Pathogenesis and differential diagnosis

of thrombotic microangiopathies

Zoltán Prohászka
Research Laboratory, IIIrd Department of Medicine,
Semmelweis University, Budapest

[email protected]; 2016-02-23
 Classification of anemia according to etiology
Decreased production Loss of blood Hemolysis
•Iron deficient •Acute •Membrane defect
•Megaloblastic •Chronic •Herediter spherocytosis
•Cobalamin (B12) •Herediter elliptocytosis
•Folic acid •Paroxysmalis nocturnalis
hemoglobinuria (PNH)
•Other forms
•Anemia of chronic •Hemoglobinopathies
diseases •Sickle cell disease
(kidney, hepar, cardiac…) •Thalassemia
•Morrow infiltration •metabolic changes
•Aplastic anemia •G6PD, PKD, other
•Various rare forms •Acquired, extrinsic
•Intoxication (pl. plummet)
•Physical (heat, trauma,
vessel malformation,
mechanic valves,
paravalvular leaks)
•Microangiopathic
hemolysis
•Infection (mycoplasma,
clostridium)
•Immun (auto-, iso-, drug)
 History >> physical examination >>

Laboratory differentialdiagnosis of anemias

(<complete blood count, urine, clincal chemistry, blood film, morrow, others>)
Microcyter Normocyter Macrocyter
•Iron deficiency •Morrow OK •Megaloblastic
•Anemia of chronic •Blood production in •Non-megaloblastic
diseases morrow depressed
•Thalassemia
•Sideroblastic an.
 L. Dóra, born: 1988
• History and family history negative, no medication.
• Weakness, palpitation, headache since 5 days. No fever,
no bleeding.
• Pallor, P 100/min, suffusions on skin

Complete blood count

•WBC 7,16 G/L (12% Mo)
•RBC 1,64 T/L
•HGB 53 g/L
•HCT 0,16
•MCV 95 fL
•MCH 32 pg
•MCHC 338 g/L
•PLT 23 G/L
•RDW-CV 23,2%
 L. Dóra, szül: 1988
• History and family history negative, no medication.
• Weakness, palpitation, headache since 5 days. No fever, no bleeding.
• Pallor, P 100/min, suffusions on skin

Complete blood count Bone morrow? Clincal chemistry

•WBC 7,16 G/L (12% Mo) •Retikulocytes 296 G/L •Total bilirubine 33,7 umol/L
•RBC 1,64 T/L •Indirect bi 8,6
•HGB 53 g/L •Crea 60 umol/L
•GOT 56 U/L
•HCT 0,16
•GPT 142 U/L
•MCV 95 fL •LDH 1714 U/L
•MCH 32 pg •Hapltoglobin 0,1 g/L
•MCHC 338 g/L
•PLT 23 G/L •Blood film: fragmentocytes
•d. Coombs: neg.
 Complexity of the diagnosis: thrombotic microangiopathy
(TMA)

• Goal: the right patient should receive the right therapy in due time

• Considerations on:
– Timeline (acute- remission- relapse- long term management)
– Therapy decisions (acute- upfront; remission- conclusive; relapse-
prevention)
– Alternate causes (definitive TMA, probable TMA, possible TMA)
– Risks of therapy (infants, gestation, peri- or post tx)
– Implementation/Access to therapy (PI, PEX, eculizumab)
 Initial, clinical diagnosis of HUS/TTP syndrome,
i.e. thrombotic microangiopathies
(hospital day 1)
• Intravasal hemolysis with
fragmentocytes, Coombs-negative
– Laboratory signs: increased LDH,
decreased hemoglobin and
haptoglobin, free plasma hemoglobin,
increase of indirect bilirubine
• Low platelet-count
– Exclusion of EDTA-induced decrease
of platelet number,
– Verified by investigation of blood-smear
• Various presence of clinical signs:
– Various neurological symptomps
– Acute renal failure www.med-ed.virginia.edu/courses/path/innes/images/rcdjpegs/rcd
Slide thanks to Dr. Kline Bolton, UVA.
– Other
• No requirement of fever

• HUS: Hemolytic uremic syndrome

• TTP: Thrombotic thrombocytopenic
purpura
 Initial, clinical diagnosis of HUS/TTP syndrome
(hospital day 1)
• Intravasal hemolysis with • Simplified classification of HUS/TTP (1-2)

fragmentocytes, Coombs-negative • Infection-related HUS

– Laboratory signs: increased LDH, – EHEC D+HUS ‘typical’
decreased hemoglobin and
haptoglobin, free plasma
hemoglobin, increase of indirect
bilirubine
• Low platelet-count
– Exclusion of EDTA-induced
decrease of platelet number,
– Verified by investigation of blood-
smear
• Various presence of clinical signs:
– Neurological or renal
• No requirement of fever

