3. Introduction
History
• Heparin causing thrombocytopenia was reported by several investigators in 1940s
• Thrombotic complications with heparin therapy was observed by Weisamn and Tobin in
1958 but did not mention thrombocytopenia
• Link between thrombocytopenia and thrombosis was first noted by Rhodes and his
colleagues in 1973
4. Introduction
Heparin
• One of the oldest biological drugs, discovered in 1916
• A highly sulphated glycosaminoglycan of different chain lengths
• Has the highest negative charge density of any known biological molecule
• Causes anticoagulation by activating naturally occurring anticoagulant AT-III
• Different sources of heparin are present; porcine, bovine and other mamals
• MW can vary from 1800Da to 30000Da; UFH-15000Da and LMWH-<8000Da
5. Introduction
Heparin
• Also occurs naturally in basophil and mast cell
• Parenteral anticoagulant used in many surgical and medical conditions
• ACS, Atrial fibrillation
• DVT and PE
• Stroke
• Cardiac bypass surgery and orthopaedic surgeries
• Anticoagulant of choice in pregnant women
6. Nomecleture
• Two types of HIT emerged in the 1980s
• First group had early onset and mild thrombocytopenia, asymptomatic with no
heparin dependent IgG antibodies this was referred to as Type 1 HIT now referred to
as non immune heparin associated thrombocytopenia
• Second group had delayed onset severe thrombocytopenia with presence of heparin
dependent IgG antibody this was referred to as Type 2 HIT i.e immune- mediated
HIT
7. Pathogenesis
1
• Heparin binds to platelets via GPIIb/IIIa complex
• Leads to signalling and activation of PI3k
2
• Low dose ADP release and causes aggregation of platelets
• Heparin enhances proaggregation affect of S. aureus, collagen
• It enhances aggregation in hyperactive platelets(sepsis,PVD, anorexia nervosa)
• Greater activation with UFH than LMWH
3 • Platelet aggregates are cleared by RES
• Leading to thrombocytopenia
Type 1 HIT
8. Pathogenesis
Type 2 HIT
• PF4 a positively charged tetrameric protein released from α-granules of platelets on
activation, it belongs to CXX chemokine family
• Heparin causes slight platelet activation leading to PF4 release
• Heparin binds strongly to PF4 this decreases the charges on these proteins, bringing PF4
tetramers closer to each other leading to immune response
• HIT antibodies IgG binds to platelets receptor FcγII leading to strong platelet activation
9. Pathogenesis
Type 2 HIT
• PF4 binds to endothelial heparin sulfate which leads to TF release
• PF4 can also activate monocyte
• Eventually leading to thrombin activation and thrombosis
• Ab coated platelet are taken up by macrophages and RES leading to thrombocytopenia
• PF4 binds to bacterial surface leading to Ab production
• PF4/heparin Ab is a misdirected bacterial defence system by our body
11. Pathogenesis
Type 2 HIT - Immune response
• IgG Ab is the predominant Ab
• develops rapidly within days of heparin exposure
• Lacks primary IgM response
• 50% of CABG and 20-30% orthpedic Sx patient develop IgG Ab at 2-3 weeks post first
time heparin exposure
• Lacks memory B-cells
• Clinically symptomatic HIT patients almost always have IgG type anti PF4/heparin Ab
• Titres of these antibodies drop rapidly within weeks, once heparin is stopped
12. Pathogenesis
Type 2 HIT Immune response
• IgM type Ab are also found but
• They lack procoagulant properties, unlike IgG IgM do not bind to FcγII receptors on
platelet
• IgA and IgM Can be found in upto 30% when patients are normally screened post
heparin exposure
13. Frequency
HIT Type 1
• Difficult to assess as patient might have already activated platelets from cormorbid
conditions; surgery, infection etc
• Some early studies suggest it might be as high as 30%
HIT Type 2
• 0.2-10%
• Bovine UFH>porcine UFH>LMWH>fondaparinux
• Surgery(major>minor)>medical>Obs/Gyn/paediatric
• Female>male
14. Frequency
HIT Type 2
• Dose duration
• Ab develop from D4 to peak at D7, most patient develop HIT while receiving heparin
• Risk increases with ≥5 of heparin therapy
• Heplock and heparin coated device have been implicated in development of anti
PF4/Heparin Ab
15. Clinical Features
HIT Type 1
• Mild decrease in platelets to about 100,000-150,000 , rarely below 80,000
• Returns to normal despite continuation of heparin
• In post op patients its difficult to differentiate HIT from perioperative hemodilution
HIT Type 2
• Thrombocytopenia develops 5-10 days after heparin exposure
• 50% fall from base line in the next 2-3 days
• If fall occurs in <24hrs- termed rapid onset HIT
16. Clinical Features
HIT Type 2
• Platelets rarely falls <10,000 unlike other causes DITP
• Platelets recovers to >150,000 on stopping heparin therapy within 4 days with 90% patient
recover within 1 week
• Resistant HIT occurs when it persist upto 30 days
• Thrombotic complications
• Develops in 70% of HIT Type 2 patient
• Severe and extensive
• Even in severe thrombocytopenia(<20,000) bleeding rarely occurs
18. Clinical Features
HIT Type 2
• Thrombotic complications
• Venous thrombosis
• More likely in CABG patients, DVT is most common, PE can occur
• Limb gangrene and phlegmasia cerulean dolen- uncommon without HIT
• Associated with coumarin therapy particularly in supratherapeutic INRs(INR>4)
• Arterial thrombosis
• More common in distal aorta and lower limb arteries
• Less commonly leading to stroke acute MI and upper limb involvement
20. Clinical Features
HIT Type 2
• DIC- develops in 5-10% of these patients
