Congenital and Acquired Hemolytic Anemias

2002
Ware
1. A four year old Caucasian male is referred to you for jaundice and possible
hemolytic anemia. The referring pediatrician says that the child has always
had mild scleral icterus and now has a palpable spleen. Recent laboratory
studies include the following: hemoglobin concentration = 9.8 gm/dL;
absolute reticulocyte count = 313 x 10
9
/L; total bilirubin = 4.0 mg/dL (direct
fraction = 0.4 mg/dL, indirect fraction = 3.6 mg/dL). A hemoglobin
electrophoresis on cellulose acetate shows predominantly hemoglobin A but
several additional faint bands that could not be identified. Which of the
following tests would be the most informative?

A. Osmotic fragility testing
B. Hemoglobin electrophoresis on acid citrate
C. Autohemolysis testing
D. Heinz body preparation
E. Quantitative G6PD assay

2. A ten week old African American male is referred to you for evaluation and
management of an abnormal FS newborn hemoglobinopathy screen. Family
testing reveals that the father has HbAS, while the mother has only HbA. You
repeat the infants studies and the lab now reports an FSA pattern on
hemoglobin electrophoresis. The most likely diagnosis that explains this
infants laboratory studies is:

A. Non-paternity
B.
+
thalassemia
C.
0
thalassemia
D. Sickle cell trait
E. Sickle cell anemia

3. A nine year old Caucasian female is referred to you for evaluation of anemia
found on a routine physical examination. She is active and the fastest person
on her soccer team. Your exam reveals mild icterus, a palpable spleen 2 cm
below the left costal margin, and the following laboratory studies:
hemoglobin concentration = 8.8 gm/dL; reticulocyte count = 423 x 10
9
/L;
MCV = 91 fL. On peripheral blood smear you note polychromasia, with
several echinocytes and an occasional spherocyte. There is no family history
of blood dyscrasia. The most likely diagnosis is:

A. Congenital spherocytosis from a spontaneous mutation
B. 5 nucleotidase deficiency
C. G6PD deficiency with extreme lyonization
D. Hereditary elliptocytosis
E. Pyruvate kinase deficiency

4. A previously well three year old Caucasian male presents with pallor, fatigue,
and dark urine. One day earlier, he was evaluated by his local pediatrician
who prescribed amoxicillin for a possible urinary tract infection, based on
dark amber urine and a urine dipstick with 3+ blood. At your evaluation, you
learn that the child has not been on any other medications and there is no
family history of blood disorders. His exam shows pallor, minimal scleral
icterus, and no hepatosplenomegaly. Hemoglobin concentration = 5.5 gm/dL;
reticulocyte count = 178 x 10
9
/L; WBC = 12.1 x 10
9
/L with a left shift;
platelets = 291 x 10
9
/L. The urine is reddish brown with 2+ protein and 4+
blood on dipstick, and 3 RBCs and 3 WBCs per high power field on
microscopic analysis. You suspect an autoimmune hemolytic process as the
etiology for this sudden-onsent hemoglobinuria, but the direct antiglobulin test
(DAT) performed at room temperature is negative. To test this child for a
Donath-Landsteiner antibody, you would need to do which of the following:

A. Repeat the DAT at 4
o
C
B. Repeat the DAT at 37
o
C
C. Collect the blood at 37
o
C, then test the serum at 37
o
C in an indirect
antiglobulin test
D. Collect the blood at 37
o
C, then incubate serum and cells at 4
o
C and then
37
o
C
E. Collect the blood at 37
o
C and repeat the DAT at 37
o
C

5. A term 3.1 kilogram female infant is noted to have jaundice at 18 hours of
life. The mother is A+ and the baby is O+, and the infants DAT is negative.
Laboratory evaluation on the baby reveals the following: hemoglobin = 14.9
gm/dL; total bilirubin = 15 mg/dL (all indirect). The peripheral blood smear
shows numerous spherocytes. The infant does well with phototherapy and
supportive care, and is discharged home at 4 days of life. Three weeks later,
the infant is thriving with a hemoglobin concentration of 13.4 gm/dL and total
bilirubin concentration of 3.1 mg/dL. What is the best diagnostic test to help
establish the etiology of this infants hemolysis?

A. Repeat DAT on the infants RBCs
B. Screening of the maternal serum for RBC antibodies
C. Tagged RBC study
D. Osmotic fragility testing at 1 month of life
E. Osmotic fragility testing at 6-12 months of life

6. A pediatrician calls you for telephone advice about a 16 month old African-
American male with microcytic hypochromic anemia. The anemia was
detected on routine screening at one year of life, and several months of oral
iron supplementation has not changed the blood counts. Latest laboratory
evaluation reveals hemoglobin concentration = 9.7 gm/dL; MCV = 69 fL;
reticulocyte count = 80 x 10
9
/L. The hemoglobin electrophoresis shows only
normal HbA and the serum ferritin = 51 ng/ml. Review of the newborn
hemoglobinopathy screen reveals an FA pattern and 4% Hb Barts. The
correct diagnosis and follow-up is:

A. Hemoglobin H disease that warrants a prompt appointment for transfusion
therapy
B. Hemoglobin H disease that needs no further follow-up
C.
D. t needs education and counseling only
E.

7. A previously healthy 16 year old Caucasian male is referred to you for
evaluation of abnormal laboratory studies. He is an active football player who
was evaluated for a pre-sports physical. Review of systems reveals only
occasional abdominal pain. The abnormal laboratory studies include:
hemoglobin concentration = 9.9 gm/dL; MCV = 104 fL; reticulocyte count =
117 x 10
9
/L; WBC = 3.0 with 26% polys, 58% lymphs, 15% monocytes, and
1% eosinophil. The platelet count is 119 x 10
9
/L. Urinalysis specific gravity
= 1.009, pH = 6.5, 2+ blood on dipstick, occasional casts but no RBCs or
WBCs on microscopic analysis. The best diagnostic test to order is which of
the following:

A. CD59 testing on erythrocytes
B. Hepatitis B serologies
C. Direct platelet antibody measurement
D. Erythrocyte adenosine deaminase activity
E. Direct antiglobulin test

8. A six week old Pakistani male is referred to you for evaluation of an abnormal
HbF only result on newborn hemoglobinopathy screening. The infant
appears healthy, and the only abnormality you detect on physician
examination is a palpable spleen tip 1-2 cm below the left costal margin. The
infants hemoglobin concentration is 9.5 gm/dL. Repeat hemoglobin
electrophoresis confirms HbF only. The mothers electrophoresis reveals
HbA with 6.1% HbA
2
, but the father is unavailable for testing. The most
likely diagnosis is:

A.
B.
C. assemia
D.
E.

9.
2

2
) has a decreased affinity for oxygen in
which of the following settings?

A. Carbon monoxide intoxication
B. Exposure to sulfa drugs
C. Increased 2,3 DPG
D. Methemoglobinemia
E. Alkalosis

10. You are called by a local pediatrician regarding a five year old African-
American male with recurrent otitis media. The physician is currently treating
him for his fourth episode of otitis media over the past 6 months. The doctor
would like to prescribe trimethoprim/sulfamethoxazole prophylaxis, but is
unsure about its safety since the G6PD status of the child is unknown. Which
of the following responses is correct?

A. All African-American children should be tested for G6PD activity before
initiating sulfa therapy
B. African-American males and females should be tested if there is a family
history of hemolysis after sulfa exposure
C. Only African-American males should be tested before using sulfa
D. No testing is warranted since the African-American variant of G6PD is
relatively mild
E. No testing is warranted since measurement of G6PD activity does not
predict hemolysis

11. Hemoglobin Constant Spring is best described as which of the following:

A. -chain variant with a 2 kb DNA deletion
B. -chain variant with a point mutation in the promoter
C. -chain variant with a point mutation at the stop codon
D. -
E. -chain variant with increased HbF levels

12. Hemoglobin E is best described as which of the following:

A. Thalassemia found in Southeast Asia
B. Thalassemia found in Mediterranean regions
C. Hemoglobinopathy found in Southeast Asia
D. Hemoglobinopathy found in Mediterranean regions
E. Thalassemic hemoglobinopathy

13. What is the
0
thalassemia trait?

A. Hb F on newborn hemoglobinopathy screening
B. Hb A
2
on newborn hemoglobinopathy screening
C. Hb Barts on newborn hemoglobinopathy screening
D. Hb A
2
after one year of life
E. Hb Barts after one year of life

14. Compared to normal erythrocytes, spherocytes have:
A. Increased osmotic fragility at all saline concentrations
B. Increased osmotic fragility at 0.5% saline
C. Decreased osmotic fragility at 0.9% saline
D. Increased levels of 2,3 DPG
E. More Howell-Jolly bodies

15. Hemoglobin H is most commonly present in which of the following
situations?
A.
B.
C.
D.
0 +
thalassemia intermedia
E.

16. In the deoxygenated state, the mutation in sickle hemoglobin primarily affects
which of the following interactions?

A.
B.
C. Hemoglobin tetramer to Hemoglobin tetramer
D. Heme ring to hemoglobin
E. Erythrocyte to erythrocyte

2004
Ware
A ten week old African American male is referred for evaluation and management of
an abnormal FS newborn hemoglobinopathy screen. Family testing reveals that the
father has HbAS, while the mother has only HbA. You repeat the infants studies and
the lab now reports an FSA pattern on hemoglobin electrophoresis. The most likely
diagnosis for this infant that explains these laboratory studies is:

A. Non-paternity
B.
+
thalassemia
C.
0
thalassemia
D. Sickle cell trait
E. Sickle cell anemia



2. A six week old Pakistani male is referred to you for evaluation of an abnormal HbF
only result on newborn hemoglobinopathy screening. The infant appears healthy,
and the only abnormality you detect on physician examination is a palpable spleen tip
1-2 cm below the left costal margin. The infants hemoglobin concentration is 9.5
gm/dL. Repeat hemoglobin electrophoresis confirms HbF only. The mothers
electrophoresis reveals HbA with 6.1% HbA
2
, but the father is currently unavailable
for testing. The most likely diagnosis is:

A.
B.
C.
D.
E.



3.
2

2
) has a decreased affinity for oxygen in which of the
following settings?

