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A Practical Guide to Laboratory Haemostasis

 

Data Interpretation: Platelet Function Testing



Introduction

The following questions will allow you to work through a number of scenarios that relate to platelet function testing.

Question 1
Study the following aggregation traces and establish the diagnosis.
Remember there may be more than one possible answer for each set of aggregation traces.

If you click HERE this will load an enlarged version of these aggregation traces in a new window.

The control traces are shown in blue and the patient's traces in red.



1. What are the possible diagnoses that would explain these abnormalities?
2. What additional tests might you request?

Click HERE for the Answers
The most obvious abnormalities in this series of aggregation traces is a lack of aggregation to all agonists except ristocetin [- although agglutination with ristocetin is not complete.]

There are two possible explanations:

1. Glanzmann's Thrombasthenia [GTT] in which there is a defect in the GpIIb/IIIa receptor.

2. Afibrinogenaemia. Remember fibrinogen is required for normal platelet function and in the absence of fibrinogen platelets cannot aggregate. Fibrinogen is not required for the action of ristocetin and so some agglutination [remember not aggregation as this requires the binding of fibrinogen to the GpIIb/IIIa receptor] is seen but this is only the primary wave as the secondary wave requires activation of the GpIIb/IIIa receptor and the binding of fibrinogen.


Tests that you might request would include:

1. Clauss Fibrinogen - one would hope that before embarking upon platelet aggregation tests, a fibrinogen assay would have been performed.
2. Flow cytometry to look in more detail at the GpIIb/IIIa receptor.
3. Mutational analysis may be useful when the diagnosis of GTT [as is the diagnosis in this case] has been confirmed.

When the diagnosis has been established, family studies and genetic counselling are important. Individuals with GTT and who have been exposed to platelets in the past can develop antibodies to the missing Guppy/IIIa receptor which makes them less responsive to further platelet infusions. Screening such individuals for GpIIb/IIIa antibodies is important.


Question 2
Study the following aggregation traces and establish the diagnosis.
Remember there may be more than one possible answer for each set of aggregation traces.

If you click HERE this will load an enlarged version of these aggregation traces in a new window.

The control traces are shown in blue and the patient's traces in red.




1. What are the possible diagnoses that would explain these abnormalities?

Click here for Part 2
These aggregation traces are essentially the inverse of those seen in Q1. That is aggregation to all agonists except ristocetin. This suggests that the problem lies with the GpIb receptor. Remember the GpIb receptor is involved in the binding of Von Willebrand factor and therefore this pattern of traces would suggest either Bernard Soulier Syndrome [BSS] or VWD [probably severe Type 1 or Type 3.] The binding of platelets to VWF via the GpIb receptor is critical for the binding of binding of platelets to the damaged vascular endothelium.

1. You suspect a diagnosis of BSS or VWD. How would you distinguish between these three possibilities?

A. Measure Von Willebrand Factor levels - again you should measure these before embarking upon platelet aggregation testing.
B. Flow cytometry using a series of monoclonal antibodies specific for the GpIb receptor.
C. Mutational analysis may be of value although you should have made the diagnosis before embarking upon this so that you know which gene to look at.


Question 3
Study the following aggregation traces and establish the diagnosis.
Remember there may be more than one possible answer for each set of aggregation traces.

If you click HERE this will load an enlarged version of these aggregation traces in a new window.

The control traces are shown in blue and the patient's traces in red.



1. What are the possible diagnoses that would explain these abnormalities?

Click here for Part 2


You suspect a diagnosis of either Storage Pool Disorder or an abnormality of the platelet nucleotide release. What additional test will you request?
The obvious abnormalities in these series of aggregation traces are the lack of second wave aggregation with ADP and adrenaline [remember these are weak agonists] but in addition the aggregation with collagen and ristocetin is abnormal [reduced.]
This suggests either a platelet storage pool disorder or an abnormality of platelet granule release.
In these cases additional tests to clarify the diagnosis would include:
measurement of platelet nucleotides [ADP, ATP and the ADP:ATP ratio]
Electron microscopy to look for the presence or absence of platelet granules
Flow analysis to look for platelet granules [mepacrine binding]
Lumiaggregometry to look at platelet granule release.


