Data Interpretation - Factor Assays

Practical-Haemostasis.com

Data Interpretation:
Factor Assays

Question 1

Answers to Question 1

Question 2

Answers to Question 2

Question 3

Factor V Assay

Factor VIII Assay

Answers to Question 3

Question 4

Answers to Question 4

Question 5

Answers to Question 5

Question 6

Answers to Question 6

Question 7

Answers to Question 7

Question 8

Answers to Question 8

Question 9

Answers to Question 10

Question 10

Answers to Question 10

Question 11

Question 12

Answers to Question 12

Question 13

Answers to Question 13

Question 14

Answers to Question 14

Question 15

Answers to Question 15

Part II

Part 1:

Part 2:

Shown below are the data for a factor V assay and a factor VIII assay.

Part 3:

Part 1:

Part 2.

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 was 94% (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

All clotting times are in seconds.

Click this LINK to take you to a site that will allow you to generate and print your own graph paper.

A. If the concentration of FX in the reference plasma was 112% (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?

The graph below shows a plot of the data from the above question.

In the graph shown the dilutions are plotted on the X-axis and the PT clotting times (in seconds) on the Y-axis.
This is a PT-based assay and so you use Log-Log paper.
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.
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].

	Activity if reference plasma = 100%	Activity if reference plasma = 94%	Activity if reference plasma = 112%	Activity if reference plasma = 0.90 IU/ml
Patient 1	18%	17%	20%	0.16 IU/ml
Patient 2	200%	188%	224%	1.80 IU/ml
Patient 3	<1%	<1%	<1%	<1 IU/ml
Patient 4	40%	38%	45%	0.36 IU/ml

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

All clotting times are in seconds.

Click this LINK to take you to a site that will allow you to generate and print your own graph paper.

If the concentration of FVIII in the standard was 0.89 IU/ml what would your answers be?

The graph below shows a plot of the data from the above question.

In the graph shown 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.
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.

	Activity if reference plasma = 100%	Activity if reference plasma = 94 IU/dl	Activity if reference plasma =0.89 IU/ml
Patient 1	45%	42 IU/dl	0.40 IU/ml
Patient 2	1%	~1 IU/dl	~0.01 IU/ml
Patient 3	20%	19 IU/dl	0.18 IU/ml
Patient 4	190%	179 IU/dl	1.70 IU/ml

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	2.9g/L	1.5-4.0g/L
Thrombin Time	13s	10-13s

How would you proceed with the investigation of this patient?

Plot the data and derive the concentrations of these two factors.

Click this LINK to take you to a site that will allow you to generate and print your own graph paper.

Dilutions	Clotting Time (s) Control	Clotting Time (s) Patient
1/10	23	60
1/20	38	92
1/40	59	160

NB Control Factor V concentration 85 IU/dl.

Dilutions	Clotting Time (s) Control	Clotting Time (s) Patient
1/10	35s	50s
1/20	43s	57s
1/40	50	64
1/100	59s	74s

NB Control Factor VIII concentration 90 IU/dl

The diagnosis of combined deficiency of factor V and VIII is confirmed.

What is the underlying molecular basis for this disorder?

Part 1:

Shown below is a plot of the Factor V assay.

The FV:C in this patient is 22 IU/dl.

Part 2:

Shown below is a plot of the Factor VIII assay.

The FVIII:C in this patient is 23 IU/dL.

Remember that APTT based assays are plotted on Log-Lin paper.
The FVIII for the patient assumed that the 1/10 dilution for the reference plasma has a value of 1 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.

What is the molecular basis underlying this disorder?