A. Carbon monoxide intoxication
B. Exposure to sulfa drugs
C. Increased 2,3 DPG
D. Methemoglobinemia
E. Alkalosis




4. A pediatrician calls you for telephone advice about a 26 month old African-American
male with microcytic hypochromic anemia. The anemia was detected on routine
screening at one year of life, and intermittent trials of oral iron supplementation have
not improved the blood counts. Latest laboratory evaluation reveals hemoglobin
concentration = 9.7 gm/dL; MCV = 66 fL; reticulocyte count = 80 x 10
9
/L. The
hemoglobin electrophoresis shows only normal HbA and the serum ferritin = 51
ng/ml. Review of the newborn hemoglobinopathy screen reveals an FA pattern and a
fast band. The most likely diagnosis and follow-up is:

A. rants a prompt appointment for
transfusion therapy
B. -up
C.
D.
E. On



5. Hemoglobin Constant Spring is best described as which of the following:

A. -chain variant with a 2 kb DNA deletion
B. -chain variant with a point mutation in the promoter
C. -chain variant with a point mutation at the stop codon
D. -
E. -chain variant with increased HbF levels



6. Homozygous Hemoglobin E is best described as which of the following:

A. Thalassemia found in Southeast Asia
B. Thalassemia found in Mediterranean regions
C. Hemoglobinopathy found in Southeast Asia
D. Hemoglobinopathy found in Mediterranean regions
E. Thalassemic hemoglobinopathy



7.
0
thalassemia trait?

A. Hb F on newborn hemoglobinopathy screening
B. Hb A
2
on newborn hemoglobinopathy screening
C. Hb Barts on newborn hemoglobinopathy screening
D. Elevated Hb A
2
after one year of life
E. Presence of Hb Barts after one year of life




8. Hemoglobin H is most easily identified in which of the following situations?
A. 3-g
B.
C.
D.
0 +
thalassemia intermedia
E.



9. In the deoxygenated state, the mutation in sickle hemoglobin primarily affects which
of the following interactions?

A.
B.
C. Hemoglobin tetramer to Hemoglobin tetramer
D. Heme ring to hemoglobin
E. Erythrocyte to erythrocyte










ANSWER KEY:

1. B
2. A
3. C
4. D
5. C
6. E
7. D
8. A
9. C







Questions for Pediatric Hematology/Oncology Board Review 2004
Hemolytic Anemia Russell E. Ware, MD, PhD



1. A four year old Caucasian male is referred for jaundice and possible hemolytic
anemia. The referring pediatrician says that the child has always had mild scleral
icterus and now has a palpable spleen. Recent laboratory studies include the
following: hemoglobin concentration = 9.8 gm/dL; absolute reticulocyte count = 313
x 10
9
/L; total bilirubin = 4.0 mg/dL (direct fraction = 0.4 mg/dL, indirect fraction =
3.6 mg/dL). A hemoglobin electrophoresis on cellulose acetate shows predominantly
hemoglobin A but several additional faint bands that could not be identified. Which
of the following tests would be the most informative?

A. Osmotic fragility testing
B. Hemoglobin electrophoresis on acid citrate
C. Autohemolysis testing
D. Heinz body preparation
E. Quantitative G6PD assay



2. A nine year old Caucasian female is referred for evaluation of anemia found on a
routine physical examination. She is active and the fastest person on her soccer team.
Your exam reveals mild icterus, a palpable spleen 2 cm below the left costal margin,
and the following laboratory studies: hemoglobin concentration = 8.8 gm/dL;
reticulocyte count = 423 x 10
9
/L; MCV = 91 fL. On peripheral blood smear you note
polychromasia, with several echinocytes and an occasional spherocyte. There is no
known family history of blood dyscrasia. The most likely diagnosis is:

A. Congenital spherocytosis
B. 5 nucleotidase deficiency
C. G6PD deficiency with extreme lyonization
D. Hereditary elliptocytosis
E. Pyruvate kinase deficiency



3. Compared to normal erythrocytes, spherocytes have:

A. Increased osmotic fragility at all saline concentrations
B. Increased osmotic fragility at 0.5% saline
C. Decreased osmotic fragility at 0.9% saline
D. Increased levels of 2,3 DPG
E. More Howell-Jolly bodies



4. You are called by a local pediatrician regarding a five year old African-American
male with recurrent otitis media. The physician is currently treating him for his
fourth episode of otitis media over the past 6 months. The doctor would like to
prescribe trimethoprim/sulfamethoxazole prophylaxis, but is unsure about its safety
since the G6PD status of the child is unknown. Which of the following responses is
correct?

A. All African-American children should be tested for G6PD activity before
initiating sulfa therapy
B. African-American males and females should be tested if there is a family
history of hemolysis after sulfa exposure
C. Routine testing is not warranted since the African-American variant of
G6PD is relatively mild
D. Only African-American males should be tested before using sulfa
E. No testing is warranted since measurement of G6PD activity does not
predict hemolysis



5. A previously well three year old Caucasian male presents with pallor, fatigue, and
dark urine. One day earlier, he was evaluated by his local pediatrician who
prescribed amoxicillin for a possible urinary tract infection, based on dark amber
urine and a urine dipstick with 3+ blood. At your evaluation, you learn that the child
has not been on any other medications and there is no family history of blood
disorders. His exam shows pallor, minimal scleral icterus, and no
hepatosplenomegaly. Hemoglobin concentration = 5.5 gm/dL; reticulocyte count =
178 x 10
9
/L; WBC = 12.1 x 10
9
/L with a left shift; platelets = 291 x 10
9
/L. The urine
is reddish brown with 2+ protein and 4+ blood on dipstick, and 3 RBCs and 3
WBCs per high power field on microscopic analysis. You suspect an autoimmune
hemolytic process as the etiology for this sudden-onsent hemoglobinuria, but the
direct antiglobulin test (DAT) performed at room temperature is negative. To test this
child for a Donath-Landsteiner antibody, you would need to do which of the
following:

A. Collect the blood at 37
o
C and repeat the DAT at 37
o
C
B. Collect the blood at 37
o
C; incubate serum and cells at 4
o
C, then 37
o
C
C. Repeat the DAT at 4
o
C
D. Collect the blood at 4
o
C and repeat the DAT at 37
o
C
E. Collect the blood at 37
o
C, then test the serum at 37
o
C in an indirect
antiglobulin test



6. A term 3.1 kilogram Hispanic female infant is noted to have jaundice at 18 hours of
life. The mothers blood group is A+ and the babys is O+; the infants DAT is
negative. Laboratory evaluation on the baby reveals the following: hemoglobin =
14.9 gm/dL; total bilirubin = 15 mg/dL (all indirect). The peripheral blood smear
shows numerous spherocytes. The infant does well with phototherapy and supportive
care, and is discharged home at 4 days of life. Three weeks later, the infant is
thriving with a hemoglobin concentration of 13.4 gm/dL and total bilirubin
concentration of 3.1 mg/dL. What is the best diagnostic test to help establish the
etiology of this infants hemolysis?

A. Repeat DAT on the infants RBCs
B. Screening of the maternal serum for RBC antibodies
C. Tagged RBC study
D. Osmotic fragility testing at 1 month of life
E. Osmotic fragility testing at 6-12 months of life


7. A previously healthy 16 year old Caucasian male is referred to you for evaluation of
abnormal laboratory studies. He is an active football player who was evaluated for a
pre-sports physical. Review of systems reveals only occasional abdominal pain. The
abnormal laboratory studies include: hemoglobin concentration = 9.9 gm/dL; MCV =
104 fL; reticulocyte count = 117 x 10
9
/L; WBC = 3.0 with 26% polys, 58% lymphs,
15% monocytes, and 1% eosinophil. The platelet count is 119 x 10
9
/L. Urinalysis
specific gravity = 1.009, pH = 6.5, 2+ blood on dipstick, occasional casts but no
RBCs or WBCs on microscopic analysis. Which of the following diagnostic test
would be reasonable to order next:

A. CD59 testing on erythrocytes
B. Hepatitis B serologies
C. Direct platelet antibody measurement
D. Erythrocyte adenosine deaminase activity
E. Direct antiglobulin test







ANSWER KEY:


1. D
2. E
3. B
4. C
5. B
6. E
7. A



2006
Zimmerman

1. Which of the following improves oxygen delivery to tissues?
a. Increased concentration of fetal hemoglobin
b. Increased production of 2,3-DPG
c. Increased pH
d. Hypothermia
e. Methemoglobinemia

ANSWER: b
Fetal hemoglobin binds less 2,3-DPG than adult hemoglobin, thus effectively shifting the
oxygen dissociation curve to the left. This provides an advantage in utero for shunting
oxygen from the mother to fetus. This left shift of the oxygen dissociation curve results
in tighter binding of oxygen and decreased tissue oxygen delivery. Shifting the oxygen
dissociation curve to the right such that at a given pO2, the hemoglobin is less saturated
results in increased tissue oxygen delivery. The curve is right shifted by increased
temperature, increased production of 2,3-DPG in the Rapoport Luebering shunt, by
increased acid production (decreased pH). Methemoglobin is the derivative of
hemoglobin in which the iron is oxidized from the ferrous (2+) to ferric (3+) state, which
leads to left shift in the oxygen dissociation curve with increased oxygen affinity and thus
decreased oxygen delivery.

2. A previously healthy 6 year old girl presents to her local physician with complaint of
pallor, fatigue, and yellow eyes following a viral infection 10 days ago. Her exam is
remarkable for scleral icterus, mild tachycardia, a II/VI systolic ejection murmur, and a
spleen palpable at the left costal margin. Complete blood count reveals a hemoglobin of
6.1 gm/dL, MCV of 104fL, WBC of 20K, and platelet count of 460K. There is no family
history of anemia and she had a normal CBC just before starting kindergarten last year.
Her blood smear is shown:
Which of the following would be the most appropriate treatment?

a. Plasmapheresis
b. Transfusion of 1 unit of PRBCs immediately
c. Rituximab, 375 mg/m2 weekly times 4 weeks
d. Prednisone, 2 mg/kg/day PO
e. IVIG 0.4 mg/kg IV x 5 days




ANSWER: d Based on the history and peripheral blood smear, this patient most likely
has autoimmune hemolytic anemia, likely related to her recent viral illness. The Direct
Antiglobulin test would likely be positive with the polyspecific serum as well as anti-IgG.
Some warm reactive erythrocyte autoantibodies also fix complement, however. Although
she is mildly tachycardic, she does not have a gallop and has an acceptable hemoglobin
concentration. Steroids, then provide the most appropriate treatment for her, reserving
the use of PRBCs for worsening anemia or increased symptoms. IVIG is not generally
useful for the treatment of AIHA. Plasmapheresis is most helpful for IgM mediated
diseases, and only for severe, refractory IgG mediated diseases when a brief response is
needed. Although there have been case reports of success using Rituximab for refractory
AIHA, it is not first line therapy and would therefore not be appropriate in this scenario.

