Microangiopathy short review- a classical description of various thrombotic microangiopathies including TTP, HUS, PIH , malignancies etc.,

This presentation gives a detailed description of thrombotic micro-angiopathies and classification with recent advances

1. Microangiopathy

2. Consensus A-Systematic review, RCT-well designed B-RCT with limitations C-case control or cohort studies ; case series D- expert opinion ; case reports 1-applicable to most subjects , benefit more than harm 2-insufficient evidence, not applicable to all ; uncertain

3. “ Thrombotic microangiopathy is a general term for the combination of microangiopathic hemolytic anemia and thrombocytopenia, often accompanied by signs and symptoms consistent with disseminated microvascular thrombosis ” - Williams hematology

4. Outline Introduction – Microangiopathy Classification DIC TTP HUS-typical atypical HUS Secondary thrombotic microangiopathy Take home points

5. Thrombotic microangiopathy is characterized by microvascular thrombosis and platelet consumption leading to thrombocytopenia and consequent hemolytic anemia leading to multiple organ dysfunction It can affect the coagulation pathways or can happen without affecting it

6. Classification of Thrombotic microangiopathy Thrombotic thrombocytopenic purpura Congenital TTP Hemolytic uremic syndrome -Shiga toxin associated Atypical Hemolytic uremic syndrome Secondary TMA DIC Infection related Transplant associated Malignancy  Pregnancy associated Autoimmune disorders- SLE, APLA, Sjogren Drugs  Cobalamin metabolism defects Malignant hypertension Mechanical Hemolysis

7. Disseminated intravascular coagulation Acquired syndrome Characterized by Systemic intravascular activation of coagulation, due to thrombin activation . ** lung, heart, liver , brain, kidney

8. Common causes of DIC Systemic infections Malignancy Trauma Obstetric complications Vascular malformations Envenomation : snake and insects bite Immunological reactions : transfusion reactions , GVHD

9. Imbalance in hemostasis

10. Lab values Prolonged PT, aPTT** and elevated FDP, D-Dimer CBC- anemia with thrombocytopenia, rapidly decreasing Monitor every 6-8 hrs ** P/S- schistocytes DIC is a disorder affecting coagulation

11. FDP vs D-dimer FDP are produced before the formation of cross linked fibrin and they are suggestive of activation of coagulation pathway only. More sensitive assay While D-dimer is formed from clots in the blood vessels. More specific assay

12. ISTH scoring for diagnosis of DIC 93% Sensitivity and 98% specificity in predicting DIH

13. Treatment Treat the underlying condition In patients with on going bleeding or who are at high risk of bleeding with <50,000/mm3- platelet and FFP No platelet/FFP transfusion in non –bleeding patients Isolated PT/aPTT Prolongation – not an indication to transfuse

14. Treatment Treat the underlying condition Bleeding management- active bleeding / at risk of bleeding FFP/platelet concentrates are to be used PT >1.5 times normal/ Platelet less than 10,000 Fibrinogen less than 100- needs cryoprecipitate 2-3 U of FFP along with 10U are sufficient Platelet concentrates 1-2U/10kg body weight Clotting factor concentrates not recommended Cryoprecipitate should not be used alone

15. Chronic, low grade DIC- giant hemangioma, metastatic carcinoma, dead fetus syndrome Plasma FDP, D-dimer are increased PT, aPTT and fibrinogen could be normal or high Normal platelet count Characterized by thrombosis more than bleeding Low dose of continuous infusion of heparin(5-10 U/kg/h) For patients with bleeding EACA or tranexamic acid can be used **(inhibit fibrin degradation)

16. Chronic low grade DIC Low grade activation of coagulation without florid bleeding manifestation Thrombotic>>>fibrinolytic Increased D-dimer Platelet , PT could be normal Treatment- low dose heparin e.g. Giant haemangioma, metastatic adenocarcinoma

