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A Practical Guide to Laboratory Haemostasis

 

Data Interpretation: Screening Tests - Answers



Introduction

The following questions will allow you to work through a number of scenarios.

Question 1
A 2-year-old girl is brought to your clinic. Developmentally normal, her parents had noticed her to be bruising easily.

Coagulation tests show:

Test Patient Reference Range
PT 13s 11-14s
APTT 105s 23-35s
Fibrinogen (Clauss) 2.7g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s
1. What additional questions might you ask the parents?
2. Briefly outline how you would investigate the clotting abnormalities.
3. What are the possible explanations for these results?

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You should ask whether there is any family history of note e.g. bleeding disorder
You should construct a family pedigree and establish any consanguinity.


1. You repeat the tests and confirm the abnormality.
This excludes the results as being laboratory error.

2. The prolonged PT and APTT correct in a mix with normal plasma.
This excludes an inhibitor [although this would be uncommon/rare in a child of this age] and makes it much more likely to be a factor deficiency.

What factor assays would you request and why?
The APTT is prolonged and the PT is normal. FXII deficiency will prolong the APTT but is not associated with a bleeding tendency and so you should request FVIII, FIX and FXI assays. You might also request VWF assays but you could wait until the result of the FVIII assay is available.
FXIII deficiency will not prolong the PT or the APTT and so if you suspect this you must specifically request a FXIII screen.


You might also request a karyotype.


Click here for Part 3 and Part 4

You request FVIII, IX and XI assays.
The FIX and FXI assays are normal but the FVIII assay is <1 IU/dl.
The karyotype is normal.
Von Willebrand Factor assays are normal.

1. What is your differential diagnosis?
The differential diagnosis is either severe haemophilia A or von Willebrand disease. The FVIII at <1 IU/dl is too low for 2N VWD and so it would need to be either severe Type 1 VWD or Type 3. However, the VWF assays are normal. The FVIII is also too low for combined FV and FVIII deficiency and in addition the PT is normal so excludes this.

2. What mechanisms might explain this finding?
There are several possibilities:
a. Mother is a carrier of severe Haemophilia A and the father has haemophilia A so that the affected female inherits an abnormal X chromosome from each parent.
b. Turners syndrome or a Turners mosaic] [i.e. XO or XX/XO.] However, in the case described the karyotype is normal and the affected female appears phenotypically normal.
c. Complete androgen insensitive: In this disorder a lack of androgen receptors can give rise to phenotypically, apparently normal females although genotypically they are XY and so a mutation in the F8 or F9 genes will be evident as there is only one X chromosome.

d. Extreme Lyonisation: Remember inactivation of one X chromosome through a process of methylation, occurs in all female embryos between 64-128 cell stage. Each subsequent cell derived from this original cell retains the same pattern of inactivation. The process of the inactivation is coordinated by a gene known as the Xist gene. Mutations in this gene have been associated with extreme Lyonisation.

This case is designed to illustrate the latter. The mother is a carrier of severe haemophilia A [heterozygous for the F8 intron 1 gene inversion] and her partner is phenotypically normal. However, their daughter has inherited a mutation in the X chromosome from her father with a mutation in the Xist gene that leads to extreme Lyonisation and hence severe haemophilia A as the only functionally active X chromosome is derived from the mother and this contains a F8 gene mutation.

Extreme Lyonisation occurring by a random process is so rare that it is unlikely to provide the explanation in this case. In a female that is heterozygous for a mutation in the F8 or F9 gene, the chances of inactivating the other X chromosome in the 64-128 cell stage that does not contain the mutation, is almost zero.



Question 2
A 45-year-old man is referred for further investigation following the finding of an abnormal coagulation profile. He had contacted his GP having developed easy bruising and epistaxes. His health had previously been excellent apart from a recent chest infection for which he had been prescribed amoxicillin.

Investigations show:

Test Patient Reference Range
PT 45s 11-14s
APTT 79s 23-35s
Fibrinogen (Clauss) 2.9g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s

1. What are the abnormalities and what do you think are the possible explanations.
2. What additional tests would you request?

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1. You repeat the tests and confirm the abnormality.
This excludes the results as being laboratory error.

2. The prolonged PT and APTT do not correct in a mix with normal plasma.
This suggests an inhibitor although it is clearly an unusual inhibitor as both the PT and APTT are prolonged. A lupus anticoagulant [LA] is a possibility although because the reagents employed in the PT contain significant amounts of phospholipid, it is unusual to find a prolonged PT with a LA.

What factor assays would you request and why?
The APTT and the PT are both prolonged. The most relevant factor assays are, therefore Factors V, X, II.
You might also consider measuring several vitamin K dependent factors to establish if this a multiple clotting factor deficiency but remember the PT and APTT do not correct with normal plasma suggesting this is an inhibitor rather than multiple clotting factor deficiencies. Similarly liver disease or afibrinogenaemia might prolong both the PT and APTT but in these cases, the laboratory abnormalities would correct in a mix with normal plasma.


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You request a FV, FX and FII assay.
The FX and FII assays are normal but the FV assay is 3 IU/dl.

What is your diagnosis and why?
This patient has an acquired FV inhibitor secondary to his antibiotic therapy. This is rare and seen only with antibiotics that contain a β-lactam ring. Such patients do not usually bleed and the inhibitor usually disappears when the antibiotic is discontinued.


Question 3
A 23-year-old woman 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) 2.6g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s

1. What questions might you ask this lady that would be of relevance?
2. How would you proceed with the investigation of this patient?

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1. This lady is of Iranian origin. Her parents are first cousins.
The only history of note is that she had a brother who died from an intra-cranial bleed shortly after birth.
2. The prolonged PT and APTT correct in a 50:50 mix with normal plasma.

