266 SECTION III • ThE ANEmIAS
Hemoglobin electrophoresis demonstrates an increase in HbA for b@thalassemia intermedia is the ability to maintain a hemoglobin
2
of 3.5–7.0% with a mean of 5.5%. Newborns have a normal HbA level associated with a comfortable survival without requiring regular
2
32
34
concentration of 0.27 { 0.02%. HbF is normal in approximately transfusions. The need for transfusions is defined by the quality of
half of the patients and increased in the other half (Figure 14-10). 69,70 life, not the hemoglobin level per se. Some patients with a hemoglo-
If HbF exceeds 5%, however, the individual has probably inherited bin level of 7 g/dL can be relatively symptom free, but others with a
an HPFH gene (see “Hereditary Persistance of Fetal Hemoglobin”) in hemoglobin of 9 g/dL can have clinical symptoms related to ineffec-
addition to the b@thalassemia gene. Vital stains to detect Heinz bodies tive erythropoiesis. Clinical symptoms often intensify during periods
are usually negative. In summary, b@thalassemia minor is indicated of physiological stress as with pregnancy and infection and can require
when the MCH is 627 pg and the HbA is 73.5%. short-term transfusion therapy. Even in the absence of regular trans-
2
Nucleic acid–based techniques can be performed to identify the fusions, some patients develop progressive iron overload and require
type of mutation present and validate the heterozygous inheritance iron-chelation therapy.
pattern but are of limited diagnostic value. Such information, however,
can be helpful in counseling prospective parents with b@thalassemia laboratory Results
minor. The CBC reflects a moderate microcytic hypochromic anemia with
a hemoglobin value range of 7–10 g/dL (Figure 14-11 ■). In milder
cases, patients express only a slight reduction in hemoglobin values.
CheCkpoint 14-7 The erythrocyte count disproportionately higher than the hemoglo-
bin values and often approaches normal.
In b@thalassemia, what erythrocyte parameter on the CBC Target cells are the predominant poikilocytes observed. Baso-
differs significantly from that found in iron deficiency?
philic stippling and nucleated red blood cells are also present. The
bone marrow shows hypochromic erythroblasts in the context of
erythroid hyperplasia. However, bone marrow examination is not
Treatment and prognosis needed for diagnosis.
Patients generally do not require treatment if they maintain good Hemoglobin electrophoresis patterns in patients with the more
0 +
+ +
health and nutrition. They are generally asymptomatic except during severe forms of b@thalassemia intermedia (b b and b b ) are
periods of physiologic stress, and they have a normal life expectancy. nearly indistinguishable from those observed in the milder forms of
b@thalassemia major. Patients express elevated HbA (5–10%) and
2
B@Thalassemia Intermedia HbF (30–75%) with the remainder being HbA. Milder versions of
b@thalassemia intermedia produce lower HbA (73.2%) and HbF
+
+
+
0
0
(B /B , B /B , B /B) levels (1.5–12.0%) (Figure 14-10). Although hemoglobin electropho-
2
expected Genotypes resis is helpful in diagnosing b@thalassemia intermedia, differentiation
All three patterns of inheritance—homozygous, double heterozy- from b@thalassemia major and minor is a clinical decision.
gous, and heterozygous—can produce b@thalassemia intermedia
(Table 14-5). The homozygous and double heterozygous forms rep- Treatment and prognosis
resent a mutation in both b@alleles, resulting in a moderate reduction Splenomegaly is common. Functional hyposplenism leads to infec-
in b@chain synthesis. Patients who inherit a mutation of one b@gene in tions requiring regular interventions with antibiotic therapy. Chela-
conjunction with a normal b@gene occasionally exhibit clinical symp- tion therapy may be warranted to combat iron overload, which tends
toms significant enough to be classified as b@thalassemia intermedia
rather than b@thalassemia minor.
Patients with b@thalassemia who co-inherit a@thalassemia or
HPFH can actually experience milder symptoms as compared with
those with pure b@thalassemia. In both cases, excess free a@chain
accumulation and precipitation are reduced, decreasing the ineffec-
tive erythropoiesis and extravascular hemolysis responsible for much
of the pathology. In the case of co-inherited b@thalassemia and HPFH,
the overexpressed g@chains combine with the excessive a@chains to
produce HbF, which reduces a@chain precipitation.
clinical Findings
Patients with b@thalassemia intermedia present with symptoms of
intermediate clinical severity between severe b@thalassemia major and ■ FiGuRe 14-11 Patients with b@thalassemia minor show
0
+
mild b@thalassemia minor. The b /b @genotype produces the greatest minimal morphologic abnormalities to include microcyto-
reduction in b@chain synthesis but has a variable clinical presenta- sis with target cells. The CBC in this patient showed the
tion; some patients have symptoms of b@thalassemia major, whereas following results: Hb 11.1 g/dL; RBC count 5.2 * 10 /L;
12
others have a much milder clinical phenotype. Patients who inherit MCV 61 fL; MCH 20.2 pg; MCHC 33 g/L (Wright-Giemsa
0
the b /b@genotype generally exhibit mild symptoms. The criterion stain; 1000* magnification).
M14_MCKE6011_03_SE_C14.indd 266 01/08/14 12:51 pm
ChAPTEr 14 • ThALASSEmIA 267
to develop later than in patients with b@thalassemia major. Most globin gene and in combination with structural hemoglobin disorders
patients have a normal life span. such as sickle cell anemia and HbC disease (Table 14-6 ★).
SC
B@Thalassemia Minima (B /B) DB@Thalassemia
b@Thalassemia minima is a form of asymptomatic b@thalassemia, d@, b@thalassemia (db@thalassemia) is a rare thalassemia observed
exhibits no major laboratory abnormalities, and is only defined by a primarily in patients of Greek, African, Italian, and Arabian ances-
mildly imbalanced a@ to non@a globin chain synthesis ratio. The dis- try whose production of both b@ and d@chains is affected. The
+
0
order is usually discovered serendipitously during family studies. The db@mutation can be categorized into two genotypes, db and db .
0
SC
gene has been given the designation b for silent carrier. The geno- The db designation indicates a complete lack of synthesis of both b@
71
SC
+
type used to describe a patient with b@thalassemia minima is b /b. and d@chains from a given chromosome, whereas the db @genotype
SC
Homozygosity for b or combination of the silent allele with other indicates a reduction in b@ and d@chain synthesis. The absence of b@
b@thal genes results in a mild form of thalassemia intermedia. and d@chains is most often due to deletion of the structural b@ and
d@gene complex. One or both of the g@genes remain, resulting in
0 66
0
100% HbF in homozygous db /db . However, increased g@chain
production fails to fully compensate for the loss of b@chain produc-
tion, resulting in a thalassemic phenotype and anemia.
case sTudy (continued from 261) In db@thalassemia, the g@chain synthesis is less than in HPFH
0
but more than in homozygous b @thalassemia. Clinically, the disease
Additional tests were performed on John’s blood to is classified as thalassemia intermedia and rarely requires blood trans-
determine the cause of his anemia.
fusions except in cases of physiological stress such as pregnancy or
hemoglobin electrophoresis iron panel infection. However, because of the impaired hemoglobin synthesis,
hbA 66% Serum iron 92 mcg/dL most patients with db@thalassemia have a mild hypochromic, micro-
1% TIBC 310 mcg/dL cytic anemia. Patients have slight hepatosplenomegaly and some bone
hbA 2
hbF 1% Serum ferritin 88 ng/mL changes associated with chronic erythroid hyperplasia. Hemolysis
hb Bart’s 8% Iron saturation 33% probably contributes to the anemia because both reticulocytes and
hbh 24% bilirubin are elevated.
0
The heterozygous form of db@thalassemia (db /b) is not iden-
5. Is the hemoglobin electrophoresis normal or abnormal? tified with any specific clinical finding. There is no anemia or sple-
6. If abnormal, list hemoglobins that are elevated, nomegaly. The hematological picture, however, is similar to that of
decreased, or abnormally present. b@thalassemia minor with microcytic, hypochromic erythrocytes.
7. If abnormal, which globin chain(s) is (are) decreased? HbA is normal or slightly decreased, whereas HbF is increased to
2
5–20%. HbA is usually 690% (Table 14-6).
8. If abnormal, which globin chains are produced in
excess?
9. Is the iron panel normal or abnormal? GDB@Thalassemia
This rare form of thalassemia (g@, d@, b@thalassemia has several vari-
10. If the iron tests are abnormal, list those outside the
reference interval and indicate whether they are ele- ants and is characterized by deletion or inactivation of the entire
33
vated or decreased. b@gene complex. Deletion of the g@, d@, and b@genes would result
in the absence of all normal adult hemoglobin production from that
11. If the iron tests are abnormal, state the disorder(s) chromosome. Therefore, only the heterozygous state has been encoun-
consistent with the abnormal iron panel.
tered because a homozygous condition would be incompatible with
12. Given all the data supplied, what is the definitive life. Although neonates have severe hemolytic anemia, as the children
diagnosis of John’s anemia? grow, the disease evolves to a mild form of b@thalassemia.
Hemoglobin Constant Spring
Hemoglobin Constant Spring (HbCS) is a hemoglobin tetramer
oTheR Thalassemias and formed from the combination of two structurally abnormal a@chains,
each elongated by 31 amino acids at the carboxy-terminal end, and
Thalassemia-like condiTions two normal b@chains. This genetic mutation is common in Thailand.
CS
In theory, any globin gene can be mutated, resulting in a reduction The chromosome with the a gene carries one normal a@gene. Thus,
in the synthesis of globin chains and the corresponding hemoglo- the homozygous HbCS individual has two normal a@genes, one on
bin. Individuals have been observed with deficiencies in each of the each chromosome, and the heterozygous HbCS carrier has three nor-
normal adult globin chains. However, the thalassemias that involve mal a@genes.
globin chains other than a and b are relatively benign in their clinical The elongated a@chains of HbCS are thought to result from a
course because they are not constituents of the major adult hemoglo- mutation of the a@chain termination codon by a single base substitu-
72
bin, HbA. Thalassemias have been observed involving more than one tion. The abnormal a@chains are synthesized at very low levels ( 1%
M14_MCKE6011_03_SE_C14.indd 267 01/08/14 12:51 pm
268 SECTION III • ThE ANEmIAS
★ TaBle 14-6 Characteristics of Other Thalassemias and Thalassemia-Like Conditions
disorder defect ancestry cBc/diff hb electrophoresis other
db@thalassemia homo- Deletion of db@gene Greek, African, Italian, micro/hypo rBCs hb T T hbA, Thalassemia intermedia
0
0
zygous (db /db ) complex Arabian 10–12 g/dL
T ThbA 2 , c c hbF
0
heterozygous (db /b) Same as homozygous No anemia T hbA, Thalassemia minor
N@ThbA 2 , c hbF
gdb@thalassemia het- Deletion or inactivation mediterranean regions Birth: marked anemia; ThbA, Thalassemia minor
0
erozygous (gdb /b) of b@gene complex Adult: Slight anemia
ThbA 2 , T hbF
homozygous Same as heterozygous Unable to observe No adult hb Incompatible with life
0
0
(gdb /gdb )
Constant Spring Long a@chain, mutated Thailand micro/hypo rBCs, hb Bart’s at birth, c c
homozygous stop codon, reduced hb 9–11 g/dL, c hbCS,
CS
CS
(aa /aa ) synthesis reticulocytes ThbA, N@hbA 2 and
hbF
heterozygous Same as homozygous Normal c hbCS Can be seen with a@thal2
CS
(aa /aa) trait
hPFh homozygous Deletion or inactivation Greek, Swiss, Black mild micro/hypo rBCs, 100% hbF Two variants— pancellular,
0
0
(db /db ) of db@gene complex no anemia, c cRBCs, heterocellular
hb 14–18 g/dL Asymptomatic
0
heterozygous (db /b) Same as homozygous Near normal c hbF, Asymptomatic
T hbA, T hbA 2
hb Lepore db@hybrid chain European marked micro/hypo No hbA, Thalassemia major or
homozygous rBCs, hb 4–11 g/dL, No hbA 2 , intermedia
(db Lepore /db Lepore ) c c c anisopoikilocytosis
c c hb Lepore, c c hbF
heterozygous Same as homozygous Slight micro/hypo RBCs, T hbA, Thalassemia minor
Lepore
(db /b) hb 12 g/dL
T hbA 2 c hb Lepore
c c c = marked increase; c c = moderate increase; c = slight increase; T T = moderate decrease; T = slight decrease; N = normal
of the output compared with a normal a gene) because of reduced CheCkpoint 14-8
stability of the elongated mRNA. The result is an overall deficiency of
a@chain synthesis producing an a@thalassemia@like phenotype. Why is gdb@thalassemia more severe than db@thalassemia
and CS-thalassemia?
clinical Findings
The homozygous state is phenotypically similar to a@thalassemia
minor. A slight anemia accompanied by mild jaundice and spleno- Hereditary Persistence of Fetal
megaly is typical. Heterozygotes show no clinical abnormalities.
Hemoglobin (HPFH)
laboratory Results Hereditary persistence of fetal hemoglobin (HPFH) is actually a group
In homozygotes, clinical findings are similar to a relatively mild form of heterogeneous disorders caused by either the absence of d@ and
of HbH disease with a mild microcytic hypochromic anemia. Hemo- b@chain synthesis or a loss of suppression of the g@globin gene, result-
globin electrophoresis demonstrates the presence of Hb Bart’s at birth. ing in an increased g@chain production into adult life. In homozygotes,
In homozygous adults, HbCS makes up 5–7% of the hemoglobin, and the result is an absence of HbA and HbA with 100% of hemoglobin
2
HbA and HbF are normal. The remainder of hemoglobin is HbA production being HbF. Hemoglobin F production continues at high
2
(Figure 14-2). levels throughout life, preventing the clinical symptoms and hema-
Heterozygotes show no hematological abnormalities, but a small tological abnormalities associated with thalassemia. The condition
amount of HbCS (0.2–1.7%) can be found on electrophoresis. HbA occurs in 0.1% of African Americans.
2
and HbF are normal with the remainder being HbA (Figure 14-2).
In some areas, the coexistence of HbCS with a@thalassemia trait Genetics
CS
(- -/aa ) can be found. The clinical findings are similar to those HPFH is characterized by either deletion or inactivation of the b@ and
of HbH disease. Hemoglobin electrophoresis characteristically shows d@structural gene complex, mutations in the g@globin gene promoter
73
HbA, HbH, Hb Bart’s, HbA , and 1.5–2.5% HbCS (Figure 14-2; region affecting the binding of transcription factors, 74–76 or muta-
2
Table 14-6). tion of gene inhibitor proteins. 77–81 Four potential mechanisms that
M14_MCKE6011_03_SE_C14.indd 268 01/08/14 12:51 pm
ChAPTEr 14 • ThALASSEmIA 269
12
12
explain the increase in g@globin gene expression have been reported. Erythrocytosis ranging from 6 * 10 /L to 7 * 10 /L occurs
Removal of the d and b promoters: (1) relieves competition with the as the result of the higher oxygen affinity of HbF as compared with
g@gene promoters for transcription factor activation or (2) removes to HbA. Corresponding high hemoglobin levels from 14.8–18.2 g/
the g@gene inhibitor–binding sites; (3) mutations might juxtapose b@ dL are also typical in HPFH. Erythrocytes are microcytic and slightly
gene enhancer sequences to g@genes, facilitating activation of g@genes; hypochromic with a mean MCV of 75 fL and a mean MCH of 25.0 pg.
and (4) mutated transcription factors increase activation or inhibit There is a mild degree of anisocytosis and poikilocytosis. The reticu-
4
suppression of the g@globin gene. Most a@chains combine with the locyte count range is 1–2%. It is doubtful that this disorder has any
available g@chains to produce HbF. Consequently, no accumulation significant degree of hemolysis because the reticulocyte count, biliru-
and no precipitation of excess a@chains occur. In HPFH, increased bin, and haptoglobin levels are normal. Electrophoresis demonstrates
production of g@chains with the corresponding elevations of HbF 100% HbF.
compensates for the reduction in HbA and HbA synthesis and dif-
2
ferentiates it from db@thalassemia in which only modest elevations heterozygotes
in HbF are observed. Heterozygous HPFH is usually found incidentally through family
studies. Patients present with a slightly elevated erythrocyte count
variants with the corresponding elevation of the hematocrit and a slightly
HPFH can be categorized into two major groups, pancellular and decreased MCH (27 pg). HbF is 10–30% of the total hemoglobin.
heterocellular, based on the distribution of HbF in erythrocytes. The HbA is decreased to 1–2% and the remainder is HbA. In the pres-
2
HbF distribution patterns can be visualized using the acid elution stain ence of iron deficiency, HbF levels are lower (Table 14-6).
developed by Kleihauer and Betke. Pancellular refers to the obser-
vance of HbF in most of the erythrocytes; in the heterocellular ver- Hemoglobin Lepore
sion, HbF is concentrated in a small subset of erythrocytes. incidence and affected individuals
Several different types of HPFH have been described: the Black, Hemoglobin Lepore was first described in 1958 as a structural hemo-
G
Greek, and Swiss types. In the Black and Swiss types, both g@ and globin variant with hematological changes and clinical manifestations
A g@chains are produced in approximately equal amounts. The Greek resembling those of thalassemia. 73,82 The disorder is widely distrib-
A
G
form is characterized by production of both g@ and g@chains, but uted throughout the world but is especially common in middle and
A
most HbF is made up of the g@chains. Both the Black and Greek eastern Europe.
types have the characteristic pancellular distribution pattern of HbF
in erythrocytes. The Swiss form exhibits a heterocellular distribution Genetics
of HbF, which results from a hereditary increase (3%) in the number In Hb Lepore, the non@a@chain is a d/b@globin hybrid in which
of fetal (F) cells 21,22 (Table 14-7 ★). the N-terminal end of a d@chain is fused to the C-terminal end of a
When present, the pancellular distribution of HbF in erythro-
cytes helps to distinguish this disorder from other disorders associated b@chain. The variant hybrid genes are thought to occur during meio-
sis from an aberrant crossover event resulting in recombination of
with an increase in HbF. Most other disorders with elevated HbF levels misaligned d@ and b@genes on separate chromosomes. The result
present with the heterocellular distribution pattern. of the unequal crossover event is two fusion genes, the d/b@Lepore
It has been suggested that the various categories of HPFH actu-
ally represent a continuum of a spectrum of b@thalassemias with and the b/d@anti@Lepore genes. The d/b@Lepore fusion gene is tran-
scribed and translated into the d/b@fusion globin chain, two of which
0
homozygous b @thalassemia at one end where the lack of b@chain combine with two a@chains to form Hb Lepore. The chromosome
synthesis is poorly compensated for by g@chain production and with containing the b/d@anti@Lepore fusion gene still contains intact b@
pancellular HPFH at the other end where the lack of b@chain synthesis and d@genes that are synthesized normally to form HbA and HbA ,
is almost completely compensated for by g@chain production. 2
respectively. Progeny bear genes from the involved chromosome that
are neither fully paternal nor fully maternal. Because the recombina-
homozygotes tion event occurred in the germ cells, the newly formed chromosomes
Homozygous HPFH is asymptomatic, including no evidence of become a permanent part of the family’s gene pool. Hb Lepore is stable
abnormal growth patterns or splenomegaly. The abundant g@chains and has normal functional properties except for a slight increase in
combine with the normal a@chains to produce HbF. oxygen affinity.
pathophysiology
★ TaBle 14-7 Characteristics of HPFH Variants The pathophysiology of hemoglobin Lepore is similar to that of
b@thalassemia. No intact b@gene is present on the chromosome car-
Types of g@chains distribution of hbF in rying the Lepore fusion gene, so b@chain synthesis is absent. The Hb
hpFh Type produced erythrocytes
Lepore gene is under the influence of the d@gene promoter, which
Black G g@ and g@ Pancellular limits synthesis of the Lepore hybrid chain to approximately 2.5%
A
A
Swiss G g@ and g@ heterocellular of normal b@chain production. Thus, the abnormal Lepore chains
A
Greek Primarily g@ Pancellular are inadequately synthesized, leading to an excess of a@chains. In
the homozygous state, no normal b@chains or d@chains would be
M14_MCKE6011_03_SE_C14.indd 269 01/08/14 12:51 pm
270 SECTION III • ThE ANEmIAS
some b@chains, resulting in HbA synthesis and few to no thalas-
semia symptoms. The most common example is HbS/b@thalassemia
83
accounting for 1 in 1667 births among African Americans. This has
also been reported in patients of Greek, Turkish, Indian, North Ameri-
84
can, Mediterranean, and Romanian ancestry. Three clinical severi-
+
0
ties have been identified: HbS/b @Type 1 (severe), HbS/b @Type 1
+
(moderate), and HbS/b @Type 2 (asymptomatic). Differentiating
this combination disorder from sickle cell disease (HbSS) and trait
(HbAS) is sometimes difficult, but a comparison of b:a ratio can
be helpful (Web Table 14-1). In sickle cell disorders, the b:a ratio is
approximately 1:1, whereas it is closer to 0.5:1 in HbS/b@thalassemia. 85
■ FiGuRe 14-12 Peripheral blood smear from a patient Combination disorders are more complex when the structural
with hemoglobin Lepore. Note the anisocytosis, poikilo- hemoglobin gene mutation is on the b@gene and the thalassemia
cytosis, and microcytosis (Wright-Giemsa stain; 1000* mutation involves the a@gene because different chromosomes are
magnification). involved. These patients can be either homozygous or heterozygous
for the structural hemoglobin variant and coexpress any of the pos-
sible a@gene combinations.
synthesized to combine with the a@chains being produced, thus, no
HbA or HbA . The more severely affected children can become trans- The severity of the combination disorder is directly proportional
2
fusion dependent and develop complications of hemosiderosis. The to the total number of affected genes and ranges from moderate to
86–88
combination of ineffective erythropoiesis, decreased HbA, increased asymptomatic. Coexistent a@thalassemia decreases synthesis
oxygen affinity of HbF, and chronic extravascular hemolysis pro- of a@chains resulting in fewer a@globin chains available to combine
duces a microcytic hypochromic anemia that is classified clinically as with the structurally abnormal b@chain. Thus, the concentration of
b@thalassemia major in the more severe cases and as b@thalassemia the structural variant (in heterozygotes) is usually decreased because
intermedia in the remaining cases. As with b@thalassemia major, the limited number of a@chains preferentially combines with the nor-
symptoms emerge within the first few years of life. mal b@ or d@ (or g@) chains. The percentage of HbA, HbF, and HbA
2
Patients with heterozygous Hb Lepore are asymptomatic and increase relative to a typical heterozygote, reducing the abnormal
classified clinically as b@thalassemia minor. The blood picture is simi- pathophysiology associated with the particular structural hemoglo-
lar to that seen in b@thalassemia minor. bin variant inherited.
S S
In the case of a homozogous b b individual with an
a@thalassemia syndrome, the clinical severity of sickling is often
laboratory Results
In the homozygous state, hematologic findings are similar to those of reduced because of a net decrease in MCHC, which reduces the ten-
dency of HbS to produce cell sickling. Thus, cell hemolysis and the
b@thalassemia major. There is no detectable HbA or A on hemoglo- clinical symptoms associated with occlusion of the microvasculature
2
bin electrophoresis, Hb Lepore ranges from 8–30%, and the remain- decrease (Chapter 13). HbF has also been shown to decrease the sick-
der consists of HbF. Hemoglobin electrophoresis must be interpreted ling process. The wide variety of clinical severities seen in sickle
13
with caution because Hb Lepore comigrates with HbS on cellulose cell anemia can be partially related to the high incidence of a@ and
acetate and agarose at an alkaline pH and with HbA on citrate agar b@thalassemia in the same population (Figure 14-13 ■).
at an acid pH.
In the heterozygous state, hematologic findings are similar to
thalassemia minor (Figure 14-12 ■): Hemoglobin electrophoresis
reveals a mean Hb Lepore concentration of 10%; HbA is decreased
2
with a mean of 2%; HbF is usually slightly elevated to 2–3%; and HbA
makes up the remainder (Table 14-6).
