Basic coagulation techniques and Quality control issues

Dr. Shrimati ShettyDeputy Director

National Institute of Immunohematology ( ICMR)KEM Hospital, Mumbai

Final Diagnosis of a bleeding disorder

Anticoagulant

0.129 M tri sodium Citrate at a ratio of 9 parts blood to 1 part anticoagulant is used for all coagulation tests.

Why not EDTA or Heparin?

EDTA irreversibly chelates Ca ions

Heparin activates antithrombin which is an inhibitor of coagulation

Anticoagulant…

If the HCT is above 55% Anticoagulant vol. [x] = 100 – hematocrit x

total vol. of anticoagulated blood required Example: Patient hematocrit = 60%

100 - 60 x 5.0 = 0.37 mL

Preanalytical variables

Sample hemolysis/ lipaemic sample Improper proportion of Anticoagulant to blood Prolonged time interval before testing Difficult punctures Freeze thawing the samples

Coagulation Tests

Screening coagulation tests Confirmatory Tests

When a bleeding patient walks in, what should be the initial tests to be performed?

Screening tests Peripheral smear ( Bernards Soulier syndrome, macrothrombocytopenia,

leukemia , thrombocytopenia )

Complete Blood Count ( BSS, MTCP, Leukemias, thrombocytopenia)

PT APTT TT FXIII screening

Only PT is abnormal

Congenital Causes

Factor VII deficiency

Acquired Causes

Liver disease

Warfarin

DIC

Inhibitors

LA

Malignancy

PT - INR

PT measures the deficiency of all VK dependent coagulation factors and also the integrity of extrinsic pathway

INR = [Patient PT]ISI

[Control PT]

Only APTT is abnormal

Congenital Causes

Factor VIII/ IX/XI/XII contact factors

Acquired Causes

Liver disease

Warfarin

DIC

Heparin

Inhibitors to factors,

LA

PT & APTT prolonged

Congenital causes

i.Combined deficiency of V & VIII

ii. Factor X deficiency

iii. Factor V deficiency

iv. Multiple VK dependent clotting factor deficiency

Acquired causes

Liver disease

Warfarin

DIC

Heparin

Inhibitors to factors

vitamin K deficiency

PT, APTT, TT prolonged

Congenital causes

i. Afibrinogenemia/ dysfibrinogenemia

ii. Factor II deficiency

Acquired causes

DIC

Liver disease

Screening for F XIII deficiency

Clot solubility test Clot formation with either thrombin or CaCl2 Solubility of the clot using 2% acetic acid , 1% mono

chloroacetic acid or urea After 24 hours , the clot is observed for solubility Different sensitivities with different clotting reagents &

solvents Severe factor deficiency sometimes gets misdiagnosed as

F XIII deficiency

ELISA test is the sensitive assay for detecting F XIII deficiency

Confirmatory tests-Factor Assays

Reagents required

Normal pooled plasma or unicalibrator

Factor Deficient plasma

APTT reagent

CaCl2

Factor assays….

Factor VIII/IX/XI/XII – APTT based

Factor II/VII – PT mode

Factor V/X – can be both PT/APTT mode

Factor VIII Graph

Interpretation of factor results

At birth, activities of the vitamin K dependent factors II, VII, IX, and X and the concentrations of the contact factors XI and XII are reduced to about 50% of normal adult values. The levels of the factors V, VIII, XIII, and fibrinogen are similar to adult values

Plasma concentrations of the naturally occurring anticoagulant proteins (antithrombin, protein C, and protein S) are significantly lower at birth than during the adult years

Most blood coagulation factors and fibrinogen increase during pregnancy. Factor (F) XI is the only blood coagulation factor that decreases.