Remember that a history of menorrhagia may not actually indicate excessive menstrual blood loss. Studies in the UK have shown that assessment of menorrhagia by both patients and clinicians is very subjective and often at variance with measured blood loss. It can be of value to assess menstrual losses more accurately using for example a pictorial bleeding chart. Other evidence of inappropriate haemorrhage should be sought, with particular attention to its’ frequency, nature, onset, precipitants and impact and whether there were any birth complications.

Which factor assays would you request and why?
A careful family history with particular attention to the pattern of inheritance of any bleeding disorder and consanguinity. Remember when interpreting family histories the possibility of non-paternity. The ethnic origin of the patient may be relevant as some diseases are commoner in certain groups (e.g. factor X deficiency in Iranians).
Afibrinogenaemia or a dysfibrinogenaemia could rise to these results but you are told that the Clauss fibrinogen is norma as is the thrombin time, so excluding a functional abnormality of fibrinogen.
Don't forget warfarin or one of the rodenticides that inhibit vitamin K, or true vitamin K deficiency could give rise to these results.

So - as both the PT and the APTT are prolonged and they correct in a mix with normal plasma - you should consider measuring:
 - Factors II, V, X - clotting factors in the 'common' pathway
 - VIII - to exclude combined FV and FVIII deficiency [you have already measured FV]
 - FIX - to exclude a mutation within one of the genes involved in vitamin K metabolism [you have already measured FII and FX]

 

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You request a FX assay - the raw data for a PT-based FX assay is shown below:

 
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

The FX reference standard has a concentration of 100%.

1. What is the diagnosis?
So - you can make a very rough estimate of the FX level that comparing the clotting times for the patient and the reference plasma. A 1/100 dilution of the reference plasma gives a clotting time of 54s whilst a 1/10 dilution of the patient plasma gives a similar time. So the patient is likely to have a FX that is ~10X less than that of the reference plasma i.e. 10%
FX assays can be plotting using either a PT or an APTT-based method. Here you are given the results for a PT-based FX assay.



So the result is that this lady has mild FX deficiency with a FX level of 17%

2. What assays are available to measure FX levels in plasma
There are 5 assays for FX:
 - PT-based method
 - APTT-based method
 - Chromogenic FX assay
 - Russell Viper Venom FX assay
 - Immunological FXI assay



Question 4
A 45-year-old man presents with an extensive above knee DVT extending into the iliac veins. He is otherwise well with no past medical history of note.

His pre-anticoagulation screen shows:

Test Patient Reference Range
PT 14s 11-14s
APTT >120s 23-35s
Fibrinogen (Clauss) 3.2g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s

1. What is the most likely diagnosis?

Click here for Part 2


1. The prolonged APTT corrects in a mix with normal plasma.
2. What factor assays would you request?

The abnormality here is a marked prolongation of the APTT. The age and previous health of the patient make an inherited bleeding disorder unlikely. The history of DVT also makes an acquired bleeding disorder unlikely although not impossible.
The most likely explanation for both the prolonged clotting time and the DVT is an antiphospholipid antibody but FXII deficiency would produce similar findings i.e. a prolonged APTT but no history of bleeding.

FINE PRINT
1. These findings could be unrelated. The patient could have an inherited coagulation factor disorder without haemorrhage (e.g. HMWK, kallikrein, factor XII and factor XI deficiency may prolong the APTT but not produce bleeding) and have had a DVT for an unrelated reason.
2. Another possibility to consider is that the ‘DVT’ is actually a calf muscle bleed and the patient has acquired factor VIII deficiency.

 

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Factor XII:C <1 IU/dl.
Do you think the FXII deficiency is clinically important?

Factor XII deficiency would produce similar clotting results and although heterozygous FXII deficiency is not associated with a thrombophilic tendency, it is possible that severe FXII deficiency may do so. Factor XII can activate fibrinolysis and a deficiency may, therefore, lead to defective fibrinolysis and potentially an increased risk of thrombosis. There is, great debate about the significant of hypofibrinolysis as a risk factor for thrombosis.


If you elect to treat this patient with unfractionated heparin how would you monitor this?

You are, of course unlikely to treat this patient with unfractionated heparin - the only reasons being if the patient have impaired renal function or if you were concerned about an increased risk of bleeding as UFH has a short T½ and in addition its anticoagulant activity can be readily reversed with protamine sulphate,
However, if you do elect to treat this patient with UFH for his DVT then you need to monitor it using an anti-Xa assay which is calibrated for UFH.


Question 5
A 10-day-old baby, previously well, breast fed and born at home is found by his parents unconscious and bleeding from mouth and gums. The only history of note is that the mother had had a major post-partum haemorrhage and had required emergency admission to hospital.

A coagulation screen shows:

Test Patient Reference Range
PT 102s 11-14s
APTT >120s 23-35s
Fibrinogen (Clauss) 1.9g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s

1. What is the most likely diagnosis?
2. How would you confirm this?
3. Why does this occur?

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The obvious abnormalities here are of a grossly prolonged APTT and PT. These could be acquired or inherited abnormalities but given the gross prolongation of both tests but the absence of any birth complications, such as umbilical stump haemorrhage, an acquired disorder is more likely.
This is Vitamin K deficiency. In the 'drama' of the mothers sudden admission to hospital vitamin K was not administered to the child and it was then forgotten.