3. Which of the following enzyme disorders is associated with neurological symptoms?

a. Pyruvate kinase deficiency
b. Glucose-6-Phosphate dehydrogenase deficiency
c. Triose phosphate isomerase deficiency
d. Pyrimidine 5-nucleotidase deficiency
e. Hexokinase deficiency

ANSWER: c. Triose phosphate isomerase (TPI) is an enzyme in the Embden-Myerhof
pathway, catalyzing the interconversion of glyceraldehyde-3-phosphate and DHAP
(dihydroxyacetone phosphate). Deficiency of TPI is a rare autosomal disorder,
characterized by hemolytic anemia, neonatal hyperbilirubinemia, progressive
neurological dysfunction, increased susceptibility to infection, and cardiomyopathy.
Pyruvate kinase converts phosphoenolpyruvate to pyruvate in the glycolytic pathway.
PK deficiency is the most common cause of hemolytic anemia due to defective glycolysis
and is inherited as an autosomal recessive disorder. Glucose-6-phosphate dehydrogenase
deficiency is inherited as an X-linked disorder and is necessary for the production of
NADPH in the Hexose Monophosphate Shunt. NADPH is used for production of
glutathione which helps to prevent oxidative damage to erythrocytes. Pyrimidine-5-
nucleotidase participates in RNA degradation in reticulocytes. Deficiency of pyrimidine-
5-nucleotidase is inherited in an autosomal recessive manner and is associated with the
presence of basophilic stippling in the erythrocytes. Lead is also a powerful inhibitor of
pyrimidine-5-nucleotidase, and is also associated with basophilic stippling on the
peripheral smear. Hexokinase deficiency is a rare autosomal recessive disorder
associated with variable degrees of hemolysis.

4. Which of the following relationships is incorrect?
a. Howell-Jolly bodies Sickle Cell Anemia
b. Heinz bodies Glucose-6-phosphate dehydrogenase Deficiency
c. Basophilic stippling Pyrimidine-5nucleotidase Deficiency
d. Pappenheimer bodies Hemoglobin Kln
e. Ring forms Malaria

ANSWER: d. Howell Jolly bodies are nuclear remnants that are seen most
commonly in patients with congenital, surgical, or functional asplenia, as seen in
patients with sickle cell anemia, usually by 2 years of age. Heinz bodies occur when
hemoglobin precipitates, damaging the red cell membrane and can be visualized
following supravital stain with methyl violet. They are present in disorders of
unstable hemoglobin, G6PD deficiency, and Hemoglobin H disease (3 gene deletion
alpha thalassemia). Basophilic stippling reflects residual RNA in the erythrocyte and
can be seen in patients with lead toxicity, thalassemia (or any other ineffective
erythropoiesis), and Pyrimidine-5-nucleotidase deficiency. Pappenheimer bodies are
composed of degenerating cellular remnants which contain iron and are most likely
related to accelerated red cell division, or impaired hemoglobin synthesis. They can
be seen in patients with severe anemia, with thalassemia, and status post splenectomy
in otherwise normal individuals. Hb Kln is an unstable hemoglobin, so would be
most likely to lead to formation of Heinz bodies, not Pappenheimer bodies. Ring
forms are the trophozoites that can be seen as erythrocyte inclusions in association
with malaria.

5. A 4 year old boy is referred to a Gastroenterologist for evaluation of persistent
jaundice. The family reports that the boy had neonatal jaundice in the first day of life,
treated with phototherapy. He has always had mild scleral icterus, but recently they
noted it to be a bit more prominent, prompting the GI evaluation. The laboratory
evaluation reveals a Total bilirubin of 1.7 mg/dL, AST of 65 IU/L, ALT of 30 IU/L,
Hemoglobin of 11.2 gm/dL, MCV of 84 fL, MCHC of 36.5%, and reticulocyte count
of 230,000 (6%). Urinalysis is negative. There is no family history of anemia,
gallstones, or splenomegaly. The Gastroenterologist calls you for assistance in
determining which test to order next?
a. Autohemolysis
b Indirect antiglobulin test
c. Osmotic fragility testing
d. PNH screen
e. Heinz body preparation

ANSWER: c. Based on the history and laboratory results, this patient most likely
has Hereditary Spherocytosis, which would be diagnosed with Osmotic Fragility
testing. Patients with HS often have a history of neonatal jaundice, occurring usually
in the first 24 hours of life. They may have exaggerated and prolonged jaundice as
well. Often there is a family history of anemia, early gallstones, or splenectomy, but
lack of a family history does not eliminate this diagnosis, since approximately of
patients will have either an autosomal recessive form of HS or a spontaneous
mutation. An MCHC > 36% is also very suggestive (almost diagnostic) of
spheroctyosis. Osmotic fragility testing is helpful since erythrocytes from patients
with HS are more sensitive to lysis under osmotic stresses. Caution must be taken in
interpreting the results in patients under 6-12 months of age since the macrocytosis of
infants may lead to falsely normal results. The autohemolysis test is most useful in
detection of RBC enzyme disorders. Indirect antiglobulin test would not be useful in
this case since it is used to detect antibodies circulating in the serum of the individual.
It is important to be sure the Direct Antiglobulin Test is negative before performing
osmotic fragility testing, since spherocytes from any cause, including autoimmune
hemolytic anemia, will demonstrate increased sensitivity to osmotic stress. In this
case, however, given the history since birth, acquired AIHA is not very likely. The
history and laboratory parameters are not typical of PNH, thus the PNH screen is less
likely to be useful. A Heinz body preparation would be useful if you suspected an
unstable hemoglobin or Hb H disease (3 gene deletion alpha thalassemia), but again,
these diagnoses are unlikely given the Hgb, MCV, and MCHC.


6. A 5 yo girl presents to her pediatrician with acute onset of pallor and fatigue.
Laboratory evaluation reveals a hemogloboin concentration of 5.2 gm/dL, MCV of
90fL, Reticulocyte count of 325K. Her urine is dark brown in color with 2+ blood on
dipstick. Her Direct Antiglobulin Test reveals 3+ polyspecific, negative IgG and 3+
C3. Monospot is negative. Cold agglutinin titers are also negative. Stools are guiaic
negative.

Which of the following laboratory tests are most likely to reveal the diagnosis?
a. Absence of CD55 and CD59 on erythrocytes by flow cytometry
b. Detection of an antibody with anti I specificity in the patients serum
c. Detection of a warm reactive panagglutinin in the patients serum
d. Detection of a Donath-Landsteiner antibody in the patients serum
e. Increased susceptibility to lysis on osmotic fragility testing

ANSWER: d. This presentation is most consistent with the diagnosis of Paroxysmal
Cold Hemoglobinuria (PCH) which is characterized by a cold reactive IgG antibody
that binds at 4 C in the extremities or the face and then activates complement when
warmed to 37 C centrally. PCH is usually an acute, self-limited illness and often
occurs after infectious illnesses such as upper respiratory infections, varicella,
mumps, influenza, or infectious mononucleosis. The diagnostic testing requires that
the blood sample be kept warm until delivered to the laboratory, where it will be
tested at both 4 C and 37 C for presence of a biphasic hemolysin, the Donath-
Landsteiner antibody, which is usually directed against antigens of the P antigen
system, with lysis occurring when the sample is warmed.
Paroxysmal nocturnal hemoglobinuria (PNH) would be diagnosed by the absence
of CD55 or CD59 on the cells, but would not be common at this age and would not be
associated with a positive DAT. Patients with cold agglutinin disease often have IgM
antibodies with anti-I specificity, but in this case, the cold agglutinin titers were
negative. Osmotic fragility testing can be abnormal in any disorder with production
of spherocytes, including warm antibody mediated immune hemolytic disease as well
as hereditary spherocytosis, but is not likely useful in this scenario.

7. Which of the following are most likely to result in a patient with hemolytic disease
of the newborn born to a G0P0 mom?

a. Group O+ mom with Group A+ infant
b. Group O- mom with Group B+ infant
c. Group A- mom with Group A+ infant
d. Group O- mom with Group O+ infant
e. Group A+ mom with Group B+ infant

ANSWER: a. Hemolytic disease of the newborn occurs when there is transplacental
passage of maternal antibody directed against fetal red cell antigens not shared by the
mom. The antibodies that cross the placenta are IgG antibodies and may be against
either the ABO group or against the Rh antigens. Although ABO incompatibility is
more common than Rh disease, it is usually less severe. ABO setup occurs
typically with a Group O mom and a Group A or B infant since individuals with
group O blood make naturally occurring anti-A and anti-B isohemagglutinins. It is
very uncommon in incompatible infants born to Group A or B mothers, probably
because the naturally occurring isohemagglutinins in these individuals are more likely
IgM than IgG, and clear any fetal cells that might get into the maternal circulation,
but cannot cross the placenta. HDN occurs more commonly with Group O moms and
Group A infants, than with Group B infants, perhaps related to the density of the
antigens on fetal red cells. When the mother is Rh- and the infant Rh+ there is also
the possibility of Rh sensitization, but that does not occur in the first born child since
Rh sensitization requires exposure of maternal circulation to Rh+ cells for antibodies
to form. To prevent this exposure, Rh negative mothers are given antiD
immunoglobulin (RhoGam) at 28 weeks and at delivery. Doses of RhoGam should
also be given after abortions, amniocentesis, or any other trauma likely to expose the
mother to fetal blood cells.

8. A 16 yo black female presents to the ER with complaints of fatigue and yellow
eyes. She reports a severe headache for the last 24 hours and her mother is
concerned that she is not acting like herself. She has no significant past medical
history prior to this acute illness. She had a temperature to 102 F on admission to the
Emergency Department, BP 148/92, HR 120, RR 20. Laboratory evaluation reveals:
Hemoglobin concentration of 6.7 gm/dL, Reticulocyte count of 408K, MCV 92fL,
WBC 19.4K, Platelets 72K, BUN 24 mg/dL, Creatinine 2.1 mg/dL, LDH 2076 IU/L,
Total Bilirubin 1.4 mg/dL. Review of the peripheral blood smear reveals
schistocytes, polychromasia, nucleated RBCs, and large platelets.

Which of the following therapies would you initiate as soon as possible?

a. IVIG, 1 gm/kg daily for 2 days
b. Prednisone 2 mg/kg/day for 1 month
c. Plasmapheresis
d. Infusion of FFP, 10 cc/kg x 1
e. Platelet transfusion x 1

ANSWER: c. This clinical scenario is most consistent with a diagnosis of
Thrombotic Thrombocytopenic Purpura (TTP), with presenting symptoms of fever,
hemolytic anemia, thrombocytopenia, renal insufficiency, and mild neurologic
symptoms. The appropriate therapy is plasmapheresis until laboratory parameters
improve. TTP occurs when there is an absence of the von Willebrand factor cleaving
protease, which can be a congenital absence of the protein or due to an acquired
inhibitor to the protease, which is more likely in this scenario. Plasmapheresis helps
remove the inhibitor and replaces the missing protease, found in FFP. If you were
unable to begin plasmapheresis, infusion of FFP might be temporizing measure until
the patient could be moved to a facility capable of performing plasmapheresis.
Steroids are sometimes used in patients refractory to plasmapheresis, but are not first
line therapy.

9. Which of the following laboratory profiles is most consistent with hemolytic
anemia?

a. Decreased hemoglobin, decreased reticulocyte count, increased MCV, increased
bilirubin, decreased haptoglobin

b. Decreased hemoglobin, increased reticulocyte count, decreased MCV, increased
bilirubin, decreased haptoglobin

c. Normal hemoglobin, increased reticulocyte count, normal MCV, increased
bilirubin, increased haptoglobin

d. Normal hemoglobin, increased reticulocyte count, normal MCV, increased
bilirubin, decreased haptoglobin

e. Decreased hemoglobin, normal reticulocyte count, high MCV, normal bilirubin,
normal haptoglobin.

