Factor assays

Girum T.
 Factor assays
• Investigation of bleeding disorder resulting
from coagulation factor deficiency or defect
• Indicated by the screening tests
• Parallel line bioassays of coagulation factors
 General principle of parallel line bioassay

• If two materials containing the same

coagulation factor
• Assayed in a specific assay system in a range of
dilutions
• Clotting time plotted against the plasma
concentration
• In a double log paper, a curve with straight
middle section is obtained
 parallel line bioassay…
• Dilutions of test and standard materials
• Two straight parallel lines
• Distance between the two lines represent
potency of the factored assay
• If the test line is to the right of the standard, it
contains less of the factor than the standard
• If it is to the left, it contains more
 1-Stage PT-Based Factor Assays

• To establish the levels of V, VII, X and II

• Measuring the degree of correction of the
prothrombin time (PT) when plasma is added
to a plasma sample(substrate plasma)
specifically deficient in the factor to be
measured
1-Stage PT-Based Factor Assays
 Method
• You need a sharp pencil, a clear ruler, possibly
an eraser (!) and some Log-Log graph paper
• Prepare dilutions of a reference plasma
standard and the test plasma in buffered
saline e.g. 1/5, 1/10, 1/20, 1/40, 1/80 etc.
Dilution 1/10 1/20 1/30 1/40 1/80 1/100 1/1000
% activity 100 50% 33% 25% 12.5% 10% 1%
%
 Method
• equal volume [commonly 0.1ml] of the test or
reference plasma is incubated with an equal
volume of the factor deficient plasma at 37°C
• Perform PT on each of these mixtures
• The clotting times for each dilution are
recorded
• Plot the results – usually on Log-Log paper
Log-Log paper
Reference plasma plotted
Test plasma plotted
Driving factor activity
 One stage assay of factor VII
• The assay compares the ability of dilutions of
the patients plasma and standard plasma to
correct the PT of the substrate plasma
• Reagents
– PPP
– Standard/reference plasma
– Factor VII deficient plasma(substrate plasma)
– Barbitone buffer saline
– Thromboplastin
 Method

• Prepare dilutions of the test and standard

plasma
• Mix 0.1ml of the dilutions with 0.1 ml
deficient(substrate) plasma
• Add Thromboplastin and start the stopwatch
• Plot the graph and read the concentration
• NR: 0.5-2.0u/ml
 What test next?
• If you find a low FV result (and similarly if you find a
low FVIII result) you should request a FVIII (or FV)
assays
• If you find a low FVII result make sure that the test
was performed using human TF in the PT
• If you find a low level of a vitamin K dependent
clotting factor - consider the possibility that the
patient is on warfarin, has true vitamin K deficiency or
may have a mutation within the genes involved in
encoding the proteins involved in the vitamin K cycle.
 1-Stage APTT-Based Factor Assays

• widely used for the measurement of factors

VIII, IX, XI and XI
• Two key principles
– The APTT has a clear mathematical relationship
with the concentration of coagulation factors
– If the only variable altering the APTT of a sample
relative to a normal reference plasma is the level
of a specific coagulation factor
 Methods
• Reagents
– APTT reagents
– Standard/reference plasma
– Factor deficient plasma (substrate plasma)
– Patient Plasma
 Methods

• Serial dilutions of a standard reference mixed

with an equal volume of substrate plasma
• An APTT performed
• The clotting times for the APTT are then
plotted against dilution on Log-Lin graph
• The test plasma is treated the same way as
the reference plasma
• Read the concentration
 Interpretation

• In general the level of factors VIII and IX

correlate with bleeding risk.
• Severe haemophilia A or B (Factor VIII or IX <1
IU/dL [%])
• Moderate haemophilia A or B (Factor VIII or IX
is >1 IU/dL but less than <5 IU/dL [%])
• Mild haemophilia A or B (Factor VIII or IX is
between 5-30 IU/dL [%])
 Reference Ranges

• May be expressed as either a percentage or in

IU/dl
• Many factors, including factors VIII, IX, X, have
reference ranges of 50-150% alternatively
expressed as 0.50-1.50 IU/ml or 50-150 IU/dl
• The range for factor XI is narrower at 0.65 –
1.25 IU/ml.
 2-Stage & Chromogenic Factor Assays

• The 1-stage factor FVIII assay is relatively

simple to perform and accurate
• However, it does have limitations including
susceptibility to interference from preactivation
of factor VIII or anti-phospholipid antibodies
• In addition some F8 mutations can lead to
discrepant 1-stage/2-stage [chromogenic] FVIII
assay results
 2 stage…
• There are two alternatives
• Both based around an initial step to produce
factor Xa in a quantity proportional to the
amount of factor VIII present
• A second step to assay the amount of factor
Xa and so deduce the amount of factor VIII
present
 2-Stage FVIII Assay