The levels of vitamin K dependent factors are physiologically low at birth. Factors that contribute to this deficiency include:

  • Low vitamin K stores at birth
  • Poor placental transfer of vitamin K
  • Low levels of vitamin K in breast milk [but not in cows milk]
  • Sterility of the fetal gut

Most commercial infant formulas contain supplemental vitamin K and so vitamin K deficiency associated bleeding is almost exclusively a problem of breast fed infants. Bleeding most commonly occurs from the umbilicus, mucous membranes, GI tract, circumcision sites and venepunctures. Intracranial bleeding is uncommon but is the major cause of mortality and long-term morbidity. Bleeding as a result of vitamin K deficiency may occur any time from birth to several weeks afterwards.

Another possibility is disseminated intravascular coagulation (the fibrinogen is often low in this condition but a low normal fibrinogen does not exclude it) as a result of sepsis. Children of this age may deteriorate very quickly with infection so the history of being previously well does not exclude this.

FINE PRINT
Other possibilities include:
1. A deficiency of factors V, X, or II.
2. Combined factor V + VIII deficiency. However, the PT and APTT are not as prolonged as this results shown above as the levels of FV and FVIII are not completely absent.
3. An inherited deficiency of one of the enzymes involved in the gamma carboxylation of the vitamin K dependent clotting factors. These deficiencies prevent formation of active forms of the vitamin K dependent factors (a similar effect to warfarin) and present very early in life, usually with intracerebral haemorrhage This is rare but frequent in countries where consanguineous marriages are common.


Question 6
The following results were found in a 32-year-old woman as part of a 'Well Woman Screen.'

Test Patient Reference Range
PT 12s 11-14s
APTT 95s 23-35s
Fibrinogen (Clauss) 3.9g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s

1. List additional tests you would perform following the findings shown above to clarify the diagnosis

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The prolonged APTT corrects in a mix with normal plasma.
What is the most likely diagnosis?

The only abnormality is a prolongation of the APTT.

A 50:50 mix of patient and a normal plasma should be tested. If the APTT fully corrects this indicates a factor deficiency. If there is no or only partial correction this suggests the presence of an inhibitor of coagulation.

Individual factor levels. Most laboratories to do not screen for deficiencies of HMWK or kallikrein when investigating a prolonged APTT as they are are not associated with a bleeding disorder. It is sufficient to exclude the clinically significant factor deficiencies.

If a factor deficiency is suggested by the mixing study then individual factors can be assayed. If there is factor VIII deficiency von Willebrand factor assays should be performed, including a factor VIII binding assay (for Type 2N VWD) or genetic studies.

If an inhibitor is suggested then the silica clotting time [SCT] and dilute Russell’s viper venom time [dRVVT] can be employed to detect the presence of an lupus anticoagulant. Inhibitors of individual clotting factors can be assessed by individual factor assays.

This patient has Factor XII deficiency with a level of 5%. It is important to make this diagnosis and to reassure the patient. It is possible that this lady may require surgery in the future and delays can be prevented if the the cause of the prolonged APTT has been established.


Question 7
What might explain the laboratory findings shown below?

Test Patient Reference Range
PT 65s 11-14s
APTT 75s 23-35s
Fibrinogen (Clauss) 0.8g/L 1.5-4.0g/L
Thrombin Time 47s 10-13s

There are multiple causes for these clotting abnormalities:
Liver disease - reduced production of coagulation factors and abnormal fibrinogen production
Hypo/dysfibrinogenaemia - inherited disorders of quantity and function
Hyperfibrinolysis e.g. following administration of streptokinase
Massive blood transfusion without adequate replacement of clotting factors including fibrinogen.


As always it is it the history that is important and this was a patient with a Mallory Weiss tear and who following a major haematemesis had received red cells without either FFP or platelet support.


Question 8
A clotting screen on a woman of Turkish origin shows the following results:


Test Patient Reference Range
PT 35s 11-14s
APTT 56s 23-35s
Fibrinogen (Clauss) 2.8g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s

1. What other tests would you request and why?

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1. The PT and APTT correct in a mix with normal plasma
2. What factor assays would you request?
3. Is this ethnic origin of the lady important?

Causes of the prolonged PT and APTT include:
Deficiencies of Vitamin K
Vitamin K antagonists e.g. warfarin, rodenticides
Liver disease

A careful bleeding, family and drug history is important.
Evidence of inappropriate haemorrhage should be sought, with particular attention to its’ frequency.
Is the patient on any anticoagulants?
Other questions could explore evidence of malabsorption, poor diet or liver disease.

Useful laboratory tests include assay of the levels of Factors V, X, II and liver function tests. If levels of multiple factors are low and administration of vitamin K antagonists is suspected (e.g. Munchausen syndrome with warfarin consumption or self-harm with a rodenticide) then some reference laboratories can assay levels of PIVKAs (proteins induced by vitamin K absence) which may help with the diagnosis.

This patient had Factor X deficiency.

FINE PRINT
Other rare possibilities include:
a. 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.
b. An deficiency of gamma carboxylase or vitamin K epoxide reductase leading to a reduction in all the vitamin K dependent clotting factors.

Finally - the ethnic origin of the patient is clearly important as some recessive disorders are more frequent in countries where consanguineous relationships are common.


Question 9
A 56-year-old woman with a suspected carcinoma of the bowel is admitted for surgery. A pre-operative clotting screen shows:

Test Patient Reference Range
PT 14s 11-14s
APTT 76s 23-35s

1. What other tests would you request and why?

Click here for Part 2

Clinical details are very helpful in evaluating cases like this and further information (e.g. bleeding history, heparin exposure, previous investigations) should be sought if possible.

A 50:50 mix of patient and normal plasma is a useful initial test. An APTT is performed on this mix and if the clotting time is normal or near normal (within 4s of the control) then a factor deficiency is likely. If there is no or little correction then an inhibitor of coagulation e.g. lupus anticoagulant, or an acquired factor inhibitor is present.