The severely anemic cases of Hb Lepore require a regular transfu-
sion protocol from early childhood. Splenectomy can be performed in
an attempt to lessen the degree of anemia.
Combination Disorders
Occasionally an individual is doubly heterozygous for a structural
hemoglobin variant and thalassemia, inheriting one of each of the two
abnormalities from each parent. The most common structural hemo-
globin variants involved in combination disorders are HbS, HbC, and
HbE. When a structural variant is inherited with a b@thalassemia ■ FiGuRe 14-13 Peripheral blood smear from a patient
gene, the severity of the combination disorder depends on the type of with hemoglobin S and a@thalassemia. The cells are micro-
0
b@gene mutation. Patients expressing the b @gene produce no HbA cytic. There are acanthocytes and cells with pointed ends
+
and experience moderate to severe symptoms. The b @gene produces present (Wright-Giemsa stain; 1000* magnification).
M14_MCKE6011_03_SE_C14.indd 270 01/08/14 12:51 pm
ChAPTEr 14 • ThALASSEmIA 271
Laboratory diagnosis is accomplished by applying the tech-
niques used to identify each of the disorders individually. Patients
present with a mild microcytic, hypochromic anemia with target cells
and the poikilocytes associated with the inherited structural hemo-
globin variant. Patients who inherit HbS can have sickle cells, and
those with HbC sometimes show HbC crystals. Hemoglobin electro-
phoresis is helpful in resolving the structural hemoglobin variant and
shows the quantitative changes in normal and abnormal hemoglo-
bins associated with a@ and b@thalassemias. In HbS@a@thalassemia,
the concentration of HbS is inversely proportional to the number of
a@gene deletions. HbS concentration in heterozygotes is 35% with
one a@gene deletion, 28% with two a@deletions, and 20% with ■ FiGuRe 14-14 Peripheral blood smear from a patient
three a@gene deletions. 89 with hemoglobin E and a@thalassemia. The RBCs are
When less common structural variants (HbE, HbO, HbD, etc.) microcytic, hypochromic, and target cells are present
are coexpressed with thalassemias, more testing could be necessary (Wright-Giemsa stain; 1000* magnification).
because they comigrate with HbS or HbC on hemoglobin electro-
phoresis (Figure 14-2). Molecular techniques including automated
sequencing, dot-blot analysis, or allele-specific amplification can be Due to the significant variations in clinical expression of the
used in these cases (Chapter 43). 90 various hemoglobin structural variants and b@thalassemia alleles, it
The incidence of double heterozygotes expressing both HbE and
b@thalassemia is increasing in Southeast Asia, and many patients are is suggested that identification of these conditions should go beyond
92
hematologic analysis and hemoglobin electrophoresis. A diagnosis
presenting with symptoms that rival b@thalassemia major in sever- defined at the molecular level to identify the genetic mutation can lead
91
ity. Approximately 3000 people with both mutations are born each to better clinical management of the disease. A summary of the dif-
year in Thailand, and many others are diagnosed in other parts of ferentiating characteristics of combination thalassemia and hemoglo-
Southeast Asia, India, and Burma. In Thailand, Laos, and Cambodia, binopathy disorders can be found in Web Table 14-1.
HbE penetrance is approximately 50%. HbE heterozygotes usually
have relatively mild disease; therefore, HbE/b@thal double heterozy-
92
gotes might be expected to have a moderately severe disease. How-
ever, the HbE mutation at the 26th position of the b@globin gene
activates a cryptic splice site at codon 25, resulting in significantly CheCkpoint 14-9
reduced b@chain production. Therefore, in double heterozygotes In combination disorders of structural hb variants and
E
0
A
of HbE and b @thal, few to no b @ or b @chains are produced. This thalassemia, why is a@thalassemia inherited with sickle
results in the accumulation of a@chains that precipitate within the cell trait less severe than b@thalassemia coexpressed with
erythrocytes, producing hemolysis as in b@thalassemia major. 92,93 A sickle cell trait?
+
more moderate anemia can result if the b @thalassemia gene is inher-
ited with HbE. In this form of the disease, there are HbE, HbA, HbF,
and HbA . The HbA, however, is less than would be expected in HbE
2
+
trait, and the anemia is more severe. When the b @thalassemia gene
E
is inherited with the b @gene, the result is microcytic, hypochromic diFFeRenTial diaGnosis
anemia with significant poikilocytosis, and nucleated erythrocytes. oF Thalassemia
Hemoglobin E can also be found in combination with Clinical signs, symptoms, and CBC results are strikingly similar
a@thalassemia (Figure 14-14 ■). This combination produces a more in microcytic, hypochromic anemias regardless of the etiology of
severe anemia than does HbE alone. The amount of HbE depends on the anemia, making the clinical diagnosis difficult. Differentiating
whether the patient is heterozygous or homozygous for HbE and the the various thalassemias is even more difficult because they are all
a@thalassemia genotype inherited (Chapter 13). inherited and occur in similar nationalities. Additional laboratory
Double heterozygotes for sickle cell and HPFH (deletion variant) tests are therefore crucial in making the differential diagnosis. 94,95
exhibit a mild form of sickle cell trait with no occurrence of crises Web Table 14-2 summarizes the tests used to differentiate thalassemia
or anemia. It has been suggested that the surprisingly favorable clini- from other anemias.
cal picture is related to the distribution of HbF in erythrocytes. The
peripheral blood smear shows anisocytosis and target cells, and the
sodium metabisulfite and solubility tests are positive. Hemoglobin
electrophoresis produces a pattern that is easily confused with that CheCkpoint 14-10
of sickle cell anemia. Only HbS, HbF, and HbA are present with HbF
2
levels of 15–35%. HbA is normal or reduced. Family studies are help- Which laboratory tests should be done first to differenti-
2
ful in identifying the double heterozygous state. ate thalassemia and iron deficiency?
M14_MCKE6011_03_SE_C14.indd 271 01/08/14 12:51 pm
272 SECTION III • ThE ANEmIAS
Summary
Thalassemias result from genetic defects that affect the produc- The thalassemias generally produce a microcytic, hypo-
tion of globin chains. Any of the globin chains can be affected, chromic anemia with changes in the concentrations of hbF,
but the most clinically significant are a@ and b@chain defects. The hbA 2 , and hbA. hbF and hbA 2 are elevated in b@thalassemia
clinical severity of the disease is related to the number of mutated and decreased in a@thalassemia with decreased concentrations
genes and the type of genetic defect. The human diploid genome of hbA in both. In the more severe a@thalassemias, hbh (b 4 )
normally has four a@genes, so in a@thalassemia, from one to four and hb Bart’s (g 4 ) can be detected. Erythrocyte morphology is
of the a@genes can be deleted. If only one gene is affected, the similar in both major forms of thalassemia and in iron-deficiency
condition is not clinically or hematologically apparent, but if two anemia; however, hemoglobin electrophoresis and iron stud-
or three are affected, both clinical and hematological abnormali- ies assist in differentiating these two entities. Some structural
ties of mild or moderate severity, respectively, occur. Deletion of hemoglobin variants are synthesized in decreased quantities
all four a@genes is incompatible with life. There are two b@alleles (i.e., hb Lepore, hb Constant Spring, hbE) and have clinical
and three b@thalassemia gene defects; one causes a complete and morphologic similarities to thalassemias. molecular tech-
0
absence of b@chain production (b @thalassemia), one causes vari- niques (Chapter 43) are now available to identify the genetic
+
ably decreased synthesis of b@chain production (b @thalassemia), mutation in the globin gene but are not always necessary for
and the third minimally affects b@chain production (silent carrier diagnostic purposes.
SC
or b ). This results in seven potential b@thalassemia genotypes Current therapies are improving, and medical access in
+ +
SC
0 +
SC
SC
0
+
0 0
(b b , b b , b b , b b, b b, b /b , b /b) that are categorized underdeveloped countries is expanding. As a result of these
into four clinical severities (b@thalassemia major, intermedia, advances, the general health and quality of life are improving
minor, and silent carrier) ranging from severe to asymptomatic. for patients with thalassemia.
Review Questions
Level i 3. What is the typical morphologic classification of erythro-
cytes in thalassemia? (Objective 3)
1. The statement that best defines thalassemia is: (Objective 1)
A. macrocytic, normochromic
A. qualitative disorder of hemoglobin synthesis derived
primarily from a genetic point mutation in one or more B. normocytic, normochromic
globin genes C. microcytic, hyperchromic
B. disorder of inappropriate iron metabolism due to D. microcytic, hypochromic
abnormal transferrin
4. Select the disorder that is an a@thalassemia. (Objectives
C. quantitative disorder of hemoglobin synthesis result- 5a, 5b)
ing from deletional and nondeletional mutations of
globin genes A. hbh disease
B. Cooley’s anemia
D. single amino acid substitution in a globin chain affect-
ing the function of hemoglobin C. hPFh
2. Which of the following statements is false for a patient D. hb Lepore
with thalassemia but true in certain hemoglobinopathies? 5. a@Thalassemia is characterized by: (Objective 5c)
(Objective 2)
A. deletion of b@genes
A. Abnormal hemoglobin will polymerize inside erythro-
cytes, altering red cell shape. B. amino acid substitutions in the a@chain
C. excess a@chain production
B. Novel hemoglobins composed of abnormal combi-
nations of normal globin chains can be detected on D. deletion of a@genes
hemoglobin electrophoresis.
6. Which nationality is most likely to be affected by thalas-
C. Elevations in embryonic and fetal hemoglobins can be semia? (Objectives 5b, 6a)
observed. A. Chinese
D. The amino acid sequence of the globin chains of the B. South American Indians
abnormal hemoglobins is normal.
C. Southeast Asians
D. Europeans
M14_MCKE6011_03_SE_C14.indd 272 01/08/14 12:51 pm
ChAPTEr 14 • ThALASSEmIA 273
7. Which of the following laboratory results would be 3. Which pathophysiologic event is involved in the pathogen-
expected in a patient with a@thalassemia? (Objective 5e) esis of HbH disease? (Objective 4b)
A. mCh = 32 pg A. hbh has a higher affinity for oxygen that hampers
oxygen release.
B. mCV = 70 fL
B. hbh is an embryonic hemoglobin that is not present
C. stomatocytes at birth.
D. increased hbA C. hbh cannot bind and transport oxygen.
8. The pathogenesis of b@thalassemia includes: (Objective 6b) D. Polymerization of hbh alters erythrocyte shape.
A. decreased production of b@chains 4. The genetic designation heterozygous a @thal@1/normal
0
B. abnormal structure of a@chains refers to: (Objective 4a)
C. bone marrow hypoproliferation A. a@thalassemia minor
B. Cooley’s anemia
D. decreased synthesis of erythropoietin
9. In b@thalassemia major, hemoglobin electrophoresis will C. hbh disease
show: (Objective 6d) D. silent carrier
A. reduced hbF 5. The single best laboratory test to distinguish b@thalassemia
minor from a@thalassemia, iron-deficiency anemia, HPFH,
B. reduced hbA 2
and hemoglobinopathies is: (Objective 8)
C. reduced hbA
A. hemoglobin solubility
D. increased hbh
B. serum iron
10. Select the thalassemia type in which the patient survives C. heinz body stain
and presents with an abnormal hemoglobin that is sensi-
tive to oxidation and precipitates in red cells after incuba- D. hbA 2 level
tion with brilliant cresyl blue. (Objective 6d)
6. Hemoglobin Constant Spring can best be described as:
A. hydrops fetalis (Objective 7c)
B. hbh disease A. deletion of three a@genes
C. b@thalassemia minor B. two normal b@chains and two elongated a@chains
D. silent carrier C. two normal a@chains and two b/g@fusion chains
D. continued synthesis of g@chains throughout adult life
Level ii 7. Select the statement that best describes hereditary persis-
tence of fetal hemoglobin. (Objective 7d)
1. a@Thalassemia most commonly results from which of the
following genetic lesions? (Objective 1) A. The homozygous state is incompatible with life.
A. gene deletion B. hbF is elevated in adults.
B. promoter mutation C. It results from the deletion of the g@gene.
C. termination codon mutation D. It is a form of b@thalassemia.
D. splice site mutation 8. A 4-year-old male patient has a microcytic, hypochromic
anemia. Hemoglobin electrophoresis shows 46% HbS, 49%
2. Why is hydrops fetalis incompatible with life? HbA, 3.5% HbA 2 , 1.5% HbF. His parents have no symptoms
(Objective 4b) of anemia. What are his parents’ most likely phenotypes?
(Objectives 5, 7)
A. Life cannot exist without hbA.
A. sickle cell trait and b@thalassemia major
B. Lack of embryonic hemoglobins precludes fetal
development. B. sickle cell anemia and a@thalassemia
C. All three normal adult hemoglobins contain a@chains. C. sickle cell anemia and heterozygous b@thalassemia
D. Fetal hemoglobin is essential to sustain life after D. sickle cell trait and normal
birth.
M14_MCKE6011_03_SE_C14.indd 273 01/08/14 12:51 pm
274 SECTION III • ThE ANEmIAS
9. Which of the following combination disorders would 10. A 28-year-old female from Laos who had a hemoglobin
exhibit more severe symptoms? (Objective 7f) of 11.2 g/dL was diagnosed with iron-deficiency anemia.
She was given iron supplements. Her reticulocyte count
A. hbS and b@thalassemia minor
increased from 4% to 5% after 6 days of treatment. Six
B. hbC trait and a@thalassemia minor months later, she returned for a follow-up CBC. Her hemo-
globin was 11.5 g/dL, and the red cells were microcytic
C. hPFh and b@thalassemia minor (75 fL), normochromic. What reflex test should be done?
D. hbS and hPFh (Objective 8)
A. hemoglobin electrophoresis
B. serum iron
C. bone marrow
D. serum ferritin
Companion Resources
http://www.pearsonhighered.com/healthprofessionsresources/
The reader is encouraged to access and use the companion resources created for this chapter. Find additional information to help
organize information and figures to help understand concepts.
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M14_MCKE6011_03_SE_C14.indd 276 01/08/14 12:51 pm
Megaloblastic and
15 Nonmegaloblastic
Macrocytic Anemias
Joel Hubbard, Phd
Stacey robinSon, MS
Objectives—Level I
At the end of this unit of study, the student should be able to: Chapter Outline
1. Explain the cause and process of megaloblastic maturation in the bone Objectives—Level I and Level II 277
marrow.
2. Describe the body’s requirements for vitamin B 12 and folate and their Key Terms 278
physiologic roles. Background Basics 278
3. List the laboratory tests used to confirm a diagnosis of cobalamin
deficiency and give expected results. Case Study 278
4. List the laboratory tests used to confirm a diagnosis of folic acid Overview 279
deficiency and give expected results.
5. Recognize the six most common disorders associated with Introduction 279
a macrocytic anemia. Megaloblastic Anemia 279
6. Name four causes of a cobalamin deficiency and give two distinguishing
clinical or laboratory characteristics of each. Macrocytic Anemia Without
7. Describe the etiology and pathophysiology of pernicious anemia, Megaloblastosis 296
including clinical symptoms and clinical subtypes. Summary 298
8. Name three causes of a folate deficiency and give two distinguishing
clinical or laboratory characteristics of each. Review Questions 299
9. Differentiate the pathophysiology and peripheral blood findings of non- Companion Resources 300
megaloblastic macrocytic anemia from those of megaloblastic anemias.
10. Summarize the typical blood picture seen with a folate or cobalamin References 301
deficiency.
Objectives—Level II
At the end of this unit of study, the student should be able to:
1. Summarize the process of cobalamin and folic acid metabolism and
explain how a deficiency can result in megaloblastosis.
2. Compare macrocytosis associated with a normoblastic marrow and
macrocytosis associated with a megaloblastic marrow on the basis of
physiological defect, and differentiate them based on the laboratory
blood picture.
3. Summarize the mechanism of maturation defects that lead to megalo-
blastosis and recognize the morphologic blood cell abnormalities.
(continued)
M15_MCKE6011_03_SE_C15.indd 277 01/08/14 1:38 pm
278 SECTION III • ThE ANEmIAS
Objectives—Level II (continued) Key Terms
4. Compare and contrast the various clinical forms Achlorhydria Intrinsic factor (IF)
and causes of a cobalamin and folate deficiency Cobalamin Megaloblastic
on the basis of clinical symptoms and laboratory Demyelination Nuclear-cytoplasmic
results. Dyspepsia asynchrony
5. Categorize the causes and clinical variations of pernicious Folate Pernicious anemia (PA)
anemia. Folic acid Schilling test
6. Compare and contrast the various clinical forms and Glossitis
causes of a folic acid deficiency.
7. Choose and briefly explain four laboratory tests used Background Basics
to identify the cause of a macrocytic anemia; give the
expected results of these four tests in a patient with an The information in this chapter builds on the concepts learned
autoantibody directed against intrinsic factor. in previous chapters. To maximize your learning experience,
8. Assess Schilling’s test results and provide a differential you should review these concepts before starting this unit of
diagnosis. study:
9. Compare and contrast the causes of macrocytosis that level i
have a normoblastic marrow. • Describe the maturation process of erythrocytes in the marrow.
10. Construct an algorithm of laboratory testing to distin- (Chapter 5)
guish between a megaloblastic anemia and a macrocytic,
normoblastic anemia. • Outline the functional and morphologic classification of ane-
11. Evaluate a case study from a patient with anemia. mia and list the basic laboratory tests to diagnose anemia.
Determine the most probable diagnosis from the (Chapter 11)
medical history and laboratory results. level ii
• Summarize the concepts of cell development, regulation, and
the process of cell division. (Chapter 2)
• List and describe the laboratory tests used in differential
diagnosis of anemia. (Chapter 11)
caSe Study
We will refer to this case study throughout the chapter. The following abnormal erythrocyte morphology was
Kathy, a 36-year-old female, experienced a recent 35-lb reported:
weight loss. her tongue was red and fissured. She also
complained of chronic fatigue and shortness of breath Macrocytes 2+
upon exertion. Physical examination suggested signs of Anisocytosis 3+
jaundice and increased numbness and a tingling sensa- Poikilocytosis 2+
tion of fingers and toes. She was hospitalized with the Ovalocytes 1+
general diagnosis of moderate anemia, jaundice, and Basophilic stippling 1+
neurological symptoms. her admitting CBC demon- Occasional howell-Jolly bodies
strated the following laboratory results:
Consider the reflex tests that might be important in iden-
differential tifying the etiology of this anemia.
9
WBC 4.5 * 10 /L Lymphs 36.0%
12
RBC 2.50 * 10 /L Monos 3.6%
hb 10.0 g/dL (100.0 g/L) Neutrophils 59.4%
hct 31% (0.31 L/L) Eosinophils 1.0%
MCV 124 fL Basophils 0.0%
mCh 40.50 pg NRBCs/100 WBCs 5.0%
mChC 32.70 g/dL Moderate
RDW 21.20 hypersegmented
9
PLT 155 * 10 /L neutrophils
M15_MCKE6011_03_SE_C15.indd 278 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 279
overview ★ table 15-1 Conditions Associated with
This chapter is a study of the macrocytic anemias, which can be mega- Megaloblastic and Nonmegaloblastic
loblastic or nonmegaloblastic. The first part of the chapter discusses (Normoblastic) Macrocytic Anemias
the megaloblastic anemias beginning with a description of the clinical Megaloblastic normoblastic
and laboratory findings. Because megaloblastic anemia is most often
due to deficiencies or abnormal metabolism of folate or cobalamin Folate deficiency Alcoholism
(vitamin B ), the metabolism of these vitamins is discussed in detail. Nutritional deficiency Liver disease
12
The latter part of the chapter reviews the causes of nonmegaloblastic Increased requirement (pregnancy) Shift reticulocytosis in hemolytic
anemia or hemorrhage
macrocytic anemia and compares the laboratory test results in non- Intestinal malabsorption hypothyroidism
megaloblastic and megaloblastic anemia. The laboratory professional Drug inhibition Aplastic anemia
can often identify diagnostic clues of megaloblastic anemia on review Cobalamin deficiency Obstructive jaundice
of a blood smear. Pernicious anemia
Small bowel resection Splenectomy
Gastrectomy Pregnancy
introduction Intestinal malabsorption Artifactual:
Macrocytic anemias are characterized by large erythrocytes (mean Nutritional deficiency hyperglycemia
MCV 7100 fL) with an increased MCH and a normal hemoglobin Increased requirement (pregnancy) cold agglutinins
content (MCHC). This is an important group of anemias because Transcobalamin deficiency leukocytosis
macrocytosis is frequently a sign of a disease process that can result Nitrous oxide abuse
in significant morbidity if left untreated. Other causes
Macrocytosis is found in 2.5–4.0% of adults who have a routine Chemotherapy with metabolic
1
complete blood count. In up to 60% of cases, macrocytosis is not inhibitors
2
accompanied by anemia, but isolated macrocytosis should always be Orotic aciduria
investigated. Macrocytosis without anemia can indicate early folate or Congenital dyserythropoietic
cobalamin (vitamin B ) deficiency because macrocytosis precedes anemia (CDA)
12
the development of anemia in these disorders.
Macrocytosis detected by automated cell counters is not always
apparent microscopically on stained blood smears. In some cases, the
erythrocyte size on automated counters is falsely elevated due to hyper- MegaloblaStic aneMia
glycemia, cold agglutinins, and extreme leukocytosis. These causes of Although very little was known about the function or origin of blood
false macrocytosis need to be differentiated from true macrocytosis. cells before the twentieth century, some perceptive individuals began
The most common cause of true macrocytosis is alcohol- to make associations between anemia and other clinical signs in
ism. Other causes include folate and cobalamin deficiencies, drugs patients. In 1822, J. S. Coombe, a Scottish physician, made the initial
including chemotherapy, reticulocytosis due to hemolysis or bleeding, clinical description of a patient who appeared to have megaloblastic
myelodysplasia, liver disease, and hypothyroidism. 2 anemia. He was the first to suggest that this anemia might be related
3
Macrocytic anemias are generally classified as megaloblastic to dyspepsia. In 1855, Thomas Addison reported his description of
or nonmegaloblastic (normoblastic), depending on morpho- a macrocytic anemia, but he made no reference to the typical micro-
4
logic characteristics of erythroid precursors in the bone marrow scopic blood findings. The discovery and description of the abnor-
(Table 15-1 ★). The megaloblastic anemias are the result of a defect mal erythroid precursors in the bone marrow associated with this
in DNA synthesis. Frequently there is an arrest in the S phase of the anemia were made possible by the advent of triacid stains. Paul Ehrlich
cell cycle and to a lesser extent during other phases of the cell cycle is credited with coining the term megaloblast in 1891 to describe the
due to delayed nuclear development. RNA and protein synthesis, large abnormal precursors in megaloblastic anemia. 5
however, are relatively unimpaired. The result is unbalanced cell Megaloblastic anemia is classified as a nuclear maturation defect.
growth and impaired cell division characterized by erythroblasts Anemia is attributed primarily to a large degree of ineffective eryth-
with distinct abnormal morphologic features. The nucleus appears ropoiesis resulting from disrupted DNA synthesis. The anemia was
immature with a fine particulate chromatin pattern, whereas the called megaloblastic in an attempt to describe the giant, abnormal-
cytoplasm is increased and relatively more mature (referred to as appearing erythroid precursors (megaloblasts) in the bone marrow.
nuclear- cytoplasmic asynchrony). These cells are referred to The generic word megaloblast describes any maturation stage of
morphologically as megaloblastic. All proliferating cells are affected. the megaloblastic erythroid series (i.e., polychromatic megaloblast).
