Platelets Flashcards
Thrombocytopenia - definitions
Normal 150- 450
Mild 100-149
Mod 50-99
Severe <50
Approach to thrombocytopenia
Confirm:
Preanalytical
- diluted sample, WBIT, underfilled,
- clotted sample
- plt clumping or satellitism
Analytical
- large plts counted as WBC
Post analytical
Review blood film:
- plt clumping, fibrin strands
- giant platelets +/- WBC inclusions
Causes of thrombocytopenia
Decreased production
increased destruction
- Immune vs non immune
Splenic sequestration
Platelet function testing
PFA-200 (screening tool)
Lumiaggregometry
Specialized testing:
- Electron microscopy
>For loss of alpha and dense granules
- Flow cytometry
>for loss of GPIb (CD42) in Bernard Soulier
>loss of GPIIb/IIIa (CD41, CD61) in Glanzmann thrombasthenia
Platelet function
- Adhesion (to the subendothelium)
- Activation (shape change, mediated by phosphorylation of receptor pathways and calcium mobilization)
- Aggregation (binding of fibrinogen to GPIIb/IIIa - a key event in Platelet aggregometry method)
GPIb/V/IX
(GP1b/5/9)
plt ADHESION
Binds to vWF/collagen
Disease:
VWD and Bernard Soulier
GPIIb/IIIa
plt AGGREGATION
Via fibrinogen
Disease:
Glanzman thromasthenia
Hypo/afibrinogenaemia
Alpha granules
LARGE molecules (vWF, fibrinogen, PF$, growth factors)
Disease:
GREY platelet syndrome
Dense granules
SMALL molecules (ADP, 5HT, Ca2)
Disease:
Chediak Higashi syndrome
Hermansky Pudlak
Actin and mysoin
plt ACTIVATION and SHAPE change
Actin failure = Wiskott Aldrich
Myosin failure= May Hegglin
Macrothrombocytopenia
May-Hegglin/MYH9 related disease
Bernard-Soulier
vWD type 2B or platelet type
Grey platelet syndrome
Conditions of increased platelet turnover (e.g. ITP)
Microthrombocytopenia
Wiskott-Aldrich syndrome
X-linked thrombocytopenia
PFA - 200
Sample: whole blood citrate collection
Principle: whole blood aspirated through cartridges through an aperture at high shear rates that are coated with either ADP/collagen or epinephrine/collagen at high sheer stress.
INNOVANCE PFA P2Y - detects blockade in patients on clopidogrel/ticagrelor.
Force of sheer stress and agonists –> stimulate vWF binding, plt attachment/activatiion and aggregation causing a formed platelet plug.
Platelet plug blocks flow –> occlusion of blood flow.
Measured as the closure time.
Prolonged closure times
- VWD: Type 2A,2B, 2M & 3
- plt disorders - GT, BSS
- drugs - aspirin/herbal medicine
NOT sensitive for some type 1/2N VWD or moderate platelet function disorders or granule defects.
Cannot RULE out diseases.
PFA -200 pre analytical considerations
fixed citrate concentration
<4 hours from collection
Decrease in Hct = prolonged closure time
Plt <100 = prolongs CT
Low VWF levels = prolongs CT (e.g. O group have lower vWF levels)
Aspirin/NSAIDs -> prolong CEPI; clopidogrel effect unpredictable
?CBP; ?liver disease ?uraemia = prolongs CT
PFA -200 advantages
Quick - 10 minutes
Only small volumes required
More physiologic as tests at high shear
Relatively insensitive to clotting factor deficiencies
Better standardized screening tests than the previously performed bleeding time.
Platelet aggregometry
Current “gold standard” test of platelet function
Standard is “turbidometric” aggregometry which uses Platelet Rich Plasma.
Measures the ability of various platelet agonists to induce in vitro activation and platelet aggregation.
Sample: citrate tube, PRP
Reagents:
- at least 5 different agonists:
ADP, Ristocetin, Collagen, Arachidonic acid, Epinephrine
> PRP is stirred in a cuvette at 37°C and the cuvette sits between a light source and a photocell.
agonist added the platelets aggregate and solution absorbs less light –> transmission increases and this is detected by the photocell.
QC: control PPP and PRP are rune in parallel with each agonist.
Aggregation curve
Baseline
Addition of an Agonist - this results in a change in platelet shape and hence a drop in the baseline absorbance
Primary wave aggregation - activation of receptors
Secondary wave aggregation - granule release
Common Platelet aggregometry patterns
- No response to all aggregating agents EXCEPT high dose ristocetin: GT, afibrinogenaemia
- Response to low dose ristocetin - type 2b vWD/PT-vWD –> perform mix to distingusih
- Normal response to all aggregating agents except ristocetin (low and high): BSS or vWD
- Absent AA and reduced 2 agg with ADP/epinephrine - TXA receptor defect/Aspirin
- Reduced 2 agg with ADP/epinephrine normal AA - storage pool disorder ?grey plt disorder
- Reduced to low dose collagen - GPVI defect , BTK inhibitor
- Weak ADP, reduced collagen and adrenaline with loss of biphasic at low doses - P2Y12 defect/clopidogrel
Platelet aggregometry - pre analytical errors
- Fasting 6h
- large bore 19-21G needle
- Discard first tube
- Release the tourniquet when the first tube starts to fill
- Collect citrate before EDTA or heparin tubes
- Ensure correct filling of tubes (>90% filling)
- 6x gentle inverstions (avoid vigorous shaking)
- Avoid pneumatic tube systems for transport
Preparation of platelet-rich plasma (PRP) requires centrifugation at ~ 10 min x 200g without braking, plt count aim150-600 G/L*
Store samples at room temperature until analysis and use within 4 h of collection
Medications/diet
- Avoid smoking and caffeine prior
- Herbs/spices – cumin, garlic, onion, ginger
- NSAIDs/aspirin/antiplatelets (10 days prior)
- SSRIs
- Frusemide, CCB, beta blockers
- Antibiotics (beta lactams, HCQ)
- Lipaemia
- Vitamin C deficiency
- Vitamin E intake
Plt agg - low dos vs high dos ristocetin
Ristocetin at high doses - causes platelet agglutination through the vWF and GPIb/V/IX complex.
