Acquired hemolytic anemia

JG Kelton H Chan N Heddle S Whittaker

10

Introduction Drug-induced hemolytic anemia

Drug-induced autoantibody
Clinical presentation and Drug (hapten) dependent antibody
laboratory features Innocent bystander
Pathogenesis of immune and Oxidative injury to red cells
non-immune hemolytic anemias
Non-immune hemolytic anemia
Immune hemolytic anemia
Infection-induced hemolytic anemia
Autoimmune hemolytic anemia
Warm autoimmune hemolytic anemia Mechanical trauma to red cells
(WAHA) Thrombotic thrombocytopenic
Immune specificity of WAHA purpura – hemolytic uremic syndrome
The diagnostic evaluation of a patient Cardiac hemolysis
with suspected WAHA External impact on the red cells
Management of patients with WAHA Thermal damage of red cells
Cold autoimmune hemolytic anemia Osmotic damage of red cells
Paroxysmal cold hemoglobinuria
Miscellaneous causes of hemolytic anemia
Alloimmune hemolytic anemia Paroxysmal nocturnal hemoglobinuria
Transfusion reactions due to Venom-induced hemolytic anemia
immune-mediated hemolysis Toxin-induced hemolytic anemia
Hemolytic disease of the newborn Hemolytic anemia in organ dysfunction

185
10 Blood and bone marrow pathology

acute abdominal conditions or musculoskeletal diseases);

Introduction cardiovascular symptoms such as dyspnea, angina and
As described earlier, hemolysis is a process characterized tachycardia; or non-specific complaints of generalized
by accelerated red cell destruction, which can be com- malaise or dizziness. In some cases, patients can be
pensated for if the body steps up production of new red asymptomatic and their pallor is detected by family
blood cells. However, if red cell destruction surpasses members or other individuals. Jaundice and brownish
production, hemolytic anemia could result. Hemolytic discolored urine are also typical of acute hemolysis.
anemia is traditionally categorized by cause as either Sometimes, in massive acute hemolysis, shock and renal
congenital or acquired. Congenital hemolytic anemia has failure can occur.
previously been presented in detail; therefore, this chapter Determining whether the hemolytic anemia has an
is restricted to a discussion of acquired hemolytic anemia. intravascular or extravascular origin can be helpful when
The term ‘acquired hemolytic anemia’ was first coined establishing the diagnosis. The differential diagnosis of
in the early 1900s1 and it is now commonly used to extravascular hemolysis is more extensive than that of
describe hemolytic anemia triggered by extrinsic factors intravascular hemolysis with the five most common intra-
such as immune disorders, drugs, infections, mechanical vascular hemolytic disorders being: cold autoimmune
trauma to red cells, exposure to toxins, and other mis- hemolysis; malaria; drug-induced hemolysis; major ABO
cellaneous causes. Generally, acquired hemolytic anemia blood group incompatibility; and paroxysmal nocturnal
can be classified as immune hemolytic anemia (auto- hemoglobinuria. Table 10.1 provides a summary of the
immune, alloimmune or drug-induced) and non-immune laboratory results typically seen in intravascular and
hemolytic anemia (infection-induced, mechanical trauma, extravascular hemolysis.
and other miscellaneous causes, including paroxysmal The diagnostic pathway and related laboratory tests
nocturnal hemoglobinuria). In this chapter, the various used to determine whether a patient has a hemolytic
categories of acquired hemolytic anemia are reviewed anemia are presented in Chapter 6 of this book. Laboratory
including pathogenesis, clinical presentation, treatment test information is discussed in this section as they pertain
and management. to specific acquired hemolytic anemia. The laboratory
tests that are typically reviewed when diagnosing
acquired hemolytic anemia are: peripheral blood film
examination, reticulocyte count, bilirubin, haptoglobin,
Clinical presentation and hemoglobinemia, methemalbumin and hemopexin, hemo-
globinuria and hemosiderinuria, lactate dehydrogenase
laboratory features (LDH) and aspartate aminotransferase (AST), bone mar-
Similar to other anemia, the symptoms of acquired row examination, direct antiglobulin test (Coombs’ test),
hemolytic anemia generally include: fever, abdominal and determination of red cell lifespan using radioactive
pain, back pain or pain in the limbs (which may mimic isotope-labeled red cells.

Table 10.1 Typical features that distinguish between intravascular and extravascular hemolysis
Intravascular hemolysis Extravascular hemolysis
Mechanism Red cell destruction in the intravascular Red cells are recognized as foreign or become
compartment resulting in hemoglobin being more rigid and are sequestered in the spleen with
released into the plasma subsequent phagocytosis
Possible causes Complement, toxins, membrane defects, enzyme Immunoglobulin, complement, membrane defects
deficiencies, drugs
Laboratory feature
Hemoglobinemia Present Absent/present in severe cases
Hemoglobinuria Present Absent/present in severe cases
Haptoglobin Reduced or absent Normal or reduced
Methemalbumin Present Absent
Hemosidinuria Present Absent
LDH Grossly elevated Elevated
Jaundice Present Present
Splenomegaly Absent Present
Blood film Schistocytes, helmet cells, fragmented red cells Spherocytes, erythrophagocytosis

