British Journal of Haematology, 2000, 111, 1010±1022
Review
PURE RED CELL APLASIA
Pure red cell aplasia (PRCA), a disorder first described in
1922 (Kaznelson, 1922), can be characterized as an
anaemia with the almost complete absence of red-cell
precursors in the bone marrow, but essentially normal
granulopoiesis and megakaryopoiesis. Typical bone marrow
findings in PRCA are shown in Fig 1. The reticulocyte count
is low while the platelet count, leucocyte count and
leucocyte differential are normal. Clinically, the patients
present with symptoms of severe anaemia in the absence of
haemorrhagic phenomena. Depending on the cause, the
course can be acute and self-limiting or chronic with rare
spontaneous remissions.
The pathophysiology of PRCA is heterogeneous, as
summarized in Table I. There is a `congenital' form
(Diamond±Blackfan anaemia) where most cases appear to
be a result of several genetic defects predominantly affecting
the erythropoietic lineage. Acquired PRCA induced by
parvovirus B19 infection typically produces an acute selflimiting disease, called `transient aplastic crisis' (TAC). In
immunosuppressed individuals, B19 infection may result in
a more chronic type of bone marrow failure, clinically
apparent as PRCA. However, most cases of PRCA are
autoimmune-mediated. Various autoimmune mechanisms
of PRCA have been described (Table I), such as antibodies
against erythroblasts or erythropoietin. T cells or natural
killer (NK) cells have been proposed to secrete factors
selectively inhibiting erythroid colonies in the bone marrow
or directly lysing erythroblasts. Lysis of erythroblasts by T
cells could result from `classic' T-cell receptor (TCR)mediated antigen recognition. In addition, PRCA can be
mediated by major histocompatibility complex (MHC)
unrestricted effector-target cell recognition, as erythroid
progenitors progressively lose expression of MHC class I and,
thus, become susceptible to destruction by NK-type cells.
This mechanism is similar to NK-mediated lysis of tumour
cells following loss of HLA class I by the tumour cells.
Autoimmune PRCA can occur as a primary `idiopathic'
form or be associated with (i) infections, (ii) autoimmune
disease, and (iii) neoplasias such as thymoma, lymphoma or
carcinoma. Specific situations under which immunemediated PRCA has been described are pregnancy and
post-allogeneic bone marrow or stem cell transplantation, in
which recipient antibody against incompatible donor ABO
blood group antigens may inhibit red-cell regeneration.
Moreover, most cases of transient erythroblastopenia of
childhood (TEC), an acute self-limiting form of PRCA, are
Correspondence: Dr Paul Fisch, Department of Pathology, University
of Freiburg Medical Centre, Albertstr. 19, 79104 Freiburg,
Germany. E-mail: [email protected]
1010
probably caused by humoral immune mechanisms, aetiologically induced following infection with an unknown virus
that is distinct from B19 parvovirus. Rarely, PRCA may
represent the initial manifestation of a preleukaemic
syndrome. Finally, PRCA has been associated with several
drugs and toxins, documented by the demonstration that
the anaemia remits shortly after removal of the causative
agent. PRCA has been also studied in cats, either as a rare
form occurring spontaneously that resolves following
immunosuppressive therapy (Stokol & Blue, 1999) and
another form induced by subgroup C feline leukaemia
retrovirus that is thought to be mediated by a direct
cytopathic effect of the virus (Dean et al, 1992), rather than
by antibody and T cells (Abkowitz et al, 1987). This article
will review the present knowledge on the aetiology of the
diverse types of PRCA with particular attention to the cases
associated with expansions of large granular lymphocytes
(LGLs) (Lacy et al, 1996; Handgretinger et al, 1999).
Pure red cell aplasia in childhood
Diamond±Blackfan anaemia. Following the first report on
red cell aplasia in infancy (Josephs, 1936), four more cases
were presented (Diamond & Blackfan, 1938) and several
names proposed, including congenital hypoplastic anaemia,
chronic congenital aregenerative anaemia or erythrogenesis
imperfecta. The diagnostic criteria for Diamond±Blackfan
anaemia (DBA) include normochromic, at times macrocytic,
anaemia developing early in childhood, a normocellular
bone marrow with a selective deficiency of red-cell
precursors beyond the level of proerythroblasts, reticulocytopenia, normal or slightly decreased leucocyte counts and
normal, increased or reduced platelet counts. The incidence
of DBA has been reported to be five per million live births
with a manifestation in the neonatal period or in early
infancy (Ball et al, 1996). Approximately three-quarters of
the cases are sporadic, but dominant or recessive inheritance of DBA has been reported in different families
(Diamond et al, 1976). Some patients have chromosomal
abnormalities (Tartaglia et al, 1966). Physical abnormalities
are present in about 30% of the patients (Diamond et al,
1976). They include short stature, atrial or ventricular
septal defects, urogenital abnormalities, microcephaly, cleft
palate, micrognathia, macroglossia and a deformed thumb
(Alter, 1978; Diamond, 1978). Further haematological
findings are increased levels of HbF (Diamond, 1978),
erythrocyte adenosine deaminase (an enzyme of the purine
salvage pathway) in most patients (Glader et al, 1983) and
erythropoietin.
Earlier reports, suggesting an immune cell-mediated
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Review 1011
Fig 1. Bone marrow histology of a patient with PRCA associated with thymoma. In the aplastic phase (A and B) there is a complete absence of
red-cell precursors, as documented by immunohistochemistry using an antibody against haemoglobin with a positive reaction in some partly
squashed erythrocytes (A). Most myeloid cells, except for the eosinophils (marked by arrows) stain positive for naphthol-AS-d-chloroacetate
esterase (B). Upon recovery of erythropoiesis following removal of the thymoma (C and D), the anti-haemoglobin immunohistochemistry reaction
clearly visualizes erythroblasts (C). These cells are also detectable as the naphthol-AS-D-chloroacetate esterase-negative cell clusters (D). (Fig 1A±
D, magnification 370).
