Guidelines for laboratory investigations of platelets disorders SUGGESTIVE HISTORY Bleeding history 2 or more of the following without blood

od transfusion 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) LABORATORY INVESTIGATIONS

1. PRE-ANALYTICAL VARIABLES 1.1 Specimen collection 1.1.1 Venipuncture Samples for platelet function studies; from fasting, resting and abstained from smoking and caffeine on the day of testing. Medications affect platelet function, e.g. non-steroidal anti-inflammatory drugs be postponed for 10-14 days after the last dose. Herbal remedies, garlic, alcohol may cause acquired platelet dysfunction. Collect blood using a standardised, atraumatic protocol, with minimal stasis, using needles between 19 and 21 gauge. The first 3-5ml of blood should not be used for platelet function tests 1.1.2 Anticoagulants Blood is 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 - 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 1. prothrombin time (PT) and activated partial thromboplastin time (aPTT), 2. von Willebrand Factor (VWF) screening tests (VWF:Ag, VWF:RCo and F:VIII:C) and measurement of platelet number. 3. 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. 4. Platelet adhesion testing: Closure time by the Platelet Function Analyser (PFA-100) is a screening test and 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 Platelet aggregation test (Light transmission aggregometry (LTA)) 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, and as platelets aggregate light transmittance increases and results are plotted on moving graph paper. 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).
ADP 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. 2. 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). 3. 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). 4. 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). 5. Thrombin May induce mono or biphasic aggregation. 6. Adrenaline May induce biphasic response; 1ry due to plat changes in shape and 2 nd 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 1.

ADP N

Adrenaline N

Collagen 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 Prim (at high concentrations, full irreversible aggregation) Prim Red

Absent Prim Prim

Absent Prim Prim

Primary aggregation N N

N N Reduced ADP

Glanzmanns Aspirin or COX deficiency Storage pool defect dense

Prim Red

Prim Red

Prim N

N Reduced

Red cell contamination Low platelets

Plasma never clears Slow and weak aggregation Eg. MPD Check if there is spontaneous aggregation

N Increased at low conc

Abs

N N

Absent/ abberant adrenaline receptor Hyperaggregation

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

Heredity Platelet count Platelet size PFA 100 L-Epi

ADP

Col Risto (RIPA) AA A23187

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

AD AR AR AR AR

Low Low N N Low

N Large N N Large

Inc Inc Inc Inc or Norm Inc or Norm

N N 0 1 N

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

N N 0 R 0

INC 0 N 1 N

N N 0 1/0 N N 0 R

N AR AR AR Normal N

1 1/N Rapidly reversible

R R

N N

R/0 R/0

N/R N

Normal N

+ N

+ N

+ N

+ N

+ N

3.2 Flow cytometry Flow cytometry is used in the investigation of Glanzmann thrombasthenia, BernardSoulier 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. 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 release-defect. 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 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 M ADP, 2 g./ml collagen, 5 M epinephrine, 1.0 mM 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) 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.

Hereditary platelet function defects

Pathophysiology

Others
Plt Count.: Thrombocytopenia due to reduced platelet survival Plt size: Giant (Increased plt nucleotides) pseudo-lymphocyte apperance Plt function: Normal aggregation except reduced in ristocetin (not corrected by addition of vWF) MK: numerous, multinucleated, vaculated Invest: Flow for GP1b detection, BT, C/P: mucocutaenous & visceral hge, menorrhagia, epistaxis. Plt Count.: Thrombocytopenia Plt size: Giant Plt function: agglutinate spontaneously in normal plasma (have vWF) without adding ristocetin. Normal aggregation with other agonists. C/P: mucocutaenous hge. ttt: Important to distinguish from type IIb VWD as treated with platelets rather than VWF concentrates

Adhesion defects to subendothelium Bernard Soulier syndrome (BSS) AR Deficiency of GP Ib/V/IX complex 1
- GpIb decreased binding to vWF reduced adherence to endothelium & plt survival - GpV (w involved in thrombin plt interaction) defect in prothrombin consumption but with the of phosphatidyl serine exposure thrombin generation by platelets. - GpIX (is a receptor for drug dependent Ab) - Mutations in platelet GP Ib resulting in increased sensitivity to ristocetin - Reduced HMW vWF multimers hge.