186 LDH, lactate dehydrogenase.

 Acquired hemolytic anemia 10
plastic syndrome,2 or graft-versus-host disease. In one
Pathogenesis of immune and study, neoplasia-related AIHA was the most common
non-immune hemolytic anemias secondary cause (233/1834 patients; 2.7%) followed by
drug-induced hemolysis (140/1834 patients; 7.6%).2 For
In this chapter, immune hemolytic anemia is classified as
diagnostic evaluations, it is important to determine the
autoimmune, alloimmune and drug-induced; whereas,
autoantibody idiotype. Autoantibodies may readily be
non-immune hemolytic anemia is presented under the
separated into warm antibody or cold antibody based on
headings of infection-induced, mechanical trauma and
the thermal range of the antibody and this classification
other miscellaneous causes. Note that different causes of
serves as the framework of the following discussion.
hemolytic anemia can overlap. For example, drug-induced
hemolysis often overlaps with immune hemolysis because
drugs can cause hemolysis either by immune mechanism Warm autoimmune hemolytic anemia
or by direct damage to the red cells. (WAHA)
Warm autoimmune hemolytic anemia (WAHA) accounts
for more than 70% of AIHAs3 and it is caused by
Immune hemolytic anemia production of an autoantibody directed against the
patient’s red cells. Most frequently, the autoantibody is
Immune hemolytic anemia is the most common form of IgG (particularly IgG1 and IgG3 subclass),4 although IgM
acquired hemolytic anemia and it can be classified as and IgA can be detected in addition to IgG.5 IgA warm
autoimmune, alloimmune or drug-induced. Irrespective autoantibody is rare, and most antiglobulin reagents
of the etiology, the typical feature in the peripheral blood are unable to detect this immunoglobulin. Warm auto-
smear is microspherocytosis (Fig. 10.1). antibody (IgG) may cause extravascular hemolysis
through one of two mechanisms: (1) Fc receptor-mediated
immune adherence; and (2) complement mediated
Autoimmune hemolytic anemia hemolysis. Most warm autoantibodies do not cause
Autoimmune hemolytic anemia (AIHA) involves the pre- autoagglutination or intravascular hemolysis.
mature destruction of red blood cells by autoantibodies.
AIHA may be idiopathic (no underlying cause identified) Fc receptor (FcR)-mediated immune
or secondary to underlying malignancy or disorder such adherence
as lymphoid malignancy, connective tissue disorders, in-
fection, medication, vaccinations, HIV-induced, myelodys- Antibody-coated red cells can be removed from the circu-
lation by two different mechanisms: phagocytosis and
cell lysis. During the phagocytic process, macrophages
engulf and lyse red cells by formation of oxygen radicals
in the cytoplasm; whereas, for cytotoxic cell lysis, the
target cell is destroyed by the lysosomal enzyme released
by the phagocytic cells.6
The phagocytic process is facilitated by the deposition
of opsonins, including antibody or C3b, on the antigen.
The immune effector cells have receptors (FcR) on the cell
surface for the Fc portion of antibodies. There are at least
three different classes of IgG FcR on the macrophages
(FcγRI, FcγRII and FcγRIII). FcγRII and FcγRIII bind to IgG
oligomer7,8 but FcγRI binds only to monomeric IgG.9,10
FcγRII and FcγRIII binds IgG3 oligomer and IgG1
oligomer with the highest affinity.9 The high affinity of
IgG3 to FcRs might explain why IgG3 is the most efficient
warm antibody in extravascular hemolysis in vivo.3,11
Fig. 10.1 Peripheral blood smear showing microspherocytosis, The in vivo functions of FcγRIII include phagocytosis,
nucleated red cell, and red cell clumps in a patient with immune endocytosis and antibody-dependent cell-mediated
mediated hemolysis. The spherocytes lack central halo of normal
red cells and they are smaller than the nucleus of a normal cytotoxicity.12,13 FcγRII is an inhibitory receptor and
lymphocyte. Wright stain, × 1000, oil field. acts as a negative regulator of B-cell and mast cell 187
10 Blood and bone marrow pathology