Question 4
Using the data shown below for a VWF:RCo assay using platelets - calculate the VWF:RCo activity in the two plasma samples 1 and 2 [the two samples are shown at the bottom].

1. Assume the reference plasma has a VWF:RCo of 100%.
2. Remember to plot your slopes on double log paper with the dilutions on the X-axis and the slopes on the Y axis.


 

If you click HERE this will load an enlarged version of this graph in a new window.

Click HERE for the Answers
1. Remember - the slope is calculated by plotting the tangent of the slope and them measuring the distance from the origin to where the tangent crosses the X or Y axis. The distance on the Y axis is then divided by the distance on the X-axis and the slope derived. For example if for the normal plasma sample, the distance from the origin to where the tangent crosses intercepts the X-axis is 12mm and the distance on the Y axis is 44mm then the slope is 44/12 = 4.

2. If we calculate the slopes (S) of each of these traces (Y/X) derived from serial dilutions of normal plasma and plot these against dilution on double-log paper - then we end up with a straight line from which the VWF:RCo activity of an unknown plasma sample can be calculated [see below]:

In Plasma Sample - 1, there is no aggregation [more correctly no agglutination] and so the VWF:RCo activity is <1%. Remember we are told the reference plasma has a VWF:RCo activity of 100% and so we must report the results in %.

In Plasma Sample - 2, the slope is 0.65 and so from our standard curve we can calculate that this equates to a VWF:RCo activity of ~8.5%.

We can check that this is correct because the slope of Plasma Sample 2 lies between that of the 1/8 and 1/16 dilution of our normal plasma sample.

Finally - we are using neat patient plasma in Plasma Samples 1 and 2 and so there is no need to make any corrections for dilution.








Question 5

These traces are from a patient with suspected Von Willebrand Disease. What do they show and what is the diagnosis?

Figure 1: Patient Plasma + Control Platelets


Figure 2: Patient Platelets + Control Plasma


Click here for Part 2

When patient plasma is mixed with normal plasma there is agglutination with ristocetin at a concentration of 1.5mg/ml but not at 0.5mg/mL. This suggests that there is not a problem with the patient's VWF and that he/she does not have Type 2B VWD.
In contrast when the patient's platelets
are mixed with normal plasma there is agglutination with ristocetin at both concentrations indicating a problem with the patient's platelets i.e. pseudo platelet type VWD.

What is the underlying molecular abnormality in this disorder
The abnormality in platelet-type VWD is a mutation in the GpIb receptor [classically in the hinge region of the protein] that leads to a conformational change in the GpIb receptor and a gain of function. The abnormal receptor leads to an increased interaction with VWF, a fall in VWF levels and to a decreased/borderline low platelet count.
The disorder can also, therefore, be diagnosed by molecular analysis of the GPIBA gene. To date 4 mutations within the GPIBA gene have been reported: Gly233Val, Met239Val, Gly233Ser, 27bp deletion.


The importance in making this distinction lies in the fact that the treatments are different - platelet transfusions for platelet-type VWD and a FVIII-VWF concentrate [or a high purity VWF concentrate] in cases of 2B VWD.


Question 6
Study the following aggregation traces and establish the diagnosis.
Remember there may be more than one possible answer for each set of aggregation traces.




Click here for Part 2

These aggregation traces were obtained from a 12-year-old male referred for surgical correction of a complex congenital heart problem. He gave a history of bleeding after dental surgery. There was no family history of note and his parents were not related. His platelet count was 107 x 109/L and the MPV was 11.6fL. A blood film showed the presence of giant platelets.

The most obvious abnormalities in this series of aggregation traces is a lack of aggregation [more correctly agglutination] to ristocetin.

These findings are suggestive of Bernard Soulier Syndrome [BSS] in which there is a defect in the platelet GpIb receptor.

Additional tests that you might request would include:
1. Flow cytometry to look in more detail at the GpIb receptor.
2. Mutational analysis may be useful when the diagnosis of BSS has been confirmed.