The basis for the nonmegaloblastic anemias is not always as Other nucleated cells of the marrow are also typically abnormal.
well defined but is often related to an increase in membrane lipids. About 95% of megaloblastic anemias are caused by deficiencies
The macrocytes in nonmegaloblastic macrocytic anemia are usually of either vitamin B (cobalamin) or folic acid, vitamins necessary as
12
round, but in megaloblastic anemia, they are oval (Figure 15-1a, b ■). coenzymes for nucleic acid synthesis. In the majority of cases, cobalamin
A flow chart for laboratory analysis to help distinguish causes of deficiency is secondary to a deficiency of intrinsic factor (IF), a pro-
macrocytic anemia is shown in Figure 15-2 ■. tein necessary for absorption of cobalamin, rather than to a nutritional
M15_MCKE6011_03_SE_C15.indd 279 01/08/14 1:38 pm
280 SECTION III • ThE ANEmIAS
a b
■ Figure 15-1 (a) Peripheral blood film from a patient with pernicious anemia. Note
the anisocytosis with oval macrocytes and the nucleated red blood cell with a Howell-Jolly
body. (Wright-Giemsa stain; 1000* magnification). (b) Peripheral blood film from a patient
with normoblastic, macrocytic anemia. Compare the size of the RBCs with the lymphocyte.
The RBCs are primarily round macrocytes. (Wright-Giemsa stain, 1000* magnification).
deficiency of the vitamin. Folic acid deficiency, on the other hand, is most with typical anemic symptoms of lethargy, weakness, and a yellow or
often due to an inadequate dietary intake. Inherited disorders affecting waxy pallor. Dyspeptic symptoms are common. Glossitis with a beefy
DNA synthesis or vitamin metabolism are rare causes of megaloblastosis. red tongue, or more commonly a smooth pale tongue, is characteristic.
Loss of weight and loss of appetite are common complaints. In
pernicious anemia (see “Pernicious Anemia”), atrophy of the gastric
CheCkpoint 15-1 parietal cells causes decreased secretion of intrinsic factor and hydro-
Explain why patients with cobalamin or folate deficiency chloric acid. Bouts of diarrhea can result from epithelial changes in
have megaloblastic maturation. the gastrointestinal tract.
Neurological disturbances occur only in cobalamin deficiency, not
in folic acid deficiency. They are the most serious and dangerous clinical
signs because neurological damage can be permanent if the deficiency
Clinical Findings is not treated promptly. The patient’s initial complaints occasionally are
The onset of megaloblastic anemia is usually insidious; because the related to neurological dysfunction rather than to anemia. Neurological
anemia develops slowly, it produces few symptoms until the hemo- damage has been reported to occur even before anemia or macrocytosis
globin and hematocrit are significantly depressed. Patients can present in some cases, particularly in elderly people. The bone marrow, however,
Hb & Hct
MCV 100 fL
Increased Bleeding, hemolysis, or
Reticulocyte count response to therapy for
anemia
Normal, low
Blood smear morphology: Macrocytes absent Consider false increase in MCV
Macro-ovalocytes No due to cold agglutinins or
Dacryocytes hyperglycemia
Howell-Jolly bodies
Hypersegmentation
Round macrocytes Suspect normoblastic
Yes macrocytic anemia
Serum cobalamin assay;
go to Figure 15-5
Liver disease;
alcoholism;
aplastic anemia
■ Figure 15-2 Algorithm for the differential diagnosis of the megaloblastic
anemias from other macrocytic anemias.
Hb = hemoglobin; Hct = hematocrit; MCV = mean cell volume
M15_MCKE6011_03_SE_C15.indd 280 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 281
usually reveals megaloblastic changes even in the absence of anemia. can precede the development of anemia by months to years. 7–9 On
Tingling, numbness, and weakness of the extremities reflect peripheral the other hand, the MCV can remain within the reference interval.
neuropathy. Loss of vibratory and position (proprioceptive) sensations Epithelial changes in the gastrointestinal tract can cause iron
in the lower extremities can cause the patient to have an abnormal gait. absorption to be impaired. If an iron deficiency (which character-
The patient’s relatives sometimes note mental disturbances such as loss istically produces a microcytic, hypochromic anemia) coexists with
of memory, depression, and irritability. Megaloblastic madness is a megaloblastic anemia, macrocytosis can be masked, and the MCV
10
term used to describe severe psychotic manifestations of cobalamin can be in the normal range. Other conditions that have been shown
deficiency. A patient with severe anemia occasionally is asymptom- to coexist with megaloblastic anemia in the absence of an increased
atic, which is probably a reflection of a very slowly developing anemia. MCV include thalassemia, chronic renal insufficiency, and chronic
9
It has been suggested that cobalamin deficiency should be suspected inflammation or infection. Sometimes these coexisting causes of
in all patients who have an unexplained anemia and/or neurological anemia are not recognized until after the megaloblastic anemia has
disturbances or in individuals who are at risk of developing a deficiency been treated. It has been suggested that if coexisting iron deficiency,
such as elderly people or those with intestinal diseases. 6 thalassemia, or chronic disease is suspected, patient medical history,
racial/ethnic background, and previous MCV should be considered. 11
Hematologic parameters vary considerably (Table 15-2 ★). The
CheCkpoint 15-2 hemoglobin and erythrocyte count range from normal to very low.
12
Patients with megaloblastic anemia often present with a The erythrocyte count is occasionally 61 * 10 /L. However, ane-
yellow or waxy pallor. What is the diagnostic significance mia is not always evident. In one study of 100 patients with confirmed
12
of this clinical symptom? cobalamin deficiency, only 29% had a hemoglobin of 612 g/dL. This
is significant because neurologic symptoms can be present even if the
13
MCV and/or hematocrit are normal. Because the abnormality is a
nuclear maturation defect, the megaloblastic anemias affect all three
Laboratory Findings blood cell lineages: erythrocytes, leukocytes, and platelets. This is unlike
Laboratory tests are critical to a diagnosis of megaloblastic anemia. most other anemias that typically involve only erythrocytes. The leuko-
The routine CBC with a review of the blood smear gives important cyte count can be decreased due to an absolute neutropenia. Platelets
diagnostic clues and helps in selecting reflex tests. can also be decreased but do not usually fall below 100 * 10 /L. The
9
relative reticulocyte count (percentage) is usually normal; however,
Peripheral blood because of the severe anemia, the corrected reticulocyte count is 62%,
Megaloblastic anemia is typically a macrocytic, normochromic the absolute reticulocyte count is low, and RPI is 62 (Chapter 11).
anemia. The MCV is usually 7100 fL and can reach a volume of The distinguishing features of megaloblastic anemia on the
140 fL. However, an increased MCV is not specific for megaloblas- stained blood smear include the triad of oval macrocytes (macro-
tic anemia. The MCH is increased because of the large cell volume, ovalocytes), Howell-Jolly bodies, and hypersegmented neutrophils
but the MCHC is normal. In cobalamin deficiency, a macrocytosis (Figure 15-1a). Anisocytosis is moderate to marked with normocytes
★ table 15-2 Comparison of Common Laboratory Values in Megaloblastic and Nonmegaloblastic Macrocytosis
laboratory value Megaloblastic Macrocytosis nonmegaloblastic Macrocytosis
WBC count Decreased Normal
Platelet count Decreased Normal
RBC count Decreased Decreased
hemoglobin Decreased Decreased
hematocrit Decreased Decreased
MCV Usually 7110 fL 7100 fL
RBC morphology Ovalocytes, howell-Jolly bodies, polychromasia Polychromasia, target cells, and stomatocytes (liver disease),
schistocytes (hemolytic anemias)
hypersegmentation of neutrophils Present Absent
Reticulocyte count Normal to decreased Normal, decreased, or increased
Serum cobalamin Decreased in cobalamin deficiency Usually normal
Serum folate Decreased in folate deficiency Normal (except in alcoholism when it can be decreased)
FIGLU Increased in folate deficiency Normal
MMA Increased in B 12 deficiency Normal
homocysteine Increased Normal
Serum bilirubin Increased Normal to increased
LD Increased Normal to increased
MCV = mean corpuscular volume; FIGLU = formiminoglutamic acid; MMA = methylmalonicacid; LD = lactic dehydrogenase
M15_MCKE6011_03_SE_C15.indd 281 01/08/14 1:38 pm
282 SECTION III • ThE ANEmIAS
caSe Study (continued from page 278)
Based on the initial CBC results, further testing was
ordered with the following results:
B 12 (cobalamin) 50 pg/mL Low
Folate 10.3 ng/mL Normal
Total billirubin 2.5 mg/dL high
Direct billirubin 0.8 mg/dL Normal
AST 35 U/mL Normal
ALT 30 U/mL Normal
■ Figure 15-3 Hypersegmented neutrophils from the Examination of a bone marrow aspirate revealed an erythro-
peripheral blood of a patient with pernicious anemia. blastic hyperplasia with megaloblastic erythroblasts.
(Wright-Giemsa stain; 1000* magnification) 1. What is the morphologic classification of the patient’s
anemia?
and a few microcytes in addition to the macrocytes. Poikilocytosis 2. Based on the information obtained so far, what is the
can be striking and is usually more so when the anemia is severe. most likely defect?
Polychromatophilia and megaloblastic erythroblasts can be seen, 3. What is the significance of the AST/ALT results?
especially when the anemia is severe, indicating the futile attempt of 4. What further testing can be done to obtain a definitive
the bone marrow to increase peripheral erythrocyte mass. Cabot rings diagnosis?
occasionally can be seen in erythrocytes.
Granulocytes and platelets can also show changes evident
of abnormal hematopoiesis. Hypersegmented neutrophils can be behind cytoplasmic maturation (Figure 15-4 ■). The nucleus
found in megaloblastic anemia even in the absence of macrocytosis of the megaloblast contains loose, open chromatin that stains
(Figure 15-3 ■). Finding 5% or more neutrophils with five lobes or poorly; cytoplasmic development continues in a normal fashion.
one neutrophil with six or more lobes is considered hypersegmenta- At each stage of development, the cells contain more cytoplasm
tion. This finding of hypersegmented neutrophils is considered highly with a more mature appearance relative to the size and matu-
sensitive and specific for megaloblastic anemia. Therefore, hyperseg- rity of the nucleus (resulting in a decreased nuclear:cytoplasmic
mented neutrophils offer an important clue to megaloblastic anemia in [N:C] ratio).
the face of a coexisting disease that tends to keep erythrocyte volume The megaloblastic features are more easily noted in later stages of
6100 fL. One study showed that in patients with renal disease, iron
deficiency, or chronic disease with a normal or decreased MCV and 1% erythroid development, especially at the polychromatophilic stage in
which the presence of hemoglobin mixed with RNA gives the cytoplasm
hypersegmented neutrophils, 94% had vitamin B or folic acid defi- the gray-blue color typical of this erythroid precursor. The polychromato-
12
8
ciency. If 5% hypersegmented neutrophils were counted, the incidence philic megaloblast nucleus, however, still has an open (lacy) chromatin
of the vitamin B or folic acid deficiency increased to 98%. Hyper- pattern more typical of an earlier stage of development.
12
segmented neutrophils tend to be larger than normal neutrophils. A Leukocytes and platelets also show typical features of a nuclear
mild shift to the left with large hypogranular bands can also be noted. maturation defect as well as ineffective leukopoiesis and thrombo-
Platelets can be large, especially when the platelet count is decreased.
poiesis. Giant metamyelocytes and bands with loose, open chromatin
in the nuclei are diagnostic (Figure 15-4a). The myelocytes can show
CheCkpoint 15-3 poor granulation as do more mature stages. Megakaryocytes can be
decreased, normal, or increased. Maturation, however, is distinctly
Why are abnormalities of leukocytes and platelets present abnormal. Larger than normal forms can be found with separation of
in megaloblastic anemia? nuclear lobes and nuclear fragments.
other laboratory Findings
bone Marrow If CBC results suggest megaloblastic anemia, further testing is
If physical examination, patient history, and peripheral blood necessary to distinguish the cause. Although no major medical orga-
findings suggest megaloblastic anemia, a bone marrow exami- nization has published guidelines for reflex testing, the most common
nation can help establish a definitive diagnosis. In megaloblas- next step is to measure serum cobalamin and serum or red cell folate.
tic states, the bone marrow is hypercellular with megaloblastic Laboratories use different methods (chemiluminescence, radioassay)
erythroid precursors and a decreased M:E ratio. In a long-standing to measure cobalamin, so there is no “gold standard” to use as a ref-
anemia, red marrow can expand into the long bones. About half erence interval. Generally values 6150 pg/mL are consistent with
the erythroid precursors typically show megaloblastic changes. cobalamin deficiency, whereas levels 7400 pg/mL suggest adequate
Megaloblasts are large nucleated erythroid precursors that display cobalamin. Borderline levels (150–400 pg/mL) can be associated
nuclear-cytoplasmic asynchrony with nuclear maturation lagging with cobalamin deficiency.
M15_MCKE6011_03_SE_C15.indd 282 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 283
a b
■ Figure 15-4 (a) Basophilic and orthochromatic megaloblasts in the bone marrow from a
patient with pernicious anemia. Note the large size of the cells, the open chromatin network in
the nuclei, and the presence of Howell-Jolly bodies in the orthochromatic megaloblasts. Note
the nuclear-cytoplasmic asynchrony. There is a large band neutrophil and a segmented neutro-
phil with at least five nuclear lobes. (Bone marrow; Wright-Giemsa stain; 1000* magnification).
(b) Nuclear-cytoplasmic asynchrony in the bone marrow. Note the large size of the cells (bone
marrow; Wright-Giemsa stain; 1000* magnification).
Measurement of erythrocyte folate is not influenced as much by The large degree of ineffective erythropoiesis results in
recent dietary changes as is serum folate and gives an accurate estimate hemolysis in the marrow and an increase in plasma iron turnover,
of the average folate levels over the preceding several months. 14,15 On serum iron, indirect bilirubin, and urobilinogen. The characteristic
the other hand, if there is a cobalamin deficiency, folate will leak out of marked increase in fractions 1 and 2 of serum lactic dehydrogenase
the cells, which will give a false low red cell folate and false increased (LD) is partially caused by the destruction of megaloblasts rich in
serum folate. In addition, red cell folate is measured by folate-binding LD. The increase is roughly proportional to the degree of anemia.
protein assays that rely on chemiluminescence methodology. These Haptoglobin, uric acid, and alkaline phosphatase are decreased.
methods show considerable analytic variability. Therefore, the less Additional tests are discussed in the following sections.
expensive serum folate measurement is preferred for initial testing.
If serum folate is 74 ng/mL, folate deficiency can be ruled out. CheCkpoint 15-4
Early megaloblastic changes can be detected by testing for
methylmalonic acid (MMA) and homocysteine levels in the blood. What abnormal morphological findings on a stained
Tests for these metabolites are intermediates in folate and cobala- blood smear compose the triad in megaloblastic anemia?
min metabolism and are elevated early in functional vitamin defi-
ciencies. Tests for these metabolites are more sensitive than serum
cobalamin levels and increase earlier than a drop in the cobalamin Folate
level. By performing tests for both MMA and homocysteine, it is A folate deficiency must be considered in the differential diagnosis of
possible to differentiate cobalamin deficiency from folate deficiency. macrocytosis associated with megaloblastic anemia.
Homocysteine is elevated in folate deficiency, whereas MMA is usually
normal. On the other hand, both homocysteine and MMA are elevated Structure and Function
in cobalamin deficiency. An increase in both MMA and homocysteine Folic acid is the parent substance of a large group of compounds known
is also found in combined cobalamin and folate deficiencies. In these as folates. Chemically, folic acid is known as pteroylmonoglutamate
cases, clinical information is important to help establish a differential (PteGlu). Structurally, folic acid is composed of three parts: (1) pteridine,
diagnosis. A block in the metabolism of histidine to glutamic acid a nitrogen-containing ring, (2) a ring of p-amino-benzoic acid, and
occurs in folic acid deficiency and causes increased urinary excretion (3) a glutamic acid residue (Figure 15-6 ■). The term folic acid refers to
of formiminoglutamic acid (FIGLU), an intermediate metabolite, this base structure (inert form) and to the commercially available syn-
after the administration of histidine. These metabolites return to thetic form used for the fortification of food supplies and for dietary
normal levels when the appropriate vitamin is given to the patient. supplements. Folic acid itself does not occur naturally. Folates can vary
It is recommended that clinicians first use the lower cost tests of in their oxidation status, their one-carbon substituent unit, and in their
serum cobalamin and serum folate to diagnose cobalamin and folate number of glutamic acid residues. Tetrahydrofolate (THF or FH ), the
4
deficiencies and use the higher cost MMA and homocysteine tests if active form, is produced by a four-hydrogen reduction of the pteridine
16
cobalamin and folate test results are not definitive (Figure 15-5 ■). ring. Dihydrofolate (DHF) is an intermediate in this reaction.
M15_MCKE6011_03_SE_C15.indd 283 01/08/14 1:38 pm
284 SECTION III • ThE ANEmIAS
Megaloblastic hematologic changes
and/or neurologic abnormalities
Cobalamin assay
<150 pg/mL 150–400 pg/mL >400 pg/mL
Serum MMA assay
Serum RBC or serum
folate assay
Homocysteine assay
Both MMA & MMA elevated, Both MMA & MMA normal,
homocysteine homocysteine homocysteine homocysteine Decreased Normal
are elevated normal are normal elevated
Consider further evaluation Consider further evaluation
Cobalamin deficiency Folate deficiency
with bone marrow with bone marrow
■ Figure 15-5 Algorithm that can be used to determine the cause of megaloblastic anemia
using laboratory tests. Analysis can begin with serum cobalamin assay. If results are in the low
normal range (150–400 pg/mL) or the patient has unexplained neurologic symptoms, mea-
surement of cobalamin metabolic intermediates, methylmalonic acid (MMA), and homocyste-
ine is suggested. If both are elevated, cobalamin deficiency is considered. If MMA is normal
and homocysteine is elevated, folate deficiency is probable. RBC or serum folate also can be
measured, especially if cobalamin is within the reference interval. If these test results conflict
with the clinical diagnosis, therapeutic trials with cobalamin can be used. Bone marrow is
rarely performed for diagnostic purposes but can be necessary in some cases. The bone mar-
row shows megaloblastic features in both folate and cobalamin deficiencies.
The function of THF is to transfer one-carbon compounds from cobalamin blocks the synthesis of the nucleotide dTTP. Folates also
10
donor molecules to acceptor molecules in intermediary metabo- transport formate (as N @formyl THF) for purine synthesis.
lism. In this capacity, folate serves a vital role in the metabolism of 2. The metabolism of histidine to glutamic acid requires THF
nucleotides and amino acids: (Figure 15-7b ■, Table 15-3). The intermediate metabolite of this
1. The carbon transfer reaction involved in the de novo synthesis of reaction is formiminoglutamic acid (FIGLU), which requires THF
DNA is initiated when the carbon side chain of serine is transferred for conversion to glutamic acid. A deficiency of folate blocks this
10
5
to THF to form N , N @methylene THF (Figure 15-7a ■; reaction, resulting in an increase in FIGLU excretion.
Table 15-3 ★). This carbon is then transferred to deoxyuridilate 3. The synthesis of methionine from homocysteine requires the
5
(dUMP) to form deoxythymidylate (dTMP), a pyrimidine of DNA. donation of a methyl group from N @methyl THF and the action
In this reaction, THF is oxidized to DHF. The DHF is reduced back of cobalamin as a cofactor, resulting in methionine and THF
5
10
to THF by DHF reductase and then to N , N @methylene THF (Figure 15-7a). A deficiency of either folate or cobalamin blocks
by the serine reaction discussed earlier. A deficiency of folate or this reaction, resulting in an increase in homocysteine levels.
N O
N 4 5
3 6 CH 2 NH C NH CH CH 2 CH 2 COOH
9 10
2 7 COOH
1 8
NH 2 N N
Pteridine p-Amino-benzoic acid Glutamic acid
■ Figure 15-6 Molecular structure of the folic acid molecule.
M15_MCKE6011_03_SE_C15.indd 284 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 285
★ table 15-3 Metabolic Reactions Requiring Folic Acid Coenzymes
System reaction
5
10
Serine 4 Glycine Ser + ThF 4 N , N @methylene ThF + Gly
10
Thymidylate synthesis dUMP + N ,N @methylene ThF S DhF + dTMP
5
5
histidine catabolism Formininoglutamate + ThF S N @formimino ThF + glutamic acid
5
Methionine synthesis homocysteine + N @methyl ThF S ThF + Methionine
Metabolism glycosyl-phosphatidylinositol [GPI]-anchored receptor; Chapter 17).
Folate is present in food and is synthesized by microorganisms. This receptor is upregulated through transcriptional, translational,
Most folate in food is in the conjugated polyglutamate form. It is and post-translational mechanisms when intracellular and extracel-
deconjugated in the intestine to a monoglutamate prior to absorp- lular folate is decreased. Translational receptor upregulation involves
tion. Absorption can take place throughout the small intestine but the covalent binding of accumulated homocysteine (which occurs
is especially significant in the proximal jejunum. Once taken up by when there is a folate deficiency) with a protein called heterogeneous
5
the intestinal epithelial cell, the folate is reduced to N @methyl THF, nuclear ribonucleoprotein-E1 (hnRNP-E1). 17
5
5
the primary circulating form of THF in the blood. N @methyl THF Once inside the cell, N @methyl THF must be demethylated and
is distributed throughout the body via the blood and attaches to cells reconjugated by the addition of seven or eight glutamic acid residues
by means of specific receptors called cell surface folate receptor@a (a to keep it from leaking out again. Demethylation is a reaction that
Deconjugation, reduction, methylation
5
a Dietary folate N -methyl THF
Homocysteine
Cobalamin (MeCbl)
Methionine synthase
Methionine SAM
Tetrahydrofolate (THF)
Serine
NADPH + H +
Dihydrofolate reductase
Glycine Serine hydroxymethyl
transferase
PLP (Vitamin B 6 )
5
10
N ,N -methylene THF Dihydrofolate (DHF)
dUMP dTMP dTTP DNA
THF
5
b Histidine Urocanic acid Formiminoglutamic acid (FIGLU) Glutamic acid + N -formimino-THF
Methylmalonic acid Hydrolase Cobalamin (AdoCbl)
c Homocysteine Propionyl CoA Methylmalonyl CoA Succinyl CoA
(MMA) Mutase
■ Figure 15-7 Biochemical reactions using folic acid, (adenosyl-cobalamin) in the conversion of methylmalonic acid
cobalamin, and derivatives. (a) The role of folate and (MMA) to succinyl CoA. With deficiencies of cobalamin and/or
cobalamin in the synthesis of DNA. A deficiency of meth- folate, the intermediates MMA, homocysteine, and FIGLU are
ylcobalamin causes a failure of methylation of homocys- trapped and increase in concentration. Tests to detect cobalamin
teine, which leads to a reduction in tetrahydrofolate (THF) deficiency include direct measurement of cobalamin and tests
5
and the trapping of the folate as N @methyl THF. This to detect increased MMA and homocysteine, metabolic inter-
10
5
causes a deficiency of N ,N @methylene THF, a coenzyme mediates that require cobalamin for metabolism. Tests to detect
needed in the synthesis of dTTP, a nucleotide of DNA. folic acid deficiency include direct measurement of folate or
(PLP = pyridoxal@5@phosphate) (b) The role of folate measurement of homocysteine and FIGLU, intermediates that
in the catabolism of histidine. (c) The role of vitamin B 12 require folate for metabolism.