GT/ afibrinogenemia = Absent or markedly impaired aggregation to all agonists except
Ristocetin. Ristocetin-induced agglutination shows only primary wave - aggregation cannot occur because fibrinogen cannot bind. Afibrinogenaemia gives similar results.
Bernard-Soulier syndrome/ vWD = Absent or markedly reduced platelet agglutination with high dose Ristocetin.
Congenital platelet disorders (grouped)
- Abnormalities of receptors for adhesive proteins:
- GP1b-V-IX = Bernard Soulier Syndrome or Platelet type vWD
- GP2b/3a = Glanzman thrombasthenia - Abnormalities of receptors for soluble agonists:
- Collagen receptor, P2Y12 or TXA2 receptor - Primary secretion defects
- Storage pool disease
4. Abnormalities of procoagulant function
- Scott’s syndrome (phospholipid scrambling)
5. Abnormalities in the cytoskeleton
- Wiskott-Aldrich syndrome (actin)
- MYH9-related disorders (myosin)
Bernard Soulier Syndrome
RARE 1 in a million
ADHESION defect
Defect in GPIB/IX/V
Why thrombocytopenia? The GPIbIX/V complex on platelets is the major locus for platelet sialic acid residues, which can shorten platelet survival
Variable clinical symptoms – fatal haemorrhage rare
Diagnosis:
- Macrothrombocytopenia
- reduced aggregation to ristocetin
- flow to detect surface GP1b
- genetic testing for 1b gene
No specific treatment – may require platelet transfusion , TXA
Limited role for DDAVP
rFVIIa (BSH recommendation)
Glanzmann Thrombasthenia
RARE 1 in million
Aggregation defects in general:
GPIIb/IIIa defect (important in primary and secondary aggregation)
Symptoms:
life-long spontaneous easy bruising, epistaxis and gum bleeding, often requiring platelet transfusions
Investigations:
- NORMAL plt count and morph
- Aggregation impaired with all physical agonists, EXCEPT high dose risto
- Flow for surface GP2b/3A
- Genetic testing – allow for DNA based carrier detection
Treatment
- Platelet transfusion
Storage pool disease
Platelet storage pool diseases are a heterogeneous group of disorders associated with an abnormal presence or contents of intracytoplasmatic platelet granules, causing a mild to moderate bleeding diathesis characterised mainly by mucocutaneous bleeding (epistaxis, menorrhagia, and easy bruising)
ALPHA granule deficiency
- contain BIG molecules (vWF, fibrinogen, factor V)
Gray platelet syndrome
> mod thrombocytopenia, MACRO plts
> assoc with early MF
> Dx -> electron microscopy
DENSE granule deficiency
- contain SMALL molecules (ADP/serotonin)
Chediak Higashi
> Dx -> electron microscopy
MYH9 related disorders
Defect in nonmuscle myosin heavy chain class IIA
Disrupts maturation and fragmentation of megas –> LARGE plts
Dohle like inclusions in neutrophils (myosin heavy chains)
Clinical sx:
- bleeding/early cataracts/deafness/nephritis
Lab:
- mild MACROthrombocytopenia
Treatment - usually not required if asymptomatic
Thrombocytopenias associated with BM failure syndromes
Fanconi anaemia
Congenital amegakaryocytic thrombocytopenia (CAMT)
- C-mpl receptor
Thrombocytopenia and absent radii (TAR)
- RBM8A gene
Radioulnar synostossis with amegakaryocytic thrombocytopenia (RUSAT)
Neonatal thrombocytopenia
Platelet refractoriness
inability or repeated failure to achieve a sustained an adequate platelet increment post transfusion from random donors
PPI <10 performed at 10-60 minutes post on more than 2 occasions
Non-immune - 80%
>sepsis, infection, splenomegaly, fever, drugs
Immune - 20%
>HPA or HLA Abs
> drug dependent Abs
>ABO Abs
if ?Immune –> step is to try a logical approach to unit selection ABO compatible plt in first instance
(high titres of A and B antibodies can clear platelets)
Consider apheresis plts
Immune:
- most commonly due to HLA class 1 antibodies
plt express HLA-A and B.
-@ risk if previous pregnancies or repeated transfuson
- Can also be due to HPA > A antigen most common less antigenic than HLA w lower rates of alloimmunisation
Management
> if HLA class 1 antibodies present
use HLA compatible plt
if poor response test for HPA antibodies and if present give HPA compatible units
> if HLA class 1 negative
consider non-immune causes
move onto HPA testing
HLA typing
- done via luminex Labtype SSO
- detects DNA sequence variation by the presence of absence of nucleotide probe binding to PCR product
- 100+ beads per sample
- each bead has a unique HLA sequence SSO probe bound to it
- beads incubated with patient DNA
- the PCR product hybridises with bead
- beads run through flow cytometer to determine pt HLA type
HLA antibody screening
- done by luminex technology LabScreen
- commercial product using labelled beads
- each bead coated with purified HLA antigens
- HLA antibodies bind to bead, and a secondary PE-labelled anti-human IgG attaches and is read via flow cytometry
- allows the presence and specificity of HLA antibodies to be defined