Table I. Pure red-cell aplasia.
`Congenital' form (Diamond±Blackfan anaemia)
Acquired PRCA
Parvovirus B19 infection
Transient aplastic crisis (TAC) in patients with shortened erythrocyte life span
Chronic type of bone marrow failure in immunosupressed patients
Immunological suppression of erythropoiesis
Antibody mediated
Antibody against red-cell progenitors
Transient erythroblastopenia in childhood (TEC)
Adult form
ABO-incompatibility following bone marrow transplantation
Antibody against erythropoietin
ab or gd T cell-mediated
T-helper cell-mediated antibody production
MHC-restricted, recognition of red-cell progenitors
MHC-unrestricted recognition of red-cell progenitors
NK cell- or T cell-mediated
MHC-unrestricted cytotoxicity
Associated with pregnancy
Associated with certain drugs and toxins
Initial manifestation of a pre-leukaemic syndrome
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pathogenesis of DBA (Hoffman et al, 1976), have not been
confirmed by others (Freedman & Saunders, 1978; Nathan
et al, 1978a). Current evidence suggests more than one
pathogenic mechanism for the erythroid failure in DBA,
suggesting that DBA is composed of more than one disorder
(McGuckin et al, 1995; Giri et al, 2000; Willig et al, 2000). It
appears that the erythroid progenitor compartment is
intrinsically defective in DBA as marrow cultures have
shown reduced or absent erythroid blast-forming units
(BFU-E) or erythroid colony-forming units (CFU-E) in most,
although not in all, DBA patients (Freedman et al, 1976;
Nathan et al, 1978a; Tsai et al, 1989; Santucci et al, 1999).
Erythroid progenitors in some DBA patients appear to
display a reduced sensitivity to erythropoietin that could be
corrected by the addition of glucocorticoids in vitro (Chan
et al, 1982a). This may provide an explanation for the
empirical response of 60±70% of the DBA patients to
steroids (Diamond et al, 1976; Chan et al, 1982b; Janov et al,
1996). However, `spontaneous remissions' are known to
occur in 20±30% of DBA cases (Diamond et al, 1976; Janov
et al, 1996), for the most part in patients with a family
history of DBA that were treated before the era of steroid
therapy. Although such patients with DBA in remission no
longer require transfusion therapy, they are not cured as
their red cells maintain fetal characteristics (Alter &
Nathan, 1979). Thus, such patients may have a more
silent clinical manifestation of the underlying defects.
Different in vitro assays of DBA bone marrow, as well as
molecular studies, have failed to demonstrate a direct role
for different growth factors or their receptors in the
pathogenesis of DBA (Tsai et al, 1989; Bagnara et al,
1991; McGuckin et al, 1995; Dianzani et al, 1996).
Nevertheless, clinical trials with interleukin (IL)-3 observed
an improvement of erythropoiesis in some DBA patients
(Dunbar et al, 1991), while erythropoietin seemed to be
ineffective in vivo (Niemeyer et al, 1991). Stem cell factor has
not been tested in DBA thus far. However, as there is a
significantly increased risk of DBA patients developing
haematological malignancies (as high as 23% by the end
of the fourth decade) (Janov et al, 1996), therapeutic
approaches of DBA with growth factors are not without
risks. In patients with `spontaneous' or steroid induced
remissions there remains an increased risk of developing
leukaemia (Janov et al, 1996). Normal haematopoiesis in
DBA patients can be restored by bone marrow allografting
(Greinix et al, 1993). Genetic mapping studies localized a
major DBA locus to chromosome 19q13.2 on the basis of (i)
the identification of a balanced reciprocal translocation
X;19 in a singular DBA patient (Gustavsson et al, 1997a),
(ii) linkage analysis in different DBA families that documented the involvement of chromosome 19q13 in the
majority of familial cases (Gustavsson et al, 1997b), and (iii)
microdeletions on chromosome 19q13, associated with a
few of the sporadic cases of the disease (Gustavsson et al,
1998). The gene encoding the ribosomal protein S19
(RSP19) was identified at the breakpoint of the reciprocal
X;19 chromosome translocation in the singular DBA case
cited above and mutations of RSP19 were documented in 10
out of 40 unrelated DBA patients, including four out of 19
of the sporadic cases analysed (Draptchinskaia et al, 1999).
There is a possibility that haploinsufficiency for the RSP19
protein results in a protein synthesis defect in some tissues
with high proliferative activity and this may also explain
some of the variable clinical outcomes and hereditary
features of DBA. However, in some of the familial cases the
DBA candidate region on 19q13 was excluded by the
segregation of marker alleles (Gustavsson et al, 1998) and
the majority of sporadic cases do not seem to have RSP19
mutations (Draptchinskaia et al, 1999). Thus, mutations in
RSP19 cause DBA in a subset of patients, but other causes of
DBA remain to be identified. The current knowledge on DBA
was the subject of a recent detailed review (Willig et al,
2000).
Transient erythroblastopenia of childhood. Transient erythroblastopenia of childhood (TEC) is an acquired anaemia in
previously healthy children. More than 80% of the patients
are 1 year of age or older at diagnosis. The children have a
temporary reticulocytopenia and, typically, the bone marrow
shows erythroblastopenia with normal white blood and
platelet counts (Ware & Kinney, 1991; Freedman, 1993).
However, significant neutropenia and hypocellular marrows
have also been observed in many patients with TEC that may
be as a result of a common pathogenic mechanism to that
producing the anaemia (Rogers et al, 1989; Skeppner &
Wranne, 1993; Cherrick et al, 1994). In addition, increased
numbers of lymphoid cells with a common pre-B-acute
lymphoblastic leukaemia (ALL) phenotype have been
noticed in the bone marrow of some patients with TEC,
thus this should not be misdiagnosed as acute leukaemia
(Foot et al, 1990). Transient central neurological changes
have been described in a few TEC cases (Green et al, 1986).