M15_MCKE6011_03_SE_C15.indd 285 01/08/14 1:38 pm
286 SECTION III • ThE ANEmIAS
requires cobalamin and methionine synthase (Figure 15-7a). Thus, a the inability to convert deoxyuridine monophosphate (dUMP) to
deficiency of cobalamin traps folate in its methylated form and blocks deoxythymidine monophosphate (dTMP), the precursor of dTTP.
the formation of other forms of THF. This is commonly referred to as Subsequently, the dUMP is phosphorylated to the triphosphate form
the folate trap or methyl trap. Although free THF is easily conjugated (dUTP). DNA polymerase does not effectively distinguish dUTP from
within cells, methyl-THF is not; consequently, much of the methyl- dTTP, and dUTP is erroneously incorporated into the DNA of folate-
THF taken up by a cobalamin-deficient cell leaks out before addi- deficient cells. The DNA “proof-reading” function of the cells recog-
14
tional glutamates can be added. The cells in cobalamin deficiency nizes the mistake and tries to repair the DNA by replacing uridine
are unable to retain their folate, leading to tissue folate depletion. In with thymidine, but the repair attempt fails due to the lack of available
5
the demethylation of N @methyl THF by cobalamin, homocysteine dTTP. The result is ultimately DNA fragmentation and cell death by
14
is methylated to methionine, a precursor of S-adenosylmethionine apoptosis. All rapidly dividing cells—including erythrocyte, leuko-
(SAM) (Table 15-3). This reaction requires methionine synthase and cyte, and platelet precursors and intestinal epithelium—are affected by
cobalamin. SAM is thought to be critical to nervous system function. a folate deficiency. Surviving hematopoietic cells show characteristic
megaloblastic changes.
requirements The bone marrow can exhibit a three-fold increase in erythro-
Folate is present in most foods including eggs, milk, yeast, mushrooms, poiesis, but the peripheral blood reticulocyte count is low, indicating
and liver but is especially abundant in green leafy vegetables (from a large degree of ineffective erythropoiesis. Increased apoptosis of the
which it gets its name). It is also synthesized by microorganisms. The blood cell precursors leads to increased heme catabolism and iron
vitamin is destroyed by heat; thus, when food is overcooked, much of turnover, signs of hemolysis, jaundice, and pancytopenia. Extramed-
the folate is destroyed. Ascorbate protects folate from oxidation and, ullary hemolysis also occurs, and circulating red cell survival can be
when present, can protect folate to some extent from heat degrada- decreased by 30–50%. The compounds requiring folic acid as a cofac-
tion. The recommended daily dietary allowance of food folic acid tor for metabolism (homocysteine and FIGLU) accumulate. Mildly
for adults is 400 mcg (mg), of which about 50–80% is absorbed in elevated levels of homocysteine are considered a major risk factor for
the intestine. This is adequate to provide the minimum daily require- atherosclerosis and venous thrombosis (Chapter 35).
ment of 50 mcg/day needed to sustain normal metabolism. The liver The clinical findings of folate deficiency develop sequentially.
stores from 5 to 10 mg of folate, which is sufficient to provide the daily Serum folate decreases within 1 to 2 weeks of onset of a folate deficiency.
requirement for three to six months if folate is omitted from the diet. Hypersegmented neutrophils are the first morphologic change and
Folate plays an important role in normal embryogenesis. The occur at about 2 weeks. The urinary excretion of FIGLU increases next
folate receptor@a is activated early in embryonic stem cells and at about 13 weeks, and anemia appears last at about 19–20 weeks.
increases as the need for folate increases. Observations have revealed
a high incidence of low folate levels in women who give birth to causes of Folate deficiency
babies with neural tube defects (NTD) compared with women who Folate deficiency can occur as the result of an inadequate dietary
give birth to normal babies. Experimental studies suggest perturba- intake, an increased requirement, malabsorption in the small intestine,
tions in folate receptor@a may be involved in these neural tube closure or drug inhibition (Table 15-4 ★).
17
abnormalities. Several studies have shown that supplementation of
folate during pregnancy can reduce the rate of occurrence of these inadequate diet
birth defects by as much as one-half. In an effort to reduce NTDs, the The most common cause of folate deficiency is an inadequate dietary
U.S. government required the fortifications of grain products with intake of folic acid. This is seen most often in poor and elderly people
18
folic acid beginning in the fall of 1997. The goal was to increase the who fail to obtain enough of the appropriate foods to maintain ade-
daily dietary folate to 100 mcg per person. It has been suggested that quate folic acid intake. People who are alcoholics whose diet consists
pregnant women need an intake of about 800 mcg/day. mainly of large quantities of ethanol have a deficiency of many vita-
Folate can become depleted quickly in conditions with rapid mins in addition to folic acid. Complicating the folate deficiency in
cell turnover such as sickle cell anemia and other hemolytic anemias,
during growth, pregnancy, and lactation. Thus, the daily requirement ★ table 15-4 Causes of Folate Deficiency
in patients with these conditions is increased.
cause examples
CheCkpoint 15-5 Inadequate diet Low income, elderly with limited function/
income, alcoholics
hal had small bowel resection due to carcinoma. Explain Increased requirement Diseases/conditions associated with rapid cell
why he is at high risk for folate deficiency. turnover (sickle cell anemia, thalassemia, leuke-
mias, other malignancies, pregnancy, infancy)
Malabsorption Ileitis, tropical sprue, nontropical sprue,
blind loop syndrome
Pathophysiology of Folate deficiency
5
10
Folate deficiency results in decreased synthesis of N , N @methylene Drug inhibition Oral contraceptives, long-term anticoagu-
lant therapy, phenobarbital, primidone,
THF, which is needed as a cofactor in DNA synthesis. Consequently, phenytoin, antimetabolite chemotherapy
there is a marked slowing of DNA synthesis, and the S phase of the Biologic competition Bacterial overgrowth in small intestine
cell cycle is prolonged. The impairment of DNA synthesis is due to
M15_MCKE6011_03_SE_C15.indd 286 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 287
alcoholic individuals, the ethanol appears to impair release of folate ★ table 15-5 Causes of False Increases and
from the liver and can be toxic to erythroid precursors. Erythroid Decreases in Folate Levels
precursors in alcoholism are frequently vacuolated. In alcoholic indi-
viduals who have liver disease but have an adequate diet, the anemia erythrocyte Folate Serum Folate
is macrocytic but not megaloblastic. False increase Early folate deficiency Recent increase in dietary
folate
increased requirement
In individuals with increased cell replication, the normal daily Reticulocytosis hemolysis of blood sample
intake of folic acid may not be sufficient to maintain normal DNA Recent RBC Coexisting cobalamin
deficiency
transfusion
synthesis. Without folate supplements, folate stores can be rapidly False decrease Cobalamin deficiency Recent low dietary intake
depleted. This occurs in hemolytic anemias such as sickle cell anemia Recent alcohol Gallium or technetium
and thalassemia, in myeloproliferative diseases such as leukemia, and consumption administration
in metastatic cancers. Anemia in pregnancy is common and can be
caused by deficiencies of iron and/or folic acid. The deficiency of folic
acid is related to the limited reserves of this nutrient and a 5–10-fold in cobalamin deficiency due to folate trapping as methyl-THF. Epithelial
increased demand for its use created by the growing fetus. Prophylactic changes in the GI tract that accompany cobalamin deficiency can lead
folic acid supplements are usually prescribed during pregnancy. 19 to malabsorption of folic acid in which case both serum and erythrocyte
Malabsorption folate levels are decreased.
Intestinal diseases affecting the upper small intestine, which interfere Competitive folate-binding assays (indirect immunoassays)
with the absorption of nutrients, can cause a folate deficiency. The using chemiluminescence methods have replaced microbiological
most common conditions of this type include ileitis, tropical sprue, assays for both serum and RBC folate.
and nontropical sprue. The blind loop syndrome associated with an
overgrowth of bacteria can cause a folate deficiency because the bac- Cobalamin (Vitamin B )
12
teria preferentially utilize the folate. Cobalamin is required for DNA synthesis and neurologic function.
drug inhibition Specific diagnosis of cobalamin deficiency is critical because associ-
Megaloblastic anemia has also been associated with certain drugs ated neurologic damage can be irreversible. Pernicious anemia is a
including oral contraceptives, long-term anticoagulant drugs, phe- specific form of cobalamin deficiency and will be discussed in detail
nobarbital, primidone, and phenytoin. Anemia occasionally is not later in this chapter.
present even though serum and erythrocyte folate are depressed.
Structure and Function
CheCkpoint 15-6 Vitamin B is a commonly used generic term for a family of cobala-
12
min vitamins in which ligands can be chelated to cobalt. The more
What is the most common cause of folate deficiency, and accurate terminology when referring to this family of vitamins is
in what groups of individuals is it usually found? cobalamin. Vitamin B refers specifically to the therapeutic form
12
of crystalline cobalamin that contains the ligand cyanide (cyanoco-
balamin), a form not naturally found in the body but used for treating
laboratory analysis of Folate deficiency cobalamin deficiency. In hematology literature, the terms cobalamin
Both serum and erythrocyte folate levels are decreased in folate defi- and vitamin B are often used interchangably. In this text we will use
12
ciency. Serum folate reflects the folic acid intake over the last several the term cobalamin.
days, whereas erythrocyte folate reflects the folate available when the Cobalamin is structurally classified as a corrinoid, a family of
red cell was maturing in the bone marrow and reflects the net folate compounds with a corrin ring. The molecule has three portions:
level over the preceding several months. Serum folate can be falsely (1) a corrin ring composed of four reduced pyrrole groups with a
increased with even slight hemolysis of the sample. Low serum folate cobalt at the center (cobalamin gets it name from the central cobalt),
can indicate an imminent folic acid deficiency and precedes erythro- (2) a nucleotide that lies almost perpendicular to the ring attached to
cyte folate deficiency. 15 the cobalt, and (3) various ligands (a b@group) attached to the cobalt
Care must be taken in interpreting folate results because both on the opposite side of the ring from the nucleotide (Figure 15-8 ■).
serum and erythrocyte folate can be falsely increased or decreased in a The b@group in cobalamin is cyanide, methyl, adenosyl, or hydroxyl.
variety of conditions (Table 15-5 ★). Neither serum (methyl-THF) nor Adenosylcobalamin (AdoCbl) and methylcobalamin (MeCbl) act
erythrocyte folate (heterogeneous folate mixture with various polyglu- as coenzymes in biological reactions. Hydroxycobalamin (OHCbl)
tamate chain lengths) is a good indicator of folate stores if there is a and cyanocobalamin (CnCbl) are not metabolically active forms of
deficiency of cobalamin. A cobalamin deficiency leads to the accumula- cobalamin but can be converted to the active methyl and adenosyl
tion of methyl-THF because the donation of the methyl group to homo- forms by tissue enzymes.
cysteine and the release of free THF is impaired. In addition, cobalamin As mentioned previously, methionine is formed from homo-
5
is required for normal transfer of methyl-THF to the cells and for keep- cysteine when a methyl group is transferred from N @methyl THF
ing the folate in the cell as conjugated folate. Thus, serum folate can be to homocysteine in a cobalamin-dependent reaction catalyzed by
falsely increased (by 20–30%) and erythrocyte folate falsely decreased methionine synthase (Figure 15-7a). For this reaction, the vitamin
M15_MCKE6011_03_SE_C15.indd 287 01/08/14 1:38 pm
288 SECTION III • ThE ANEmIAS
–
–
CH , –CH , CH , or deoxyadenosyl HC, releasing cobalamin. The released cobalamin quickly binds to
3
intrinsic factor (IF), which resists pancreatic degradation. Intrinsic
factor is a glycoprotein secreted by parietal cells of the gastric mucosa
NH OCH CH 2 CH 3 CH 3 CH OCNH in response to the presence of food, vagal stimulation, histamine, and
2
2
NH COCH 2 C 2 2 gastrin. IF binds cobalamin with a 1:1 stoichiometry and is required
2
CH CH CONH 2 for the intestinal absorption of cobalamin. The IF–cobalamin com-
2
2
CH 3 N N plex resists digestion and passes through the jejunum into the ileum
CH 3 Co CH where it binds the specific IF receptor (cubulin) on the microvilli of
N N ileal mucosal cells (Figure 15-9 ■). Binding requires a pH of 5.4 or
14
CH 3
higher as well as calcium. Following attachment of the IF–cobalamin
C CH 3 complex to cubulin, the entire complex is taken into the mucosal cell by
OC CH CH 2 CH 3 CH 3 CH 2 CH CONH 2 endocytosis. Cobalamin is released from IF and enters the portal blood
2
2
14
while IF is degraded. The IF-receptors are recycled to the microvilli of
the ileum where they participate in absorption of more IF–cobalamin
Nucleotide complexes. Large oral doses of cobalamin can be absorbed by simple
(dimethylbenzimidazole derivative) diffusion without IF (see the section on “Therapy” ).
■ Figure 15-8 Molecular structure of the cobalamin transport
molecule.
Proteins that transport cobalamin in the blood are transcobalamin
(previously known as transcobalamin II) and haptocorrins
must be in the MeCbl form. A cobalamin deficiency traps newly (previously known as transcobalamin I and transcobalamin III or
5
acquired folate in the N @methyl THF form (the form in which the R-proteins). These plasma haptocorrins belong to the same family of
5
cell acquires folate from the plasma). N @methyl THF is not efficiently proteins as the gastric haptocorrin.
conjugated with glutamic acids and thus leaks back out of cells (see the Transcobalamin (TC) is produced in many types of cells, includ-
discussion of folate metabolism). The result is an intracellular func- ing hepatocytes, enterocytes, macrophages, and hematopoietic precur-
tional deficiency of folate and a block of DNA synthesis. sors in the bone marrow. Although TC carries only a small fraction of
AdoCbl is required for only one mammalian reaction: the conver- the total cobalamin in the plasma, it binds 90% of the newly absorbed
sion of methylmalonyl CoA to succinyl CoA (Figure 15-7c). AdoCbl cobalamin and is the primary plasma protein that mediates transfer of
acts as a coenzyme with methylmalonyl CoA mutase in this reaction. cobalamin into the tissues. TC also binds corrins that are chemically
Increased urinary excretion of methylmalonic acid (MMA), a precursor similar to cobalamin but have no known function in mammalians
of methylmalonyl-CoA, is a diagnostic aid in cobalamin deficiency. (cobalamin analogues). The TC–cobalamin complex is thought to be
14
formed within the ileal mucosal cells and released to the blood. This
transport complex disappears rapidly from blood (T of 6–9 minutes)
l/2
CheCkpoint 15-7 as it is taken up by cells in the liver, bone marrow, and other dividing
A patient has the following results: vitamin B 12 50 pg/mL, cells that have specific receptors for TC (TCblR, CD320). The TC–
serum folate 4 ng/dL, RBC folate 100 ng/mL. Interpret cobalamin complex is internalized by receptor-mediated endocytosis.
these results. Once inside the cell, cobalamin is released from TC and utilized, and
the TC is degraded. Congenital deficiency of TC produces a severe
megaloblastic anemia in infancy. However, serum cobalamin concen-
tration in this condition is normal.
Metabolism The functions of the haptocorrins, a group of immunologically
Defects in any of the steps of cobalamin metabolism can lead to a related proteins, are less well understood. They are synthesized by
cobalamin deficiency and megaloblastic anemia. The laboratory plays the mucosal cells of the organs that secrete them and by phagocytes.
a critical role in both assessing the level of the vitamin and determin- They are found in body fluids including plasma, saliva, amniotic fluid,
ing the cause of low levels. Thus, it is important to understand the milk, and gastric juice. Haptocorrins (HC) bind 70–90% of circulat-
metabolism of this important nutrient. ing cobalamin forming the cobalamin–HC complex. Cobalamins
absorption undergo a significant amount of enterohepatic recirculation. The
Cobalamin is present in most foods of animal origin including milk, cobalamin–HC complex attaches to an asialoglycoprotein receptor
eggs, and meat. The vitamin complex is released from food by peptic on hepatocytes. Clearance of HC from plasma is much slower (T
1/2
digestion at the low pH in the stomach and binds tightly to a hapto- 9–10 days) than that of TC. Within the hepatocyte HC is degraded
corrin (HC)-like protein, a cobalamin binding protein secreted in the and cobalamin is excreted into the bile where it binds to haptocorrin.
saliva and in the stomach. This binding protein was previously known This HC– cobalamin complex enters the intestine where the cobala-
as R-protein, named because of its rapid electrophoretic mobility min is released from HC by pancreatic proteases and bound by IF in
compared with the mobility of intrinsic factor. HC is the preferred the same manner as dietary cobalamin.
binding protein for cobalamin released from food. The HC–cobalamin HC binds ligands less specifically and more tightly than TC.
complex protects the cobalamin from degradation by the hydrochloric It binds a wide variety of corrinoids in addition to cobalamin.
acid in the stomach. In the duodenum, pancreatic proteases degrade These analogues are transported by HC to the liver, metabolized,
M15_MCKE6011_03_SE_C15.indd 288 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 289
Cbl food
complex
IF Stomach (secretes HC and IF)
HC Cbl
HC Cbl
Duodenum HC
(haptocorrin degraded)
Cbl
Cbl IF Cbl IF
Cbl IF
Cbl IF
Ileum receptors IF
Cbl IF on cells Cbl
Cbl TC Cbl TC
leaves the
cell to enter
the plasma
Mucosal lining
cells
■ Figure 15-9 Assimilation of cobalamin.
HC = haptocorrin; IF = intrinsic factor; TC = transcobalamin; Cbl = Cobalamin
and excreted in the bile. The analogues bind poorly to IF and therefore Pathophysiology of cobalamin deficiency
are not absorbed but are excreted in the feces. Thus, HC can serve to Deficiency of cobalamin is reflected by (1) impaired DNA synthesis
clear the body of nonphysiologic cobalamin analogues. (megaloblastic anemia) and (2) defective fatty acid degradation (neu-
In plasma, HC is largely produced by granulocytes. Haptocor- rologic symptoms).
rins are increased in myeloproliferative disorders, presumably due to impaired dna Synthesis
excess proliferation of granulocytes.
A deficiency in either cobalamin or folic acid results in impaired
production of methylene-THF, a defect in thymidylate synthesis, and
requirements ultimately a defect in DNA synthesis (Figure 15-7a). This produces
About 3–5 mcg of cobalamin per day is needed to maintain normal megaloblastic anemia and epithelial cell abnormalities. All dividing
biochemical functions. It is estimated that only about 70% of cobalamin cells including the hematopoietic cells in the bone marrow are affected.
intake is absorbed, which suggests that the diet should include 5–7 mcg defective Fatty acid degradation
of the vitamin per day. This amount is available in a regular “balanced” AdoCbl is a cofactor in the conversion of methylmalonyl CoA to suc-
diet but not in a strict vegetarian diet. Cobalamin stores ( 5000 mcg) cinyl CoA. In cobalamin deficiency, there is a defect in degradation of
are sufficient to provide the normal daily requirement for about propionyl CoA to methylmalonyl CoA and, finally, to succinyl CoA.
1000 days. Therefore, it takes several years to develop a deficiency if no As propionyl CoA accumulates, it is used as a primer for fatty acid syn-
cobalamin is absorbed from the diet. About half of the vitamin is stored thesis, replacing the usual primer acetyl CoA. This results in fatty acids
in the liver, and the rest is located in the heart and kidneys. 20
with an odd number of carbons. These odd-chain fatty acids are incor-
porated into neuronal membranes, causing disruption of membrane
CheCkpoint 15-8 function. It is probable that demyelination (destruction, removal, or
loss of the lipid substance that forms a myelin sheath around the axons
Explain why there is a megaloblastic anemia in transco- of nerve fibers), a characteristic finding in cobalamin deficiency, is a
balamin deficiency when the serum vitamin B 12 concentra- result of this erroneous fatty acid synthesis.
tion is normal. A critical feature of demyelination in cobalamin deficiency
is neurological disease. Peripheral nerves are most often affected,
M15_MCKE6011_03_SE_C15.indd 289 01/08/14 1:38 pm
290 SECTION III • ThE ANEmIAS
presenting initially as motor and sensory neuropathy. The brain and ★ table 15-6 Causes of Cobalamin Deficiency
spinal cord can also be affected leading to dementia, spastic paraly- and Associated Conditions
sis, and other serious neurological disturbances. Neurologic damage
has been known to occur occasionally without any sign of anemia or cause associated conditions
21
macrocytosis, making accurate diagnosis difficult but critical. The Malabsorption Pernicious anemia (lack of IF)
bone marrow, however, always shows megaloblastic hematopoiesis. Gastrectomy or gastric bypass
Neurological disease might not be totally reversible but, if treated early, Crohn’s disease
can be partially resolved. Neurological disease does not occur in folate Tropical sprue
deficiency. Administration of synthetic folic acid corrects the anemia Celiac disease
of cobalamin deficiency but does not halt or reverse neurological Surgical resection of the ileum
disease because synthetic folic acid, unlike dietary folate, is reduced Imerslund-Grääsbeck disease
directly to THF without the requirement of cobalamin as a cofactor. Pancreatic insufficiency
The THF can correct the megaloblastosis, but because there is a bypass Drugs (colchicine, neomycin, p-aminosalicylic
of the cobalamin-dependent reaction of conversion of homocyste- acid, or omeprazole)
ine to methionine, SAM—a metabolite considered critical to nervous Blind loop syndrome
system function—is not formed. Thus, it is essential to differentiate Diverticulitis
folate deficiency and cobalamin deficiency so that appropriate treat- Biologic competition Intestinal parasite (i.e., Diphyllobothrium
ment can be given. latum)
Gastritis and abnormalities of the gastrointestinal epithelium Leishmaniasis
secondary to cobalamin deficiency can interfere with the absorption Bacterial overgrowth
of folic acid and iron, complicating the anemia. Nutritional deficiency Strict vegetarian diets
Pregnant women on a poor diet
Malnutrition
CheCkpoint 15-9 Impaired utilization Transcobalamin deficiency
Explain why severe cobalamin deficiency sometimes Nitrous oxide inhalation
presents with neurological disease.
against intrinsic factor are found in 75% of PA patients and are
highly specific for PA. These IF antibodies are of two types: blocking
causes of cobalamin deficiency and binding. Type I, or blocking antibodies, are antibodies to IF and
Cobalamin deficiency has many causes, including lack of intrinsic prevent formation of the IF–cobalamin complex. Type II, or binding
factor (pernicious anemia), malabsorption, nutritional deficiency, antibodies, are directed against the IF–cobalamin complex and pre-
and impaired utilization by tissues due to defective or absent transport vent the IF–cobalamin complex from binding to ileal receptors. Bind-
proteins or enzymes (Table 15-6 ★). ing antibodies are found in about half the sera that contain blocking
Pernicious anemia antibodies. A number of findings suggest that the immune destruction
Pernicious anemia (PA) is a specific term used to define the megalo- of the gastric mucosa is not antibody mediated but more likely T-cell
blastic anemia caused by an absence of IF secondary to gastric atrophy. mediated. Patients with agammaglobulinemia have a higher than
An absence of IF leads to cobalamin deficiency because the vitamin expected incidence of PA. Also, lymphocytes from PA patients have
cannot be absorbed in its absence. PA is the most common cause of been shown to be hyperresponsive to gastric antigens. 14
cobalamin deficiency, accounting for 85% of all deficiencies. Atrophy of Pernicious anemia frequently occurs with other autoimmune
gastric parietal cells is demonstrated by finding achlorhydria of gastric diseases such as Graves disease and Hashimoto’s thyroiditis, type I
juice after histamine stimulation (these cells produce HCl as well as IF). diabetes, and Addison’s disease. In addition, a predisposition to PA can
PA is generally a disease of older adults, usually occurring after 40 years be inherited. Relatives of patients with PA have a higher incidence of
of age. This anemia is seen more commonly among people of Northern antiparietal cell antibodies and anti-intrinsic factor antibodies than
European background, especially Great Britain and Scandinavia, but can the general population, even in the absence of overt PA. 14
be found in all ethnic groups. More women than men are affected, and Juvenile pernicious anemia is rare in children. It can occur sec-
some patients have prematurely graying or whitening hair. Although no ondary to a variety of conditions, including a congenital deficiency
particular genetic abnormality has been identified, a positive family his- or abnormality of IF (the more common type, with lack of IF but
tory of PA increases the risk of developing it by 20-fold. The incidence otherwise normal gastric secretion), or more rarely, true PA of child-
of gastric carcinoma in patients with PA is increased. 22,23 hood. True PA of childhood has an absence of intrinsic factor, gastric
PA is an autoimmune disease. The gastric atrophy is thought to atrophy, decreased gastric secretion, and antibodies against IF and
result from immune destruction of the acid-secreting portion of the parietal cells. Megaloblastic anemia in childhood from malabsorp-
14
gastric mucosa. Up to 90% of PA patients have antibodies against tion of cobalamin can also be due to a congenital deficiency of TC, a
24
parietal cells. However, these antibodies are not specific for perni- congenital gastric haptocorrin deficiency, or selective malabsorption
cious anemia and are also found in patients with gastritis, thyroid of cobalamin (Imerslund-Gräsbeck disease). The latter can be due to
disease, and Addison’s disease. On the other hand, serum antibodies abnormal cubulin receptors in the ileum.
