| COMPLEMENT DISORDERS AND HEMATOLOGIC CONSEQUENCES |

Atypical hemolytic uremic syndrome

Vahid Afshar-Kharghan

Section of Benign Hematology, MD Anderson Cancer Center, Houston, TX

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy (TMA) that affects multiple organs and the
kidneys in particular. aHUS can be sporadic or familial and is most commonly caused by dysregulation of the alternative
complement pathway. The initial attack of aHUS can occur at any age, and is associated with a high rate of progression to
end stage renal disease. Many aHUS patients relapse in the native or transplanted kidneys, and require close monitoring
and long-term management. Availability of anticomplement therapy has revolutionized the management of aHUS, and
can change the natural course of aHUS by inducing hematologic remission, improving or stabilizing kidney functions,

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and preventing graft failure. As a result, it is important to succeed in the challenging task of differentiating aHUS from
other TMAs and initiate adequate treatment early during the course of disease. Considering the high cost of currently
available anticomplement therapy, it is important also from a financial point of view to accurately diagnose aHUS early
during the course of disease and determine the necessary length of therapy. This highlights the need for development of
precise complement functional and genetic studies with rapid turnaround time.

or Shiga-like toxin-producing enteropathogenic bacteria, most

Learning Objectives commonly Stx-producing Escherichia coli (STEC). Typical HUS is
• To understand the regulation of the complement system and also known as Stx-HUS or STEC-HUS, and will be referred to as
role of complement dysregulation in the pathogenesis of STEC-HUS in the rest of this study. STEC-HUS is an abrupt severe
aHUS illness. One week after the development of bloody diarrhea, ~15% of
• To become familiar with a diagnostic algorithm for aHUS children develop acute renal failure,3 but long-term renal impairment
• To understand how to treat aHUS and to monitor the thera- in survivors is not common. Rarely, urinary tract infection by STEC,
peutic response of aHUS patients without diarrhea, can cause STEC-HUS.

In addition to E coli, invasive pneumococcal infections can result

in HUS, via generating neuroaminidase that removes Thomsen-
Definition Friedenreich antigen (T antigen) from the surface of erythrocytes,
Thrombotic microangiopathies (TMA) are a group of disorders platelets, and glomerular endothelial cells. Preformed immuno-
characterized by injury and occlusion of microvessels resulting in globulin M natural antibodies bind to the exposed T antigen and
tissue ischemia and organ damage. TMA can be categorized initiate complement-mediated tissue damage. As a result of binding
according to their etiology (hereditary or acquired), age of onset, of natural antibodies to exposed T antigen on red blood cells, most
being sporadic or familial, involved organs, clinical association and patients with pneumococcal HUS have a positive direct Coombs test
manifestations, and prognosis. Overlap between these categories for C3. Pneumococcal infection- associated HUS has a worse
make a definite diagnosis challenging, and justify the initial con- prognosis than STEC-associated HUS with a mortality rate close
sideration of different therapeutic approaches.1,2 to 50%.4

Thrombotic thrombocytopenic purpura (TTP) is a distinct category aHUS is less common than STEC-HUS, and only ~10% of HUS
of TMA, caused by a severe reduction in the function of ADAMTS13 cases are aHUS.5,6 In comparison with STEC-HUS, aHUS has
(a disintegrin and metalloproteinase with thrombospondin type 1 a more insidious onset with fluctuating clinical and laboratory ab-
motif 13) (,10% activity), due to blocking ADAMTS13 autoan- normalities. aHUS is not associated with STEC infection, but can be
tibodies or in 5% to 10% of patients due to loss-of-function mutations triggered by infections (especially in children), pregnancy, or drugs.
in ADAMTS13 (Upshaw-Shulman syndrome). TTP is a systemic aHUS can be further classified into the primary aHUS that is as-
disorder with microthrombi in several organs, including the heart, sociated with dysregulation of the alternative complement pathway,
central nervous system, and to a lesser degree, the kidneys. and secondary aHUS (cancer-, chemotherapy-, solid-organ transplant-
or hematopoietic stem cell transplant- [HSCT], pregnancy-, or au-
Hemolytic uremic syndrome (HUS) is another category of TMA that toimmune disorder-associated). Whether complement dysregulation
is associated with microthrombi mainly in the kidneys; and can be has a role in the pathogenesis of secondary HUS is an interesting
classified into typical HUS and atypical HUS (aHUS). Majority of question that requires additional studies. In this study, we will focus
HUS patients are children (mostly .6 months of age), diagnosed on primary aHUS, and on complement-mediated tissue injury as the
with typical HUS as a sequela of an infection with Shiga-toxin (Stx) main pathogenic mechanism of primary aHUS.