17. Thrombotic thrombocytopenic purpura Systemic microvascular thrombosis with predominantly central nervous system manifestations constitutes TTP Due to impaired inhibition of VWF by ADAMTS13 Does not affect coagulation pathways 95% -ADAMTS13 autoantibody 5%- loss of ADAMTS13 Diagnosis- <10% ADAMTS13

18. Platelet adhesion, activation and aggregation

19. von Willebrand factor

20. Pathogenesis of TTP Under conditions of high fluid shear stress, VWF factors bind to the exposed endothelium. Platelets bind to the vWF in the endothelium thereby leading to enhanced adhesion, activation and aggregation of platelets. At the same time the vwf multimer is stretched and the tyr -met bond in A2 domain is exposed to ADAMTS13 in the plasma, which cleaves it and thereby causing release of any adherent platelets

21. ADAMTS13 deficiency in TTP is caused by polyclonal autoantibodies against ADAMTS13,usually IgG but rarely IgA or IgM (ADAMTS13 inhibitors). With no activity of ADAMTS13 , vwf goes uninhibited causing platelet microthrombi all over the circulation leading to MAHA+thrombocytopenia and subsequent organ failure, without affecting the process of coagulation and coagulation factors Accepted level of ADAMST13 to prevent microangiopathic hemolytic anemia is 10%

22. Autoimmune inhibitors of ADAMTS13 >95% of TTP cases, accounted by inhibitors of ADAMTS13 , triggered by rather an innocuous infection in genetically susceptible heterozygous individuals HLA DRB1*11 10 to 40% ANA positive** Ticlopidine, clopidogrel **

23. ADAMTS13 mutations Heterozygous or less commonly homozygous mutations of ADAMTS13 gene on chromosome 9q34

24. Epidemiology Median age : 30 to 40 years Female to male ratio- 2:1 Role of stressor:Women have a tendency to present late during pregnancy or peripartum

25. Clinical triad

26. Clinical syndrome Acute or insidious Asymptomatic ADAMTS13 deficiency

27. Could be asymptomatic with thrombocytopenia( vs ITP) Thrombosis without MAHA or thrombocytopenia Trigger- infection , pregnancy Heart, brain ,lungs , adrenals, pancreas , skin , spleen and marrow are affected Kidney is spared**

28. Thrombocytopenia without hemolytic anemia- atypical Could be misdiagnosed as TTP** Macro vascular thrombosis without MAHA- CAD,CVA(50%) Antecedent upper respiratory illness or flu-like illness Nausea, vomiting and diarrhea Renal functions are relatively spared. Why?*** Systemic micro vascular thrombosis affecting the heart, brain, pancreas, adrenals, skin , spleen and marrow Pancreatitis, mesenteric ischemia

29. Neurological symptoms of TTP Headache Visual disturbances Vertigo Personality changes Confusion Lethargy Syncope Seizures Aphasia Hemiparesis

30. Advanced renal failure Hypertension Pleural effusion Pericardial effusion ARDS Ascites Anasarca WHY??

31. Lab Anemia- increased LDH , negative coomb’s test Thrombocytopenia Normal PT , aPTT Increased D-DIMER ADAMTS13 <10%

32. Lab features Anemia- hemolytic; Elevated LDH, unconjugated hyper bilirubinemia, low haptoglobin , negative coomb’s test*** Mean Hb: 8mg/dL Thrombocytopenia Mean platelet level : 20,000/mm3 Renal micro vascular injury Micro hematuria , granular or RBC casts and proteinuria Serum creatinine seldom above 2 mg/dl

33. Coagulation profile PT, aPTT , fibrinogen levels normal PS Schistocytes Mean 8.3%( 1-18.4%) ADAMTS13 less than 10% Investigations to rule out secondary causes of TMA MRI brain

