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

The results of factor assays are show below:

Factor Assay	Patient	Reference Range
FVIII	0.23 IU/mL	0.45 - 1.49 IU/mL
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?

Click here for Part 3

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]
Immunoglobulins	IgG 4.2g/L IgM 17.3g/L IgA 2.2g/L	IgG 6 - 13g/L IgM 0.4 - 2.2g/L IgA 0.8 - 3.7g/L
Renal & LFTS Calcium	Normal

1. What is the diagnosis?
2. Are there any additional tests you might request?

Click here for Part 4

Shown below are two images from a bone marrow aspiriate on this patient. What do these show and what is the diagnosis?

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

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:

Test	Patient	Reference Range
PT	12s	10.6-12.4s
APTT	95s	21-32s
Fibrinogen (Clauss)	3.1g/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

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

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
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
Protein electrophoresis	M Band 17g/L IgG Κappa
Immunoglobulins	IgG 22g/L IgM 2.3g/L IgA 2.1g/L	IgG 6 - 13g/L IgM 0.4 - 2.2g/L IgA 0.8 - 3.7g/L
Renal & LFTS Calcium	Normal

1. What are the possible diagnoses?
2. Are there any additional tests you might request?

Click here for Part 4

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?

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:

Test	Patient	Reference Range
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?

Click here for Part 2

The results of a Factor XIII screen are shown below:

5M Urea FXIII Screening Test

Patient: Complete lysis at 24 hours

Control: No lysis at 24 hours

What tests would you request next?
What is the sensitivity of a FXIII screening assay?

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:

Test	Patient	Reference Range
PT	48s	11-14s
APTT	39	28-34.5s
Fibrinogen (Clauss)	3.9g/L	1.9-3.7 g/L
Thrombin Time	14s	12-14s

On the basis of these tests what you do next?

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The results of factor assays are shown below:

Factor Assay	Patient [IU/dL]	Reference Range [IU/dL]
Factor II	103	70-130
Factor V	98	75-155
Factor VII	34	70-130
Factor VIII	167	75-155
Factor IX	99	75-135
Factor X	32	70-130
Factor XI	86	75-155

What do you think might explain these findings?
What would you request next?

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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?

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 2	22s		28s			34s
Patient 3	180s			182s			182s
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?

Question 6

The following are the clotting times [in seconds] for a 1-stage APTT Factor VIII assay.

Plot the following factor VIII data and calculate the factor VIII:C levels.
The concentration of FVIII in the reference plasma is 94 IU/dl.

	Dilutions
	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
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?

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:

Test	Patient	Reference Range
PT	34s	11-14s
APTT	82s	23-35s
Fibrinogen (Clauss)	2.9g/L	1.5-4.0g/L
Thrombin Time	13s	10-13s

How would you proceed with the investigation of this patient?

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You request factor V and VIII assays. The raw data for these assays is shown below.
Plot the data and derive the concentrations of these two factors.

Factor V Assay

Dilutions	Patient PT [s]	Reference 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

Dilutions	Patient APTT [s]	Reference APTT [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.

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The diagnosis of combined FV and FVIII deficiency is confirmed.

1. What is the molecular basis for this disorder?
2. How would you manage her menorrhagia?

Question 8

A 7-year-old boy is investigated with prolonged bleeding following dental surgery. A coagulation screen is requested:

Test	Patient	Reference Range
PT	23s	11-14s
APTT	56s	28-34.5s
Fibrinogen (Clauss)	2.1g/L	1.9-3.7 g/L
Thrombin Time	13s	12-14.2s

i. What factor assays would you request and why?

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Shown below are the data for a PT-based factor X assay. From this data derive the patient’s factor X level.

Dilutions	Patient PT [s]	Reference PT [s]
1/10	35	20
1/20	42	24
1/40		28
1/80	59	34
1/100	62	35

Is there any value in performing an APTT-based FX assay?

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There are 5 methods for assaying Factor X - what are they?

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:

Test	Patient	Reference Range
PT	12s	11-14s
APTT	90s	23-35s
Fibrinogen (Clauss)	2.9g/L	1.5-4.0g/L
Thrombin Time	13s	10-13s
Full Blood Count	Normal

1. What tests would you request and why?

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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.