ANSWER: d. Despite the normal hemoglobin in scenario (d), which suggests
compensated hemolysis, the increased reticulocyte count, increased bilirubin, and
decreased haptoglobin are most consistent with a diagnosis of hemolytic anemia. For
scenario (a), the decreased reticulocyte count is not typical. For scenario (b), the
decreased MCV is not typical of hemolytic anemia. For scenario (c), the increased
haptoglobin, although an acute phase reactant, is less suggestive of hemolytic anemia.
For scenario (e), the normal reticulocyte count, high MCV, and normal haptoglobin
suggest the possibility of bone marrow failure or megaloblastic anemia rather than
hemolytic anemia.

10. Which of the following statements about warm reactive antibodies against
erythrocytes is incorrect?

a. They are usually of IgG specificity
b. They bind best at 37C.
c. They are most commonly of anti-I specificity.
d. They result in clearance of the antibody coated erythrocytes by the spleen.
e. They can fix complement.

ANSWER: c. Warm reactive antibodies are of IgG specificity and react best at body
temperature of 37 C. They typically result in extravascular hemolysis with clearance
of antibody coated erythrocytes by the reticuloendothelial system, primarily the
spleen and liver. Some IgG antibodies can fix complement, including some warm
reactive antibodies as well as the cold-reactive biphasic hemolysin, the Donath-
Landsteiner antibody. Most warm reactive antibodies are against Rh antigens, but
can also be anti-U, anti-Kell, anti-Jk
a
or Fy
a
. Antibodies of anti-I specificity are most
typically cold reactive IgM antibodies.
2009
Congenital and Acquired Hemolytic Anemias
Ellis J. Neufeld, MD PhD

1. A 15-year-old girl presents with 3-day history of fevers, abdominal pain, fatigue,
pallor, and 1 day of severe headache. She was previously well, has consumed no red
meat in weeks, and has had neither diarrhea nor urinary symptoms. Nobody in the
family has had anything like this in the past. Laboratory studies reveal Hb 7.1 g/dl,
reticulocytes 14%, platelets 60k/mm3, normal WBC count and differential. The
prothrombin time is normal, as is the PTT. The peripheral smear features numerous
schistocytes, occasional nucleated RBC, and large platelets. Chemistry studies
include total bilirubin 2.5 mg/dl (1.9 indirect, 0.6 direct), LDH 1400U/l, and
creatinine 1.1 mg/dl. A pregnancy test is negative. Stool studies and antinuclear
antibody studies are pending. Which of the following approaches would be most
correct in this clinical scenario?
A. Send and wait for ADAMTS13 enzyme and inhibitor studies. This could be TTP
and the etiology should be proven before initiation of therapy.
B. Microangiopathy and thrombocytopenia suggest DIC, therefore, admit to the ICU
for supportive care and launch a search for etiology, e.g. infection or malignancy.
C. Initiate treatment for TTP immediately, with plasmapheresis (+/- concomitant
steroids) because the diagnosis of TTP best fits this scenario, the diagnosis is
clinical, and the disorder can be life threatening. Send ADAMTS13 studies but
dont wait.
D. Follow renal function and provide supportive care for HUS, which is more
common than TTP in pediatrics, and initiate a search for E coli O57H7 strains,
which produce HUS-inducing toxins.
E. Transfuse for symptomatic anemia but only after saving blood for enzymatic
studies because a congenital hemolytic anemia is likely.

Answer: C
Explanation: Initiate treatment for TTP immediately, with plasmapheresis (+/-
concomitant steroids), because the diagnosis of TTP best fits this scenario, the diagnosis
is clinical, and the disorder can be life threatening. Send ADAMTS13 studies but dont
wait. This scenario is very typical for TTP, which can be life-threatening. In this case,
primary antibody-mediated TTP is likely because there is no history of drugs, pregnancy,
or congenital causes.
Answer A is incorrect because the screening tests for the antibody to the von
Willebrand cleaving protease, and the functional consequences of the antibody (low
levels) are not good enough or fast enough in 2009 to be used for real-time diagnosis,
though they are helpful in research settings. Answer B is incorrect because the normal PT
and PTT rule out DIC, although the smears cannot necessarily be differentiated. Answer
D is incorrect as a management strategy, although it is true that taking pediatrics as a
whole, HUS is more common than TTP. In post-pubertal patients with no clinical
exposures to suggest HUS, it is prudent to start down the TTP road immediately, and in
variant cases, to pursue supportive studies even as pheresis and often steroids are begun.
Answer E would be correct for many kinds of hemolytic anemias, but this is the wrong
clinical scenario based on the clinical history and the lab findings which support
intravascular hemolysis.


2. A newborn infant female presents with jaundice on the second day of life, with total
bilirubin 14 mg/dl, and hemoglobin 12 g/dl. She is nursing vigorously and otherwise
well. The peripheral smear reveals numerous bizarre forms, as if the cells had been
through a Waring blender, with schistocytes, tiny fragments, many ovalocytes,
marked polychromasia, and NRBCs 60 per 100 WBC. Mothers blood type is O
positive, the child is A negative. The DAT is negative. The reticulocyte count is 14%.
Family history reveals that the G1P1 mother has been told she has elliptocytosis,
that she was transfused in the newborn period, and has been well ever since. A quick
trip to the chart archives of the hospital discovers mothers old chart and confirms her
story. Both parents are of Sicilian descent, and not known to be related. Father has no
history of jaundice. Which of the following statements about this case is correct?
A. Although the mother had elliptocytosis, this newborn smear seems (by
description) to be much too severe for ordinary autosomal domininat HS due to
spectin mutations. This must be a recessive Ankyirin mutation affecting vertical
interactions in the membrane.
B. Ordinary (mild) dominant HE is often more severe in the newborn period. It is
possible that the newborn will have the same clinical course as mother, although a
more severe course (due to a silent mutation from father, for example) cant be
ruled out until the clinical course is followed for months or longer.
C. The severity of early jaundice strongly suggests that there is not only
elliptocytosis, but also an antibody-mediated hemolysis due to anti-A.
D. The smear as described is most suggestive of severe G6PD deficiency, which
would account for the jaundice.
E. The Sicilian ancestry would support the likelihood of beta thalassemia major,
even without consanguinity, because thalassemia alleles are extremely common in
Southern Italy.

Answer: B
Explanation: Ordinary (mild) dominant HE is often more severe in the newborn period.
It is possible that the newborn will have the same clinical course as mother, although a
more severe course (due to a silent mutation from father, for example) cant be ruled out
until the clinical course is followed for months or longer.
Answer A is incorrect because vertical interaction problems in the red cell
membrane cause spherocytosis (loss of membrane) whereas lateral interactions cause
elliptocytosis. Answer C is incorrect because the DAT is negative (despite the A-O
setup), and ABO antibodies are rarely of substantial import in primiparous mothers.
When they are significant, the DAT should be discernable, even if subtle. ABO
incompatibility is much more common than anti-A or B hemolysis. Answer D is incorrect
because the smear is not really compatible with G6PD as the major problem, and this is a
girl with an apparently unaffected father. Answer E is incorrect because beta
hemoglobinopathies are essentially never apparent at birth, and only manifest as beta is
expressed during the first year or more of life. The smear doesnt suggest thalassemia
either. Severe hypochromia and microcytosis is present in symptomatic thalassemia
syndromes (at an appropriate age).
This particular patient did have dominant elliptocytosis, but went on to have
severe, transfusion-dependent hemolysis and eventually evidence of myopathy, with
dramatically elevated serum CPK but low serum Aldolase. She proved to be a compound
heterozygote for two distinct mutations in the Aldolase A gene, for which her parents
were asymptomatic carriers. She ultimately succumbed to severe rhabdomyolysis with a
febrile illness. See Yao et al., Blood, 2004. Sometimes patients have more than one
hemolytic disorder!


3. A 17-year-old with no significant past medical history presents after her pediatrician
notes that her lips are blue at an annual check up. Oxymetry readings are 84% in
room air. A quick emergency room visit leads to the following data: Hb 15.3 g/dl.
PaO
2
98 by a blood gas machine. The blood is chocolate-brown in color, as noted by
the lab techs. The retic count is 2.1%. Methemoglobin by co-oximetry is 12% (normal
less than 1%). The peripheral smear reveals normal morphology. The patients father
is a dentist, and his office is off the garage of the family home. She works at an ice
cream parlor. She denies recreational drug use. She started smoking cigarettes at age
15, and now smokes pack a day. Which of the following statements is true?
A. The markedly elevated methb could be due to either a congenital deficiency of
cytochrome b5 reductase, or surreptitious use of nitrous oxide or other nitrates, to
which she may have access at home or at her job.
B. The role of the cytochrome reductase is to keep Hb in its oxidized (Fe+3) state.
C. Hemoglobin M rarely presents as cyanosis.
D. High affinity hemoglobins must be kept in the differential diagnosis of
methemoglobin.
E. Low affinity hemoglobins cause not only cyanosis but poor tissue oxygenation.

Answer: A
Explanation: The markedly elevated methb could be due to either a congenital
deficiency of cytochrome b5 reductase, or surreptitious use of nitrous oxide or other
nitrates, to which she may have access at home or at her job.
The differential diagnosis for met hb includes drugs, defects in the system that
keeps hemoglobin REDUCED (not oxidized) and abnormal hemoglobins which oxidize
spontaneously. Answer B is wrong because the reductase helps keep hemoglobin in its
reduced, Fe2+ state. Answer C is wrong as this disorder often causes cyanosis, though it
is rare. Answer D is incorrect because high affinity hemoglobins cause poor tissue
oxygen delivery, but the blood is very red, saturations is not falsely measured as low, and
cyanosis is not seen. Answer D is only half right. Cyanosis is found with some low
affinity hemoglobins, but tissue oxygen delivery is very good at the capillary-tissue
boundary.


4. A 3-year-old girl has had transfusion-dependent anemia since age 6 months. She is
found to have an unstable hemoglobin by sequence analysis (Hb Hammersmith, beta
Phe42Ser). She has bony deformity from extramedullary hematopoiesis and marked
splenomegaly. Her urine is tinged pink with no red cells due to brisk intravascular
hemolysis. Her hemoglobin is 7 g/dl 4 weeks after a transfusion, and her reticulocyte
count is 18%. A Heinz body prep is positive. Nucleated reds number 35/100 WBC.
Which of the following statements is correct?
A. As in Hereditary Spherocytosis, anemia will be entirely ameliorated by
splenectomy and her gallstone risk will be reduced.
B. As in pyruvate kinase deficiency, splenectomy may result in an apparent increase
in reticulocytosis.
C. This diagnosis might have been made by newborn screening (by electrophoresis,
isoelectric focusing, or HPLC), just as can be done with sickle trait, C trait, and
other beta hemoglobinopathies.
D. A decision about splenectomy should take into account growth status, transfusion
requirements, interval change in spleen size, and potential long-term risks of
infection and thrombosis.
E. Heinz bodies are nuclear remnants which increase in all hemolytic anemias.