34. Differentials Disseminated intravascular coagulation STEC-HUS Infections(viral, bacterial and fungal) Atypical HUS Transplant associated- GVHD, chemotherapy, radiation Malignancy Pregnancy associated( preeclampsia, eclampsia , HELLP) SLE,APLA, Scleroderma Drugs- Ticlopidine , clopidogrel , cyclosporine, tacrolimus , gemcitabine Malignant hypertension Prosthetic heart valve

35. Treatment PLEX/plasma infusion Plasma exchange and plasma infusion are the only therapies with proven efficacy, with mortality rates of 10% and 20% respectively 1-1.5 plasma volume/day, until counts become normal , then start tapering

36. What is plasma volume? Body weight*(1-hct)*0.065 Why 1-1.5 plasma volumes/day? When PLEX should be stopped? When thrombocytopenia is resolving and platelet count is more than 1.5 lakhs for 2 days , start decreasing to alternate days and then stop if counts are static or improving in trend

37. Concept of serial ADAMTS13 levels monitoring ** Plasma infusion is used as an emergency alternative to exchange as the later requires use of logistics, but with disadvantage of volume overload(20 to 40ml/kg/day) Replacement fluid- FFP, cryoprecipitate, cryosupernatant **

38. Plasma therapy does not have an effect on autoimmunity. All patients will have at least one relapse during lifetime

39. 2.Rituximab- used preemptively to prevent antibody production 3.Steroids 4.Platelets- only after plasma exchange. Why?? 5.Splenectomy

40. 2. Glucocorticoids - IV methyl prednisolone for 3 days or 1mg/kg prednisolone till plasma exchange and tapering after that 3 .Platelet transfusion - associated with acute deterioration and death in TTP. If need to be given in life threatening situations, preferably after plasma exchange Absolutely detrimental before plasma exchange**

41. 4. Rituximab -375mg/m2 weekly for 4 weeks For patients with no response to PLEX. Given as prophylaxis to prevent relapse after remission. Complete response in 1-3 weeks . Given with PLEX ,it decreases the time to response and prevents future relapse

42. Splenectomy can result in lasting remissions, prevent future relapses by removing the potential site of antibody production. Cyclosporine, IV cyclophosphamide, oral azathioprine and MMF have also been used but with minimal efficacy

43. ISTH panel recommendation-PLEX+STEROIDS+RITUXIMAB** RITUXIMAB is recommended for prophylaxis in patients who have high risk for relapse- persistently low ADAMTS13 levels Hence continuous monitoring of ADAMTS13 helps in predicting future relapses and timely intervention**

44. Congenital TTP(UPSHAW SCHULMAN SYNDROME) <5% of all TTP cases Inherited deficiency of ADAMTS13( homozygous or heterozygous mutations- ch9q34) AR, M:F- 1:1 Neonates – hyperbilirubinemia / thrombocytopenia and sometimes with neonatal distress, with no ABO/Rh incompatibility Plasma infusion is the treatment of choice . Blood transfusion is given in neonates and it helps***

45. Congenital TTP Due to deficiency of ADAMTS13(<5%) M:F-1:1

46. Kidneys are affected** CKD is common in neonatal form?? Plasma infusion is the therapy of choice Labs similar as acquired TTP Twice weekly plasma therapy

47. 50% have chronic relapsing form of TMA, while others develop symptoms during early twenties. Triggers- infections, pregnancy and other inflammatory states Unlike acquired TTP, AKI with proteinuria and hematuria, deranged RFT does occur, which progresses to CKD in chronic relapsing disease Basis of development of CKD in congenital TTP versus acquired TTP

48. Pregnancy is associated with increased VWF levels in 3 rd trimester /postpartum, thus acting as a trigger. TTP is associated with stillbirth, abortion in pregnancy. ADAMTS13 < 5%, characteristic of hereditary TTP Lab similar to acquired TTP Therapy- Plasma infusion 7.5-10ml/kg** , plasma exchange reserved for CKD. Why ??? In hereditary TTP, complications like stroke, renal failure are unpredictable hence maintenance plasma therapy twice weekly is required.