activation.14,15 On the other hand, FcγRI exclusively Complement activation is controlled by a number
mediates in vitro cytotoxic activity.16 of regulatory mechanisms. Some complement fragments
Apart from IgG, the presence of other classes of anti- can become enzymatically cleaved and the cleavage
red cell antibodies (IgM and IgA) is a major determining products, such as C3d or C4d, are incapable of further
factor of hemolysis.4 IgM, apart from complement activation. The complement inhibitors on cell membrane
activation, may synergistically enhance IgG-mediated include decay accelerating factor (DAF),21 C8-binding
hemolysis. It has been demonstrated that patients with protein,22,23 membrane cofactor protein, CR1/CD35
red cells coated with IgG and IgM have more severe protein and P18/CD59 protein.24 Other proteins such as
hemolysis than those with IgG alone as these patients C1 inhibitors, C4 binding protein, factor H and factor I
have lower level of haptoglobin.5 IgA autoantibodies, are circulating in the plasma. The complement cascade is
although rare, probably induce hemolysis via FcR in arrested and early complement protein fragments are
the same way as IgG.2 However, the exact relationship bound to the red cell membrane. The complement-coated
between FcR and the different combinations of immuno- red cells are then recognized by complement receptors on
globulin classes remains unclear. Circulating monocytes, hepatic Kupffer’s cells. It has been demonstrated that
K-cells and granulocytes also have FcR; however, the role about 550–800 bound C3b molecules are required to
of these cells in the pathogenesis of hemolysis is un- trigger hepatic clearance of red cells. As these receptors
certain, although it has been postulated that they may are less efficient than the splenic macrophage FcRs,
play a role in patients with immune hemolytic anemia complement-sensitized red cells often escape the phago-
refractory to splenectomy.17 cytic or cytotoxic process and circulate in the peripheral
The amount of antibody bounded on the red cell blood with normal or slight reduction in red cell survival.
surface also determines the mechanism of FcR-mediated Because C3d or C4d coated red cells have less binding
hemolysis. When the amount of antibody on the red cell sites for other complement proteins, these cells are in fact
membrane is low, the phagocytic process is predominant.2 protected from further hemolysis. However, C3d and
However, because IgG1 is a less efficient mediator of C4d coated cells are less deformable than normal ery-
phagocytosis than IgG3, IgG1 coated red cells are not throcytes; hence, the complement proteins may augment
always rapidly destroyed. IgG-mediated phagocytosis.25–27 Consequently, the extra-
Under normal conditions, a typical splenic macrophage vascular hemolytic process may be enhanced by the
has 30 000–40 000 FcR per cell.18 Concomitant infection or presence of complement on the cell, whereby complement
immunization worsens the hemolysis by increasing the acts as an opsonin to facilitate the phagocytic process.
number and affinity of FcR, possibly through cytokines
such as gamma interferon.19 Circulating neutrophils prob-
ably have only a marginal role in autoimmune phago-
Immune specificity of WAHA
cytosis, however, they may become important during In initial serologic testing, most warm autoantibodies
infection.20 are panagglutinins reacting with all red cells. These
In summary, the FcR-mediated cell destruction processes panagglutinins can be classified into different categories
depend on a number of factors: the specific immune based on the agglutinating pattern with certain Rh
characteristics of the immunoglobulins; the amount of phenotypes: (1) the antibodies react with all red cells with
antibody bound to the red cells; and the overall activity a normal Rh phenotype but not with those having Rhnull
of the macrophages in the reticuloendothelial system. phenotype, or with those having partially deleted Rh
antigens; (2) the antibodies are specific to part of the Rh
antigen system (i.e. react with normal Rh phenotype and
Complement-mediated hemolysis
cells with partially deleted antigens); and (3) the anti-
The activation of the complement cascade, up to and bodies react to all cells including Rhnull cells.28 The first
including the terminal pathway, brings about the for- two categories, which are specific to the Rh antigen
mation of a membrane attack complex and intravascular system, account for 50–70% of warm autoantibodies29 as
hemolysis. To activate the complement pathways, two most warm autoantibodies react with all red cells except
FcRs must be spatially close together. IgM is the most those of the Rhnull phenotype. In one series of 150 persons
efficient antibody class in the activation of complement; with warm autoantibodies, only four were specific to
however, in warm autoimmune hemolysis, IgG on the only a single Rh antigen (i.e. anti-e or anti-c specificity).30
red cell membrane occasionally may be of sufficient den- Because Rh antigens have a relatively low density on the
sity to activate complement. Of the IgG subclass, IgG3 red cell membrane, autoantibodies against Rh antigens
188 and IgG1 bind readily to C1q, but not IgG4. do not activate complement.
 Acquired hemolytic anemia 10
Autoantibodies not showing Rh specificity can some- IgG1 appears to be the predominant IgG subclass detected.
times show specificity for other red cell antigens. Using IgG1 has been shown to be less efficient than IgG3 for
immunoprecipitation methods, up to 50% of warm auto- activating complement,36 which may explain the lack of
antibodies precipitate band 3 and glycophorin A.31,32 hemolysis. In about 2% of patients with WAHA, the DAT
Band 3, an anion transport protein, requires a portion of is negative (i.e. Coomb’s negative autoimmune hemolytic
glycophorin A to form Wrb antigen. Interestingly, Dib, an anemia). There are at least two possible explanations: (1)
alloimmune antigen of Diego system, is also carried on the level of red cell antibody sensitization is below the
protein band 3. Compared with antigen Wrb, Dib is a rare sensitivity of the DAT; or (2) hemolytic anemia caused
target for autoantibodies.30 The glycosylation and the by IgA or other immunogloblulins37–39 that would not be
complexity of the Wrb and Rh antigen system may detected by conventional antiglobulin reagents. Variable
implicate their immunogenicity.30 expression of red cell antigen during the course of
Autoantibody formation can also impact red cell anti- disease40 may also play a role in Coomb’s negative auto-
genic expression.33 Decreased expression of blood group immune hemolytic anemia.
antigens in the following systems have been described: The lower limit of immunoglobulin detection using the
Kell, Rh, MNS, Duffy, and the Kidd system.33,34 The DAT is estimated to be about 100–300 antibody molecules
mechanism of this phenomenon is unknown, but it may per red cell.41 Tests such as 125I-radioimmune direct anti-
be an effect to protect against hemolysis. globulin test,42 enzyme-linked direct antiglobulin test,43,44
Some warm autoantibodies appear to have specificity and two-stage immunoradiometric assay with 125I-
against certain red cell antigens on initial testing, but the staphylococcal protein A45 may increase the sensitivity of
antibodies can still be absorbed by red cells lacking the detecting antibodies on the red cells of patients with
corresponding antigen. In addition, elutes made from DAT-negative autoimmune hemolytic anemia.3 However,
the red cells used for this absorption procedure again the significance of these findings is uncertain as it has
demonstrate immune specificity against that particular been shown that clinical hemolysis is unlikely to occur if
antigen. The cause of pseudo-specificity of warm auto- the number of IgG1 molecules is less than 1000 per cell.46
antibodies is unclear. Clinically, the pseudo-specificity of Special techniques for detecting IgG, such as assays using
the autoantibodies is not directly associated with the staphylococcal protein A or functional assays (e.g. the
severity of hemolysis, although the serologic results may monocyte-phagocytic test), are sometimes useful in these
lead to diagnostic confusion. Sometimes, autoantibodies cases; however, these assays are difficult to perform and
of real specificity and of pseudo-specificity may coexist in they are not routinely available. In some cases, concen-
the same patient.33,35 This is of practical importance in trated elutes may demonstrate the presence of antibody
selecting compatible blood for transfusing patients with even though the DAT is negative. Before the diagnosis of
warm autoimmune hemolytic anemia. Autoantibodies of Coombs’ negative autoimmune hemolytic anemia is
pseudo-specificity have been described in blood group made, other non-immune causes of hemolysis must be
systems such as Kell, Duffy, Kidd and MNS systems.33 excluded.
The indirect antiglobulin test (IDAT) will detect
autoantibody in the patient’s serum in 57.4% of patients
The diagnostic evaluation of a patient with a routine antibody screen, and up to 88.9% of cases
when enzyme-treated red cells are used.47 If the patient
with suspected WAHA requires transfusion, the challenge for the laboratory is
With suspected WAHA, the most useful test to detect the detection of alloantibodies when warm autoanti-
warm antibodies is the direct antiglobulin test (DAT) bodies are present. A warm autoabsorption technique
with polyclonal or monoclonal antibodies specific for IgG will remove autoantibody and leave alloantibody to be
or C3d (also called the Coombs’ test). However, the detected. Alternatively, a titration technique can be useful
presence of antibodies and/or complement on the red if the alloantibody has a higher titer than the autoantibody.
cell membrane does not necessarily result in hemolysis
and the strength of reactivity of the DAT is not directly
related to the clinical severity of hemolysis. A positive
Management of patients with WAHA
DAT without evidence of hemolysis occurs in about one The fundamental therapy for WAHA is to suppress auto-
per 10 000 healthy blood donors. antibody production. However, it usually takes time for
IgG and C3 are detected on the red cells in 50% of the immunosuppressive therapy to exert its full therapeutic
cases; IgG alone in 23% of cases and C3 in 27% of the cases. effect. Hence, acute supportive measures may be required
When IgG is detected on red cells of normal individuals, in patients with acute hemolytic events. Treatment of 189
10 Blood and bone marrow pathology

