Overview

• Platelets– Normal Physiology– Categories of Thrombocytopenias

• ITP• TTP• HIT

• Thrombocytopathy• Thrombocytosis

Normal Physiology-Production and Number

Platelets are normally made in the bone marrow from progenitor cells known as megakaryocytes.

Normal platelet lifespan is 10d. Every day, 1/10 of platelet pool is replenished.

Normal platelet count is between 150 and 450 x 109/l.

Platelet Response

•Platelets provide phospholipid scaffold for thrombin generation.

•Platelets adhere

to vessel wall, then

aggregate, leading

to formation of a

platelet plug

Platelets physiology

PLT contain: mitochondria, glycoprotein containing granules, lysosomes, and:

2 types of granules:

-granules: fibrinogen, von Willebrand f., fibronectin, f. V, PLT f. 4, -thromboglobulin, thrombospondin, PDGF

dense granules: serotonin, calcium, ADP, ATP

PLT surface: GpIb: receptor for vWf

GpIIb-IIIa: receptor for fibrinogen, fibronectin, vitronectin

Platelet disorders

Thrombocytopenia

Thrombocytopathy

Combined

Thrombocytosis

Platelet disorders

Thrombocytopenia

Thrombocytopathy

Combined

Acquired

Congenital/inherited

Platelet disorders

Thrombocytopenia

Thrombocytopathy

Combined

Acquired

Congenital/inherited

Symptoms: Asymptomatic Bleeding Thrombosis

Thrombocytopenias

PLT < lower limit of the range, usualy < 130 x 109/l

ITP definition: PLT < 100 x 109/l Severe thrombocytopenia: PLT ≤ 20 x 109/l

Congenital (rare): Fanconi´s s., Wiskott-Aldrich´s s., May-Hegglin a., Bernard-Soulier´s s., amegakaryocytic trombocytopenia. Often include thrombocytopathy

Acquired: frequent

Thrombocytopenia:5 broad categories of causes

• Pseudothrombocytopenia

• Underproduction (AL, MDS, congenital TP, infiltration of bone marrow)

• Peripheral Destruction or consumption (ITP, TTP, DIC)

• Splenic sequestration (hypersplenism)

• Other: dilution (multiple erytrocyte transfusions etc.)

Pseudothrombocytopenia

•

Pseudothrombocytopenia• Artificial platelet clumping in the tube with EDTA

• Platelet clumping is of no clinical significance

• No increased risk of bleeding or clotting

• How to confirm:

1. Clumps in light microscopy 2. Repeated blood count test using citrate or Heparin as anticoagulant agent reveal normal PLT count

Thrombocytopenia due to Peripheral Destruction

Non-immune mechanisms: Platelet activation and consumption: e.g. TTP and DIC

Immune Mechanisms: ITP antibody-mediated platelet destruction may be primary, secondary, or drug-

induced.

ITP – Immune Thrombocytopenia (in the past: „Idiopathic Thrombocytopenic Purpura“) Definition: isolated thrombocytopenia with no clinically apparent associated conditions or other causes of thrombocytopenia.

Etiology: autoantibodies directed against platelets coat platelet surface. IgG-coated platelets are taken up by RE system.

Incidence: approximately 100 per million; half of these are children. In adults, two peaks: one are young (<40) with female predominance, one are older (>60), no gender predominance.

ITP – Immune Thrombocytopenia (in the past: „Idiopathic Thrombocytopenic Purpura“) Definition: isolated thrombocytopenia with no clinically apparent associated conditions or other causes of thrombocytopenia. PLT < 100x109/l

Etiology: autoantibodies directed against platelets coat platelet surface. IgG-coated platelets are taken up by RE system.

Incidence: approximately 100 per million; half of these are children. In adults, two peaks: one are young (<40) with female predominance, one are older (>60), no gender predominance.

