Data Interpretation - Factor Assays - Answers

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Data Interpretation: Factor Assays
Introduction
This section covers factor assays but strays into other areas and so is not specific to factor assays.
Question 1
A 72-year-old man is admitted through Accident and Emergency with a 2-week history of easy bruising. He was on no
drugs at the time of his visit to hospital. Shown below are the results of his initial investigations:
Test Patient Reference Range

PT 13s 10.6-12.4s
APTT 105s 21-32s
Fibrinogen (Clauss) 3.9g/L 2-4g/L
1. What are the possible diagnoses taking into account the patient's history.
2. What additional investigations would you request?
Click here for Part 2
1. The history suggests that the problem is of recent onset and therefore this excludes an inherited bleeding
disorder. The prolonged APTT and normal PT would suggest a deficiency of factors VIII, IX, XI or XII. Of these
the most likely is FVIII deficiency i.e. Acquired haemophilia A. A Factor VIII assay would, therefore, be a logical
test to perform.
2. You should also screen for a lupus anticoagulant [SCT and dRVVT.] This can cause bleeding problems in some
patients either because of thrombocytopaenia or a low FII level [prothrombin deficiency.] It will also prolong the
APTT. We are not given the platelet count and so this remains a possibility. The PT is normal and so acquired
prothrombin deficiency is unlikely.
3. You should request a full blood count to check the platelet count.
The results of factor assays are show below:
Factor Assay Patient Reference Range

FVIII 0.23 IU/mL 0.45 - 1.49 IU/mL
http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

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FIX 1.18 IU/mL 0.50 - 1.51 IU/mL
FXI 1.01 IU/mL 0.50 - 1.50 IU/mL
FXII 1.67 IU/mL 0.45 - 1.49 IU/mL
1. What additional tests might you request and why?
1. You should request a FVIII inhibitor screen to exclude or confirm a diagnosis of acquired haemophilia A.
2. You should also request Von Willebrand Factor assays to exclude/confirm a diagnosis of acquired Von
Willebrand Syndrome [AVWS.]
Shown below are the results of additional investigations on this patient:
Assay Patient Reference Range

VWF:Ag 0.21 IU.mL 0.48 - 1.55 IU/mL
VWF:Act 0.19 IU/mL 0.50 - 1.50 IU/mL
FVIII:C 0.19 IU/mL 0.45 - 1.49 IU/mL
Protein electrophoresis M Band [IgM 11.3g/L]
IgG 4.2g/L IgG 6 - 13g/L
Immunoglobulins IgM 17.3g/L IgM 0.4 - 2.2g/L
IgA 2.2g/L IgA 0.8 - 3.7g/L
Renal & LFTS
Normal
Calcium
1. What is the diagnosis?

The diagnosis is Acquired Von Willebrand Syndrome [AVWS.] Syndrome is used to distinguish it from the
inherited form of VWD.
2. Are there any additional tests you might request?

The IgM is raised although there is no evidence of immuneparesis You should request protein electrophoresis and
consider the possibility of an MGUS, lymphoma or Waldenströms macroglobulinaemia.
Protein electrophoresis identified an monoclonal band [M band] in this patient of 13g/L.

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Shown below are two images from a bone marrow aspiriate on this patient. What do these show and what is the
diagnosis?
These marrow aspirate shows an infitrate of lymphoplasmacytoid cells consistent with a diagnosis of
Waldenströms macroglobulinaemia.

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Question 2
A 52-year-old man is admitted through Accident and Emergency with a short history of haematuria. He was on no drugs
at the time of his visit to hospital. Shown below are the results of his initial investigations:
How would you manage this patient if he needed a cholecystectomy?

Test
IV Patient with AVWSReference
immunoglobulin is the treatment of choice for patients secondaryRange
to an IgG paraprotein but is of
PT 12s 10.6-12.4s
no benefit in patients with AVWS secondary to an IgM paraprotein. The options for this patient include plasma
exchange
APTT to remove the paraprotein, a VWF-containing95s concentrate either
21-32san intermediate purity FVIII
concentrate of a high
Fibrinogen (Clauss) purity VWF concentrate. There
3.1g/Lare advantages to the latter as it replaces VWF without
2-4g/L
excessive rises in FVIII.
You
1. mayare
What also
theelect to treat
possible the Waldernströms
diagnoses macroglobulinaemia.
taking into account the patient's history.
2. What additional investigations would you request?
In summary, this patient has AVWS secondary to an IgM Paraprotein and Waldernströms macroglobulinaemia .
This case is very similar to the first but there are some differences.
1. The history suggests that the problem is of recent onset and therefore this excludes an inherited bleeding
disorder. The prolonged APTT and normal PT would suggest a deficiency of factors VIII, IX, XI or XII. Of these
the most likely is FVIII deficiency i.e. Acquired haemophilia A. A Factor VIII assay would, therefore, be a logical
test to perform.
2. You should also screen for a lupus anticoagulant [SCT and dRVVT.] This can cause bleeding problems in some
patients either because of thrombocytopaenia or a low FII level [prothrombin deficiency.] It will also prolong the
APTT. We are not given the platelet count and so this remains a possibility. The PT is normal and so acquired
prothrombin deficiency is unlikely.
3. You should request a full blood count to check the platelet count.

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The results of factor assays are show below:
Factor Assay Patient Reference Range

FVIII 0.12 IU/mL 0.45 - 1.49 IU/mL
FIX 0.79 IU/mL 0.50 - 1.51 IU/mL
FXI 1.23 IU/mL 0.50 - 1.50 IU/mL
FXII 1.10 IU/mL 0.45 - 1.49 IU/mL
Shown below are the results of additional investigations on this patient:
Assay Patient Reference Range

VWF:Ag 0.13 IU.mL 0.48 - 1.55 IU/mL
VWF:Act 0.03 IU/mL 0.50 - 1.50 IU/mL
FVIII:C 0.11 IU/mL 0.45 - 1.49 IU/mL
M Band 17g/L
Protein electrophoresis
IgG Κappa
IgG 22g/L IgG 6 - 13g/L
Immunoglobulins IgM 2.3g/L IgM 0.4 - 2.2g/L
IgA 2.1g/L IgA 0.8 - 3.7g/L
Renal & LFTS
Normal
Calcium
1. What are the possible diagnoses?

The diagnosis is Acquired Von Willebrand Syndrome [AVWS.]
2. Are there any additional tests you might request?

The IgG is raised although there is no evidence of immuneparesis You should request protein electrophoresis and
consider the possibility of an MGUS or myeloma. Protein electrophoresis identified an monoclonal band [M
band] in this patient of 19g/L.
A bone marrow aspirate and trephine showed no evidence of myeloma and a skeletal survey did not reveal any
lytic lesions.
3. How would you manage this patient if he needed a cholecystectomy?

IV immunoglobulin [IVIG] is the treatment of choice for patients with AVWS secondary to an IgG paraprotein.
However the effect of IVIG on raising FVIII and VWF levels is not immediate and in an emergency a VWF-
containing concentrate either an intermediate purity FVIII concentrate of a high purity VWF concentrate is
required. There are advantages to the latter as it replaces VWF without excessive rises in FVIII.

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In summary, this patient has AVWS secondary to an IgG paraprotein and MGUS.
He is admitted 6 weeks later with a major GI haemorrhage. Endoscopy shows diffuse bleeding from the stomach but no
obvious ulcer.
How would you manage this patient?
In this situation you need to elevate the FVIII and VWF levels rapidly. Therefore you need to administer a FVIII
and VWF-containing concentrate. However, this will be cleared rapidly and repeat dosing will be necessary with
the dose and frequency being guided by FVIII and VWF levels. Administering IVIG will be of value but its effect
is not immediate.
Question 3
A 6-week-old male baby is found at home unconscious. He is admitted to hospital and investigations show a large
intracerebral haemorrhage. A series of haemostasis investigations are performed and these are show below:

PT 14s 11-14s
APTT 35s 28-34.5s
Fibrinogen (Clauss) 3.5g/L 1.9-3.7 g/L
1. What additional tests would you request?
The results of a Factor XIII screen are shown below:
Patient:
Complete
lysis at
5M Urea 24 hours
FXIII
Screening Control:
Test
No lysis
at 24

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hours
What tests would you request next?

In this situation you must request a FXIII assay. The screening test is highly suggestive of FXIII deficiency i.e.
there is accelerated clot lysis due to defective cross-linking of the fibrin clot.
What is the sensitivity of a FXIII screening assay?