In some of the patients, a preceding viral illness had been
observed, but a common viral aetiology, including parvovirus B19 association, could not be found. TEC can occur in
siblings simultaneously (Labotka et al, 1981). It appears
that most cases of TEC are as a result of antibodies against
red-cell progenitors by a mechanism similar to autoimmune
thrombocytopenic purpura or autoimmune haemolytic
anaemia in childhood (Freedman, 1993). TEC can be
differentiated from DBA by the typically older age of onset,
the lack of associated anomalies, the normocytic type of
anaemia in TEC vs. the mostly macrocytic type in DBA, the
normal levels of HbF and red-cell adenosine deaminase
activity and the spontaneous recovery in TEC, usually
within 4±8 weeks without recurrence (Freedman, 1993).
However, in children presenting with TEC in the first year of
life this distinction may be difficult at times (Ware & Kinney,
1991). TEC is generally a disease with a good prognosis and
most children recover fully with a blood transfusion alone
(Skeppner & Wranne, 1993). Interestingly, a recent study
from Sweden suggested that TEC may also involve
hereditary factors (Skeppner et al, 1998).
Parvovirus B19-associated PRCA
The first report on this condition was probably that of a 42year-old man suffering from recurrent jaundice, slowly
increasing splenomegaly, enhanced urobilinuria and pigmented cholelithiasis (Minkowski, 1900). Other members of
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Fig 2. Typical `gigantoproerythroblasts' in
parvovirus B19-associated PRCA. (A) Bone
marrow histology with several gigantoproerythroblasts (marked by asterisks) that
resemble Hodgkin cells. A small regressive
erythroblast (marked with an arrow)
typically contains the immunoreactive B19antigen. (B) Bone marrow cytology of
another patient with B19 infection showing
two gigantoproerythroblasts (marked by
asterisks). (Fig 2A, magnification 750; Fig
2B, magnification 400).
his family in three generations were affected by the same
syndrome. This patient died following a short episode of
influenza-like symptoms, severe malaise and lobar pneumonia. This early description was followed by repeated causerelated reports on patients suffering from congenital
haemolytic diseases (such as familial spherocytosis, sickle
cell anaemia, acquired immune haemolytic anaemia,
hereditary elliptocytosis, thalassaemia, glucose-6-phosphate
dehydrogenase and pyruvate kinase deficiencies, etc.) who
developed severe acute anaemia combined with fever,
anorexia, nausea, vomiting, headache, abdominal pain
and chills (Horne et al, 1945; Dameshek & Bloom, 1948;
Owren, 1948; Heilmeyer, 1950). The haemoglobin concentrations fell as low as 5 g/dl, sometimes requiring transfusions, with most patients recovering from their severe illness
within 10 d. A meticulous analysis of six cases within
families with congenital haemolytic anaemias deduced that
these `haemolytic crises' were as a result of a sudden
cessation of red blood cell production (Owren, 1948). This
disproved the initial idea that the crises were because of
enhanced haemolysis. Subsequently, it was reported (Gasser,
1950) that a febrile infection triggered these `transient
aplastic crises' in patients with chronic haemolytic disorders
and that this type of infection even provoked mostly
clinically inapparent acute erythroblastic aplasias in normal
subjects. The causative agent, parvovirus B19, was identified in 1981 as the infective cause of outbreaks of aplastic
crises in sickle cell anaemia (Pattison et al, 1981; Serjeant
et al, 1981). B19 infection studies in normal volunteers
confirmed that a 5±10 d erythropoietic aplasia does not
produce significant anaemia in healthy subjects whose red
cells have a life span of about 120 d (Potter et al, 1987). In
contrast, in patients with shortened erythrocyte survival
(e.g. 15 d in familial spherocytosis), B19 infection produces
a transient decompensation of the erythropoietic system
with its clinical manifestation as severe anaemia. B19induced aplastic crisis may also occur in patients with noninherited forms of haemolysis, such as iron deficiency
anaemia (Kudoh et al, 1994).
Parvovirus B19 was found to be a rather small (15±
28 nm) non-enveloped single-stranded DNA virus related to
several animal parvoviruses (Cossart et al, 1975). It is
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transmitted via the respiratory tract or, rarely, by blood
products. The main target of B19 infection is the erythroid
progenitor cell and the basis of this erythroid tropism is the
B19 cellular receptor globoside, known as the blood group P
antigens (P, P1 and Pk antigens; Brown & Young, 1995,
1997). Very rare individuals with erythrocytes lacking the P
antigens (p phenotype) cannot be infected with parvovirus
B19. Most infections with parvovirus B19 are clinically
inapparent, with up to 15% of children below 5 years, 50±
60% of young adults and up to 90% of elderly individuals
being seropositive. The clinical manifestations of B19
infection include erythema infectiosum (`fifth disease'), an
acute polyarthropathy syndrome, hydrops fetalis, the
`transient aplastic crisis' (TAC) described above and a
more chronic type of bone marrow failure manifesting as
a chronic pure red cell aplasia (Kurtzman et al, 1989;
Frickhofen et al, 1994). Only the latter two will be further
discussed in this review.