M15_MCKE6011_03_SE_C15.indd 290 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 291
other causes of Malabsorption ★ table 15-7 Causes of False Decrease and False
Pernicious anemia is only one specific cause of cobalamin malabsorp- Normal/Increase in Serum Cobalamin Levels
tion, which also can be caused by a loss of IF secondary to gastrectomy
or secondary to diseases that prevent binding of the IF–cobalamin False decrease False normal/increase
complex in the ileum. An iron deficiency usually precedes cobalamin Folate deficiency Cobalamin treatment
deficiency in patients who have had a gastrectomy. Diseases that can Pregnancy (last trimester) Liver disease
affect the absorption of the IF–cobalamin complex in the ileum include Oral contraceptive use Nitrous oxide exposure
Crohn’s disease, tropical sprue, celiac disease, and surgical resection of Multiple myeloma Chronic myelogenous leukemia
the ileum. In Imerslund-Gräsbeck disease, the IF receptors (cubulin) Elderly Polycythemia vera
are missing or abnormal, causing a form of juvenile megaloblastic haptocorrin deficiency Transcobalamin deficiency
anemia. Patients with severe pancreatic insufficiency experience a lack
of absorption of cobalamin because the vitamin cannot be released
from HC and transferred to IF. Normally, pancreatic proteases are studies have shown that serum cobalamin does not always detect
responsible for degrading HC. Certain medications can interfere with a cobalamin deficiency, and further testing could be necessary if a
intestinal absorption. In addition, conditions that allow a buildup of deficiency is suspected. Furthermore, serum cobalamin can appear
bacteria in the small bowel can cause a cobalamin deficiency as the falsely decreased or falsely normal/increased in some conditions
bacteria preferentially take up the vitamin before it reaches the ileum. (Table 15-7 ★). Cobalamin deficiency can be masked by folate
This situation occurs in the blind loop syndrome and in diverticuli- therapy.
tis. Infestation with the fish tapeworm Diphyllobothrium latum can A specific test that measures the increased excretion of methyl-
cause a deficiency as the worm accumulates the vitamin avidly. malonic acid (MMA) in the urine indirectly indicates a decrease in
nutritional deficiency cobalamin concentration. Up to 40% of patients may have increased
22
Dietary deficiency of cobalamin is rare in the United States. Food from MMA levels in the urine but normal cobalamin levels. These
animal sources, especially liver, is rich in cobalamin. Strict vegetarian patients, however, show laboratory and clinical evidence of cobalamin
diets, however, do not supply cobalamin and individuals following deficiency. The only condition in which MMA is increased in addition
these diets can develop a deficiency over a period of years. Occa- to cobalamin deficiency is congenital methylmalonic aciduria. This
sionally, pregnant women with a poor diet can develop a deficiency condition is caused by complete or partial deficiencies of enzymes and
presumably due to an increased demand by the developing fetus. cofactors involved in the conversion of methlmalonyl CoA to succinyl
However, folic acid deficiency is a more common cause of megalo- CoA. There is no associated hyperhomocysteinemia or megaloblastic
blastic anemia in pregnancy due to the lower preexisting stores of this anemia. Determination of MMA concentration also is useful in distin-
nutrient. guishing a cobalamin deficiency and folate deficiency.
Homocysteine is increased in the plasma of patients with cobala-
other causes min or folate deficiency. Monitoring serum levels can be an early
26
Transcobalamin deficiency produces megaloblastic anemia because detector of cobalamin deficiency, and recent studies have concluded
it is the major transport protein responsible for delivering the vita- that MMA and homocysteine are the most sensitive and specific indi-
min to the tissues. In TC deficiency, cobalamin is absorbed normally, cators of deficiency. 26–29 Some studies have shown that in addition to
and serum cobalamin levels are normal because of the relatively high being a good indicator of cobalamin deficiency, increased homocys-
amount bound to HC. Tissue cobalamin deficiency, including mega- teine levels are associated with a three-fold increase risk in myocardial
loblastic anemia, develops, however, because the cellular receptors infarction as well as venous thrombosis. Increased concentrations
28
recognize only TC-bound, not the haptocorrin-bound cobalamin. 14 of both analytes can be found in many patients with normal serum
Nitrous oxide, N O (“laughing gas”), abuse has been reported cobalamin concentrations. Some clinicians recommend initial
28
2
to result in a cobalamin deficiency and megaloblastic anemias. N O testing for serum cobalamin levels and following up low or border-
2
rapidly inactivates methionine synthetase, for which cobalamin is a line normal results with MMA and homocysteine measurements.
29
coenzyme. Cobalamin cleaves N O and at the same time is oxidized Others have found that MMA and homocysteine measurements are
2
to an inert form. This leads to a rapid deficiency.
more sensitive tests of early pernicious anemia, preceding hematologic
abnormalities and decreased serum cobalamin levels, and recommend
CheCkpoint 15-10 them as a superior testing regimen (Figure 15-7 ■).
Homocysteine and MMA tests also are helpful in determining a
Why is pernicious anemia considered an autoimmune patient’s response to treatment with cobalamin and folate. The serum
disorder? MMA level remains increased in cobalamin-deficient patients treated
inappropriately with folate. Folate-deficient patients treated inappro-
priately with cobalamin have increased serum homocysteine level. See
laboratory analysis of cobalamin deficiency Table 15-8 ★ for a list of the advantages and disadvantages of labora-
Laboratory diagnosis of pernicious anemia and/or cobalamin defi- tory tests used to diagnose cobalamin deficiency. 6
ciency usually begins with a serum cobalamin assay. If the cobalamin The serum cobalamin and MMA assays establish the existence
level is decreased and there are clinical symptoms of a deficiency, no of a cobalamin deficiency but do not provide a distinction between
25
further testing is warranted before therapy is initiated. However, PA and other causes of cobalamin deficiency. Gastric analysis and the
M15_MCKE6011_03_SE_C15.indd 291 01/08/14 1:38 pm
292 SECTION III • ThE ANEmIAS
★ table 15-8 Tests Used to Diagnose Cobalamin Deficiency a
test advantages/disadvantages
Cobalamin/serum high sensitivity; widely available; variation in reference intervals according to method of
analysis; measures both hC and TC although only holo-TC is available for cells; falsely
decreased in folate and hC deficiency
Methylmalonic acid (MMA)/urine high sensitivity and specificity; increased concentration helps differentiate cobalamin from
folate deficiency; increased level may precede hematologic abnormalities; helpful in deter-
mining patient response to cobalamin treatment; falsely increased in reduced renal func-
tion; not readily available; expensive
homocysteine/plasma high sensitivity; low specificity; increased level may precede hematologic abnormalities;
helpful in determining patient response to cobalamin treatment; does not differentiate
cobalamin deficiency from folate deficiency; falsely increased in reduced renal function,
hypothyroidism, hypovolemia
holo-TC Sensitivity and specificity similar to cobalamin; measure of cobalamin available to tissue;
falsely increased in liver disease and in reduced renal function; complicated interpretation
(see section Laboratory Analysis of Cobalamin Deficiency”); not a readily available test
Complete blood count with differential high sensitivity of macrocytosis with megaloblastic changes; specificity of macrocytosis
without megaloblastic changes is low; not readily recognizable in subclinical cobalamin
deficiency
a Sensitivity and specificity of these tests may be lower in subclinical cobalamin deficiency; subclinical deficiency has no known clinical expression and diagnosis
depends on biochemical markers; recommended diagnostic test combination for cobalamin deficiency is serum cobalamin and MMA. Both should be abnormal
for a diagnosis of cobalamin deficiency.
holo@hC = Transcobalamin II with cobalamin attached
Schilling test are more useful in establishing the specific diagnosis of PA borderline or low and MMA is increased, the diagnosis of PA should
but are rarely performed anymore. A recommended testing algorithm be confirmed with testing for intrinsic factor blocking antibody
30
for laboratory diagnosis of PA is shown in Figure 15-10 ■. The (IFBA) and serum gastrin levels. The serum gastrin test may be used
order of testing can improve the efficiency of proper diagnosis by to replace the Schilling test (see the following discussion). Gastrin is
first measuring serum cobalamin and MMA. If serum cobalamin is usually markedly increased in PA due to gastric atrophy. In contrast
Cobalamin Assay, Serum
>400 ng/L <150 ng/L
150–400 ng/L Intrinsic factor blocking antibody
≤0.4 nmol/mL Methylmalonic acid >0.4 nmol/mL Positive Indeterminate Negative
Result does not imply Result does not imply a
cobalamin deficiency. deficiency of cobalamin Result consistent
No further testing at the cellular level. with pernicious
suggested. No further testing suggested. anemia. Gastrin
>200 pg/mL ≤200 pg/mL
Result consistent Result does not
with pernicious suggest
anemia. pernicious anemia.
■ Figure 15-10 Testing algorithm for the diagnosis of pernicious anemia.
M15_MCKE6011_03_SE_C15.indd 292 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 293
to the Schilling test, the gastrin test does not require radioisotopes and ★ table 15-9 Conditions Associated with Abnormal
is less expensive. Schilling Test Results
Measurement of holo-transcobalamin (holo-TC), the fraction of
plasma cobalamin bound to TC, can be an early marker for cobalamin corrected by intrinsic not corrected by intrinsic Factor
deficiency. Because cellular receptors specifically mediate the uptake of Factor in Part ii in Part ii
holo-TC but not haptocorrin-bound cobalamin (holo-HC), holo-TC Pernicious anemia Crohn’s disease
could provide a more accurate view of patients’ cobalamin status than total Abnormal intrinsic factor Tropical sprue
31
cobalamin levels that include both holo-TC and holo-HC. Low holo- molecule Celiac disease
TC levels, however, may only be temporary or may even remain at the hereditary intrinsic factor Surgical resection of the ileum
subclinical level rather than progress to pathologic levels. The interpreta- deficiency Imerslund-Gräsbeck disease
tion of holo-TC values is complicated by the fact that it is unclear whether Gastrectomy Pancreatic insufficiency
low results reflect insufficient availability to tissues or some impairment in Drugs
32
the ability of the holo-TC complex to leave the ileal mucosal cells. Previ- Blind loop syndrome
ously tested by enzyme-linked immunosorbent and radioimmunoassay Diverticulitis
techniques, holo-TC is now available as an automated monoclonal anti- Intestinal parasite— Diphyllobothrium
body assay with a reference interval of approximately 20–125 pmol/L. 33 latum
gastric analysis Transcobalamin deficiency
Because atrophy of the parietal cells is a universal feature of PA, an
absence of free HCl in gastric juice after histamine stimulation is the results. Incontinence or inability to empty the bladder gives false
indicative of PA. Parietal cells secrete both HCl and intrinsic factor; low values even when absorption was normal. Spuriously low urinary
thus an absence of HCl is indirect evidence for lack of IF. After hista- excretion in part II can also be due to the inability to absorb IF@B
mine stimulation in patients with pernicious anemia, the pH fails to because of megaloblastoid epithelial changes in the gut. 12
fall below 3.5, and gastric volume, pepsin, and rennin are decreased. The Schilling test is seldom used now because of the difficulties
Approximately 80% of cases of PA have increased gastrin. Testing for in using radioisotopes and the inconvenience of the test for the patient.
anti-intrinsic factor antibodies, serum gastrin, and serum pepsinogin An additional problem is that the absorption of crystalline vitamin B
12
A and C together as a group provides a sensitive indication of gastric used in the oral dose can differ from the absorption of cobalamin in
atrophy and pernicious anemia. 33 food. 15,34 For these reasons, MMA and homocysteine are better mark-
Schilling test ers for early detection of PA or cobalamin deficiency.
The Schilling test is a definitive test useful in distinguishing cobalamin On the other hand, the Schilling test has advantages. First, the
deficiency due to malabsorption, dietary deficiency, or absence of IF. The test results can suggest what further testing (e.g., stool analysis for
test measures the amount of an oral dose of radioactively labeled crys- D. latum) should be done. Second, results can help identify how to
35
talline vitamin B that is absorbed in the gut and excreted in the urine. treat the clinical condition and the duration of treatment.
12
57
The patient is given a dose of Co@labeled B orally with or followed
12
within two hours by an intramuscular injection of unlabeled vitamin caSe Study (continued from page 282)
B . The injection is termed the flushing dose; its purpose is to saturate
12
all cobalamin receptors in the tissues. Thus, any of the labeled oral dose A Schilling test and antibody testing had the following
absorbed in the gut and passing into the blood will be in excess of avail- results:
able receptor uptake. The excess is filtered by the kidney and appears in
the urine. Urine is collected for 24 hours, and its radioactivity is deter- Schilling test Part I, without intrinsic factor, 1%
mined. If 77.5% of the standard oral dose is excreted, absorption is said Part II, with intrinsic factor, 8%
to be normal. Excretion in PA and in malabsorption syndromes Intrinsic-factor-blocking Positive to a titer of 1:6400
antibodies
is 67.5% because the labeled oral vitamin B is not absorbed.
12
If excretion is 67.5%, part II of the Schilling test is performed to 5. What is this patient’s definitive diagnosis?
distinguish between PA and other causes of malabsorption. In part
II, the oral dose of labeled B is accompanied by a dose of intrinsic
12
factor. The remainder of the test is the same as in part I. If part II cobaSorb test
shows 77.5% excretion, absorption is considered normal with the The CobaSorb test is an alternative test designed to measure the
lack of absorption in part I due to the lack of IF. The diagnosis is PA. If absorption of cobalamin. 6,36 There are three parts to this test in which
part II is abnormal, the patient likely has another malabsorption defect the patient is given nonradioactive cobalamin either alone or with
such as sprue (Table 15-9 ★). recombinant IF or haptocorrin. Blood levels of holo-TC and holo-
Several points must be considered when interpreting the results HC are measured before and after administration of the cobalamin.
of a Schilling test. First, the test results are not valid with the presence An increase in the concentrations of these plasma transport proteins
of renal disease. The patient may have been able to absorb the vitamin saturated with cobalamin after administration of the cobalamin is
but cannot filter the excess vitamin efficiently because of abnormal reflective of active cobalamin absorption. Refer to Table 15-10 ★ for
kidney function. Second, incomplete collection of urine invalidates a list of tests used to investigate the cause of a cobalamin deficiency.
M15_MCKE6011_03_SE_C15.indd 293 01/08/14 1:38 pm
294 SECTION III • ThE ANEmIAS
★ table 15-10 Tests Used to Investigate the Cause of Cobalamin Deficiency a
test advantages/disadvantages
Schilling test Gold standard for measure of cobalamin absorption; high specificity for PA; requires
handling and administration of radioactive cobalamin; requires collection of a 24 hour
urine sample; false positive with reduced renal function; decreased availability of the
test
IF blocking antibodies high specificity for lack of IF; low sensitivity; pathognomonic for pA
Pepsinogen and gastrin high sensitivity; low specificity for pA; mirrors gastric function; increased in gastric
atrophy
Parietal cell antibodies May be present in PA; low specificity
CobaSorb Alternative to Schilling test; reflects absorption of cobalamin; use in routine practice
needs further evaluation
a These tests are used primarily to evaluate cobalamin deficiency due to pernicious anemia. Additional testing may be necessary to evaluate other causes of
cobalamin deficiency. Patient history is also important in diagnosis.
IF = intrinsic factor; PA = pernicious anemia; holo@TC = TCII with attached cobalamin
CheCkpoint 15-11 Specific therapy can reverse the peripheral neuropathy of
cobalamin deficiency, but spinal cord damage is usually irrevers-
What two lab tests are the most specific indicators of ible. Pernicious anemia must be treated with lifelong monthly
cobalamin deficiency? parenteral doses of hydroxycobalamin (OHCbl) because of these
patients’ inability to absorb oral cobalamin. Recently, it was reported
that large doses of cobalamin therapy (usually 1000–2000 mcg/day)
administered orally could be feasible if the patient is followed care-
24
fully. The oral treatment can be better tolerated and less expen-
37
therapy sive. The rationale behind oral therapy using large doses of vitamin
Therapeutic trials in megaloblastic anemia using physiologic doses is that a small amount (from 1 to 3%) of the vitamin is absorbed by
of either vitamin B or folic acid produce a reticulocyte response diffusion without IF.
12
only if the specific vitamin that is deficient is being administered.
For instance, small doses (1 mcg) of vitamin B given daily produce
12
a reticulocyte response in cobalamin deficiency but not in folic acid caSe Study (continued from page 293)
deficiency. On the other hand, large therapeutic doses of cobalamin
or folic acid can induce a partial response to the other vitamin defi- 6. how would the diagnosis change if the special testing
ciency as well as the specific deficiency. results were as follows?
Generally, it is best to determine which deficiency exists and Schilling test Part I, without intrinsic factor, 1%
to treat the patient with the specific deficient vitamin. Large doses Part II, with intrinsic factor, 3%
of folic acid will correct the anemia in cobalamin deficiency but Intrinsic-factor-blocking Negative
do not correct or halt demyelination and neurologic disease. This antibodies
makes diagnosis and specific therapy in cobalamin deficiency crit-
ical. Specific therapy causes a rise in the reticulocyte count after 7. What would you predict this patient’s reticulocyte
the fourth day of therapy. Reticulocytosis peaks at about 5–8 days count to be?
and returns to normal after 2 weeks. The degree of reticulocytosis
is proportional to the severity of the anemia with more striking
reticulocytosis in patients with severe anemia. The hemoglobin Other Megaloblastic Anemias
rises about 2–3 g/dL every 2 weeks until normal levels are reached. A megaloblastic anemia occasionally is associated with drugs,
The marrow responds quickly to therapy, as evidenced by pronor- congenital enzyme deficiencies, or other hematopoietic diseases.
moblasts (normal) appearing within 4–6 hours and nearly com-
plete recovery of erythroid morphologic abnormalities within 2–4 drugs
days. Granulocyte abnormalities disappear more slowly. Hyper- A large number of drugs that act as metabolic inhibitors can cause
segmented neutrophils can usually be found for 12–14 days after megaloblastosis (Table 15-11 ★). Some of these drugs are used in
therapy begins. chemotherapy for malignancy. Although aimed at eliminating rapidly
M15_MCKE6011_03_SE_C15.indd 294 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 295
proliferating malignant cells, these drugs are not selective. Any normal
proliferating cells, including hematopoietic cells, are also affected.
enzyme deficiencies
Methionine synthase reductase (MSR) deficiency is a rare autosomal
recessive disorder. A deficiency of this enzyme leads to a dysfunc-
tion of folate/cobalamin metabolism and results in hyperhomocys-
38
teinemia, hypomethioninemia, and megaloblastic anemia. MSR is
necessary for the reductive activation of methionine synthase and the
resultant folate-cobalamin-dependent conversion of homocysteine
to methionine.
■ Figure 15-11 Peripheral blood from a case of con-
congenital deficiencies genital dyserythropoietic anemia type II (CDA-II). There
A congenital deficiency is a physiological aberration present at birth. is anisocytosis with microcytic, hypochromic cells as well
The following congenital deficiencies result in megaloblastosis. as macrocytes and normocytes. The nucleated cell is
orotic aciduria an orthochromic normoblast showing lobulation of the
Inborn defects in enzymes required for pyrimidine synthesis or nucleus (Wright-Giemsa stain; 1000* magnification).
folate metabolism can result in megaloblastic anemia. Orotic acid-
uria is a rare autosomal recessive disorder in which there is a failure
to convert orotic acid to uridylic acid. The result is excessive excre- (Table 15-12 ★). There are three types: CDA I, CDA II, and CDA III.
tion of orotic acid. Children with this disorder also fail to grow and Types I and II are inherited as autosomal recessive disorders, and Type
develop normally. The condition responds to treatment with oral III is inherited in an autosomal dominant fashion. Red cell multinucle-
uridine. arity in the bone marrow and secondary siderosis are recognized in all
congenital dyserythropoietic anemia types; however, megaloblastic erythroid precursors are present only in
Congenital dyserythropoietic anemia (CDA) is actually a hetero- Type I and Type III.
geneous group of refractory, congenital anemias characterized • CDA I Bone marrow erythroblasts are megaloblastic and often
by both abnormal erythropoiesis and ineffective erythropoiesis binucleate with incomplete division of nuclear segments. The
incomplete nuclear division is characterized by internuclear chro-
★ table 15-11 Drugs That Can Cause matin bridges.
Megaloblastosis • CDA II Bone marrow precursors are not megaloblastic but are
typically multinucleated with up to seven nuclei (Figure 15-11 ■).
dna base inhibitors
Type II is distinguished by a positive acidified serum test (Ham test)
Pyrimidine Purine antimetabolites other but a negative sucrose hemolysis test. In the Ham test, only about
Azauridine Acyclovir Cytosine Azacytidine 30% of normal sera are effective in lysing CDA II cells. This type
Adenosine arabinoside Cyclophosphamide has also been termed hereditary erythroblastic multinuclearity
arabinoside Fluorocytidine Zidovudine (AZT) with positive acidified serum test (HEMPAS). CDA II is the most
Azathioprine Fluorouracil common of the three types of CDA.
Gancyclovir hydroxyurea
• CDA III This type of CDA is morphologically distinct from
Mercaptopurine Methotrexate Types I and II because of the presence of giant erythroblasts (up to
Thioguanine 50 mcM) containing up to 16 nuclei. Sometimes the erythrocytes
Vidarabine are agglutinated by anti-I and anti-i antibodies.
★ table 15-12 Comparison of Congenital Dyserythropoietic Anemia (CDA) Types
characteristics cda i cda ii cda iii
Inheritance Autosomal recessive Autosomal recessive Autosomal dominant
RBC multinuclearity Present Present Present
Number of nuclei 2 Up to 7 Up to 16
Siderosis Present Present Present
Megaloblastosis Present Absent Present
Other characteristics Incomplete nuclear division positive ham test (hEmpAS) RBC agglutination by anti-I and anti-i antibodies
M15_MCKE6011_03_SE_C15.indd 295 01/08/14 1:38 pm
296 SECTION III • ThE ANEmIAS
other Hematopoietic diseases Alcoholism
The myelodysplastic syndromes are a group of stem cell disor- Alcohol abuse is one of the most common causes of nonanemic mac-
ders characterized by peripheral blood cytopenias and dyshema- rocytosis. It has been suggested that all patients with macrocytosis
topoiesis. Erythroid precursors in the bone marrow frequently should be questioned about their alcohol consumption. Macro-
39
exhibit megaloblastic-like changes. There is occasionally a non- cytosis associated with alcoholism is usually multifactorial and can
megaloblastic macrocytic anemia. These diseases will be discussed be megaloblastic. Macrocytosis is probably the result of one or more
in Chapter 25. of four causes: (l) folate deficiency due to decreased dietary intake,
(2) reticulocytosis associated with hemolysis or gastrointestinal bleed-
ing, (3) associated liver disease, and (4) alcohol toxicity.
CheCkpoint 15-12 Folate deficiency associated with a megaloblastic anemia is the
most common cause of the macrocytosis found in hospitalized alco-
Which clinical type of CDA gives a positive ham test result holic patients. The deficiency probably results from poor dietary hab-
and presents with a normoblastic marrow?
its, although ethanol also appears to interfere with folate metabolism.