The FXIII screening assay may be relatively insensitive to FXIII deficiency when the FXIII levels are >10% of
normal.
Question 4
A 4-year-old boy with multiple developmental problems, is referred by the paediatricians for investigation of a potential
bleeding disorder. He had a long history of easy bruising and had bled after minor ENT surgery. There was no family
history of note. He was followed up by the paediatricians because of various congenital abnormalities and
developmental delay.
Investigations showed:

PT 48s 11-14s
APTT 39 28-34.5s
Thrombin Time 14s 12-14s
On the basis of these tests what you do next?
The results of factor assays are shown below:
Reference
Factor Patient
Range
Assay [IU/dL]
[IU/dL]
Factor
103 70-130
II
Factor
98 75-155
V
Factor
34 70-130
VII
Factor
167 75-155
VIII
Factor
99 75-135
IX
Factor
32 70-130
X
Factor
86 75-155
XI
What do you think might explain these findings?

These results show low FVII and FX levels. The genes for both these proteins are located on the long arm of

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chromosome 13v[13q34.] Partial deletion of this region is associated with various congenital abnormalities in
addition to low levels of FVII and FX. In fact the genes were originally mapped to chromosome 13 by analysis of
a family with FVII and FXI deficiencies - see references.
What
Clickwould
here you request
for Part 3 next?
A chromosomal karyotype.
Karyotype analysis shows a partial deletion of the long arm of chromosome 13 [13q34].
Does this fit in with your explanation for the low factor assays and if so why?
Yes - see previous .
Question 5
Plot the following factor X assay data [from a PT-based assay] and calculate the factor X levels for the four plasma
samples. The concentration of FX:C in the reference plasma is 94 IU/dL.
Dilutions
1/10 1/20 1/30 1/40 1/80 1/100 1/1000
Reference plasma 25s 32s 38s 59s

Patient 1 35s 54s 82s
Patient 4 30s 34s 39s 45s
A. If the concentration of FX in the reference plasma was 112 IU/dL - what would your answers be?
B. If the concentration of FX in the reference plasma was 0.90 IU/ml - what would your answers be?
Click HERE for the answers

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1. In the graph shown above the dilutions are plotted on the X-axis and the PT clotting times (in seconds) on
the Y-axis.
2. This is a PT-based assay and so you use Log-Log paper.
3. In this example we have used the 1/10 dilution as 100% activity (100 IU/dl) and then calculated the activity
in the various patient samples from this graph.
4. In the plasma sample from patient 3 - the clotting times are all grossly prolonged and show little change
with increasing dilutions. As we have said earlier -'you cannot dilute nothing' and so the factor level in this
case must be <1% [<1 IU/dL].
Question 6
FX:C if Reference Plasma FX:C if Reference Plasma FX:C if Reference Plasma
The following are the clotting times [in seconds] for a 1-stage APTT Factor VIII assay.
Activity = 94 IU/dL Activity = 112 IU/dL Activity = 0.90 IU/mL
Patient
Plot 1
the following 16VIII
factor IU/dL 19VIII:C
data and calculate the factor IU/dL levels. 0.15 IU/mL
Patient
The 2
concentration 188 IU/dL
of FVIII in the reference plasma is 94 224 IU/dL
IU/dl. 1.80 IU/mL
Patient 3 <1 IU/dL <1 IU/dL <1 IU/mL
Patient 4 40 IU/dL 48 IU/dL Dilutions 0.39 IU/mL
1/5 1/10 1/20 1/40 1/80 1/100
Reference plasma 41 53 66 82
Patient 1 55 79 96
Patient 2 108 120

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Patient 3 70 82 107
Patient 4 30 42 55 71
If the concentration of FVIII in the standard was 0.89 IU/ml what would your answers be?
Click HERE for the answer
1. In the graph shown below the dilutions are plotted on the X-axis and the APTT clotting times (in seconds)
on the Y-axis. This is an APTT-based assay and so you use Log-Lin paper.

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2. In this example we have used the 1/10 dilution as 100% activity (100 IU/dl) and then calculated the activity
in the various patient samples from this graph.
A correction for the actual FVIII:C in the reference plasma (89 IU/dl) is then made to each of the factor
assays that you have derived from the graph e.g. if the factor level in the plasma sample from the graph was
100 IU/dl then the actual concentration is: [concentration Reference plasma] /100 x [concentration in the
test sample] = 89 IU/dl.
FVIII:C Reference Plasma = FVIII:C Reference Plasma = FVIII:C Reference Plasma =

100% 94 IU/dL 0.89 IU/mL
Patient 1 45% 43 IU/dL 0.40 IU/mL
Patient 2 1% 1 IU/dL 0,01 IU/mL
Question 7
A 23-year-old woman of Iranian descent presents to her GP with menorrhagia. The GP requests a coagulation screen and
the results of this are shown below:

PT 34s 11-14s
APTT 82s 23-35s
Fibrinogen (Clauss) 2.9g/L 1.5-4.0g/L
How would you proceed with the investigation of this patient?
You request factor V and VIII assays. The raw data for these assays are shown below.
Plot the data and derive the concentrations of these two factors.
Factor V Assay
Patient Reference
Dilutions
PT [s] PT [s]
1/10 60 23
1/20 92 38
1/40 160 59
Reference Factor V:C concentration 85 IU/dL.
Factor VIII Assay

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Patient
Reference
Dilutions APTT
APTT [s]
[s]
1/10 50 35
1/20 57 43
1/40 64 50
1/100 74 59
Reference Factor VIII:C concentration 90 IU/dL.
Factor V Assay
The FV:C in this patient is 22 IU/dl.
Factor VIII Assay

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The FVIII:C in this patient is 23 IU/dL.
1. Remember that APTT based assays are plotted on Log-Lin paper.
2. The FVIII assay for the patient assumed that the 1/10 dilution for the reference plasma has a value of 100
IU/dl [100 IU/dl] but in this case it does not. Therefore, the corrected value is 90/100 x 25 = 23 IU/dl.
The diagnosis of combined deficiency of factor V and VIII is confirmed.
The diagnosis of combined FV and FVIII deficiency is confirmed.

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1. What is the molecular basis for this disorder?
Combined factor V + VIII deficiency. The phenotype can be very mild and present later in life. If factor V
deficiency is found then factor VIII levels should also be assayed. Abnormalities of two genes have been identified
as causes of combined factor V +VIII deficiency - LMAN1 and MCFD2 and these can be sequenced if combined
FV and FVIII deficiency is suspected. For more information see References.
2. How would you manage her menorrhagia?

Most cases will respond to the use of the combined oral contraceptive pill [COCP] or to tranexamic acid. In some
women a progesterone-coated IUD can be helpful.
Most cases do not need replacement treatment with FV and FVIII.
Question 8
A 7-year-old boy is investigated with prolonged bleeding following dental surgery. A coagulation screen is requested:

PT 23s 11-14s
APTT 56s 28-34.5s
Thrombin Time 13s 12-14.2s
i. What factor assays would you request and why?
A prolongation of both the PT and APTT suggests either a common pathway deficiency [e.g. II, V or
X] or multiple deficiencies [V & VIII or a deficiency of all the vitamin K deficient clotting factors.]
Oral anticoagulation is unlikely in a 7-year-old boy but should be considered in older age groups.
ii. Shown below are the data for a PT-based factor X assay. From this data derive the patient’s factor X level.
Patient Reference
Dilutions
PT [s] PT [s]
1/10 35 20
1/20 42 24
1/40 28
1/80 59 34
1/100 62 35

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The FX level in the patient is 10 IU/dL. We are not given the reference plasma FX concentration and so I have
assumed it to be100 IU/dL.
You could get an estimate of the FX level in the patient by looking at the clotting times in the patient and the
reference plasma and seeing at which plasma dilution in the patient gives a similar clotting time to the reference.
So a 1/100 dilution of the reference plasma gives an APTT of 35s but for the patient an APTT of 35s is seen with a
dilution of 1/10. Therefore the patient's FX level must be 1/10 that of the reference plasma i.e. 10 IU/dL.
Is there any value in performing an APTT-based FX assay?

