Guidelines for laboratory investigations of platelets disorders Suggestive history ISTH 2 or more of the following without blood transfusion

on or 1 or more of the following with blood transfusion or 1 symptom recurring on 3 distinct occasions Nose bleeding >10 minutes in absence of trauma Cutaneous bleeding with no or minimal trauma Prolonged bleeding from trivial wounds >15 minutes Oral cavity, including tooth extraction, post tonsillectomy etc. bleeding requiring medical attention Spontaneous GI bleeding Menorrhagia or bleeding from other mucosal sites Positive family history 2 or more distinct bleeding sites A single bleed so severe as to require blood transfusion Need to stop certain drugs before testing (aspirin, clopidogrel, NSAIDs, antihistamines, beta-blockers, penicillin / cephalosporins. Aminophylline and some foods e.g. garlic)

PRE-ANALYTICAL VARIABLES 1.1 Specimen collection 1.1.1 Venipuncture Samples for platelet function studies should only be collected from fasting and resting subjects who have refrained from smoking and caffeine ingestion on the day of testing. Medications affect platelet function, e.g. non-steroidal anti-inflammatory drugs be deferred for 10-14 days after the last dose. Herbal remedies, garlic, alcohol and certain foods may also cause acquired platelet dysfunction. Collect blood using a standardised, atraumatic protocol, with minimal stasis Use needles between 19 and 21 gauge; evacuated tube systems or syringes are acceptable 1.1.2 Anticoagulants The first 3-5ml of blood should not be used for platelet function tests (2C) Blood should be collected into a 1/10 volume of trisodium citrate (105-109 mmol/L) 1.1.3 Specimen processing All specimens must be maintained at room temperature (20-25C) and should not be placed on ice, in a refrigerator or a water bath.

The time delay between collection, transport and analysis should ideally be preferably between 30 minutes and 2 hours but not more than 4 hours. 2 TESTS AND ASSAYS i. Measurement of platelet number and size ii. Global screening tests of platelet haemostatic function iii. Specific assays of platelet haemostatic function i. Measurement of platelet number and size - Perform a full blood count on all patients - In samples with abnormalities in platelet count or size distribution (as indicated by an automated analyser), a blood film should be examined ii. Global screening tests of platelet haemostatic function prothrombin time (PT) and activated partial thromboplastin time (aPTT), von Willebrand Factor (VWF) screening tests (VWF:Ag, VWF:RCo and F:VIII:C) and measurement of platelet number. Template bleeding time is not recommended as it is subjective and is influenced by patient variables unrelated to haemostasis, such as age, gender, haematocrit, vascular pattern, skin thickness and skin temperature.
Platelet adhesion testing

Closure time by the Platelet Function Analyser (PFA-100) provides an optional screening test, but the test is not diagnostic or sensitive for mild platelet disorders. The PFA-100 device is a test system in which citrated whole blood is aspirated at high shear rates (5000-6000s-1) through disposable cartridges containing an aperture coated with either collagen and epinephrine (CEPI) or collagen and ADP (CADP). These agonists trigger platelet adhesion, activation and aggregation leading to rapid occlusion of the aperture and cessation of blood flow.
Affected by Platelet count Haematocrit Diet Aspirin vWF levels need to measure vWF levels if abnormal result

3 SPECIFIC ASSAYS OF PLATELET FUNCTION 3.1 Light transmission aggregometry (LTA)

Platelet aggregation test

 Assay based on measuring the decrease in light absorbance that occurs in platelet rich plasma when platelets aggregate. Aggregometer is set with platelet poor plasma to demonstrate 100% light transmittance, and platelet rich plasma is used to set the baseline at 0%. Different agonists are then added to separate test aliquots, and as platelets aggregate light transmittance increases and results are plotted on moving graph paper. Agonists include ADP, adrenaline, collagen and ristocetin, (arachidonic acid U46619 (thromboxane receptor agonist), TRAP (thrombin receptor activating peptide), heparin (HIT)) Recommended that full dose response curves are obtained with each agonist In thrombocytopenic samples (<120) it is best to adjust the control sample to the same platelet count or perform studies on washed platelets in which the count can be adjusted (neither technique is perfect) Limited sensitivity for storage pool disorders (25% will have normal platelet aggregation)