The reduced erythrocyte survival with a corresponding reticu-
locytosis has been associated with chronic gastrointestinal bleeding
secondary to hepatic dysfunction (decreased coagulation proteins) or
Macrocytic aneMia witHout thrombocytopenia, hypersplenism from increased portal and splenic
MegaloblaStoSiS vein pressure, pooling of cells in splenomegaly, and altered erythrocyte
The typical findings of megaloblastic anemia are not evident in other membranes caused by abnormal blood lipid content in liver disease
macrocytic anemias. The macrocytes in macrocytic anemias without (Chapter 17). Stomatocytes are associated with acute alcoholism, but
megaloblastosis are usually not as pronounced and are usually round there appears to be no abnormal cation permeability, and hemolysis
rather than oval as seen in megaloblastic anemia (Figure 15-1a, b). of these cells is not significant.
Hypersegmented neutrophils are not present, and leukocytes and Liver disease is common in alcoholic individuals; typical hemato-
platelets are quantitatively normal. Jaundice, glossitis, and neuropa- logic findings associated with this disease are discussed in the following
thy, the typical clinical findings in megaloblastosis, are absent. The section. Even when anemia is absent, most alcoholic individuals have a
cause of the macrocytosis without megaloblastosis is unknown in mild macrocytosis (100–110 fL) unrelated to liver disease or folate defi-
many cases. In some cases the macrocytes can be due to an increase ciency. This can be caused by a direct toxic effect of ethanol on developing
in membrane lipids or to a delay in erythroblast maturation. Some erythroblasts. Vacuolization of red cell precursors, similar to that seen in
diseases associated with nonmegaloblastic macrocytic anemia are patients taking chloramphenicol, is a common finding after prolonged
listed in Table 15-13 ★. Three of the most common—alcoholism, alcohol ingestion. If alcohol intake is eliminated, the cells gradually assume
liver disease, and reticulocytosis (stimulated erythropoiesis)—are their normal size, and the bone marrow changes disappear. The associa-
discussed in this section. tion of a sideroblastic anemia and alcoholism is discussed in Chapter 12.
The multiple pathologies of this type of anemia result in the pos-
sibility of a variety of abnormal hematologic findings. Thus, it is pos-
★ table 15-13 Conditions Associated with sible to have a blood picture resembling that of megaloblastic anemia,
Nonmegaloblastic Macrocytosis chronic hemolysis, chronic or acute blood loss, liver disease, or (more
than likely) a combination of these conditions. Alcohol can also cause
condition cause of Macrocytosis disordered heme synthesis as discussed in Chapter 12.
Alcoholism Direct toxic effect of alcohol on
erythroid precursors Liver Disease
Reticulocytosis associated with
hemolysis or GI bleeding The most common condition associated with a nonmegaloblastic
macrocytic anemia is liver disease (including alcoholic cirrhosis).
Liver disease: Abnormal RBC
membrane lipid composition The causes of this anemia are multifactorial and include hemolysis,
Can also be megaloblastic from impaired bone marrow response, folate deficiency, and blood loss
folate deficiency (Table 15-14 ★). Although macrocytic anemia is the most common
Liver disease Increased RBC membrane lipids form of anemia in liver disease, occurring in 750% of the patients,
hemolysis or Reticulocytosis associated with normocytic or microcytic anemia can also be found depending on
posthemorrhagic anemia stimulated erythropoiesis the predominant pathologic mechanism.
hypothyroidism Unknown Erythrocyte survival appears to be significantly shortened in alco-
Aplastic anemia Unknown holic liver disease, infectious hepatitis, biliary cirrhosis, and obstructive
Artifactual Cold agglutinin disease jaundice. The reason for this is unknown. Cross-transfusion studies in
Severe hyperglycemia which patient cells are infused into normal individuals demonstrate
RBC clumping an increase in patient cell survival. This suggests that an extracellular
Swelling of RBCs factor is probably responsible for cell hemolysis. The spleen is thought
to play an important role in sequestration and hemolysis in individuals
M15_MCKE6011_03_SE_C15.indd 296 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 297
★ table 15-14 Causes and Characteristics of Anemia in Liver Disease
causes anemia type characteristics
Abnormal liver function Macrocytic MCV normal to increased
Increased RBC membrane cholesterol resulting in target cells and acanthocytes
Normal to slightly increased reticulocytes
Folic acid deficiency Macrocytic MCV increased
Pancytopenia
Ovalocytes and teardrop cells
Normal to decreased reticulocytes
Functional folate deficiency
hemolysis Normocytic or macrocytic MCV normal to increased
Spur cells and schistocytes
Increased reticulocytes
hypersplenism Normocytic MCV normal
Pancytopenia
Increased reticulocytes
Portal hypertension
Marrow hypoproliferation Normocytic MCV normal
Pancytopenia
Decreased reticulocytes
Associated with renal disease or alcohol suppression
Chronic blood loss Microcytic MCV decreased
Iron deficiency
Leukocyte and platelet counts slightly increased
Reticulocytes increased
Commonly associated with gastrointestinal bleeding
with splenomegaly or hypersplenism. In some cases, hemolysis is well and lipids. Therefore, the anemia is a minor finding among the abnor-
compensated for by an increase in erythropoiesis and there is no malities associated with this organ’s dysfunction.
anemia. In some patients with alcoholic liver disease, a heavy drink- The anemia is usually mild with an average hemoglobin concentra-
ing spree produces a brisk but transient hemolysis. These patients also tion of about 12 g/dL. With complications, the anemia can be severe. The
show abnormal liver function and have markedly increased levels of erythrocytes can appear normocytic, macrocytic (usually not 7115 fL
plasma triglycerides. MCV), or microcytic. A discrepancy between the MCV and the appear-
Abnormalities in erythrocyte membrane lipid composition ance of the cells microscopically often occurs. In these cases, thin, round
are common in hepatitis, cirrhosis, and obstructive jaundice. Both macrocytes (as determined by diameter) with target cell formation are
cholesterol and phospholipid are increased, resulting in cells with found on the blood smear, but the MCV is within normal limits. The retic-
an increased surface area–to–volume ratio. This abnormality is not ulocyte count can be increased, but the RPI is usually 62 unless hemo-
thought to cause decreased cell survival. In contrast, in severe lysis is a significant factor. Thrombocytopenia is a frequent finding, and
hepatocellular disease, erythrocyte membranes have an excess of platelet function can be abnormal. Various nonspecific leukocyte abnor-
cholesterol relative to phospholipid, which decreases the erythrocyte malities have been described including neutropenia, neutrophilia, and
deformability. This membrane lipid imbalance is associated with the lymphopenia. The bone marrow is either normocellular or hypercellular,
formation of spur cells in which the erythrocyte exhibits spikelike pro- often with erythroid hyperplasia. The precursors are qualitatively nor-
jections. These cells have a pronounced shortened life span leading to mal unless folic acid deficiency is present. In this case, megaloblastosis is
an anemia termed spur cell anemia (Chapter 17). apparent with the typical associated blood abnormalities.
Kinetic iron studies have revealed that the bone marrow response Other laboratory tests of liver function are variably abnormal
in liver disease can be impaired. It has been proposed that liver disease including increased serum bilirubin and increased hepatic enzymes.
can affect the production of erythropoietin because this organ has Tests for carbohydrate and lipid metabolites are frequently abnormal
40
been shown to be an important extrarenal source of the hormone. depending on the degree of liver disease.
In alcoholic cirrhosis, the alcohol may have a direct suppressive effect
on the bone marrow. CheCkpoint 15-13
Clinical findings and symptoms in liver disease are secondary
to the abnormalities in liver function. The liver is involved in many What are the causes of macrocytosis seen in alcoholism?
essential metabolic reactions and in the synthesis of many proteins
M15_MCKE6011_03_SE_C15.indd 297 01/08/14 1:38 pm
298 SECTION III • ThE ANEmIAS
Stimulated Erythropoiesis With a decrease in thyroid hormone (i.e., hypothyroidism), tissue
oxygen requirement is reduced and the kidneys adapt appropriately.
Increased erythropoietin (stimulation) in the presence of an adequate
iron supply (e.g., autoimmune hemolytic anemia) can result in the The net result is a decrease in the production of EPO and, correspond-
release of shift reticulocytes from the bone marrow. These cells are larger ingly, erythrocytes. Thus although the hemoglobin concentration is
than normal with an MCV as high as 130 fL. A reticulocyte count and reduced, the tissue oxygenation is adequate. This type of “anemia” often
examination of the blood smear allow distinction of this macrocytic presents as a macrocytic, normochromic anemia but can also be a
entity from megaloblastic anemia. In the presence of large numbers normocytic, normochromic anemia. The anemia can be complicated
12
of shift reticulocytes, polychromasia is markedly increased. The WBC by iron, folic acid, or B deficiency, and the blood picture can reflect
count is slightly increased, and the platelet count is normal. In addition, these forms of anemia.
the oval macrocytes typical of megaloblastic anemia are not present in
conditions associated with increased erythropoietin stimulation. CheCkpoint 15-14
What are three clinical or laboratory findings (in addition
Hypothyroidism to assessing the bone marrow) that can distinguish a non-
Anemia of hypothyroidism presents as a mild to moderate ane- megaloblastic macrocytic anemia from a megaloblastic
mia with a normal reticulocyte count. Thyroid hormone regulates anemia?
cellular metabolic rate and therefore tissue oxygen requirement.
Summary
Macrocytosis due to megaloblastic anemia must be differen- tetrahydrofolate. This product is an important cofactor needed
tiated from macrocytosis with a normoblastic marrow. The for the production of DNA thymidine.
laboratory test profile of a patient with megaloblastic anemia Defective production of intrinsic factor is the most com-
commonly indicates pancytopenia. The blood smear reveals mon cause of cobalamin deficiency (pernicious anemia [PA]),
macro-ovalocytes (i.e., large ovalocytes), poikilocytosis, tear- which is caused by failure of the gastric mucosa to secrete IF. PA
drop cells, howell-Jolly bodies, and neutrophil hypersegmen- most commonly occurs in people after 40 years of age. Central
tation. The marrow is characterized by megaloblastosis of nervous system symptoms can be present in advanced cases.
precursor cells due to a block in thymidine production. Because Laboratory tests used to diagnose PA include serum cobalamin,
thymidine is one of the four DNA bases, the deficiency leads to MMA, IFBA, serum gastrin, and the Schilling test. Other causes
a diminished capacity for DNA synthesis and a block in mitosis. of cobalamin deficiency include gastrectomy, malabsorption
The marrow is hypercellular, but erythropoiesis is ineffective. diseases such as Crohn’s disease, and drugs. Megaloblastic ane-
Causes of megaloblastosis are nearly always due to cobalamin mia rarely results from chemotherapeutic drugs or congenital
or folic acid deficiencies. enzyme deficiencies or with other hematopoietic diseases such
Folate is primarily acquired from the diet. The liver is as congenital dyserythropoietic anemia.
the main storage site of folic acid. Folate deficiency results Serum and RBC folate can be measured to diagnose folate
5
10
in decreased synthesis of N , N @methylene ThF, a cofactor deficiency, but the tests do not reliably indicate folate stores in
in DNA synthesis. Consequently, DNA synthesis slows. Clini- the presence of cobalamin deficiency. Cobalamin levels can be
cally, symptoms from inadequate dietary folate can occur within directly assessed by measuring serum cobalamin or indirectly
months as compared with years after onset of a cobalamin by measuring MMA in the urine/serum/plasma or homocysteine
deficiency. in plasma.
Vitamin B 12 (i.e., cyanocobalamin) absorption occurs in Macrocytosis due to megaloblastic anemia must be dif-
the small intestine. Dietary cobalamin is released from diges- ferentiated from macrocytosis with a normoblastic marrow.
tion of animal proteins in meats and bound by gastric hapto- Normoblastic, macrocytic anemias can result from acute blood
corrins and subsequently intrinsic factor (IF). Once absorbed, loss or hemolysis due to shift reticulocytosis from the marrow.
cobalamin is bound to specific plasma proteins known as Alcohol abuse is one of the most common causes of normo-
transcobalamin and haptocorrins. Normal serum cobalamin blastic macrocytosis. Liver disease, often resulting from alcohol
values are highly variable based on age and sex. Cobalamin abuse, is also commonly associated with macrocytosis without
function is related to DNA synthesis because cobalamin is a a megaloblastic marrow.
vital cofactor in the conversion of methyl tetrahydrofolate to
M15_MCKE6011_03_SE_C15.indd 298 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 299
Review Questions
Level i 7. A deficiency of cobalamin leads to impaired: (Objective 1)
1. The most common cause of macrocytosis is: (Objective 5) A. folic acid synthesis
A. folate deficiency B. DNA synthesis
B. alcoholism C. intrinsic factor secretion
C. liver disease D. absorption of folate
D. pernicious anemia 8. Laboratory diagnosis of pernicious anemia can include
which of the following? (Objective 3)
2. In the majority of cases, cobalamin deficiency is due to a
deficiency of: (Objective 6) A. urinary FIGLU
A. intrinsic factor B. WBC count
B. vitamin B 6 C. gastric analysis
C. folate D. LDh
D. methylmalonic acid 9. Alcoholic individuals commonly develop a macrocytic ane-
mia due to: (Objective 8)
3. Which of the following is the best clue in diagnosing mega-
loblastic anemia? (Objective 3) A. folate deficiency
A. decreased hemoglobin and hematocrit B. increased blood cholesterol levels
B. leukocytosis C. development of autoantibodies against intrinsic factor
C. hypersegmented neutrophils D. intestinal malabsorption of cobalamin
D. poikilocytosis 10. Anemia due to liver disease is often associated with which
of the following RBC morphological forms? (Objective 9)
4. Increases in urinary excretion of formiminoglutamic
acid (FIGLU) most likely indicate which of the following? A. ovalocytes
(Objective 4)
B. microcytes
A. cobalamin deficiency
C. spur cells
B. autoantibodies to intrinsic factor
D. teardrop cells
C. folic acid deficiency
Level ii
D. hemolysis
5. The liver stores enough folate to meet daily requirement 1. If a patient presents with anemia, macrocytosis, pancy-
needs for how long? (Objective 2) topenia, and malnutrition, which of the following should
be investigated first as a possible cause of the anemia?
A. 1 month (Objectives 4, 12)
B. 6–8 weeks A. pernicious anemia
C. 2 years B. folic acid deficiency
D. 3–6 months C. cobalamin deficiency
6. Which of the following conditions increases the daily D. celiac disease
requirement for cobalamin? (Objective 2)
2. Which laboratory tests are most sensitive to decreased
A. pregnancy levels of cobalamin? (Objectives 8, 11)
B. aplastic anemia A. red cell and serum folate
C. hypothyroidism B. serum cobalamin and red cell folate
D. splenectomy C. transcobalamin assays
D. MMA and homocysteine
M15_MCKE6011_03_SE_C15.indd 299 01/08/14 1:38 pm
300 SECTION III • ThE ANEmIAS
3. Which of the following can be found in a patient with 8. If a patient excretes *7.5% of a radioactively tagged crystal-
megaloblastic anemia? (Objectives 2, 8, 11) line B 12 dose after 24 hours both before and after administra-
tion of a dose of oral intrinsic factor, which of the following is
A. giant metamyelocytes and hypolobulated a possible diagnosis? (Objective 9)
neutrophils
A. pernicious anemia
B. howell-Jolly bodies and pappenheimer bodies
B. liver disease
C. hypersegmented neutrophils and oval
macrocytes C. gastrectomy
D. hypochromic macrocytes and thrombocytosis D. celiac disease
4. The metabolic function of tetrahydrofolate is to: (Objective 1) 9. Which type of congenital dyserythropoietic anemia (CDA)
presents with giant multinucleated erythrocytes in the
A. synthesize methionine
marrow? (Objective 10)
B. transfer carbon units from donors to receptors
A. CDA I
C. serve as a cofactor with cobalamin in the synthesis of B. CDA II
thymidylate
C. CDA III
D. synthesize intrinsic factor
D. CDA IV
5. Folic acid deficiency can be caused by: (Objective 6)
10. A 48-year-old Caucasian female experiencing fatigue, loss
A. alcoholism
of appetite, and weight loss over a period of three months
B. chronic blood loss was seen by her physician. A medical history revealed she
had a history of alcohol abuse. An initial laboratory workup
C. strict vegetarian diet demonstrated that she was anemic, had a leukocyte count
9
D. vitamin B 6 deficiency of 3 * 10 /L, and had an MCV of 119 fL. Macro- ovalocytes
and neutrophil hypersegmentation were noted on her
6. A lack of intrinsic factor could be due to: (Objective 7) blood smear evaluation. Based on the initial laboratory
A. gastrectomy test results, her physician obtained a serum cobalamin and
folate workup. Results were as follows:
B. cobalamin deficiency
Serum cobalamin 550 pg/mL
C. folate deficiency Serum folate 4.0 ng/mL
D. large bowel resection RBC folate 105 ng/mL
7. Which of the following is more typical of nonmegaloblastic Based on her clinical history and laboratory results, the best
than megaloblastic anemia? (Objectives 2, 3) possible diagnosis is which of the following? (Objective 12)
A. oval macrocytes A. pernicious anemia
B. round macrocytes B. folate deficiency
C. howell-Jolly bodies C. primary cobalamin deficiency
D. hypersegmented neutrophil D. anemia of liver disease
Companion Resources
http://www.pearsonhighered.com/healthprofessionsresources/
The reader is encouraged to access and use the companion resources created for this chapter. Find additional information to help
organize information and figures to help understand concepts.
The views expressed in this chapter are those of the author and do not necessarily reflect the official policy or position of the Depart-
ment of the Army, the Department of Defense, or the U.S. Government.
M15_MCKE6011_03_SE_C15.indd 300 01/08/14 1:38 pm
ChApTEr 15 • mEgALOBLASTIC AND NONmEgALOBLASTIC mACrOCyTIC ANEmIAS 301
References
1. hoggarth K. macrocytic anaemias. Practitioner. 1993;237(1525):331–32,335. 23. Carson-Dewitt RS. Pernicious anemia. In Gale Encyclopedia of Medicine.
2. Colon-Otero g, menke D, hook CC. A practical approach to the differential Farmington hills, mI: The Thomson Corp.; 2002;2224.
diagnosis and evaluation of the adult patient with macrocytic anemia. 24. Oh R, Brown OL. Vitamin B 12 deficiency. Am Fam Phys. 2003;67(5):979–92.
Med Clin North Am. 1992;76:581–97. 25. Legan rC, Zawistoski KJ. Update on vitamin B 12 deficiency. Am Fam
3. Kass L. pernicious anemia. In: Smith Lh Jr, ed. Major Problems in Internal Physician. 2011;83(12):1425–30.
Medicine. Vol. 7. Philadelphia: W.B. Saunders; 1976:1–62. 26. Carmel r, melnyk S, James SJ. Cobalamin deficiency with and without
4. Addison T. On the Constitutional and Local Effects of Disease of the neurologic abnormalities: differences in homocysteine and methionine
Suprarenal Capsules. London, England: Sam highley; 1855:43. metabolism. Blood. 2003;101(8)3302–8.
5. Ehrlich P. Farbenanalytische Untersuchungen zur Histologie und Klinik des 27. Welch gN, Loscalzo J. homocysteine and atherothrombosis. N Engl J Med.
Blutes. Berlin, germany: A. hirschwald; 1891:137. 1998;338:1042–50.
6. hvas A, Nexo E. Diagnosis and treatment of vitamin B 12 deficiency. 28. ridker pm, hennekens Ch, Selhub J et al. Interrelation of hyperhomocyst(e)
An update. Haematologica. 2006;91:1506–12. inemia, factor V Leiden, and risk of future venous thromboembolism.
7. Carmel R. Macrocytosis, mild anemia, and delay in the diagnosis of Circulation. 1997;95:1777–82.
pernicious anemia. Arch Intern Med. 1979;139:47–50. 29. holleland g, Schneede J, Ueland pm et al. Cobalamin deficiency in general
8. hall CA. Vitamin B 12 deficiency and early rise in mean corpuscular volume. practice: assessment of the diagnostic utility and cost-benefit analysis
JAMA. 1981;245:1144–46. of methylmalonic acid determination in relation to current diagnostic
9. Spivak JL. masked megaloblastic anemia. Arch Intern Med. 1982;142: strategies. Clin Chem. 1999;45:189–98.
2111–14. 30. Mayo Clinic. Pernicious anemia cascade, serum. www.mayomedicallaboratories
10. Carmel r, Weiner Jm, Johnson CS. Iron deficiency occurs frequently in .com. 03 mar. 2005. Web; accessed July 21, 2014.
patients with pernicious anemia. JAMA. 1987;257:1081–83. 31. Valente E, Scott Jm, Ueland pm et al. Diagnostic accuracy of
11. Chan CWJ, Liu Syh, Kho CJB et al. Diagnostic clues to megaloblastic holotranscobalamin, methylmalonic acid, serum cobalamin, and
anemia without macrocytosis. Intern J Lab Hematol. 2007;29(3):163–71. other indicators of tissue vitamin B 12 status in the elderly. Clin Chem.
12. Pruthi RK, Tafferi A. Pernicious anemia revisited. Mayo Clin Proc. 2011;57(6):856–63.
1994;69:144–50. 32. Carmel R. Biomarkers of cobalamin (vitamin B 12 ) status in the epidemiologic
13. Lindenbaum J, healton EB, Savage Dg et al. Neuropsychiatric disorders setting: a critical overview of context, applications, and performance
caused by cobalamin deficiency in the absence of anemia or macrocytosis. characteristics of cobalamin, methylmalonic acid, and holotranscobalamin II.
N Engl J Med. 1988;318:1720–28. Am J Clin Nutr. 2011; 94(1):348S–58S.
14. Green R. Folate, cobalamin, and megaloblastic anemias. In: Kaushansky 33. Nexo E, hoffmann-Lucke E. holotranscobalamin, a marker of vitamin
K, Lichtman MA, Beutler E et al., eds. In Williams Hematology, 8th ed. B 12 status: analytical aspects and clinical utility. Am J Clin Nutr.
New york: mcgraw-hill; 2010; 533–63. 2011;94(1):359S–65S.
15. Klee GG. Cobalamin and folate evaluation: Measurement of methyl- 34. Kumar V. Pernicious anemia. MLO. 2007;39(2):28–30.
malonic acid and homocysteine vs vitamin B 12 and folate. Clin Chem. 35. Norman EJ, morrison JA. Screening elderly populations for cobalamin
2000;46(8 Pt 2):1277–83. (vitamin B 12 ) deficiency using the urinary methylmalonic acid assay by gas
16. Schrier SL. Diagnosis and treatment of vitamin B 12 and folate deficiency. chromatography mass spectrometry. Am J Med. 1993;94:589–94.
UpToDate©. www.uptodate.com; accessed July 22, 2013. 36. havas Am, morkbak AL, Nexo E. plasma holotranscobalamin compared
17. Antony AC megaloblastic anemias. In: r hoffman, EJ Benz Jr, LE Silberstein with plasma cobalamins for assessment of vitamin B 12 absorption; optimiza-
et al., eds. Hematology Basic Principles and Practice, 6th ed. Philadelphia: tion of a non-radioactive vitamin B 12 absorption test (CobSorb). Clin Chim
Saunders; 2013:491–524. Chapter 37. Acta. 2007;376(1–2):150–54.
18. Jacques pF, Selhub J, Bostom Ag et al. The effect of folic acid fortification 37. Balaman Z, Kadikoylu g, yukselen V et al. Oral versus intramuscular
on plasma folate and total homocysteine concentrations. N Engl J Med. cobalamin treatment in megaloblastic anemia: a single-center, prospective,
1999;340:1449–54. randomized open-label study. Clin Ther. 2003;25(12):3124-34.