The most common cause of thrombocytopenia

ITP: terminology

Newly diagnosed ITP: within 3 months from

diagnosis Persistent ITP: between 3 to 12

months from diagnosis. Chronic ITP: lasting for more than 12

months

Severe ITP

Presence of bleeding symptoms at presentation sufficient to mandate

treatment, or occurrence of new bleeding symptoms requiring additional therapeutic intervention with a different platelet-enhancing agent or an increased

Autoantobodies in ITP: 75% GpIIb/IIIa (CD41) (αβ-integriny)

GpIbIX (CD42) HIV positive: anti-IIIa

12- lipooxygenase

Arach. acid

NADPH oxidase

Superoxides + free 02 radicals

Autoantobodies in ITP: 75% GpIIb/IIIa (CD41) (αβ-integriny)

GpIbIX (CD42) HIV positive: anti-IIIa

12- lipooxygenase

Arach. acid

NADPH oxidase

Superoxides + free 02 radicals

OpsonisationADCC

Ab mediated autocytolysis

ITP – pathogenesis

+

differentiation & maturation of MGKC apoptosis of megakaryocyte

Antibodies

T LYMPHOCYTES

Shorter PLT lifespan

Relative thrombopoetin deficit in ITP

23

Normal(n=96)

Aplasticanemia

ITP(n=45)

0

100

200

300

400

0

250

500

750

1000

1250

1500

1750Mean PLT count (x 109/L)Maximal eTPO level (pg/mL)

PLT

cou

nt (

x 10

9 /L)

eTP

O (pg/m

L)

(n=23)

Podle: Nichol. Stem Cells 1998;16(suppl 2):165–175

ITP = increased PLT destruction and suboptimal PLT production

Adapted from: Kuter et al. Thrombopoiesisand Thrombopoietins: Molecular, cellular, preclinical and clinical biology. Totowa, NJ: Humana Press Inc;1997

Role of lymphocytes in pathogenesis of ITP

• Cytokine profile TH1/Th0 corelates with disease activity

• Regulatory T cells reduced + not functional

• T cells can directly influence MGKCs and decrease PLT production

• 25% ITP without detected antibodies: „T cell - mediated toxicity“

B lymfocytes in ITP

• Autoantibodies production

BAFF (B cell activating factor): TNF family

- Produced and released by monocytes a T lymphocytes

- increases lifespan of B and T lymphocytes decreases apoptosis

- BAFF itself increases apoptosis in

thrombocytes - mRNA BAFF in ITP patients s ITP is increased

Monocytes and Tregs in ITP

CD4

CD25 Foxp3

CD28

Ag

IL-2

TGFβ

MONO

TPO-R A

CD4

PLT -

Ig-

+

ITP - Diagnosis

ITP is a Diagnosis of Exclusion (diagnosis per exclusionem)

No specific laboratory test can confirm diagnosis of the ITP

Need to exclude other causes of thrombocytopenia

Primary/secondary ITP

SLE, systemic lupus erythematosus; APS. antiphospholipid antibody syndrome; CVID, common variable immunodeficiency; CLL, chronic lymphocytic leukaemia; ALPS, autoimmune lymphoproliferative syndrome

Cines D et al. Blood2009;113:6511–6521

Primary/secondary ITP

SLE, systemic lupus erythematosus; APS. antiphospholipid antibody syndrome; CVID, common variable immunodeficiency; CLL, chronic lymphocytic leukaemia; ALPS, autoimmune lymphoproliferative syndrome

Cines D et al. Blood2009;113:6511–6521

Evaluation of Patient with Low Platelets

History Has the patient ever had a normal platelet count?

Family history Carefully review medications, including Over the

counter meds. Antibiotics, quinine, anti-seizure medications

Ask about other conditions which may be associated with low platelets Liver Disease/hepatitis Thyroid Disease - both hypo- and hyper- Infections: viral, rickettsial Pregnancy

Consider other conditions which may be associated with ITP Lupus erythematosus, CLL, lymphoma,

antiphospholipid syndrome

Evaluation of Patient with Low Platelets

Physical Evaluate for lymphadenopathy and splenomegaly Look for signs of bleeding Blood blisters and oral petechiae, ie “Wet Purpura”

best harbinger of intracranial hemorrhage Laboratory Data

Other blood counts should be normal. Check B12 and folate levels. Look at peripheral blood smear to exclude

pseudothrombocytopenia, exclude TTP (especially if anemia also present.)