Conflict-of-interest disclosure: V.A.-K. is on the Board of Directors or on an advisory committee for Alexion.
Off-label drug use: None disclosed.

Hematology 2016 217

Table 1. Genotype-phenotype correlation in aHUS targeted cells. CD59 is a membrane protein that prevents the as-
sembly of C5b-9 on the cell surface, a negative complement regu-
Recurrence
Frequency ESRD/death Relapse in transplanted latory function that is also performed by soluble proteins clusterin
(%) (%) (%) kidney and vitronectin. The final results of complement attack are opso-
nization of targeted cells by covalently bound C3b for phagocytosis,
CFH 23-27 63-80 20-40 80-90 membrane perforation and cell lysis by C5b-9, and chemotaxis of
CFI 3-8 50-70 20-40 70-80 neutrophils and macrophages guided by the gradient of anaphyla-
C3 2-8 43-60 20-40 40-50
toxins. The extensive damage that can be inflicted by the alternative
MCP 5-9 17-20 80 15-20
CFB 1-2 70 70 88
complement pathway on cells explain the necessity for a tight com-
Anti-FH Ab 3-6 30 60* 20 plement regulation under physiologic conditions (Figure 1); and the
10† pathologic consequences (endothelial damage and platelet activa-
THBD 0-5 60 — — tion) of complement dysregulation.
Combined 4 — — —
No mutation 33-54 27 20-40 27
Regulatory proteins of the alternative complement
Based on the data provided in Noris et al,8 Noris and Remuzzi,11 Bu et al,13 and Loirat pathway involved in the pathogenesis of aHUS

