THROMBOCYTOPENIA Decreased production Increased destruction Sequestration Pseudothrombocytopenia

PSEUDOTHROMBOCYTOPENIA Artifactually low platelet count due to in vitro clumping of platelets Usually caused by antibodies that bind platelets only in presence of chelating agent (EDTA) Seen in healthy individuals and in a variety of disease states Diagnosis: Marked fluctuations in platelet count without apparent cause Thrombocytopenia disproprotionate to symptoms Clumped platelets on blood smear "Platelet satellitism" - platelets stuck to WBC Abnormal platelet/leukocyte histograms Platelet count varies with different anticoagulants

PSEUDOTHROMBOCYTOPENIA Anticoagulant-dependent clumping of platelets in pseudothrombocytopenia. Platelet clumping in EDTA No clumping in heparin

PSEUDOTHROMBOCYTOPENIA Anticoagulant-dependent clumping of platelets in pseudothrombocytopenia. Platelet “satellitism” Blood 2012;119:4100

DECREASED PLATELET PRODUCTION Marrow failure (pancytopenia) aplastic anemia, chemotherapy, toxins B-12, folate or (rarely) iron deficiency Viral infection Drugs that can selectively reduce platelet production Alcohol Estrogens Thiazides Chlorpropamide Interferon Amegakaryocytic thrombocytopenia myelodysplasia (pre-leukemia) immune? (related to aplastic anemia) Cyclic thrombocytopenia (rare) Inherited thrombocytopenia

INHERITED THROMBOCYTOPENIA Congenital aplastic anemia/Fanconi's anemia Congenital amegakaryocytic thrombocytopenia Thrombocytopenia with absent radius syndrome (recessive) Bernard-Soulier syndrome (recessive, giant platelets, defective function, GPIb defect) May-Hegglin anomaly and related disorders (dominant, leukocyte inclusions, giant platelets, MYH-9 mutation) Other autosomal dominant syndromes (platelets may be normal or giant) Wiskott-Aldrich syndrome (X-linked, immunodeficiency, eczema)

May-Hegglin anomaly PMN inclusions Peripheral blood smear in a patient with May-Hegglin anomaly, a form of dominantly-inherited thrombocytopenia characterized by giant platelets and neutrophil inclusions. Giant platelets (macrothrombocytopenia)

Bernard-Soulier syndrome Wiskott-Aldrich syndrome Macrothrombocyte Microthrombocytes Drachman, Blood 2004:103:390

THROMBOCYTOPENIA AND PREGNANCY Benign thrombocytopenia of pregnancy Occurs in up to 5% of term pregnancies Accounts for about 75% of cases of thrombocytopenia Asymptomatic, mild, occurs late in gestation Microangiopathy (Preeclampsia/eclampsia, HELLP) ITP (? increased incidence in pregnancy)

INCREASED PLATELET CONSUMPTION Immune destruction Intravascular coagulation (DIC or localized) Microangiopathy Damage by bacterial enzymes, etc

IMMUNE PLATELET DESTRUCTION Autoimmune (ITP) Childhood Adult Drug-induced Heparin Quinine, others Immune complex (infection, etc) Alloimmune Post-transfusion purpura Neonatal purpura

POST-TRANSFUSION PURPURA Caused by re-exposure to foreign platelet antigen via blood product transfusion Almost all cases in multiparous women HPA-1a (formerly PLA1) antigen most commonly involved (present in 98% of population) Antibodies cause destruction of patient’s own platelets by uncertain mechanism Possibly due to coating of patient’s platelet by donor platelet microparticles Typically presents as sudden onset of severe thrombocytopenia (<10K) 5-7 days after transfusion Consider this diagnosis whenever there is sudden, severe, unexplained drop in platelet count in hospitalized patient Diagnosis: clinical suspicion, demonstrate anti HPA-1a (or other) alloantibodies in serum Treatment: IVIG, avoid giving HPA-1a positive platelets; wash RBC to remove passenger platelets