If there is a possibility that the patient is receiving unfractionated heparin or if it is unclear if the patient has been exposure to unfractionated heparin status, then a thrombin time/reptilase time can be performed [A phone call maybe easier to perform to clarify if the patient is on unfractionated heparin.] If this is normal then exposure to unfractionated heparin is not the cause of the prolonged APTT. If the thrombin time is prolonged then a reptilase time should be performed. If this is normal it confirms unfractionated heparin as the cause of the prolonged APTT. An alternative is to add a heparin neutralising agent to the plasma (e.g. Hepzyme) and repeat the APTT. Again, a normal result confirms unfractionated heparin as the cause of the prolonged APTT.
[The TEG machines includes Heparinase-coated cups that remove any influence that UFH may make to the coagulation profile. This can be very useful in monitoring patients on cardio-pulmonary bypass receiving UFH and in whom the underlying coagulation status needs to be established.]

Individual factor levels. Most laboratories to do not screen for deficiencies of HMWK or kallikrein when investigating a prolonged APTT as as they are are not associated with a bleeding tendency and the deficient plasmas are expensive to purchase. It is sufficient to exclude the clinically significant factor deficiencies.

If an inhibitor is suggested but heparin is excluded then the silica clotting time [SCT] and dilute Russell viper venom time [dRVVT] can be used to detect the presence of a lupus anticoagulant. Inhibitors of individual clotting factors can be assessed by individual factor assays.

Investigations in this patient showed the presence of a lupus anticoagulant.

The surgeons are concerned that this lady will bleed during surgery because of this. What will you tell them?

If the PT and platelet count are normal, then causes of bleeding associated with the lupus anticoagulant have been excluded and the patient should not bleed. Occasionally Lupus anticoagulants can be associated with a low factor VIII and factor assays are important to exclude this. If the tests show normal factor assays, the surgeons should be reassured and the patient should receive pharmacological thromboprophylaxis with for example a low molecular weight heparin.


Question 10
A 23-year-old woman develops a DVT. There is no other personal or family history of note. She is not on the oral contraceptive pill.

Test Patient Reference Range
PT 14s 11-14s
APTT 95s 23-35s

1. Comment upon the results of the PT and APTT.
2. What tests would you request next?

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1. Results show the presence of a lupus anticoagulant.
2. Briefly outline the tests you would perform to establish the presence or absence of a lupus anticoagulant.

Demonstration of the presence of an antiphospholipid antibody by coagulation tests requires:
a) Prolongation of a phospholipid dependent coagulation test AND
b) Correction of that prolongation by adding an excess of phospholipid or otherwise eliminating the effect of any APL OR
c) comparison to a phospholipid independent confirmatory test.

When screening for a lupus anticoagulant, two tests based on different principals should be used. Currently the ISTH SSC working party on antiphospholipid antibodies recommends the Silica Clotting Time [SCT] and the dilute Russell Viper Venom Time [dRVVT].


Question 11
A 53-year-old man is admitted unconscious to casualty. His coagulation results are shown below:

Test Patient Reference Range
PT >120s 11-14s
APTT >120s 23-35s
Fibrinogen (Clauss) 2.01g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s
1. What is the most likely explanation for these findings?

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He has a midline sternotomy scar and has been taking warfarin for many years.
These results are consistent with someone who has an overdose of warfarin either intentionally or unintentionally.

1. How does warfarin work?
Warfarin is a racemic mixture of two optically active isomers - the R and S forms. Normally, vitamin K is converted to vitamin K epoxide in the liver. This epoxide is then reduced by the enzyme vitamin K epoxide reductase. The reduced form of vitamin K epoxide is necessary for the synthesis of factors II, VII, IX and X, as well as Proteins C & S. Warfarin inhibits the enzyme vitamin K epoxide reductase in the liver and so prevents/limits gamma carboxylation of the vitamin K dependent factors and inhibits coagulation from occurring. Although one might predict that the immunological levels of the vitamin K dependent clotting factors would be normal, in fact these are reduced and it seems probable that the partially carboxylated forms of the the vitamin K dependent clotting factors have a shorter half-life.

2. How do we monitor warfarin and why do we use this test?
Warfarin is monitored using the INR [International Normalised Ratio]. The International Normalised Ratio (INR) is the PT ratio of a test sample compared to a normal PT (derived from the log mean normal prothrombin time (LMNPT) of 20 normal donors) corrected for the sensitivity of the thromboplastin [Tissue Factor] used in the test.
It is the Prothrombin Time Ratio obtained using the first WHO Reference Thromboplastin which was assigned an ISI of 1.0.
The use of the INR corrects for any differences in sensitivities that the sources of Tissue Factor used in the PT may have.

3. How would you address the coagulation abnormalities in this patient?
This patient is unconscious and the concern is that he has had an intracranial bleed. He should receive rapid reversal of his presumed warfarin toxicity with a prothrombin complex concentrate [PCC] together with iv vitamin K. Fresh Frozen Plasma [FFP] is an inefficient method for reversing warfarin and should not be used unless PCCs are not available.


Question 12
Comment upon the following results in an 18-year-old asymptomatic individual who is admitted with acute appendicitis.

Test Patient Reference Range
PT 45s 11-14s
APTT 46s 23-35s
Fibrinogen (Clauss) 0.8g/L 1.5-4.0g/L
Thrombin Time 45s 10-13s
Reptilase Time 51s 11-14s

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1. What additional assay would you request and why?

You might request a fibrinogen antigen to confirm the presence of a dysfibrinogenaemia.