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et al.14 Mutations can be detected in ~50% to 60% of aHUS patients, with
Ab, antibody; CFB, factor B; CFH, factor H; CFI, factor I.
a majority of these mutations involving complement regulatory
*Without immunosuppression.
†With immunosuppression. proteins (FH, FI, and MCP) and a small percentage of gain-of-
function mutations in complement proteins C3 and FB.6,11 Lack of
identifiable mutations in complement proteins does not rule out the
Regardless of the etiology of HUS, the clinical and pathologic possibility of aHUS, as became clear after the detection of mutations
manifestations of STEC-HUS and aHUS are similar. Clinical in coagulation proteins and metabolic enzymes in aHUS patients.
manifestations of HUS include intravascular hemolysis, thrombo-
cytopenia, and kidney failure. Pathologic findings in kidneys are FH protein family
consistent with endothelial injury in microvessels and glomeruli, About half of detected mutations in aHUS are located in CFH (25%
including endothelial swelling and proliferation, widening of sub- of all aHUS patients).6,11 FH is the main regulatory protein of the
endothelial space, mesengiolysis, and fibrin-rich thrombi in capil- alternative complement pathway and is functional both in plasma and
laries and arterioles.7 on the cell surface.16,17 FH is composed of 20 homologous structural
domains, known as short consensus repeats (SCR). Despite the
In the past 3 decades, various mutations in complement and com- structural similarities, the SCRs have distinct functional roles. The
plement regulatory proteins (loss-of-function mutations in Factor H N-terminal SCRs are necessary and sufficient for the regulation of C3
[FH], FI, membrane cofactor protein (MCP), and deletions in FH- in the fluid phase.18 The C-terminal SCRs 19 and 20 are necessary
related proteins; and gain-of-function mutations in C3 and FB) have for FH binding to glycosoaminoglycans on membrane, and hence
been discovered in 50% to 60% of aHUS patients.8-12 The genotype- complement regulation on cell surfaces (Figure 1). FH prevents
clinical phenotype correlations in aHUS will be discussed in this autolysis of C3 in plasma, and together with FI, degrades C3b
study (Table 1).8,11,13,14 However, it is important to emphasize that deposited on the cell surfaces to iC3b. Most aHUS mutations in FH
the anticomplement therapy is effective in aHUS associated with are heterozygous, located in the region of the gene encoding SCRs 19
different genotypes, and have changed the natural course of aHUS. and 20, and do not change the quantity of FH in plasma, but reduce its
activity on the cell membrane. Mutant FH molecules are unable to
Alternative complement pathways adhere to the cell surface and cannot regulate complement activation,
There are 3 complement pathways: classical, alternative, and lectin resulting in complement-induced damage and microthrombi, espe-
pathways. The 3 pathways differ in their initiation steps, but all cially in the renal microvasculature.
converge on the formation of a C3 convertase that is essential for
propagation of complement activation. The classical or lectin Several complement regulatory genes including genes encoding
pathways require recognition of an antigen by an antibody or FH and FH-related proteins (FHRP1-5) are located in proximity to
mannose-binding lectin, respectively, and subsequent activation of one another on chromosome 1, in a region known as the region of
C4 by either C1 complex (classical pathway) or a C1-like complex complement activation.19 The proximity of CFH and FHRP1-5
(lectin pathway). The alternative complement system is a component provides the opportunity for genetic recombination, resulting in
of innate immunity and comprises several plasma and cell surface deletions or hybrid genes. Heterozygous FHRP1-3 deletion is
proteins.15 For activation of the alternative pathway, there is no need a relatively common polymorphism with a frequency of 2% to 9%
for a specific antigen recognition, and merely the presence of (Europeans) to 16% (Africans) in the general population.20 Ho-
a “permissive” surface that does not actively inhibit complement mozygous FHRP1-3 deletion is detected in patients with aHUS, most
activation suffices to activate the alternative pathway (Figure 1). commonly associated with anti-FH antibodies. About 80% of aHUS
Complement activation products C3a and C5a are generated by patients with anti-FH antibodies have concomitant FHRP-1 deletion.
cleavage of C3 by the C3 convertase and cleavage of C5 by C5a Anti-FH antibodies result in acquired FH deficiency and are de-
convertase. C3a and C5a are anaphylatoxins with discrete cellular tectable in 3% to 8% of aHUS patients, mostly in children ,16 years
receptors, that when occupied by their cognate ligand, trigger of age.6,11,21
a number of biological responses including chemotaxis of myeloid
cells and degranulation of mast cells. The other complement acti- FI
vation product is MAC (C5b-9) that perforates the cell membrane, FI is a plasma serine protease that prevents activation of the alter-
and after reaching an adequate density on cell membrane, lyses the native and classic complement pathways by degrading C3b to iC3b,

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Figure 1. Activation and regulation of the alternative complement pathway. A small amount of plasma C3 is continuously converted to C3b that is highly active
and binds to cell surface via forming thiol bonds. Under normal conditions, C3b is rapidly degraded in plasma by FI and FH (FI cofactor), or on the cell surface
by FI and glycosoaminoglycan-bound FH, membrane protein CD46 (or MCP), or CD35. Degradation of C3b to an inactive product (iC3b) on the cell
membrane by membrane-bound FH, MCP, or CD35 requires FI, and is known as cofactor activity. IC3b cannot participate in any further complement
activation. If not inactivated, C3b is able to bind to an activation product of FB (Bb) produced by FD-mediated cleavage of FB, and generate C3bBb (C3
convertase). C3 convertase is the engine of the complement pathways and deposit additional C3b molecules on membrane, which in turn amplify complement
activation. C3 convertase is an important target for complement regulatory proteins. FH prevents the formation of C3 convertase and dissociate C3
convertase by competing with FBb for binding to C3b. This negative regulatory activity of FH is known as decay accelerating activity. If C3bBb remains intact,
the complex is stabilized by FP (properdin) and binds to additional C3b to generate C3bBbC3b, which is also known as C5 convertase. C5 convertase
activates C5 to generate C5b, which in turn binds to C6, C7, and C8, forming the C5b-8 complex that stably inserts into the lipid bilayer of the cell. Next, the
C5b-8 complex binds and polymerizes multiple molecules of C9 (C5b-9n), forming cytolytic MAC of complement on the cell surface. CFB, factor B; CFD,
factor D; CFH, factor H; CFI, factor I; GAG, glycosoaminoglycans; MAC, membrane attack complex; P, properdin. Reprinted from The University of Texas MD
Anderson Cancer Center with permission.

