Anemia

Laboratory
Assessment of
Anemia
Jerick G. Dela Cruz
Iron recycling
•Macrophages carry a receptor
for the haptoglobin-
hemoglobin complex
•hepatocytes have a
hemopexin-heme receptor
Iron
Iron
Hematocrit
Hematocrit
Red Blood Cell
Indeces
Red Blood Cell
Indices
•Average volume
•Hemoglobin content
•Concentration of
hemoglobin
Red Blood Cell
Indices
•MCV
•MCH
•MCHC
Red Blood Cell
Indeces
MCH?
MCHC?
Reticulocytes
Reticulocytes
•Assess BM activity
•Compensation for
anemia
Reticulocytes
ARC
• Actual number of reticulocytes in 1 L or 1 uL
of blood
ARC
• Absolute reticulocyte count
• Actual number of reticulocytes in 1 L or 1 uL
of blood
CRC
• Corrected Reticulocyte count
• specimens with a low hematocrit
• the percentage of reticulocytes may be
falsely elevated because the whole blood
contains fewer RBCs.
CRC
• specimens with a low hematocrit
• the percentage of reticulocytes may be
falsely elevated because the whole blood
contains fewer RBCs.
RPI
•Reticulocyte Production Index
•Shift reticulocytes
•Lose reticula in 2 – 3 days
instead of 1 day in the
peripheral blood
•Retics move to peripheral
blood earlier than usual
RPI
•Shift reticulocytes
•Lose reticula in 2 – 3 days
instead of 1 day in the
peripheral blood
•Retics move to peripheral
blood earlier than usual
RPI
RPI
RDW
•Red Cell distribution
width
•Visualizes difference in
RBC size
RDW
•Red Cell distribution
width
•Visualizes difference in
RBC size
Anemia
Anemia
•“anaimia”
•Hemoglobin or RBC
count
•Decreased oxygen-
carrying capacity
Anemia
•Not a disease but an
underlying
manifestation of a
condition or
deficiency
Anemia
•Patient history is vital
•Diet, drug ingestion,
chemicals, occupation,
etc
•Fatigue
•Shortness of breath
Anemia
•Patient history is vital
•Diet, drug ingestion,
chemicals, occupation,
etc
•Fatigue
•Shortness of breath
Mechanisms of Anemia
•Ineffective
Erythropoiesis or
Insufficient
Erythropoiesis
•Blood loss or hemolysis
Ineffective Erythropoiesis
•Precursor cells are
defective
•Apoptosis
Ineffective Erythropoiesis
•Megaloblastic
anemia
•Thalassemia
•Sideroblastic anemia
Insufficient Erythropoiesis
•Decreased precursors in
the BM
•Iron deficiency
•Renal disease
•Autoimmune dse
(aplastic anemia)
•Infection
Insufficient Erythropoiesis
•Metastatic tumors
•Myelophthisic
anemia
•Leukemia
Blood loss or
hemolysis
•Traumatic injury
•Chronic blood loss
Laboratory diagnosis
Disorders of Iron
kinetics and Heme
Metabolism
Iron-related Disorders
Anemia may result whenever:
•red blood cell (RBC)
production is impaired,
•RBC life span is shortened,
•loss of RBCs from the body
•Iron-restricted anemia
•IDA
•Anemia of Chronic
Inflammation
•Iron-restricted anemia
•IDA
•Anemia of Chronic
Inflammation
•Protoporphyrin synthesis
blockage
•Sideroblastic anemia
•Hemachromatosis
•Excess accumulation of
iron usually without
anemia
•Iron-loading anemias
•involve chronic erythroid
hyperplasia as is seen in
the hemoglobinopathies
and thalassemias
Iron Deficiency
Anemia