Send coagulation screens (PT/PTT) to exclude DIC Send HIV, hepatitis serologies and TSH H. pylori: antigen in stools or breath urease test

Consider doing a bone marrow biopsy and smear Obligatory in pts >60 years of age

Megakaryocytes should be present.

Thrombocytopenia-How low is too low?

100,000 - 50,000: no symptoms No treatment generally required.

50,000 - 20,000: first symptoms Generally need to begin therapy

20,000-10,000: may be life-threatening Sometimes requires admission

<10,000: risk for spontaneous intracranial hemorrhage, usually requires inpatient treatment

Need for PLT counts if normal PLT function

Dental prophylaxis (descaling, deep cleaning) ≥20–30 × 109/l

Simple extractions ≥30 × 109/lComplex extractions ≥50 × 109/lRegional dental block ≥30 × 109/lMinor surgery ≥50 × 109/lMajor surgery ≥80 × 109/lMajor neurosurgery ≥100 × 109/lVaginal delivery: 50 x 109/l

ITP: Serious and fatal bleeding depending on age

Podle: Cohen et al. Arch Intern Med 2000;160:1630–1638

Age

Ble

edin

g ris

k in

5 y

ears

0

10

20

30

40

50

60

70

80

90

100

<40 40–60 >60

Fatal

|Non- fatal

Management of ITP: Asymptomatic Adult

If platelet count is >40-50 x109/l, no therapy is required. Check platelet counts at designated intervals.

If platelet count is < 20-30 x109/l , begin therapy with corticosteroids.

Stop all NSAIDS and ASA to improve platelet function.

Corticosteroids in ITP

1. Prednison: 0,5 – 2mg/kg of BW/day

1. Or: Methylprednisolone i.v. 1g/D for 3-5 days followed by oral Prednison

2. Or: Dexamethason 40mg p.o.(i.v.) D1-4

CAVE: in pregnancy only Prednison 20mg/day and less and No corticosteroids in the 1st trimester!!

Initial Management of ITPAdult with Symptomatic Purpura

If platelet count is >10, treat with prednisone alone

If suspicious intracranial or GI bleeding (ulcer etc.), treat with itravenous immunoglobulins IVIg 1g/kg/d x 2d. - may require admission

If patient has severe bleeding and life is in danger: may need platelet transfusions

Along with prednisone, add Calcium and Vitamin D to prevent bone loss.

Subsequent Management of ITPAdult with Symptomatic Purpura

Follow platelet counts daily until >20, then can d/c patient with close follow-up

Once platelet count normalizes, commence a slow steroid taper over 6-8 weeks.

1/3 of adults will go into remission. 2/3 of patients will relapse during or

after steroid taper.

Management of Relapsed ITP• Once the patient relapses, may need to use

steroids to increase the platelet count out of the danger range, but THIS CANNOT SUBSTITUTE FOR DEFINITIVE THERAPY.

• Prednisone is now a crutch to support a dangerously low platelet count. Long corticosteroid treatment should be avoided.

• Options today include: 1. splenectomy, if contraindicated (including

patient´s refusal): 2. TPO mimetics (Romiplostim, Eltrombopag). 3. In the past: intermittent treatment with anti-

D immune globulin.

Management of Relapsed ITP: Splenectomy Splenectomy is effective in 2/3 of

patients, leading to normal platelet counts.

Can be performed via open method or laparoscopically.

Need to vaccinate against encapsulated bacteria 2 weeks before procedure.

May need steroids and/or IVIg before procedure to boost platelet counts preoperatively.

Management of Relapsed ITPAnti-D Immune Globulin

Can be used as a substitute for IVIg for maintenance therapy

Especially useful in patients with contraindications to splenectomy.

Coats red cells with IgG and allows red cells to serve as decoy for splenic macrophages.

Patient must be Rh positive. Not effective after splenectomy. Designed to cause hemolytic anemia--Hgb

may drop as much as 3g/dl. Intermittent dosing may allow patients to

avoid splenectomy.