B19-mediated TAC may be rarely associated with changes
in the other blood lineages, varying degrees of neutropenia
and thrombocytopenia. As a characteristic finding, the bone
marrow contains abnormally large proerythroblasts, `gigantoproerythroblasts', that appear towards the end of the
aplastic phase (Fig 2) (Gasser, 1950; Schaefer, 1992). These
cells contain large vesicular nuclei with loosely distributed
chromatin and prominent, inclusion body-like nucleoli,
surrounded by a basophilic cytoplasm. Some of these blasts
may somehow resemble Hodgkin cells (Fig 2). As the
anaemia resolves, usually between the fifth and tenth day
after onset, the gigantoproerythroblasts completely disappear from the bone marrow and are followed by regenerating normoblasts. This is mostly as a result of a humoral
protective immune response to the virus, virus-specific
immunoglobulin (Ig)M and IgG antibodies to the viral
capsid proteins (Brown & Young, 1995). It appears that
only antibodies against the 83 kd minor capsid species VP1
are virus-neutralizing antibodies, while the early antibody
response against the 58 kd major capsid protein VP2 is
insufficient to clear the infection. A protective antibody
response generally results in life-long immunity against
reinfection. Because of their bizarre aspect, gigantoproerythroblasts are to some extent diagnostic for the parvovirus
B19 infection. However, immunostaining for the B19
antigen fails to demonstrate the presence of virus in these
cells. Positive results are seen in smaller regressive
erythroblasts with a dense ring of chromatin that surrounds
an inclusion-like central part of the nucleus harbouring the
immunoreactive B19 antigen (Fig 2A). These decaying
erythroblastic virocytes become visible for only a very
short time at the early phase of the infection. At the time of
bone marrow assessment, the erythroblastic virocytes have
just disappeared in many cases with gigantoproerythroblasts being the only hallmark of the previous cytolytic viral
infection. When B19 infection in pregnancy leads to fatal
hydrops fetalis, erythroblastic virocytes are easily detectable
by immunostaining in various organs. We propose that
these gigantoproerythroblasts in B19 infection represent
very early red-cell precursors that appear after clearance of
the B19 virus as evidence of regenerating erythropoiesis.
Persisting B19 infection can occur in a wide variety of
conditions of immunosuppression [congenital immunodeficiency, human immunodeficiency virus (HIV) infection,
lymphoproliferative disorders, post organ transplant, etc.],
even in cases of only minimal or clinically previously
unrecognized immune dysfunction (Kurtzman et al, 1989;
Frickhofen et al, 1994). In such patients, the presentation
may be as a chronic PRCA or, more rarely, as pancytopenia
(Brown & Young, 1995). The bone marrow examination in
chronic B19-induced PRCA also shows gigantoproerythroblasts, although in some cases they cannot be found. B19specific antibody is absent or low and directed against the
major capsid protein VP2. Typically, BFU-E and CFU-E are
decreased and the patients' sera inhibit normal BFU-E, CFUE, but not granulocyte-macrophage CFU (CFU-GM), colony
formation because of viraemia. The diagnosis is established
by the demonstration of viral DNA in the serum by nucleic
acid hybridization assays. Although polymerase chain
reaction (PCR) is much more sensitive it carries a high
risk of contamination and false-positive results (Brown &
Young, 1997). In chronic B19 infections, antibody studies
often do not produce clear results, although high titres of
B19 IgG make a diagnosis of a persistent infection
improbable. As indicated above, fetal infection with parvovirus can lead to a miscarriage, hydrops fetalis and to
infants born with chronic anaemia (Brown et al, 1994,
1995). Thus, a maternally transmitted B19 infection should
be suspected in infants with congenital red cell aplasia.
Although the patients may be seriously ill, parvovirus
B19-induced `aplastic crisis' in immunocompetent individuals is generally a self-limiting problem, usually requiring
only supportive therapy including blood transfusions.
Chronic B19-associated PRCA has been successfully treated
by the repeated administration of immunoglobulin until the
virus is no longer detectable by PCR (Kurtzman et al, 1989;
Brown & Young, 1995). Patients generally respond within
2 weeks of treatment, but should be monitored for
recurrence of viraemia.
Immunologically mediated PRCA
PRCA has been associated with autoimmune diseases such
as rheumatoid arthritis (Dessypris et al, 1984; Rodrigues
et al, 1988) and systemic lupus erythematosus (Cassileth &
Myers, 1973; Dainiak et al, 1980). In addition, red cell
aplasia occurred as a paraneoplastic syndrome (Field et al,
1968), complicating thymoma (Jacobs et al, 1959; Roland,
1964), large granular lymphocyte expansions (Loughran &
Starkebaum, 1987), chronic lymphocytic leukaemia (Mangan et al, 1982; Chikkappa et al, 1986), Hodgkin's disease
(Morgan et al, 1978) and diverse carcinomas (Mitchell et al,
1971). Although early observations suggested that up to
50% of patients with PRCA had thymomas (Jacobs et al,
1959; Roland, 1964; Krantz, 1973), more recent studies
found thymomas in less than 10% of PRCA patients (Charles
et al, 1996; Lacy et al, 1996). An autoimmune mechanism
of the anaemia was suggested by remissions of the red cell
aplasia following resection of the thymoma in about 25% of
cases (Zeok et al, 1979) and the response of some PRCA
patients to steroids (Finkel et al, 1967), cyclophosphamide
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Fig 3. Possible mechanisms of PRCA directly mediated by T- or NKLGLs. A. T-LGLs expressing the TCR ab may utilize the TCR to
directly recognize (`1'-signal) a red-cell peptide presented by an
HLA class I molecule. At the same time, killer cell inhibitory
receptors expressed by the LGLs fail to deliver an `off '-signal (`±'signal) to the killer cells because red-cell precursors express low
levels of HLA class I. B. T-LGLs expressing a TCR gd may utilize the
TCR to directly recognize (`1'-signal) a ligand expressed by myeloid
and by red cell precursors. The myeloid cells express normal levels of
HLA class I molecules that bind to the killer cell inhibitory receptors
on the T-LGLs and deliver off-signals (`±' signals) to the killer cells,
while cytolysis of red-cell precursors expressing low levels of HLA
class I is not inhibited. C. Similarly, MHC-unrestricted cytotoxicity
by NK-LGLs and T-LGLs may be inhibited against HLA class I1
myeloid precursors, but not the HLA class I-deficient erythroid
progenitors. NK-LGLs do not express a TCR, but may be positively
triggered by circulating antibody against red-cell antigens, activating NK receptors and adhesion molecules (not shown). T-LGLs of
ab2 or gd-type may use this same mechanism of NK-mediated
lysis. Then, the TCR would not be directly involved in the
recognition of red-cell progenitors.