Malignancies

Interpretation

Should we go ahead with factor assays even when screening tests are normal?

yes, in case of any clinical indication we should do the specific factors even if PT/APTT is normal

NPP

20-25 healthy donors , blood group matched Compare it with unicalibrator with known

factor values Never use a single individual sample as

control

Deficient Plasma& APTT reagents

Should have 0% FVIII Negative for TTD/inhibitors Different APTT reagents have different

sensitivities

Factor Assays

Severe <1% factor Moderate 1-5% Mild 6-40%

About 5% of the patients are clinically mild despite having <1% factor

Can we diagnose a patient as HA or HB without doing factor assays?

Mixing Studies

BaSO4 adsorbed normal plasma, deficient in factors VII, IX, X and prothrombin

Aged normal serum, deficient in factors V and VIII, prothrombin, and fibrinogen

Mixing Studies….

Result Interpretation

NPP+Adsorbed Plasma

Correction F VIII deficiency

NPP+ Aged Serum

No Correction

NPP+Adsorbed Plasma

No Correction F IX deficiency

NPP+ Aged Serum

Correction

Mixing Studies…..

Mixture Result Interpretation

NPP + F VIII Def Plasma

Correction F IX deficiency

NPP + F IX Def Plasma

Correction F VIII deficiency

Platelet Aggregation tests

Highly variable results Diet, Medication, physical activity Platelet receptor studies to confirm diagnosis Always confirm diagnosis by other tests

Never give a diagnosis based on platelet aggregation alone!

Disorders diagnosed by platelet aggregation and receptor studies

Von Willebrand disease Glanzmanns thrombasthenia Bernard Soulier syndrome Storage pool defect Cyclooxygenase deficiency others

What are the other supporting tests?

Platelet receptor studies using antibodies specific for platelet receptors

GP 1b/IX for diagnosis of BSS (CD 42)

GP IIb/IIIa for diagnosis of GT( CD41, CD61)

collagen receptors ( CD36)

Diagnosis of VWD

Platelet aggregation with Ristocetin ( 1.25 mg/ml) absent or reduced

Type IIb shows increased aggregation with 0.5 mg/ml whereas in normal cases there is no aggregation

VWF by Electrophoresis sensitive only for severe def ELISA is the test of conirmation RCof , collagen binding ELISA and Multimer analysis

to subtype

Diagnosis of BSS

Giant platelets in peripheral smear Normal or reduced platelet count Absence of aggregation with 1.25 mg/ml

risticetin Absence of GP 1b/IX receptors by flow

cytometry

Diagnosis of GT

Absence of aggregation with 6uM ADP, 4ug/mL collagen and 0.75mM arachdonic acid

Absence of GP IIb/IIIa receptors by flow cytometry

Storage pool defect

Primary phase aggregation with all agonists

Screening for inhibitors ( Mixing studies)

NPP and patient plasma mixed and APTT performed at 0 hour, 1 hour and 2 hour

Should exclude Lupus anticoagulants FVIII inhibitors are generally progressive ,

FIX inhibitors/LA immediate acting

Screening for inhibitors

0 hr 1 hr 2 hrs

NPP

Patient

Separately incubated

Incubated Mix

The Bethesda Assay

Specialized Investigations

Thromboelastography

Thromboelastography

Thrombinoscope

Thrombinoscope

PFA 100

Quality control exercises

IQC EQC

Some examples

Case 1

Peripheral smear- giant platelets seen

Platelet count 130X103/uL

PT – C14 Secs/ P 15 secs ;

APTT – C29 secs/P 32 secs;

TT – C 16 secs/P 15 secs

F XIII - N

RIPA – Ristocetin 5%

ADP, collagen, AA 90-100%

GP1b/IX receptors – highly reduced

Diagnosis : BSS

Some examples …..

Case 2

PS – normal

Platelets 260X 103/ul

PT C14 secs/ P 32 secs

APTT C 30 secs P 58 secs

TT C 15 secs/ P 16 secs

F XIII - N

FX 96%; F V 15%

F VIII 8%

Diagnosis: Combined deficiency of F V and VIII

Thank you