Fc carrier - part of Ig

Podle: Bussel et al. N Engl J Med 2006;355:1672–1681

Peptide domain

Romiplostim: structure

Bussel et al. N Engl J Med;355:1672–1681. Copyright © 2006 Massachusetts Medical Society. All rights reserved.

Long lasting response to Romiplostim vs placebo

Dlo

uhod

obá

odpo

věď

na

Rom

iplo

stim

(%

)

(p=.0013) (p<.0001) (p<.0001)

05 2

38

61

49

0

10

20

30

40

50

60

70

80

Splenectomized Not splenectomized All

Placebo Romiplostim

Podle: Kuter et al. Lancet 2008;371:395–403

Overall response to romiplostimem

Odp

ověď

na

Rom

iplo

stim

(%

)

(p<.0001) (p<.0001) (p<.0001)

0

147

79

8883

0

20

40

60

80

100

Splenectomized Not splenctomized All

Placebo Romiplostim

Podle: Kuter et al. Lancet 2008;371:395–403This article was published in Lancet, 371:395–403, Kuter et al. Efficacy of romiplostim in patients with chronic immune

thrombocytopenic purpura: a double-blind randomised controlled trial. Copyright Elsevier 2008

Need for salvage therapyP

acie

nti,

kteř

í pot

řebo

vali

zách

rann

ou lé

čbu

(%)

(p=.0175) (p=.0004) (p<.0001)

5762 60

26

1722

0

20

40

60

80

100

Splenectomized Not - splenectomized All

Placebo Romiplostim

Podle: Kuter et al. Lancet 2008;371:395–403 This article was published in Lancet, 371:395–403, Kuter et al. Efficacy of romiplostim in patients with chronic immunethrombocytopenic purpura: a double-blind randomised controlled trial. Copyright Elsevier 2008

Management of Relapsed ITPAnti-D Immune Globulin Can be used as a substitute for IVIg for

maintenance therapy Especially useful in patients with

contraindications to splenectomy. Coats red cells with IgG and allows red cells

to serve as decoy for splenic macrophages. Patient must be Rh positive. Not effective after splenectomy. Designed to cause hemolytic anemia--Hgb

may drop as much as 3g/dl. Intermittent dosing may allow patients to

avoid splenectomy.

No more available: induced several cases of fatal haemolytic anaemia

Case 1

A 18 y.o. female high school student presents with a rash over her lower extremities. She had a viral illness with pharyngitis 3 weeks ago. She has no other medical problems, and she takes no medications.

Physical examination reveals petechiae over the shins.

Platelet count is 1 x 109/l (!). The patient was admitted to the hospital

and was given DEX 40mg i.v. D1-4

Case 2 A 40 y.o. man presents with epistaxis to

the ER. He has no medical problems, and he takes no medications. He works as a big truck driver and has no occupational exposures. He is married and has 2 children.

Physical examination is remarkable only

for epistaxis and scattered petechiae.

The platelet count is 28 x109/l The patient is given PDN 1mg/kg/D as

outpatient but not able to drive a truck

Case 3

A 46 y.o. woman is found to have a platelet count of 20 x109/l on routine laboratory testing. She has some easy bruising and gum bleeding, but admits to not flossing.

She has no PMHx, and is on no medications. She works as gym instructor in a fitness center.

She is started on 1 mg/kg of prednisone. Adviced not to do gym lessons.

Management of Refractory ITP One third of patients will have an inadequate

response to splenectomy. Management of these patients is a chalange

and involves accepting that they have a chronic, incurable condition.

Target platelet counts should be lower--aim for about 30K or absence of bleeding.

Today:

Agonists of TPO receptor (TPO agonists):ROMIPLOSTIM, ELTROMBOPAG

In the past:

Management of Refractory ITP One third of patients will have an inadequate

response to splenectomy. Management of these patients is a chalange

and involves accepting that they have a chronic, incurable condition.

Target platelet counts should be lower--aim for about 30K or absence of bleeding.