(Marmont et al, 1975) and splenectomy (Eisemann &
Damashek, 1954). Several studies published in recent
years helped to elucidate diverse immunological mechanisms that can explain red-cell progenitor destruction in
individual patients.
PRCA mediated by antibody. Initial demonstrations of
factors in the patients' sera that inhibited erythropoiesis in
vivo (Jepson & Lowenstein, 1968) and in vitro (Krantz & Kao,
1967) were followed by the demonstration that these
inhibitors could be IgG antibodies directed against erythroblasts (Krantz & Kao, 1969). These antibodies either
inhibited haemoglobin synthesis (Krantz & Kao, 1967) or
they were complement-binding and directly cytotoxic for
erythroblasts in vitro (Zaentz & Krantz, 1973). Expanding on
these experiments, immunosuppressive drugs were used for
the treatment of PRCA (Krantz & Kao, 1969). Later studies
of another patient who did not respond to immunosuppressive therapy revealed that such antibodies could specifically
block differentiation of BFU-E in vitro (Messner et al, 1981).
The pathogenic role of such antibodies was strongly
suggested by the demonstration that plasmapheresis was
effective to induce remission in this patient (Messner et al,
1981). In other patients, the inhibitory antibodies were
found to be directed against erythropoietin (Marmont et al,
1975; Peschle et al, 1975; Casadevall et al, 1996). While
erythropoietin levels in other patients with PRCA are
typically elevated for the degree of anaemia, they are low
in PRCA patients with erythropoietin-specific antibodies. In
vitro inhibition of erythroid colonies could be reversed by the
addition of purified erythropoietin and the patient's serum
could immunoprecipitate purified erythropoietin (Casadevall et al, 1996). These observations gave final proof for this
pathogenic mechanism.
In a minority of cases, antibody-mediated PRCA could
spontaneously remit with supportive therapy alone,
although this could take more than a year (Clark et al,
1984). Such a self-limiting course is reminiscent of transient
erythroblastopenia in children that is also believed to be
antibody-mediated and that may be associated with a viral
infection. Thus, some cases of adult autoimmune PRCA
might be induced by antibodies produced following a viral or
bacterial infection that might cross-react with erythroid
precursor cells or erythropoietin. Antibody-mediated PRCA
has been described in association with thymoma, systemic
lupus erythematosus, rheumatoid arthritis, Hodgkin's disease and other diseases. However, only few of these studies
unambiguously demonstrated a serum immunoglobulin as
the mechanism of inhibition of erythropoiesis (Al-Mondhiry
et al, 1971; Cassileth & Myers, 1973; Morgan et al, 1978;
Dessypris et al, 1984). In some of the earlier studies,
parvovirus B19 viraemia itself might have been misinterpreted as a circulating cytotoxic erythropoiesis inhibitor
(Zaentz et al, 1975). The possible immunological mechanism of antibody-mediated inhibition of erythropoiesis
includes direct complement-mediated lysis of red-cell
progenitors and the formation of immune complexes with
erythropoietin that result in functional inactivation and the
removal of erythropoietin from the circulation. Although
this has not yet been demonstrated experimentally, antierythroblast antibodies that are not directly cytotoxic might
impair red-cell progenitor maturation by blocking the
erythropoietin receptor or another red-cell signalling pathway. It should be kept in mind that T-helper cells (Th2 cells)
could play a role in the production of such autoantibodies.
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This might explain why inhibitory humoral factors have
been detected in the serum of some patients with a disturbed
T-cell function, such as patients suffering from thymoma,
Hodgkin's disease and diverse autoimmune diseases. Moreover, this could be why some patients with apparently
antibody-mediated PRCA responded to anti-thymocyte
globulin (Marmont et al, 1975).
Occasionally, ABO-incompatibility between donor and
recipient following allogeneic bone marrow or stem cell
transplantation can result in antibody-mediated PRCA with
elevated titres of the incompatible agglutinins (GmuÈr et al,
1990). Red-cell engraftment and reticulocyte recovery may
occur spontaneously within weeks or several months as the
agglutinin titres fall, but there are reports of successful
treatment of this condition with erythropoietin, increased
immunosuppressive therapy or plasmapheresis (GmuÈr et al,
1990; Heyll et al, 1991).
PRCA mediated by T cells and NK cells. In patients with
PRCA in whom a plasma inhibitor of erythropoiesis cannot
be demonstrated, lymphocyte-mediated inhibition of erythropoiesis is the most probable mechanism of pathogenesis.
Originally, investigations in a patient with T-cell chronic
lymphocytic leukaemia showed that the patient's malignant
T lymphocytes suppressed erythroid colony formation by
allogeneic human bone marrow cells (Hoffman et al, 1978).
Pretreatment of the patient's bone marrow T cells with antithymocyte globulin and complement reversed this suppression and markedly augmented autologous erythropoiesis in
vitro. Subsequent studies demonstrated that T cells with
receptors for the Fc portion of the IgG molecule (CD16), the
so-called Tg cells from patients with B-cell chronic
lymphocytic leukaemia (B-CLL), suppressed erythroid colony formation in vitro (Nagasawa et al, 1981). Initially, it
was controversial whether this was an active suppressive
effect of these Tg cells or whether this phenomenon was
owing to the lack of normal T-cell help in vitro because these
cultures originated from patients with B-CLL (Nathan et al,
1978b). However, subsequent studies demonstrated that
removal of the Tg cells by E-rosetting markedly augmented
erythroid colony growth, suggesting that the Tg cells
actively suppressed erythropoiesis (Mangan et al, 1982).