Question 13
A 28-year-old woman with Crohn's disease is found to have the following clotting abnormalities:

Test Patient Reference Range
PT
>120
10.6-12.4s
APTT
105s
21-32s
Fibrinogen (Clauss) 1.9g/L 2-4g/L
Inhibitor Screen
 Immediate Mix
 2 hour incubation

29s
28.3s
 
1. What additional tests would you request?

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1. You repeat the tests and confirm the abnormality.
2. You request 50:50 mix correction tests - the results of which are shown below:

Test Patient Reference Range
PT
50:50 Mix NP
>120s
14.2s
10.6-12.4s

APTT
50:50 Mix NP
107s
27.7s
21-32s

Fibrinogen (Clauss) 1.9g/L 2-4g/L

What factor assays would you request and why?

The possibilities include either Vitamin K deficiency, a defect in the common pathway e.g. FII, V or X deficiency, or a possible combined FV and FVIII deficiency. The history of Crohn's disease should raise the possibility of vitamin K deficiency.

 

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Below are the results of the factor assays you may have requested.

Factor Assay Patient Reference Range
FII <0.01 IU/mL 0.5 - 1.5 IU/mL
FV 1.13 IU/mL 0.5 - 1.5 IU/mL
FVII <0.01 IU/mL 0.63 - 1.71 IU/mL
FIX <0.01 IU/mL 0.55 - 1.58 IU/mL
FX <0.01 IU/mL 0.59 - 1.69 IU/mL
FXI 0.74 IU/mL 0.70 - 1.50 IU/mL

What is the most likely diagnosis?

1. Vitamin K deficiency
2. An inherited deficiency of one of the enzymes involved in the gamma carboxylation of the vitamin K dependent clotting factors. These deficiencies prevent formation of active forms of the vitamin K dependent factors (a similar effect to warfarin) and present very early in life, usually with intracerebral haemorrhage.
3. Patient is taking warfarin or a warfarin-like drug.

Why is liver disease an unlikely diagnosis?
The Factor V assay is normal as is the FXI assay and both are synthesised in the liver. FV is also found in platelets but the factor V in platelets is derived from the plasma FV.

This patient had vitamin K deficiency secondary to malabsorption as a consequence of her Crohn's disease.


Question 14
A 22-year-old man is admitted for an arthroscopy of his right knee. He has no previous medical history of note but investigations showed the following:


Test Patient Reference Range
PT 13s 11-14s
APTT >120s 23-35s
Fibrinogen (Clauss) 2.7g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s
1. What is the most likely diagnosis?

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1. You suspect a contact factor deficiency as there is no history of note but request the following:

Factor VIII, IX, XI and XII assays - Normal .
Lupus anticoagulant screen - Negative.


What is your diagnosis?

Contact Factor Deficiency e.g. Pre-Kallikrein Deficiency. The APTT can be made more sensitive to a contact Factor deficiency by reducing the incubation time e.g. to 2 minutes than 5 minutes of 10 minutes.
It would also be of value to repeat the APTT to ensure it was not a problem with how the test was performed!


Question 15
A 23-year-old woman is 34 weeks pregnant and found to have the following results:

Test Patient Reference Range
Hb 10.9 g/dL 13.5 -16.5g/dL
WCC 9.7 x 109/L 4 - 11 x 109/L
Platelets 98 x 109/L 150 - 400 x 109/L
MCV 94 fL 80 -98fL
MPV 12.1 fL 7.5 - 11.5 fL
1. What do you think the most likely diagnosis is?
2. Are there are additional tests you might request?

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In a pregnant woman in the last trimester of pregnancy, the most likely diagnosis is Gestational Thrombocytopaenia [GT]. The platelet count is perhaps lower than one might expect to see in GT and therefore the following might be sensible tests to consider:
1. Repeat platelet count + film to look for platelet clumping and red cell fragments
2. Platelet count in a citrate-containing anticoagulant rather than EDTA as EDTA can cause in vitro platelet clumping in some individuals
3. You might consider performing an ANA, ENA and a screen for anti-phospholipid antibodies. If the ENA is positive it important to screen for anti-Ro and anti-La antibodies which can have implications for the baby.
4. It would also be sensible to check renal and liver function; to check the urine and BP.


Finally - you should monitor the platelet count at least every 2 weeks and ensure that if returns to normal after delivery. If the platelet count count falls to <80 then an immunological platelet count can be useful as impedance methods for counting platelets may underestimate the true platelet count when the MPV is raised as in this case.


Don't forget to look and see if the platelet count was low in a previous pregnancy if this data is available.



Question 16
A 23-year-old woman is 9 weeks pregnant and admitted with a miscarriage. Her investigations show:

Test Patient Reference Range
PT 21s 11-14s
APTT 39s 23-35s
Fibrinogen (Clauss) 0.42g/L 1.5-4.0g/L
Thrombin Time 35s 10-13s
Reptilase Time 43s 11-14s
1. Comment upon the results of these findings.
2. What additional tests would you request?

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You suspect DIC but when she has recovered her clotting abnormalities are essentially unchanged. The platelet count is normal.

Fibrinogen antigen may be a test to consider.


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You request a Fibrinogen Antigen which is reported as 3.4g/L.
1. What is the diagnosis.
2. What might you consider in a future pregnancy?

In fact this lady has a long history of recurrent miscarriages secondary to hypofibrinogenaemia. In a future pregnancy you should consider supplementing the pregnancy with fibrinogen.

Don't forget to construct a family pedigree and screen other 'at risk' family members.


Question 17
An 18-year-old woman with nephrotic syndrome is admitted for a renal biopsy. Shown below are the results of a coagulation screen.

Comment upon the results of these tests and what do you think may be the explanation?