THROMBOCYTOPENIA AND INFECTION Immune complex-mediated platelet destruction Childhood ITP Bacterial sepsis Hepatitis C, other viral infections Activation of coagulation cascade Sepsis with DIC Vascular/endothelial cell damage Viral hemorrhagic fevers Rocky Mountain Spotted Fever Damage to platelet membrane components by bacterial enzymes (eg, S pneumoniae sialidase) Decreased platelet production Viral infections (EBV, measles) Mixed production defect/immune consumption HIV infection

DRUG-INDUCED IMMUNE THROMBOCYTOPENIA Quinine type: Drug binds to platelet membrane Antibody binds to drug Exposed Fc portion targets platelet for destruction Thrombocytopenia typically severe, with bleeding Some patients develop microangiopathy with renal failure (HUS) Heparin type: Heparin-platelet factor 4 complex forms on platelet surface Antibody binds to complex Fc portion of antibody binds to platelet/monocyte Fc receptor Platelet and monocyte activation triggered Thrombocytopenia typically mild to moderate Associated with thrombosis in many patients

DRUGS MOST LIKELY TO CAUSE THROMBOCYTOPENIA * Hematology 2009;153

ITP Childhood form (most < 10 yrs old) May follow viral infection, vaccination Peak incidence in fall & winter ~50% receive some treatment ≥75% in remission within 6 mo Adult form No prodrome Chronic, recurrences common Spontaneous remission rate about 5%

Childhood (acute) ITP Adult (chronic) ITP

Epidemiology (adults) ITP Epidemiology (adults) Prevalence: 1-10/100,000 Slightly more common in women Peak incidence at age 60+ About 40% have an associated disorder

ITP: associated disorders SLE Antiphospholipid syndrome CLL Large granular lymphocyte syndrome Autoimmune hemolytic anemia (Evans syndrome) Common variable immune deficiency Autoimmune lymphoproliferative disorder (ALPS) Autoimmune thyroid disease Sarcoidosis Carcinomas Lymphoma H pylori infection Following stem cell or organ transplantation Following vaccination

ITP IS A RELATIVELY BENIGN DISEASE A PROSPECTIVE STUDY OF ITP IN 245 ADULTS Overall incidence 1.6/100,000/yr Median age 56, F:M 1.2:1 12% presented with bleeding, 28% asymptomatic 18% needed no treatment Only 12% needed splenectomy 63% in remission (plts > 100K) at end of followup period (6-78 mo, median 60) 87% had at least partial remission (plts >30K) and freedom from symptoms 1.6% died from complications of disease or its treatment Age-specific incidence This study challenges the notion of ITP as a chronic disease that preferentially affects women. The age of patients was also higher than in many other published series. Brit J Haematol 2003;122:966

ITP Pathogenesis ITP plasma induces thrombocytopenia in normal subjects Platelet-reactive autoantibodies present in most cases Often specific for a platelet membrane glycoprotein Antibody coated platelets cleared by tissue macrophages Most destruction in spleen (extravascular) Most subjects have compensatory increase in platelet production Impaired production in some (many?) patients Intramedullary destruction? Enhanced TPO clearance?

Splenectomy specimen from a patient with chronic ITP Splenectomy specimen from a patient with chronic ITP. Left: (40x) Activated lymphoid follicles with expanded marginal zone (short arrow), a well-defined dark mantle zone (long arrow), and a germinal center containing activated lymphocytes (arrowhead). Right: (1000x) PAS stain showing histiocytes with phagocytized platelet polysachharides (arrows). Transfusion 2005;45:287

Infusion of ITP plasma into a normal person causes dose-dependent thrombocytopenia Infusion of ITP plasma into normal subject causes dose-dependent thrombocytopenia.