Subsequently, the Tg cells were renamed large granular
lymphocytes (LGLs) and several groups found that expansions of LGLs may be the disorder most commonly
associated with PRCA (Abkowitz et al, 1986; Levitt et al,
1988; Tefferi et al, 1994; Charles et al, 1996; Lacy et al,
1996). These LGLs may be of T-cell type or of natural killer
(NK)-cell type (Loughran, 1993). T-LGLs express CD3 and a
T-cell receptor (TCR) of ab- (in the majority of cases) or gdtype. In contrast, NK-LGLs are CD32 and, consequently, do
not express a TCR at the cell surface. Typically, these NKLGLs do not rearrange the TCR genes a, b, g and d.
Non-malignant LGLs typically display MHC unrestricted
cytolytic activity against HLA class I-deficient NK-sensitive
tumour cells, such as the erythroleukaemia K562. In some,
although not all, patients with clonal LGL proliferations of the
CD31 (T-LGL)- and the CD32 (NK-LGL)-type, the LGLs are
directly cytotoxic against K562 cells in vitro when freshly
isolated (Partanen et al, 1984; Kaufmann et al, 1987;
Handgretinger et al, 1999). If these fresh LGLs are not
cytotoxic against K562 cells, cytotoxicity can be induced by
antibody cross-linking the TCR of the T-LGLs with the Fc
receptor on target cells, or by antibody cross-linking the Fc
receptor on the T-LGLs or NK-LGLs (the CD16 molecule) with
any specific ligand for the antibody on the target cells
(Loughran et al, 1987). As in vitro cytotoxicity by the clonal
LGLs against K562 cells may no longer be detectable once the
LGLs have been cryopreserved, some of the published studies
on the function of leukaemic LGLs may not have adequately
assessed the LGLs cytolytic abilities. Thus, in principle, most
neoplastic LGLs may bear a functional cytotoxic machinery
that is able to destroy erythroblasts in vivo. This may be
mediated by similar mechanisms as fresh LGLs kill the
erythroleukaemia K562 in vitro (Partanen et al, 1984;
Handgretinger et al, 1999). Triggering of cytolysis against
erythroblasts could occur (i) via the TCR of ab- or of gd-type
that could recognize unknown ligands expressed by erythroid
progenitors (Fig 3A and B), (ii) via antibodies against red-cell
progenitors binding to CD16 on the LGLs (Tg cells), or (iii) by
some novel triggering molecules, analogous to those recently
cloned on NK cells (Pessino et al, 1998).
Recently, a case of PRCA and a clonal expansion of T-LGLs
of gd-type has been described where the malignant LGLs
were shown to carry functional inhibitory MHC class I
receptors (Handgretinger et al, 1999). These killer cell
inhibitory receptors (KIRs) include several receptors specific
for diverse MHC class I antigens, such as the lectin-type
receptors CD94-NKG2A and B, as well as the receptors that
are members of the immunoglobulin superfamily (Long,
1999). KIRs inhibit the lytic machinery of the killer cell
when the target cell expresses the HLA class I antigen to
which the particular KIR binds (Fig 3). When tumour cells
lose HLA class I antigens as an `escape' mechanism to avoid
recognition by classical MHC class I-restricted CD81 T cells,
they can be destroyed by cytotoxic MHC-unrestricted killer
cells expressing KIRs. Besides immunosurveillance against
transformed cells, another biological role of the KIRs on T
cells and NK cells is the prevention of autoimmunity against
cells with normal levels of HLA class I expression (Fisch et al,
2000). This mechanism probably induced the PRCA in the
patient with the gd T-LGL proliferation and KIR expression
described recently (Handgretinger et al, 1999). The KIRs
inhibited cytolysis by the LGLs of myeloid cells that express
normal HLA class I levels, but not the cytolysis of red-cell
progenitors that are in the process of progressively losing
HLA class I (Fig 3). In addition to this `lack of inhibition' by
diminished HLA class I expression, TCR-mediated recognition of an unknown ligand on the red-cell progenitors by the
T-LGLs might positively trigger red-cell destruction (Fisch
et al, 2000). In principle, this ligand may also be expressed
on other haematopoietic cell types, but high levels of HLA
class I expression would protect such other target cells from
the attack by T cells (Fig 3B). This possibility may apply
particularly for the LGLs of gd-type as gd T cells are believed
to recognize ubiquitous ligands without restriction by the
MHC. However, a model of `lack of inhibition' of erythroblast
cytolysis by KIR1 LGLs would be also valid for NK-LGLs that
do not express a TCR, but may be stimulated via antibody
q 2000 Blackwell Science Ltd, British Journal of Haematology 111: 1010±1022
Review 1017
against red-cell progenitors or by other activating molecules
expressed by erythroid cells (Fig 3C).
At present, it is unclear how many of the PRCA cases
associated with LGL proliferations indeed express functional
KIRs. However, as most of the normal counterparts of the in
vivo-expanded LGLs (the NK cells, the NK-ab T cells and
many gd T cells) carry KIRs, it would be surprising if most of
the malignant LGLs do not express these inhibitory HLA
class I receptors. Besides direct perforin-mediated cytotoxicity, such KIRs could also inhibit lymphokine production by
the LGLs, such as tumour necrosis factor b (D'Andrea et al,
1996). Thus, PRCA in some patients with associated LGL
expansions may not be induced by perforin-mediated
cytotoxicity, but by LGL-lymphokine secretion that is not
inhibited via the KIRs upon interaction of the LGL with the
proerythroblasts. Similarly, as for killer cell-mediated
cytotoxicity, this would require close physical contact of
the LGLs with the red-cell progenitors in the bone marrow.
One early study concluded that the suppression of
erythropoiesis by T cells of the LGL-type could be MHCrestricted (Lipton et al, 1983), thus suggesting that T cells
could recognize erythroblasts by classical (TCR)-mediated
co-recognition of erythroblast peptides presented by the
autologous MHC (Fig 3A). This remains a distinct possibility
for individual patients although it has never been conclusively demonstrated. However, this would not be in direct
confrontation to the KIR-induced pathophysiology of PRCA
explained above because KIRs can also downmodulate
classical TCR-mediated antigen recognition (Long, 1999).