Pseudo or platelet VWD

AD

3 1

Defect in reactivity to collagen Glanzmanns thombasthenia

Defect in GPIa/IIa

Aggregation defects - reduced GPIIb/IIIa (CD41/ CD61) Fail to form aggregates Type 1 and type 2 Type 1: 0-<10% GP IIb/IIIa complexes and absent platelet fibrinogen, absent clot retraction Type 2: up to 30% GP IIb/IIIa with reduced platelet fibrinogen, reduced clot retraction - tyrosine phosphorylation processes w Plt Count.: normal Plt size: normal Plt function: Abnormal aggregation in all except ristocetin, In ristocetin, may have a primary response only Prolonged PFA100 Invest: Flow for GPIIb/IIIa detection, BT, C/P: mucocutaenous hge. ttt: :platelet transfusions, rVIIa can be used to arrest bleeding Acquired form due to auto-Abs to GPIIb/IIIa (rare)

are depended on GP IIb Storage pool defects

Dense granule disorders ( SPD) contain (ATP, ADP, calcium pyrophosphate and serotonin) Idiopathic deficiency/ AD -in number and contents of dense bodies 1 storage pool disease - has mutation on chromosome 10 Hermansky-Pudlak syndrome AR -Total absence of dense bodies or in number 2 and function of dense bodies

Aggregation is absent by collagen & decreased by ADP, adrenaline and arachidonic acid - haemorrhagic tendency & BT - Oculocutaneous albinism - accumulation of ceroid like pigment in macrophages w stimulates PDGF pulmonary fibrosis - Commonest genetic disorder mutations in HPS1 (10q) & ADTB3A (5q). - ttt with DDAVP

Chediak-Higashi syndrome

AR

- Reduced or irregular dense bodies - mutation in LYST gene (1q)

Wiskott Aldrich syndrome

XR

TAR (thrombocytopenia with absent radius) syndrome

- Reduced dense bodies & defective ADP metabolism - in T-lymphocyte sialoglycoprotein (leucosialin) CD43 - GP Ib in plt - mutation in WASP gene above disorders - 1q21.1 deletion

- mild or no he but may br marked due to increased fibrinolysis - Oculocutaneous albinism, hair abnormalities, severe infections. - Classical finding is very large peroxidase-positive cytoplasmic granules in neutrophils, monocytes, fibroblast & melanocyte but not in plt and rarely in MK. - Death usually in first decade. - characterized by eczema, microthrombocytopenia, immune deficiency, and bloody diarrhea (secondary to the thrombocytopenia).

- characterized by the absence of the radius bone and a dramatically reduced platelet count. - may include heart, kidney and knee problems, frequently lactose intolerance and often thumb hypoplasia Large and agranular, thrombocytopenia Contents of alpha granules are absent eg. PF4, PDGF, VWF, fibrinogen Platelet nucleotides are normal EM demonstrates reduced or absent granules Aggregation reduced response to collagen and ADP Associated with myelofibrosis due to PDGF P selectin retained and flow will be positive for this if platelets activated first Platelets appear grey on blood film Treat. with DDAVP +/- platelets - thrombocytopenia, giant abnormal alpha granules due to fuse normal granules but cant release their contents normally - micro-MK with abnormalmaturation Associated mental retardation, cardiac abnormalities, craniofacial abnormalities very rare - Low factor V within platelet -granules, but normal plasma levels - Low multimerin w stabilize FV and its release so, its FV release he. - Defective procoagulant activity due to failure in assembly of prothrombinase complex Urokinase plasminogen is released in large quantities upon plt activation, therefore Unresponsive to platelet transfusion, treat with anti-

Alpha granules defect (CD62P = P-selectin) contain PDGF and PF4 - Decreased alpha granules with absent contents Grey platelet syndrome 1 1- proteins necessary for cell contact as ( vWF, fibronectin, thrombospondin) 2- factors necessary for thrombin generation (FVa) or for fibrin generation (fibrinogen, PAI)

Paris-Trousseasu or Jacobson syndrome

AD

- mutation in chromosome 2

Quebec platelet syndrome or factor V quebec

AD

FV and multimerin in alpha granules

fibrinolytics No characteristic aggregometry pattern Combined alpha and dense granule disorders Rare - gene defect currently unknown

Platelet activation defects (Platelet receptors and signal transduction pathways)