19. MRC Vitamin Study Research Group. Prevention of neural tube 38. Wilson A, Leclerc D, Rosenblatt DS et al. Molecular basis for methionine
defects: results of the Medical Research Council Vitamin Study. Lancet. synthase reductase deficiency in patients belonging to the cblE comple-
1991;338(8760):131–37. mentation group of disorders in folate/cobalamin metabolism. Hu Mol
20. Chanarin I. Megaloblastic anaemia, cobalamin, and folate. J Clin Pathol. Gene. 1999;8:2009–16.
1987;40:978–84. 39. Seppa K, Sillanaukee P, Saarni M. Blood count and hematologic morphol-
21. Elin rJ, Winter WE. methylmalonic acid: a test whose time has come. ogy in nonanemic macrocytosis: differences between alcohol abuse and
Arc Path Lab Med. 2001;125(6):824–27. pernicious anemia. Alcohol. 1993;10:343–47.
22. hsing AW, hansson L-E, mcLaughlin JK, et al. pernicious anemia and subse- 40. Katz R, Cooper GW, Gordon AS, Zanjani ED. Studies on the site of
quent cancer: a population-based cohort study. Cancer. 1993;71:745–50. production of erythropoietin. Ann NY Acad Sci. 1968;149:120–27.
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16 Hypoproliferative
Anemias
Rebecca Laudicina, Phd
Objectives—Level I
At the end of this unit of study, the student should be able to: Chapter Outline
1. Define hypoproliferative anemia. Objectives—Level I and Level II 302
2. Cite the diagnostic criteria for aplastic anemia.
3. Describe the epidemiology and etiology of aplastic anemia. Key Terms 303
4. Explain the pathophysiology of aplastic anemia. Background Basics 303
5. Compare and contrast acquired and inherited aplastic anemia.
6. List the major clinical and laboratory characteristics of aplastic Case Study 303
anemia. Overview 303
7. Identify environmental factors associated with the development of
aplastic anemia. Introduction 303
8. Describe the etiology, bone marrow, and peripheral blood in pure red Aplastic Anemia 303
cell aplasia.
9. Identify peripheral blood findings associated with the following: Pure Red Cell Aplasia 310
aplastic anemia, pure red cell aplasia, and anemia due to renal disease. Other Hypoproliferative Anemias 312
Summary 313
Objectives—Level II
At the end of this unit of study, the student should be able to: Review Questions 314
1. Propose and explain possible causes of aplastic anemia. Companion Resources 316
2. Discuss prognosis in aplastic anemia. References 316
3. Compare methods of treatment and management of patients with
aplastic anemia.
4. Contrast aplastic anemia with other causes of pancytopenia on the
basis of clinical findings and peripheral blood and bone marrow
findings.
5. Compare and contrast pure red cell aplasia with aplastic anemia and
other causes of erythroid hypoproliferation.
6. Compare and contrast major characteristics of Diamond-Blackfan
syndrome and transient erythroblastopenia of childhood.
7. Explain the pathophysiology of anemia due to renal disease.
8. Evaluate laboratory test results and medical history of a clinical case for
a patient with hypoproliferative anemia, suggest additional laboratory
tests if appropriate, and a possible diagnosis.
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Chapter 16 • hypoproliferative anemias 303
Key Terms
Aplasia Dyshematopoiesis Hypoproliferative Pancytopenia
Aplastic anemia fanconi anemia (fa) Idiopathic Pure red cell aplasia
Diamond-Blackfan anemia Hypocellularity myelophthisis
(DBA) Hypoplasia
Background Basics
The information in this chapter builds on the concepts learned • outline the classification of anemias. (Chapter 11)
in previous chapters. To maximize your learning experience, you • explain the process of hematopoiesis. (Chapter 4)
should review these concepts before starting this unit of study:
Level ii
Level i • explain the concepts of stem cell renewal and differentiation.
• Describe basic laboratory procedures used to screen for and (Chapter 4)
assess anemia. (Chapters 10, 11) • Describe the normal bone marrow structure and cellularity and
• identify abnormal values and results for basic hematologic the process of bone marrow examination. (Chapters 3, 38)
laboratory procedures. (Chapter 10)
marrow hypocellularity. Much of the area in the bone marrow
case study normally occupied by hematopoietic tissue is replaced by fat. The
terms aplastic, aplasia, and hypoplastic refer to a bone marrow with
We will address this case study throughout the chapter.
an overall decrease in hematopoietic cellularity. If there is hypopla-
Rachael, a 13-year-old female, was admitted to the sia of only one of the hematopoietic lineages, the terms erythroid,
hospital with complaints of progressive weakness and myeloid, or megakaryocytic hypoplasia should be used to define
shortness of breath with minimal physical effort. She has the specific entity.
experienced recurrent fevers reaching 102°f. physical The hematopoietic defect is due to depletion, damage, or inhibi-
examination revealed a well-developed adolescent with tion of hematopoietic stem cells (HSCs) and/or hematopoietic pro-
good nutritional status and in no acute distress. There genitor cells (HPCs). Either the unipotent erythroid progenitor cell
was no lymphadenopathy or organomegaly. many pete- or a multilineage hematopoietic progenitor cell can be affected. The
chial hemorrhages covered her chest and legs. Several peripheral blood findings provide important clues to help identify
bruises were found on her legs and thighs. Laboratory the bone marrow abnormality. If only the erythroid progenitor cells
tests were ordered upon admission. (BFU-E, CFU-E) are affected, platelets and leukocytes remain nor-
Consider the diagnostic possibilities in this case and mal, and the diagnosis is pure red cell aplasia. More commonly, the
how laboratory tests can be used to assist in differential multilineage hematopoietic precursor cells (HSC, MPP, CMP, CFU-
diagnosis.
GEMM, etc.) are affected, resulting in pancytopenia (decreases of all
three cell lineages), and the diagnosis is aplastic anemia.
aPLastic anemia
OveRview The term aplastic anemia is used to describe the condition of pan-
Finding cytopenias in the peripheral blood often suggests the presence cytopenia that is associated with a hypocellular bone marrow. The
of a serious medical condition. Although there are a variety of causes, mature blood cells that are produced in aplastic anemia (AA) usu-
bone marrow hypoproliferation is one of the most serious. This chap- ally appear normal. Aplasia of the bone marrow is only one of several
ter discusses the acquired and inherited anemias that are associated possible causes of peripheral blood pancytopenia, but pancytopenia
with bone marrow hypoproliferation. The first section describes the due to causes other than AA can result in morphologically abnormal
classification of hypoproliferative anemias followed by discussions of blood or bone marrow cells. Whereas AA is usually characterized by
aplastic anemia and pure red cell aplasia. These anemias are contrasted pancytopenia, granulocyte, platelet, and erythrocyte, levels may not
to other causes of cytopenias. be depressed uniformly.
Diagnostic criteria for severe aplastic anemia are included in
Table 16-1 ★. Additional diagnostic criteria are based on disease
intROductiOn severity. With disease progression, concentrations of cells in all three
1
The hypoproliferative anemias are a heterogeneous group of cell lineages eventually become further depleted. This reflects an
acquired and inherited disorders in which there is a normocytic or impaired proliferative capacity of the marrow stem cells, which lose
macrocytic, normochromic anemia associated with chronic bone their ability for normal cellular renewal.
M16_MCKE6011_03_SE_C16.indd 303 01/08/14 1:41 pm
304 seCtion iii • the anemias
★ tabLe 16-1 Diagnostic Criteria for Aplastic Anemia interferon@g (IFN@g) and tumor necrosis factor (TNF), substances
4
known to inhibit hematopoiesis. One mechanism of inhibition is
Bone marrow cellularity 625%, plus two of the following: induction of apoptosis, resulting in cell death. Both IFN@g and TNF
9
Granulocyte count 60.5 * 10 /L induce overexpression of Fas (a cell membrane receptor) by HSCs and
9
Platelet count 620 * 10 /L HPCs. When the Fas receptor is activated by binding with its ligand,
Anemia with corrected reticulocyte count 61% apoptosis is initiated (Chapter 2). It is estimated that the number of
9
(absolute concentration 640 * 10 /L) HSCs in severe acquired AA is decreased to 61% of normal. In tis-
sue culture, the addition of lymphocytes taken from patients with AA
decreases hematopoietic cell production, and their removal improves
the production of hematopoietic cells, thereby supporting the immu-
case study (continued from page 303) nosuppression theory. 7
Flow cytometric analysis of lymphocyte subpopulations in
1. Select laboratory tests appropriate for screening for acquired aplastic anemia has revealed a marked increase in activated
+
aplastic anemia. CD8 lymphocytes. The cause of activation of T lymphocytes in AA
2. Justify the selection of laboratory screening tests remains unclear, although it may be associated with viral infections or
based on Rachael’s clinical signs and symptoms. medical drugs in small numbers of cases. There is also evidence sup-
porting a genetic basis of aberrant cytotoxic T lymphocyte activation.
An increased incidence of AA is associated with the presence of the
class II histocompatibility antigen DR2, which is also linked to other
CheCkpoint 16-1 autoimmune disorders. Aplastic anemia patients positive for this anti-
gen respond well to immunosuppressive therapy. 8
An anemic patient has a (corrected) reticulocyte count The success of hematopoietic stem cell transplants (SCT) in many
of 1.5%, hemoglobin of 100 g/L, hematocrit of 0.30 L/L, patients with AA indicates that the pancytopenia can be corrected by
9
total neutrophil count of 0.4 * 10 /L, and a platelet count repopulation of the marrow with normal HSCs and immune cells. The
9
of 30 * 10 /L. Is it likely that this patient has aplastic aberrant clone of T lymphocytes is eliminated through the pretrans-
anemia? plant conditioning regimen and subsequently replaced with normal
cells, thus lending further support to the immune basis of aplastic
anemia. 4
Defective telomere maintenance in HSCs and HPCs has been
Epidemiology observed in some patients with AA and may contribute to its patho-
The anemias described in this chapter are rare, occurring at a fre- physiology. Telomeres are the ends of chromosomes consisting of 500–
quency of two cases per million. The actual prevalence of aplastic ane- 2000 tandem repeats of the hexanucleotide TTAGGG. The telomere
mia may be somewhat higher due to underdiagnosis and imprecision ends of chromosomes shorten with each cell division (Chapter 2).
1
in applying diagnostic standards. A geographic variation in incidence Maintenance of telomere length occurs through the action of the
occurs with 2–3 times more cases in Asia than in western countries. It enzyme telomerase. Mutations in telomerase-related genes, including
is believed that this variation is related to environmental and occupa- telomerase reverse transcriptase (TERT), telomerase RNA template
2
tional factors. Individual susceptibility could also play a role. Aplastic (TERC), and shelterin (TINF) result in shortened telomeres, reduced
anemia is most commonly seen in 15–25-year-olds with two smaller proliferation of hematopoietic precursors, and increased apoptosis.
affected groups consisting of 2–5 year olds and adults older than 60. Furthermore, shortened telomeres may predispose to malignant trans-
The occurrence of AA in young children is mostly due to inherited formation, which is observed in some patients with AA. Additional
forms, whereas the occurrence in adults is most frequently due to environmental or genetic factors can also contribute to telomere
acquired forms, although there are exceptions. 1,3 shortening. Shortened telomeres and the mutations associated with
them have been observed in patients with both acquired and inherited
Pathophysiology forms of aplastic anemia. 9
Recent attention has been focused on immunologic suppression of
4
hematopoiesis as a cause of acquired aplastic anemia. Response of Classification and Etiology
marrow failure to immunosuppressive therapy has led to the char- Aplastic anemia can be classified as either acquired or inherited
acterization of most cases of acquired AA as an immune-mediated (Table 16-2 ★). Historically, much attention has focused on an asso-
5
disease. The immune mechanism responsible is thought to involve ciation between acquired AA and environmental exposures. Drugs,
the suppression of HSC growth and differentiation by abnormal cyto- chemicals, radiation, infectious agents, and other factors have been
toxic T lymphocytes. This is supported by finding that treatment of linked to the development of acquired AA, which can be temporary
the majority of AA patients with immunosuppressive drugs, includ- or persistent. In most cases, no environmental link can be identified,
ing antithymocyte globulin (ATG) and cyclosporine results in clinical and the cause is said to be idiopathic. The immune pathophysiologic
improvement. 6 model (discussed in the previous section) provides a unifying basis
Immunosuppressive therapy presumably eliminates an abnor- for understanding the disorder regardless of the presence or absence
mal population of activated cytotoxic T lymphocytes that produces of environmental factors. Although acquired AA is more common
M16_MCKE6011_03_SE_C16.indd 304 01/08/14 1:41 pm
Chapter 16 • hypoproliferative anemias 305
★ tabLe 16-2 Classification of Hypoproliferative Anemias
aplastic anemia Pure Red cell aplasia Other Hypoproliferative anemias
acquired Transitory infections Anemia of chronic renal disease
Idiopathic Acquired pure red cell aplasia Anemia associated with endocrine
Drugs: chloramphenicol, phenylbutazone, gold compounds, Acute: infections, transient erythrobla- abnormalities
sulfa drugs, antihistamines, antithyroid, tetracyclines, penicillin, stopenia of childhood (TEC), drugs
methylphenylethylhydantoin Chronic: thymoma, autoimmune disorders
Chemical agents: benzene, insecticides, hair dye, carbon tetra-
chloride, chemotherapeutics (vincristine, busulfan, etc.), arsenic
Ionizing radiation
Biological agents: parovirus, infectious mononucleosis, infec-
tious hepatitis, measles, influenza, errors of amino acid metabo-
lism, starvation
Pregnancy
Paroxysmal nocturnal hemoglobinuria
inherited inherited
fanconi anemia Diamond-Blackfan anemia
familial aplastic anemia
Dyskeratosis congenital
Congenital amegakarocytic thrombocytopenia
Shwachman-Diamond syndrome
in adults, it is also an infrequent cause of aplasia in children. Both United States, indicated that an elevated risk of developing AA was
idiopathic and other acquired forms of AA are discussed in the next associated with exposure to only a small number of substances, includ-
sections. ing sulfonamides, thiazide diuretics, and mebendazole. In the Thai
Inherited forms of AA result in chronic failure of the bone mar- study, an increased risk was not found for chloramphenicol, a drug
10
row, which may or may not be present at birth. A congenital condition frequently implicated in case reports of AA. Other drugs that have
is one that is manifest early in life, often at birth, but that is not neces- been implicated include gold, anticonvulsants, nonsteroidal analgesics,
1
sarily inherited; it can be caused by acquired factors (e.g., maternal antiprotozoals, and antithyroid medications. Most individuals taking
exposure to an environmental toxin while pregnant). Inherited AA such medications, however, do not develop AA. One possible explana-
is quite rare and is often associated with other congenital anomalies. tion is that persons with diminished P-glycoprotein, an efflux pump
that is the product of the multi-drug resistance gene MDR-1, may have
excessive accumulation of drugs that can increase susceptibility to HSC
11
case study (continued from page 304) damage. In cases associated with drug exposure, the pathophysiology
is thought to involve an abnormal immune response to the HSC.
3. Evaluate the relationship between Rachael’s age and The widespread use of toxic chemical agents in industry and
the likelihood that she has aplastic anemia. agriculture is probably responsible for some cases of bone marrow
4. If aplastic anemia is present, would you expect her to aplasia. Benzene derivatives are well established as a cause of bone
have an idiopathic or secondary form? Explain your marrow suppression. Although most cases develop within a few weeks
answer. after exposure, some occur after months or years of chronic exposure.
Although stem cells can be damaged, the main toxic effect of benzene
is usually expressed on transient stages of committed proliferating
precursor cells. Elevated risk of AA was documented in the Thai study
acquired Forms of aplastic anemia for persons exposed to benzene and other solvents as well as agricul-
idiopathic tural pesticides such as organophosphates and DDT and for persons
The majority of cases of aplastic anemia cannot be linked to an envi- who drink nonbottled water in rural regions. 10
ronmental factor and are referred to as idiopathic. It is possible that Most of the cytotoxic drugs used in chemotherapy of malignant
previous exposure to an unrecognized agent or event could be respon- diseases kill rapidly proliferating cells. However, the drugs do not distin-
sible for stimulating the immune system. guish between malignant and normal cells. Therefore, all proliferating cells
drugs and chemical agents are damaged, including normal cells of the hematopoietic compartment.
Recent research has shown that exposure to drugs or chemical agents Although quiescent (G ) stem cells are spared from immediate drug
0
is rarely associated with aplastic anemia, although historically these exposure, repeated doses of the drug over a long period of time can even-
associations were given prominent attention. A study conducted in tually deplete the remaining HSCs as they enter the proliferating pool. In
Thailand, where the incidence of AA is 2–3 times higher than in the most cases, timely withdrawal of the drug leads to hematopoietic recovery.
M16_MCKE6011_03_SE_C16.indd 305 01/08/14 1:41 pm
306 seCtion iii • the anemias
ionizing Radiation PNH is an acquired stem cell disease in which a blood cell mem-
Aplastic anemia has been encountered in persons exposed to ionizing brane abnormality increases the cells’ susceptibility to in vivo comple-
radiation in industrial accidents, military nuclear tests, and therapeutic ment-mediated hemolysis. PNH is caused by mutations in the PIGA
regimens for malignancy. Ionizing radiation is directly toxic to HSCs gene resulting in the absence of a glycolipid that serves to attach and
and HPCs, and the effects are dose dependent. Small doses affect all anchor proteins to the cell membrane. Although considerable varia-
cells but are especially destructive to rapidly proliferating cells. The tion in clinical manifestations occurs, the typical picture in severe
bone marrow can recover from sublethal doses of irradiation because cases is pancytopenia and marrow hypoplasia. Between 40–50% of
quiescent stem cells are induced to begin proliferating after exposure- patients with an initial diagnosis of AA develop an abnormal eryth-
induced depletion of more differentiated progeny. Therapy in these rocyte population similar to that seen in PNH, but they do not usually
cases is mostly supportive and needed only until hematopoietic func- develop the full clinical manifestations of PNH (intravascular hemoly-
14
tion is restored. With high doses (74000 rads), bone marrow aplasia sis and venous thrombosis). PNH is a prominent, late complication
and peripheral blood pancytopenia are usually permanent. in many AA patients who have received immunosuppressive ATG
15
therapy. PNH is discussed in detail in Chapter 20.
infectious agents
Viral and bacterial infections can be followed by a transient cytopenia.
The aplasia can be limited to the erythroid elements or can include all
three cell lineages. Aplasia has been described in patients after recov-
ery from infectious mononucleosis, tuberculosis, and hepatitis but is case study (continued from page 305)
typically transient. Hepatitis-associated AA occurs most frequently in
males and does not appear to be caused by any of the known hepatitis for the past 3 months, rachael’s family physician
12
viruses. It is often fatal if untreated. An aplastic crisis in patients with has been following her recovery from viral hepatitis.
hereditary hemolytic anemias is commonly associated with human Her recovery was uneventful; her liver enzyme lev-
parvovirus infection. This aplasia, however, is limited to the erythroid els returned to normal within 2 months. She has no
lineage. other past medical history. There is no family history
In Epstein-Barr viral infections as well as other viral infections, of hematologic disorders.
the virus can infect the HSC, triggering an immune response. Cyto- 5. What aspect of this patient’s history could be associ-
toxic lymphocytes then destroy the virus-infected stem cell. Other ated with the occurrence of aplastic anemia?
mechanisms of stem cell damage in viral infections have been postu-
lated, including direct cytotoxicity of the virus and inhibition of cel-
lular proliferation and differentiation. 4
metabolic inherited aplastic anemia
The rare inborn errors of amino acid metabolism, which result in A number of disorders have a congenital predisposition for develop-
accumulation of ketones and glycine, have been associated with aplas- ing AA, as described below.
tic anemia. Fanconi anemia
Starvation or protein deficiency results in hypoproliferative ane- Fanconi anemia (FA) is an autosomal recessive (or rarely X-linked
mia after about 3 months of deprivation. Starvation that is not self- recessive) disorder resulting from a variety of molecular defects and is
induced usually occurs in areas where other endemic pathologies are characterized by childhood-onset aplastic anemia, abnormal chromo-
also present, such as parasitic infection and blood loss. Thus, the causes somal fragility, and an increased predisposition to developing leuke-
of this anemia can be multifactorial. mia and cancer. FA has a prevalence of about 1–5 per million persons
Decreased hormonal stimulation of hematopoietic precursor in North America with a frequency of the heterozygous carrier state
cells is important primarily as a factor in erythroid hypoplasia. Renal of 1 in 300. Patients can have a complex assortment of congenital
16
disease and endocrine diseases are examples of hypoproliferation anomalies in addition to progressive bone marrow hypoplasia. The
caused by a decrease in erythropoietin. congenital defects include dysplasia of bones, renal abnormalities, and
A life-threatening pancytopenia rarely can occur during preg- other organ malformations as well as mental retardation, dwarfism,
nancy. The condition usually remits after delivery or abortion. It has microcephaly, hypogonadism, and skin hyperpigmentation. Some
been suggested that the aplasia may be related to estrogen inhibition patients lack congenital defects and may not be diagnosed until AA
of stem cell proliferation. 13 develops, which occasionally may not occur until adulthood. 17
Aplastic anemia eventually develops in 90% of FA patients. The
association with clonal disorders hematological manifestations are generally slowly progressive from
Even prior to the widespread use of immunosuppressive therapy birth and need to be monitored closely. Clinical signs of pancytopenia
for AA, patients occasionally developed clonal disorders, including usually appear between the ages of 5 and 10 years with the median
3
paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplastic age at diagnosis of 6.5 years. Anemia is usually macrocytic (although
syndromes (MDS). Thus, the development of these disorders in AA it can be normocytic) with macrocytosis often preceding anemia.
patients is not necessarily related to the treatment regimens currently Erythrocytes often show increased levels of Hemoglobin F and the i
used. As many as 15% of patients treated with immunosuppressive antigen (reflecting hematologic stress and the development of eryth-
therapy for aplastic anemia may develop MDS. 1 rocytes from earlier erythroid progenitor cells). Leukopenia primarily
M16_MCKE6011_03_SE_C16.indd 306 01/08/14 1:41 pm
Chapter 16 • hypoproliferative anemias 307
involves the granulocytes. Thrombocytopenia often precedes anemia Patients with Shwachman-Diamond Syndrome (SDS) can present
and leukopenia. Androgen therapy can reverse the pancytopenia for with some of the same congenital anomalies, including AA as is seen in
several years in about 50% of FA patients. G-CSF therapy can help FA patients. However, they also have exocrine pancreatic dysfunction
increase neutrophil counts. and malabsorption syndrome and do not display increased chromo-
HSCT offers the only possible curative treatment for bone mar- somal fragility and defective DNA repair processes. This disorder is due
row failure in FA patients. In patients with HLA-matched sibling to mutations of the SBDS gene, although the exact pathogenesis of the
donors, 5-year survival is close to 75%. Stem cells from HLA-matched disorder is still unknown. Diagnosis is confirmed by genetic testing. 3
unrelated donors have also been used. Because FA patients are acutely
sensitive to chemotherapeutic agents and radiation, pretransplant
conditioning protocols must be altered to reduce toxicity that could case study (continued from page 306)
increase the risk of developing a clonal disorder. Although HSCT can
cure the marrow failure in FA patients, the increased risk of nonhe- 6. Is it likely that Rachael has an inherited form of aplastic
matologic malignancies remains. anemia? Explain your answer.
The risk for developing cancer in FA is high and increases with
age. Acute nonlymphocytic leukemia and solid tumors are common
complications. Leukemia is difficult to treat, and survival is poor. With
improved treatment options, the median survival for FA patients is Clinical Findings
now 29 years of age. 3 The onset of symptoms in AA is usually insidious and related to
Karyotyping of FA cells shows increased spontaneous chromo- the cytopenias. Common initial signs are bleeding accompanied by
somal breakage, gaps, rearrangements, exchanges, and duplications. petechial and mucosal hemorrhages and infection. Pallor, fatigue,
Diagnosis of FA involves exposing blood lymphocytes or skin fibro- and cardiopulmonary complications can be present as the anemia
blasts to DNA crosslinking agents such as mitomycin C, diepoxybu- progresses.