Sample preparation for LTA Citrated blood samples are centrifuged to prepare platelet rich plasma (PRP) and platelet poor plasma (PPP). To prepare PRP, whole blood is centrifuged at 170-200 g for 10 minutes. Autologous PPP is prepared by centrifugation (after removal of PRP or using whole samples) at least 1500 g for at least 15 minutes. A plastic pipette should be used to separate the top 2/3rds of PRP or PPP. Icteric, lipaemic, red cell contaminated and haemolysed samples should not be tested. Agonists for LTA ADP, epinephrine, collagen (type I, tendon), arachidonic acid and ristocetin are the traditional baseline panel of agonists for LTA. An extended panel of agonists include gamma Thrombin, Thrombin Receptor Activating Peptides (TRAP), Collagen-Related Peptide, endoperoxide analogue U46619 and calcium ionophore A23187 Most laboratories perform dose response curves for ADP (0.5 20 M), collagen (1.0 5.0 g/ml) and epinephrine (0.5 -10 M).
Platelet aggregation Assay based on measuring the decrease in light absorbance that occurs in platelet rich plasma when platelets aggregate. Aggregometer is set with platelet poor plasma to demonstrate 100% light transmittance, and platelet rich plasma is used to set the baseline at 0%. Different agonists are then added to separate test aliquots, and as platelets aggregate light transmittance increases and results are plotted on moving graph paper. Agonists include ADP, adrenaline, collagen and ristocetin, (arachidonic acid U46619 (thromboxane receptor agonist), TRAP (thrombin receptor activating peptide), heparin (HIT)) Recommended that full dose response curves are obtained with each agonist In thrombocytopenic samples (<120) it is best to adjust the control sample to the same platelet count or perform studies on washed platelets in which the count can be adjusted (neither technique is perfect)

 ADP

Limited sensitivity for storage pool disorders (25% will have normal platelet aggregation)

Platelet response to ADP depends on its concentration: Low concentrations of ADP (0.2 m) cause primary or reversible aggregation (descent limb due to aggregation, then plateau, then ascent due to disaggregation). ADP initially binds its receptor and releases intracellular Ca which causes a shape change (reflected by a small initial change in absorbance), Fibrinogen then adds to the cell to cell contact and reversible aggregation occurs. Intermediate concentrations of ADP (0.4 m) cause an irreversible secondary wave aggregation which is associated with the release of dense and alpha-granules as a result of activation of the arachidonic acid pathway. High concentrations make 1ry and 2ry waves merge into one irreversible. Collagen This results in a single wave of aggregation after a lag phase, which results from ADP release after activation of the arachidonic acid pathway (absent in Glanzmans dis., disorder of release, asprin). Ristocetin This reacts with vWF and the membrane receptor (GP Ib-IX) to induce platelets to clump together (agglutination) and does not activate any of the aggregation pathways. Gives single wave (absent with defect of vWF or GP Ib-IX receptor (BSS). Arachidonic acid AA induces thromboxan A2 (TX A2) generation and granule release even if there is a defect of agonist binding to the surface membrane or the phospholiase induced release of endogenous arachidonate. Aggregation only impaired if further steps in the pathway are impaired such as inhibition of cyclooxygenae (aspirin effect). Thrombin May induce mono or biphasic aggregation. Adrenaline May induce biphasic response; 1ry due to plat changes in shape and 2nd due to ADP release. Pitfalls Centrifugation red cell contamination may cause apparent incomplete aggregation Time for 30 minutes after PRP preparation, platelets are unresponsive to agonists Platelet count low counts may cause slow/ weak aggregation pH - <7.7 inhibits aggregation, >8.0 enhances aggregation mixing speed - <800 or >1200rpm slows aggregation haematocrit temperature dirty cuvette/ air bubbles in cuvette ADP N Adrenaline Collagen N N Ristocetin Absent Platelet nucletoides Increased (large platelets) Diagnosis Bernard Soulier Nucleotides normal in vWD Flow for GPIb Flow for IIbIIIa Can also use AA to distinguish Prim/ absent = SPD Red = asp

Abs Prim

Absent Prim

Absent Prim Prim

Primary aggregation N N

N N Reduced ADP

Glanzmanns Aspirin or COX deficiency Storage pool defect dense

Prim (at high Prim concentrations, full irreversible aggregation)

Prim Red

Prim Red

Prim Red

Prim N

N Reduced

Red cell contamination Low platelets

Abs

Increased at low conc

Increased at Increased at Increased at low conc low conc low conc

Plasma never clears Slow and weak aggregation Absent/ abberant Eg. MPD adrenaline receptor Hyperaggregation Check if there is spontaneous aggregation

Heredity Adhesion Pseudo vWD (increased affinitiy for GpIb) Bernard-soulier (absent GpIb) Aggregation Glanzmann (abnormal IIbIIIa) Storage pool granule (HP / CHS) -granule (GPS) Signalling Cyclooxygenase deficiency (aspirin) Thromboxane ADP Phospholipid Scott syndrome Ehlers-Danlos