1
Defect in reactivity to ADP - A defect in ADP pathway, + possible decrease in ADP receptor (P2Y12). - Gp IIb IIIa are normal - impaired ADP aggregation and ADP induced fibrinogen and vWF binding. - fibrin clot retraction induced by ADP is absent - decreased response to thrombin and collagen to lesser extent as they ADP dependent. - Ticlopedine and clopidogrel decrease plt reactivity to ADP as targets (P2Y12) receptor. 10 cases described - impaired aggregation and secretion in response to epinephrine. - aggregation to ADP and collagen are normal. - Decreased aggregation to all agonists - Normal thromboxane A2 and prostaglandin synthesis Plt Count.: Thrombocytopenia Plt size: Giant Plt function: thrombin mediated is low but defers from BSS as it has normal ristocitin aggregation. N.B. - Some drugs interfere with this step as Ca inhibitors - Asprin inhibits prostaglandin metabolism & thromboxane A2 synthesis. Hge of variable severity, prolonged BT - aggregation by arachidonic acid is nill - decreased aggregation by ADP, thrombin and collagen - impaired aggregation to arachidonic acid with preserved ristocetin response - defect in Thrombaxane synthase or COX produce similar pattern but have preserved aggregation to prostaglandin or synthetic thromboxane. Due to decreased surface exposure of phosphatidylserine (PS) defective binding of FVa, FXa. Aggregation is normal Flow to demonstrate absent PS on surface of activated platelets Prothrombin consumption index test is

2 3 4 5

Collagen receptor defects Defect in reactivity to epinephrine Defect in calcium metabolism Montreal platelet syndrome

Defects in GPVI or GPIa/IIa - there is decrease in 2 adrenoreceptors There is a defect in calcium mobilization from storage vesicles - the defect is in Ca activated neutral protinase (calpin). - Caplin is calcium-dependent, proteolytic enzymes

Defect in prostaglandin metaboism Thrombaxane A2 receptor defectd

Cyclooxygenase deficiency affects synthesis of thromboxane A2 more than prostaglandins as the later can be synthesized by endoperoxidases (side pathway).

Defects in platelet coagulant activities Scott syndrome AR 1

There is failure of scramblase activity, once platelet activated allows phosphatidylserine to move from the inner to the outer membrane w permits availability of plt factor 3 (PF3), FVa, FXa. Scramblase is a protein responsible for the translocation of phospholipids between the two monolayers of a lipid bilayer Thrombocytopathies hereditary connective tissue disorders Osteogenesis imperfecta , response to collagen, ADP and PF3. 1 Marfan syndrome, Ehlers Abnormal connective tissue itself Danlos syndrome

-show excessive hge.

Hereditary thrombocytopenia
Pathophysiology

Others
MK absent, Pancytopenia. Patients have congenital defects, commonly short stature, abnormalities of the skin, arms, head, eyes, kidneys, and ears, gonadal atrophy and developmental disabilities. Liable to develop AML MK absent, severe in plt count Absent both radii, cardiac & renal disorders facial hemangiomas, cows milk intolerance. Severe thrombocytopenia MK are absent Radio-ulnar abnormal fusion Management platelet support + HSCT Macro -hrombocytopenia (20-130) with varying platelet dysfunction (usually mild), MK normal in number & shape. Often associated with Granulocyte inclusions (Dohle-like bodies) Phenotype very variable may be associated with other congenital abnormalities including: Glomerulonephritis Sensorineural deafness Cataracts

Fanconi syndrome

2 3

Thrombocytopenia with absent radii (TAR) Amegakaryocytic thrombocytopenia with radioulnar synostosis Hereditary macrothrombocytopenias (MYH9 mutations) May-Heggelin anomaly Fechtner syndrome Sebastian syndrome Epstein syndrome Alport syndrome Alport syndrome (one of the above group)

AR & XL 2% AR

Abnormalities in several genes FANCA, -B, -C, -E, F, -G, -L and M leave them unable to repair DNA.

There is a defect in signal transduction (thrombopoietin - c-mpl signaling pathway) mutations in HOX A11

AD

AD

MYH9 gene (chr22) mutations (non-muscle myosin II-A heavy chain involved in cytoskeleton in megas, platelets and other tissues)

AD

MYH9 gene (chr22) mutations

Macro -hrombocytopenia
Glomerulonephritis Sensorineural deafness Cataracts Microthrombocytopenia, eczema and immunodeficiency (T and B cell) Platelet dysfunction (storage pool disorder or lack of GP Ib &Ia) MK are present AI disorders esp haemolytic anaemia and vasculitis Malignancy (usually lymphoreticular) Severe thrombocytopenia MK are absent

Wiskott-Aldrich Syndrome

XL

WAS gene mutation

Congenital amegakaryocytic thrombocytopenia

AR

mutations in MPL abnormal function or expression of the thrombopoietin receptor