(1) Besbas et al, 2006, Kidney International

(2) Ariceta et al, 2009, Pediatric Nephrology
 Clinical course of thrombotic microangiopathies (HUS/TTP syndrome)
Predisposition, Acute disease episode,
direct cause of disease hemolysis, thrombocytopenia Complication, outcome

D+HUS Shiga-like toxin

producing bacteria,
gastroenteritis
Rarely

aHUS

P-HUS

TTP
(Moschcowitz sy)

Congenital TTP
(Upshaw-Schulman sy)

Secondary
HUS/TTP
www.eurosurveillance.org
Numbers of diarrhea positive and HUS
cases over time (2011)

Peak of
exposition

Frank C, NEJM 2011; Jun 22

 Initial, clinical diagnosis of HUS/TTP syndrome
(hospital day 1)
• Intravasal hemolysis with • Simplified classification of HUS/TTP (1-2)

fragmentocytes, Coombs-negative • Infection-related HUS

– Laboratory signs: increased LDH, – EHEC D+HUS
decreased hemoglobin and
haptoglobin, free plasma – Pneumococcus P-HUS
hemoglobin, increase of indirect – Influenza Neur-HUS
bilirubine • Complement-related HUS
• Low platelet-count – Mutations aHUS, factor deficient
– Exclusion of EDTA-induced – Autoimmune anti-FH positive
decrease of platelet number,
• ADAMTS13 deficient TTP
– Verified by investigation of blood-
smear – Autoimmune anti-ADAMTS13 pos
– Mutations Upshaw-Schulman sy
• Various presence of clinical signs:
– Neurological or renal • Secondary forms, other rare entity
• No requirement of fever

(1) Besbas et al, 2006, Kidney International

(2) Ariceta et al, 2009, Pediatric Nephrology
 Clinical course of thrombotic microangiopathies (HUS/TTP syndrome)
Predisposition, Acute disease episode,
direct cause of disease hamolysis, thrombocytopenia Complication, outcome

D+HUS Shiga-like toxin

producing bacteria,
gastroenteritis
Rarely

aHUS Complement deficiency

(Mutation, autoantibody)

ESRD, dialysis, tx

P-HUS

TTP
(Moschcowitz sy)

Congenital TTP
(Upshaw-Schulman sy)

Secondary
HUS/TTP
 Simplified schema of the complement system
Classical pathway (Immune complexes) Alternative pathway (Spontaneous C3 activation)
Factor B and Factor D
Regulators:
C3 activation
C1-inhibitor, C4-binding protein, Factor I
Regulators:
MCP, DAF, Factor H and Factor I
Lectin pathway (Carbohydrate structures)
Alternative pathway
amplification
C3bBbP
Opsonization
Antigen presentation
Antibody production
C5 activation

Regulators:
CD59, S protein and Clusterin
Anaphylatoxins C3a, C5a
Inflammation
Chemotaxis C5-C9
Terminal Pathway
•Cascade
•Activation: whole pathway
•Missing regulation: characteristic picture
Lysis
Cellular damages
Induction of apoptosis
 Simplified schema of the complement system
Classical pathway (Immunecomplexes) Alternative pathway (Spontaneous C3 activation)
Factor B and Factor D
Regulators:
C3 activation
C1-inhibitor, C4-binding protein, Factor I
Regulators:
MCP, DAF, Factor H and Factor I
Lectin pathway (Carbohydrate structures)
Alternative pathway
amplification
C3bBbP
Opsonization
Antigen presentation
Antibody production
C5 activation

Regulators:
CD59, S protein and Clusterin
Anaphylatoxins C3a, C5a
Inflammation
Chemotaxis C5-C9
Terminal Pathway

Lysis
Cellular damages
Induction of apoptosis
 Pathogenesis of complement mediated atypical
HUS
• Predisposition, rare genetic variants
– Mutations of complement alternative pathway regulators
(CFH>MCP>CFI>>THMB>CFB>C3>CFHR5>others)
• Predisposition, frequent genetic variants („complotype”)
– Haplotypes
• CFH H3/H8
• MCPggaac
– Copy-number variations
• CFHR1-3 deletion
• Predisposition, autoantibodies
– Development of anti-Factor H autoantibodies, based on genetic
predisposition (CFHR1 deletion)
• Direct disease-precipitating trigger
– Infections
– Pregnancy
 Clinical course of thrombotic microangiopathies (HUS/TTP syndrome)
Predisposition, Acute disease episode,
direct cause of disease hamolysis, thrombocytopenia Complication, outcome

D+HUS Shiga-like toxin

producing bacteria,
gastroenteritis
Rarely

aHUS Complement deficiency

(Mutation, autoantibody)

ESRD, dialysis, tx

Invasive pneumococcus
P-HUS infection ?