1,3
tane (DEB), or cisplatin, which amplify chromosomal breakage. Hepatosplenomegaly and lymphadenopathy are absent. Spleno-
Molecular techniques can be used to identify specific mutations. Test- megaly has occasionally been noted in later stages of the disease, but
ing for FA is performed when warranted based on clinical findings if found in the early stages, the diagnosis of aplastic anemia should be
and/or abnormal blood counts, usually in infants and children. questioned.
The molecular defects in FA are heterogeneous. To date, muta-
tions in 13 genes that are associated with the FA phenotype have been Laboratory Findings
identified. Several of the FA-identified proteins function in a com- Laboratory studies of peripheral blood and bone marrow are essential
mon cellular DNA repair mechanism, which is activated in response if a diagnosis of aplastic anemia is suspected.
to DNA damage. Defective FA proteins are thus responsible for the
genetic instability associated with DNA damage, resulting in increased Peripheral blood
1,3
apoptosis of hematopoietic stem cells and AA. In addition to genetic Pancytopenia is typical. Although the degree of severity can vary, the
defects leading to defective DNA repair and DNA instability, TNFa diagnosis of AA should be questioned unless the leukocyte count,
is elevated in most patients. The telomeres of the HSC and HPC are erythrocyte count, and platelet count are all below the reference inter-
shortened, suggesting an abnormally high proliferative rate, probably vals. Hemoglobin is usually 670 g/L. Erythrocytes appear normocytic
induced by the marrow’s attempt to replace damaged HSCs/HPCs and and normochromic, or they can be slightly macrocytic. The presence
ultimately lead to HSC/HPC premature senescence. 9 of nucleated erythrocytes and teardrops is not typical of AA but sug-
Other causes of inherited aplastic anemia gests marrow replacement (myelophthisic anemia). Myelodysplastic
Several other rare disorders have a predisposition to developing AA. syndrome, rather than AA, is suggested by the presence of dysplastic
Dyskeratosis congenita (DC) is an inherited bone marrow failure syn- neutrophils and other abnormal cells. The relative reticulocyte count
drome, resulting in AA in 50% of cases. The physical abnormalities (%) can be misleading due to the severe anemia. Therefore, the reticu-
and mutations in patients with DC differ from those seen in patients locyte count should always be determined in absolute concentration
with FA. Most DC patients have dystrophic nails and leukoplakia of and/or be corrected for anemia before interpretation. The absolute
9
the oral mucosa, conditions not seen in FA. Mutations in several genes reticulocyte count is usually 625 * 10 /L. The corrected reticulo-
are associated with DC. Markedly reduced telomerase activity, telo- cyte count is 61%. Most often, thrombocytopenia is present at the
mere shortening, early HSC/HPC senescence, and a reduced stem cell time of diagnosis. Neutropenia precedes leukopenia; initially, lympho-
compartment are characteristic. There are multiple subtypes of DC cyte and monocyte counts are normal. Because of the neutropenia,
with varying clinical features. 3 the differential count reflects a relative lymphocytosis. When the leu-
9
Congenital amegakaryocytic thrombocytopenia (CAT) presents in kocyte count is below 1.5 * 10 /L, an absolute lymphocytopenia is
the neonatal period with isolated thrombocytopenia due to reduced also present. The band to segmented neutrophil ratio is increased, and
or absent marrow megakaryocytes. Aplastic anemia subsequently occasionally more immature forms are found. Neutrophil granules
develops in 45% of patients, usually in the first years of life. The are frequently larger than normal and stain a dark red; these granules
defect is a mutation in the MPL gene, which codes for the thrombo- should be distinguished from toxic granules, which are bluish black.
3
poietin (TPO) receptor. TPO is required for maintenance of HSC Flow cytometry of the peripheral blood can be ordered to detect
18
viability (Chapter 4). CD59+ cells when PNH is suspected (Chapter 20).
M16_MCKE6011_03_SE_C16.indd 307 01/08/14 1:41 pm
308 seCtion iii • the anemias
case study (continued from page 307) case study
7. Correlate Rachael’s clinical findings of weakness and Rachael was referred to a hematologist who ordered
shortness of breath as well as petechial hemorrhages a bone marrow examination. The aspirate obtained
and bruises with her laboratory screening results, was inadequate for evaluation due to lack of marrow
which follow. spicules. Only a single site was aspirated. Touch preps
Admission laboratory data for patient: made from the biopsy showed a markedly hypocel-
lular marrow with very few hematopoietic cells. Cells
12
RBC 2.42 * 10 /L present consisted of lymphocytes, plasma cells, and
Hb 71 g/L stromal cells. There were no malignant cells present.
Hct 0.24 L/L 14. Compare these results with those expected for a
9
PLT 8.0 * 10 /L person with aplastic anemia.
9
WBC 1.2 * 10 /L 15. Interpret the significance of the lack of malignant
Differential
Segmented neutrophils 2% cells and hematopoietic blasts.
Lymphocytes 94% 16. Suggest a way to improve the validity of bone marrow
monocytes 4% examination results for this patient.
Uncorrected reticulocyte count 0.7%
8. Evaluate each of Rachael’s laboratory results by com-
paring them to reference intervals. Other Laboratory Findings
9. Which of Rachael’s routine laboratory results are con- Other abnormal findings are not specific for aplastic anemia but are
sistent with those expected for aplastic anemia? frequently found associated with the disease. Hemoglobin F can be
10. Classify the morphologic type of anemia. increased, especially in children. Erythropoietin is often increased,
particularly when compared with the erythropoietin levels in patients
11. Calculate the absolute lymphocyte count. Are with similar degrees of anemia. Serum iron is increased with 750%
Rachael’s lymphocytes truly elevated as suggested saturation of transferrin, reflecting erythroid hypoplasia. The clear-
by the relative lymphocyte count?
59
ance rate of iron (Fe ) from the plasma is decreased because of the
12. Correct the reticulocyte count. Why is this step decrease in iron utilization by a hypoactive marrow. Patients who are
important? younger than age 50 should be screened for FA using tests for chromo-
18
13. Calculate the absolute reticulocyte count. somal breakage. Results of these tests will be normal in other forms
of inherited AA and in acquired/idiopathic forms of AA.
Prognosis and Therapy
Recent advances in treatment have tempered the previously grim prog-
bone marrow nosis of patients diagnosed with aplastic anemia. HSCT and immu-
Examination of the bone marrow is necessary to differentiate aplastic nosuppressive therapy (IST) have greatly improved survival. Presently,
anemia from other diseases accompanied by pancytopenia. In AA, the
bone marrow is hypocellular with 770% fat (Figure 16-1 ■). Thus, it
is often difficult to obtain an adequate sample. Bone marrow infiltra-
tion with granulomas or cancer cells can lead to fibrosis, also resulting
in a hypocellular dry tap on aspiration. Both aspiration and biopsy are
18
needed for a correct diagnosis. It is recommended that several dif-
ferent sites be aspirated because focal sampling of the marrow can be
misleading. Some areas of acellular stroma and fat can be infiltrated
with clusters of lymphocytes, plasma cells, and reticulum cells. Areas
of residual hematopoietic tissue termed hot spots can be found pri-
marily early in the disease but can occasionally be found in severe
refractory cases. Iron staining reveals many iron granules in macro-
phages, but granules are rarely seen in normoblasts. Flow cytometry
should be performed; the percentage of CD34+ cells in the bone mar- ■ FiguRe 16-1 Bone marrow preparation from a patient
row in AA is typically 60.3%. Bone marrow karyotyping is useful for with aplastic anemia shows marked hypocellularity (10%).
differentiating hypocellular forms of myelodysplastic syndromes from The patches of cells remaining are primarily lymphocytes
aplastic anemia. 1 (Wright-Giemsa stain; 100* magnification).
M16_MCKE6011_03_SE_C16.indd 308 01/08/14 1:42 pm
Chapter 16 • hypoproliferative anemias 309
19
the 5-year survival rate is 79%. Choice of definitive therapy for severe Differentiation of Aplastic Anemia
acquired AA depends on the age of the patient and availability of a from other Causes of Pancytopenia
matched donor. HSCT is recommended for patients up to age 45 who Pancytopenia can be associated with disorders other than AA, but these
have a matched sibling donor, although some recommendations extend disorders differ in that the pancytopenia is not the result of a defect in
the age limit to age 55. HSCT is also recommended for patients up to stem cell proliferation. Rather, the bone marrow can be normocellular,
age 21 with a fully compatible HLA-matched unrelated donor. IST is hypercellular, or infiltrated with abnormal cellular elements.
recommended for patients without a matched related donor, the situa-
tion faced by the majority of patients with aplastic anemia. 20
Before beginning HSCT or IST, putative causative drugs should myelophthisic anemia
be withdrawn or the patient removed from a hazardous environment. Myelophthisis signifies marrow replacement or infiltration by fibrotic,
The immediate treatment is often supportive with the administration granulomatous, or neoplastic cells (Chapter 21). The abnormal replace-
of erythrocytes, platelets, and antibiotics. Granulocyte transfusions ment cells reduce normal hematopoiesis and disrupt the normal bone
can be given to severely neutropenic patients with life-threatening marrow architecture, allowing the release of immature cells into the
18
sepsis. To avoid alloimmunization, transfusion should be mini- peripheral blood. Anemia can be accompanied by normal, increased,
mized if HSCT is anticipated. Irradiated and/or leukocyte-reduced or decreased leukocyte and platelet counts. The most characteristic find-
blood products can be ordered (Chapter 1). Hematopoietic growth ings are a leukoerythroblastic reaction and a moderate to marked
factors such as G-CSF generally do not have a beneficial effect on poikilocytosis. Dacryocytes are common, as are large bizarre plate-
patients with AA and may increase the risk of MDS. 5 lets. By contrast, nucleated erythrocytes and significant morphologic
HSCT using cells collected from bone marrow has become a rela- changes are almost never found in the peripheral blood in AA. Myelo-
tively common procedure and is curative in many patients with aplastic phthisic anemia is associated with diffuse cancer of the prostate, breast,
anemia. Bone marrow is preferred over peripheral blood stem cells due and stomach and is typical of myelofibrosis and lipid storage disorders.
to the increased risk of graft-vs-host disease with the latter. Antithymo-
cyte globulin (ATG) and cyclosporine are typically used as precondi- myelodysplastic syndromes
tioning regimens to suppress the immune response of the recipient. Myelodysplastic syndromes (MDS) are a group of hematologi-
20
Preconditioning is nonmyeloablative. The current 5-year survival cal disorders that have a propensity to terminate in acute leukemia
rate is 77% for HLA-matched sibling donors. Survival rates are high- (Chapter 25). The principal peripheral blood findings are pancyto-
4
est for children and for patients who have been minimally transfused. penia, bicytopenia, or isolated cytopenias with reticulocytopenia. A
Treatment complications and post-transplant mortality remain high, macrocytic anemia is common. The bone marrow, however, is usu-
especially in older patients; therefore, the probability for long-term cure ally normocellular or hypercellular with various degrees of qualitative
must be weighed against the inherent risks of complications, including abnormalities of one or more cell lineages (dyshematopoiesis). Signs
graft-vs-host disease and early and late toxicities of the conditioning of dyshematopoiesis are also reflected in morphologic abnormalities
regimen. Although engraftment of HSCs is successful in many cases, of one or more cell lineages in the peripheral blood. These qualitative
some transplants, even when performed between identical twins, do abnormalities of peripheral blood cells are useful in differentiating
not correct the AA. These unsuccessful transplants suggest that the cytopenias due to true hypoproliferation of stem cells (AA) from cyto-
donor HSC growth is suppressed by the same immune mechanism penias due to ineffective erythropoiesis.
that induced the original aplasia (Chapter 29). An additional constraint A subgroup of patients with MDS can have a hypocellular mar-
to HSCT is that matched sibling donors are available for only 20–30% row. This condition is referred to as hypoplastic MDS (HMDS). In
of patients with aplastic anemia. Combined, intensive immunosup- these cases, differentiation of MDS and AA can be complicated if
pressive therapy (IST) using ATG in combination with cyclosporine there are few bone marrow cells from a biopsy or aspirate to exam-
has become standard treatment for those patients with acquired ine. If granulocytic and megakaryocytic dysplasias are seen and/or if
severe aplastic anemia who lack a suitable bone marrow donor. IST an abnormal karyotype consistent with MDS is present, HMDS can
is effective in restoring hematopoiesis in 60–90% of patients who are be diagnosed. However, if only erythroid dysplasia is present, the evi-
over 45 or who lack an HLA-matched sibling donor. 4,18 More favorable dence is insufficient to differentiate HMDS and AA because erythroid
outcomes are observed in children. Relapse requiring additional IST dysplasia can be found in some cases of AA. 15
occurs in 30–40% of patients. About 15% of patients treated with IST
develop clonal disorders such as PNH, leukemia, and myelodysplastic congenital dyserythropoietic anemia
syndromes. 4 Congenital dyserythropoietic anemia (CDA) is a very rare familial refrac-
tory anemia characterized by both abnormal and ineffective eryth-
ropoiesis. The bone marrow is normocellular or hypercellular, but the
case study (continued from page 308) peripheral blood is pancytopenic. In contrast to the normal erythroid
precursors found in aplastic anemia, erythrocyte precursors in the bone
17. Appraise the prognosis for Rachael. marrow of CDA patients exhibit multinuclearity, and myeloblasts and
18. Predict a treatment regimen. promyelocytes are increased. The anemia can be normocytic, but most
often it is macrocytic. The three types of CDA are discussed in Chapter 15.
M16_MCKE6011_03_SE_C16.indd 309 01/08/14 1:42 pm
310 seCtion iii • the anemias
Hypersplenism ★ tabLe 16-3 Laboratory Findings in Pure Red
Hypersplenism due to a variety of causes can result in a decrease Cell Aplasia
of one or more cellular elements of the blood as these elements
become pooled and sequestered in the spleen. In this condition, the test Findings
bone marrow is hyperplastic, corresponding to the peripheral blood Erythrocyte count, Decreased
cytopenia. Anemia is accompanied by a reticulocytosis as opposed hematocrit, and
hemoglobin
to the reticulocytopenia found in the true hypoproliferative anemias.
Granulocytopenia can be accompanied by a shift to the left in these Reticulocyte count Severely decreased
cells. Splenomegaly and other findings of the underlying disease are Leukocyte count normal or possibly increased
important in diagnosing this disorder. Splenectomy, although not Platelet count normal or possibly increased
always advisable, corrects the cytopenias. Bilirubin Low or normal
Bone marrow Absence of erythroid cells if examined early in
disease; if examined later in disease progression,
Other increase in young erythroid cells, which can be
Deficiency of cobalamin or folic acid can be accompanied by mistaken for an erythroid maturation arrest. If the
pancytopenia (Chapter 15). The bone marrow in these cases, however, patient is followed, however, these cells show
normal maturation and differentiation. There is
reveals normocellularity or hypercellularity with megaloblastic normal myelopoiesis and granulopoiesis.
changes. In the peripheral blood, hypersegmented neutrophils and
Howell-Jolly bodies are typical but are not found in pancytopenia of
aplastic anemia. other cancers, autoimmune disorders, infection following admin-
istration of certain drugs, immunosuppressive therapy, and post-
21
transplantation. Transient erythroblastopenia of childhood (TEC)
CheCkpoint 16-2 is an acquired, self-limiting form of erythroid hypoplasia found in
A pancytopenic patient has a presumptive diagnosis of children. Inherited forms of PRCA, including Diamond-Blackfan
aplastic anemia. While performing a smear review, you anemia, are discussed in the section “Diamond-Blackfan Anemia.”
observe the presence of several types of poikilocytes, The underlying pathophysiology of PRCA is variable and depends on
dysmorphic neutrophils, and large agranular platelets. Do associated clinical conditions. Treatment of the underlying condition
these findings support the presumptive diagnosis? If not, can resolve the anemia, but unresponsive cases are considered to have
22
which disorder(s) would be more likely for this patient? an immune basis. Refer to Table 16-3 ★ for results of laboratory
tests in PRCA.
Acquired Acute Pure Red Cell Aplasia
case study (continued from page 309) Viral (parvovirus, Epstein-Barr virus, and viral hepatitis) and/or
bacterial infections can be associated with a temporary suppression
19. What other hematologic conditions must be ruled of erythropoiesis. B19 parvovirus infects erythroid progenitor cells,
out for Rachael? lyses the target cell, and blocks erythropoiesis. Infection, as well as
20. What laboratory test is most beneficial in differen- the block to erythropoiesis, is terminated by the production of neu-
tiating aplastic anemia from these other disorders? tralizing antibodies to the virus. In individuals who have a normal
Compare the expected results of AA with those of erythrocyte life span and hemoglobin level, temporary erythroid
the other disorders. hypoproliferation is not noticed. However, if the erythrocyte life
span is decreased due to an underlying condition, the complica-
tion of sudden erythroid hypoproliferation (aplastic crisis) can be
life threatening. These aplastic crises are most frequently noted in
PuRe Red ceLL aPLasia patients with hemolytic anemias including sickle cell anemia, par-
Pure red cell aplasia (PRCA) is a heterogeneous group of disorders oxysmal nocturnal hemoglobinuria, and autoimmune hemolytic
characterized by a selective decrease in erythroid precursor cells in the anemias.
marrow and by peripheral blood anemia. The anemia can be severe The aplastic crisis cases brought to the attention of a physician
and is typically normocytic, normochromic. PRCA is thought to probably represent only a minor fraction of the actual occurrence of
occur due to selective hypoproliferation of the committed erythroid temporary erythroid aplasia. The aplastic crises are often preceded
progenitor cell. The term aplastic anemia should be avoided in by fever with upper respiratory and intestinal complaints. Several
describing PRCA because there is no disturbance of granulopoiesis members of the same family are frequently affected with the illness.
or thrombopoiesis. Reticulocytes can be present but are 61% when Patients with concurrent hemolytic anemia have a rapid onset of
corrected for the degree of anemia. PRCA can be acquired (acute or lethargy and pallor. Patients without hemolytic anemia may seek
chronic) or inherited and can affect any age group. Acquired PRCA medical attention because of the primary illness, and anemia is only
can develop in patients with thymoma, hematopoietic neoplasms, an incidental finding.
M16_MCKE6011_03_SE_C16.indd 310 01/08/14 1:42 pm
Chapter 16 • hypoproliferative anemias 311
If the anemia is severe, supportive therapy of packed erythrocyte of cases have a mutation of the RPS19 gene (which encodes for a pro-
transfusions may be necessary until spontaneous recovery occurs. tein involved in ribosome assembly). Other genes mutated in patients
PRCA associated with viral hepatitis has a poor prognosis. with DBA have been identified and although the basic defect in most
Acute erythroid hypoplasia also occurs with the administration cases appears to be defective ribosome biogenesis, the exact functional
of some drugs and chemicals. After removal of the inciting agent, defect in all mutations is not clear. A wide range of physical abnor-
normal erythropoiesis usually resumes. malities can be observed in approximately 50% of DBA patients, and
21
Transient erythroblastopenia of childhood (TEC) is a form of patients are predisposed to occurrences of cancer. Many cases have
acquired acute pure red cell aplasia, but because of the importance no familial pattern, suggesting spontaneous mutations or acquired
of distinguishing this pediatric anemia from Diamond-Blackfan disease.
anemia (DBA), TEC will be discussed with DBA in the section The anemia in DBA is not due to a deficiency of erythro-
“ Diamond-Blackfan Anemia.” poietin (EPO) because EPO levels are consistently increased
and actually higher than expected for the degree of anemia. The
Chronic Acquired Pure Red Cell Aplasia EPO is active, and no antibodies directed against this cytokine
are found, nor are there apparent mutations in either EPO or the
An acquired selective depression of erythroid precursors is a rare EPO receptor. The probable defect in DBA is an intrinsic defect of
disorder encountered in middle-aged adults. This disease usually erythroid progenitor cells, resulting in their inability to respond
occurs in association with thymoma, autoimmune hemolytic normally to inducers of proliferation and differentiation. Hence,
anemia, systemic lupus erythematosus, rheumatoid arthritis, or there is typically a deficit of erythroid precursors in the marrow
hematologic neoplasms (MDS, large granular lymphocytic leu- of patients with DBA. Physical findings and symptoms are those
21
kemia, chronic lymphoid leukemia, Hodgkin disease). The high associated with anemia or related to the congenital defects. Anemia
incidence of autoimmune disorders associated with this disorder is severe with erythroid hypoplasia in an otherwise normocellular
suggests that an immunologic mechanism could be responsible for bone marrow.
the red cell aplasia. Cytotoxic antibodies to erythropoietin-sensitive Diagnostic criteria for DBA are included in Table 16-4 ★.
21
cells in the marrow and to erythropoietin have been demonstrated Testing for RPS19 mutations is helpful in only about 20–25% of cases.
in some cases. More commonly, the mechanism appears to be a Testing for the chromosomal breakage associated with FA is negative.
T cell-mediated immunosuppression of erythropoiesis. In some Vitamin B and folate levels are normal. Fetal-like erythrocytes are
12
cases, PRCA may be the initial presenting sign of the underlying present; hemoglobin F is increased to 5–25%, and the i antigen is
disorder. 21 increased. The presence of fetal-like erythrocytes is not particularly
Clinical findings are nonspecific. Pallor is usually the only useful in diagnosing DBA in children younger than 1 year of age
physical finding. Therapies include transfusion with packed because children in this age group normally possess erythrocytes with
erythrocytes, thymectomy if the thymus is enlarged, and fetal characteristics. Serum iron and serum ferritin are increased, and
immunosuppression. Various drugs, including corticosteroids, transferrin is 100% saturated.
cyclosporine, cytotoxic agents, ATG, and monoclonal antibodies Therapy includes erythrocyte transfusions and administration of
against B lymphocytes (rituximab/anti-CD20, alemtuzumab/ adrenal corticosteroids. Up to half of DBA patients develop prolonged
anti-CD52), have been used to suppress the immune response. remission, but most eventually require additional therapy. Transfusion
Androgens or erythropoietin to stimulate erythrocyte produc- dependence requires iron-chelation therapy (deferroximine). Most
tion is rarely used. In drug-induced red cell aplasia, withdrawal of deaths are due to complications of therapy such as hemosiderosis.
the putative agent is indicated. Approximately 80% of patients with DBA must be distinguished from transient erythroblastopenia
acquired PRCA have a spontaneous remission or remission induced of childhood (TEC), a temporary suppression of erythropoiesis
by immunosuppression. About half will relapse, but with additional that frequently occurs after a viral infection in otherwise nor-
immunosuppression, 80% will enter a second remission. By retreating mal children (Table 16-5 ★). The age of onset of TEC ranges
relapsing patients or continuing maintenance immunosuppression, from 1 month to 10 years with most cases occurring in children
22
many can be maintained transfusion-free for years. HSCT is rarely over the age of 2. Progressive pallor in a previously healthy child
indicated for acquired pure red cell aplasia.
Diamond-Blackfan Syndrome
★ tabLe 16-4 Diagnostic Criteria for
Diamond-Blackfan anemia (DBA) is a rare congenital pro- Diamond- Blackfan Anemia
gressive erythrocyte aplasia that occurs in very young children.
Anemia is present at birth in 25% of cases, and in 98% of cases, macrocytic (or normocytic) normochromic anemia in first year of life
21
diagnosis is made within the first year of life. Unlike Fanconi Reticulocytopenia
anemia, there is no leukopenia or thrombocytopenia. The anemia normocellular marrow with marked erythroid hypoplasia
is typically severe. Increased serum erythropoietin (EPO)
DBA is actually a diverse family of diseases with a common normal or slightly decreased leukocyte count
hematologic phenotype. Evidence exists for both autosomal domi- normal or increased platelet count
nant and autosomal recessive modes of inheritance. About 20–25%
M16_MCKE6011_03_SE_C16.indd 311 01/08/14 1:42 pm
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Clinical Laboratory Hematology (3rd Edition) ( PDFDrive )
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