and through additional cleavages of iC3b, to C3d and C3dg. C3b Approximately half of these mutations result in dysfunctional FI with
degradation products (ic3b, C3d, and C3dg) cannot bind to FB and normal plasma quantities.6,11
cannot generate C3 convertase. FI protease activity depends on the
concurrent binding to C3b of a cofactor, either FH, CD35, or CD46. CD46 or MCP
FH binds to C3b (on cell surface or in plasma) and FI, and provides MCP is a membrane protein expressed on nucleated cells and
the platform on which FI cleaves C3b. Mutations in FI are het- regulates both the alternative and classical complement pathways
erozygous and detected in 4% to 8% of patients with aHUS. by serving as a cofactor for FI for cleavage of C3b and C4b. Gene

Hematology 2016 219

encoding MCP is also located in the region of complement activation of vitamins B12, B2, and folate, despite the presence of normal
of chromosome 1q3.2. Most MCP mutations detected in aHUS are plasma levels of these vitamins in most of the patients.28
located in 4 extracellular SCRs, and the majority of these mutations
decrease the expression of MCP that can be detected by flow
Clinical presentation and clinical course
cytometry on white blood cells in the peripheral blood. MCP mu-
aHUS is rare but has a high mortality rate, high risk of progressing to
tations are detected in ~5% to 9% of aHUS patients and are asso-
ESRD, and high risk of relapse after kidney transplant. aHUS results
ciated with frequent relapses, but overall, a mild clinical course.
in intravascular hemolysis, thrombocytopenia, and renal failure in
Majority of aHUS patients with MCP mutations have a complete
a majority of patients. Although commonly assumed to be a pediatric
recovery from their initial aHUS attack, and among them only 6% to
disorder, ~40% to 60% of aHUS cases occur in individuals .18
19% progress to develop end-stage renal disease (ESRD) and very
years of age.29 Thrombocytopenia can be moderate with 42% of
few develop relapse after kidney transplant.6,11
children and 27% of adults having platelet counts ,50 000/mL.
Fifteen percent of aHUS patients have normal platelet counts.6 On
C3 and FB the other hand, renal failure is severe and the majority of patients
In addition to mutations in complement regulatory proteins, muta- require hemodialysis (59% of children and 81% of adults). In ad-
tions in C3 and FB are detected in 2% to 8% and 1% to 2% of patients dition to an elevated creatinine, hematuria, proteinuria, edema, and

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with aHUS, respectively. C3 mutations reduce binding of C3b to FH, hypertension are other manifestations of renal damage in aHUS.
and FB mutations increases the affinity of FB to C3b, resulting in Extra-renal findings are seen in 20% to 25% of aHUS patients in-
a more stable C3 convertase and hyperactivation of the alternative volving the cardiovascular system, central nervous system (seizure,
complement pathway. Patients with aHUS associated with C3 or FB focal neurologic finding, and altered mental status), and the gas-
mutations have low plasma C3 levels, and have an aggressive clinical trointestinal tract (diarrhea, pancreatitis, and hepatitis).6 Although it
course with .88% developing ESRD, and ~80% relapse in the was suggested that the presence of a platelet count .30 000/mL and
transplanted kidney grafts.6,22,23 a serum creatinine level .2.25 mg/mL eliminates the possibility of
a severely deficient ADAMTS13 activity as the cause of TMA,
Mutations in noncomplement genes in aHUS patients additional studies provide a more complicated clinical picture. For
Thrombomodulin (THBD) example, aHUS associated with MCP mutations can mimic TTP,30
THBD is a membrane protein on endothelial cells that enhances and there are anecdotal reports of aHUS patients with normal kidney
thrombin-mediated activation of protein C and activation of car- functions with mainly neurologic findings.31
boxypeptidase B (also known as thrombin activatable fibrinolysis
inhibitor). Carboxypeptidase B inactivates anaphylatoxins (C3a and An additional factor that complicates the distinction between aHUS
C5a) by removing an arginine residue. In addition to its role in and other TMAs are the triggering events that often precedes an
coagulation, THBD may participate in complement regulation, episode of aHUS and are also frequently seen in other TMAs, such as
THBD enhances FI-mediated inactivation of C3b, and promotes the an infection or a pregnancy. Preceding or concurrent infections, as
generation of thrombin activatable fibrinolysis inhibitor that in turn a result of various pathogens, are common in children with aHUS.6
inactivates anaphylatoxins.24 Mutations in THBD can be detected in Differential diagnosis of TMA in pregnancy is particularly chal-
~5% of patients with aHUS.24 lenging. The clinical and laboratory findings initially consistent with
a diagnosis of HELLP syndrome (hemolysis, elevated liver enzymes,
Diacylglycerol kinase « (DGKE) and thrombocytopenia) can evolve into TTP or aHUS.32 To make
DGKE is present in endothelial cells, podocytes, and platelets, and matters more complicated, mutations in complement regulatory
terminates arachidonic acid-containing diacylglycerol-mediated sig- proteins were reported in patients with a diagnosis of HELLP
naling. Mutations in DGKE were identified in infantile recessive syndrome.32 Although TTP can occur in the second or third trimester
aHUS, with multiple relapses and proteinuria, and are usually un- of pregnancy, aHUS most commonly occurs in the postpartum
responsive to anticomplement therapy.25,26 period,34 with the highest frequency after the second pregnancy.

