HUS and TTP Testing. Kenneth D. Friedman, M.D. Director, Hemostasis Reference Lab BloodCenter of Wisconsin, Milwaukee WI

Transcription

1 HUS and TTP Testing Kenneth D. Friedman, M.D. Director, Hemostasis Reference Lab BloodCenter of Wisconsin, Milwaukee WI

2 Disclosures Relevant Financial Relationships Consultant: Ablynx, Bayer, CSL Behring, Shire Speaker: Alexion, Off-label drug usage None

3 Objectives Be able to list a broad differential diagnosis of thrombotic microangiopathy (TMA) Understand the laboratory characteristics and utility of ADAMTS13 assay Review laboratory studies used in evaluation of infection and complement-mediated TMA

4 Thrombotic microangiopathy Convergence of multiple pathologic mechanisms DIC TMA (TTP/i-TMA/c-TMA) HTN Common mechanism is microvascular obstruction 4 Can all have overlapping and similar clinical presentations

5 Thrombotic Microangiopathy Convergence of clinical picture Microvascular occlusion syndrome Small vessel platelet aggregates Consumptive Thrombocytopenia Red Cell Fragmentation Hemolytic Anemia, hyperbilirubinemia Multiple organ ischemia Clinical symptoms relate to vascular ischemia 5

6 TMA: Supporting lab data Supporting Laboratory Studies RESULT CBC with smear Schistocytes LDH: lactate dehydrogenase Coagulation studies nl DAT: direct Coombs' test neg Haptoglobin Bilirubin (unconjugated) Creatinine, BUN +/- 6

7 Diverse causes of microangiopathic blood film Thrombotic Microangiopathy (TMA) Thrombotic Thrombocytopenic Purpura (TTP) Idiopathic, congenital, secondary Hemolytic Uremic Syndrome (HUS) Infection-TMA (STEC+HUS and others), CM-TMA (ahus) Severe hypertension DIC, sepsis Vasculitis (SLE, Scleroderma, Antiphospholipid, etc) HELLP syndrome or Preeclampsia Disseminated malignancy Stem-cell transplantation Intravascular prosthetic devices LV assist device, prosthetic valve dysfunction

8 TMA Modified from Nester CM et al. Mol Immunol 2015 Sep;67(1):31-42.

9 TTP: Pathogenesis Absent ADAMTS13 à Platelet/VWF thrombi TPE replaces ADAMTS13 and depletes auto-antibody Immunosuppresion suppresses auto-antibody production Sadler JE: Hematology 2015; 2015:631-6

10 Assays of ADAMTS13 ADAMTS13 activity: Mix patient plasma with VWF-like substrate Assess degradation of substrate Considered diagnostic of TTP if ADAMTS13 < 10% ADAMTS13 Inhibitor: Mix patient plasma with source of ADAMTS13 Assess residual ADAMTS13 activity Sensitivity is only 45 90% depending on method Inhibitor Unit calculation similar to F-VIII BU 1 U inhibits half of protein activity in a 1:1 mix Anti-ADAMTS13 IgG Useful in cases where ADAMTS13 <10% and inhibitor negative? Specificity Positive serology observed in patients with SLE and APLA Commercial kits detect clinically irrelevant antibody in 10-15% of normal controls For Review see Peyvandi F, et al. J Thrombo Haemost 2010; 8:

11 ADAMTS13 activity Two VWF73 methods FRETS Method Cleavage of substrate abolished quenching, permitting fluorescence detection ELISA method ADAMTS13 cleaved substrate is recognized by monoclonal antibody N10. Kokame, et al:br J Haematol 2005:129 (1), Kato S, et al., Transfusion 2006:46,

12 ADAMTS13 Activity Diagnostic value in clinical TTP TTP is confirmed by severe ADAMTS13 deficiency 1 What level of ADAMTS13 activity is Severe Deficiency? 2 ADAMTS13 < 5% is more specific ADAMTS13 < 10% is more sensitive All but 1 relapsing TTP patient in Oklahoma series had < 10% But Some patients with malignancy, sepsis and HIV also < 10% Some patients with TMA that responded to plasma exchange had ADAMTS13 activity > 10% at presentation. Some authors suggest using 15-20% cut-off 1,3 1 Scully M. Hematology : George JN, et al. J Clin Apheresis 2008; 23: Li A, et al. J Thromb Haemost 2017; 16:

13 Diagnostic specificity: Pre-analytic interfering substances EDTA sample is unacceptable! ADAMTS13 <5%, Strong inhibitor effect Severe hemolysis in sample (Studt JD, et al: Blood 2005) Free hemoglobin inhibits ADAMTS13 activity Icteric sample (Meyer SC, et al: JTH 2007) Bilirubin absorbs fluorescence of Nma Reduced ADAMTS13 activity reported by FRET Positive inhibitor due to bilirubin (not autoantibody) Less important if total bilirubin < 6 Free Hemoglobin Inhibitor Titer mg/dl BU 200 < to > Bilirubin (mg/dl)

14 Severe ADAMTS13 deficiency is rare in secondary thrombotic microangiopathy Cancer Hematopoietic stem cell transplantation Solid organ transplantation Preeclampsia Systemic infection Infection associated -TMA (STEC+HUS) Complement Mediated-TMA (atypical HUS)

15 Testing ADAMTS13 activity after TPE is initiated Percent patients with ADAMTS13 < 10% 100% 89% 83% 78% 59% Serial measurement of ADAMTS13 activity ADAMTS13 activity measured by SELDI-TOF mass spectrometry Conclude: ADAMTS13 measured after initiation of TPE still offers diagnostic value Wu N, et al. Transfusion 2015; 55:18-24

16 Value of ADAMTS13 activity in TTP at presentation Diagnostic value at presentation Idiopathic TTP: Severe deficiency strongly supports a diagnosis of TTP Observing an inhibitor probably improves specificity Present in ~45-90% of idiopathic TTP at presentation Cross-over syndromes: Systemic Lupus Erythematosus: Severe deficiency of ADAMTS13 may indicate a role for apheresis Pregnancy: Important to distinguish TTP from HELLP syndrome and ahus Severe ADAMTS13 deficiency indicates TTP complicating pregnancy

17 Clinical distinction of TTP from other TMA is imprecise TTP STEC-HUS ahus Sadler JE. Hematology 2015; 2105:631-7

18 Scoring tools improve clinical decision making Association of routine labs with severe ADAMTS13 deficiency in TMA Lab Parameter Adjusted Odds Ratio 95% CI p value Creatinine 2.3 mg/dl < Platelet 30,000/uL < Coppo P, et al Plos ONE 2010; 5: e doi: Bendapudi PK, et al. Lancet Haematol 2017; 4:e157-e164.

19 Safety of withholding TPE when ADAMTS13 > 10% Retrospective selection from 186 patients (71 TPE/115 no TPE) Propensity matching performed to select 59 TPE and 59 no TPE Result: 90 day mortality ratio 0.88 (CI , P =0.72) Conclude: Routine use of TPE in a diverse cohort of patients with TMA and ADAMTS13 >10% did not significantly improve outcomes Li A, et al. Transfusion 2016; 56:

20 Value of ADAMTS13 activity in TTP at presentation Prognostic value Value at initial presentation Severe deficiency defines a subpopulation of TMA patients Most likely to have TTP More favorable response to plasma exchange therapy Value in longer term follow-up If have severe ADAMTS13 deficiency at presentation ~ 35% chance of relapse Rate of relapse increases with duration of follow-up Persistent ADAMTS13 deficiency in remission is a risk marker for relapse Should the goal of therapy be suppression of auto-antibody and restoration of the ADAMTS13 level? Sadler JE. Hematology 2015, 2015; Peyvandi, F. et al. Haematologica 2008;93: Peyvandi F, et al. J Thrombo Haemost 2010; 8: , Hovinga-Kremer JA, et al. Blood 2010; 115:

21 Congenital ADAMTS13 Deficiency Autosomal recessive inheritance Severely deficient ADAMTS13, no inhibitor/antibody May have family history of TTP Mutation analysis can confirm diagnosis Clinical course is extremely variable 1,2 Neonatal presentation ~ 45% Infancy/childhood presentation ~ 30% Adult presentations include ~ 25% TTP associated with pregnancy, infection, or idiopathic 25%% who present with pregnancy-related TTP have congenital deficiency 3 If index case found, family screening is recommended radamts13 is in clinical trail 1 Fujimura Y, Matsumoto M. Int Med 2010; 49: Fujimura Y, et al: J Thromb Haemost 2011; 9: Moatti-Cohen et al: Blood 2012; 119:

22 Summary: ADAMTS13 assay Early lab assay of ADAMTS13 clinically is useful A very low ADAMTS13 level supports a diagnosis of TTP ADAMTS13 assay of prognostic value Low activity at presentation or in remission is a risk factor for relapse Some consider ADAMTS13 level a therapeutic driver Supporting data is phase II and uncontrolled

23 STEC+ HUS One of the forms of Infection-associated TMA STEC: Shiga toxin producing E. coli Verotoxin binds globotriaosylceramide (Gb 3 ) on cell High concentrations inhibit ribosomal function Low concentrations have dramatic effects on gene regulation Consequences of Shiga toxin Local toxic effects on colonic cells à pain and bloody diarrhea Systemic toxin effects à systemic disease Renal endothelial cell are particularly sensitive to toxin à thrombosis Younger children are more susceptible 25% of survivors have long-term renal sequel CNS dysfunction occurs in more severe cases Zoja C, et al. Pediatr Nephol 2010; 25: Petruzziello TN et al. Kidney International 2009; 75 (suppl 112) s17-s19)

24 STEC+ HUS What about this is typical 1. Recognizable prodromal illness Diarrhea which is often bloody and/or quite painful TTP does not have prodromal diarrhea; abdominal pain is concurrent 2. Time course is often instructive Median time to HUS is 1 week, rarely more than 2 3. Epidemiology consistent with infectious agent 4. Corroborative tests helpful No coagulation evidence for DIC Stool studies often informative, especially if obtained early Culture evidence of precipitating E. Coli (O157:H7, O111. O104:H4, others) EIA test often positive for Shiga toxin ADAMTS13 activity is not severely decreased Tarr PI. Kidney International 2009; 75 (suppl 112) S29-S32

25 Atypical HUS Complement Medicated TMA A systemic disease which leads to thrombosis in multiple vascular beds and subsequent organ dysfunction Atypical in that: There is not the typical bloody diarrhea prodrome 1 However, trigger events occur in 22-50% of patients 2 Pregnancy may be the trigger event in 8 20% of adult cases 2 Disease may be episodic with recurrent flairs of activity 1 Familial cases usually do not present concurrently 2 Most cases appear sporadic 2 Progression to renal failure is common 1,2 ADAMTS13 activity is not severely decreased 1 Waters AM, Licht C. Pediatr. Nephrol 2011; 26: Noris M, Remuzzi G. N Engl J Med 2009; 361:

26 Complement-Mediated TMA Uncontrolled Complement Activation C3a and C5a C5-9 MAC3 Vascular Permeability Organ Dysfunction Ischemia Endothelial Injury Intimal Thickening Thrombosis Thrombocytopenia Stenosis Shear Stress Red Cell Fragmentation Modified from Tsai H-M. Am. J Med 2013;; 126:

27 Complement Medicated TMA Lectin Pathway Classical Pathway C3 Tick C3 + H 2 O Alternative Pathway Amplification C3b, Bb C3a Inactivation TM + FII àtafi C3b C5 Inactivation CFH + CFI CFH+ MCP TM + CFH + CFI ic3b Anaphylatoxin - Pro-inflammatory - Endothelial activation - Leukocyte activation C5a C5b Membrane Attack Complex - Cell Lysis - Inflammation - Thrombosis

28 CM-TMA Genetics of uncontrolled complement activation Gene Defect Frequency Rate of quantitative deficiency Remission rate with plasma therapy Rate of ESRD/Death Factor H 20-30% 25% 60% 70 80% Factor I 4-10% 40% 30 40% 60 80% CFHR1 & 3 with antibody 6% % 30 40% MCP (CD46) 10 15% 75% Not indicated < 20% Thrombomodulin 5% -- 60% 60% C3 5 10% 40 50% 60% Factor B 1 2% 30% 70% Notes: Heterozygous mutation is sufficient to cause disease No mutation found in up to 50% of cases Disease has variable penetrance: Half of carriers are disease free Waters AM, Licht C. Pediatr. Nephrol 2011; 26:41-57 Norris M, et al. Clin J Am Soc Nephol 2010; 5:

29 Complement-Medicated TMA Phenotypic Assays Serum C3 and C4 May distinguish classical from alternative pathway activation C3 variably decreased in atypical HUS Neutralizing autoantibody against Factor H Associated with homozygous deletion of CFHR1 Deletion is found in 2-5% of population Not identified by genetic testing, requires serum specimen MCP expression by flow cytometry on PBMCs Expression typically ~50% decreased in heterozygous pts But acquired mild deficiency may occur as a consequence of medical illness May be normal in qualitative defects

30 Evaluation of Complement in TMA: Potential benefits of Genetic Testing May help confirm a clinical diagnosis Provides some prognostic data Progression to end-stage renal disease CFH > CFI, CFB, C3, No mutation > MCP Transplant recurrence CFH, CFB, C3 > Factor I, No mutation >> MCP, DGKE May be useful to eliminate family donors

31 Evaluation of Complement in TMA: Challenges of Genetic Testing Logistic issues: Slow turn around: Does not inform acute management Expensive and may not be covered by insurance Medical Issues Fail to identify mutation in ~50% of patients with ahus Does not exclude ahus Does not indicate lack of efficacy of complementtargeted therapy (Legendre et al, NEJM 2013) Variants identified may not be biologically relevant Predictive algorithms disagree on pathogenicity Problem compounded by incomplete penetrance Asymptomatic family members with same mutation? Relevance without functional testing

32 Evaluation of Complement in TMA: Complement deposition on endothelial cells 1 ADP-activated endothelial cells incubated with sera from ahus pts C3, C5b-9 deposition detected by confocal microscopy Modified Ham test 2 Novel functional tests PIGA deficient TF-1 cells incubated with sera from ahus pts Colorimetric determination of cell viability 1 Noris M, et al. Blood 2014: 124: Gavriilaki, E, et al. Blood 2015; 125:

33 Coagulation driven-tma: DGKε Mutations in DGKε described in a small cohort of patients with atypical HUS Autosomal recessive with nearly complete penetrance Intracellular enzyme: endothelial cells, podocytes, and platelets Reduces activation of prothrombotic PKC Presented < 1 year of life, and had persistent urinary abnormalities (microscopic hematuria and proteinuria) Does not demonstrate recurrence after transplantation Lemaire M et al, Nature Genetics 2013

34 Lab evaluation if suspect ahus ahus: a clinical diagnosis, lab evaluation is encouraged 1-3 Investigate for STEC+ HUS Measure ADAMTS13 activity Send for levels of C3, C4, CFH, CFI, and autoantibody to Factor H Most ahus patients have normal C3 and C4 levels 4 Factor H levels are often normal in ahus patients with CFH mutations 5 Consider assay of alternate pathway function (APH50) Flow cytometry of mononuclear cells for CD46 Low levels may not be specific for ahus Genetic mutation analysis: CFH, CFI, CFHR1-5, CFB, MCP, C3, THBD, DGKε Test is expensive, turn around is slow and result requires interpretation 1 Waters AM, Licht C. Pediatr. Nephrol 2011; 26: Noris M, Remuzzi G. N Engl J Med 2009; 361: Kavanagh D, Goodship THJ. Hematology 2011: Ariceta G, et al: Pediatr Nephrol 2009; 24: Noris M, et al: CJASN 2010; 10:

35 An Approach to Thrombotic Microangiopathy Modified from Zuber J, et al. Nat Rev Nephrol doi: /nrneph Thrombocytopenia + MAHA Non TTP/HUS diagnosis: - STEC, T-activation, vasculitis, cancer, stem-cell transplant, DIC, hypertension, HELLP, CAPS, vascular devices History & laboratory tests Co-morbidity ADAMTS13 Activity Stools for shiga toxins, Urine pneumococcal Ag Autoimmune serology (ANA, APLA) DIC panel, Coombs tests Blood cultures & HIV studies as indicated Imaging studies, BM, tissue biopsy as indicated Plasma exchange No severe ADAMTS13 deficiency Complement Factors CFH CFI CFB MCP expression Anti CFH Genetic testing Severe ADAMTS13 deficiency Inhibitor assay Ab assay Serial ADAMTS13 ADAMTS13 genetics Not TTP/CM-TMA Specific management CM-TMA: Eculizumab Acquired TTP: Plasma exchange Immunotherapy Hereditary TTP: Plasma Therapy

36 Questions Can we play now?