DR11/DQ3 haplotype
Anti-ADAMTS13
TTP
(Moschcowitz sy) Neurological defect

ADAMTS13 mutation
Congenital TTP
(Upshaw-Schulman sy) Neurological defect

Secondary
?
HUS/TTP
Underlying disease, if treatable, good outcomes
 The cause of TTP are the pro-coagulant changes of in
microvessels

• ADAMTS13 metalloprotease cleaves von Willebrand Factor to small

(4-6-8) oligomers
• In case of ADaMTS13 deificency ultralarge vWF multimers remain
attached to the endothelial surface
• ADAMTS13 deficiency may be related to
– Mutation of ADAMTS13 (congenital form, ultrare, Upshaw-Schulman sy)
– Autoantibodies (acquired form, Moschcowitz sy, most frequent)
– Consumption
The cause of TTP are the pro-coagulant changes of
in microvessels
• Ép funkció

ADAMTS13

non-processed processed oligomers

ULVWF
 ADAMTS13 sérülés

ADAMTS13 mutation ADAMTS13 inhibitory autoantibody

Y
Thrombocyta adhesion

Thrombocyta activation

ULVWF Thrombocyta degranulation

Thrombocyta aggregation
Thrombus
Endothel-
activation
Complement-
activation
 Illustrative cases, adults
• Case #1 (BSI, HUN59) • Case #2 (BI, HUN238)
• 37 y old female, no major • 46 y old male, no major diseases
diseases • Abdominal pain, vomiting and
• Abdominal pain, nasal bleeding non-bloody diarrhea, dark urine
and headache • On the day of hospitalization
• On the day of hospitalization oliguria, increased creatinine and
coma, focal neurological signs BUN levels
• Intracranial bleeding (pons) • Se Bi: 46 umol/L, WBC 10 G/L,
• Se Bi: 21 umol/L, WBC 21 G/L, Hgb 103 g/L, Plt 2 G/L, Crea 408
Hgb 93 g/L, Plt 3 G/L, Crea 295 umol/L, LDH 4161 U/mL
umol/L, LDH 278 U/mL • EHEC testing negative
• Plasmapheresis and SoluMedrol • Plasmapheresis and SoluMedrol
therapy initiated, working therapy initiated, working
diagnosis: HUS/TTP syndrome diagnosis: HUS/TTP syndrome
 Illustrative cases, peripartum
• Case #3 (MZ HUN1) • Case #4 (MN HUN246)
• 21 y old female with first • 20 y old female with first
uncomplicated pregnancy until uncomplicated pregnancy
week 40 • Postpartum HUS syndrome
• HELLP syndrome, urgent • Renal biopsy: TMA
cesarean section • Severe hypertension,
• Se Bi: 72 umol/L, WBC 11 G/L, encephalopathy
Hgb 65 g/L, Plt 35 G/L, Crea 54 • Plasmapheresis (26 sessions),
umol/L, LDH 990 U/mL corticosteroid, rituximab
• Critical clinical status despite
plasmapheresis, corticosteroids
• Revision of diagnosis: HUS/TTP?
 Illustrative cases, children
• Case #5 (GL HUN193) • Case #6 (KM HUN200)
• 8 months old boy • 8 y old boy
• Pallor, weakness, no fever, no • Weakness, vomiting, abdominal
diarrhea pain, icterus, dark urine, no fever,
• Hgb 62 g/L, Plt 42 G/L, WBC 11 no diarrhea
G/L, Crea 196 umol/L, LDH 4250 • Hgb 106 g/L, Plt 44 G/L, WBC 7,8
U/mL G/L, Crea 159 umol/L, LDH 4300
• Critical clinical status, worsening U/mL, EHEC testing negative
renal and heart function • Stable clinical status
• Multiple plasma infusions, • Multiple plasma infusions and
Initial diagnosis: HUS plasmapheresis
Initial diagnosis: HUS
 Illustrative cases, summary
Variable Case #1 Case #2 Case #3 Case #4 Case #5 Case #6

Age, 37 y old 46 y old 21 y old 20 y old 8 months 8 y old boy

gender female male female female old boy
Dominating Neurological Acute renal Postpartum Postpartum Acute renal Acute renal
clinical signs failure Low Acute renal failure failure
symptoms (coma) platelet, failure
bleeding
Clinicians’ TTP HUS HELLP HUS aHUS aHUS
impression
 Initial, life-threatening therapy
• PE with FFP is still the most effective treatment available for TTP (1)
– Rituximab (or cyclophosphamide) is indicated for patients with chronic
relapsing disease (autoimmune)
– Anti-platelet therapy and immunosuppression may also be considered

• For HUS, most of the RCTs focused on typical HUS and showed
that supportive therapy including dialysis is still the most effective
treatment (1)