Plasminogen (PLG) Finally, the interactions between the complement system and he-
PLG is a component of the fibrinolytic system, and upon conversion mostatic factors,35-38 evident by the presence of dual-function
to plasmin can degrade fibrinogen and fibrin. PLG also cleaves proteins in the complement system and hemostasis, provide the
several complement proteins, and might have a role in the regulation real possibility that, in some patients, the pathogenesis of TMA
of the complement system. Mutations in PLG have been reported in involves abnormalities in both systems (2-hit hypothesis) such as
~5% of white American aHUS patients.13 concurrent mutations in both complement proteins and hemostatic
proteins (coagulation proteins or ADAMTS13),39,40 or complement
dysregulation in patients with a low ADAMTS13 activity level.41,42
C cobalamin (Cbl C) deficiency
Cbl C deficiency is a rare condition that is caused by the deficiency of
methylmalonic aciduria and homocysteinuria type C, and inherited Genotype and phenotype correlations in aHUS
as an autosomal recessive disorder.6,27 Cbl C deficiency results in Mutations in FH, MCP, FI, C3, FB, PLG, THBD, and DGKE,
hyperhomocysteinemia, reduced methionine concentration in blood, deletion of FHRP1-3 (usually concurrent with the presence of anti-
and methylmalonicaciduria. Cbl C deficiency is usually detected in FH antibodies), and Cbl C deficiency are detected in aHUS patients.
the first year of life in association with neurologic findings, such as It is important to mention that most of the detected mutations in
hypotonia and seizure. Adult onset of symptoms is rare and can aHUS are heterozygous. Penetrance of mutations in familial aHUS is
manifest as psychosis, ataxia, and cognitive impairment. In all age not complete (~50%) and only about half of the individuals that carry
groups, hemolysis and kidney failure can be detected. Treatment of an abnormal mutation would develop the clinical manifestations of
Cbl C deficiency includes the administration of pharmacologic doses aHUS.6 One possible explanation is that the presence of high-risk

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Figure 2. Diagnostic and therapeutic algorithm in TMAs. Role of eculizumab in the treatment of secondary aHUS (cancer-, chemotherapy-, solid-organ
transplant- or HSCT-, pregnancy-, or autoimmune disorder-associated) have not been established and is out of the scope of this study. *In patients
with anti-FH antibody, immunosuppressive reagents (steroid, rituximab, cyclophosphamide, and azathioprine) should be added to eculizumab. ELISA,
enzyme-linked immunosorbent assay; LDH, lactate dehydrogenase; PCR, polymerase chain reaction.