Test Patient Reference Range
PT 13s  
APTT 35s  
Fibrinogen (Clauss) 3.8 g/L  
Thrombin Time 23s  
Reptilase Time 25s  

1. Comment upon the results of these findings

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1. These results are consistent with hypo-albuminaemia.

The prolongation of the Thrombin Time and Reptilase Time in the context of a low serum albumin was first reported with nephrotic syndrome but was subsequently found in patients with HIV.


2.How could you confirm that these abnormalities are due to the low albumin?

You can supplement the plasma with albumin and see if this corrects the clotting abnormalities [which if it is due to a low serum albumin it will.] You can also correct the low albumin levels in the patient and see if the clotting abnormalities correct [they usually do.]

3. What advice would you give to the nephrologists regarding the risk of bleeding associated with these abnormalities and what pre-biopsy treatment would you advise?

This is an acquired abnormality and affected patients do no usually bleed and in fact may have hyper aggregable platelets. The advice to the nephrologists is to do nothing - she does not require any treatment prior to her biopsy
.


Question 18
A 25-year-old woman is admitted through A & E with a 5-day history of easy bruising. Her admission was precipitated by a grand mal convulsion which she suffered at home.

Test Patient Reference Range
Hb 7.6 g/dL 11.5-13.5g/dL
WCC 11.9 x 109/L 4 -11 x 109/L
Platelets 10 x 109/L 150 - 400 x 109/L
PT 13s 11-14s
APTT 35.6s 23-35s
Fibrinogen 2.1g/L 1.5-4.0g/L

1. What is you differential diagnosis?
2. What additional tests would you request?

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1. A repeat FBC confirms these abnormalities
2. The LDH is elevated at 2389 U/dL
3. The Troponin T is raised
4. The creatinine is raised at 389 µmol/mL
5. A blood film is shown below:



What is the diagnosis?

The most likely diagnosis is Thrombotic Thrombocytopaenia Purpural [TTP].

Are there any additional tests you would request?

1.You should measuring the ADAMTS13 activity and screening for the presence of an inhibitor to ADAMTS13 i.e. an autoantibody.


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The ADAMTS13 activity results are shown below:

Test Patient Reference Range
ADAMTS13 Activity <5% 65-125%
ADAMTS13 Inhibitor Assay 28 AU/mL <11 AU/mL


What does this suggest?

These results are consistent with a diagnosis of TTP secondary to the presence of an inhibitory antibody to ADAMTS13. The ADAMTS13 activity is extremely low and there is evidence of an inhibitory antibody.
Remember - there are two types of antibodies seen in TTP. The first is an inhibitory antibody that as the name suggests inhibits the function of the metalloprotease ADAMTS13 whilst the second is a non-inhibitory antibody which accelerates the clearance of ADAMTS13.


What are the principles of treatment in this disorder?

1. Plasma exchange with fresh frozen plasma to remove the autoantibody and replace ADAMTS13.
2. High dose steroids to suppress the inhibitor antibody. Rituximab has been shown to be of value in preventing relapses in TTP.
3. The major cause of death in TTP is due to cardiac arrhythmias. Individuals with an elevated LDH and Troponin level should be monitored in a Coronary Care or Intensive Care Unit.


Question 19
Briefly outline the roles for the following snake venoms:

Venom Mode of Action
Botrocetin Botrocetin is a snake venom isolated from Bothrops jararaca which alters the conformation of VWF and increases it affinity for GpIb. The Botrocetin assay of VWF is similar to that of the ristocetin cofactor assay but some patients have been reported with 100% botrocetin cofactor activity and 0% ristocetin cofactor activity.
Ristocetin Ristocetin is an antibiotic obtained from Amycolatopsis lurida but was found to cause thrombocytopaenia and so removed from the market. It causes thrombocytopaenia by inducing platelet agglutination but only in the presence of VWF.
Russell Viper Venom Isolated from Daboia russelii and is a specific activator of factors V and X. It is used the dRVVT as a screening test for a Lupus anticoagulant.
Textarin Textarin, a protein fraction of Pseudonaja textilis venom (Australian Eastern brown snake), activates prothrombin in the presence of PL, factor V and calcium ions.
Ecarin Isolated from Echis carinatus and is a Prothrombin activator [generates meizothrombin]. Ecarin will activate prothrombin in the absence of any cofactors. It forms the basis for the assay of direct thrombin inhibitors and in addition forms the basis for a screening test for a lupus anticoagulant.
Botox Botulinum toxin is a protein produced by the bacterium Clostridium botulinum, and is extremely neurotoxic. It is used medically to treat a number of disorders but its major role appears to be for cosmetic purposes. It does not as far as we are aware have any role in the haemostasis lab.
Ancrod A defibrinogenating agent derived from the venom of the Malayan pit viper [Agkistrodon rhodostoma also called Calloselasma rhodostomar.] It also has an action on platelet function.
Protac Isolated from Agkistrodon contortrix and is a specific activator of Protein C to Activated Protein C [APC]. It forms the basis for the chromogenic PC assay and is also used in a functional PS assay.
Reptilase Isolated from Bothrops atrox and is an activator of fibrinogen. It is unaffected by the presence of unfractionated heparin [in contrast to the thrombin time] and so is often used to establish the presence of UFH in a sample with a prolonged APTT.


Question 20
A 3-month old baby is admitted to hospital unconscious. A CT scan shows a large intra-cerebral bleed. The results of clotting tests are shown below:

Test Patient Reference Range
PT 14s 11-14s
APTT 32.8s 23-35s
Fibrinogen 3.1g/L 2-4 g/L
FXIII screen [5M urea] Control: No lysis at 24 hours
Patient: Lysis at 24 hours
1. Comment upon the results of these tests?
2. What questions might you ask the parents?