Platelet count Low Low N N Low

Platelet size N Large N N Large

PFA 100

LEpi N N 0 1 N

ADP

Col

Risto AA A23187 (RIPA) INC 0 N 1 N N N 0 1/0 N N 0 R

AD AR AR AR AR

Inc Inc Inc Inc or Norm Inc or Norm

N N 0 High dose, full irreversible aggregation N/0

N N 0 R 0

N AR AR AR Normal N

1 1/N Rapidly reversible

R R

N N

R/0 N/R R/0 N

Normal N

+ N

+ N

+ N

+ N

+ N

3.2 Flow cytometry Flow cytometry is used in the investigation or confirmation of Glanzmann thrombasthenia, Bernard Soulier Syndrome and Scott syndrome (1C); investigate abnormalities in the collagen (GpVI and GpIa/IIa) and thrombin receptors (PAR-1) 3.3 Measurement of total and released nucleotides It is an additional diagnostic tool with aggregometry for determining whether there is any specific deficiency in dense granule numbers or their content (e.g. storage pool disease), or specific defect(s) in degranulation (e.g. release defects),using bioluminescent assays. There are two nucleotide pools within the platelet:- the metabolic pool and the dense granular/storage pool, the latter comprising about 60% of the total content. The ratio of ATP:ADP is therefore of fundamental diagnostic importance. Any storage defects are associated with a decrease in the amount of stored and released ADP with an increased ratio of

ATP:ADP. Normal ADP levels and ATP:ADP ratios but decreased ADP release are indicative of a releasedefect. Serotonin (5-HT) is actively taken up and stored within the platelet dense granules and it is possible to measure the uptake and release of radiolabelled serotonin into and from the platelets with standardised assays. 3.4 Whole blood aggregometry In impedance aggregometry, whole blood is stirred at 37C and aggregation is detected by the accretion of platelets to the surface of two fine, metal, wire electrodes. Adherent platelets increase the electrical impedance between the electrodes, which can be displayed as a wave of aggregation. Impedance aggregation measurements in whole blood may be influenced by: haematocrit (>0.35 L/L), platelet count, and elevated white cell count.
3.5 Genetic analysis Wiskott Aldrich Offered to some families with severe platelet function disorders to allow prenatal diagnosis

FIGURE 1: Illustration of how LTA patterns can be used to diagnose a range of rare platelet defects. Note that these are generalised illustrations and actual patterns may differ slightly between patients with similar defects. The 0% baseline (bottom of y axes) has been set with undiluted PRP and the 100% aggregation (top of y axes) limit set with autologous PPP. After establishment of a stable baseline for a few minutes the following agonists were added to these final

concentrations, 10 arachidonic acid and low (0.5-0.7 mg/ml) and high dose (1.2-1.5 mg/ml) ristocetin. Aggregation was then monitored for up to 10 minutes (x axes from left to right) Example tracings are shown for a normal subject, a patient with Glanzmann thrombasthenia (GT), patients with VWD or Bernard Soulier Syndrome (BSS), Type 2B VWD/pseudo-VWD, GpVI deficiency, P2Y12 deficiency, an aspirin-like defect, storage pool and release defects and a P2Y1 defect. Normals will give an initial shape change (slight negative deflection) followed by a rapid and irreversible aggregation response to high concentrations of ADP. Lower doses can be used to determine the threshold for secondary aggregation. High dose collagen will give a characteristic lag phase followed by a shape change (slight negative deflection) followed by a rapid and irreversible aggregation. Epinephrine will give the classical biphasic response (primary and secondary aggregation) with no shape change. Arachidonic acid will give a shape change followed by full aggregation. Only high dose ristocetin will give a normal agglutination response in normals. The GT patient shows no aggregation to any agonist except high dose ristocetin (which is reversible). Flow cytometry and molecular biology can then be used to confirm the defect in samples (but not BSS) will be correctable after addition of a source VWF to the plasma. VWD should be confirmed with a VWF panel of tests/molecular biology and BSS confirmed by flow cytometry and molecular biology. Type 2B and pseudo-VWD show the gain of function with a response to low dose ristocetin. A VWF panel/molecular biology will confirm type 2B VWD and molecular biology will confirm the diagnosis of pseudo-VWD in the GpIb gene. Gp VI deficiency gives no response to collagen which can be confirmed using CRP, by flow cytometry (see extended

panel of agonists) or molecular biology. A P2Y12 defect shows a reduced and 30 reversible response to ADP. In a homozygous P2Y12 defect, only minimal primary aggregation to all concentrations of ADP will be present. In a heterozygous P2Y12 defect there will be absence of secondary aggregation but disaggregation tends to begin < 10 -like defect

will show reduced secondary aggregation responses to ADP and epinephrine and absent arachidonic acid aggregation. A defect in the thromboxane receptor can then be checked for using U46619 (see extended panel of agonists). Patients with storage pool disease or release defects will give an identical pattern as the aspirinlike defect except giving a good primary aggregation response to arachidonic acid. The P2Y defect pattern is based upon in vitro response to anti-P2Y1 antagonists as no patients with a P2Y1 defect have ever been described. This figure was significantly modified with permission from Figure 12.3, page 115 in Chapter 12 Diagnostic Assessment of platelet function by P.Nurden and A. Nurden in Quality in Laboratory Hemostasis and Thrombosis edited by Kitchen S, Olson JD & Preston FE and published by Wiley-Blackwell in 2009.

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