Today:

Agonists of TPO receptor (TPO agonists):ROMIPLOSTIM, ELTROMBOPAG If not effective

In the past:

Treatment of Refractory ITP Immunosuppressive agents

Cyclophosphamide Azathioprine Rituximab (anti-CD20) – off label Cyclosporin A Mycofenolate mofetil

Adjunct agents Danazol Examine & Treat Helicobacter pylori

Other - Vincaalkaloids - Alemtuztumab (anti-CD52)

Stem cell transplant

Drugs Commonly Implicated in Thrombocytopenia

Beta-lactam antibiotics. Trimethoprim-sulfamethoxazole and

other sulfa drugs. Vancomycin. Quinine/quinidine. Heparin. Abciximab (ReoPro®). H2 blockers If a patient’s platelets fall, ALL

unnecessary drugs need to be stopped.

Case 4

A 60 y.o. woman presented with bleeding from her nose and mouth and gums.

PMHx – Hypertension, DM Medications - glucotrol, glucophage,

HCTZ, quinine for leg cramps PEx - petechiae over limbs and trunk,

blood blisters in mouth, epistaxis. Platelet count 2 x109/l

Case 4

Pt admitted to hospital, quinine stopped, patient treated with platelet transfusions and IVIg.

Platelet count rose to normal over the next 5-6 days.

Eight months later, thrombocytopenia recurred, and patient admitted to taking quinine again for recurrent leg cramps.

Drug Induced ITP

Usually, removing the offending agent is enough to allow the platelets to rise on their own.

If platelets are severely low, platelet transfusions may be required.

IVIg is particularly helpful in quinine-induced ITP.

Case 5 A 65 y.o. male smoker with a h/o

peripheral vascular disease presented to the ER with unstable angina. He was admitted to the hospital and placed on heparin. Platelet count on admission was 450. Cardiac catheterization showed severe 3-vessel coronary disease, and the patient was scheduled for CABG which occurred on hospital day #7.

Pre-op platelet count was 200. Post-op platelet count was 90.

Case 5 On hospital day #12, the patient

developed acute left leg swelling and a DVT was diagnosed by ultrasound. Platelet count was 150. The patient was started on IV heparin. The next day, he developed a pulseless left leg and had a platelet count of 30.

While in vascular radiology, he developed acute chest pain and suffered a cardiac arrest and subsequently died. Autopsy showed occlusion of all of his bypass grafts.

Evan´s syndrome

Immune thrombocytopenia (ITP)

+

Autoimmune hemolytic anemia (AIHA)

Heparin-Induced Thrombocytopenia (HIT) Seen in 1-3% of patients treated with

heparin Usually, 7-10 d after heparin started,

platelets fall by at least 1/3 to 1/2. Can occur earlier in patients who have been

previously exposed to heparin, even as SQ injections.

Less often but still risk exists with low molecular weight heparin

Caused by antibodies against the complex of heparin and PF4. These antibodies activate platelets.

Can lead, paradoxically, to THROMBOSIS, in up to half of patients.

More common in patients with vascular disease

Alternate Presentations of HIT/T Small drop in platelet count

(especially with skin necrosis) Earlier onset thrombocytopenia with

heparin re-exposure Delayed-onset thrombocytopenia/

thrombosis after stopping heparin Thrombosis after heparin exposure

HIT/T treatment

1. IF PLATELETS FALL ON HEPARIN, STOP HEPARIN IMMEDIATELY.

2. Stop heparin3. Stop heparin4. Use a different anticoagulant

1. Fondaparinux (Arixtra®)2. Lepirudin3. Argatroban

Case 6 A 36 y.o.woman presented in the summer with

fever, cefalea, nausea, vomiting and ppsychomotoric deterioration. Admitted to ICU of Neurology dept with provisional dg. Viral encephalitis – tickborn. On Hospital day 2, hemoglobin and platelet count both noted to drop. By hospital day 4, Hgb 70 g/l, Plts 12 x109/l. PT/PTT normal. Bilirubin 87 umol/l.

CSF clear from infection The patient recovered after 20

plasmaexchanges.