When the diagnosis has been established, family studies and genetic counselling are important.


What additional diagnosis must you consider?
In the context of someone with this diagnosis and congenital heart disease, you should consider a diagnosis of the Velocardiofacial syndrome. This is a disorder that arises due to a microdeletion of q11.2 on the long arm of chromosome 22. Approximately 10% of patients inherit this abnormality from their parents and in the reminder it is a new mutation.

GpIb is a heterodimeric transmembrane protein consisting of a disulfide-linked 140 kD alpha chain and 22 kD beta chain. It is part of the GpIb-V-IX system that constitutes the receptor for von Willebrand factor. The GpIb alpha chain provides the VWF binding site, and GpIb beta contributes to surface expression of the receptor and participates in transmembrane signalling through phosphorylation of its intracellular domain. Mutations in the GpIb beta subunit have been associated with Bernard-Soulier syndrome, velocardiofacial syndrome and giant platelet disorder. The gene for GpIb beta maps to 22q11.21.


Question 7

These aggregation traces were obtained from a 23-year-old woman referred for investigation of life-long menorrhagia and prolonged bleeding after dental extraction.

Test Patient Reference Range
PT 11.2s 11.5-13.5s
APTT 34s 28-34.5s
FVIII:C 59 IU/dL 50-150 IU/dL
VWF:Ag
VWF:RCo
56 IU/dL
24 IU/dL
50-150 IU/dL
50-150 IU/dL
Platelets 79 x 109/L 150-400 x 109/L



1. What diagnoses would you consider?
2. What the treatment options for this woman?

Click HERE for the Answers

The most obvious abnormality here is agglutination with both standard dose and low dose ristocetin suggesting a diagnosis of either 2B VWD or platelet-type VWD.
The abnormality in platelet-type VWD is a mutation in the GpIb receptor [classically in the hinge region of the protein] that leads to a conformational change in the GpIb receptor and a gain of function. The abnormal receptor leads to an increased interaction with VWF, a fall in VWF levels and to a decreased/borderline low platelet count.
The disorder can also, therefore, be diagnosed by molecular analysis of the GPIBA gene. To date 4 mutations within the GPIBA gene have been reported: Gly233Val, Met239Val, Gly233Ser, 27bp deletion.

The importance in making this distinction lies in the fact that the treatments are different - platelet transfusions for platelet-type VWD and a FVIII-VWF concentrate [or a high purity VWF concentrate] in cases of 2B VWD.


Question 8
These aggregation traces were obtained from a 33-year-old woman referred for investigation of life-long menorrhagia and prolonged bleeding after dental surgery. There was a family history suggestive of a bleeding disorder but no conclusion had ever been reached.

The referring hospital had performed a PFA100 and the closure time using both ADP/Collagen and ADP/Adrenaline were prolonged.


1. What the major abnormalities?
2. What is the most likely diagnosis?

Click here for Part 2


You suspect either Storage Pool Disorder or a Primary Secretion Defect - how will you distinguish between the two?

The obvious abnormalities in these series of aggregation traces are the lack of second wave aggregation with ADP and adrenaline [remember these are weak agonists] but in addition the aggregation with collagen and ristocetin is abnormal [reduced.]
This suggests either platelet storage pool disorder or an abnormality of platelet granule release.
In these cases additional tests to clarify the diagnosis would include:
measurement of platelet nucleotides [ADP, ATP and the ADP:ATP ratio]
Electron microscopy to look for the presence or absence of platelet granules
Flow analysis to look at platelet granules [mepacrine binding]
Lumiaggregometry to look at platelet granule ADP release.

Remember there are two types of granules in platelets: Dense granules and alpha-granules.


Question 9
A 67-year-old man has coronary angiography and develops a large haematoma at the catheter insertion site. He has the following tests performed prior to stenting.

What do these tests show?
What are possible explanations for these findings?