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1. The parents are first cousins and were born in North Africa.
2, Their first child died shortly after birth from cause unknown. They have two other children with no apparent problems.
3. What additional tests might you request?
The FXIII screen is abnormal with clot lysis at <24 hours. This is highly suggestive of FXIII deficiency and in this situation you must request a FXIII assay.


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You request a FXIII:B ELISA which shows a value of 110 U/dL [Reference Range: 60-130 U/dL].

Are there any additional tests you would request?

You must request as FXIII:A ELISA or functional FXIII assay. FXIII:B is a carrier for XIII:A and so the assay can be normal in some cases of severe FXIII.
In fact this child had severe FXIII deficiency. Treatment was given with FXIII concentrate to correct the abnormality. There is a high incidence of intracranial haemorrhage in FXIII deficiency and such children must start prophylactic treatment with FXIII concentrate soon after delivery.


Question 21
A 47-year-old male is admitted for a partial nephrectomy. His pre-operative screen shows:

Test Patient Reference Range
PT 12.5s 11-14s
APTT 42s 23-35s
Fibrinogen 3.4 g/L 2-4g/L
PFA-100 Collagen-ADP Cartridge: Normal closure times 85-113s

1. What additional tests would you request?

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1. You request FVIII, FIX, FXI and Von Willebrand Factor assays.

The FVIII assay is 6.2 IU/dL and this confirmed on repeat testing.
VWF Assays [VWF:Ag and VWF:Act] are normal.

2. What is the diagnosis and how would you manage his surgery?

Don't forget you should exclude Type 2N VWD which historically was misdiagnosed as mild Haemophilia A. In fact this patient has mild Haemophilia A. In view of the fact that he has mild HA but requires prolonged treatment, DDAVP is inappropriate and he should receive a recombinant FVIII concentrate.


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1. You diagnose mild Haemophilia A and elect to treat him with a recombinant FVIII to cover his surgery rather then DDAVP.

Don't forget you should exclude Type 2N VWD which historically was misdiagnosed as mild Haemophilia A.

The surgery is uneventful and after 5 days his rFVIII is stopped and he is sent home. He represents some 3 weeks later with widespread subcutaneous bruising on his arms and legs which came on suddenly with no history of trauma.

Investigations show:

Test Patient Reference Range
PT 12.5s 11-14s
APTT 133s 23-35s
Fibrinogen 3.4 g/L 2-4g/L
FVIII:C < 1 IU/dL 50-150 IU/dL

1. What is the most likely explanation for these findings?

He has developed a FVIII inhibitor in response to his FVIII replacement therapy that was used to cover his surgery. The inhibitor antibody induced by the exogenous FVIII has cross-reacted with his own endogenous FVIII and converted his phenotype from that of an individual with mild HA to to a severe HA.


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1.You suspect he has an inhibitor which is confirmed. He has a titre of 12 Bethesda Units [Bu]. How will you manage him?

Therapeutic options include rVIIa or FEIBA until his bleeding has stopped and healing is complete. It is probable that in the absence of any exogenous FVIII, his inhibitor level will fall and his own endogenous FVIII levels will rise.

2. If he requires surgery again in 2 years how would you manage him?
This is difficult and depends upon the nature of the surgery. If he requires minor surgery then DDAVP ± Tranexamic acid is a possibility. For more invasive surgery you may elect to treat him with FVIII for a short period of time recognising that his inhibitor will return and you will then require to switch him to an alternative treatment such as rVIIa. FEIBA is a possibility but does contain small amounts of FVIII:Ag and may precipitate the return of his inhibitor. You may elect to treat him at the outset with rVIIa to prevent the recurrence of his inhibitor and a change in his phenotype.





Question 22

A 24-year-old student presents to the Emergency Department with a short history of easy bruising and frank haematuria. On examination he has widespread bruises but there is little else to find.
There was no past medical history or family history suggestive of a bleeding disorder.

His blood tests show:

Test Patient Reference Range
PT >120s 11-14s
APTT >120s 23-35s
Fibrinogen (Clauss) 3.2g/L 1.5-4.0g/L
Thrombin Time 13s 10-13s
Hb 8.9 g/dL 11.5-13.5g/dL
Platelets 256 x 109/L 150-400 x 109/L

1. What questions might you ask this student?
2. What are the possible diagnoses?

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1. He does not admit to taking any anticoagulant drugs. His diet is good and he has not been abroad recently.
2. The prolonged PT and APTT correct in a mix with normal plasma.

3. What factor assays would you request?

 

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His vitamin K dependent clotting factors [II, VII, IX & X + Proteins C and S] are all <5% of normal.

What does this make you think?

This is strongly suggestive of vitamin K deficiency or ingestion of a vitamin K antagonist such as warfarin.

 

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On further questioning he admits that he have 'taken' some rat poison a few days earlier but he is not too sure how much.

How does rat poison affect blood clotting and outline the principles of management of such a case?
Rodenticides are long-acting vitamin K antagonists.

Brodifacoum is frequently found in rat poisons [rodenticides] and is a 4-hydroxycoumarin anticoagulant, with a mode of action similar to that of warfarin. However, in contrast to warfarin and due to to its potency and long T½, it is known as a 'superwarfarin.' Brodifacoum may also increase the permeability of blood vessels which exacerbates the bleeding tendency. Initial treatment of such patients requires replacement of the depleted clotting factors [Prothrombin complex concentrate or if not available FFP] together with large doses of vitamin K. Correcting the coagulation disorder with a PCC may not improve the low levels of PC and PS and may make the patient thrombogenic.
Vitamin K is often needed in large doses for prolonged periods of time to correct the coagulation abnormalities.