haplotypes in FH (CFHtgtggt) or MCP (MCPggac) genes increases diagnostic possibilities, and initiate laboratory studies that can
the risk of disease in patients carrying aHUS mutations.19,43 identify the diagnosis and guide the long-term management. Un-
fortunately, several important laboratory results will not be available
Genetic studies in aHUS patients have diagnostic and prognostic immediately, and may take from a few days (ADAMTS13 activity
values, and might affect the natural course of disease and the long-term level, CH50, C3, C4, FH and FI levels, anti-FH antibody level) to
management of the patients. One important shortcoming of genetic a few weeks or months (genetic studies on complement genes) to be
studies is that a specific mutation can be identified in only about half of completed. Clinicians need to start treatment based on clinical and
aHUS patients. Prognosis of aHUS patients depends on the presence of available laboratory clues, and modify the treatment according to the
ESRD, extra-renal involvement, frequency of recurrence after kidney therapeutic response and laboratory results as they become available
transplant, and the time lag between the onset of symptoms and ini- (Figure 2).
tiation of therapy. About 50% of aHUS patients develop ESRD, with
an overall mortality rate of 25%.6 However, the death or ESRD rates The clinical manifestations of aHUS,8,9,44 similar to other TMAs, are
vary significantly among aHUS patients according to their mutations; caused by anemia and acute kidney failure, and include pallor, fatigue,
with FH, FB, C3, and FI mutations being associated with the worst failure to thrive, edema, and drowsiness. Hypertension, either new-
rates (60% to 80% death or ESRD) and MCP mutations with the best onset or worsening of a previously well-controlled disease, is an
rates (30% to 40% death or ESRD)11 (Table 1). Prior to the availability important finding that should be considered as a diagnostic red flag.
and prophylactic use of anticomplement reagent, the rate of recurrence Extra-renal manifestations, including cardiac events, seizure, diffuse
in transplanted kidneys in patients with FH and FI mutations were as or focal neurologic findings, abdominal pain, and vomiting occur in
high 80% to 90%. This rate was lowest in patients with MCP mutations ~20% of aHUS patients.6 Concurrent infections are not rare, and might
and CHFR1-3 deletion (20%), and intermediate for the other mutations. also affect the clinical presentation. Laboratory findings are consistent
with intravascular hemolysis (anemia, elevated reticulocyte counts,
Diagnosis reduced haptoglobin, elevated lactate dehydrogenase, hemoglobinuria,
At the time of presentation, the etiology of TMA in most patients is negative Coombs tests [except in the case of pneumococcal
not clear, and the clinicians need to consider several different HUS]) and microangiopathy (thrombocytopenia and evidence of

Hematology 2016 221

microangiopathic hemolytic anemia with fragmented red blood cells and normal C4 are consistent with the activation of the alternative
in the peripheral blood smear). Abnormal kidney function tests are complement pathway in aHUS. One should consider that ~35% of
one of the main laboratory abnormalities, and include elevated patients with proven diagnosis of aHUS have normal C3 levels.6
creatinine, microscopic hematuria, and proteinuria that can be in the Most patients with aHUS caused by FB or C3 mutations have low
range of nephrotic syndrome. serum C3 levels. Majority of patients with FH mutations have
a normal quantity of FH in the serum, and ~40% of aHUS patients
Although the presence of comorbidities causing secondary TMA with FI mutations have lower levels of FI.11
(such as cancer, HIV infection, pregnancy, solid organ transplant, or
HSCT) complicates the initial manifestation of TMA, the most The results of genetic studies in aHUS patients are the last results to
important diagnostic step is to consider the possibility of aHUS in the become available. These results will confirm the diagnosis of aHUS
differential diagnosis. The initial attack of aHUS may occur at any and impact the long-term management of patients, such as the length
age and in both genders.45 In infants, the possibility of hereditary of anticomplement therapy and decisions about kidney transplant.
causes including mutations in genes encoding complement proteins, Genetic studies are conducted in the specialized reference labora-
ADAMTS13, DGKE, and methylmalonic aciduria and homocysteinuria tories that should be contacted to obtain detailed information about
type C, should be considered. However, the development of TMA in the processing of the collected blood samples. Collection of 10 mL

