Data Interpretation:
Screening Tests - Answers

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

A. What other questions might you ask the parents?
B. Briefly outline how you would investigate the clotting abnormalities.

 

Answers for Question 1

The only abnormality is the marked prolongation of the APTT. This suggests a disorder of the ‘intrinsic pathway’, i.e. HMWK, Kallikrein and factors XII, XI, IX, VIII (See APTT))  The age of the child and the history of bruising must raise suspicion of an inherited bleeding disorder. One should also consider the possibility of non-accidental injury [NAI] although in this case there is an explanation for the bruising.

A. Questions to ask include:

    1. Other evidence of inappropriate haemorrhage should be sought, with particular attention to its frequency, nature, onset, precipitants, site and impact and whether there were any birth complications (e.g. umbilical stump bleeding - this is not unique to FXIII deficiency.)
    2. A careful family history with particular attention to the pattern of inheritance of any bleeding disorder. This is useful to determine the inheritance pattern (e.g. X linked recessive for factor IX or VIII deficiency, autosomal recessive for Type 3 von Willebrands disease).
    3. Whether the parents are consanguineous. Consanguinity increases the likelihood of an inherited disorder.
    4. The ethnic origin of the patient may be relevant as some diseases are commoner in certain groups e.g. Factor XI deficiency in Ashkenazi Jews.

      Remember when interpreting family histories to consider the possibility of non-paternity. Studies have shown an incidence as high as 10% in Western society so any given family history may not be wholly relevant to the child.

B. How to investigate further

  1. A 50:50 mix of patient and known 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.
  2. Individual factor levels. Most laboratories to do not screen for deficiencies of HMWK or kallikrein when investigating a prolonged APTT as as they are considered to have limited clinical significance. Varying the incubation times in the APTT can make the APTT more or less sensitive to deficiencies of HMWK or kallikrein or FXII and may suggests a deficiency of these factors. They are not associated with a bleeding tendency and so can be excluded in this case.
    It is sufficient to exclude the clinically significant factor deficiencies.
  3. If there is factor VIII deficiency then von Willebrand factor assays should be performed, including a factor VIII binding assay (for Type 2N VWD). In the 21st Century it is often easier to undertake a screen of the VWF gene for mutations associated with Type 2N VWD although mutations associated with 2N VWD have been reported in a number of exons within the gene [see VWF Mutational Database].
  4. If there is a deficiency of factor VIII or IX then the child’s karyotype should be established as Haemophilia A or B in a female may indicate Turner’s syndrome (46, XO) or a Turner's Mosaic (46, X0, XX).
  5. Other possible explanations for this finding are extreme Lyonisation. In such cases the mother is usually a carrier of an abnormal F8 or F9 gene and the father carries a mutation in his X-chromosome that is lethal to all cells that express it. Therefore, only cells that express maternal F8 gene survive.
    A mutation within the Xist gene has been reported to be associated with extreme Lyonisation in some females with severe haemophilia A.
  6. Inheritance of an abnormal gene from each parent i.e. the father has Haemophilia A or B and the mother is a carrier.
  7. Complete androgen sensitivity can be associated with haemophilia in a phenotypically normal female. Such individuals have an XY genotype but as they lack the receptor for androgens, they appear female. If their X chromosome contains a mutation within the F8 or F9 gene then they can have haemophilia even though they appear phenotypically female.
  8. A full blood count should also be performed to determine if sufficient haemorrhage has occurred to cause anaemia and to verify that the other significant element of haemostasis, the platelet count, is normal.
  9. Antiphospholipid antibodies e.g. a Lupus anticoagulant [LA] may cause an isolated prolonged APTT and this has been reported in children. The history of bruising suggests a bleeding disorder and although children of this age do bruise naturally as they are active but have not fully developed motor co-ordination.
    LAs have been associated with bleeding due either to thrombocytopaenia or to low prothrombin [Factor II] levels - this is rare.

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

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

Answers for Question 2

The abnormalities here are of a prolonged APTT and PT. The age of the patient and previous good health makes an inherited bleeding disorder unlikely. The most likely explanation for these findings is, therefore, an acquired disorder.