• LMWH can be associated with necrotising skin lesion even without thrombocytopenia
• Heparin resistance develops due to high levels of PF4 and other heparin binding protiens
• Acute anaphylactoid reactions
• Due to HIT Ab- occurs on administration of IV heparin
• Patient develops head ache, chest pain, dyspnea from 10-30 minutes after IV heparin
• Transient drop in platelets occurs during this time
21. Clinical Features
HIT Type 2- other terminologis
• Subclinical HIT- where patient has recovered from HIT but has persistent HIT Ab, this
patient is at risk of HIT recurrence on heparin re-exposure
• HIT Variants- HIT Ab activate platelets in absence of heparin- sometime referred to as
autoimmune HIT
• Delyed onset HIT- HIT develops 5 days after heparin has been with drawn
• Reftactory HIT- Thrombocytopenia lasting for weeks after stopping heparin
• Spontaneous HIT- Rare, occurs in the absence of recent heparin exposure
22. Diagnosis
Clinical Diagnosis
0 to 3 points Low probability
4 to 5 points Intermediate
probability
6 to 8 points High probability
Pretest probability of heparin-induced thrombocytopenia
(4 Ts score)
24. Diagnosis
Lab Diagnosis- Functional tests
• 14C-serotonin release assay(SRA)
• Pooled PRP from normal donors + 14C serotonin is incubated at 37 C for 30 mins for platelets to
take up the serotonin
• These platelets are washed with tyrodes solution and 300,000 count platelet suspension is
prepared
• 75µl platelets+ 25µl Pt serum+ 5µl heparin/buffer
• Heparin/buffer is made of different concentrations 0.1U/ml, 0.2U/ml, 0.3U/ml….. and a
higher concentration of 100U/ml
• Normal serum as negative control
• Weak HIT serum as positive control
25. Diagnosis
Lab Diagnosis- Functional tests
• 14C-serotonin release assay(SRA)
• Mixtures are placed in a microtitre plate which is agitated for 1hr after which the
reaction is topped and stopper EDTA/PBS is added
• The mixture is centrifuged and the supernatant is assayed for 14C-serotonin release
• >20% release is considered significant
• Results are positive when 14C-serotonin release occurs in heparin of therapeutic
range(0.1U/ml to 0.3U/ml) and negative in higher heparin concentration
26. Diagnosis
Lab Diagnosis- Functional tests
• Heparin induced platelet activation(HIPA)
• Similar to SRA
• Platelet from 4 healthy donors as source of PRP each tested separately
• Hirudin is added to inhibit thrombin from platelet activation
• Platelet are incubated with heat inactivated patient serum and heparin/buffer
• Mixture is placed in a magnetic stirrer at 500rpm for 45 minutes
• The reaction is read every 5 minutes for aggregation(end point), read on an aggregometer
• Aggregation positive in test serum with 2 out of 4 donors is considered positive (of heparin
concentrations within therapeutic range)
27. Diagnosis
Lab Diagnosis- Functional tests
• Platelet aggregation test(PAT)
• PRP + patient plasma + heparin/buffer is used
• Aggregation is the end point which is read on a platelet aggregometer
• Aggregation>20% is considered positive
28. Diagnosis
Lab Diagnosis- Functional tests
• Other tests
• ATP release test
• Platelet derived microparticle release
• Flow cytometry to detect platelet membrane changes
• Whole blood aggregometry
29. Diagnosis
Lab Diagnosis- Functional tests
• Limitations
• Trace thrombin can activate platelets
• Variability of response to various donor platelet of the anti PF4/heparin Ab
• PRP activation during washing steps
• Dilution of IgG during mixture preparation
30. Diagnosis
Lab Diagnosis- Immunoassays
• Solid phase enzyme immunoassay(EIA)
• Microtitre plates well coated with PF4+heparin are used
• Patient serum is added the plate is incubated for 1hr at RT
• Absorbance is read at 450nm
• Positive cut off is 3SD above mean absorbance of a large normal patient serum
• Fluid phase EIA
31. Diagnosis
Lab Diagnosis- Immunoassays
• Particle gel immunoassay(PaGIA)
• Similar to serology gel cards
• PF4/heparin coated polysterene microbeads(red) is used 20µl
• 10µl Pt serum is added and incubated for 5mins then centrifuged
32. Diagnosis
Lab Diagnosis- Immunoassays
• Instrumentation labroratorie(IL) assays
• These are automated assays
• One is based on agglutination of latex particles
• Another is based on chemiluminiscence
34. Treatment
HIT Type 1
• No specific treatment is required
• Differentiation from immune HIT can be difficult
• Decision to continue or withdraw heparin should be made carefully after carefull
evaluation of clinical picture and lab results- it’s the clinicians call
35. Treatment
HIT Type 2
• Discontinue heparin
• Start non heparin anticoagulants, avoid Vitk antagonists
• Long acting antithrombin dependent inhibitors of Fxa (fondaparinux, danaparoid)
• Short acting direct thrombin inhibitors(r-hirudin, argatroban, bivalirudin)
• Minimise platelet transfusion
• Thrombosis may require thrombolytic therapy or embolectomy
• Warfarin is avoided until platelet recovers to >150000
36. Treatment
HIT Type 2
• Re exposure to heparin
• Not recommended/avoided even after antibody disappears
• In some cases re exposure it is warranted -Cardiac/vascular surgery with antibody not
detectable or non platelet activating
• Familiarity of surgeons/anaesthetist and perfusionist with heparin
• Lack of short acting anticoagulant with an antidode and proven track record of
heparin in cardiac/vascular surgeries
• Low risk of HIT especially when use of UFH is restricted to surgery itself