49. TTP in pregnancy Same as others, treat with PLEX Pregnancy is often a trigger, also for atypical HUS 30% ADAMTS13 reduction in pregnancy, 65% near term Microvascular thrombosis affecting maternal and fetal morbidity Role of Rituximab***(class C drug) Serial monitoring of ADAMTS13** Plasma infusion in cTTP patients

50. Hemolytic uremic syndrome Diagnosis MAHA- anemia Hb<10, schistocytes >2,HCT<30% , and LDH>450 or very low haptoglobin Thrombocytopenia – less than 1,50,000 AKI- Serum Creatinine more than 50% of normal Exclude DIC,TTP, Tropical infections**

51. Hemolytic uremic syndrome-Shiga toxin associated Caused by Shiga toxin producing enterohemorrhagic E.coli of O157:H7 serotype worldwide Associated with favorable prognosis than TTP Only supportive care** S.dysenteriae common in India

52. Epidemiology Children less than 5 years, not less than 6 months 80% due to O157:H7 strain, 20% by non-0157:H5 and Shigella dysenteriae type 1(worldwide) Feco -oral contamination <100 organisms enough to cause the outbreak Need not be associated with diarrhea all times Both sporadic and epidemics possible

53. Epidemiology in India: consensus guidelines doesn’t suggest E.coli as a causative factor of diarrheal illness in India. Shiga associated HUS is prevalent The incidence has decreased because of improved standards of hygiene The capacity to diagnosis the disease is limited

54. Pathogenesis Colonization of intestinal epithelium and release of Shiga toxin

55. A shiga -toxin molecule comprises an A-subunit and a pentameric B subunit Shiga-toxin molecule binds via B subunit to Gb3( globotriaosylceramide ) expressed on endothelial cells in kidney Subsequently the toxin is endocytosed and A subunit is retrogradely transported to protein synthesis machinery via Golgi-apparatus A subunit deadenylates 28s subunit of 60S RNA leading to inhibition of protein synthesis and cytotoxicity/apoptosis

56. Inhibition of protein synthesis by deadenylating 28s RNA of 60S subunit ribosomal RNA, leading to cytotoxicity in kidney

57. Shiga toxins also activate the alternate complement pathway** Cytotoxicity leads to endothelial cell swelling, abnormal vascular permeability and cell death Contributes to capillary leak phenotype Activation of coagulation cascade*** Capillary leak like prodrome without MAHA***

58. Cerebral edema Pleural effusion Pericardial effusion Ascites Mesenteric or intestinal edema Anasarca In the absence of MAHA- capillary leak phenotype

59. Clinical manifestations 15%**** Disease of variable spectrum Diarrhea after 2 to 12 days of ingestion, followed by bloody diarrhea 1 to 3 days later Old age, prior antibiotic usage, bloody diarrhea, high TLC, long duration of diarrhea - Grave prognosis Diarrhea subsides, HUS begins

60. Extrarenal 50%*** Cardiovascular- MI, CCF, pericardial or pleural effusion Neurological- cerebral edema , seizures , ischemic stroke GIT- bowel perforation , mesenteric ischemia, pancreatitis Suggestive of endothelial dysfunction and microvascular thrombosis throughout the systemic circulation**** Older individuals are more prone for extrarenal manifestations

61. Laboratory parameters Hb-low Thrombocytopenia++ after bloody diarrhea, before RFT derangement Renal injury- Raised U/Cr, hypertension(vascular), hematuria, proteinuria, oliguria (ATN). Coagulation parameters-Normal PT/aPTT Raised FDP ADAMTS13 normal***

62. How to diagnose? Stool culture- sorbitol MacConkey agar PCR or ELISA for Shiga-toxin detects non O157:H7 also** Serology by paired samples Positive stool culture+ either PCR or ELISA or IgM ++ constitutes confirmed infection Why not stool culture alone??? Most sensitive and specific investigation for screening??