Some rare FX variants may give different levels based upon the method of assay. A functional assay should
always be performed in preference to an immunological assay as a dysfunctional protein may be present in
normal concentration.
There are 5 methods for assaying Factor X - what are they?
There are 5 ways in which FX can be assayed:

- PT-based functional assay
- APTT-based functional assay
- Russell Viper Venom (RVV)-based functional assay

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- Immunological FX assay - which measures FX:Ag
- FX chromogenic assay
Question 9
A developmentally normal 2-year-old girl is referred with a history of easy bruising, haematoma formation after
vaccination and more recently of a probable left knee haemarthrosis. A coagulation screen shows:

PT 12s 11-14s
APTT 90s 23-35s
Full Blood Count Normal
1. What tests would you request and why?
The finding of a prolonged APTT suggests either:
- Factor VIII, IX, XI or XII deficiency - but if we assume the bleeding problems are due to a clotting factor
deficiency - then we can exclude FXII deficiency as this does not result in a bleeding disorder.
- Lupus anticoagulant - this rarely leads to bleeding problems unless there is significant thrombocytopaenia or
Factor II [prothrombin] - deficiency. We can exclude both of these as the Full Blood Count is reported to be
normal and the Prothrombin Time is normal.
1. You request Factor VIII, IX and XI assays.

2. Shown below are the data for a 1-stage factor VIII assay. From this data derive a factor VIII result for the patient.
Patient
Reference
Dilutions APTT
APTT [s]
[s]
1/10 42 29
1/20 47 34
1/40 52 39
1/100 58 45
Factor VIII:C Reference 104 IU/dL.

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3. What additional tests would you request and why?
4. What additional questions might you ask the parents?
1. You request Von Willebrand Factor Assays - they are normal.

2. You request a FV assay - although the PT is not prolonged. The FV is 110 IU/dL.
3. A karyotype shows: 46,XX - with no obvious chromosomal abnormalities.
4. The parents are unrelated. The father is well with a normal PT and APTT. The mother tells you that her father had
severe haemophilia A and was shown to be hemizygous for the F8 intron 22 inversion.
How would you explain these findings and how would you investigate further?
The FVIII:C in this patient is ~16 IU/dl from the graph but the concentration of FVIII:C in the reference plasma
is 104 IU/dl - therefore the corrected value is 104/100 x 16 = ~16 IU/dl.
What are the possibilities to explain these findings?
- Type 1 Von Willebrand Disease (VWD)

- Mild Haemophilia A due to a mutation in one of the two F8 genes and Lyonisation of the other F8 gene on the
other X chromosome.
- Turners Syndrome (XO) or other karyotypic abnormality affecting one of the two X chromosomes
- Inheritance of the 2 separate F8 gene mutations either homozygous or compound heterozygous one on each X
chromosome.
- Androgen insensitivity i.e. she is XY and has a mutation in the F8 gene
In fact this patient has extreme Lyonisation. Her mother is a carrier of a mutation associated with severe
haemophilia A.

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Question 10
An 18-year-old male patient with factor VII deficiency has a factor VII assay performed.
Calculate the FVII levels in the patient.
The results of his parents are also shown. Calculate their FVII levels.
Index
Case Mother Father Reference
Dilutions
PT PT [s] PT [s] PT [s]
[s]
1/10 55 36 36 29
1/100 68 50 50 41
1/1000 82 64 64 56
All clotting times are in seconds. FVII standard: 115 IU/dl.

What can you infer from these family studies? What might you ask the parents?

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Question 11
A 56-year-old male with moderate Haemophilia A diagnosed 16 years ago requires surgery to repair an inguinal hernia.
In advance of this he has a pre-operative screen performed:
The FVII:C in the index case is ~ 1 IU/dl and with a correction for the standard reference plasma FVII level [115
IU/dl]
Test the true FVII level is ~1 IU/dl. Patient Reference Range
PT FVII levels in both parents is ~20 IU/dl from the
The 12s graph and with 11-14s
a correction for the standard reference
APTT FVII level [115 IU/dl] the true FVII level is33s
plasma 23 IU/dl. 23-35s
What can you
Thrombin Time infer from these family studies? What might
13s you ask the parents?
10-13s
It is probable that both parents are related and you should construct a careful pedigree. Genetic analysis will be
useful in establishing that the parents are heterozygous for the same mutation [as they are] and that the index
case
1. Areisyou
homozyous
surprisedfor
by this
thesemutation
results? [he is].
Finally, genetic counselling is important in this case as the parents have a1:4 chance of having similarly affected
children.
Yes the APTT is normal and in moderate haemophilia A, one would expect the APTT to be prolonged due to the
low FVIII levels.
2. What might explain this?
i. Could be an error in the laboratory - therefore repeat.

ii. The patient may have been misdiagnosed when originally tested. Factor assays are significantly more precise
today than they were 16 years ago.
iii. He has a mutation associated with a 1-stage 2-stage discrepancy and the original assay was performed using a
2-stage assay but the most recent using a 1-stage assay.

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Below is data from a 1-stage APTT FVIII assay.

Derive the FVIII:C from this assay.
Patient
Reference
Dilutions APTT
APTT [s]
[s]
1/10 37 35
1/40 44 42
1/160 49 47
Factor VIII:C Reference 100 IU/dL.
Are you surprised by this result?

Yes and no! It supports the possibility that this patient has a F8 gene mutation associated with a 1-stage/2-stage
FVIII assay discrepancy.

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What would you request next?
2-stage or chromogenic FVIII Assay.
Below is data from a chromogenic FVIII assay. Derive the FVIII:C from this assay.
FVIII:C
Concentration
[IU/dL]
150 100 50 0
Absorbance
[405nm]
0.47 0.32 0.19 0.04
Reference
Plasma
Absorbance Patient: 0.06
1. What is the basis for a chromogenic FVIII Assay?

The assay is similar to the two-stage FVIII assay in that it involves an incubation step to generate FXa and a
second stage to determine the amount of FXa produced. In the chromogenic assay case amount of FXa is
measured by its action on a highly specific chromogenic substrate and since the colour intensity produced is
directly proportional to the amount of FXa, which in turn is directly proportional to the amount of FVIII, the
FVIII levels may be calculated from the absorbance of the sample at a specific wavelength (the optimal
absorbance wavelength for the chromophore produced by FXa cleavage of the chromogen, e.g. 405nm for the
commonly used S-2765 chromogen).
2. How do you explain these findings.

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The original assay used to diagnose haemophilia in this patient was a 2-stage assay and not a 1-stage assay. The
chromogenic assay suggests a diagnosis of mild haemophilia A. This is an uncommon but well described
phenomenon. The chromogenic and 2-stage FVIII:C give similar results in such patients and significantly lower
than the 1-stage assay. The importance in this lies in that many labs perform only a 1-stage assay and so may
miss mild cases of haemophilia A. Therefore, is a bleeding history suggests a bleeding disorder and the 1-stage
assay is normal, a 2-stage or chromogenic FVIII should be performed. Plotting the data from the above and
deriving the FVIII:C in the patient indicates a value of 10 IU/dl by the chromogenic assay. From the 1-stage
FVIII:C assay the level was 67 IU/dl. As we are not given a reference value for this - we can assume it is 100
IU/dL.
The finding of a normal APTT in someone with a history of moderate haemophilia A diagnosed some years ago
should suggest the possibility of a variant F8 gene in which there is a discrepancy between the 1-stage and 2-
stage/chromogenic FVIII assays. This case highlights the importance of the clinical history. The chromogenic
FVIII assay should be used to guide clotting factor replacement in this patient and not the 1-stage FVIII assay
which as you will see below is normal.
3. What is the molecular basis for this?