The estimated average fatal dose of Brodifacoum for an adult man is about 15mg [without treatment.]



Question 23

A 6-year-old boy is brought to the Emergency Department having dropped a book onto his foot and which resulted in a large bruise. His parents commented that he seemed to bruise more easily than other boys of his age and more so that than their other son.

His blood tests show:

Test Patient Reference Range
PT 15.2s 11-14s
APTT 79s 23-35s
Fibrinogen (Clauss) 2.9g/L 1.5-4.0g/L
Thrombin Time 12.1s 10-13s
Platelets 319 x 109/L 150-400 x 109/L

1. What questions might you ask this student?
2. What are the possible diagnoses?

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1. There was no past medical history of note and no family history suggestive of a bleeding disorder. The parents are not related.

2. You request VIII, IX, XI and Von Willebrand Factor assays - the results of which are shown below.

Factor Patient Reference Range Comments
FVIII 0.92 IU/mL 0.57 -1.40 IU/mL Non-parallel
FIX 0.67 IU/mL 0.7 - 1.55 IU/mL Non-parallel
FXI 0.87 IU/mL 0.72 - 1.52 IU/mL Non-parallel
VWF:Ag 0.65 IU/mL 0.53 - 1.49 IU/mL  
VWF:Act 0.63 IU/mL 0.53 - 1.49 IU/mL  


What additional tests would you request and why?
The next most logical tests are:
1. Screen for a lupus anticoagulant.
2. You could also measure the 'common' pathway clotting factors - II, V and X in view of the prolonged PT and APTT. However note the PT is only marginally prolonged.

 

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You request a screen for a lupus anticoagulant - the results of which are shown below:

Test Patient Reference Range
dRVVT ratio 2.72 <1.20
Phospholipid Correction 44.5% <15%
SCT ratio 1.78 <1.24
Phospholipid Correction 19.5% <15%

What do you think the diagnosis is?
These tests are consistent with a lupus anticoagulant.

Are there any additional tests you might request (clearly the answer is yes!!) and why?
You should consider measuring the Factor II [Prothrombin] levels.

 


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In view of the prolonged PT and APTT you request a factor II assay which is: 0.36 IU/mL [Reference Range: 0.53 - 1.38 IU/mL].

What would your final diagnosis be?
Lupus anticoagulant with probable acquired Factor II deficiency. It is possible that the low Factor II levels are not secondary to the lupus anticoagulant but an inherited disorder. This could be confirmed by sequencing the Factor II [F2] gene for a mutation or observing and seeing if the Factor II levels improve if the lupus anticoagulant disappears. Remember lupus anticoagulants are common in children.

Do these findings provide an explanation for the child's history of easy bruising?
Possibly - although the Factor II levels are not particularly low.


Question 24

A 6-month-old boy is admitted to hospital with a short history of vomiting and lethargy which had started 24 hours previously. A CT scan shows evidence of a large subdural haematoma with midline shift.

His blood tests show:

Test Patient Reference Range
PT 38s 11-14s
APTT 109s 23-35s
Fibrinogen (Clauss) 2.9g/L 1.5-4.0g/L
Platelets 160 x 109/L 150-400 x 109/L

1. What questions might you ask the parents?
2. What are the possible diagnoses?

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1. There was no past medical history of note and no family history suggestive of a bleeding disorder.
The parents are first cousins.

2. You request a series of factor assays based upon the PT and APTT data - the results of which are shown below:

Factor Patient Reference Range
FII 0.08 IU/mL 0.57 -1.40 IU/mL
FVII 0.22 IU/mL 0.7 - 1.55 IU/mL
FIX 0.28 IU/mL 0.72 - 1.52 IU/mL
FX 0.15 IU/mL 0.53 - 1.49 IU/mL

These results suggest:
1. Vitamin K deficiency
2. A mutation in the VKORC1 gene leading to a deficiency in all the VK-dependent clotting factors

3. Liver disease although the fibrinogen is normal.

What additional tests would you request and why?
A Factor V assay would be useful as it is synthesised by the liver but not VK-dependent.

 

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You request a factor V assay which is 0.79 IU/mL [Reference Range: 0.57 -1.40 IU/mL].

You might also measure the levels of other Vitamin K dependent factors [Protein C, Protein S, Protein Z] - which you would find are also low.

The parents are from Iran and are first cousins.

What would explain these findings?
These results are consistent with a deficiency of Vitamin K or a mutation in the VKORC1 gene.

How would you treat this child?
1. A Prothrombin complex concentrate [PCC] will rapidly correct the low levels of the VK-dependent clotting factors. Fresh Frozen Plasma [FFP] could be given if a PCC was not available and in practice is likely to be given first.

2. This should be given together with i.v. Vitamin K

 

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The child has no evidence of malabsorption and appears well nourished.
Liver function tests are normal.

The parents refuse to allow you to take a blood sample from them to perform any clotting factor assays.

You elect to treat the child with Fresh Frozen Plasma [FFP] + Vitamin K and the clotting factor abnormalities return to normal. However, 3 weeks later the child is noted to be bruising easily and repeat clotting factor assays are similar to those at presentation.

What do you think the diagnosis is?
The diagnosis is likely to be a mutation in the VKORCI gene leading to a deficiency of all the vitamin K-dependent proteins.

How will you mange this child in the long-term?
Vitamin K which often has to be given in large doses and frequently.


What else might this child show as a consequence of this disorder?

Such patients often show other non-haemostatic symptoms due to defective γ-carboxylation of proteins other than the clotting factors. Developmental and skeletal abnormalities resembling those seen in warfarin embryopathy and include stippling of the long bone epiphyses and shortness of finger distal phalanges .





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