Clinical features and diagnosis ITP Clinical features and diagnosis Petechiae, purpura, sometimes mucosal bleeding Major internal/intracranial bleeding rare Mortality rate < 5% Absence of constitutional symptoms or splenomegaly Other blood counts and coagulation parameters normal No schistocytes or platelet clumps on blood smear Marrow shows normal or increased megakaryocytes (optional) Rule out other causes of thrombocytopenia (HIV, etc)

Confirmatory laboratory testing ITP Confirmatory laboratory testing Serum antiplatelet antibody assay (poor sensitivity) Test for specific anti-platelet glycoprotein antibodies (more specific, negative in 10-30%) Confirmatory testing not necessary in typical cases

ITP IN CHILDREN Management Platelets > 20-30 K, no bleeding: no Rx 30-70% recover within 3 weeks Platelets < 10K, or < 20K with significant bleeding: IVIg or corticosteroids prednisone, 1-2 mg/kg/day single dose IVIg 0.8-1g/kg as effective as repeated dosing Splenectomy reserved for chronic ITP (> 12 mo) or refractory disease with life-threatening bleeding pre-immunize with pneumococcal, H. influenzae and meningococcal vaccines

Treatment of newly diagnosed adults ITP Treatment of newly diagnosed adults Indications: Platelets < 20-30K Active bleeding or high bleeding risk, platelets <50K First line treatment options: Glucocorticoids IVIG Anti-Rh globulin (WinRho) Splenectomy Rituximab? Hospitalization often not necessary

Glucocorticoid therapy ITP Glucocorticoid therapy Mechanism of action: Slows platelet destruction, reduces autoantibody production Prednisone, 1-2 mg/kg/day (single daily dose) Begin slow taper after 2-4 weeks (if patient responds) Consider alternative therapy if no response within 3-4 weeks About 2/3 of patients respond (plts > 50K) within 1 week Most patients relapse when steroids withdrawn Advantages: high response rate, outpatient therapy Disadvantages: steroid toxicity (increases with time and dose), high relapse rate

Prednisone attenuates the effect of ITP plasma infusion on platelet count Prednisone blunts the decrease in platelet count caused by ITP plasma infusion.

ITP Splenectomy Sustained remission in 2/3 of patients Almost all responses occur within 7-10 days of splenectomy Operative mortality < 1% Severe intra- and postoperative hemorrhage rare (about 1% of patients) Laparoscopic splenectomy usually preferable technique Advantage: High sustained response/cure rate Disadvantages: Operative risk (mainly older pts with comorbid disease); post-splenectomy sepsis (fatality rate 1/1500 patient-yrs); increased risk of cardiovascular events Indication: Steroid failure or relapse after steroid Rx (persistent severe thrombocytopenia or significant bleeding)

Splenectomy attenuates the effect of ITP plasma infusion on platelet count More antibody needed to cause thrombocytopenia in asplenic subject.

LAPAROSCOPIC SPLENECTOMY IS THE PROCEDURE OF CHOICE IN ADULT ITP A Systematic Review 66% complete response rate (2632 patients) No preoperative characteristic consistently predicted response Type of procedure Mortality Complication rate Open 1% 12.9% Laparoscopic 0.2% 9.6% Blood 2004;104:2623

Intravenous immunoglobulin therapy ITP Intravenous immunoglobulin therapy Possible mechanisms of action: Slowed platelet consumption by Fc receptor blockade Accelerated autoantibody catabolism Reduced autoantibody production Dose: 0.4 g/kg/d x 5 days (alternative: 1 g/kg/d x 2 days) About 75% response rate, usually within a few days to a week Over 75% of responders return to pre-treatment levels within a month Advantages: rapid acting, low toxicity Disadvantages: high cost, short duration of benefit, high relapse rate Indications: Lifethreatening bleeding; pre-operative correction of platelet count, steroids contraindicated or ineffective

Treatment options for relapsed or refractory disease ITP Treatment options for relapsed or refractory disease No treatment (platelets > 10-20K, no major bleeding or bleeding risk) Corticosteroids (low-dose, or pulse high dose) I.V. Ig (frequent Rx, very expensive) Accessory splenectomy Vinca alkaloids Danazol Colchicine Eradication of H. pylori, if present Rituximab Thrombopoiesis-stimuating drugs Romiplostim (Nplate) Eltrombopag (Promacta) Accessory spleen