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siblings within a short time period also raises the possibility of ex- EDTA-anticoagulated whole blood (2 purple-top tubes) and 10 mL
posure to Stx and STEC-HUS. Pregnancy is an important trigger for of coagulated blood (red-top tubes) would be enough to complete
TMA, and the initial presentation of TMA in the postpartum period genetic and functional complement studies.48
should raise the possibility of aHUS.
Treatment
Upon the initial suspicion of TMA, additional laboratory studies
should be ordered (Figure 2). The goal of laboratory evaluation in the Plasma therapy
short-term is to identify TTP and STEC-HUS patients. The next step Infusion of plasma or plasma exchange has been reported to be
is to identify complement dysregulation and specially the presence of effective in improving hematologic parameters in patients with
anti-FH antibody. The last step is to detect aHUS mutations. Severely aHUS, and as a prophylaxis against relapse after kidney transplant.11
reduced ADAMTS13 activity level (,10%) is the first result that Despite improvement in the hematologic parameters in 50% of aHUS
determines the nature of TMA by identifying TTP patients. The patients with plasma therapy, ~48% of children and 67% of adult
presence of Stx in stool samples (detected by either enzyme-linked aHUS patients die or develop ESRD despite plasma therapy.14
immunosorbent assay or polymerase chain reaction) and positive Guidelines for the management of pediatric49 and adult patients50
Sorbitol-MacConkey stool culture (detecting E coli O157:H7, which with aHUS recommended initiation of plasma therapy soon after the
is the most common STEC), confirms the diagnosis of STEC-HUS. diagnosis of aHUS is suspected (daily exchange of 1.5 to 23 of
Although rare, a urine culture should be obtained to rule out STEC plasma volume or daily plasma infusion of 20 to 30 mL/kg of body
infection in the urine. In TMA patients with invasive pneumococcal weight). These guidelines were developed prior to the availability of
infections (pneumonia, empyema, or meningitis) and positive direct anticomplement therapy, and one should consider treatment with an
Coombs test, the possibility of pneumococcal HUS should be anticomplement reagent as the first-line therapy in patients with
considered. The second group of results that will be available are a certain diagnosis of aHUS, such as in patients with relapsed aHUS
the titer of anti-FH antibody, functional complement assays, and or in a sibling of a patient with a known diagnosis of aHUS. The
therapeutic response to plasma therapy depends on the aHUS-
quantifications of individual complement proteins. The presence of
anti-FH antibody alters the management of aHUS and mandates the causing mutations.9,11,44 Patients with C3 or THBD mutations
addition of immunosuppressive therapy. The titer of anti-FH anti- and anti-FH antibodies are more responsive to plasma therapy, and
body can be monitored to determine the length of therapy. Functional patients with FH or FI mutations are less responsive. In patients with
complement assays include CH50, AP50, and the FH functional anti-FH antibody, immunosuppressive reagents (steroid, rituximab,
assay.46 CH50 (antibody-coated sheep erythrocyte lysis assay) de- cyclophosphamide, and azathioprine) should be added to plasma
termines the total complement activity (classic and alternative exchange.14 MCP is a membrane protein, and plasma therapy is not
pathways) and is normal in aHUS patients. AP50 (rabbit erythrocyte effective in aHUS patients with MCP mutations. In fact, most of the
lysis assay) mainly determines the alternative complement activity and aHUS patients with MCP mutations will have complete remission
is reduced in aHUS patients due to the consumption of alternative with or without plasma therapy.
pathway proteins. In aHUS patients, one would expect normal CH50
and low AP50 values. In the FH functional assay (uncoated sheep In practice at the time of the initial presentation, every patient with
erythrocyte lysis assay), FH present in normal serum binds to uncoated a clinical diagnosis of TMA should be started on plasma exchange.
sheep erythrocytes and prevents lysis. In aHUS caused by FH mu- Plasma exchange should be continued for 5 days or until
tations or anti-FH antibodies, uncoated sheep erythrocytes lyse in the ADAMTS13 activity assay and Stx assay results become available
presence of patient’s serum. More recently, a modified Ham test has (Figure 2). If ADAMTS13 level is .10% or if Stx is detected, plasma
been proposed as a functional assay of the alternative complement exchange can be discontinued. In patients with ADAMTS13 ac-
pathway to distinguish aHUS from TTP, STEC-HUS, and dis- tivity .10% and negative results for Stx, if hemolysis and throm-
seminated intravascular coagulation.47 If proven by additional bocytopenia do not improve after 5 days of plasma therapy,
studies to be superior to traditional functional complement assays, anticomplement therapy should be considered.
a modified Ham assay can be an important step forward in the di-
agnostic evaluation of TMA. Quantification of C3, C4, FH, and FI by Eculizumab
enzyme-linked immunosorbent assay or western blotting, and MCP Eculizumab is a humanized immunoglobulin G2 monoclonal anti-
by flow cytometry on mononuclear cells are usually conducted in the body against complement C5 (Figure 3) with a plasma t1/2 of 11
reference laboratories together with the genetic studies. A low C3 hours that blocks complement activation for 2 to 3 weeks after

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Figure 3. Mechanism-of-action of eculizumab, a humanized monoclonal antibody that binds to C5 with a high affinity and inhibits cleavage of C5 by C5
convertase, and prevents generation of C5a and formation of C5b-9 MAC.