  1. A careful history and examination should be undertaken. As always the history is of utmost importance!
  2. Warfarin administration, vitamin K deficiency (e.g. diet, malabsorption) and liver disease can all produce the abnormalities given. The patient’s illness could compromise anticoagulant control and amoxicillin may potentiate the effect of warfarin. The PT is more prolonged than the APTT and this makes warfarin less likely than other possible causes for these clotting abnormalities.
  3. If the conditions listed above are excluded then deficiencies of Factors V, X, or II should be considered and can be assayed. Some haematologists would perform mixing studies with equal proportions of patient and known normal plasma before individual factor assays. If the APTT or PT correct then this indicates a factor deficiency.
  4. In fact this patient has an acquired Factor V inhibitor secondary to his antibiotics. FV inhibitors are rare but have been reported in association with β-lactam containing antibiotics In general these inhibitors are not associated with major bleeding problems and resolve when the drug withdrawn.

Fine print:
Massive transfusion could also produce these coagulation abnormalities though the fibrinogen is often also reduced and the clinical scenario does not suggest this.

Antiphospholipid antibody, the development of which may follow infective episodes, can rarely cause acquired prothrombin deficiency which would produce the coagulation abnormalities and bleeding noted.

There is one other very rare condition to consider - combined factor V + VIII deficiency. Only approximately 100 cases have been reported but the phenotype may be very mild and present later in life.

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?


Answers for Question 3

The abnormalities here are of a prolonged APTT and PT. The age of the patient and the fact no comment is made on previous bleeding history leave open the possibility of either an acquired or inherited disorder.

a) A careful bleeding, family and drug history are essential.

  1. 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.
  2. 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)
  3. Is the patient on any anticoagulants?
  4. Other questions could explore evidence of malabsorption, a poor diet or liver disease.
  5. Clotting factor abnormalities to explain these findings include, Factor X deficiency, Factor V deficiency, Combined FV and FVIII deficiency, Factor II [Prothrombin] deficiency. A deficiency in one of the genes involved in Vitamin K metabolism could also lead to this phenotype.
    Many haematologists would perform mixing studies with equal proportions of patient and known normal plasma before individual factor assays. If the APTT or PT correct then this indicates a factor deficiency.

This lady is of Iranian extraction and her parents are first cousins. She has severe Factor X deficiency with a homozygous mutation within her F10 gene. Both her parents are heterozygous for the same mutation.

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

A. What are the possible explanations for these findings?

Answers for Question 4

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.

a. The most likely explanation for both the prolonged clotting time and the DVT is an antiphospholipid antibody i.e. a lupus anticoagulant which is what his patient had.

b. 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.

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.
3. Alternatively, the patient could have factor XI, IX or VIII deficiency (accounting for the prolonged APTT) and have had excessive replacement therapy (e.g. prior to operation or following trauma) which resulted in transient high coagulation factor levels and thrombosis


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 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

A. What is the most likely explanation for these findings?
B. Why does this occur?

Answers to Question 5

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 slightly more likely.

a) This is Vitamin K deficiency. In the drama of 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:

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 (e.g. overnight) 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 and not as prolonged as this as the levels of FV and FVIII are not completely absent.

3. An inherited deficiency of gamma carboxylase or vitamin K epoxide reductase. The deficiencies of gamma carboxylase or vitamin K epoxide reductase 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 commoner in countries where consanguineous marriage is found.

 

Question 6

The following results were found in a 32-year-old woman as part of a Well Woman Screen. There was no personal or family history suggestive of a bleeding disorder.

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

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

 

Answers to Question 6

The only abnormality is a prolongation of the APTT.

  1. A 50:50 mix of patient and known 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.
  2. Individual factor levels. Most laboratories to do not screen for deficiencies of HMWK or kallikrein when investigating a prolonged APTT as as they are considered to have clinical significance. It is sufficient to exclude the clinically significant factor deficiencies.
  3. If a factor deficiency is suggested by the mixing study then individual factors can be assayed. It is sufficient to exclude the clinically significant factor deficiencies. If there is factor VIII deficiency von Willebrands factor assays should be performed, including factor VIII binding assay (for Type 2N VWD).
  4. If an inhibitor is suggested then a number of tests such as the silica clotting time and dilute Russell’s viper venom time (DRVVT) can be employed to detect the presence of an antiphospholipid antibody. Inhibitors of individual clotting factors can be assessed by individual factor assay and further mixing studies with known quantities of factors VIII, IX or XI.

This patient has Factor XII deficiency with a level of 5%.

 

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

 

Answers to Question 7

There are multiple causes for these clotting abnormalities:


Question 8

A clotting screen on a woman of Iranian descent 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

A. What coagulation abnormalities might explain these findings.
B. What additional tests would you perform?

 

Answers to Question 8

a) Causes include :

b) A careful bleeding, family and drug history is important.

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) then some reference laboratories can assay levels of PIVKAs (proteins induced by vitamin K absence) which may help.