Answer: D
Explanation: A decision about splenectomy should take into account growth status,
transfusion requirements, interval change in spleen size, and potential long-term risks of
infection and thrombosis.
Answer A is incorrect for most hemolytic states except HS. However, transfusion
requirements and growth problems may be partially ameliorated by splenectomy. Answer
B is probably incorrect because PK deficiency is unique for the death of reticulated forms
in the spleen, and paradoxical increase in the periphery after splenectomy. Answer C is
wrong on two fronts. First, the amino acid substitution HbHammersmith is isoelectric (no
change in charge) so that it may not be distinguishable by some methods, further, very
unstable hemoglobins are hard to detect in the periphery, particularly when beta is a small
minority in the newborn period.
Answer E is wrong. Howell-Jolly bodies are nuclear remnants. Heinz bodies are
precipitated hemoglobin seen only with supravital stains.


5. A previously healthy 5-year-old boy has sudden onset of dark urine, pallor, and
tachycardia a week after a respiratory illness with pronounced cough and low-grade
fever, treated with azithromycin. On presentation, he is pale and has a heart rate of
140/min. His spleen is just below the costal margin. His hemoglobin is 5.5 g/dl,
reticulocytes 12%, bilirubin 5.2 mg/dl, with direct fraction 0.3 mg/dl. His DAT is
positive for complement C3, and negative for IgG. You suspect either cold agglutinin
disease or a Donath Landsteiner antibody (paroxysmal cold hemoglobinuria). The
blood bank receives from your student a warm blood sample to be spun promptly.
They report a cold-reacting IgG of high thermal amplitude, which fixes
complement upon warming. Which of the following statements is correct about this
case?
A. Because this is not a cold agglutinin, there is no need to use a blood warmer, and
cold will not be a factor for the patient upon discharge.
B. Donath Landsteiner antibodies are readily removed by plasmapheresis.
C. The DAT (Coombs) reagent must be defective if the IgG cant be detected on the
cells, as the blood bank found it there on the specific testing.
D. PCH in children nearly always resolves spontaneously and may not respond well
to steroids.
E. Extravascular hemolysis is the rule in PCH and leads to impressive splenomegaly
in some cases.

Answer: D
Explanation: Answer A is wrong because cold-reacting IgGs do bind better in the cold
and will fix complement when they get warm centrally. Fastidious attention to keeping
the extremities warm, and to warming blood will help. Answer B is wrong because IgGs
distribute in extravascular space as well as intravascular. Thus, IgM is easy to remove by
pheresis, but IgG much less so. Answer C is wrong. This lab scenario defines the Donath
Landsteiner antibody. DAT reagents are used at room temperature, and if all the coated
cells have lysed from complement the cells wont agglutinate with the Coombs reagent.
Answer E is wrong because the hemolysis in this circumstance (in contrast to most IgGs
which dont fix complement well) is often entirely intravascular, and the spleen may be
indifferent.


6. Three months after liver transplantation for TPN-induced cholestatic liver disease, a
4-year-old boy becomes pale and jaundiced. His ALT remains in its stable range of
50-70 U/L, but his hemoglobin has dropped to 7 g/dl, his bilirubin is 5.8 mg/dl with
1.2 mg/dl direct. The reticulocyte count is 6%. He is immunosuppressed with
tacroimus and on a tapering dose of prednisone, currently 0.25 mg/kg/day. The liver,
from a cadaveric donor, was blood type O+, as is the patient. Direct antiglobulin test
is positive for a warm-reacting IgG antibody of broad (untype-able) specificity.
Which of the following statements is correct about this scenario?
A. The positive DAT rules out drug-related mechanisms of hemolysis.
B. AIHA following solid organ transplant may respond to changes in the
immunosuppresion regimen.
C. When the blood bank cant determine a specific target antigen, transfusion is
precluded by positive cross-match.
D. The elevated direct bilirubin suggests that intravascular hemolysis may be part of
the problem.
E. The Donor/recipient ABO blood type match rules out immune hemolysis based
on host-vs-graft hematopoiesis or graft-vs host equivalents.

Answer: B
Explanation: Tacrolimus may be the offending agent and various substitutes or increases
in other immunosuppressants may be helpful.
A is wrong because it is not uncommon for drugs to cause AIHA by one of
several mechanisms, including haptenizing effects such as penicillins, and
immunomodulation, such as tacrolimus. C is incorrect. Blood banks may provide least
badly matched blood for emergencies, and hemolysis wont necessarily be worse than
that seen for endogenous bl ood. D is incorrect. The direct hyperbilirubinemia depends on
hepatic excretion of conjugated bilirubin, not on the site of hemolysis. E is wrong
because there are many potential blood group interactions besides ABO. In principle, if
either donor or host lacked a common blood antigen, this scenario might result. This is
more commonly an issue in hematopoietic stem cell transplantation.


7. The monoclonal antibody therapeutic, Eculizumab, blocks hemolysis in paroxysmal
nocturnal hemoglobinuria by blocking C5 binding to C3 coated red cells (in PNH,
loss of PI-linked proteins includes loss of complement protection mechanisms).
Without the terminal components of complement, the cells no longer lyse but
circulate coated with C3. At the American Society of Hematology meetings in 2008,
Eculizumab investigators reported that some patients on this drug get a secondary
hemolytic anemia. A reasonable explanation is splenic clearance of C3-coated red
cells. Because PNH is a clonal disorder, patients are chimeric for normal red cells and
PNH red cells without the protective mechanism. Which of the following is a
reasonable prediction about the enhanced hemolysis in these patients?
A. The DAT will be positive for C3 in both clones in the chimeric patients, namely
PNH clones(CD59 neg) and normal (CD59+) red cells.
B. The DAT will be positive for C3 only on PNH cells in Eculizumab treated
patients.
C. The DAT for C3 is always positive in PNH patients, with or without the new
agent.
D. Steroids would not be effective for extravascular hemolysis of C3 coated red
cells, by analogy to AIHA.

Answer: B
Explanation: A is incorrect because the normal (non-PNH cells) wont accumulate
complement, just as normal individuals should not have a C3+ DAT. Choice C is
incorrect, because complement fixation usually lyses the C3 coated cells, so they are not
detectable by the DAT reagent. The antiC5 antibody blocks this lysis, and allows
detection of the coated cells. D is incorrect in that by analogy to AIHA, steroids should
be very effective, and indeed, this is the case.


8. Consider a 6-year-old Caucasian girl who is admitted to the hospital for acute onset of
dark urine and anemia after eating fresh fava beans at a farmers market. She has
impressive indirect hyperbilirubinemia and requires a transfusion for Hb 5 g/dl.
Which of the following statements is true?
A. For a girl to be affected with symptomatic G6PD deficiency (an X-linked
recessive trait), one of her parents has most likely passed on the gene (affected
father or carrier mother) but it need not be the case that both carry the gene.
B. Her G6PD level should be measured immediately upon hospitalization, or else
one would run the risk of missing the deficiency when the fava bean effect
dissipates.
C. If she wasnt jaundiced in the newborn period, this cant be G6PD deficiency.
D. If anemia this severe results from eating fava beans, then this patient probably has
chronic hemolytic anemia all the time.
E. G6PD deficiency is equally severe in all populations in which it arose (Asia,
Mediterranean, Sub-Saharan Africa).

Answer: A
Explanation: All women who carry a sex-linked mutant allele are chimeric for normal
and abnormal red cells. Unequal X-chromosome inactivation can lead to more than half
of red cell precursors carrying the abnormal gene, and such women may be symptomatic
under stress. B is incorrect. It is best to wait for RBC recovery to test the enzyme, as for
some variants, newer cells have much more enzyme. C is incorrect because the kinetics
of bilirubin production and clearance vary within individuals, and the fresh fava beans
may have been a greater stress for this patient than was birth. D is incorrect for the same
reason as C. The acute stress may be more of a problem than the oxidant burden of daily
life. E is incorrect. The African variant is milder than the other two regions of high G6PD
prevalence.


9. A 2-week-old boy is so pale at his pediatricians visit that he gets a CBC, which
reveals Hb 6.0 g/dl, retics 2%, MCV 99 fl. The MCHC is mildly elevated at 37 g/dl.
The smear has a few spherocytes, moderate anisocytosis, and some poikilocytosis. He
was mildly jaundiced as a newborn, maximum bilirubin 12 mg/dl and no blood type
mismatch setup was noted (mother and patient O negative). The bilirubin is now
normal, as are the WBC, differential, and platelet counts. The child otherwise seems
to be thriving. Family history is negative for transfusions, splenectomy,
cholecystectomy, and positive for neonatal jaundice requiring only lights in a cousin.
Which of the following statements is correct?
A. This cant be hereditary spherocytosis, as the reticulocyte count is too low to
suggest hemolysis.
B. This cant be hereditary spherocytosis, as the jaundice history is much too mild.
C. This has a good chance of representing hereditary spherocytosis during the
neonatal nadir period when reticulocyte counts arent helpful.
D. He should have a non-incubated osmotic fragility test right away, before you give
him a transfusion.
E. Testing the parents should confirm or rule out HS in this case.

Answer: C
Explanation: Pronounced anemia a couple of weeks after birth is very common in HS
for reasons not entirely clear. The mutation does not need to be severe to cause this
effect, and about 1/3 of HS patients have new mutations so that testing the family may
not suffice (so E is incorrect). A is incorrect because the nadir renders retic counts
relatively useless for several weeks. Normal goes down to zero. B is incorrect because the
kinetics of newborn jaundice vary from individual to individual. D is incorrect because
the incubated test is much more sensitive, and the test has relatively poor predictive
characteristics in the newborn period compared to a few months later, though if positive
now, an incubated test would be conclusive.


10. A 12-year-old boy with a history of ITP 3 years ago presents with 2-week history of
fatigue and pallor, and is found to have tachycardia and modest splenomegaly (2 cm
below the costal margin). His growth has been good. His diet is varied. Hes had no
fevers, night sweats, adenopathy, or GI symptoms. His labs include a hemoglobin of
6 g/dl, MCV 97 fl, plts 68,000/mm3, LDH 1100 U/L, uric acid normal, and minimally
elevated indirect bilirubin 2 mg/dl. The retic count is 9%. The smear shows no
schistocytes, no teardrops or blasts, and lots of polychromatophilic red cells. Some
giant platelets are seen. Anisocytosis is present. Hypersegmentation is absent. DAT is
positive for IgG. You suspect Evans syndrome, perhaps related to underlying
immunologic disturbance. His prior ITP responded promptly to 5 days of
prednisone, but hes not been seen much in the interim. Which of the following
statements is most likely to be true about this patient?
A. He should promptly have a bone marrow to rule out leukemia, and certainly
before he receives steroids.
B. The macrocytosis suggests a primary nutritional or bowel absorption problem.
C. His apparent AIHA is likely to respond to prednisone, but the kinetics of response
may be different for red cells and platelets, and higher doses and longer course are
indicated than when he had ITP alone in the past.
D. This cant be due to a mutation in the Fas system (i.e., autoimmune
lymphoproliferative syndrome, ALPS) because he has no massive adenopathy.
E. The AIHA is likely to be short-lived/self resolving. No therapy is necessary.