For a review of this area see:

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.
Neerman-Arbez, M., Johnson, K.M., Morris, M.A., McVey, J.H., Peyvandi, F., Nichols, W.C., Ginsburg, D., Rossier, C., Antonarakis, S.E. & Tuddenham, E.G. (1999) Molecular analysis of the ERGIC-53 gene in 35 families with combined factor V-factor VIII deficiency. Blood, 93, 2253-2260.
Peyvandi, F., Duga, S., Akhavan, S. & Mannucci, P.M. (2002) Rare coagulation deficiencies. Haemophilia, 8, 308-321.
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.
Nyfeler, B., Kamiya, Y., Boehlen, F., Yamamoto, K., Kato, K., de Moerloose, P., Hauri, H.P. & Neerman-Arbez, M. (2008) Deletion of 3 residues from the C-terminus of MCFD2 affects binding to ERGIC-53 and causes combined factor V and factor VIII deficiency. Blood, 111, 1299-1301.
Seligsohn, U. & Ginsburg, D. (2006) Deciphering the mystery of combined factor V and factor VIII deficiency. J Thromb Haemost, 4, 927-931.
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.
Mohanty, D., Ghosh, K., Shetty, S., Spreafico, M., Garagiola, I. & Peyvandi, F. (2005) Mutations in the MCFD2 gene and a novel mutation in the LMAN1 gene in Indian families with combined deficiency of factor V and VIII. Am J Hematol, 79, 262-266.
Zhang, B., Kaufman, R.J. & Ginsburg, D. (2005) LMAN1 and MCFD2 form a cargo receptor complex and interact with coagulation factor VIII in the early secretory pathway. J Biol Chem, 280, 25881-25886

A 7-year-old boy is investigated with prolonged bleeding following dental surgery. A coagulation screen is requested and he is found to have a prolonged PT and APTT.

Click this LINK to take you to a site that will allow you to generate and print your own graph paper.

i. What factor assays would you request and why?
ii. Shown below are the data for a PT-based factor X assay. From this data derive the patient’s factor X level.
iii. Is there any value in performing an APTT-based FX assay?
iv. There are 5 methods for assaying FX - what are they?

Dilutions	1/10	1/20	1/40	1/80	1/100
Standard	20s	24s	28s	34s	35s
Patient	35s	42s		59s	62s

NB Factor X Standard 110 IU/dl.

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.

This graph shows a PT-based FX assay.

Remember the Factor X Standard is 110 IU/dl and so the result for the patient has to be corrected for this. The factor X level from this graph is ~10 IU/dl and taking into the account the concentration of reference which is 110 IU/dl the true FX level is 110/100 x 10 = 11 IU/dl.

iii. 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.

iv. 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
Immunological FX assay - which measures FX:Ag
FX chromogenic assay

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	2.9g/L	1.5-4.0g/L
Thrombin Time	13s	10-13s
Full Blood Count	Normal

i. What factor assays would you request and why?
ii. Shown below are the data for a 1-stage factor VIII assay. From this data derive a factor VIII result for the patient.

Click this LINK to take you to a site that will allow you to generate and print your own graph paper.

Dilutions	1/10	1/20	1/40	1/100
Standard Reference Plasma	29s	49s	59s	72s
Patient	68s	78s	88s	101s

NB Factor VIII Standard 104 IU/dl.

What are the possibilities to explain these findings?

i. What factor assays 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.

ii.. Shown below are the data for a 1-stage factor VIII assay. From this data derive a factor VIII result for the patient.

The graph below shows the results of the FVIII assay:

The FVIII:C in this patient is ~11 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 11 = 11.4 i.e. ~11 IU/dl.

What are the possibilities to explain these findings?

Type 3 Von Willebrands 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
Extreme Lyonisation

In fact this patient has extreme Lyonisation. Her mother is a carrier of a mutation associated with severe haemophilia A and the patients has inherited a second mutation [on the X chromosome] from her father that is lethal to all the cells that express it. As a result only the F8 gene inherited from her mother and which carries a emulation associated with severe haemophilia A is expressed.

An 18-year-old male patient with factor VII deficiency has a factor VII assay performed.

Calculate the FVII levels in the patient.

Click this LINK to take you to a site that will allow you to generate and print your own graph paper.

	Dilution
	1/10	1/100	1/1000
Standard	29	41	56
Patient	55	68	82
Father	36	50	64
Mother	36	50	64

All clotting times are in seconds
FVII standard: 115 IU/dl.