• The „Guideline for the investigation and initial therapy of diarrhea-

negative hemolytic uremic syndrome” article indicates initiation of
PE with FFP in the first 24 hours (2)
– Eculizumab can be considered for proven cases of complement-
mediated aHUS
– Pulse cyclophosphamid and rituximab can be considered for anti-FH
autoantibody mediated aHUS

(1) Michael et al, The Cochrane Library 2009, Issue 1

(2) Ariceta et al, 2009, Ped Nephrol
 Illustrative cases, summary
Variable Case #1 Case #2 Case #3 Case #4 Case #5 Case #6

Age, 37 y old 46 y old 21 y old 20 y old 8 months 8 y old boy

gender female male female female old boy
Dominating Neurological Acute renal Postpartum Postpartum Acute renal Acute renal
clinical signs failure Low Acute renal failure failure
symptoms (coma) platelet, failure
bleeding
Clinicians’ TTP HUS HELLP HUS aHUS aHUS
impression
Initial PEX, S PEX, S PEX, S PEX, S, R FFP, S PEX, S
therapy
 Therapeutic dilemma on day ~3-6
• Judgment of initial response to plasma therapy, steroids
• Results of initial laboratory testing, exclusion of:
– Sepsis, DIC
– Anti-phospholipid syndrome and SLE
– Other causes of hemolysis and low platelet numbers
– Secondary HUS/TTP
 Therapeutic dilemma on day ~3-6
• Judgment of initial response to plasma therapy, steroids Failing or
• Results of initial laboratory testing, exclusion of: insufficient
– Sepsis, DIC
– Anti-phospholipid syndrome and SLE
– Other causes of hemolysis and low platelet numbers No alternative
– Secondary HUS/TTP diagnosis
• Where next with HUS/TTP on hospital day 3-6?
– Is there a reliable laboratory test to classify patients into the following groups in a
short time?
• D+HUS
• aHUS (factor deficient or autoimmune)
• TTP (ADAMTS13 deficient with or without autoantibodies)
– How to decide?
• Intensify immunosuppression (Cy, R) for autoimmune disease (Ab+ aHUS, Ab+TTP)?
• Intensify factor supply/ targeted inhibition of complement (factor deficient TTP or aHUS)?
 Guideline for the investigation of aHUS (1)

• Clinical recognition of aHUS • Laboratory recognition of aHUS

– No recent diarrhea – Complement C3
– Recent diarrhea but any one of the – Complement FI and FH
following – Anti-FH autoantibody
• Age <6months – MCP (CD46) surface expression
• Insidious onset
– Genetic analysis (CFH, CFI, CD46,
• Relapse of HUS
CFB, C3, THBD2)
• Suspected previous HUS
• Previous unexplained anaemia • Exclusion of TTP
• HUS post-transplantation of any – ADAMTS13 activity measurement
organ – Anti-ADAMTS13 autoantibody
• Asynchronous family history of HUS determination

(1) Ariceta et al, 2009, Ped Nephrol

 Illustrative cases, summary
Variable Case #1 Case #2 Case #3 Case #4 Case #5 Case #6

Age, gender 37 y old 46 y old 21 y old 20 y old 8 months old 8 y old boy
female male female female boy

Clinician’s’ impression TTP HUS HELLP HUS aHUS aHUS

ADAMTS13 activity (67- 112 0 0 43 27 76

147%)
ADAMTS13 No Yes Yes No No No
autoantibody
Complement C3 (0.9-1.8 1.45 1.0 0.75 0.58 0.36 0.69
g/L)
Factor B:Ag (70-130%) 183 56 100 66 65 13

AP activity (70-105%) 96 102 24 21 5 49

Anti-FH IgG Slight negative negative negative negative Strong

positive positive
Final diagnosis Non- TTP, Ab+ Peri- Peripartum aHUS, aHUS, Ab+
ADAMTS13 partum aHUS CFH deficient
deficient TTP, Complement
TTP, ADAMTS13 deficient
aHUS? def., Ab+
Comment CFHR1-3 CFH H3 CFH H3 and FH:Ag CFHR1-3
deficient risk MCPggaac undetectable deficient
haplotype risk haplotype CFH Ser722X
heterozyg heterozygous and CFH H3
ous Mutational risk haplotype
analysis
ongoing
 Suggestions for diagnostic testing of
patients with suspicion of TMA
• Hospital day 1: verification of intravasal hemolysis and low platelet
count, blood smear, differential-diagnosis.
• Hospital day 2-5: Before plasmapheresis initiation of detailed
complement and ADAMTS13 testing
– Identification of AP over-activation and consumption, anti-FH IgG
– ADAMTS13 deficiency
• Within 1-2 months: Detailed complement investigations
– Identification of missing complement factor(s)
– Genetic analysis
 Coworkers of the Füst György Komplement Diagnosztikai Laboratórium
www.kutlab.hu

CEE Roundtable discussion, Zoltan Prohaszka 23-10-2014 Photo: Kata Tolnai