•Iron is inadequate and
without compensation
•Impaired absorption
•Chronic loss of Hemoglobin
Iron Deficiency
Anemia
• Inadequate intake
• 1 mg of iron is lost from the body,
mainly in the mitochondria of
desquamated skin and sloughed
intestinal epithelium
• Diet
• If consistently inadequate, over time
the body’s stores of iron become
depleted
Iron Deficiency
Anemia
•Increased demand vs
supply
•Especially in periods of rapid
growth and pregnancy
•Treatment via EPO
•Functional iron deficiency
Iron Deficiency
Anemia
•Mutation
•Increased hepcidin
production
•Malabsorption
•Celiac disease
Iron Deficiency
Anemia
•Diseases that decrease
stomach acidity
•Ferric → Ferrous is
impaired
Iron Deficiency
Anemia
•repeated blood
donations,
•chronic hemorrhage,
•hemolysis
Iron Deficiency
Anemia
• Chronic blood loss
• iron loss exceeds iron intake over time
• Excessive heme iron can be lost
through repeated blood donations;
chronic gastrointestinal bleeding from
ulcers, tumors, parasitosis,
diverticulitis, ulcerative colitis, or
hemorrhoids; and gastritis caused by
alcohol or aspirin ingestion
Iron Deficiency
Anemia
•Menorrhagia or uterine
tumors
•Kidney stones or tumors
•Chronic infections
•PNH
Iron Deficiency
Anemia (Pathogenesis)
Iron
Iron Deficiency
•Happens in stages as disease
progresses
Iron Deficiency
Iron Deficiency
•Stage 1
• Gradual loss of storage iron
• RBC production and development is
normal
• RBC production uses up reserves of
iron
• Ferritin drops
• Latent iron deficiency = patients
appear healthy
Iron Deficiency
•Stage 2
•Loss of storage iron
•Hemoglobin content of retics
gradually decrease (iron
restricted hematopoiesis)
•MCV, MCH, MCHC
•Hgb normal or low and RDW
may increase
Iron Deficiency
• Stage 2
• serum iron and serum ferritin levels decrease
• total iron-binding capacity (TIBC) increases
• Free erythrocyte protoporphyrin (FEP) begins to
accumulate
• transferrin receptor (sTfR) levels increase
measurably.
• Prussian blue stain of the bone marrow shows
essentially no stored iron, and iron-restricted
erythropoiesis is evident
• Hepcidin would be measurably decreased
Iron Deficiency
•Stage 3
•Anemia
•Low Hgb, Low Hct
•Storage and Transport iron levels
decreased
•RBC precursors are affected
•RBC = microcytic and hypochromic
Iron Deficiency
•Stage 3
•Anemia
•Low Hgb, Low Hct
•Storage and Transport iron levels
decreased
•RBC precursors are affected
•RBC = microcytic and hypochromic
Iron Deficiency
•Stage 3
•Very low ferritin
•FEP and sTfR increased
•EPO increased
•Hepcidin decreased
Iron Deficiency
•Stage 3
•Anemia symptoms
Iron Deficiency
• Stage 3
• Anemia symptoms
Iron Deficiency
Iron Deficiency
Anemia
• Epidemiology
• Growing children
• Gastrointestinal diseases
• Aspirin and alcohol ingestion
• Iron-lacking diet
• Loss gastric acidity
• Hookworm infection
• Trichuris trichiura, Schistosoma mansoni,
and Schistosoma haematobium
Iron Deficiency
Anemia (Diagnosis)
•No evidence for anemia? Not yet
tested.