Dilutions	Patient APTT [s]	Reference APTT [s]
1/10	42	29
1/20	47	34
1/40	52	39
1/100	58	45

Factor VIII:C Reference 104 IU/dL.

3. What additional tests would you request and why?
4. What additional questions might you ask the parents?

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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?

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.

Dilutions	Index Case PT [s]	Mother PT [s]	Father PT [s]	Reference PT [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?

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:

Test	Patient	Reference Range
PT	12s	11-14s
APTT	33s	23-35s
Fibrinogen (Clauss)	2.9g/L	1.5-4.0g/L
Thrombin Time	13s	10-13s
Full Blood Count	Normal

1. Are you surprised by these results?
2. What might explain this?

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Below is data from a 1-stage APTT FVIII assay.
Derive the FVIII:C from this assay.

Dilutions	Patient APTT [s]	Reference APTT [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?
What would you request next?

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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] Reference Plasma	0.47	0.32	0.19	0.04

Absorbance Patient: 0.06

1. What is the basis for a chromogenic FVIII Assays?
2, How do you explain these findings.
3. What is the molecular basis for this?

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 ?

Dilutions	Plasma Sample 1 APTT [s]	Plasma Sample 2 APTT [s]	Reference APTT [s]
1/10	40	37	30
1/20	43	38	33
1/40	47	40	37
1/100	52	42	42
1/160		43

Reference Factor XI:C concentration 100 IU/dL.

Question 13

Calculate the Bethesda titres for each of the following plasma samples.

Sample	Plasma Dilutions	Residual FVIII	Titre [BU/ml]
1	Undiluted	70%
2	Undiluted	40%
3	Undiluted	0.5 IU/ml
4	Undiluted	0.6 IU/ml
5	Undiluted	45 IU/dl
6	1/5 1/10 1/20	33% 55% 68%
7	1/20 1/40 1/80	35% 55% 68%
8	1/320 1/640 1/1280	0.30 IU/ml 0.52 IU/ml 0.65 IU/ml

Question 14

A 56-year-old woman of Jewish ancestry is referred for an aortic valve replacement (AVR). Her pre-operative screening tests show:

Test	Patient	Reference Range
PT	13s	11-14s
APTT	61s	23-35s
Fibrinogen	2.9g/L	1.5-4.0g/L
Thrombin Time	13s	10-13s
Full Blood Count	Normal

What tests would you perform next?

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The results of a FXI assay are shown below:

Dilutions	Patient APTT [s]	Reference APTT [s]
1/10	36	26
1/20	39	29
1/100	48	38

Reference FXI:C 100 IU/dL
1. Calculate the FXI levels in the plasma sample.
2. How will you manage her surgery?

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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:

Test	Patient	Reference Range
PT	11.8s	11-14s
APTT	85s	23-35s
Fibrinogen	2.9g/L	1.5-4.0g/L
Thrombin Time	12s	10-13s
Full Blood Count	Normal

Y0u request a further FXI:C assay - the raw data is shown below:

Dilutions	Patient APTT [s]	Reference APTT [s]
1/10	50	26
1/20	54	29
1/100	62	38

Reference FXI:C 100 IU/dL.
1. What is the FXI level.
2. How would you explain the differences in FXI:C assays.

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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.
2. What is the inhibitor level.

Dilutions	Patient APTT [s]	Reference APTT [s]
1/10	29	26
1/20	32	29
1/100	41	38

Reference FXI:C 100 IU/dL.
1. What is the FXI level.
2. How would you explain the differences in FXI:C assays.

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? Click here for Part 2 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?

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?

Click here for Part 2

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?

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.

Test	Patient	Reference Range
PT	45s	11-14s
APTT	79s	23-35s
Fibrinogen	3.2g/L	1.5-4.0g/L
Thrombin Time	13s	10-13s
Full Blood Count	Normal

Outline how you would investigate this patient. Give the reasons behind these investigations.

Click here for Part 2

The results of relevant factor assays are shown below:

Factor	Patient	Reference Range
FII	132 IU/dL	50-150 IU/dL
FV	2IU/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?

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?

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.