WAHA can be broadly divided into supportive and more alloantibody production.33 When hematologic improve-
definite treatments. ment is seen, the dose of steroid should be gradually
There should be no difficulty in finding compatible tapered over the next several months to minimize the
blood for transfusion if patients have a negative DAT. side-effects of long-term steroid therapy. In 60–70% of
However, many patients with severe hemolysis have patients, complete remission can be achieved, but for
detectable plasma antibody (panagglutinins); hence, it some patients, maintenance therapy is required. Among
may be impossible to find compatible blood for these these patients, 50% may relapse and further treatment
patients. In this case, if clinically indicated, incompatible with higher doses of steroid therapy may be beneficial. If
blood can be transfused if the laboratory takes special there is a nonresponse to steroid by the end of the first 3
care to ensure it is ABO and Rh compatible. It has been weeks, continued therapy with steroid alone is usually
reported that blood transfusion may adversely affect the ineffective. Up to 40% of patients with WAHA become
patient by introducing more alloimmune antigens that either steroid-dependent or steroid-resistant.86,87
further activate the immune system.33 None the less, Splenectomy is effective in about half of patients
transfusion therapy is still an important supportive with WAHA.88 Splenectomy removes the major site of
measure in patients whose anemia has put them at risk of antigen presentation and, in turn, reduces antibody
serious complications or death. production.33,89,90 With the advance of laparoscopic splen-
Corticosteroid therapy, initiated with the blood trans- ectomy, the incidence of severe surgical complications
fusion, may suppress the immune destruction of the has been reduced.91,92 The major long-term complication
transfused red cells.3 Rarely, other adjunct supportive of splenectomy is infection, particularly of encapsulated
measures such as oxygen, sedation, ventilator support, organisms. Therefore, all patients undergoing splen-
and hypothermia therapy may be needed. ectomy should receive vaccine immunization against
High-dose intravenous gamma-globulin (IVIgG) has Streptococcus pneumoniae, Meningococcus and possibly
been investigated as an acute treatment of WAHA.48–59 Hemophilus influenzae.
This therapy causes blockage of the reticuloendothelial Immunosuppressive therapies, including vinca
system and reduces the clearance of the IgG-sensitized alkaloids,93 azathioprine and cyclophosphamide, have
red blood cells. The use of IVIgG is well described in the been reported to be beneficial in the treatment of WAHA,
treatment of hemolysis associated with Evan’s syndrome although the therapeutic effect may be delayed for 3–6
and lymphoproliferative diseases, and during an acute months after the initiation of treatments. In a small case
phase of hemolytic crisis.60 However, the use of IVIgG is series, the response rate was reported to be about 50%.86
consistently less effective for AIHA compared to immune Patients who do experience a clinical response to this
thrombocytopenia. Consequently, a higher dose of gamma therapy may require maintenance therapy for up to 12
globulin may be needed because patients with auto- months to induce remission.
immune hemolytic anemia may have an expanded A recent study has suggested that Danazol may
reticuloendothelial system.60 The mechanism of action of induce long-lasting remission in patients with refractory
IVIgG therapy is likely due to FcR blockade,61–67 although WAHA.94–97 The possible mechanisms include: reduction
other mechanisms such as the presence of anti-idiotype in red cell bound C3d;98 immunomodulation by alter-
in the IVIgG,61,68–72 interference with T-cell signals to B- ation of T-cell subsets;99 and reduction of FcR in the
cells,73–76 activation of T-suppressor cells, and inhibition reticuloendothelial (RE) system.100 Side-effects from
of B-cell maturation77–82 have been described.33 Because Danazol include virilization effects and dose-dependant
the therapeutic effect of IVIgG is usually short-lived, hepatic toxicity.
further immunosuppressive therapy is required to main-
tain clinical remission.
Treatment with high-dose steroids, usually in the form
Cold autoimmune hemolytic anemia
of prednisone at 1–1.5 mg/kg/day, is initiated once the Cold autoimmune hemolytic anemia (CAHA), also known
diagnosis of WAHA has been confirmed. The median as cold hemagglutinin disease, is much less common
time of response is 7–10 days. The mechanism of action of than WAHA. In a large prospective series,3 391/2390
steroid therapy include: suppression of red cell clearance patients (16.4%) undergoing investigations for red cell
by the reticuloendothelial system;83 downregulation of the autoantibodies presented with cold autoantibodies while
number of FcR;84 inhibition of the release of lysosomal another 10 patients (0.4%) had both warm and cold
enzymes by macrophages; and suppression of auto- autoantibodies. In another series AIHA,101 a combination
antibody production.85 Steroid therapy can reduce the of warm and cold autoantibodies was reported in 8% of
190 concentration of autoantibody, but has no effect on patients with AIHA. These antibodies react best at cold
 Acquired hemolytic anemia 10
temperatures between 15 and 20°C. Clinically, the
specificity of cold antibody is less important than the
thermal amplitude.
The presence of a cold hemagglutinin is readily
demonstrated using the cold agglutinin test. When deter-
mining the thermal range of the antibody using this
procedure, albumin should be added as this improves
the clinical correlation. The cold agglutinin often causes
problems with ABO and Rh phenotype cross-match. To
overcome this dilemma, the red cells should be washed
with warm saline to remove the cold agglutinin. The
cross-match and antibody screen should be done using a
prewarmed sample and using monospecific anti-IgG to
avoid false-positive reactions. The DAT, using monoclonal
reagents, is usually negative for IgG, but positive for C3d
Fig. 10.2 Peripheral blood smear showing agglutination of red when using an ethylene diamine tetra-acetic acid (EDTA)
cells in a patient with Waldenström’s macroglobulinemia. The
background of the smear is bluish because of high protein
sample.
content, which contains lgM hemagglutinin. Wright stain, × 400. Most patients with CAHA have mild symptoms or
none at all. Patients are warned to avoid exposure to cold.
During a severe acute hemolytic episode, plasmapheresis
temperatures (below 30°C). When the peripheral blood of can be used to reduce the level of IgM cold autoantibody.
patients with cold hemagglutinin examined at room Plasmapheresis is especially effective in CAHA because
temperature, it typically shows agglutination of red cells IgM is confined to the intravascular space. This procedure
(Fig. 10.2). This temperature-dependent reactivity of the is best performed in a warm environment and the tubing
cold autoantibodies is of clinical significance because of the plasmapheresis machine may have to be pre-
hemolysis is unlikely to occur if the thermal reactivity is warmed. Blood transfusion, when needed, is infused at
below 30°C. Cold autoantibodies are mainly IgM (85%);3 room temperature with a controlled rate. It is still contro-
however, the IgG biphasic Donath–Landsteiner antibody versial whether the use of a blood warmer offers additional
accounts for approximately 15% of the cases, especially in benefits. If CAHA is secondary to an underlying neo-
the pediatric age group.3,102 plastic disease, chemotherapy including alkylating agents
Red cell sensitization by cold IgM usually occurs in the may reduce the production of cold autoantibody.
body extremities (e.g. fingers, ears, nose) where tem-
peratures may fall below 30°C allowing antibody binding
to the red cells. The complement cascade is activated, and
Paroxysmal cold hemoglobinuria
if the cascade proceeds to activation of the membrane Paroxysmal cold hemoglobinuria (PCH) was one of the
attack complex, hemolysis will occur. This gives rise to first recognized anemias when described in the mid-
the characteristic clinical features of Raynaud’s phenom- 1800s107 and, for years, it was commonly believed to be a
enon, acrocyanosis or gangrene. If the inhibitors of rare acquired AIHA associated with congenital syphilis.
complement stop the cascade, the red cells (now coated However, it is now recognized that PCH causes up to
with C3b) can be removed through extravascular phago- 40% of acute transient hemolytic anemia in young
cytosis. Eventually C3b is degraded to C3d on the cell children108 during viral infections such as measles,
surface. As macrophages do not have receptors for C3d, mumps, chickenpox and influenza; however, due to the
these red cells will not be cleared from the circulation. transient nature of the disease, the diagnosis of PCH may
It is this component of complement that is detected by be difficult after acute episodes. The cold biphasic IgG
the DAT. The most common autoantibody specificity in antibody (Donath–Landsteiner antibody) directs against
CAHA is to the ‘I’ blood group antigen. Auto-anti-I globoside glycosphingolipid (P antigen) and causes
typically occurs during or after Mycoplasma pneumoniae hemolysis by a unique mechanism. The antibody binds
infection. In contrast, anti-I may be formed during or to red cells at cooler temperatures in the peripheral circu-
following infectious mononucleosis. Other less frequent lation. The complement pathway is activated and intra-
autoantibody specificities include: P, Pr, A1, D, Vo, Gd vascular hemolysis occurs when red cells return to the
(glycolipid dependent gangliosides),103,104 -Lud,105 F1,106 warmer core body temperature. The Donath–Landsteiner
Ju and IA3,107 antigens. Specificity will be apparent at antibody is more potent than IgM cold agglutinin in its 191
10 Blood and bone marrow pathology