•High risk group
•Patient history
Iron Deficiency
Anemia (diagnosis)
•Screening
•CBC results = anisocytosis,
microcytic, hypochromic
•Decreased hemoglobin and
increased RDW
•NO consistent poikilocytosis**
Iron Deficiency
Anemia (diagnosis)
•Diagnostic testing
•Biochemical analyses include
assays of serum iron, TIBC,
transferrin saturation, and serum
ferritin
Iron Deficiency
Anemia (diagnosis)
•Diagnostic testing
•Biochemical analyses include
assays of serum iron, TIBC,
transferrin saturation, and serum
ferritin
Anemia of Chronic
Inflammation
Anemia of Chronic
Inflammation
•Associated with chronic
inflammatory conditions
brought about by:
•Malignancies
•RA
•Chronic infxns
• TB
• HIV
Anemia of Chronic
Inflammation
•Not the same as chronic
blood loss
•Inflammation = unifying
factor
•Sideropenia although iron
stores are plenty
Anemia of Chronic
Inflammation
•Impaired ferrokinetics
•Iron-restricted
erythropoiesis
•Impaired erythropoiesis and
shortened RBC life span
Anemia of Chronic
Inflammation
•Hepcidin = APR
•Regardless of iron stores,
hepcidin production increases
•Low iron absorption and low
iron release
Anemia of Chronic
Inflammation
•Iron is sequestered in
hepatocytes and
macrophages
Anemia of Chronic
Inflammation
•Iron is sequestered in
hepatocytes and
macrophages
Anemia of Chronic
Inflammation
•Iron sequestration during
inflammation = nonspecific
defense vs bacteria
•Bacteria uses iron = reduced
iron favorable to the body
Anemia of Chronic
Inflammation
•Lactoferrin
•Iron-binding protein in the
granules of neutrophils
•Important vs phagocytized
bacteria from using
intracellular iron
Anemia of Chronic
Inflammation
•Lactoferrin
•protection for the phagocyte
from oxidized iron that forms
when reactive oxygen species
(ROS) are produced during
phagocytosis
Anemia of Chronic
Inflammation
•Infxn and inflammation →
neutrophil death → Lactoferrin
in blood → scavenges iron →
binds to MACs and liver cells → if
hepcidin level is high → iron
remains sequestered
Anemia of Chronic
Inflammation
•Diminished Erythropoiesis
Anemia of Chronic
Inflammation
•Diminished Erythropoiesis
Anemia of Chronic
Inflammation
•Shortened RBC lifespan
•Extracellular mechanism
•Inflammation → increased
hemophagocytic
macrophages
Anemia of Chronic
Inflammation
•Laboratory Diagnosis
•Mild anemia
•8-10 g/dl hemoglobin
concentration
•Without reticulocytosis
•Microcytosis and
hypochromia = not specific
Anemia of Chronic
Inflammation
•Leukocytosis, thrombocytosis
or both
•Ferritin is an APR
•Decreased hemoglobin of
retics = iron restricted
erythroipoiesis
Anemia of Chronic
Inflammation
•sTfR = normal
•Bone marrow =
hypoproliferation of RBCs,
Prussian blue staining
Anemia of Chronic
Inflammation
•Diagnostic Dilemma:
•High ferritin
•Iron deficiency vs latent iron
deficiency vs anemia of
chronic inflammation
Anemia of Chronic
Inflammation
•Diagnostic Dilemma:
•High ferritin
•Iron deficiency vs latent iron
deficiency vs anemia of
chronic inflammation
Anemia of Chronic
Inflammation
•Treatment
•Therapeutic EPO + iron
•Cure underlying causes
•Anti-hepcidin therapies
Sideroblastic
anemia
Sideroblastic Anemia
•Protoporphyrin production is not
complete
•Microcytic hypochromic but with
elevated iron in bone marrow
•Prussian blue stain in BM smear
• Erythroblasts with iron deposits in the
mitochondria
•Ringed sideroblasts
complete
mitochondria
complete
mitochondria
•Acquired: Lead Poisoning
•CNS and hematologic system
•Impaired mental development
and anemia
•Peripheral neuropathy with
abdominal cramping and
vomiting or seizures
•Lead vs Porphyrin synthesis
1. VS ALA dehydratase
LEAD
2. VS Ferrochelatase/Heme Synthase
•ALA = urine
•FEP/ZPP = RBC
•Lead Poisoning
•Normocytic, normochromic
•Chronic exposure = Microcytic,
hypochromic
•Retic count in acute poisoning =
elevated
•Impaired PPS (oxidant stress)
•Lead Poisoning
•Erythroid hyperplasia
•Basophilic stippling = classic
finding
•Lead inhibits pyrimidine 5'-
nucleotidase
•Breakdown of rRNA in retics
•Lead Poisoning
•Aggregated ribosomes
aggregate
•Stippling is heavier vs
other anemias = Truly
punctate basophilia
•Lead Poisoning
•Aggregated ribosomes
aggregate
•Stippling is heavier vs
other anemias = Truly
punctate basophilia
•Lead Poisoning
•calcium disodium edetate
(CaNa2EDTA)
•Dimercaprol are often used to
chelate the lead = excreted in
urine
•Lead poisoning = acquired
sideroblastic anemia and acquired
porphyria
•Porphyria
•Impaired production of porphyrin
component of Heme
•May be acquired or hereditary
•Most often refer to hereditary
conditions vs protoporphyrin
production
•Porphyria
•Impaired production of porphyrin
component of Heme
•May be acquired or hereditary
•Most often refer to hereditary
conditions vs protoporphyrin
production
• Porphyrins
• Porphyrins leak from cells as they age or
die
• Excreted in urine and feces and also
deposits in body tissue
• Deposition in skin = photosensitivity,
severe burns when exposed
• bone marrow specimen the
erythroblasts will be bright red under a
fluorescent microscope
Iron Overload
•Iron Overload
•XS iron accumulation in all cells
•Hereditary hemochromatosis
•Secondary to chronic anemia and
their treatments
•lipids, proteins, nucleic acids, and
heme iron become oxidized.