Amino-acid substitutions in the A1, A2 and A3 domains of factor VIII have been described in affected individuals
with this phenotype. The mutation is thought to lead to instability of the FVIII that is highlighted in the 2-stage
and chromogenic assay with a longer incubation period that allows the FVIII to dissociate whereas in the 1-stage
assay this does not occur and the FVIII is relatively well preserved due to the shorter incubation times and hence
generates a normal FVIII assay result.
Question 12
Shown in the table below are the data for an APTT-based factor XI assay performed on 2 separate plasma samples
[Plasma 1 and Plasma 2.]
Plot the data for the FXI reference plasma and draw a best fit line. For the two plasma samples plot the data but do not
attempt to draw a best fit line.
1. Calculate the FXI levels for each of the five dilutions in the two plasma samples.
2, Explain your findings.
3. What would you do next ?
Plasma Plasma
Sample Sample
Reference
Dilutions 1 2
APTT [s]
APTT APTT
[s] [s]
1/10 40 37 30
1/20 43 38 33
1/40 47 40 37
1/100 52 42 42

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1/160 43
Reference Factor XI:C concentration 100 IU/dl.
1. Shown below is a graph of the FXI:C data. Note that the line of plasma 1 is parallel to the reference plasma but
that of plasma 2 is not.
The factor XI:C for each of the dilutions plotted is shown in the table below:
Dilutions
1/10 1/20 1/40 1/100 1/160
Reference Plasma 30 IU/dL 33 IU/dL 37 IU/dL 42 IU/dL
7x2 3.5 x 4 1.5 x 10
Plasma Sample 1 15 IU/dL
= 14 IU/dL = 14 IU/dL = 10 IU/dL
20 x 2 15 x 2 9 x 10 7 x 16
Plasma Sample 2 25 IU/dL
= 40 IU/dL = 60 IU/dL = 90 IU/dL = 112 IU/dL
If we take the 1/10 dilution of the reference plasma to have a FXI:C of 100 IU/dL then Plasma 1 has a FXI:C level
of ~13IU/dL The value is similar to for all dilutions.
However, for plasma 2 the dilutions give quite different FXI:C results - this patient has a strong lupus
anticoagulant. As you can see increasing the dilutions results in a higher FXI:C level as the inhibitor is diluted
out. This case also illustrates the problem with single point factor assays - if you had chosen only a single dilution
e.g. 1/20 - then you would derive a FXI:C of 40% which is incorrect.
Finally - in plasma 2 - repeat assays should be performed with an APTT reagent that is insensitive to the presence
of the lupus anticoagulant

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Question 13
Calculate the Bethesda titres for each of the following plasma samples.
Sample Plasma Dilutions Residual FVIII Titre [BU/ml]

1 Undiluted 70% 0.5 Bu
2 Undiluted 40% 1.3 Bu
3 Undiluted 0.5 IU/ml 1.0 Bu
4 Undiluted 0.6 IU/ml 0.74 Bu
5 Undiluted 45 IU/dl 1.15 Bu
1/5 33%
6 1/10 55% 7.7 Bu
1/20 68%
1/20 35%
7 1/40 55% ~31 Bu
1/80 68%
1/320 0.30 IU/ml
8 1/640 0.52 IU/ml 608 Bu
1/1280 0.65 IU/ml
For samples 6-9 we have taken the residual FVIII:C closest to 50% to calculate the inhibitor titre - don't forget to
take into account the dilution of the original plasma sample. An alternative approach is to calculate the inhibitor
titres for each of the dilutions and take the average of the 3.
Question 14
A 56-year-old woman of Jewish ancestry is referred for an aortic valve replacement (AVR). Her pre-operative screening
tests show:

PT 13s 11-14s
APTT 61s 23-35s
Fibrinogen 2.9g/L 1.5-4.0g/L
What tests would you perform next?
The APTT is prolonged but the PT is normal. This suggests a deficiency of either FVIII, FIX, FXI, FXII, or a

Page 24 of 47
lupus anticoagulant. However, the clinical data that indicates Jewish ancestry for this patient should make you
think of FXI deficiency which is significantly more common in Jewish individuals.
The results of a FXI assay are shown below:
Patient
Reference
Dilutions APTT
APTT [s]
[s]
1/10 36 26
1/20 39 29
1/100 48 38
Reference FXI:C 100 IU/dL [although the graph shows 100%!]
1. Calculate the FXI levels in the plasma sample [Plasma Sample 1]. The FXI:C is 15 Id/L.
2. How will you manage her surgery?

You elect to treat her with a FXI concentrate pre-operatively so that she can be monitored on cardio-pulmonary
bypass using the ACT. You also suggest a porcine valve rather than a metallic valve to minimise the period of oral
anticoagulation that would be necessary.

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This lady has an uneventful AVR. She is supported through the surgery and the post-operative period with FXI
concentrate. 12 days later she begins to ooze from her wound from her chest drain sites. A repeat clotting screen shows:

PT 11.8s 11-14s
APTT 85s 23-35s
Y0u request a further FXI:C assay - the raw data is shown below:
Patient
Reference
Dilutions APTT
APTT [s]
[s]
1/10 50 26
1/20 54 29
1/100 62 38
Reference FXI:C 100 IU/dL [although the graph shows 100%!]

1. What is the FXI level [Plasma Sample 2]. The FXI:C is significantly lower than the pre-operative samples at 1
IU/dL.

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An inhibitor assay is undertaken - the results of which are shown below:

1. Calculate the residual FXI:C. This was based upon a 1:20 dilution of plasma.
The residue FXI is 57 IU/dL [57%].
Patient
Reference
Dilutions APTT
APTT [s]
[s] you explain the differences in FXI:C assays.
2. How would
1/10 29 26
She has
1/20 developed
32 a FXI inhibitor in response to the FXI concentrate. These are rare and classically seen in
29
individuals with severe FXI deficiency and in association with mutations that can be predicted to result in a
1/100 absence
complete 41 of38FXI e.g. the Type II mutation [exon 5, Glu117-->Stop]. In some cases these patients can be
managed very successfully with rFVIIa concentrate.
Reference FXI:C 100 IU/dL [although shown in the graph as 100!].

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The inhibitor level is calculated from the observation that for FVIII inhibitors, 1Bu is the amount of inhibitor
that will inactivate 50% of a clotting factor during an incubation period. FXI inhibitors appear to be immediate
acting and do not require an incubation period as FVIII inhibitor assays do. They appear to be more like FIX
inhibitors in this respect.
In this patient a 1:20 dilution of the patient plasma had a residual FIX:C of 57 IU/dL. Therefore, from the graph
below this equates to a titre of 0.7 Bu but this must be multiplied by 20 because of the dilution and so the final
value is 14 Bu.
2. How would you explain the differences in FXI:C assays.

The patient has developed a FXI inhibitor.

Page 28 of 47
Question 15
A 23-year-old male is diagnosed with mild haemophilia A (VIII:C 12 IU/dL) following prolonged bleeding after dental
surgery. He requires further dental surgery and you elect to treat him with DDAVP and tranexamic acid.
1. How do DDAVP and tranexamic acid work – illustrate with a diagram if you find this easier.
2. What are the side effects of DDAVP and how do we minimise these?
1. Tranexamic acid [TA] is a synthetic derivative of the amino acid lysine and a potent fibrinolytic inhibitor. TA
binds to so blocks the high affinity lysine binding sites on plasminogen [and subsequently plasmin when
plasminogen is activated] preventing the binding of plasmin and plasminogen to the fibrin clot or to fibrin
monomers.
Human plasminogen contains 4 to 5 lysine binding sites with low affinity for tranexamic acid and 1 with high
affinity and it the high affinity site of plasminogen that is involved in its binding to fibrin.
Tranexamic acid has a similar mechanism of action to aminocaproic acid, but is approximately 10 times more
potent in vitro. Tranexamic acid is excreted in the urine and its use is contraindicated in patients with haematuria
as it can cause clot formation in the renal tract.
2. DDAVP - in a dose dependent manner, DDAVP increases FVIII and VWF release from endothelial cells.
Interestingly DDAVP also causes the release of T-PA and formed the basis for a test similar to the venous
occlusion test and known as the DDAVP stimulation test.
DDAVP is of value in individuals with mild haemophilia A and Type 1 VWD. It may be of value in Type 2A, 2M
and 2N VWD although in the latter the rapid clearance of FVIII due the lack of a stabilising VWF results in a
very short T½. DDAVP is sometimes used in individuals with non-specific platelet function defects prior to minor
surgery. It is of no value in patients with severe haemophilia A or Type 3 VWD and as it does not increase the
levels of FIX in plasma, it is of no value in Haemophilia B.
DDAVP is a potent anti-diuretic nonapeptide and fluid retention leading to hyponatraemia is a recognised side-
effect. For these reasons, DDAVP should not be administered more than once every 24 hours. DDAVP also
exhibits tachyphylaxis that is a reduction in the amount of FVIII and VWF that is released from endothelial cells
with repeated dosing.
DDAVP can be given intravenously, subcutaneously or intra-nasally. DDAVP has been given to women at the time
of delivery as the anti-diuretic nonapeptide affect of DDAVP is mediated through the V2 vasopressin receptors
whereas in the uterus vasoconstriction and uterine contraction are related to the V1 receptor.