ability to initiate intravascular hemolysis, mainly because ing bone marrow or stem cell transplantation. Apart from
antigen/antibody affinity of the cold IgM antibody another individual’s red cells, the sources of alloantigen
decreases when red cells return to core temperatures.2,109 also can be from environmental antigens unrelated to
It has been demonstrated that IgG3 is the major immuno- erythrocytes.
globulin subclass for Donath–Landsteiner antibody.110
The definitive test for PCH is the Donath–Landsteiner
test, which can be performed as a direct or indirect Transfusion reactions due to immune-
procedure.111 The direct test is conducted by incubating a
test sample at 0°C for 1 h, and then at 37°C for an
mediated hemolysis
additional 30 min. If the Donath–Landsteiner antibody is The first transfusion reaction was recorded in 1667 by
present, it will bind to the red cells during the cold Denis.114 Today, the incidence of clinically relevant
incubation phase and hemolyse the cells during the warm immune-mediated hemolytic reactions is estimated to
phase. A positive test result is not considered validated be around one reaction per 20 000 units of blood
unless a control tube, maintained at 37°C throughout the transfused.115–119 The spectrum of clinical presentations
test, shows no hemolysis. The indirect test is done by can range from life-threatening ABO incompatibility to
mixing the patient’s serum with ABO compatible P- subclinical hemolysis depending on the magnitude of
positive red cells of a normal person in the presence of antigen–antibody interactions, the degree of complement
fresh serum as a source of complements. The indirect test activation, and the activity of the reticuloendothelial
has a much higher sensitivity than the direct test for system.
several reasons: (1) complement proteins are present in There are two major types of immune-mediated
fresh normal serum; (2) the serum-to-cell ratio can be hemolysis following blood transfusion: acute and delayed.
adjusted to increase sensitivity; and (3) the indicator cells More than 80% of acute hemolytic transfusion reactions
from an allogenic donor are more susceptible to lysis than are due to ABO incompatibility.116 The mortality rates of
the patient’s own red cells as the indicator cells are not up to 40% seen in the past115 have declined to 10%, prob-
coated with C3d. The sensitivity of the test can be increased ably reflecting improved management and support.
further by using enzyme-treated red cells. False-positive Transfusion reactions due to intravascular hemolysis are
results can occur due to IgM hemolytic antibody with a generally more severe than with extravascular hemolysis
high thermal range. False negatives occur less frequently because of the side-effects associated with complement
in the indirect Donath–Landsteiner test, although they activation which may be triggered by alloantibodies to
can occur when the antibody titer is low or if soluble Kell, Kidd and Duffy antigen systems.114–116 Patients may
globoside (P antigen) present in the fresh normal serum experience fever, chills, joint pain, shock, renal failure
causes inhibition of the antibody.102 and/or disseminated intravascular coagulation.
The management of PCH may require urgent blood Delayed hemolytic transfusion reactions may be caused
transfusions depending on the level of anemia and by primary alloimmunization but, more frequently, the
whether the patient is symptomatic. Theoretically, donor transfused red cells trigger a delayed (anamnestic) IgG-
cells from P-negative patients may minimize in vivo mediated transfusion reaction 7–14 days after the blood
hemolysis; however, P-negative blood is usually not transfusion. Although most patients have been previously
available and in most cases, transfusion with P-positive alloimmunized, over time, their antibody level falls below
blood can be beneficial. Patients with PCH can be success- detectability and it can be difficult to prevent this form
fully transfused if the transfusion rate is slow and the of transfusion reaction.33 Sometimes, the only clinical
patient is kept warm and closely monitored.112 The use of evidence of this reaction is a positive DAT.
a blood warmer may be beneficial for some patients. It One of the major dilemmas for clinicians in the manage-
has been shown that the removal of complement proteins ment of post-transfusion reactions is how to differentiate
from donor plasma by washing red cells113 and by using immune-mediated transfusion reactions from pseudo-
steroid therapy have not been beneficial.102,108,112 hemolytic transfusion reactions. Non-immune hemolytic
anemia can occur if transfused red cells are subject
to excessive thermal, osmotic, mechanical or chemical
Alloimmune hemolytic anemia insults. Clinically, febrile symptoms secondary to
Alloimmune hemolytic anemia occurs when the immune hemolysis may have other causes including: (1) febrile
system is sensitized and antibodies form in response to reactions due to non-hemolytic transfusion reactions; (2)
red cell alloantigens. Typically, this occurs following a bacterially contaminated blood products; (3) transfusion-
192 blood transfusion, during or after pregnancy, or follow- related acute lung injury; or (4) severe allergic symptoms
 Acquired hemolytic anemia 10
(hypotension and shock) that mimic acute hemolytic With the advance of modern medicine, hydrops fetalis
transfusion reactions. However, the presence of cutaneous due to Rh HDN is rarely seen. Primary prevention is the
manifestations suggest an allergic pathophysiology. cornerstone of the management of Rh HDN. First, Rh-
negative women who can bear children should receive
Rh-negative blood. Second, Rh-negative pregnant woman
Hemolytic disease of the newborn should receive passive immunization with Rh immune
Hemolytic disease of the newborn (HDN), also called globulin at 28-weeks’ gestation and within 72 h of ex-
erythroblastosis fetalis, is hemolysis in the fetus caused posure to fetal D-positive red cells due to either delivery
by transplacental transfer of maternal IgG. During or an invasive procedure such as an abortion. Although
pregnancy, maternal IgG is transferred via specific the dose regimens are slightly different between North
FcRs on the placental cells.120–123 Alloimmune hemolytic America and Europe, the rule of thumb is that 10 µg of
anemia may occur in the fetus if there is a blood group anti-D should be given for each ml of Rh D positive
incompatibility between the mother and the fetus. whole blood. This dose can be decreased if intravenous
Generally, by 12 weeks, maternal IgG is detectable in the anti-D is used. Kleihauer–Betke tests or flow cytometry
fetal circulation33 and the rate of IgG transfer progressively can be useful in determining the amount of fetal cells in
increases across the pregnancy so that, at term, the fetal the maternal circulation so that the dose of Rh immune
IgG level may be equal to124 or higher33,120 than that of the globulin can be adjusted. The successful rate associated
mother’s. The antibodies against the Rh and the ABO with Rh immune globulin prophylaxis is estimated to be
antigen systems are the two major causes of hemolytic 98–99%.
disease of the newborn, with anti-D causing the most The prenatal management of women at risk of HDN
severe cases.33 With the routine use of Rh immune varies. All pregnant women should be typed for ABO
globulin, HDN is now seen more often with antibodies of and Rh D and have an antibody screen performed during
other blood group antigens. the first trimester, usually at the first visit to the
During pregnancy, most women are exposed to less obstetrician. For D-negative women without detectable
than 0.1 ml of fetal blood, and this small volume does not antibody, an antibody screening should be repeated at
cause alloimmunization. However, in a subsequent the 28th–30th week at the time that Rh immune globulin is
pregnancy, the secondary immune response may trigger given. If alloantibody is detected, titration studies should
the production of large amounts of IgG antibody and this be performed at regular intervals. The antibodies titer
could cause severe hemolysis of the fetal red cells. Hence, may be used to guide the timing of fetal monitoring and
Rh HDN seldom affects the first pregnancy. With Rh further interventions;126 however, it must be emphasized
antibodies, the severity of the disease is progressive in that the titer is only a semi-quantitative indicator. Once
each subsequent pregnancy. The risk of stillbirth in a the titer reaches a certain level (1:16–1:32), other measure-
woman with a previous history of mild Rh HDN is about ments should be considered to estimate the risk of severe
2% compared to a 70% risk in a woman with a previous HDN. The severity of HDN may be predicted by various
history of Rh alloimmunization.125 Conversely, ABO HDN methods including: (1) amniocentesis with measurement
may affect a firstborn infant as the antibodies are already of total bile pigment in the amniotic fluid;127 (2) ultra-
preformed due to environmental stimulation. The severity sonography to detect the evidence of extramedullary
of ABO HDN in a previously born fetus does not predict hematopoiesis;128,129 and (3) percutaneous umbilical blood
the severity in the next infant.33 sampling.130 However, there is no available test to identify
Stillbirth and hydrops occur in the severest cases of women whose infants are at risk of ABO HDN. ABO
HDN. Severe fetal anemia can result in extramedullary alloimmunization does not affect the fetus in utero;
erythropoiesis, gross hepatosplenomegaly, portal hyper- however, symptoms of hemolysis occur 12–24 hours after
tension and hepatic failure. More commonly, the affected birth.
fetus may present with anemia and hyperbilirubinemia There are several therapeutic interventions to reduce
within the first 24 h of life. Without proper treatment, fetal death due to severe HDN. IVIG, started at the
kernicterus may develop. On the other end of the spectrum, 10th–12th gestational week at 1 g/kg every 1–3 weeks until
positive serologic tests with no clinical findings may be delivery, is moderately effective with multifactorial
the only indication of antibody transfer to the fetus. Since effects including blockage of FcRs on the placenta and
HDN cannot be diagnosed solely by serologic tests, it fetal RE system. Anecdotal reports describe that intense
has been suggested that laboratory evidence of disease maternal plasmapheresis with IVIG replacement and/or
without clinical findings should be described simply as plasma replacement from a D-negative donor may be a
maternal–fetal blood group incompatibility.33 useful treatment between the 10th and 24th weeks, after 193
10 Blood and bone marrow pathology

