Light Transmission Aggregometry [LTA]

Platelet function testing is difficult, time consuming and prone to a wide-variety of problems due to pre-analytical variables.

Before undertaking any tests of platelet function - consider:

  1. Clinical History and examination. Some syndromes [e.g. Hermansky Pudlak syndrome, Cheddiak Higashi syndrome, Wiskott-Aldrich syndrome, Velocardiofacial Syndrome (VCFS), Noonan syndrome, MYH9-related disorders] are associated with abnormal platelet function and you may get some idea of the diagnosis from the clinical history and examination.
  2. Drug History - there are a large number of drugs and especially food substances that can interfere with platelet function.
  3. Full Blood Count (FBC) and Blood Film
    1. Consider pseudothrombocytopenia often due to cold reacting platelet agglutinins or to platelet satellitism.  Approximately 0.1% of the healthy population show EDTA-induced pseudothrombocytopenia and it is important to exclude this before undertaking more extensive tests of platelet function.  Similar findings have also been reported with the use of both citrate and heparin as anticoagulants.  A blood film may identify platelet clumps and provide a clue to the diagnosis.
    2. Mean Platelet Volume [MPV – reference range 7-10fL]: the MPV is an often ignored parameter of the FBC but can provide important insights into the causes of a low platelet count. 
      - It can also in some cases give a clue to the diagnosis e.g. the hereditary macrothrombocytopenias, Bernard Soulier Syndrome [BSS]
      - In individuals with an elevated MPV, an immunological-based platelet count may provide a more accurate and often significantly higher platelet count.
      - The MPV can be an indication of platelet turnover – an increased MPV indicating accelerated platelet clearance as in ITP or gestational thrombocytopenia.
      - The MPV may be reduced in cases of Wiskott-Aldrich Syndrome and in some cases of bone marrow failure.
    3. An examination of the blood film and platelet morphology can be useful in both establishing a diagnosis of pseudothrombocytopenia but also in establishing a primary platelet problem e.g. Gray Platelet Syndrome.  In some cases of thrombocytopenia e.g. May Hegglin anomaly – the blood film may show the presence of Döhle bodies [light blue-gray, oval, basophilic, leukocyte inclusions located in the peripheral cytoplasm of neutrophils.]

Platelet Aggregation Testing: Born Aggregometry

Principles

Platelet aggregation testing measures the ability of various agonists to platelets to induce in vitro activation and platelet-to-platelet activation. Classically Born aggregometry uses platelet rich plasma [PRP] but whole blood aggregometry can be also used.

In the Born aggregometer, PRP is stirred in a cuvette at 37°C and the cuvette sits between a light course and a photocell. When an agonist is added the platelets aggregate and absorb less light and so the transmission increases and this is detected by the photocell.

You can also see the principles of Born aggregometry as an animation on this site [http://www.platelet-research.org/3/aggregometry.htm]



Pre-analytical variables.

    1. Drugs: Drugs which can interfere with platelet function include aspirin and anti-inflammatory drugs, specific anti-platelet drugs including clopidogrel and imidazole.  However, there are numerous other drugs whose primary role is not to inhibit platelet function but nevertheless can do so e.g. antibiotics, anti-depressants, beta-blockers etc - Click HERE for a list of drugs etc that may affect platelet function tests.
    2. Food stuffs
      - A high fat diet can lead to the presence of chylomicra in the plasma and interfere with light transmission in aggregation testing.
      - Others include garlic, turmeric and caffeine.
    3. Platelet count: In individuals with very high or low platelet counts – it may be necessary to adjust the platelet count to achieve a count in the region of 200-400 x 109/L.  For very high counts the count can be adjusted with PPP.  Platelet counts below 200 x 109/L can give rise to diminished aggregation responses.  Although it seems logical to undertake additional centrifugation in such cases to increase the platelet count, in practice this can lead to activation of platelets and is not recommended.
    4. Temperature: Blood samples for platelet aggregation testing should be stored at room temperature.
    5. pH: Platelet aggregation should be carried out at physiological pH.
    6. Platelets will only aggregate (although they may agglutinate) if fibrinogen is present and so it is important to check fibrinogen levels before undertaking platelet aggregation testing.