The results of his parents are also shown. Calculate their FVII levels.
What can you infer from these family studies?

Shown below are the results of the FVII assays:

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] the true FVII level is ~1 IU/dl.

The FVII levels in both parents is ~20 IU/dl from the graph and with a correction for the standard reference plasma FVII level [115 IU/dl] the true FVII level is 23 IU/dl.

What can you infer from these family studies?
It is probable that both parents are related.

For a review of FVII deficiency see:

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.
Mariani, G., Konkle, B.A. & Ingerslev, J. (2006) Congenital factor VII deficiency: therapy with recombinant activated factor VII -- a critical appraisal. Haemophilia, 12, 19-27.
Perry, D.J. (2002) Factor VII Deficiency. Br J Haematol, 118, 689-700.
Eigenbrot, C. (2002) Structure, function, and activation of coagulation factor VII. Curr Protein Pept Sci, 3, 287-299.

A 56-year-old male with moderate Haemophilia A diagnosed 16 years ago requires surgery.

Shown below are the results of his pre-operative clotting screen:

Test	Patient	Reference Range
PT	12s	11-14s
APTT	33s	23-35s
Fibrinogen	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?

Below is data from a 1-stage APTT FVIII assay and a chromogenic FVIII assay. Derive the FVIII:C from these two assays.

[Click this LINK to take you to a site that will allow you to generate and print your own graph paper.

1-stage FVIII:C Assay

	Dilutions
	1/10	1/40	1/160
Reference Plasma	35	42	47
Patient	37	44	49

Chromogenic FVIII:C Assay

	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?

1. Are you surprised by these results?
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.

Part 2. Below is data from a 1-stage APTT FVIII assay and a chromogenic FVIII assay. Derive the FVIII:C from these two assays.

a. 1-stage APTT FVIII assay

b. Chromogenic FVIII:C Assay

The absorbance reading for the patient was 0.06.

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%.

1. How do you explain these findings.

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.

2. What is the molecular basis for this?

See:

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.

Show in the table below are the data for a factor XI assay performed on 2 separate plasma samples [Plasma 1 and Plasma 2.]

Plot the data for the FXI reference plasma [FXI:C 100%] and draw a best fit line. For the two plasma samples plot the data but do not attempt to draw a best fit line.

Calculate the FXI levels for each of the dilutions in the two plasma samples.
Explain your findings.
What would you do next ?

	Dilutions
	1/10	1/20	1/40	1/100	1/160
Reference Plasma	30	33	37	42
Plasma 1	40	43	47	52
Plasma 2	37	38	40	42	43

[Nb Plot the data on Log-Lin paper]

Calculate the FXI:C for each of the dilutions for Plasma 1 and Plasma 2.

How would you explain these findings and what would you do next?

Calculate the FXI:C for each of the dilutions for Plasma 1 and Plasma 2.
Shown below is a graph of the FXI:C data. Note that 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	33	37	42
Plasma 1	15%	7% x 2 = 14%	3.5% x 4 = 14%	1.5% x 10 = 10%
Plasma 2	25%	20% x 2 = 40%	15% x 2 = 60%	9% x 2 = 90%	7% x 16 = 112%

If we take the 1/10 dilution of the reference plasma to have a FXI:C of 100% then Plasma 1 has a FXI:C level of ~13%. The value is similar to for all dilutions.

However, for plasma 2 the dilutions give quite different FIX: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.

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

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
6	1/5 1/10 1/20	33% 55% 68%	7.7 Bu
7	1/20 1/40 1/80	35% 55% 68%	~31 Bu
8	1/320 1/640 1/1280	0.30 IU/ml 0.52 IU/ml 0.65 IU/ml	608 Bu

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.

Don't forget you derive these figures using the graph [Inhibitor level plotted against residual factor level] shown in Figure 10. Although the original Bethesda assay was designed to measure FVIII inhibitors it is now commonly used to measure inhibitors for al clotting factor proteins.

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?

The results of a FXI assay are shown below:
Calculate the FXI levels in the plasma sample.