which intrauterine transfusion can be performed. As test;132 however, only 0.3–0.8% of patients develop
intrauterine transfusion requires specialized trained hemolytic anemia.133,134 Kelton135 showed that patients
personnel and some risk of maternal/fetal hemorrhage, with a positive DAT, but without hemolysis, have
it should only be used in the case where fetal survival significant impaired RE function. The development of a
is at risk. Donor blood should be fresh, irradiated, positive DAT in patients on α-methyldopa depends on
cytomegalovirus (CMV)-negative, and lacking the antigen the dose132 and the duration of the therapy. It typically
specific for the mother’s antibody.33 Some blood centers occurs in patients after 3 or more years of continuous
use group O blood whereas others use ABO-specific treatment.136 Hemolysis resolves after discontinuation of
blood. Preterm delivery usually occurs around the 35th the drug although serologic abnormalities usually resolve
week gestation given the risk associated with HDN. After more gradually and this can last for up to 24 months.137
delivery, phototherapy and/or exchange transfusion The autoantibody induced by α-methyldopa is IgG,138
may be used to reduce hyperbilirubinemia and the risk of and is directed against Rh antigens in many patients.139,140
kernicterus. Intravenous immunoglobulin (IVIg), directly The antibody does not activate complement; hence, the
infused to the newborn, has been shown to be beneficial DAT is positive only with IgG detected on the cells.
in reducing the need of exchange transfusion.131 If the The pattern of antibody specificities is similar to that in
infant is severely anemic, small volume transfusion may idiopathic autoimmune hemolytic anemia. Besides a
be required during the first few months of life. positive direct antiglobulin test, patients may have
positive antinuclear factor,136,141,142 lupus erythematosis
(LE) cells,136,142–145 rheumatoid factor142,145 and factor III
inhibitor.146 The underlying pathogenesis is still un-
Drug-induced hemolytic anemia known but α-methyldopa is demonstrated to produce an
Drug-induced hemolysis can be immune mediated or aberration in lymphocyte proliferation by increasing lym-
non-immune mediated. The former is categorized into phocyte c-AMP that inhibits suppressor T-cell function.147
three major groups based on different mechanisms of Other drugs including levodopa, procainamide and
action. Drugs, such as α-methyldopa, may induce true NSAIDs (e.g. mefenamic acid and diclofenac) may also
autoantibody production similar to warm autoimmune induce autoantibody similar to α-methyldopa.148
hemolytic anemia. Penicillin (hapten) binds to the red cell
membrane and stimulates antibodies that are directed at
the drug bound to the red cell. Drugs, such as quinine
and quinidine, bind to plasma proteins, antibody, and
Drug (hapten) dependent antibody
then to the red cell. The last two categories illustrate the A number of drugs bind firmly to red cell membranes,
immunologic principle that a small chemical with a possibly via covalent bonds149 and they elicit the
molecular weight under 500–1000 is unable to induce an production of IgG specific for that drug. As a rule, the
immunologic response unless it is tightly bound to a antibody binds to red cells in the presence of the drug
macromolecule such as a protein. As the antibodies can- and this causes a positive DAT result. Usually the positive
not bind to red cells in the absence of a drug, they are also DAT is a coincidental finding and only rarely will these
called drug-dependent antibodies. On the other hand, antibodies cause overt hemolysis, which is usually extra-
some drugs may directly damage the red cells or induce vascular and, very infrequently, intravascular.150 Penicillin
oxidative changes in the cells. The former mainly occurs and cephalosporin are the most common drugs that
with industrial toxins such as copper and arsine, and do stimulate antibody production by this mechanism. They
not involve antibodies (non-immune). share a common benzyl-penicillol group that acts as a
hapten.151 It should be noted that up to 97% of normal
healthy adults have antipenicillin antibody; however,
this is an IgM antibody that can be present even without
Drug-induced autoantibody prior treatment with penicillin or cephalosporin.152 The
True autoantibody induction by α-methyldopa provides occurrence of hemolytic anemia depends on the appro-
a human model for studying the mechanism underlying priate ratio of antigen and antibody. Therefore, penicillin-
autoimmune disorders. It is one of the most extensively induced hemolytic anemia typically occurs in patients
studied drug-induced autoimmune hemolytic anemia. treated with high-dose penicillin for 7–14 days via intra-
None the less, the exact mechanism of autoantibody venous route, or in patients with renal failure resulting in
formation is still unclear. Up to 20% of patients on reduced drug clearance.33,152 Hemolysis stops promptly
194 methyldopa therapy develop a positive direct antiglobulin when the drug is stopped.
 Acquired hemolytic anemia 10
Innocent bystander
Instead of binding to red cell membranes, this group of
drugs (haptens) binds to plasma proteins, and the drug–
protein complex becomes sufficiently large to trigger an
immune response.153 The drug–protein complex binds to
the surface of red cells and is further stabilized by the
drug-dependent antibodies that also have immune
specificities against alloantigens on the red cell mem-
brane. The antibody can be IgM, IgG or both. The
antibody–drug–protein complex activates the comple-
ment pathway and triggers red cell clearance. Sometimes,
other cell lines in the blood are also involved producing
neutropenia or thrombocytopenia.33,152 Examples of drugs
in this category include quinine, quinidine, sulfonamides,
sulphonylurea and thiazide. Fig. 10.3 Peripheral blood smear showing spherocytosis, bite cells
It is likely that the pathophysiology of drug-induced and blister cells (ghost cells) in a patient with drug-induced
oxidative hemolysis. Wright stain, × 1000, oil field.
hemolysis is more complex than noted previously. For
example, Phenacetin, Streptomycin, and non-steroidal
anti-inflammatory drugs can cause hemolysis by both Diagnosis of drug-induced hemolytic anemia depends
autoantibodies (drug-independent antibodies) and drug- on a detailed drug history. Demonstration of drug-
dependent antibodies.138,154–156 dependent hemagglutination in the indirect antiglobulin
test is confirmatory. However, sometimes, the diagnosis
may only be established by resolution of the hemolysis
Oxidative injury to red cells
on removal of the offending drug.
Hemoglobin binds oxygen and, consequently, it is prone
to oxidation and denaturation by oxidative agents,
particularly if the anti-oxidative protective mechanisms
in the red cell are overwhelmed. The amount of oxidative Non-immune hemolytic anemia
hemolysis is determined by the strength and the blood
level of the oxidant, and congenital deficiency of the G6PD Infection-induced hemolytic
or glutathione-dependant pathways. Older red cells are
more prone to oxidative injury by oxidants than are
anemia
young red cells. The characteristic features of oxidative Microorganisms may cause injury to red cells through
hemolysis include the formation of methemoglobin, different mechanisms such as: (1) physical invasion of red
sulfhemoglobin and Heinz bodies. Clinically, methemo- cells (e.g. malaria); (2) hemolysin secretions to damage the
globin and sulfhemoglobin may present as bluish dis- red cells directly (e.g. Clostridium perfringen) (Fig. 10.4);
coloration indistinguishable from cyanosis. Heinz bodies (3) infection that triggers formation of antibody (anti-I)
are the microscopic appearance of denatured hemo- against red cells (e.g. mycoplasma); (4) microangiopathic
globin. Moreover, examination of a peripheral blood hemolysis caused by disseminated intravascular coagu-
smear may also reveal ‘bite cells’,118,119 blister cells,114 and lation associated with infection; or (5) antibiotic therapy
eccentrocytes119 (Fig. 10.3). ‘Bite cells’ are semicircular may cause hemolysis. In some cases, multiple mechanisms
remnants of red cells after being partially phagocytosed of hemolysis coexist, which often poses a diagnostic
or after extrusion of the Heinz body from the red cells.117 challenge to the clinician.
If the denatured hemoglobin shifts to one side of the red
cell, the red cell may appear as a blister cell and this is
usually an indicator of brisk hemolysis.
Mechanical trauma to red cells
Drugs that can cause oxidative hemolysis include Mechanical trauma to red cells can occur in three
nitrofurantoin, amniosalicylic acid, dapsone and pyridium conditions: excessive shearing forces due to high-
(phenazopyridine). Very rarely, high-dose oxygen therapy pressure gradient in the circulation; direct external
can result in oxidative injury to the red cells, particularly impact; and microangiopathic thrombotic hemolysis.158
in patients with vitamin E deficiency.115,116,149,157 On examination of a peripheral blood smear, burr cells 195
10 Blood and bone marrow pathology