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He is treated with DDAVP and his 1 hour post-DDAVP FVIII:C is 89 IU/dL. He proceeds to surgery which is carried out
uneventfully. However, 4 hours following dental surgery he has profuse bleeding from the extraction sites and a repeat
FVIII:C is 14 IU/dL.
Comment upon these results?
What do you think is the problem?
He has shown a poor response to DDAVP. There are a number of possibilities:

1. He does not have mild haemophilia A but 2N VWD. We are not given the VWF assays but as we are not we
should assume they are normal. Ideally one would want to repeat these assays to confirm they were normal.
2. He has VWD associated with rapid clearance or the FVIII-VWF complex. This is a possibility but less likely
than (1) as the VWF levels are low in Type 1C [Type 1 Vicenza] VWD.
3. He has a FVIII inhibitor - seems unlikely.
In fact he has 2N VWD. Historically this was often misdiagnosed as mild haemophilia A. In anyone with a low
FVIII and normal VWF levels it important to consider 2N VWD. This is diagnosed either by a FVIII binding
assay or alternatively by looking for the specific sequences located in the VWF gene that encode the FVIII
binding site.
Finally, in all patients in whom treatment with DDAVP is a therapeutic option, a DDAVP study should be
undertaken. Had this been performed in this patient it would have shown a rapid clearance of FVIII and raised
the possibility (hopefully) of 2N VWD. Remember 2 N VWD is inherited as homozygous defect.
Question 16
A 45-year-old male is referred for further investigation following the finding of an abnormal coagulation profile. He had
contacted his GP having developed bruising and epistaxes.
His health had previously been excellent apart from a recent chest infection for which he had been prescribed
amoxicillin.

PT 45s 11-14s
APTT 79s 23-35s

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Outline how you would investigate this patient. Give the reasons behind these investigations.
The results of relevant factor assays are shown below:
Factor Patient Reference Range

FII 132 IU/dL 50-150 IU/dL
FV 2 IU/dL 50-150 IU/dL
FX 89 IU/dL 50-150 IU/dL
1. What do you think has happened and why.

2. Are there any other tests you might request?
3. Why does this problem arise?
4. How would you manage this patient?
See Question 2 [Answers]: Data Interpretation - Screening Tests.
Question 17
What are the differences between a 1-stage and a 2-stage factor VIII assay?
Why might you request a 2-stage factor VIII assay?
See Question 11 for an explanation.
It is important to remember that chromogenic FVIII assays are the method for establishing the concentration of
FVIII in FVIII concentrates.
Question 18
A 3-year-old boy with severe haemophilia B (IX:C<1 IU/dl) has a poor response to factor IX concentrate. An inhibitor
screen is performed which is positive.
1. Outline the basis of the inhibitor screen.

2. From the data provided establish the factor IX inhibitor titre in this patient.

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Dilutions Residual FIX
[%]
1/5 33
1/10 55
1/20 68
Shown below is the inhibitor graph. As you have 3 separate plasma dilutions you can either use the 1/10 dilution
which gives you a value closest to 50% residual FIX or calculate the inhibitor titre for each of the dilutions and
take the mean of the three.

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1. If we use the 1/10 dilution which gives a value closes to 50% then the inhibitor titre is 0.85 x 10 = 8.5 Bu.
2. If we calculate the inhibitor titres for all three dilutions the the inhibitor titre is:
[0.6 x 20] = 12 Bu + [0.85 x 10] = 8.5 Bu + [1.575 x 5] = 7.875 Bu. The mean of these is 9.5 Bu.
Remember individuals with Haemophilia B who develop inhibitors are rare, they may show a significant gene
deletion and they are difficult to tolerise because of problems with nephrotic syndrome and anaphylaxis to
clotting factor concentrates.
Question 19
Shown below are the results of DDVAP studies in five patients with Von Willebrand Disease.
Comment upon the results of the these studies and identify the possible VWD subtype(s).
8 hour post-
Pre- 1 hour post- 2 hour post- 4 hour post-
DDAVP
DDAVP DDAVP DDAVP DDAVP
Patient FVIII:C FVIII:C FVIII:C FVIII:C

FVIII:C
VWF:Ag VWF:Ag VWF:Ag VWF:Ag
VWF:Ag
VWF:RCo VWF:RCo VWF:RCo VWF:RCo
VWF:RCo
[IU/dL] [IU/dL] [IU/dL] [IU/dL]
[IU/dL]
30 110 50 30 28
1 10 85 42 28 22
22 80 40 27 20
20 162 100 88 67
2 15 143 96 75 60
14 144 92 73 60
20 160 100 86 67
3 15 140 95 78 62
14 140 96 74 61
1 1.5 1 <1 1
4 1 1.2 1 1 1
1 1.2 1 <1 1
18 150 40 20 17
5 62 140 95 78 62
64 140 96 74 61
110 400 320 280 200
Control 95 220 300 260 190
90 218 310 248 100

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Assume all references ranges are 50-150 IU/dL.
Nb Patient 2 showed a fall in platelet count during the study.
1 hour 2 hour 4 hour 8 hour post-

Pre-
post- post- post- DDAVP
DDAVP
DDAVP DDAVP DDAVP
Patient FVIII:C VWD Subtype
FVIII:C FVIII:C FVIII:C FVIII:C
VWF:Ag
VWF:Ag VWF:Ag VWF:Ag VWF:Ag
VWF:RCo
VWF:RCo VWF:RCo VWF:RCo VWF:RCo
[IU/dL]
[IU/dL] [IU/dL] [IU/dL] [IU/dL]
Type 1C VWD
['Vicenza-type'
VW]: Shows a
30 110 50 30 28 good rise following
1 10 85 42 28 22 DDAVP but FVIII
22 80 40 27 20 and VWF fall
rapidly due to
accelerated
clearance.
2B VWD: The fall
in platelet count
20 162 100 88 67
should suggest this
2 15 143 96 75 60
as otherwise the
14 144 92 73 60
rise in FVIII/VWF
is normal.
20 160 100 86 67
3 15 140 95 78 62 Type 1 VWD
14 140 96 74 61
1 1.5 1 <1 1 Type 3 VWD:
4 1 1.2 1 1 1 Virtually no FVIII
1 1.2 1 <1 1 or VWF
2N VWD: The
FVIII falls more
rapidly than the
18 150 40 20 17 VWF levels as it is
5 62 140 95 78 62 not protected by
64 140 96 74 61 the VWF and is,
therefore cleared
rapidly from the
plasma.
110 400 320 280 200
Control 95 220 300 260 190 Normal!
90 218 310 248 100
1. In patient 1 - what additional investigations may be of benefit?

a. Mutation analysis

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b. VWF propeptide [VWFpp] studies
2. In patient 2 - what additional investigations may be of benefit?

a. Low dose RIPA
b. Mutation analysis
Click
3. here for
In patient Part 3additional investigations may be of benefit?
3 - what
a. Multimers may be useful in showing a quantitative reduction in all multimer sizes.
1. In patient 1 - you request VWF propeptide [VWFpp] studies: Why?
Measurement of VWFpp can be of value in establishing whether low VWF levels are due to decreased synthesis
or increased clearance. In the former the VWFpp to VWF:Ag ratio is normal [i.e. close to 1] whereas in the latter
it is increased. In individuals with Type 1 VWD due to accelerated clearance of the VWF protein from the
plasma, the VWFpp to VWF:Ag ratio is increased and in the region of 4. This suggests that the VWFpp has a
normal clearance and half-life [T½] but that the T½ of the mature VWF is decreased i.e. it is cleared more
rapidly from the plasma due to a mutation within the mature VWF protein.
2. In patient 2 - you request platelet aggregation tests with low dose ristocetin [0.5mg/mL]: Why?
Ristocetin induced platelet agglutination [RIPA] is carried out on patient PRP using a low concentration of
ristocetin [~0.5µg/ml.] This low concentration of ristocetin does not cause VWF binding and agglutination of
platelets in samples from normal persons, but will do so in patients with Type 2B VWD or mutations in the
platelet VWF receptor [platelet-type or pseudo VWD.] It is, therefore, used as a screening test for 2B VWD.
3. In patient 3 - you request multimers: Why?

In general in Type 1 VWD, all multimers are present including high molecular weight [HMW] forms but are
present in reduced concentration. In the Vicenza variant of VWF [Type 1C VWD], multimeric analysis shows the
presence of ultralarge VWF multimers.
Question 20
A 2-year-old boy develops a haemarthrosis of his left knee following a minor injury. Investigations show:

PT 13s 11-14s
APTT 91s 28-34.5s
FVIII:C 1.32 IU/mL 0.50-1.45 IU/mL
1. What is the most likely diagnosis?