a single IV infusion.51,52 In 2011, eculizumab was approved by the and then 1200 mg once every other week (maintenance). Patients
US Food and Drug Administration for the treatment of aHUS, based who continue to undergo plasma exchange should receive an ad-
on the results of two phase 2 clinical trials on a total of 37 patients.53 ditional dose of 600 mg of eculizumab after each plasma exchange.
Administration of eculizumab in aHUS patients resulted in hema-
tologic remission and improvement in kidney functions, as was Every aHUS patient should receive tetravalent vaccine (preferably
evident by an increase in glomerular filtration rate, reduction in conjugated) against Neisseria meningitides (A, C, Y, W135), at least
proteinuria, and reduction in dialysis-dependency. Improvement in 2 weeks prior to the initiation of eculizumab, and a booster dose
kidney function was more pronounced if treatment with eculizumab at least 2 months after the first vaccination. If vaccination is
was initiated earlier after the diagnosis of aHUS. After 2 years of performed ,2 weeks prior to eculizumab, then patient should
treatment with eculizumab, TMA event-free status was maintained in receive prophylactic antibiotics against N meningitides for 2 weeks
the vast majority of these patients. Kidney functions remain stable in after vaccination. Immunocompromised patients (ESRD or kidney
most patients, and continued to modestly improve in aHUS patients transplant) should continue to receive prophylactic antibiotics as
started on eculizumab earlier after the initial diagnosis.54 The key long as treatment with eculizumab continues.51 Patients from North
questions about eculizumab treatment are: who should receive it, America and Europe on eculizumab should also receive vaccination
when to start it, how to monitor it, and when to stop it. Patients with against N meningitides serotype B. Additionally, an annual influenza
familial aHUS or relapsed aHUS with native or a transplanted kidney vaccination should be recommended.
should receive eculizumab. Patients with TMA, normal ADAMTS13
activity level, and negative Stx assays, and patients with TMA and The therapeutic response to eculizumab can be assessed by moni-
resistance to or dependency on plasma therapy should be candidates toring disease activity markers, such as platelet counts, lactate
for receiving eculizumab. Patient with aHUS, who undergo kidney dehydrogenase, haptoglobin, and creatinine. If eculizumab fails to
transplantation and have a high risk for recurrence based on their improve aHUS activity markers, the complement activity markers
mutations, should receive prophylactic eculizumab.51 Despite the should be measured. There are no standard ways of monitoring
possibility of complement activation in TTP, there is not enough complement activity on eculizumab. However, a few studies have
evidence to justify treatment with eculizumab simultaneously with shown that CH50, Weislab complement activity assays,52 or C5b-9
plasma exchange in TTP patients, unless genetic studies show deposition on endothelial cells in vitro55 can be used to confirm the
a concurrent mutation in complement proteins. Eculizumab should blockade of complement activity by eculizumab. Among these
not be used in aHUS caused by Cbl C deficiency or DGKE mutation. assays, CH50 is the most readily available one. Effective therapy of
Eculizumab is not shown to be effective in STEC-HUS except in aHUS with eculizumab is expected to decrease CH50 from nor-
anecdotal cases. Similarly, the role of complement activation in the mal values to ,10%.14 If disease activity markers and CH50 show
pathogenesis of secondary aHUS (post-HSCT, cancer, and drug- a suboptimal response to eculizumab, one can consider increasing
induced aHUS) is not proven, and treatment with eculizumab in the maintenance dose of eculizumab by 300 mg.55,56 The con-
secondary aHUS should be considered mainly in the frame of re- centration of eculizumab in blood has not been used as a monitoring
search protocols. Patients with a suspicion of aHUS should receive tool, but a concentration of $100 mg/mL is considered to be
eculizumab at a dose of 900 mg once weekly for 4 weeks (induction), a therapeutic level.51

Hematology 2016 223

Currently, there are no clear guidelines available about the dis- Correspondence
continuation of eculizumab therapy. However, a few facts can be Vahid Afshar-Kharghan, Section of Benign Hematology, MD Anderson
helpful in making a decision about the length of therapy with Cancer Center, 1515 Holcombe Blvd, Houston, TX 77030; e-mail:
eculizumab. There are aHUS genotypes that are associated with [email protected].
a higher risk of relapse and progression to ESRD (FH, C3, FB, and FI
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