Fig. 10.4 Peripheral blood smear showing micro-spherocytosis and Fig. 10.5 Peripheral blood smear showing fragments of red cells
cytoplasmic vacuolation in neutrophils in a patient with clostridia and thrombocytopenia in a patient with thrombotic
infection. Wright stain, × 1000, oil field. thrombocytopenic purpura. The size of red cells is approximately
equal to the size of the nucleus in a non-reactive lymphocyte.
Similar features may be present in patients with hemolytic uremic
syndrome or microangiopathic hemolysis. Wright stain, × 1000,
and schistocytes of variable shapes such as crescents, oil field.
helmets, micro-spherocytes and fragments are apparent.
Therefore, this is commonly called schistocytic hemolytic
anemia. Other non-specific features include aniso- actually be a different spectrum of the same disorder.
poikilocytosis, polychromatic macrocytosis, thrombo- Most adult cases of TTP/HUS syndrome are idiopathic.
cytopenia and/or procoagulants activation. Extravascular However, TTP/HUS syndrome may be triggered or
hemolysis is the predominant feature, although intra- associated with other disorders or conditions such as: (1)
vascular hemolysis occurs in severe cases. vaccination; (2) infection such as enterotoxin-producing
Schistocytic hemolytic anemia (SHA) is classified ac- Escherichia coli and Shigella dysenteriae; (3) human
cording to the size of the blood vessels where hemolysis immunodeficiency virus (HIV); (4) drugs (e.g. quinine,
occurs. Large-vessel SHA includes hemolysis in malig- quinidine, ticlopidine and mithramycin); (5) malignancy
nant hypertension and in patients with prosthetic heart such as adenocarcinoma; (6) bone marrow transplan-
valves. Small-vessel SHA occurs in march hemoglobinuria, tation; (7) pregnancy; and (8) collagen vascular disease.
autoimmune vasculitis, disseminated intravascular The pathogenesis of TTP/HUS syndrome is unknown.
hemolysis, and thrombotic thrombocytopenic purpura – Disseminated platelet-rich thrombi are the key pathology
hemolytic uremic syndrome (TTP/HUS). and endothelial cell apoptosis induced by plasma from
TTP patients,160 disseminated platelet activation triggered
by protein p37161 or calpain,162 and abnormally large
Thrombotic thrombocytopenic multimers of von Willebrand factor (vWF)163 have been
shown. Recently, mutations in zinc metalloproteinase
purpura – hemolytic uremic syndrome genes (ADAMTS13) have been found in familial TTP
In 1924, Moschowitz identified thrombotic thrombo- patients.164
cytopenic purpura (TTP) as observed in a 16-year-old Laboratory abnormalities of a TTP patient may
girl, while hemolytic uremic syndrome (HUS) was first include thrombocytopenia, fragmented red cells (Fig.
described by Gasser in 1955. They are characterized by 10.5), elevated LDH and renal impairment. Serial
the triad of thrombocytopenia, anemia and renal dys- measurements of platelet counts using a phase contrast
function. Fever and neurological symptoms such as microscope are important because platelet count and LDH
hemiparesis, aphasia, seizure, fluctuating mental function are the markers of TTP activity. In contrast, hemolysis
and coma are less common manifestations. Only about indicates neither the severity nor the activity of TTP.
half of the patients present with full pentad symptoms The primary treatment of TTP or HUS is plasma ex-
(anemia, thrombocytopenia, renal dysfunction, fever and change with vWF-poor plasma, and 1–1.5 times the plasma
neurological dysfunction).159 Because of the overlap in volume should be exchanged with fresh-frozen plasma
196 the clinical and pathologic features, TTP and HUS may (FFP).165 On-going trials comparing cryosupernatant or
 Acquired hemolytic anemia 10
detergent-treated FFP may provide evidence on the choice
of replacement fluids. Plasma infusion is less effective
than plasma exchange166 because less plasma volume is
being replaced.167 However, plasma infusion has been
used in familial TTP patients. Neurological symptoms may
recover within hours after plasmapheresis. LDH and
thrombocytopenia usually normalize within days. If
possible, platelet transfusions should be avoided in patients
with TTP or HUS. Antiplatelet therapy such as aspirin,
ticlopidine or dipyridamole may be considered when the
platelet count is above 50 × 109/l. Evidence of the value
of corticosteroid therapy exists only from anecdotal
studies and one retrospective study.168 Intravenous IgG,
splenectomy and vincristine should be reserved for
refractory or resistant TTP patients. Without definitive
treatment, the mortality of TTP exceeds 90%. However, if Fig. 10.6 Peripheral blood smear showing spherocytes of variable
sizes in a patient with burn injury. The red cells have also formed
treated promptly, TTP has a relatively low mortality. micro-vesicles and fine filamentous structures. Wright stain,
× 1000, oil field.