2. How would you confirm this?

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The FVIII is normal but in the context of a prolonged APTT and a haemarthrosis in a boy, you should [must]
consider FIX deficiency and request a FIX assay.
You request a Factor IX assay - the raw data for a 1-stage FIX assay are shown below. From the data provided what is
the FIX:C level in the patient?
Dilution Patient Control

1/10 78s 41s
1/50 80s 50s
1/100 93s 56s
The FIX:C of the reference preparation is 110 IU/dL.
In this boy even allowing for the Reference Plasma with a FIX:C of 110 Idol - the FIX:C is <1 IU/dL. The diagnosis is

Page 36 of 47
severe Haemophilia B.
Question 21
A 4-year-old boy is diagnosed with haemophilia B with a level of 1.2 IU/dL. He is treated on demand with factor IX
concentrate to which he responds well.
15 years later at the age of 19 years of age he comes for review and you note that he has had no bleeds over the
preceding 3 years and required no replacement FIX therapy.
1. What might explain these findings?

2. What further tests might you request?
1. A possible explanation for these findings is that he has Haemophilia B Leyden.

2. You request F9 gene sequencing. What is this likely to show?
Classically such individuals show a mutation in the promotor region of the F9 gene.
Question 22
A 22-year-old woman from North African decent is investigated for recurrent miscarriages. She gave a long history
suggestive of an inherited bleeding diathesis and this included prolonged bleeding from the umbilical stump and poor
wound healing.
1. What questions might you ask this patient?

2. What tests would you request next?
You request a panel of screening tests - the results of which are shown below:

PT 13s 11-14s
APTT 28s 28-34.5s
Fibrinogen (Clauss) 2.9 g/dL 1.9-3.7 g/L
Platelet count 367 x 109/L 150 - 400 x 109/L
PFA-100 Closure times within the reference ranges
What additional tests would you request and why?

Page 37 of 47
You must request a FXIII assay as the history is suggestive of this. FXIII deficiency is associated with a normal
PT, APT and Thrombin Time.
You request a FXIII screen [using 5M Urea]:
Test Patient Plasma Reference Plasma

Clot lysis at 1 hour + -
Clot lysis at 6 hours + -
[+ = lysis has occurred/- = lysis has not occurred]
What do these results suggest and what additional tests would you request.
These results are consistent with defective fibrin cross-linking. The reduced cross-linking leads a fibrin clot that
is susceptible to lysis in 5M Urea. You should request a FXIII assay either a FXIIIa ELISA assay or a functional
FXIII assay.
You request a FXIIIB ELISA assay and the results are reported as 'Normal'. Are you surprised by these results and if so
what additional tests would you request?
FXIIIB is a carrier for FXIIIA and so can be normal even in the presence of severe FXIII deficiency (and
deficiency of the FXIIIa subunit). You should request a FXIIIA ELISA assay or functional FXIII assay. If the
FXIIIA assay is low it may be of interest to measure FXIIIB levels but this is not essential to make the diagnosis.
Question 23
The following results were obtained from a 63-year-old male being investigated for prolonged bleeding after dental
surgery. Comment upon the results of these tests.

PT 12.2s 9.8-12.6s
APTT 43.8s 25.8-33.8s
Fibrinogen (Clauss) 2.9 g/L 1.6-3.7 g/L

Page 38 of 47
1. What tests would you perform next?
1. You confirm the results on repeat testing. He has a FVIII of 0.14 IU/ml and and VWF:Ag of 0.09 IU/mL.
These results are strongly suggestive of VWD. You must take a history to establish if he has had any previous
haemostatic challenges and if so did he bleed. If he did not then this raises the possibility that he has acquired von
Willebrand Syndrome [AVWS].
Comment upon these results which were obtained following the administration of 5000 units of an intermediate purity
Factor VIII concentrate.
Time APTT [s] VIII:C [IU/ml] VWF:Ag [IU/ml] VWF:Act [IU/ml]

25.8-33.8s [0.57-1.41] [0.53-1.49] [0.43-1.26]
Pre-sample 43.8 0.14 0.09 0.01
+ 1 hr 31.4 0.56 0.77 0.31
+ 2 hr 35.2 0.37 0.44 0.15
+ 4hr 37.2 0.26 0.24 0.05
+5 hr 38.8 0.20 0.19 0.02
+24hr 43.4 0.14 0.09 0.00
What do you think might provide an explanation for this?

Are there any other tests you would request and why?
The FVIII and VWF are cleared more rapidly than one would expect in patient with inherited VWD. These
results are consistent with AVWS. You should exclude lympho- or myeloproliferative disorders. AVWS is also
associated with solid tumours, immunological and cardiovascular disorders as well as other miscellaneous
conditions e.g. hypothyroidism.
Question 24
Your lab is undertaking factor assays as part of an international trial of a new recombinant factor VIII. You receive a
batch of plasma samples for analysis. The results of one of these samples shows:

Page 39 of 47
PT 23s 11-14s
APTT 68s 28-34.5s
VWF:Ag 1.43 IU/mL 0.5-1.45 IU/mL
VWF:Act 1.31 IU/mL 0.5-1.45 IU/mL
FIX:C 1.15 IU/mL 0.5-1.45 IU/mL
APTT50:50 Mix with normal plasma No correction in a mix with normal plasma
1. Comment upon the results of these tests?
These results are strongly suggestive of a FVIII inhibitor. There is prolongation of the FVIII, normal VWF levels
and no correction of the APTT in a mix with normal plasma.
2. What would you do next?
1. You repeat the tests on a separate sample but from the same patient and collected at the same time. The results of the
second batch of tests are shown below:

PT 12s 11-14s
APTT 32s 28-34.5s
VWF:Ag 1.43 IU/mL 0.5-1.45 IU/mL
FIX:C 1.15 IU/mL 0.5-1.45 IU/mL
APTT50:50 Mix with normal plasma Not performed
How do you explain these findings?

These results do not suggest a FVIII inhibitor. This pattern of results is seen where a sample has been mistakenly
collected into EDTA and then the plasma has been labelled as a citrate sample. The second sample as correctly
collected into citrate and there is no evidence of a FVIII inhibitor. These results highlight that you should always
repeat a test where the result is abnormal and unexpected .
Question 25

Page 40 of 47
A 67-year-old man is admitted for investigation of haematuria. His initials investigations show:

PT 10.1s 9.8-12.6s
APTT 41s 25.8-33.8s
Thrombin Time 13s 11.5-13s
VWF:Ag 0.05 IU/mL 0.72-1.52 IU/mL
1. What questions might you ask this patient that would be important?
1. He has undergone a number of surgical procedures in the past including dental extractions without problems. His last
surgical procedure was 8 years previously.
2. There was no family of note.
3. He has a monoclonal IgG paraprotein of 2.3g/L
What is the diagnosis and how will you manage this patient if he requires a prostatectomy?
This patient has acquired Von Willebrand Syndrome [AVWS] secondary to his IgG monoclonal paraprotein. For
surgery such patients can frequently be safely managed with IV Immunoglobulin but this will require to be given
24-48 hours prior to surgery to allow the FVIII and VWF levels to return to normal. In an emergency where a
rapid rise in FVIII and VWF is required IVIG can be combined with an intermediate purity FVIII concentrate.
Question 26
A 67-year-old man was diagnosed mild haemophilia A in 1973. He requires a cholecystectomy and you repeat his FVIII
levels.
Investigations show:

FVIII 1973 0.35 IU/mL 0.50 - 1.49 IU/mL
FVIII 2010 0.52 IU/mL 0.50 - 1.49 IU/mL
How would you explain these findings?
Click HERE for the Answers