63. Why not culture alone? Excretion of STEC is short lasting Prior antibiotic use Delayed referral from lower centers Most sensitive and specific investigation for screening? PCR for Shiga-toxin from stool sample

64. Management

65. Antibiotics Invasive diarrhea- culture proven- shall go with antibiotics Level 1A Ciprofloxacin, azithromycin, or cefixime for 5 days Antibiotics might increase expression of Shiga-toxin and risk of HUS

66. Why PEX?? PEX is not recommended in Shiga-toxin associated HUS, unless it is complicated by severe neurological or cardiological involvement level Why eculizumab?? Mostly conservative management- wait and watch Supportive therapy Dialysis to be done If indications of dialysis are present Mortality- 12%

67. No role for steroids, heparin, plasma infusion, Shiga-toxin binders For anemia, blood transfusion must be done to maintain hemodynamic stability Judicious hydration in the presence of AKI, to prevent volume overload**** Conservative- wait and watch*** Mortality-12%**(0 to 30%) Never recurs

68. Atypical hemolytic uremic syndrome Suspect atypical HUS when DIC, STEC and tropical causes, and other secondary causes have been carefully ruled out Presence of positive family history TTP without severe ADAMTS13 deficiency** Occurs in both adults and children M:F-1:1 for children Slight female preponderance in adults as it is triggered by pregnancy

69. Pathology – same as HUS , glomerular endothelial swelling and damage, subendothelial expansion and increased vascular permeability , accompanied by thrombosis In chronic cases, deposition of matrix by chronic inflammatory cells do occur leading to IFTA **** , hence the propensity to cause CKD unlike STEC associated HUS which does not recur

70. Pathogenesis- all components of the complement regulators and activators are involved, leading to heterogeneity in presentation Strongly familial – SNPs associated , AD inheritance . Multiple haplotypes for single gene present Activators- CFB, CFD , C3 Regulators- CFH, CFI, MCP, THBD, CFHR 1-5

71. Clinical manifestations Spontaneously or after trigger- infection, pregnancy**(postpartum) MAHA + thrombocytopenia + AKI + multiple organ dysfunction(20%) **

72. Abdominal pain, diarrhea , nausea and vomiting – acute presentation Increased vascular permeability- cerebral edema, pericardial effusion , pleural effusion, mesenteric edema , ascites CNS-headache, altered sensorium are common Exudative retinopathy- blurring of vision Focal neurological deficits not common as in TTP** Myocardial involvement and heart failure do occur Airway edema, pulmonary edema also are common Severe hypertension***

73. Laboratory values MAHA+ thrombocytopenia RFT derangement with microhematuria, proteinuria with anuria in the other end of the spectrum Normal PT, aPTT and Increased D-dimer C4 is usually normal** C3 may be decreased in 30% , but normal in most people*** Antibodies to CFH 3 to 7%** Flow cytometry for MCP DNA sequencing**- nil role in aHUS . Why???

74. Diagnosis Do all patients need genetic workup for mutations??? Transplant patients Diagnosis of exclusion*** 50% of atypical HUS have mutations in regulators/activators Compound mutations SNPs, haplotype variant Complex genotype involvement

75. Most prevalent mutation is CFH THBD mutations are associated with poor prognosis Progression to CKD is most common with CFH and C3 mutations Prevalence of antibodies to factor H is 3-7% - 50% in Indian paediatric age group***

76. Treatment of atypical HUS without anti-FH antibodies Standard of care- Eculizumab** Problem with eculizumab PEX>>>Plasma infusion***

77. PLEX

78. Eculizumab Lack of response to PLEX after 7-10 days Life threatening cardiovascular or neurological deterioration Complications due to PLEX or vascular access issues Inherited defects in complement proteins, where PLEX has failed *** has no effect in non-complement protein genetic mutations Costly- not possible in developing countries-1,50,000 rupees/vial ***RCT only on renal manifestations are present