• Involves an incubation step for production of

FXa and a second stage to determine the
amount of FXa produced.
• Components
– Activated serum (X,IX, Xia)
– Factor V
– Adsorbed patient plasma (removed II,VII,XI,X)
– Normal plasma
– CaCl2 and phospholipid
 Methods
• Adsorbed patient's plasma is mixed with activated
serum, factor V, calcium and phospholipid to
initiate coagulation and generate factor Xa
• After a fixed period of incubation an aliquot of the
mixture is added to an aliquot of normal plasma
and the time to clot formation measured
• Clotting times are plotted on double log paper and
from which the factor VIII level in the patient
sample can be derived
 Method
• The patient’s plasma is adsorbed to remove
prothrombin
• Coagulation is initiated by addition of factor XIa
and factor X and V are provided in excess
• The factor VIII level is the rate limiting step in the
formation of factor Xa
• In the second stage, a sample of the 1st stage
mixture is added to normal plasma and the time
to clot formation recorded
 Methods
• Clotting time will be dependent on the factor
Xa level in the sample from the first stage
• The factor Xa level  is, in turn, proportional to
the concentration of factor VIII in the patient’s
plasma
• The factor VIII level may, therefore, be derived
from the clotting time of the second stage
 Chromogenic FVIII Assay
• The assay is similar to the two-stage FVIII assay
• It involves an incubation step to generate FXa and
a second stage to determine the amount of FXa
produced
• In this case the amount of FXa is measured by its
action on a highly specific chromogenic substrate
• the colour intensity produced is directly
proportional to the amount of FXa, which in turn
is directly proportional to the amount of FVIII
 Chromogenic FVIII Assay
• Components
– Reagent cocktail for generating Fxa
• Contains FIXa, FX in excess, thrombin, a source of
calcium ions and phospholipid
– Chromogenic substrate
• A substance cleaved by FXa to produce a colour change
– Patient plasma
 Methods
• The patient’s plasma is incubated with the reagent
cocktail at 37°C
• FX activated to Fxa
• FVIII is the rate limiting step
• The chromogenic substrate is added
• After a second incubation period the absorbance at
a specific wavelength is measured
• Then compared to a reference curve to give the
FVIII level
Chromogenic FVIII Assay

ADCOCK et al. 2018

1-Stage Russell Viper Venom [RVV] Factor X Assay

• RVV is isolated from Daboia russelii.

• The principle of the RVV Factor X is similar to
that of the 1-stage PT-based Factor Assay
• A series of dilutions of the reference and test
plasma are made and clotted with RVV
• plotted on Log-Log paper and by comparison to
the reference FX standard the concentration of
FX in the test plasma can be determined
 RVV
• Components
– Reference or test plasma Dilutions: 1/10, 1/20,
1/140, 1/100
– RVV + Platelet substitute: Purchased commercially
– 0.025M Calcium Chloride  
– FX deficient substrate plasma: Purchased
commercially
 Methods
• FX deficient substrate plasma is mixed with an
the diluted reference or test plasma
• incubated at 37°C for 30s and then the RVV-
platelet substitute is added
• clotting is initiated by the addition of 0.025M
Calcium Chloride and the time to clot formation
recorded
• The results are plotted on Log-Log paper and the
results derived as for the 1-stage PT-based assay.
 Interpretation
• Causes of low factor X
– vitamin K deficiency and in patients on vitamin K
antagonists
– Congenital Factor X deficiency
– Liver disease
– Amyloidosis due to absorption of FX onto the
amyloid fibrils
– Acquired factor inhibitors [rare]
– Patients with deletions of chromosome 13 [chr13q]
 Clot solubility test for FXIII
• Fibrin clot formed in the presence of factor XIII
and thrombin are stable for at least 1 hour in 5
mol/l urea
• in the absence of factor XIII dissolve rapidly
• Clot solubility test is more sensitive without
calcium
• The use of 5 M urea as described here will
detect factor XIII deficiency of up to 5 iu/dl
• Reagents
– PPP From the patient and a control subject
– Thrombin
– Urea
 Method
• In duplicate, 0.2 ml patient plasma is mixed with
0.2 ml 10 NIH unit thrombin solution
• incubated at 37°C for 20 min
• Set up a normal plasma control in the same way
• Each tube is filled with approximately 3 ml of
urea solution
• carefully dislodging the clot, and left
undisturbed at 37°C for 24 hours
 Interpretation
• The control clot, if normal, shows no sign of
dissolving after 24 hours
• However, in the absence of factor XIII, the clot
will have dissolved
• The test is reported as normal if the clot is
present and abnormal if the clot is absent
• In suspected cases photometric and ELISA assays
of factor XIII are available for quantitative
measurement