Click here for Part 2


1. What advice would you give to the cardiologists?
The obvious abnormality here is a lack of aggregation with ADP at all concentrations and impaired aggregation with adrenaline and collagen. Remember ADP, adrenaline and collagen are all dependent upon binding of ADP to the ADP receptor [primarily the P2Y12 receptor.]
This pattern of abnormalities suggest either an abnormality of the P2Y12 receptor [rare] or a drug effect such as Clopidogrel [common.]
In this case the patient was on Clopidogrel and the lack of ADP-induced platelet aggregation indicates complete blockade of the P2Y12 receptor.

In this situation - if there is bleeding - stop any relevant drug and give a pool of platelets.


Question 10
These aggregation traces were obtained from an 18-year-old student under investigation for easy bruising. She had a prolonged closure time of >300s with PFA100 [Collagen-Adrenaline cartridge].


1. What possible diagnoses would you consider?

2. To confuse you - the control and patient traces have been reversed and the control is in red and the patient in black.

Click HERE for the Answers
The obvious abnormalities are an absent response to arachidonic acid and reduced secondary wave aggregation with ADP and Adrenaline. Collagen is also reduced.

These findings suggest a defect in the Thromboxane A2 pathway. Possibilities include drugs such as aspirin, NSAIDs, food stuffs that contain salicylates or a true defect in the TxA2 pathway.


Question 11
A 25-year-old Indian woman is investigated for recurrent miscarriages and menorrhagia

Test Patient Reference Range
PT >120s 11.5 -13.2s
APTT >120s 26-32s
Fibrinogen (Clauss) Not requested 2-4g/L
Bleeding Time >20 minutes <10 minutes
PFA100
  Collagen-ADP
  Collagen-Adrenaline

>300s
>300s

<110s
<112s

Comment upon the results of these tests.
What would you do next?

Click here for Part 2


Shown below are the results of limited aggregation studies.



1. What is the most likely diagnosis?
The most likely diagnosis is afibrinogenaemia. Remember fibrinogen is needed for platelet aggregation via the GpIIb/IIIa receptor. Hopefully you would have performed a Clauss fibrinogen assay before performing platelet aggregation testing.
2. What additional tests would you request?
A Clauss fibrinogen assay
3. What are the treatment options for this woman in pregnancy?
Fibrinogen supplementation throughout the pregnancy/delivery and for several weeks post-partum.


Question 12
A 43-year-old male with no past medical history of note, is admitted to hospital with a history of recent onset of recurrent and prolonged epistaxes. The results of initial investigations are shown below:

1. Comment upon the results of these investigations.
2. What tests would you request next?

Test Patient Reference Range
Hb 8.9 g/dL 13.5-16.5g/dL
Platelets 110 x 109/L 150 - 400 x 109/L
PT 13s 11.5 -13.2s
APTT 34s 26-32s
Fibrinogen 3.4 g/L 2-4 g/L
Bleeding Time >30 minutes < 10 minutes

Click here for Part 2


You suspect an acquired platelet disorder due to the recent onset of symptoms and the prolonged bleeding time; You request platelet aggregation studies. The results of one of these tests using Ristocetin [1.5mg/ml] is shown below.
1. Comment upon the results of this.
These results show abnormal agglutination with ristocetin suggesting either Bernard Soulier Syndrome or VWD i.e. a defect in the GpIb receptor. However, the history is of recent onset indicating that this is unlikely to be an inherited disorder and therefore more likely an acquired disorder.

2. What you do next?
It would be interesting to see if normal platelets suspended in patient plasma in the presence of ristocetin shows similar effects or not. If they do i.e. no agglutination - this suggests that something in the patient plasma is inhibiting the agglutination e.g. an autoantibody.


 

Click here for Part 3

You consider the possibility of an acquired platelet disorder further - look at the following traces.
[The first aggregation trace from Part 2 is shown again to help you. An IgG fraction was prepared from the patients plasma and from a control plasma.]

What does this suggest?

These results indicate that the IgG fraction prepared from patient plasma is inhibiting the ristocetin induced platelet agglutination - this is a case of acquired BSS due to anti-GpIb autoantibody.