Dilutions	Clotting Time (s) Reference Plasma	Clotting Time (s) Patient Plasma Sample 1
1/10	26	36
1/20	29	39
1/100	38	48

NB Reference Factor XI:C concentration 100%.

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. 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

Shown below is the raw data for a repeat FXI assay. Calculate the FXI level in this plasma sample. What do you think has happened?

Dilutions	Clotting Time (s) Reference Plasma	Clotting Time (s) Patient Plasma Sample 1
1/10	26	50
1/20	29	54
1/100	38	62

NB Reference Factor XI:C concentration 100%.

An inhibitor assay is undertaken. The FXI:C assay data is shown below.

Calculate the residual FXI:C. This was based upon a 1:20 dilution of plasma.

What is the inhibitor level.

Dilutions	Clotting Time (s) Reference Plasma	Clotting Time (s) Patient Plasma Sample
1/10	26s	29s
1/20	29s	32s
1/100	38s	41s

NB Reference Factor XI:C concentration 100%.

These tests show a prolonged APTT but a normal PT. This suggests either a clotting factor deficiency (VIII, IX, XI or XII) or a lupus anticoagulant.

The results of the first FXI assay [Patient Plasma] are shown below:
Calculate the FXI levels in the plasma sample. The results of a FXI:C assay are shown below.

NB Reference Factor XI:C concentration 100%.

These results are consistent with FXI deficiency - the FXI:C level was ~15%.

Test	Patient	Reference Range
PT	111.8s	11-14s
APTT	85s	23-35s
Fibrinogen	2.9g/L	1.5-4.0g/L
Thrombin Time	12s	10-13s
Full Blood Count	Normal

Shown below are the raw data for a repeat FXI assay. Calculate the FXI level in this plasma sample. What do you think has happened?

Dilutions	Clotting Time (s) Reference Plasma	Clotting Time (s) Patient Plasma Sample 1
1/10	26	50
1/20	29	54
1/100	38	62

NB Reference Factor XI:C concentration 100%

See Graph above - Patient Plasma Sample 2. The FIX has fallen significantly and is in the region of 1%.

The Factor XI has fallen significantly and although the lines are parallel - the most likely explanation is that she has formed an inhibitor following the use of FXI concentrate given at the time of surgery and in the post-operative period. A formal FXI inhibitor screen confirms this. The FXI inhibitor level is 16Bu as shown in the graph below.

The residual FXI:C is ~57%. If we then use the graph relating residual clotting factor level to inhibitor titre and remembering that 1Bu = 50% inhibition and that the 57% residual activity represented a 1 in 20 dilution then the inhibitor titre is 0.8 x 20 = 16Bu.

Remember to allow for the dilution in the original sample that the FXI:C assay was performed on.

Factor XI inhibitors are rare but seen usually in individuals with a complete absence of FXI i.e. severe FXI deficiency. Amongst Jewish individuals there are 4 'common' F11 gene mutations [Types 1-IV].

Type	Mutation
I	G>A mutation at the 5' donor splice site exon 14
II	Glu135X Exon 5
III	Phe310Leu Exon 9
IV	14bp deletion in exon 14 and extending 11bp into intron N

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?

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.

The results of relevant factor assays are shown below: Factor II 132u/dl [Reference range: 50-150u/dl]
Factor V 2u/dl [Reference range: 50-150u/dl]
Factor X 89u/dl [Reference range: 50-150u/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?

1. What do you think has happened and why.

These results are consistent with factor V deficiency

2. Are there any other tests you might request?

You might request a 50:50 mix to see if the clotting abnormality corrects. In addition as he has no previous history suggestive of a bleeding disorder you might suspect an acquired inhibitor and request a formal inhibitor screen.

3. Why does this problem arise?

The b-lactam containing antibiotic is well recognised although rather rather cause of a factor V inhibitor Presumably there is some epitope similarity between the β-lactam ring and factor V.
Favour V inhibits have also been reported in association with topical bovine thrombin which contains bovine FV. The patient develops an antibody to the bovine FV which cross-reacts with their own factor V causing acquired FV deficiency.

