Times of 20 s and longer are definitely abnormal.

Common causes of prolonged TT Hypofibrinogenaemia as found in DIC and, more rarely, in a congenital defect or deficiency Raised concentrations of FDP, as encountered in DIC or liver disease Dysfibrinogenaemia, either inherited or acquired, in liver disease or in neonates Hypoalbuminaemia Paraproteinemia. Shortening of the TT occurs in conditions of coagulation activation.

What next If results are abnormal (Prolonged time) Prolonged aPTT indicates deficiency of FXII, FXI, FIX, FVIII, FX, FV, FII and Fibrinogen Prolonged PT indicates deficiency of FVII, FX, FV, FII and Fibrinogen A Thrombin time is done since it picks Fibrinogen deficiency.

Mixing/Correction Studies Done when PT/APTT are prolonged Mix equal volumes of Test plasma which gave the abnormal time Control plasma ( PNP -where all factors are present in normal quantity) The possibilities Correction : Deficiency of factor No correction: Inhibitor Note : Standard Mixing studies – will miss a FVIII inhibitor

Mixing study Factor Deficiency 1:1 Mixing Study with PNP + Prolonged aPTT occurs when factor < 35-40% Principle : Mixing study is done to evaluate if the prolongation of aPTT is due to a factor deficiency 1:1 Mixing Study with PNP Test Sample (Haemophilia) Pooled Normal Plasma 1: 1 Ratio mix Correctable Normal aPTT Run aPTT + 50% 0% factor 100% factor Factor Deficiency

No correction Due to - Heparin-use protamine sulphate - Lupus anticoagulant (antiphospholipid antibody) - Factor Inhibitor ( antibodies to Clotting factors) - FDP/D-Dimer

FVIII inhibitor Late acting & Time dependent Picked only on incubation , for eg, Patient – aPTT = 120 sec Control = 30 sec Mixing = 40 sec (fresh mix) (Keep the “Mix” and individual plasmas 2 hours in water bath) aPTT of Incubated Mix = 102 sec

Correction Studies Direct interpretation to therapy to patient who is bleeding with a prolongation in plasma clotting tests Non Correction of time – No benefit of transfusing FFP. Use safer products Heparin – use Protamin Sulphate Inhibitor – other agents –like FEIBA

Mixing studies – contd…. Adsorped Plasma / Factor IX def Plasma Useful in resource limited settings for correction studies The plasma thus prepared is deficient in Vitamin K related factors (FII, FVII, FIX, FX) The common adsorbing agents used are Barium sulphate & aluminum hydroxide Aged serum / Factor VIII def plasma The plasma prepared will be deficient in labile factors (FI, FII ,FV,FVIII) The serum tube to be kept in 370C water bath for 4 hours or 48 hrs in Room temperature andserum separated

For Practice aPTT Test sample Mixing with PNP Mixing with Aged serum Factor VIII def Plasma Adsorbed plasma Or factor IX def Plasma 1 Prolonged Corrected No Correction Correction 2 3 4

Answers

INHIBITOR SCREEN-Its importance The presence of an inhibitor can be determined by a simple mixing experiment using the test plasma and normal pooled plasma. This method is generally reliable but some factor VIII antibodies, which have slow reaction rates, may be missed if insufficient time is allowed for the incubation step.

Inhibitor For children How often do we screen for it For children Once every five exposure days until 20 exposure days Every 10 exposure days between 21 and 50 exposure days at least two times a year until 150 exposure days. For adults Every 6-12 monthly review Or any failure to respond to adequate factor in a previously responsive patient Recent intensive treatment Prior to surgery

Inhibitors affecting APTT may be Immediate-acting Time-dependent. Patient plasma & NPP and are incubated at 37°C for 1–2 hours, both separately and as a 50:50 mixture.

Inhibitor screen - Procedure Place 0.5ml (500 μl) of pooled normal plasma (PNP) in a test tube no.1 Place 0.5ml (500 μl) of test plasma in a test tube no.2 Mix 0.5 ml (500 μl) of PNP and 0.5 ml (500 μl) of test plasma in a test tube no.3 Place all the tubes in a water bath (370C)

Fresh mix & Incubated mix At the end of first hour mix equal amounts of PNP and test plasma and do aPTT (i.e., FRESH MIX). Do an aPTT on tube no.3 (pre mixed PNP + test plasma – i.e. INCUBATED MIX)

aPTT is done on All The APTT is then determined on the A=Patient plasma B= NPP C=Incubated mixture D=Fresh Mixture prepared from equal volumes of test and normal plasma after separate incubation (Immediate mix). aPTT is done on All

RESULTS & Interpretation If difference between fresh mix and incubated mix is >5 sec indicates the presence of Inhibitors. If difference between fresh mix and incubated mix is <5 sec considered as absence of inhibitors. If inhibitor screen positive - Proceed to Inhibitor Assay

Example of an Inhibitor screen result

APTT correction of the mix are compared Poor correction in Immediate Mix( D ) suggestive of an immediate-acting inhibitor. (Factor IX inhibitor) Poor correction in the incubated mixture ( C ) is suggestive of a time-dependent inhibitor ( Factor VIII inhibitor) Aptt of C > D Time dependent Inhibitor Confirmations and quantification of the titre is performed using the Nijmegen modified Bethesda assay Titer of ≥ 0.6 BU/ml is to be taken as clinically significant

UREA SOLUBILITY TEST FOR FACTOR XIII (Screening assay) Factor XIII or clot stabilizing factor is a transglutaminase enzyme which after activation crosslinks fibrin strands by catalyzing the formation of covalent bonds between the fibrin molecules thereby stabilizing the fibrin clot

Urea clot solubility test Principle: Thrombin and calcium (Ca) are required to activate FXIII so that it will crosslink fibrin in to a stable form. Citrate sample – Patient sample & Positive control EDTA sample – Used as Negative control Addition of 2% acetic acid or 5M urea results in the lysis of non-cross linked clots.

Procedure Add 500 uL of above-mentioned samples( Test, PC & NC) Add 500uL of 0.025M CaCl2 to each tubes Leave the tubes in water bath at 37 deg C for 30 min to form clot. After 30 min tap the tube gently to loosen clot from sides and transfer the clot into 5 ml of 5 M urea solution. Leave the tubes at room temperature for 24 hrs and look for the lysis.

Interpretation Normal citrated sample (PNP) should have an intact clot visible. Patient sample: Complete lysis of the clot in the solution indicates Factor XIII deficiency. NOTE: This test is more sensitive if only Factor XIII is less than 2.5% Factor XIII levels can also become decreased in individuals with DIC, Severe liver disease, Acute Leukemia.

References WFH , Lab manual on Diagnosis of Hemophilia and Other Bleeding Disorders, second edition Practical Hemostasis & Thrombosis Dacie & Lewis – Practical Hematology – 11th edition

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