This patient had factor X deficiency.

Fine print:
Other rare possibilities include:

 

Question 9

A 56-year-old woman with a colonic carcinoma is admitted for surgery. A pre-operative clotting screen shows:

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

What other tests would you request and why?

 

Answers to Question 9

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.

This lady has a lupus anticoagulant.

 

Question 10

A 23-year-old woman develops a DVT. There is no other personal or family history of note?

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

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

 

Answers to Question 10

The finding of a prolonged APTT and a normal PT suggests:

1. Deficiency of factors VII, IX, XI or XII.

2. An inhibitor - either a lupus-type anticoagulant or a factor inhibitor.

In the context of a woman with no other personal or family history of note and a DVT - a lupus anticoagulant is the most likely. FXII deficiency has been implicated as a risk factor for venous thromboembolic disease because it is involved in the activation of the fibrinolytic pathway and therefore deficiency of FXII would lead to defective fibrinolysis. However, there is very little if any data to support FXII deficiency as a risk factor for either venous or arterial thromboses.

In the context of these results and the history - the next investigations should be:

A. Mixing studies with normal plasma to establish the presence or absence of a missing clotting factor
B. A screen for anti-phospholipid antibodies [lupus anticoagulant and anti-cardiolipin antibodies.]

NOTE: One assumes in this question that there is objective evidence of a DVT. Some patients with an acquired inhibitor can present with signs and symptoms suggestive of a DVT but in reality this is a large haematoma secondary to defective haemostasis.

This patient had a lupus anticoagulant which was still present when tested 12 weeks later and so fulfilled the critera for the Antiphospholipid Syndrome [APS].

 

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

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

 

Answers to Question 11

The finding of a prolonged PT and APTT but a normal fibrinogen and thrombin time indicates:

1. Deficiency of a single clotting factor - this would have to be either FII, V or X.
2. Deficiency of multiple clotting factors. Deficiency of FV and VIII is rare and in general does not give risk to such prolonged clotting times. More likely is a deficiency of the Vitamin K dependent clotting factors (II, VII, IX and X). This could be due to a deficient intake of vitamin K - which is rare or because the patient is taking a vitamin K antagonist such was warfarin.

In the context of an unconscious patient - one must consider the possibility of a major intracranial bleed and urgent clarification of the clotting problems is essential. A drug history from the relatives of the patient or his GP may be invaluable. Alternatively it may be obvious why this patient is on an that this patient is taking a vitamin K antagonist e.g. prosthetic heart valve. The patient may be carrying a card indicating that he is taking a vitamin K antagonist.

Assaying the vitamin Dependent clotting factors will show low levels in a patient on a vitamin K antagonist. In this case, urgent reversal of anticoagulation is indicated with intravenous vitamin K and a clotting factor concentrate containing factors II, VII, IX and X. Fresh Frozen Plasma (FFP) is not advised in this situation.

In fact this patient had a mid-line sternotomy scar and a metallic heart valve and was on warfarin.

 

Question 12

Comment upon the following results in an 18-year-old asymptomatic individual.

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

Answers to Question 12

These findings are strongly suggestive of a dysfibrinogenemia. Many of the variant fibrinogens are asymptomatic but some may be associated with an increased risk of venous and arterial thromboses and some with bleeding.

A discrepancy of 0.5g/L between a functional assay of fibrinogen (Clauss assay) and an Immunological assay is generally considered to suggest a dysfibrinogenemia. Many dysfibrinogenemias are present in reduced concentration i.e. a hypodysfibrinogenemias and this is the explanation for the clotting abnormalities in this this patient.

 

Question 13

An 8-year-old boy is admitted with prolonged bleeding following tonsillectomy. His clotting screen shows:

Test Patient Reference Range
PT 25s 11-14s
APTT 64s 23-35s

A. What factor assays would you request and why?

 

Answers to Question 13

The finding of a prolonged PT and APTT indicates:

1. Deficiency of a single clotting factor - this would have to be either FII, V or X.
2. Deficiency of multiple clotting factors. Deficiency of FV and VIII is rare but in certain ethnic groups is more common and could explain these prolonged clotting tests.. Vitamin K deficiency again could explain these findings.

Mixing studies would establish whether this was due to an inhibitor or not. Acquired FV, FX and FII inhibitors are rare although reported in the literature - see References.

Assays for factors V, X and II should be performed. If the FV level is reduced, a FVIII assay should be performed. If the FII level is low, a formal screen for a lupus anticoagulant should be undertaken. Occasionally lupus anticoagulants can be associated with FII (Prothrombin) deficiency.