Answer: C
Explanation: Most hematologists would take a positive DAT and reassuring peripheral
blood film as evidence against leukemia in this clinical setting. Although folate and B12
deficiency are theoretically possible, the history and smear findings dont support these
as likely primary problems. D is based on the clinical versus mutation analyses of ALPS
patients. FAS pathway mutations are common in Evans syndrome, even when overt
adenopathy or other problems of ALPS are not present. In contrast to PCH and some
other pediatric immune cytopenias, the AIHA associated with Evans syndrome could be
protracted and require immunosuppression for months or longer.


11. The second child to a woman whose first infant was jaundiced has evidence of
hydrops in utero at 35 weeks. The child is delivered urgently and found to have
ascites and severe anemia, with hemoglobin of 6 g/dl. Both child and mother are
typed as O positive but the mother has a circulating anti-e (little e) andtibody and
genotyping reveals that she is E/E while the infant is E/e. the child is transfused
slowly with crossmatch compatible O negative blood (e/e). She makes a prompt
recovery. Which of the following is true about this scenario?
A. If the mother had received antiD globulin (WinRho or Rhogam) previously, this
hemolytic disease of the newborn could have been avoided.
B. The anemia and transfusion requirements could go on for 6 months or more.
C. There is a 25% chance of chronic anemia.
D. Almost invariably, the anemia will be entirely resolved by 3 months of age.
E. The child has a 50% chance of having the same problem when she has children.

Answer: D
Explanation: Anti-D does not protect against variance in Rh Ee or Cc systems, so A is
wrong. Maternal antibodies acquired passively across the placenta are nearly always gone
by 3 months when they are being constantly cleared on the infants red cells. 6 months
(choice B) is extremely unlikely. HDN is not a situation that leads to chronic anemia in
and of itself, so C is incorrect, and the child is heterozygous, so that the E/e system wont
be a problem for her children, thus E is incorrect.




2011

Congenital and Acquired Hemolytic Anemias
Ellis J. Neufeld, MD PhD

1. A 15-year-old girl presents with 3-day history of fevers, abdominal pain, fatigue,
pallor, and 1 day of severe headache. She was previously well, has consumed no red
meat in weeks, and has had neither diarrhea nor urinary symptoms. Nobody in the
family has had anything like this in the past. Laboratory studies reveal Hb 7.1 g/dl,
reticulocytes 14%, platelets 60k/mm3, normal WBC count and differential. The
prothrombin time is normal, as is the PTT. The peripheral smear features numerous
schistocytes, occasional nucleated RBC, and large platelets. Chemistry studies
include total bilirubin 2.5 mg/dl (1.9 indirect, 0.6 direct), LDH 1400U/l, and
creatinine 1.1 mg/dl. A pregnancy test is negative. Stool studies and antinuclear
antibody studies are pending. Which of the following approaches would be most
correct in this clinical scenario?
A. Send and wait for ADAMTS13 enzyme and inhibitor studies. This could be TTP
and the etiology should be proven before initiation of therapy.
B. Microangiopathy and thrombocytopenia suggest DIC, therefore, admit to the ICU
for supportive care and launch a search for etiology, e.g. infection or malignancy.
*C. Initiate treatment for TTP immediately, with plasmapheresis (+/- concomitant
steroids) because the diagnosis of TTP best fits this scenario, the diagnosis is
clinical, and the disorder can be life threatening. Send ADAMTS13 studies but
dont wait.
D. Follow renal function and provide supportive care for HUS, which is more
common than TTP in pediatrics, and initiate a search for E coli O57H7 strains,
which produce HUS-inducing toxins.
E. Transfuse for symptomatic anemia but only after saving blood for enzymatic
studies because a congenital hemolytic anemia is likely.

Answer: C
Explanation: Initiate treatment for TTP immediately, with plasmapheresis (+/-
concomitant steroids), because the diagnosis of TTP best fits this scenario, the diagnosis
is clinical, and the disorder can be life threatening. Send ADAMTS13 studies but dont
wait. This scenario is very typical for TTP, which can be life-threatening. In this case,
primary antibody-mediated TTP is likely because there is no history of drugs, pregnancy,
or congenital causes.
Answer A is incorrect because the screening tests for the antibody to the von
Willebrand cleaving protease, and the functional consequences of the antibody (low
levels) are not good enough or fast enough in 2009 to be used for real-time diagnosis,
though they are helpful in research settings. Answer B is incorrect because the normal PT
and PTT rule out DIC, although the smears cannot necessarily be differentiated. Answer
D is incorrect as a management strategy, although it is true that taking pediatrics as a
whole, HUS is more common than TTP. In post-pubertal patients with no clinical
exposures to suggest HUS, it is prudent to start down the TTP road immediately, and in
variant cases, to pursue supportive studies even as pheresis and often steroids are begun.
Answer E would be correct for many kinds of hemolytic anemias, but this is the wrong
clinical scenario based on the clinical history and the lab findings which support
intravascular hemolysis.


2. A newborn infant female presents with jaundice on the second day of life, with total
bilirubin 14 mg/dl, and hemoglobin 12 g/dl. She is nursing vigorously and otherwise
well. The peripheral smear reveals numerous bizarre forms, as if the cells had been
through a Waring blender, with schistocytes, tiny fragments, many ovalocytes,
marked polychromasia, and NRBCs 60 per 100 WBC. Mothers blood type is O
positive, the child is A negative. The DAT is negative. The reticulocyte count is 14%.
Family history reveals that the G1P1 mother has been told she has elliptocytosis,
that she was transfused in the newborn period, and has been well ever since. A quick
trip to the chart archives of the hospital discovers mothers old chart and confirms her
story. Both parents are of Sicilian descent, and not known to be related. Father has no
history of jaundice. Which of the following statements about this case is correct?
A. Although the mother had elliptocytosis, this newborn smear seems (by
description) to be much too severe for ordinary autosomal dominant HS due to
spectrin mutations. This must be a recessive Ankyrin mutation affecting vertical
interactions in the membrane.
*B. Ordinary (mild) dominant HE is often more severe in the newborn period. It is
possible that the newborn will have the same clinical course as mother, although a
more severe course (due to a silent mutation from father, for example) cant be
ruled out until the clinical course is followed for months or longer.
C. The severity of early jaundice strongly suggests that there is not only
elliptocytosis, but also an antibody-mediated hemolysis due to anti-A.
D. The smear as described is most suggestive of severe G6PD deficiency, which
would account for the jaundice.
E. The Sicilian ancestry would support the likelihood of beta thalassemia major,
even without consanguinity, because thalassemia alleles are extremely common in
Southern Italy.

Answer: B
Explanation: Ordinary (mild) dominant HE is often more severe in the newborn period.
It is possible that the newborn will have the same clinical course as mother, although a
more severe course (due to a silent mutation from father, for example) cant be ruled out
until the clinical course is followed for months or longer.
Answer A is incorrect because vertical interaction problems in the red cell
membrane cause spherocytosis (loss of membrane) whereas lateral interactions cause
elliptocytosis. Answer C is incorrect because the DAT is negative (despite the A-O
setup), and ABO antibodies are rarely of substantial import in primiparous mothers.
When they are significant, the DAT should be discernable, even if subtle. ABO
incompatibility is much more common than anti-A or B hemolysis. Answer D is incorrect
because the smear is not really compatible with G6PD as the major problem, and this is a
girl with an apparently unaffected father. Answer E is incorrect because beta
hemoglobinopathies are essentially never apparent at birth, and only manifest as beta is
expressed during the first year or more of life. The smear doesnt suggest thalassemia
either. Severe hypochromia and microcytosis is present in symptomatic thalassemia
syndromes (at an appropriate age).
This particular patient did have dominant elliptocytosis, but went on to have
severe, transfusion-dependent hemolysis and eventually evidence of myopathy, with
dramatically elevated serum CPK but low serum Aldolase. She proved to be a compound
heterozygote for two distinct mutations in the Aldolase A gene, for which her parents
were asymptomatic carriers. She ultimately succumbed to severe rhabdomyolysis with a
febrile illness. See Yao et al., Blood, 2004. Sometimes patients have more than one
hemolytic disorder!


3. A 17-year-old with no significant past medical history presents after her pediatrician
notes that her lips are blue at an annual checkup. Oxymetry readings are 84% in room
air. A quick emergency room visit leads to the following data: Hb 15.3 g/dl. PaO
2
98
by a blood gas machine. The blood is chocolate-brown in color, as noted by the lab
techs. The retic count is 2.1%. Methemoglobin by co-oximetry is 12% (normal less
than 1%). The peripheral smear reveals normal morphology. The patients father is a
dentist, and his office is off the garage of the family home. She works at an ice cream
parlor. She denies recreational drug use. She started smoking cigarettes at age 15, and
now smokes pack a day. Which of the following statements is true?
*A. The markedly elevated methb could be due to either a congenital deficiency of
cytochrome b5 reductase, or surreptitious use of nitrous oxide or other nitrates, to
which she may have access at home or at her job.
B. The role of the cytochrome reductase is to keep Hb in its oxidized (Fe+3) state.
C. Hemoglobin M rarely presents as cyanosis.
D. High affinity hemoglobins must be kept in the differential diagnosis of
methemoglobin.
E. Low affinity hemoglobins cause not only cyanosis but poor tissue oxygenation.

Answer: A
Explanation: The markedly elevated methb could be due to either a congenital
deficiency of cytochrome b5 reductase, or surreptitious use of nitrous oxide or other
nitrates, to which she may have access at home or at her job.
The differential diagnosis for met hb includes drugs, defects in the system that
keeps hemoglobin REDUCED (not oxidized) and abnormal hemoglobins which oxidize
spontaneously. Answer B is wrong because the reductase helps keep hemoglobin in its
reduced, Fe2+ state. Answer C is wrong as this disorder often causes cyanosis, though it
is rare. Answer D is incorrect because high affinity hemoglobins cause poor tissue
oxygen delivery, but the blood is very red, saturations is not falsely measured as low, and
cyanosis is not seen. Answer D is only half right. Cyanosis is found with some low
affinity hemoglobins, but tissue oxygen delivery is very good at the capillary-tissue
boundary.