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The most likely explanation is that the sample in 1973 was assayed using a 2-stage FVIII assay and that in 2010, a
1-stage assay. This is an example of 1-stage/2-stage FVIII assay discrepancy.
Click HERE to return to the top of the page.
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Try to avoid looking at the answers until you have worked through the questions.
Comments
1. You will gain maximum educational value if you work through each part of each answer before you look at the second
and subsequent parts.
2. The answers will give you some help as to the reasoning behind some of the questions.
3. The questions span disorders from common to exceptionally rare but interesting.
References
Questions 1 & 2.
1. Alvarez, M.T., Jimenez-Yuste, V., Gracia, J., Quintana, M. & Hernandez-Navarro, F. (2008) Acquired von Willebrand
syndrome. Haemophilia, 14, 856-858.
2. Arkel, Y.S., Lynch, J. & Kamiyama, M. (1994) Treatment of acquired von Willebrand syndrome with intravenous
immunoglobulin. Thromb Haemost, 72, 643-644.
3. Baxter, P.A., Nuchtern, J.G., Guillerman, R.P., Mahoney, D.H., Teruya, J., Chintagumpala, M. & Yee, D.L. (2009)
Acquired von Willebrand syndrome and Wilms tumor: not always benign. Pediatr Blood Cancer, 52, 392-394.
4. Collins, P., Budde, U., Rand, J.H., Federici, A.B. & Kessler, C.M. (2008) Epidemiology and general guidelines of the
management of acquired haemophilia and von Willebrand syndrome. Haemophilia, 14 Suppl 3, 49-55.
5. Eikenboom, J.C., Tjernberg, P., Van Marion, V. & Heering, K.J. (2007) Acquired von Willebrand syndrome: diagnostic
problems and therapeutic options. Am J Hematol, 82, 55-58.
6. Federici, A.B. (2005) Use of intravenous immunoglobulin in patients with acquired von Willebrand syndrome. Hum
Immunol, 66, 422-430.
7. Federici, A.B. (2008) Acquired von Willebrand syndrome: is it an extremely rare disorder or do we see only the tip of
the iceberg? J Thromb Haemost, 6, 565-568.
8. Federici, A.B., Stabile, F., Castaman, G., Canciani, M.T. & Mannucci, P.M. (1998) Treatment of acquired von
Willebrand syndrome in patients with monoclonal gammopathy of uncertain significance: comparison of three different
therapeutic approaches. Blood, 92, 2707-2711.

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9. Franchini, M. & Lippi, G. (2007) Acquired von Willebrand syndrome: an update. Am J Hematol, 82, 368-375.
10. Franchini, M., Lippi, G. & Favaloro, E.J. (2010) Advances in hematology. Etiology and diagnosis of acquired von
Willebrand syndrome. Clin Adv Hematol Oncol, 8, 20-24.
11. Luboshitz, J., Lubetsky, A., Schliamser, L., Kotler, A., Tamarin, I. & Inbal, A. (2001) Pharmacokinetic studies with
FVIII/von Willebrand factor concentrate can be a diagnostic tool to distinguish between subgroups of patients with
acquired von Willebrand syndrome. Thromb Haemost, 85, 806-809.
12. Mayerhofer, M., Haushofer, A., Kyrle, P.A., Chott, A., Mullner, C., Quehenberger, P., Worel, N., Traby, L. &
Eichinger, S. (2009) Mechanisms underlying acquired von Willebrand syndrome associated with an IgM paraprotein. Eur
J Clin Invest, 39, 833-836.
13. Michiels, J.J., Schroyens, W., Berneman, Z. & van der Planken, M. (2001) Acquired von Willebrand syndrome type 1
in hypothyroidism: reversal after treatment with thyroxine. Clin Appl Thromb Hemost, 7, 113-115.
14. Tefferi, A., Smock, K.J. & Divgi, A.B. (2010) Polycythemia vera-associated acquired von Willebrand syndrome
despite near-normal platelet count. Am J Hematol, 85, 545.
15. Velik-Salchner, C., Eschertzhuber, S., Streif, W., Hangler, H., Budde, U. & Fries, D. (2008) Acquired von Willebrand
syndrome in cardiac patients. J Cardiothorac Vasc Anesth, 22, 719-724.
16. Pareti, F.I., Lattuada, A., Bressi, C., Zanobini, M., Sala, A., Steffan, A. & Ruggeri, Z.M. (2000) Proteolysis of von
Willebrand factor and shear stress-induced platelet aggregation in patients with aortic valve stenosis. Circulation, 102,
1290-1295.
17. Sadler, J.E. (2003) Aortic stenosis, von Willebrand factor, and bleeding. N Engl J Med, 349, 323-325.
18. Yoshida, K., Tobe, S., Kawata, M. & Yamaguchi, M. (2006) Acquired and reversible von Willebrand disease with
high shear stress aortic valve stenosis. Ann Thorac Surg, 81, 490-494.
Question 3.
Jennings, I., Kitchen, S., Woods, T.A. & Preston, F.E. (2003) Problems relating to the laboratory diagnosis of factor XIII
deficiency: a UK NEQAS study. J Thromb Haemost, 1, 2603-2608.
Question 4.
1. Pfeiffer, R.A., Ott, R., Gilgenkrantz, S. & Alexandre, P. (1982) Deficiency of coagulation factors VII and X associated
with deletion of a chromosome 13 (q34). Evidence from two cases with 46,XY,t(13;Y)(q11;q34). Hum Genet, 62, 358-
360.
Question 5.
1. Uprichard, J. & Perry, D.J. (2002) Factor X deficiency. Blood Rev, 16, 97-110.
2. Peyvandi, F., Mannucci, P.M., Lak, M., Abdoullahi, M., Zeinali, S., Sharifian, R. & Perry, D. (1998) Congenital factor
X deficiency: spectrum of bleeding symptoms in 32 Iranian patients. Br J Haematol, 102, 626-628.
Question 7.

Page 43 of 47
1. Spreafico, M. & Peyvandi, F. (2009) Combined Factor V and Factor VIII Deficiency. Semin Thromb Hemost, 35, 390-
399.
2. Zhang, B., Spreafico, M., Zheng, C., Yang, A., Platzer, P., Callaghan, M.U., Avci, Z., Ozbek, N., Mahlangu, J., Haw,
T., Kaufman, R.J., Marchant, K., Tuddenham, E.G., Seligsohn, U., Peyvandi, F. & Ginsburg, D. (2008) Genotype-
phenotype correlation in combined deficiency of factor V and factor VIII. Blood, 111, 5592-5600.
3. Zhang, B., McGee, B., Yamaoka, J.S., Guglielmone, H., Downes, K.A., Minoldo, S., Jarchum, G., Peyvandi, F., de
Bosch, N.B., Ruiz-Saez, A., Chatelain, B., Olpinski, M., Bockenstedt, P., Sperl, W., Kaufman, R.J., Nichols, W.C.,
Tuddenham, E.G. & Ginsburg, D. (2006) Combined deficiency of factor V and factor VIII is due to mutations in either
LMAN1 or MCFD2. Blood, 107, 1903-1907.
4. Peyvandi, F., Tuddenham, E.G., Akhtari, A.M., Lak, M. & Mannucci, P.M. (1998) Bleeding symptoms in 27 Iranian
patients with the combined deficiency of factor V and factor VIII. Br J Haematol, 100, 773-776.
Question 9.
1. Bicocchi, M.P., Migeon, B.R., Pasino, M., Lanza, T., Bottini, F., Boeri, E., Molinari, A.C., Corsolini, F., Morerio, C. &
Acquila, M. (2005) Familial nonrandom inactivation linked to the X inactivation centre in heterozygotes manifesting
haemophilia A. Eur J Hum Genet, 13, 635-640.
Question 10.
1. Mariani, G. & Bernardi, F. (2009) Factor VII Deficiency. Semin Thromb Hemost, 35, 400-406.
Lapecorella, M. & Mariani, G. (2008) Factor VII deficiency: defining the clinical picture and optimizing therapeutic
options. Haemophilia, 14, 1170-1175.
Question 11.
1. Rodgers, S.E., Duncan, E.M., Barbulescu, D.M., Quinn, D.M. & Lloyd, J.V. (2007) In vitro kinetics of factor VIII
activity in patients with mild haemophilia A and a discrepancy between one-stage and two-stage factor VIII assay results.
Br J Haematol, 136, 138-145.
2. Keeling, D.M., Sukhu, K., Kemball-Cook, G., Waseem, N., Bagnall, R. & Lloyd, J.V. (1999) Diagnostic importance of
the two-stage factor VIII:C assay demonstrated by a case of mild haemophilia associated with His1954-->Leu
substitution in the factor VIII A3 domain. Br J Haematol, 105, 1123-1126.
3. Allain, J.P., Verroust, F. & Soulier, J.P. (1979) One or two stage assay for factor VIII? Lancet, 1, 1076-1077.
4. Kirkwood, T.B. & Barrowcliffe, T.W. (1978) Discrepancy between one-stage and two-stage assay of factor VIII:C. Br J
Haematol, 40, 333-338.
Question 12.
1. Bern, M.M., Sahud, M., Zhukov, O., Qu, K. & Mitchell, W., Jr. (2005) Treatment of factor XI inhibitor using
recombinant activated factor VIIa. Haemophilia, 11, 20-25.
2. Bortoli, R., Monticielo, O.A., Chakr, R.M., Palominos, P.E., Rohsig, L.M., Kohem, C.L., Xavier, R.M. & Brenol, J.C.
(2009) Acquired factor XI inhibitor in systemic lupus erythematosus--case report and literature review. Semin Arthritis
Rheum, 39, 61-65.
3. Salomon, O., Zivelin, A., Livnat, T. & Seligsohn, U. (2006) Inhibitors to Factor XI in patients with severe Factor XI
deficiency. Semin Hematol, 43, S10-12.