Cardiac hemolysis
Almost any intracardiac lesion that alters the hemo- a similar mechanism of hemolysis occurs in patients with
dynamics and generates excessive shear force to the heat stroke.
red cells can cause intravascular hemolysis. Traumatic
hemolysis may occur after cardiac surgeries such as heart
valve replacement or heart valve repair. Synthetic
Osmotic damage of red cells
material, small valvular area, and complications such as Fresh water or salt water drowning can cause hemolysis
thrombotic valve and perivalvular leaks are particularly because of abrupt osmotic changes in the pulmonary
at the risk of significant hemolysis. Hemolysis also occurs circulation.
in patients with native valvular lesion including severe
aortic stenosis, coarctation of aorta and ruptured aneurysm
of the sinus of Valsalva. In addition, aortofemoral bypass Miscellaneous causes of
has been described as being associated with traumatic hemolytic anemia
hemolysis because of the same pathophysiology.158
Paroxysmal nocturnal hemoglobinuria
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare
External impact on the red cells type of intravascular hemolysis, caused by an abnormal
When red cells flow through small vessels over the surface membrane protein. The disease arises from abnormal
of bony prominences, they are prone to external impact. clones of red cells with increased sensitivity to com-
March hemoglobinuria, a well-described but uncommon plements, particularly in an acidic environment. It is a
condition of intravascular hemolysis, typically occurs disease of pluripotent stem cells and, therefore, other
after strenuous marching or running on a hard surface in hematopoietic cells are affected. Patients with PNH can
susceptible individuals who wear thin-soled shoes. There progress to aplastic anemia and acute myeloid leukemia.
is usually no underlying intrinsic erythrocyte abnormality. For uncertain reasons, it is sometimes complicated by
This condition can be prevented by insertion of a soft thrombotic episodes at unusual sites.
inner sole. More than 20 different proteins, including complement
proteins, enzymes and various other receptors, are
missing from the surface of PNH red cells.169 Ferguson et
al170 identified glycosyl-phosphatidyl-inositol (GPI
Thermal damage of red cells anchor) as the key glycolipid structure to hold the proteins
In patients with extensive burn injury, red cell denatu- on to the red cell membrane. The defect of GPI synthesis
ration and fragmentation occur (Fig. 10.6). Less commonly, has been mapped to the mutation of pig-a gene on the X 197
10 Blood and bone marrow pathology

chromosome (Xp22.1). However, it is still unclear why Hemolytic anemia in organ

the abnormal PNH clone proliferates preferentially as
compared with other normal hematopoietic stem cells.
dysfunction
The screening test of PNH is the Ham’s test. This test Hemolysis may occur in patients with renal failure or
demonstrates that the abnormal red cells are abnormally hepatic failure. Hemolytic anemia in these conditions is
sensitive to be hemolysed by the complements in of less importance than other causes of anemia.
acidified serum. Although the Ham’s test was the gold
standard for PNH, it is semi-quantitative and not
sensitive enough to detect small numbers of PNH clones.
Acknowledgments
In addition, the sensitivity of the test is affected by recent We thank Neame P. for help with figures and Horsewood
hemolysis or blood transfusion. Flow cytometry with a P. for critical comments of the manuscript on the section
combination of monoclonal antibodies has contributed of Fc receptors.
significantly to the diagnosis of PNH. CD55 and CD59
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