Dilutions	Residual FIX [%]
1/5	33
1/10	55
1/20	68

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).

Patient	Pre-DDAVP FVIII:C VWF:Ag VWF:RCo [IU/dL]	1 hour post-DDAVP FVIII:C VWF:Ag VWF:RCo [IU/dL]	2 hour post-DDAVP FVIII:C VWF:Ag VWF:RCo [IU/dL]	4 hour post-DDAVP FVIII:C VWF:Ag VWF:RCo [IU/dL]	8 hour post-DDAVP FVIII:C VWF:Ag VWF:RCo [IU/dL]
1	30 10 22	110 85 80	50 42 40	30 28 27	28 22 20
2	20 15 14	162 143 144	100 96 92	88 75 73	67 60 60
3	20 15 14	160 140 140	100 95 96	86 78 74	67 62 61
4	1 1 1	1.5 1.2 1.2	1 1 1	<1 1 <1	1 1 1
5	18 62 64	150 140 140	40 95 96	20 78 74	17 62 61
Control	110 95 90	400 220 218	320 300 310	280 260 248	200 190 100

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

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1. In patient 1 - what additional investigations may be of benefit?

2. In patient 2 - what additional investigations may be of benefit?

3. In patient 3 - what additional investigations may be of benefit?

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1. In patient 1 - you request VWF propeptide [VWFpp] studies: Why?

2. In patient 2 - you request platelet aggregation tests with low dose ristocetin [0.5mg/mL]: Why?

3. In patient 3 - you request multimers: Why?

Question 20

A 2-year-old boy develops a haemarthrosis of his left knee following a minor injury. Investigations show:

Test	Patient	Reference Range
PT	13s	11-14s
APTT	91s	28-34.5s
Fibrinogen (Clauss)	3.6g/L	1.9-3.7 g/L
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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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	109s	82s

The FIX:C of the reference preparation is 110 IU/dL.

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 yrs 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?

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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?

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?

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You request a panel of screening tests - the results of which are shown below:

Test	Patient	Reference Range
PT	13s	11-14s
APTT	28s	28-34.5s
Fibrinogen (Clauss)	2.9 g/dL	1.9-3.7 g/L
Thrombin Time	12s	12-14s
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?

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You request a FXIII screen [using 5M Urea]:

Test	Patient Plasma	Reference Plasma
Clot lysis at 1 hour	+	-
Clot lysis at 6 hours	+	-
Clot lysis at 12 hours	+	-
Clot lysis at 24 hours	+	-

[+ = lysis has occurred/- = lysis has not occurred]

What do these results suggest and what additional tests would you request

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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?

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.

Test	Patient	Reference Range
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

1. What tests would you perform next?

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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.

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Comment upon these results which were obtained following the administration of 5000 units of an intermediate purity Factor VIII concentrate.
What do you think might provide an explanation for this.
Are there any other tests you would request and why?

Time	APTT [s] 25.8-33.8s	VIII:C [IU/ml] [0.57-1.41]	VWF:Ag [IU/ml] [0.53-1.49]	VWF:Act [IU/ml] [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

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:

Test	Patient	Reference Range
PT	23s	11-14s
APTT	68s	28-34.5s
FVIII:C	0.04 IU/mL	0.5-1.45 IU/mL
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?
2. What would you do next?

Click here for Part 2

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:

Test	Patient	Reference Range
PT	12s	11-14s
APTT	32s	28-34.5s
FVIII:C	1.12 IU/mL	0.5-1.45 IU/mL
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	Not performed

How do you explain these findings?

Question 25

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

Test	Patient	Reference Range
PT	10.1s	9.8-12.6s
APTT	41s	25.8-33.8s
Fibrinogen (Clauss)	3.9g/L	1.6-3.7 g/L
Thrombin Time	13s	11.5-13s
FVIII:C	0.30 IU/mL	0.70-1.50 IU/mL
VWF:Ag	0.05 IU/mL	0.72-1.52 IU/mL
VWF:Act	0.05 IU/mL	0.72-1.52 IU/mL

1. What questions might you ask this patient that would be important?
2. What additional tests might you request and why?

Click here for Part 2

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?

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:

Test	Patient	Reference Range
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?

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