Page 44 of 47
Question 13.
1. Kasper, C.K., Aledort, L., Aronson, D., Counts, R., Edson, J.R., van Eys, J., Fratantoni, J., Green, D., Hampton, J.,
Hilgartner, M., Levine, P., Lazerson, J., McMillan, C., Penner, J., Shapiro, S. & Shulman, N.R. (1975) Proceedings: A
more uniform measurement of factor VIII inhibitors. Thromb Diath Haemorrh, 34, 612.
Question 14.
See Question 12.
Question 15.
1. Gaucher C, Jorieux S, Mercier B, Oufkir D, Mazurier C. The "Normandy" variant of von Willebrand disease:
characterization of a point mutation in the von Willebrand factor gene. Blood. 1991;77(9):1937-41.
2. Tuley EA, Gaucher C, Jorieux S, Worrall NK, Sadler JE, Mazurier C. Expression of von Willebrand factor
"Normandy": an autosomal mutation that mimics hemophilia A. Proc Natl Acad Sci U S A. 1991;88(14):6377-81.
3. Caron C, Mazurier C, Goudemand J. Large experience with a factor VIII binding assay of plasma von Willebrand
factor using commercial reagents. Br J Haematol. 2002 Jun;117(3):716-8.
4. Miller CH, Kelley L, Green D. Diagnosis of von Willebrand disease type 2N: a simplified method for measurement of
factor VIII binding to von Willebrand factor. Am J Hematol. 1998 Aug;58(4):311-8.
Question 16.
1. Coots, M.C., Muhleman, A.F. & Glueck, H.I. (1978) Hemorrhagic death associated with a high titer factor V inhibitor.
American Journal Of Hematology, 4, 193-206.
2. Chong, L.L. & Wong, Y.C. (1985) A case of factor V inhibitor. American Journal Of Hematology, 19, 395-399.
3. Knobl, P. & Lechner, K. (1998) Acquired factor V inhibitors. Baillieres Clin Haematol, 11, 305-318.
Kirkeby, K.M. & Aronowitz, P. (2005) Acquired factor V inhibitor: a common and avoidable complication of topical
bovine thrombin application. Am J Med, 118, 805.
4. Leus, B., Devreese, K., van den Bossche, J. & Malfait, R. (2006) Factor V inhibitor: case report. Blood Coagul
Fibrinolysis, 17, 585-587.
Question 17.
See Question 11.
Question 18.
1. High, K.A. (1995) Factor IX: molecular structure, epitopes, and mutations associated with inhibitor formation. Adv
Exp Med Biol, 386, 79-86.
2. Lusher, J.M. (2000) Inhibitor antibodies to factor VIII and factor IX: management. Semin Thromb Hemost, 26, 179-
188.

Page 45 of 47
3. Chuansumrit, A., Moonsup, Y., Sirachainan, N., Benjaponpitak, S., Suebsangad, A. & Wongwerawattanakoon, P.
(2008) The use of rituximab as an adjuvant for immune tolerance therapy in a hemophilia B boy with inhibitor and
anaphylaxis to factor IX concentrate. Blood Coagul Fibrinolysis, 19, 208-211.
4. Chitlur, M., Warrier, I., Rajpurkar, M. & Lusher, J.M. (2009) Inhibitors in factor IX deficiency a report of the ISTH-
SSC international FIX inhibitor registry (1997-2006). Haemophilia, 15, 1027-1031.
5. Tengborn, L., Hansson, S., Fasth, A., Lubeck, P.O., Berg, A. & Ljung, R. (1998) Anaphylactoid reactions and nephrotic
syndrome--a considerable risk during factor IX treatment in patients with haemophilia B and inhibitors: a report on the
outcome in two brothers. Haemophilia, 4, 854-859.
Question 19.
See VWD
Question 21.
1. Morgan, G.E., Rowley, G., Green, P.M., Chisholm, M., Giannelli, F. & Brownlee, G.G. (1997) Further evidence for the
importance of an androgen response element in the factor IX promoter. Br J Haematol, 98, 79-85.
2. Hung, H.L. & High, K.A. (1993) Hepatocyte nuclear factor-iv binds to the promoters of factor-vii, factor-ix, and
factor-x. Thrombosis And Haemostasis 69, 1237-1237.
3. Hirosawa, S., Fahner, J.B., Salier, J.P., Wu, C.T., Lovrien, E.W. & Kurachi, K. (1990) Structural and functional basis of
the developmental regulation of human coagulation factor IX gene: factor IX Leyden. Proc Natl Acad Sci U S A, 87,
4421-4425.
4. Reitsma, P.H., Mandalaki, T., Kasper, C.K., Bertina, R.M. & Briet, E. (1989) Two novel point mutations correlate with
an altered developmental expression of blood coagulation factor IX (Hemophilia B Leyden phenotype). Blood, 73, 743-
746.
5. Reitsma, P.H., Bertina, R.M., van Amstel, J.K.P., Riemens, A. & Briët, E. (1988) The putative Factor IX gene promotor
in Hemophilia B Leyden. Blood, 72, 1074-1076.
Question 22.
1. Bolton-Maggs, P.H., Perry, D.J., Chalmers, E.A., Parapia, L.A., Wilde, J.T., Williams, M.D., Collins, P.W., Kitchen, S.,
Dolan, G. & Mumford, A.D. (2004) The rare coagulation disorders--review with guidelines for management from the
United Kingdom Haemophilia Centre Doctors' Organisation. Haemophilia, 10, 593-628.
2. Cunningham, M.T., Brandt, J.T., Laposata, M. & Olson, J.D. (2002) Laboratory diagnosis of dysfibrinogenemia. Arch
Pathol Lab Med, 126, 499-505.
3. Martinez, J. (1997) Congenital dysfibrinogenemia. Curr Opin Hematol, 4, 357-365.
Question 24.
1. Crist, R.A., Gibbs, K., Rodgers, G.M. & Smock, K.J. (2009) Effects of EDTA on Routine and Specialized Coagulation
Testing and an easy Method of Distinguish EDTA-Treated from Citrated Plasma Samples. Laboratory Haematology, 15,

Page 46 of 47
45-48.
Question 25.
See Question 1.
Question 26.
See Question 11.
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Practical-Haemostasis.

SCREENING FACTOR PLATELET FUNCTION THROMBOPHILIA FIBRINOLYTIC GENETIC MISCELLANEOUS

Data Interpretation: Factor Assays

Test Patient Reference Range

Click here for Part 2

The results of factor assays are show below:

Factor Assay Patient Reference Range

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

1. What additional tests might you request and why?

Click here for Part 3

Shown below are the results of additional investigations on this patient:

Assay Patient Reference Range

1. What is the diagnosis?

2. Are there any additional tests you might request?

Click here for Part 4

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

How would you manage this patient if he needed a cholecystectomy?

Click here for Part 2

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

Factor Assay Patient Reference Range

1. What additional tests might you request and why?

Click here for Part 3

Shown below are the results of additional investigations on this patient:

Assay Patient Reference Range

1. What are the possible diagnoses?

2. Are there any additional tests you might request?

3. How would you manage this patient if he needed a cholecystectomy?

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

Click here for Part 4

Test Patient Reference Range

1. What additional tests would you request?

Click here for Part 2

The results of a Factor XIII screen are shown below:

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

What tests would you request next?

What is the sensitivity of a FXIII screening assay?

Test Patient Reference Range

On the basis of these tests what you do next?

Click here for Part 2

The results of factor assays are shown below:

What do you think might explain these findings?

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

1/10 1/20 1/30 1/40 1/80 1/100 1/1000

Reference plasma 25s 32s 38s 59s

Click HERE for the answers

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

2. This is a PT-based assay and so you use Log-Log paper.

1/5 1/10 1/20 1/40 1/80 1/100

Patient 2 108 120

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

Click HERE for the answer

http://practical-haemostasis.com/Data%20Interpretation/Data%20Answers/data_interpretation_factor_assays_answers.html 03/04/2019, 11C49

FVIII:C Reference Plasma = FVIII:C Reference Plasma = FVIII:C Reference Plasma =

Test Patient Reference Range

Click here for Part 2

Reference Factor V:C concentration 85 IU/dL.