This means that one HLA class I allele may present an
antigenic peptide to T-LGLs, while other class I alleles may
inhibit cytolysis by binding to the KIRs (Fig 3A) (Ikeda et al,
1997). It is rather difficult to prove TCR-mediated MHCrestricted antigen recognition for this model experimentally,
unless the peptide antigen and the MHC-restricting element
have been identified. This would require the functional
analysis of T-cell clones recognizing autologous erythroid
progenitor cells and the cloning of the particular peptide
ligand, as has been successfully performed for some tumour
antigens (Ikeda et al, 1997). On the other hand, expression
of a TCR of ab-type does not by itself mean MHC-restricted
peptide antigen recognition, because small subpopulations
of ab T cells may recognize fundamentally different nonpeptidic epitopes that may be presented by non-classical
presenting molecules (Porcelli et al, 1996).
Most T-LGL proliferations associated with PRCA are
probably clonal (Loughran, 1993), although it is conceivable
that polyclonal expansions of T-LGLs, such as following an
immune response against an unknown pathogen, may be
associated with autoimmunity. The molecular demonstration
of clonality in some LGL patients may mean dominance of
one or a few clones within a polyclonal population of LGLs. In
addition, most patients with T-LGL expansions have a chronic
course and the demonstration of clonality by itself does not
therefore mean malignancy. `Benign' clonal T-cell expansions
have been described following viral infections and in elderly
humans (Maini et al, 1999). Indeed, there have been some
reports of clonal T-LGL proliferations that were no longer
detectable when autoimmunity developed (Loughran,
1993). Similar thoughts apply for NK-LGL expansions,
although clonality is difficult to demonstrate in NK LGL
proliferations because of the lack of TCR gene rearrangements in NK-LGLs (Tefferi et al, 1994). Some of the NK-LGL
lymphocytoses were shown to be polyclonal by molecular
analysis of the inactivation patterns of the X-chromosomes
in LGLs from women patients (Nash et al, 1993). Patients
with NK-LGL leukaemia have been reported to have an
acute leukaemia-like course requiring intensive chemotherapy and these cases seemed to be associated with
autoimmunity less often than the T-LGL expansions
(Loughran, 1993), although this more acute course was
not observed in all studies (Tefferi et al, 1994). Thus, it
appears that expansions of NK-LGLs are quite common and
that patients with massive NK lymphocytosis, chromosomal
abnormalities in the LGLs and frank leukaemia were
preferentially described in the literature (Scott et al, 1994).
Chronic expansions of LGLs may be present for many years
and associated with autoimmunity before an acute leukaemic phase may develop in some of the patients (Loughran,
1993). Thus, the majority of patients with NK-LGL and TLGL proliferations may have lower LGL expansions that only
become apparent if an absolute lymphocytosis or an
increased proportion of peripheral lymphocytes with an
LGL phenotype are specifically looked for (Scott et al, 1994).
Importantly, the expansion of LGLs can easily be missed in
the analysis of bone marrow histology of PRCA patients
because it may be rather scanty. For example, in some
patients with documented clonal T-LGL proliferations, the
clonal LGLs accounted for less than 5% of the bone marrow
cells (Lacy et al, 1996). It is possible that, in some anaemic
patients with increased levels of LGLs, not all erythroblasts
are destroyed by the LGLs and the bone marrow histology
reveals merely reduced erythropoiesis, but not a true PRCA.
Finally, it should be kept in mind that not all cases of PRCA
associated with LGL expansions are mediated immunologically, but that the associated immunodeficiency may
predispose some patients to chronic parvovirus B19
infection (Ergas et al, 1996).
In summary, PRCA development in LGL expansions may
depend on:
(i) Positive triggering of the LGLs by the erythroblasts
either via the TCR or via another activation pathway that
includes activating NK receptors and erythroblast-specific
antibody bound to CD16 of the LGLs.
(ii) Expression by the LGLs of a pattern of different KIRs
that downregulate cytolysis of cells with normal levels of
HLA class I expression, but are insufficient to protect
erythroblasts with naturally decreased class I levels.
(iii) The ability of the LGLs to secrete inhibitory
lymphokines or mediate direct or antibody-induced cytotoxicity.
(iv) A particular level of LGL expansion with infiltration of
the bone marrow. Thus, lower levels of clonal LGL
expansions that may not infiltrate the bone marrow will
not cause PRCA, even if the LGLs are cytotoxic against redcell precursors in vitro.
(v) The presence of a concomitant chronic parvovirus B
19 infection.
q 2000 Blackwell Science Ltd, British Journal of Haematology 111: 1010±1022
1018 Review
The PRCA in patients with associated clonal LGL
expansions may respond to cytotoxic chemotherapy directed
against the LGL expansion (Charles et al, 1996; Lacy et al,
1996). Similarly, in patients with PRCA apparently associated with other types of lymphoma, thymoma and
autoimmune disease, this underlying condition should be
treated. Most patients with immunologically mediated PRCA
achieve remissions if normal or increased BFU-E growth in
vitro is present (Charles et al, 1996). Steroids are generally
used as the initial immunosuppressive agent. Further
options include cyclosporin, cyclophosphamide, anti-thymocyte globulin, methotrexate and mycophenolate mofetil.
In patients with normal BFU-E growth in vitro, one should
persist with trials of drug therapies until a complete
remission is obtained (Clark et al, 1984; Charles et al,
1996). Thus, it is often difficult to contribute the eventual
response to a single agent. Splenectomy, recommended in
some of the earlier literature (Eisemann & Damashek, 1954;
Zaentz et al, 1975), appears to be of little value in more
recent series (Charles et al, 1996).