4. A 3-year-old girl has had transfusion-dependent anemia since age 6 months. She is
found to have an unstable hemoglobin by sequence analysis (Hb Hammersmith, beta
Phe42Ser). She has bony deformity from extramedullary hematopoiesis and marked
splenomegaly. Her urine is tinged pink with no red cells due to brisk intravascular
hemolysis. Her hemoglobin is 7 g/dl 4 weeks after a transfusion, and her reticulocyte
count is 18%. A Heinz body prep is positive. Nucleated reds number 35/100 WBC.
Which of the following statements is correct?
A. As in Hereditary Spherocytosis, anemia will be entirely ameliorated by
splenectomy and her gallstone risk will be reduced.
B. As in pyruvate kinase deficiency, splenectomy may result in an apparent increase
in reticulocytosis.
C. This diagnosis might have been made by newborn screening (by electrophoresis,
isoelectric focusing, or HPLC), just as can be done with sickle trait, C trait, and
other beta hemoglobinopathies.
*D. A decision about splenectomy should take into account growth status, transfusion
requirements, interval change in spleen size, and potential long-term risks of
infection and thrombosis.
E. Heinz bodies are nuclear remnants which increase in all hemolytic anemias.

Answer: D
Explanation: A decision about splenectomy should take into account growth status,
transfusion requirements, interval change in spleen size, and potential long-term risks of
infection and thrombosis.
Answer A is incorrect for most hemolytic states except HS. However, transfusion
requirements and growth problems may be partially ameliorated by splenectomy. Answer
B is probably incorrect because PK deficiency is unique for the death of reticulated forms
in the spleen, and paradoxical increase in the periphery after splenectomy. Answer C is
wrong on two fronts. First, the amino acid substitution HbHammersmith is isoelectric (no
change in charge) so that it may not be distinguishable by some methods, further, very
unstable hemoglobins are hard to detect in the periphery, particularly when beta is a small
minority in the newborn period.
Answer E is wrong. Howell-Jolly bodies are nuclear remnants. Heinz bodies are
precipitated hemoglobin seen only with supravital stains.


5. A previously healthy 5-year-old boy has sudden onset of dark urine, pallor, and
tachycardia a week after a respiratory illness with pronounced cough and low-grade
fever, treated with azithromycin. On presentation, he is pale and has a heart rate of
140/min. His spleen is just below the costal margin. His hemoglobin is 5.5 g/dl,
reticulocytes 12%, bilirubin 5.2 mg/dl, with direct fraction 0.3 mg/dl. His DAT is
positive for complement C3, and negative for IgG. You suspect either cold agglutinin
disease or a Donath Landsteiner antibody (paroxysmal cold hemoglobinuria). The
blood bank receives from your student a warm blood sample to be spun promptly.
They report a cold-reacting IgG of high thermal amplitude, which fixes
complement upon warming. Which of the following statements is correct about this
case?
A. Because this is not a cold agglutinin, there is no need to use a blood warmer, and
cold will not be a factor for the patient upon discharge.
B. Donath Landsteiner antibodies are readily removed by plasmapheresis.
C. The DAT (Coombs) reagent must be defective if the IgG cant be detected on the
cells, as the blood bank found it there on the specific testing.
*D. PCH in children nearly always resolves spontaneously and may not respond well
to steroids.
E. Extravascular hemolysis is the rule in PCH and leads to impressive splenomegaly
in some cases.

Answer: D
Explanation: Answer A is wrong because cold-reacting IgGs do bind better in the cold
and will fix complement when they get warm centrally. Fastidious attention to keeping
the extremities warm, and to warming blood will help. Answer B is wrong because IgGs
distribute in extravascular space as well as intravascular. Thus, IgM is easy to remove by
pheresis, but IgG much less so. Answer C is wrong. This lab scenario defines the Donath
Landsteiner antibody. DAT reagents are used at room temperature, and if all the coated
cells have lysed from complement the cells wont agglutinate with the Coombs reagent.
Answer E is wrong because the hemolysis in this circumstance (in contrast to most IgGs
which dont fix complement well) is often entirely intravascular, and the spleen may be
indifferent.


6. Three months after liver transplantation for TPN-induced cholestatic liver disease, a
4-year-old boy becomes pale and jaundiced. His ALT remains in its stable range of
50-70 U/L, but his hemoglobin has dropped to 7 g/dl, his bilirubin is 5.8 mg/dl with
1.2 mg/dl direct. The reticulocyte count is 6%. He is immunosuppressed with
tacrolimus and on a tapering dose of prednisone, currently 0.25 mg/kg/day. The liver,
from a cadaveric donor, was blood type O+, as is the patient. Direct antiglobulin test
is positive for a warm-reacting IgG antibody of broad (untype-able) specificity.
Which of the following statements is correct about this scenario?
A. The positive DAT rules out drug-related mechanisms of hemolysis.
*B. AIHA following solid organ transplant may respond to changes in the
immunosuppression regimen.
C. When the blood bank cant determine a specific target antigen, transfusion is
precluded by positive cross-match.
D. The elevated direct bilirubin suggests that intravascular hemolysis may be part of
the problem.
E. The Donor/recipient ABO blood type match rules out immune hemolysis based
on host-vs-graft hematopoiesis or graft-vs host equivalents.

Answer: B
Explanation: Tacrolimus may be the offending agent and various substitutes or increases
in other immunosuppressants may be helpful.
A is wrong because it is not uncommon for drugs to cause AIHA by one of
several mechanisms, including haptenizing effects such as penicillins, and
immunomodulation, such as tacrolimus. C is incorrect. Blood banks may provide least
badly matched blood for emergencies, and hemolysis wont necessarily be worse than
that seen for endogenous blood. D is incorrect. The direct hyperbilirubinemia depends on
hepatic excretion of conjugated bilirubin, not on the site of hemolysis. E is wrong
because there are many potential blood group interactions besides ABO. In principle, if
either donor or host lacked a common blood antigen, this scenario might result. This is
more commonly an issue in hematopoietic stem cell transplantation.


7. The monoclonal antibody therapeutic, Eculizumab, blocks hemolysis in paroxysmal
nocturnal hemoglobinuria by blocking C5 binding to C3 coated red cells (in PNH,
loss of PI-linked proteins includes loss of complement protection mechanisms).
Without the terminal components of complement, the cells no longer lyse but
circulate coated with C3. At the American Society of Hematology meetings in 2008,
and in subsequent reports, Eculizumab investigators reported that some patients on
this drug get a secondary hemolytic anemia. A reasonable explanation is splenic
clearance of C3-coated red cells. Because PNH is a clonal disorder, patients are
chimeric for normal red cells and PNH red cells without the protective mechanism.
Which of the following is a reasonable prediction about the enhanced hemolysis in
these patients?
A. The DAT will be positive for C3 in both clones in the chimeric patients, namely
PNH clones(CD59 neg) and normal (CD59+) red cells.
*B. The DAT will be positive for C3 only on PNH cells in Eculizumab treated
patients.
C. The DAT for C3 is always positive in PNH patients, with or without the new
agent.
D. Steroids would not be effective for extravascular hemolysis of C3 coated red
cells, by analogy to AIHA.

Answer: B
Explanation: A is incorrect because the normal (non-PNH cells) wont accumulate
complement, just as normal individuals should not have a C3+ DAT. Choice C is
incorrect, because complement fixation usually lyses the C3 coated cells, so they are not
detectable by the DAT reagent. The antiC5 antibody blocks this lysis, and allows
detection of the coated cells. D is incorrect in that by analogy to AIHA, steroids should
be very effective, and indeed, this is the case.


8. Consider a 6-year-old Caucasian girl who is admitted to the hospital for acute onset of
dark urine and anemia after eating fresh fava beans at a farmers market. She has
impressive indirect hyperbilirubinemia and requires a transfusion for Hb 5 g/dl.
Which of the following statements is true?
*A. For a girl to be affected with symptomatic G6PD deficiency (an X-linked
recessive trait), one of her parents has most likely passed on the gene (affected
father or carrier mother) but it need not be the case that both carry the gene.
B. Her G6PD level should be measured immediately upon hospitalization, or else
one would run the risk of missing the deficiency when the fava bean effect
dissipates.
C. If she wasnt jaundiced in the newborn period, this cant be G6PD deficiency.
D. If anemia this severe results from eating fava beans, then this patient probably has
chronic hemolytic anemia all the time.
E. G6PD deficiency is equally severe in all populations in which it arose (Asia,
Mediterranean, Sub-Saharan Africa).

Answer: A
Explanation: All women who carry a sex-linked mutant allele are chimeric for normal
and abnormal red cells. Unequal X-chromosome inactivation can lead to more than half
of red cell precursors carrying the abnormal gene, and such women may be symptomatic
under stress. B is incorrect. It is best to wait for RBC recovery to test the enzyme, as for
some variants, newer cells have much more enzyme. C is incorrect because the kinetics
of bilirubin production and clearance vary within individuals, and the fresh fava beans
may have been a greater stress for this patient than was birth. D is incorrect for the same
reason as C. The acute stress may be more of a problem than the oxidant burden of daily
life. E is incorrect. The African variant is milder than the other two regions of high G6PD
prevalence.


9. A 2-week-old boy is so pale at his pediatricians visit that he gets a CBC, which
reveals Hb 6.0 g/dl, retics 2%, MCV 99 fl. The MCHC is mildly elevated at 37 g/dl.
The smear has a few spherocytes, moderate anisocytosis, and some poikilocytosis. He
was mildly jaundiced as a newborn, maximum bilirubin 12 mg/dl and no blood type
mismatch setup was noted (mother and patient O negative). The bilirubin is now
normal, as are the WBC, differential, and platelet counts. The child otherwise seems
to be thriving. Family history is negative for transfusions, splenectomy,
cholecystectomy, and positive for neonatal jaundice requiring only lights in a cousin.
Which of the following statements is correct?
A. This cant be hereditary spherocytosis, as the reticulocyte count is too low to
suggest hemolysis.
B. This cant be hereditary spherocytosis, as the jaundice history is much too mild.
*C. This has a good chance of representing hereditary spherocytosis during the
neonatal nadir period when reticulocyte counts arent helpful.
D. He should have a non-incubated osmotic fragility test right away, before you give
him a transfusion.
E. Testing the parents should confirm or rule out HS in this case.

Answer: C
Explanation: Pronounced anemia a couple of weeks after birth is very common in HS
for reasons not entirely clear. The mutation does not need to be severe to cause this
effect, and about 1/3 of HS patients have new mutations so that testing the family may
not suffice (so E is incorrect). A is incorrect because the normal newborn nadir renders
retic counts relatively useless for several weeks. Normal goes down to zero. B is incorrect
because the kinetics of newborn jaundice vary from individual to individual. D is
incorrect because the incubated test is much more sensitive, and the test has relatively
poor predictive characteristics in the newborn period compared to a few months later,
though if positive now, an incubated test would be conclusive.