Thrombotic thrombocytopenic purpura (TTP)

Microangiopathic Hemolytic Anemia (MAHA) Elevated LDH, elevated bilirubin Schistocytes on the peripheral smear MUST BE PRESENT

Low platelets - MUST BE PRESENT

Fever

Neurologic Manifestations - headache, sleepiness, confusion, stupor, stroke, coma, seizures

TTP - etiology

May be associated with an antibody against or a deficiency of the protease which cleaves the ultra-high molecular weight multimers of von Willebrand’s factor. These very high molecular weight vWF multimers cause abnormal platelet activation.

Can be induced by drugs, including ticlopidine, quinine, cyclosporine, tacrolimus, mitomycin C.

Increased incidence with pregnancy or HIV

TTP - Course and Prognosis 95% fatal prior to therapy, now 5% fatal.

Treatment relies on PLASMA EXCHANGE + corticosteroids Plasma exchange is superior to plasma infusion,

but if PLEX is delayed, give FFP. Remove all inciting agents. Platelet transfusions contra-

indicated. Multiple case reports of stroke and/or death

during or immediately after platelet transfusion. Can consider giving if life-threatening

hemorrhage is present, but avoid routine platelet transfusions.

Secondary measures if no response to plasma exchange include splenectomy, vincristine

HUS - Hemolytic Uremic Syndrome Usually classified along with TTP as

“TTP/HUS” Has fewer neurologic sequelae, more

renal manifestations. More abdominal pain in symptoms Usually precipitated by diarrheal illness,

especially E. coli O157:H7 („Shigatoxin“) or Shigella

Seen more in pediatric patients, usually has better prognosis. May respond less well to plasma exchange.

Thrombocytopathy

Congenital/inherited: rare

Acquired: frequent

Congenital/inherited thrombocytopathy Bernard – Soulier´s syndrome AR inehritance GP Ib/IX defect von Willebrand´s factor not

bound adhesivity defectiveClinically: bleeding of „platelet type“, may be

dengerous and fatalDg lab: giant PLT in the blood smear, often

thromocytopenia, abnormal PLT aggregation with Ristocetin. Negative reaction with anti GP Ib/IX Ab

Therapy: Symptomatic + DDAVP, rF VIIa

Glanzman´s thrombastheniaAR inheritanceDefect ib GPIIb/IIIa fibrinogen not

bound aggregation defectiveClinically: no spontenous bleeding but

after invasion: surgery, dental, delivery, etc.

Dg lab: morfology and No. of PLT normal but aggregation with ADP, epineprine, kolagen and thrombin affected

Th: symptomatic + rFVIIa

PLT secretory dysfunction Chediak-Higashi´s sy, Wiskott-Aldrich

´s sy, Hermansky-Pudlak´s sy Defects of δ and α granules Aspirine-like disease: realease from

granules defectDg lab: pathology in PLT aggregation

with colagen, arachidonic acid, ADP.Th: Symptomatic (thrombocytes),

DDAVP, rFVIIa

Acquired thrombocytopathy

1. Diseases accociated with thrombocytopathy: Myeloprolipherative diseases Myelodysplastic syndrome Leukemias + Lymphoprolipherative disease Paraproteinemia Renal insufficiency Liver failure Autoimmune diseases Excorporeal circulation

2. Medication-related thrombocytopathy

Met. arach. acid involved: acetylsalicylic ac., corticosteroids, NSA (ibuprofen, indomethacine, phenylbutasone, thromboxane antagonists, etc.)

PLT cAMP: dipyridamol, theofylin, phospohodiesterase inhibitors,

Clopidogrel (Plavix, Thrombex) ADP receptor blocade GPIIb/IIIa inactivation aggregation

Other: penicillin, cephalosporins, dextran, tricyclic antidepressants, antihistamins, betablockers, Ca antagonists, ticlopidin, vitE, UFH, etc.

Thrombocytosis PLT > 450 x109/l Clonal thrombocytosis = ET (essential

thrombocythaemia, PMF, PV, CML) Reactive thrombocytosis: - Bacterial infections: osteomyelitis,

pneumonia, TBC, - Postsplenectomy thrombocytosis- Iron defficiency- Bleeding- Th: if >700 x109/l consider antiaggregation

Thank You very much