`Myelodysplasia-associated PRCA'
Typically, in patients with autoimmune PRCA, BFU-E and/
or CFU-E are normal if assayed in the absence of autologous
serum or autologous T cells because there is no intrinsic
defect of the haematopoietic stem cells or early red-cell
progenitors. On bone marrow morphology, patients with
PRCA typically have less than 1% of red-cell progenitors
(including proerythroblasts). Thus, in contrast to myelodysplasias, the bone marrow in PRCA should not show
evidence of dyserythropoiesis and megaloblastoid features.
In PRCA, the maturation in the other haematopoietic
lineages is normal and no cytogenetic abnormalities should
be present. Importantly, PRCA should not be diagnosed in
patients with dysmyelopoiesis, increased number of blasts,
myelofibrosis and abnormalities of the megakaryocytes. If
these criteria are not observed, myelodysplastic syndromes
and myeloproliferative disorders with evidence of erythroid
hypoplasia can be erroneously diagnosed as PRCA (Pierre,
1974; Garcia-Suarez et al, 1998; Nemoto et al, 1999). The
numbers of BFU-E and CFU-E in patients with myelodysplastic syndromes are characteristically low and cytogenetic
abnormalities may be detectable (Charles et al, 1996). Thus,
such studies may be of further help to differentiate PRCA
from stem cell disorders. Acquired PRCA is not usually a
preleukaemic disorder if cases with dysmyelopoiesis and
cytogenetic abnormalities are carefully excluded. Diagnostic
difficulties may arise in patients with parvovirus infection
where bone marrow histology may vary depending on the
stage of the disease, in patients with PRCA in remission who
are in the process of relapsing and whose bone marrow may
show signs of dyserythropoiesis (Keefer & Solanki, 1988),
and in occasional patients in whom erythroid aplasia or
hypoplasia precedes the development of abnormalities in the
other haematopoietic lineages (Pierre, 1974; Dessypris et al,
1980).
Other causes of PRCA
PRCA has been reported in association with more than 30
drugs, but most literature reports describe only one or two
patients (Ammus & Yunis, 1987; Thompson & Gales, 1996).
Drug-induced PRCA was reversible in most, although not
all, cases after the alleged agent had been discontinued.
Naturally, a causal relationship has not been clearly proven
in most cases, but for phenytoin, azathioprine and isoniazid
there seems to be sufficient evidence of causality (Thompson
& Gales, 1996). The pathogenesis of PRCA in such druginduced cases is largely unknown, but it may include direct
effects on the red-cell precursors (Yunis et al, 1967), as well
as the induction of autoimmunity (Dessypris et al, 1985).
In the early literature of anaemias, red cell hypoplasia and
aplasia associated with giant proerythroblasts has been
reported in children with severe nutritional deficiencies that
responded to riboflavin or pyridoxine (Kondi et al, 1963). It
is possible that some of these cases could have been caused
by parvovirus B19 infections in children that had a
shortened erythrocyte life span because of severe malnutrition (Kudoh et al, 1994). PRCA could be induced in
monkeys by a lack of riboflavin in the diet (Kondi et al,
1967).
Severe renal failure can be associated with marked
erythroid hypoplasia owing to low erythropoietin stimulation and a possible toxic effect of azothaemia on red-cell
progenitors (Fisher, 1998). However, this does not usually
lead to classical PRCA. Although extremely rare, the
association of PRCA with pregnancy is well established
(Baker et al, 1993) and important to recognize because of
the potential danger to the fetus. Anaemia occurs early in
pregnancy, subsides soon after delivery, may re-occur in
subsequent pregnancies and has not been transferred to the
neonates. In one such case, three episodes of PRCA that
were spontaneously reversible, occurred before any pregnancy and PRCA subsequently relapsed in three different
pregnancies with spontaneous recovery after termination of
pregnancy or normal delivery (Picot et al, 1984). The
mechanisms of PRCA in such cases remain unclear.
CONCLUDING REMARKS
This review has summarized the current knowledge on the
pathogenesis of PRCA. It seems important to stress that
PRCA should not be confused with cases of red cell
hypoplasia associated with myelodysplastic and myeloproliferative syndromes. In addition, acute or chronic parvovirus
B19 infections should be excluded in all cases of congenital
and acquired PRCA. In patients with antibody-mediated
PRCA, it would be important to determine the molecules
recognized by the antibodies on the red-cell progenitors. It
will be even more challenging to define the epitopes, if any,
recognized by T-LGLs on erythroblasts. This may provide
further clues to the autoimmune aetiology of PRCA as such
molecules could be virally encoded or cross-reactive with
microorganisms. Microdissection of LGLs from the bone
marrow may help to determine if such LGLs are clonal or
polyclonal. The concept that inhibitory receptors for HLA
class I are involved in the pathogenesis of PRCA in some or
most cases associated with LGL expansions needs further
confirmation. Thus, LGLs in the bone marrow should be
q 2000 Blackwell Science Ltd, British Journal of Haematology 111: 1010±1022
Review 1019
examined for the expression of such inhibitory receptors and
additional functional studies need to be performed using
freshly isolated LGLs from such patients. Eventually, this
could translate into therapy of PRCA, if novel pharmacological agents could signal through these inhibitory MHC
class I receptors, but without binding to the T-cell receptors.
Thus, this novel immunosuppressive therapy would block
the destructive function of LGLs without broadly inhibiting
T-cell responses.
1
Department of Pathology,
University of Freiburg Medical
Centre, Freiburg, and
2
Department of Paediatrics,
Children's University Hospital,
TuÈbingen, Germany
Paul Fisch 1
Rupert Handgretinger 2
Hans-Eckart Schaefer 1
ACKNOWLEDGMENT
This work was supported by SFB364-B4 from the Deutsche
Forschungsgemeinschaft.
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Keywords: pure red-cell aplasia, large granular lymphocytes, T lymphocytes, parvovirus B19, Diamond±Blackfan
anaemia.
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