4. How would you manage this patient?

These FV inhibitors do not usually cause any bleeding problems and so removal of the β-lactam containing antibiotic leads to resolution of the problem. Aggressive immunosuppressive therapy is not usually indicated.

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?

1-stage assay:
The 1-stage assay FVIII is based upon the activated partial thromboplastin time (APTT). A log–log plot of concentration versus clotting time gives straight parallel lines from which the FVIII:C level in test samples can be calculated. The 1-stage assay is simple and readily automated but lacks sensitivity at low FVIII:C levels. The current ISTH criteria define severe haemophilia A as a FVIII:C < 1 IU/dl but the 1-stage assay may be unable to separate a FVIII:C of 0.5 IU/dl from 1.5 IU/dl.

Two-stage assay:

i. First Stage: In the first stage of the assay a combined reagent containing CaCl₂, phospholipid, bovine factor V and human serum (a source of factors IX, X, XI and XII) is mixed with dilutions of test and standard plasma which have been adsorbed with Al(OH)₃ (to remove prothrombin). The rate of generation of factor Xa (FXa) is dependent on the FVIII:C level in the test or standard plasma: in the absence of prothrombin the reaction cannot proceed further.

ii. Second Stage: In the second stage prothrombin and fibrinogen are provided by addition of normal plasma and the time to clot formation is dependent upon the amount of FXa generated in the first stage.

The 2-stage assay is more cumbersome to perform but has increased sensitivity at low FVIII:C levels. The chromogenic FVIII assay is similar to the 2-stage FVIII assay and in many labs has now replaced the 2-stage assay. It is the recommended method for measuring FVIII potency in clotting factor concentrates.

The discrepancy between the two tests is well recognised although the mechanism is less clear. One possible explanation of the apparently lower two-stage FVIII:C levels may be that the that following proteolytic activation of FVIII to FVIIIa in the first stage of the assay, dissociation of the A2 domain from the active FVIIIa heterotrimer may be more rapid in the variant FVIIIa than in wild-type FVIIIa, leading to reduced activity as measured in the second stage. FVIII is only transiently activated during clotting in the 1-stage assay, this enhanced dissociation may have less effect or may even be normal.

When the FVIII:C from individuals with mild haemophilia A is measured by both the 1-stage and 2-stage/chromogenic assays, approximately 30% of cases with have discrepant levels. The discrepancy is important for those with a reduced 2-stage but a normal 1-stage assay as the diagnosis of haemophilia will otherwise be missed (hence the importance of a good bleeding history). In patients with a reduced 1-stage but a normal 2-stage FVII assay, such cases may be incorrectly diagnosed as having haemophilia.

Deriving a 1-stage:2-stage ratio may be useful. In general the ratio is between 0.5-2. A ratio >2 is suggestive of mild haemophilia A.

A 3-year-old boy with severe haemophilia B (IX:C<1IU/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.

	Patient Plasma Dilutions
	1/5	1/10	1/20
Residual FIX:C	33%	55%	68%

Look at the inhibitor plot [Inhibitor level plotted against residual FVIII:C] in the answer to Question 10 but substitute FIX:C for FVIII:C as in Question 14 our patient has FIX deficiency.
The value closer to 50% is the 1/10 dilution with a residual FIX:C of 55%. 55% from the graph is ~0.85 Bu - but remember this is a 1/10 dilution and so you must multiply the answer by 10 to derive the true value of 8.5 Bu.

Plot the following data from a patient with suspected Factor V deficiency. What is the factor V level?

	Dilutions
	1/10	1/20	1/40	1/100
Control	50s	68s	95s	135s
Patient	80s	115s	160s	No Data

Control FV:C 85 U/dL.

Shown below is a PT-based 1-stage FV assay.

The FV:C in the patient is 35% [35 IU/dl] and taking into account the control FV:C of 85 IU/dl, the actual FV level in the patient is 35 x 0.85 = 30% or more correctly as the standard is given in IU/dl - 30 IU/dl.

Reference Plasma