Intervention: Rituximab, 375 mg/m2 weekly x 4 doses Background: Most patients treated for ITP with corticosteroids relapse when steroids are withdrawn. Many patients can be cured by splenectomy, but some are not. Treatments for relapsed and chronic ITP and adults have variable efficacy. Subjects: 57 adults with chronic ITP, with platelet counts <30K. All had at least 2 prior ITP treatments and 31 had been splenectomized. Intervention: Rituximab, 375 mg/m2 weekly x 4 doses Outcome: Overall 54% response rate (32% with plts >150K, 22% with plts 50-150K). 89% of complete responders remained in remission a median of 72.5 weeks after treatment. Toxicity was minimal. Conclusion: Rituximab is a promising treatment for chronic/refractory ITP The most effective treatment for patients with ITP who relapse after corticosteroid treatment and/or splenectomy has not been established. This paper shows that Rituximab is effective in a majority of these patients, with minimal toxicity. British Journal of Haematology 2004; 125: 232–239

Outcomes 5 years after response to rituximab therapy in children and adults with immune thrombocytopenia Vivek L. Patel, Matthieu Mahévas, Soo Y. Lee, Roberto Stasi, Susanna Cunningham-Rundles, Bertrand Godeau, Julie Kanter, Ellis Neufeld, Tillmann Taube, Ugo Ramenghi, Shalini Shenoy, Mary J. Ward, Nino Mihatov, Vinay L. Patel, Philippe Bierling, Martin Lesser, Nichola Cooper, and James B. Bussel 26% of children and 21% of adults with ITP who had an intial response (CR or PR) to rituximab remained in remission without further treatment after 5 years No major toxicity observed Children did not relapse after 2 years, but adults did The most effective treatment for patients with ITP who relapse after corticosteroid treatment and/or splenectomy has not been established. This paper shows that Rituximab is effective in a majority of these patients, with minimal toxicity. Blood 2012;119:5989

ROMIPLOSTIM FOR CHRONIC ITP Patients: 125 patients with chronic ITP (about half had been splenectomized) Intervention: Random assignment to treatment with romiplostim (AMG 531) or placebo Endpoint: Durable platelet response (platelets at least 50K during at least 6 of last 8 wks of treatment) Outcome: 16/42 splenectomized patients had durable response with romiplostim, vs 0/21 with placebo. 25/41 non-splenectomized pts had durable response with romiplostim vs 1/21 with placebo. 20/23 pts on romiplostim stopped or reduced concurrent treatment for ITP, vs 6/16 on placebo. No significant toxicity from romiplostim Lancet 2008;371:395

Lancet 2008;371:395

Responses to oral eltrombopag in ITP Lancet 2010

LONG-TERM OUTCOMES IN ITP PATIENTS WHO FAIL SPLENECTOMY McMillan and Durette, Blood 2004;104:956-60 Most patients with “refractory” ITP eventually attain stable remission, but this takes a long time in many cases. There is a subpopulation with resistant disease that has significant morbidity and mortality. 11 deaths from ITP-related bleeding; 6 deaths from complications of Treatment (5%) Median time to remission 46 mo

EMERGENCY TREATMENT OF ITP Platelet transfusion + high dose steroids Platelet transfusion + continuous IVIG rVIIa (risk of thrombosis) Antifibrinolytics Emergent splenectomy

ITP AND H PYLORI Up to 50% of patients with ITP and concomitant H pylori infection improve after eradication of infection Confirm infection via breath test, stool antigen test or endoscopy Higher response rates in: Patients from countries with high background rates of infection Patients with less severe thrombocytopenia

ITP IN PREGNANCY Mild cases indistinguishable from gestational thrombocytopenia Rule out eclampsia, HIV etc Indications for treatment platelets < 10K platelets < 30K in 2nd/3rd trimester, or with bleeding Treatment of choice is IVIg corticosteroids may cause gestational diabetes, fetal toxicity Splenectomy for severe, refractory disease some increased risk of preterm labor; technically difficult in 3rd trimester Potential for neonatal thrombocytopenia (approx 15% incidence) consider fetal blood sampling in selected cases consider Cesarian delivery if fetal platelets < 20K