Question 13
A 23-year-old man is investigated for the possibility of an inherited platelet disorder. His full blood count is normal apart from a platelet count of 55 x 109/L. The MPV is 11.2fL and the blood film shows the presence of giant platelets.

Shown below are the results of flow analysis using 3 separate monoclonal antibodies [CD40, CD42b and CD610 - and the results are expressed as median fluorescence intensity.

Sample
Platelet Count [ x 109/L]
MPV [fL]
CD41
CD61
CD42b
Control
268
8.6
4.87
28.7
6.23
Patient
55
11.2
19.9
55.2
2.39
1. How would you explain these findings?
2. What is a possible diagnosis and how would you confirm this?


In this analysis three antibodies [CD41, CD61 and CD42b] directed against specific platelet membrane glycoproteins were used:

Antibody Specificity
CD41 CD41 recognises the platelet membrane glycoprotein GpIIb (the integrin alpha IIb chain) which is non-covalently associated with GpIIIa (the integrin beta 3 chain) to form the GpIIb/IIIa complex.
CD61 CD61 recognises the platelet membrane glycoprotein GpIIIa (the integrin beta 3 chain).
CD42b CD42b reacts with GpIb on megakaryocytes and platelets.  CD42b also inhibits ristocetin-dependent binding of Von Willebrand Factor to platelets and ristocetin-induced platelet agglutination.

Click HERE for the Answers
There is deceased fluorescence with CD42b which binds to the GpIb receptor. This suggests the possibility of Bernard Soulier Syndrome [BSS].
Remember in BSS the platelets are large [increased MPV] and therefore there will be increased expression of the GpIIb/IIIa receptor. As a consequence of this fluorescence using CD41 and CD61 which are specific for the GpIIb and GpIIIa receptors respectively will be increased as in this case. The diagnosis is BSS.


Question 14
A 32-year-old woman is admitted through the Emergency Department with a short history of easy bruising. She is slightly confused.

Test Patient Reference Range
PT 12.5s 11.5 -13.2s
APTT 33s 26-32s
Fibrinogen (Clauss) 1.9 g/L 2-4 g/L
Thrombin Time 14.5s 12.5-13.9s

1. What tests would you request?

Click here for Part 2


1. You request the following:

Test Patient Reference Range
Platelet count 34 x 109/L 150 - 400 x 109/L
Blood Film Marked red cell fragmentation noted  
LDH 2400 U/L <450 U/L
Troponin T 0.9  μg/L 0–0.01 μg/L

What is the most likely diagnosis?
How will you manager this patient?
Are there any other tests you might request?


Click here for Part 3


1. You request an HIV screen which is positive. Will this affect your management?
2. What is the significance of the raised Troponin T and LDH?


Question 15
A 28-year-old male is investigated for the possibility of an inherited bleeding disorder. Platelet aggregation testing was not available but the results of platelet membrane glycoprotein analysis by flow are show below.

Comments upon the results of these tests.
Do these tests suggest a diagnosis?




FS: Forward scatter
SS: Side scatter

In this analysis three antibodies [CD41, CD61 and CD42b] directed against specific platelet membrane glycoproteins were used:

Antibody Specificity
CD41 CD41 recognises the platelet membrane glycoprotein GpIIb (the integrin alpha IIb chain) which is non-covalently associated with GpIIIa (the integrin beta 3 chain) to form the GpIIb/IIIa complex.
CD61 CD61 recognises the platelet membrane glycoprotein GpIIIa (the integrin beta 3 chain).
CD42b CD42b reacts with GpIb on megakaryocytes and platelets.  CD42b also inhibits ristocetin-dependent binding of Von Willebrand Factor to platelets and ristocetin-induced platelet agglutination.

Click HERE for the Answers

If you look at the last panel in which platelet are analysed for the expression of CD41 and CD61, then there is virtually no fluorescence. This suggests absence of the GpIIb/IIIa receptor and the findings are consistent with a diagnosis of Glanzmann's Thrombasthenia [GTT.]

There is normal fluorescence with CD42b which is specific for the specific for GpIb receptor.



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