Preparation of Platelet Rich Plasma (PRP)

  1. Platelets are very sensitive and can be readily activated during the preparation of PRP.
  2. Anticoagulant: Venous blood with minimal venous occlusion is collected 3.2%/0.109M citrate in a ratio of 1:9 [1 part anticoagulant to 9 parts blood.] 
  3. Whole blood samples should be processed within 4 hours of collection.
  4. Blood samples for platelet aggregation testing should be stored at room temperature – cooling platelets can lead to activation.
  5. Transport samples to the laboratory at room temperature.
  6. PRP is prepared by centrifugation at 20°C for 10-15 minutes at 150-200g. The PRP is carefully removed and placed into a stoppered plastic tube. PRP should be stored at room temperature. PPP can be prepared by further centrifugation of the remaining plasma at 2700g for 15 minutes.


Agonists

Addition of a platelet agonist to the PRP leads to platelet activation, a change in their shape from discoid to spiny spheres which is associated with a transient increase in optical density. The only exceptions to this are epinephrine in which there is no shape change and ristocetin which causes platelet agglutination rather than aggregation. 

There are two types of agonists:

Strong Agonists e.g. Collagen, thrombin, TxA2: These directly induce platelet aggregation, TxA2 synthesis and platelet granule secretion.

Weak Agonists e.g. ADP & epinephrine: These induce platelet aggregation without inducing secretion. Platelet secretion can sometimes follow aggregation induced by a weak agonist, when the synthesis of endogenous TxA2 is triggered by the close platelet-to-platelet contact that occurs during platelet aggregation. Strong agonists, when used at low concentrations, may act like weak agonists, but weak agonists even at high concentrations will not act as strong agonists.

With some weak agonists [ADP and adrenaline] at critical concentrations, the platelet aggregation curve has a biphasic appearance: an initial wave of aggregation (primary wave), followed by a secondary wave of aggregation, which is usually irreversible. Secondary wave aggregation may not occur and the primary wave may disaggregate. At higher agonist concentrations (except with epinephrine) the two waves of aggregation combine and only a single wave is seen and the biphasic waveform is absent.

The aggregation response to an agonist is amplified by the production of TxA2 from membrane phospholipids and by the secretion of ADP from the dense granules.  ADP and TxA2 are aggregating agents, which, by interacting with their specific receptors, amplify the aggregation response of the platelet.

Commonly used agonists, their working concentration and mode of action are listed below. In practice many laboratories use a number of agonists and various dilutions but vary the actual agonists or agonist concentration depending upon the results of initial tests and the suspected abnormality. Not all laboratories necessarily use the concentrations shown below e.g. some labs may use collagen at 5μg/mL rather than 4μg/mL.

It is useful to consider the role of these various agonists by looking at an image of a platelet and the various receptors that are activated by the agnosits discussed below and how these interact with the platelet. The following link takes you to an image that you may find useful to consider with the table below:

Agonist

Working concentration

Comments

ADP

1, 2.5, 5μM
[Low dose]

10μM
[ High dose]

ADP binds to the ADP receptor on the surface of platelets.  Initial binding results in the release of intracellular calcium and a change in the shape of the platelet leading to the primary wave of aggregation.  The secondary wave reflects the release of ADP from platelet storage granules.

Low dose ADP induces only primary aggregation and the effect is reversible.

ADP and arachadonic acid are considered mild platelet agonists.

ADP binds to two G-protein coupled receptors: P2Y1 and P2Y12. Binding of ADP to the P2Y1 receptor induces shape change and initiates primary wave platelet aggregation through calcium mobilisation. The P2Y12 receptor is considered to be the major ADP receptor and responsible for full platelet aggregation thought the inhibition of adenyl cyclase. The P2Y12 receptor is also the target for clopidogrel.

With both ADP and Arachadonic acid - this second wave of aggregation is inhibited by aspirin and NSAID's.

1. Stable baseline
2. Addition of agonist
3. Shape change
4. Primary wave
5. Secondary wave

With high dose ADP the primary and secondary wave separation is lost and only a single wave is seen.

Collagen

1, 4μg/mL

Collagen binds to the GpVI and GpIa/IIa receptors inducing granule release, TBXA2 generation and then sustained GPIIb-IIIa activation.

The GpIa/IIa receptor is involved in platelet adhesion. The GpVI receptor is involved in platelet signaling and TBXA2 generation.


A lag phase is seen with collagen following addition of the agonist to the PRP and usually <1 minute.