10. A 12-year-old boy with a history of ITP 3 years ago presents with 2-week history of
fatigue and pallor, and is found to have tachycardia and modest splenomegaly (2 cm
below the costal margin). His growth has been good. His diet is varied. Hes had no
fevers, night sweats, adenopathy, or GI symptoms. His labs include a hemoglobin of
6 g/dl, MCV 97 fl, plts 68,000/mm3, LDH 1100 U/L, uric acid normal, and minimally
elevated indirect bilirubin 2 mg/dl. The retic count is 9%. The smear shows no
schistocytes, no teardrops or blasts, and lots of polychromatophilic red cells. Some
giant platelets are seen. Anisocytosis is present. Hypersegmentation is absent. DAT is
positive for IgG. You suspect Evans syndrome, perhaps related to underlying
immunologic disturbance. His prior ITP responded promptly to 5 days of
prednisone, but hes not been seen much in the interim. Which of the following
statements is most likely to be true about this patient?
A. He should promptly have a bone marrow to rule out leukemia, and certainly
before he receives steroids.
B. The macrocytosis suggests a primary nutritional or bowel absorption problem.
*C. His apparent AIHA is likely to respond to prednisone, but the kinetics of response
may be different for red cells and platelets, and higher doses and longer course are
indicated than when he had ITP alone in the past.
D. This cant be due to a mutation in the Fas system (i.e., autoimmune
lymphoproliferative syndrome, ALPS) because he has no massive adenopathy.
E. The AIHA is likely to be short-lived/self resolving. No therapy is necessary.

Answer: C
Explanation: Most hematologists would take a positive DAT and reassuring peripheral
blood film as evidence against leukemia in this clinical setting. Although folate and B12
deficiency are theoretically possible, the history and smear findings dont support these
as likely primary problems. D is based on the clinical versus mutation analyses of ALPS
patients. FAS pathway mutations are common in Evans syndrome, even when overt
adenopathy or other problems of ALPS are not present. In contrast to PCH and some
other pediatric immune cytopenias, the AIHA associated with Evans syndrome could be
protracted and require immunosuppression for months or longer.


11. The second child to a woman whose first infant was jaundiced has evidence of
hydrops in utero at 35 weeks. The child is delivered urgently and found to have
ascites and severe anemia, with hemoglobin of 6 g/dl. Both child and mother are
typed as O positive but the mother has a circulating anti-e (little e) antibody and
genotyping reveals that she is E/E while the infant is E/e. The child is transfused
slowly with crossmatch compatible O negative blood (e/e). She makes a prompt
recovery. Which of the following is true about this scenario?
A. If the mother had received anti-D globulin (WinRho or Rhogam) previously, this
hemolytic disease of the newborn could have been avoided.
B. The anemia and transfusion requirements could go on for 6 months or more.
C. There is a 25% chance of chronic anemia.
*D. Almost invariably, the anemia will be entirely resolved by 3 months of age.
E. The child has a 50% chance of having the same problem when she has children.

Answer: D
Explanation: Anti-D does not protect against variants in Rh Ee or Cc systems, so A is
wrong. Maternal antibodies acquired passively across the placenta are nearly always gone
by 3 months when they are being constantly cleared on the infants red cells. 6 months
(choice B) is extremely unlikely. HDN is not a situation that leads to chronic anemia in
and of itself, so C is incorrect, and the child is heterozygous, so that the E/e system wont
be a problem for her children, thus E is incorrect.

12. A previously well three year old Caucasian male presents with pallor, fatigue, and
dark urine. One day earlier, he was evaluated by his local pediatrician who
prescribed amoxicillin for a possible urinary tract infection, based on dark amber
urine and a urine dipstick with 3+ blood. At your evaluation, you learn that the child
has not been on any other medications and there is no family history of blood
disorders. His exam shows pallor, minimal scleral icterus, and no
hepatosplenomegaly. Hemoglobin concentration = 5.5 gm/dL; reticulocyte count =
178 x 10
9
/L; WBC = 12.1 x 10
9
/L with a left shift; platelets = 291 x 10
9
/L. The urine
is reddish brown with 2+ protein and 4+ blood on dipstick, and 3 RBCs and 3
WBCs per high power field on microscopic analysis. You suspect an autoimmune
hemolytic process as the etiology for this sudden-onset hemoglobinuria, but the direct
antiglobulin test (DAT) performed at room temperature is negative. To test this child
for a Donath-Landsteiner antibody, you would need to do which of the following:

A. Collect the blood at 37
o
C and repeat the DAT at 37
o
C
*B.Collect the blood at 37
o
C; incubate serum and cells at 4
o
C, then 37
o
C
C. Repeat the DAT at 4
o
C
D. Collect the blood at 4
o
C and repeat the DAT at 37
o
C
E. Collect the blood at 37
o
C, then test the serum at 37
o
C in an indirect
antiglobulin test

Answer B.
Explanation: This question gets at the mechanism of cold-reacting IgG AIHA or
Donath-Landsteiner antibody, and its detection in the laboratory. The blood must reach
the lab warm, or antibody may all be stuck in the red cell pellet. It must be tested for
binding in the cold, or binding to red cells will not be detected. The antibody will only
fix complement in the warm again. The vignette is typical for AIHA, and therefore the
negative initial DAT demands further evaluation for the phenomenon also known as
paroxysmal cold hemoglobinuria.



13. A previously healthy 16 year old Caucasian male is referred to you for evaluation of
abnormal laboratory studies. He is an active football player who was evaluated for a
pre-sports physical. Review of systems reveals only occasional abdominal pain. The
abnormal laboratory studies include: hemoglobin concentration = 9.9 gm/dL; MCV =
104 fL; reticulocyte count = 3.5%, (117 x 10
9
/L); WBC = 3.0 with 26% polys, 58%
lymphs, 15% monocytes, and 1% eosinophil. The platelet count is 119 x 10
9
/L.
Urinalysis specific gravity = 1.009, pH = 6.5, 2+ blood on dipstick, occasional casts
but no RBCs or WBCs on microscopic analysis. Which of the following diagnostic
test would be reasonable to order next:

*A. CD59 testing on erythrocytes
B. Hepatitis B serologies
C. Direct platelet antibody measurement
D. Erythrocyte adenosine deaminase activity
E. Indirect antiglobulin test

Answer: A
Explanation: Pancytopenia with evidence of intravascular hemolysis
(hemoglobinuria without red cells in the urine) must raise the possibility of
paroxysmal nocturnal hemoglobinuria, even though it is rare. A PNH screen by
flow cytometry evaluates the cell surface expression of phosphotidyl inositol-linked
proteins in the different lineages, because PNH is a clonal disorder of PIG-A
deficiency (an enzyme responsible for PI-linked proteins). The other tests listed may
play a role in assessment of aplastic anemia states or in assessment of autoimmune
cytopenias if PNH is absent.


14. A two year old male has had pallor since birth. Spasticity was first noted upon
physical examination at two months of age, and hemolysis was first noted at six months
of age, with Hb 8.9 g/dl, reticulocytes count 7%, DAT was negative, and numerous
dense, speculated cells were noted on peripheral smear. He was the product of an
uncomplicated pregnancy to unrelated parents from a geographically isolated region of
Spain. Progressive neuromuscular decline has been his main clinical problem, with
intermittent infections, including pyelonephritis and pneumonia. Which of the following
enzyme disorders is the most likely candidate for the patients disease?

a. Pyruvate kinase deficiency
b. Glucose-6-Phosphate dehydrogenase deficiency
*c. Triose phosphate isomerase deficiency
d. Pyrimidine 5-nucleotidase deficiency
e. Hexokinase deficiency

ANSWER: c.

Explanation: Triose phosphate isomerase (TPI) is an enzyme in the Embden-Myerhof
pathway, catalyzing the interconversion of glyceraldehyde-3-phosphate and DHAP
(dihydroxyacetone phosphate). Deficiency of TPI is a rare autosomal disorder,
characterized by hemolytic anemia, neonatal hyperbilirubinemia, progressive
neuromuscular dysfunction, increased susceptibility to infection, and cardiomyopathy.
Pyruvate kinase converts phosphoenolpyruvate to pyruvate in the glycolytic pathway.
PK deficiency is the most common cause of hemolytic anemia due to defective glycolysis
and is inherited as an autosomal recessive disorder, without neuromuscular disease.
Glucose-6-phosphate dehydrogenase deficiency is inherited as an X-linked disorder and
is necessary for the production of NADPH in the Hexose Monophosphate Shunt.
NADPH is used for production of glutathione which helps to prevent oxidative damage to
erythrocytes. Pyrimidine-5-nucleotidase participates in RNA degradation in
reticulocytes. Deficiency of pyrimidine-5-nucleotidase is inherited in an autosomal
recessive manner and is associated with the presence of basophilic stippling in the
erythrocytes. Lead is also a powerful inhibitor of pyrimidine-5-nucleotidase, and is also
associated with basophilic stippling on the peripheral smear. Hexokinase deficiency is a
rare autosomal recessive disorder associated with variable degrees of hemolysis.

15. A previously healthy 6 year old girl presents to her local physician with complaint of
pallor, fatigue, and yellow eyes following a viral infection 10 days ago. Her exam is
remarkable for scleral icterus, mild tachycardia, a II/VI systolic ejection murmur, and a
spleen palpable at the left costal margin. Complete blood count reveals a hemoglobin of
6.1 gm/dL, MCV of 104fL, WBC of 20K, and platelet count of 460K. There is no family
history of anemia and she had a normal CBC just before starting kindergarten last year.
Her blood smear is shown:
Which of the following would be the most appropriate treatment?

a. Plasmapheresis
b. Transfusion of 1 unit of PRBCs immediately
c. Rituximab, 375 mg/m2 weekly times 4 weeks
*d. Prednisone, 2-4 mg/kg/day PO
e. IVIG 0.4 mg/kg IV x 5 days or 1 g/kg IV x 2 days











ANSWER: d
Explanation: Based on the history and peripheral blood smear, this patient most likely
has (warm) autoimmune hemolytic anemia, likely related to her recent viral illness. The
Direct Antiglobulin test would likely be positive with the polyspecific serum as well as
anti-IgG. Some warm reactive erythrocyte autoantibodies also fix complement, however.
Although she is mildly tachycardic, she does not have a gallop and has an acceptable
hemoglobin concentration. Steroids, then provide the most appropriate treatment for her,
reserving the use of PRBCs for worsening anemia or increased symptoms. IVIG is not
generally useful for the treatment of AIHA. Plasmapheresis is most helpful for IgM
mediated diseases, and only for severe, refractory IgG mediated diseases when a brief
response is needed. Although there have now been several case reports and series of
success using Rituximab for refractory AIHA (Failing steroids or steroid wean), it is not
first line therapy and would therefore not be appropriate in this scenario. Initial dosing of
steroids varies, and some clinicians prefer doses more than 2 mg/kg/day, tapering slowly
(over weeks or months).