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

2. 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

3. 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

4. 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

5. 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)

6. 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)

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

8. 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

9. 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

11. 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 times than in our body

12. 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)

13. 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).

14. 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).

15. 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

16. 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.

17. 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

18. 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

19. 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)

20. 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

21. 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: ) .
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:

22. 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.

23. 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

24. 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.

25. 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

26. 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

28. 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

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

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

31. 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.

32. 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.

33. 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.

34. 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.

35. 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

36. 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

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

38. 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

39. 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

40. 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

41. 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

42. Answers

43. 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.

44. 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

45. 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.

46. 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)

47. 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)

48. 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

49. 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

50. Example of an Inhibitor screen result

51. 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

52. 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

53. 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.

54. 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.

55. 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.

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

57. THANK YOU