This boy has factor V deficiency.

 

Question 14

Give 7 possible explanations for the following findings

Test Patient Reference Range
PT 21s 11-14s
APTT 62s 23-35s

Nb. There are more than 7!

 

Answers to Question 14

Possible answers include:

  1. Inherited deficiency of FV, FX or FII.
  2. Inherited deficiency of FV and VIII
  3. Acquired FV, FX or FII inhibitor
  4. Lupus Anticoagulant - although it unusual to find such a prolonged PT.
  5. Liver disease
  6. DIC
  7. Dysfibrinogenemia.
  8. Vitamin K deficiency
  9. Oral vitamin K antagonists - probably not. It would be unusual to find a PT of 21s and a more prolonged APTT.

 

Question 15

A 23-year-old male is investigated for a possible bleeding disorder whilst living in Paris. He has a bleeding time performed and this is found to be significantly prolonged at 26 minutes (NR: <10 minutes).

A. Briefly outline the principles of the bleeding time.
B. What abnormalities would explain the prolonged bleeding time in this patient?

 

Answers to Question 15

1. See Bleeding Time

2. The causes of a prolonged BT are summarised in the section on the BT but in essence are:

The BT is not widely performed in the UK. It is a difficult test to standardise.

This man had ITP with a platelet count of 14 x 109/L. The BT was unecessary and one could have predicted that it would be prolonged.

 

Question 16

A thrombin time is performed in a 78-year-old man with an extensive lower limb DVT prior to starting him on unfractionated heparin.

Test Patient Reference Range
Thrombin Time 32s 10-13s

A. What is your differential diagnosis?
B. What tests would you request next?

 

Answers to Question 16

1. We are given no other clotting information which in reality would be unusual. Therefore, you should concentrate on the causes of a prolonged thrombin time and whether any of these causes may increase the risk of a DVT.

The causes of a prolonged TT are numerous (see Thrombin Time) but in this situation the important ones and therefore, the differential diagnosis are:

2. The next tests involve establishing which of the causes listed above are relevant.

In fact this patient has a very low serum albumin leading to an acquired dysfibrinogenemia. Such patients also show a prolonged reptilase time. The abnormalities can be corrected in vitro by supplementing the plasma with albumin and in vivo by correcting the serum albumin. This was originally reported in patients with nephrotic syndrome and subsequently in patients with HIV.
In practice such patients do not seem to be at risk of bleeding and there is some evidence that they may have hyperaggregable platelets and may, therefore, be at increased risk of thromboses.

 

Question 17

A 56-year-old man with a long history of COPD is admitted for surgery. His pre-operative investigations show a Hb of 19g/dl and an Hct of 0.58

His pre-operative clotting screen shows:

Test Patient Reference Range
PT 20 11-14s
APTT 47 23-35s

A. What do you think might explain these findings?
B. What would you do next?

 

Answers to Question 17

The most likely explanation in this situation is an incorrect ratio of citrate anticoagulant to blood due to the high haematocrit in the patient. In such cases, when the plasma is recalcified to initiate clotting in the PT and APTT, citrate is still present, the calcium is chelated and there is insufficient for efficient coagulation and so the clotting times are prolonged.

The correct ratio of citrate to blood is 1:9 and for patients with a high haematocrit the correct volume can be calculated from the formula:

C (ml) = 1.85 x 10-3 x (100-Hct (%)) x V (ml)

C = ml of 3.2% sodium citrate anticoagulant

Hct (%) = haematocrit of the patient in %

V = ml of whole blood in tube

For this patient and for a 10ml sample of blood, the volume of citrate (C) is:

C = 1.85 x 10-3 x (100- 59) x 10

   = 0.76ml

 

Question 18

A 41-year-old male with nephrotic syndrome is admitted for renal biopsy. His pre-biopsy clotting screen shows:

Test Patient Reference Range
PT 13 11-14s
APTT 36 23-35s
Thrombin Time 19 10-13s
Reptilase Time 23 11-14s

A. Comment upon the results of these tests.
B. What advice would you give to the nephrologists regarding the renal biopsy?

Answers to Question 18

Hypoalbuminaemia resulting in a prolonged thrombin time and reptilase time is well reported both in cases of nephrotic syndrome and also in cases of HIV. In such cases there is strong correlation between the serum albumin levels and the TT and RT. The mechanism is unclear but appears to be due to an acquired dysfibrinogenemia. Such patients do not seem to be at risk of bleeding and may be a increased risk of thrombosis due to platelet hyperaggregability.

See also Q 17.