1. Stable baseline
2. Lag phase after addition of agonist
3. Shape change
4. Single ‘secondary wave’

Ristocetin

0.5mg/mL
[Low dose]
1.5, 5mg/mL
[High dose]

Ristocetin (but not generally in low dose i.e. 0.5 mg/mL) causes platelet agglutination (and not aggregation) through the VWF and GPIb-IX-V complex.

[Platelet aggregation requires the binding of fibrinogen to the platelet via the GpIIb-IIIa complex.]

1. Stable baseline
2. Addition of agonist
3. Single agglutination/aggregation response

 

Low Dose Ristocetin (0.5mg/mL)

1. Stable baseline
2. Addition of agonist - no aggregation/agglutination is seen.

Adrenaline

5, 10μM

Adrenaline binds to the a2-adrenergic receptor on the surfaces of platelets leading to inhibition of adenyl cyclase and the release of calcium ions.

Aggregation of platelets with Adrenaline is similar to that of ADP with an initial primary wave of aggregation, the release of stored ADP from the platelet dense bodies and second wave sustained aggregation.

As with ADP - this second wave of aggregation is inhibited by aspirin and NSAIDs.

Adrenaline is considered [as is ADP] to be a weak agonist. However, defects in signaling through the a2-adrenergic have been associated with a bleeding disorder.

Absent aggregation has been noted in approximately 10-15% of apparently healthy individuals.

1. Stable baseline
2. Addition of agonist
3. Primary wave
4. Secondary wave

Arachadonic Acid

500μg/mL

Arachadonic Acid is the precursor of thromboxane A2 [TBXA2] within platelets. Arachadonic Acid is converted to TBXA2 by cyclooxygenase and thromboxane synthase. TBXA2 is a potent inducer of platelet aggregation causing granule release, more TBXA2 generation and then sustained GpIIb-IIIa activation.

1. Stable baseline
2. Addition of agonist
3. Shape change
4. Single secondary wave
Thrombin  

Thrombin is the most potent physiological activator of platelets and protease-activated receptors 1 (PAR1) and 4 (PAR4) are activated by thrombin. PAR1 and PAR4 are members of a 7-transmembrane group of G-protein coupled receptors that are activated by a single cleavage within the N-terminal domain to generate a new N-terminus which activates the G-subunits and intracellular signaling.

Thrombin induced platelet aggregation at low concentration [50nmol/L].

At this dose, thrombin is insufficient to induce full aggregation but binds to platelets and induces a shape change.

1. Base line
2. Addition of agonist
3. Shape change

At a higher concentration (100nmol/L), Thrombin induces full aggregation.

1. Base line
2. Addition of agonist
3/4. Shape change and full aggregation

Method

  1. Platelet aggregometry is performed at 37°C.

  2. The aggregometer is calibrated by:
    - A cuvette with PRP which equates to 0% light transmission
    - A second cuvette containing PPP which equates to 100% light transmission.

  3. Platelets will only aggregate if they are activated (with an agonist) and in contact with each other - so they must be stirred whilst testing is taking place. Absence of stirring will lead to an absence of, at least a significant reduction in, aggregation.

  4. A check for spontaneous platelet aggregation - [SPA: Rare in healthy individuals but seen in some cases of VWD, in some patients with diabetes, in some lipid disorders and in a variety of other disorders] should be made in all patients by placing undiluted PRP in the aggregometer and stirring for 15 minutes.  In cases of SPA, dilution of the PRP may abolish this and if the platelet count remains >200 x 109/L then aggregation testing can proceed.

  5. In general - 270μL of PRP is added to the aggregometry cuvette and warmed at 37°C until a steady baseline is achieved.  30μL of the agonist is added the response recorded. 


    The following illustration outlines the principle of Born aggregometry using ADP as an agonist.

 

Interpretation

The interpretation of platelet aggregation traces can be difficult! The attached file [Click HERE] provides a summary of the abnormalities that may be identified by platelet aggregation testing.



The Ristocetin Cofactor Assay

The Ristocetin Cofactor Assay measures the ability of a patient’s plasma to agglutinate platelets in the presence of the antibiotic Ristocetin.  The rate of Ristocetin induced agglutination is related to the concentration and functional activity of the plasma von Willebrand factor.  Ristocetin induced platelet aggregation (RIPA) is similar to the Ristocetin Cofactor Assay but RIPA measures platelet agglutination induced by Ristocetin-mediated VWF binding to the platelet Gp1b receptor but in this case the ristocetin is added directly to the patient’s platelet rich plasma and there are no serial dilutions of the plasma sample.
Originally, fresh washed platelets were sued for the measurement of plasma Ristocetin Cofactor activity but now frozen or formaldehyde-fixed platelets may be used.  It is also possible to replace the use of an aggregometer and use a microtitre plate and an ELISA plate reader although the principles remain the same.

Method


The method is similar to a factor assay.

  1. Fresh or fixed platelets are mixed with dilutions of patient plasma in a standard platelet aggregometer and a fixed concentration of ristocetin added.

  2. Patient samples are normally performed in duplicate at a single dilution.

  3. Agglutination is allowed to proceed and from the traces the slope of the curve is derived. 

  4. A standard curve is established by using a similar method but replacing patient plasma with serial dilutions of normal plasma. 

  5. The slope of the curve is plotted against dilution on double-log paper and a straight line obtained.  From this the Ristocetin cofactor activity in the patient’s plasma samples can be derived.

The following traces demonstrate this more clearly:

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:RCoF activity of an unknown plasma sample can be calculated - see below.

 

In the case of Plasma sample - 1, there is no aggregation [more correctly no agglutination] and so the VWF:RCoF activity is <1%. In Plasma sample - 2, the slope is 0.65 and so from our standard curve we can calculate that this equates to a VWF:RCoF 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 not need to make any corrections for dilution.

 

What Test Next?

On the basis of an abnormal platelet aggregation trace, you should establish if this fits in with any recognisable disorder. All abnormal results should be repeated and you may wish to undertake flow cytometry and nucleotide studies. Genetic testing can be of value in some cases.

Don't forget to establish a family pedigree - some of the rare platelet disorders are commoner in consanguineous relationships.

 

Useful Links & References

1. Remuzzi, G., et al., Platelet hyperaggregability and the nephrotic syndrome. Thromb Res, 1979. 16(3-4): p. 345-54.
2. Lages, B. and H.J. Weiss, Biphasic aggregation responses to ADP and epinephrine in some storage pool deficient platelets: relationship to the role of endogenous ADP in platelet aggregation and secretion. Thromb Haemost, 1980. 43(2): p. 147-53.
3. Guidelines on platelet function testing. The British Society for Haematology BCSH Haemostasis and Thrombosis Task Force. J Clin Pathol, 1988. 41(12): p. 1322-30.
4. Lages, B. and H.J. Weiss, Heterogeneous defects of platelet secretion and responses to weak agonists in patients with bleeding disorders. Br J Haematol, 1988. 68(1): p. 53-62.
5. Hardisty, R.M., Disorders of platelet secretion. Baillieres Clin Haematol, 1989. 2(3): p. 673-94.
6. Michelson, A.D., Flow cytometry: a clinical test of platelet function. Blood, 1996. 87(12): p. 4925-36.
7. Rao, A.K., Congenital disorders of platelet function: disorders of signal transduction and secretion. Am J Med Sci, 1998. 316(2): p. 69-76.
8. Rodgers, G.M., Overview of platelet physiology and laboratory evaluation of platelet function. Clin Obstet Gynecol, 1999. 42(2): p. 349-59.
9. Shapiro, A.D., Platelet function disorders. Haemophilia, 2000. 6 Suppl 1: p. 120-7.
10. Kottke-Marchant, K. and G. Corcoran, The laboratory diagnosis of platelet disorders. Arch Pathol Lab Med, 2002. 126(2): p. 133-46.
11. Handin, R.I., Inherited platelet disorders. Hematology Am Soc Hematol Educ Program, 2005: p. 396-402.
12. Harrison, P., Platelet function analysis. Blood Rev, 2005. 19(2): p. 111-23.
13. Bolton-Maggs, P.H., et al., A review of inherited platelet disorders with guidelines for their management on behalf of the UKHCDO. Br J Haematol, 2006. 135(5): p. 603-33.
14. Hayward, C.P., Diagnostic approach to platelet function disorders. Transfus Apher Sci, 2008. 38(1): p. 65-76.
15. Harrison, P. and A. Mumford, Screening tests of platelet function: update on their appropriate uses for diagnostic testing. Semin Thromb Hemost, 2009. 35(2): p. 150-7.
16. Mezzano, D., T. Quiroga, and J. Pereira, The level of laboratory testing required for diagnosis or exclusion of a platelet function disorder using platelet aggregation and secretion assays. Semin Thromb Hemost, 2009. 35(2): p. 242-54.

 

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.