Iron Overload
•Iron Overload
•Iron is stored first as ferritin and
then as hemosiderin within cells
•Repeated transfusions
•Treatment of anemia:
•Sickle cell and B-thalassemia
major
Iron Overload
•Iron Overload
•Hereditary hemolytic anemia
•Iron loading anemias
•Develop compensatory erythroid
hyperplasia
•Erythroblasts downregulate
hepcidin production by excreting
erythroferrone
Iron Overload
•Iron Overload
•Hereditary hemolytic anemia
•Erythroid hyperplasia ➔
increased erythroferrone ➔
decreased hepcidin ➔ increased
iron absorption and recycling
•Chronic hemolytic anemia →
excessive iron
Iron Overload
•Iron Overload
•Hereditary hemolytic
anemia
•If hemolysis is controlled,
iron loading subsides
Iron Overload
•Iron Overload
•Hemachromatosis
•Mutations in genes for proteins
controlling iron kinetics
•Iron Overload
•Hemachromatosis
Iron Overload
•Iron Overload
•Hemachromatosis
•Body continues to absorb
iron
Iron Overload
•Iron Overload
•Hemachromatosis
•Body continues to absorb
iron even when stores are
full
•Iron Overload
•Hemachromatosis
•HFE mutation
Iron Overload
•Oxygen + iron =
generation of superoxides
and other free radicals
•Cells die due to irreversible
membrane changes
Iron Overload
•Iron Overload
•Increased iron in
parenchymal cells
•Ferritin → hemosiderin
(intracellular
accumulation)
Iron Overload
•hemosiderin gives the skin a
golden color; the liver, where
cirrhosis-induced jaundice and
subsequent cancer may
develop; and the pancreas,
where damage results in
diabetes mellitus
Iron Overload
•heart muscle also is especially
vulnerable to excessive iron
deposition, which leads to
congestive heart failure
Iron Overload
•Diagnosis
•screen for the condition, diagnose
the cause of organ damage, pinpoint
the mutation for family genetic
counseling, and monitor treatment.
•Elevations of transferrin saturation
or serum ferritin can be used as a
screening test for hereditary
hemochromatosis
Iron Overload
•Diagnosis
•screen for the condition, diagnose
the cause of organ damage, pinpoint
the mutation for family genetic
counseling, and monitor treatment.
•Elevations of transferrin saturation
or serum ferritin can be used as a
screening test for hereditary
hemochromatosis
Anemias Caused by
Defects of DNA
Metabolism
Impaired DNA
Metabolism
•causes systemic effects by
impairing production of all
rapidly dividing cells of the
body
•Megaloblastic anemia
Megaloblastic Anemia
•Very large cells of the bone
marrow due to the reduction in
the number of cell divisions
•Macrocytic anemia
•Vitamin B12 (Cobalamin) and
Folic acid (Folate)
Roles of VitB12
and Folate
•Vitamin B12 (cobalamin) is an
essential nutrient consisting of a
tetrapyrrole (corrin) ring
containing cobalt that is attached
to 5,6-dimethylbenzimidazolyl
ribonucleotide
Roles of VitB12 and Folate
•Vitamin B12 is a coenzyme in two
biochemical reactions in humans. One
is isomerization of methylmalonyl
coenzyme A (CoA) to succinyl CoA,
which requires vitamin B12 (in the
deoxyadenosylcobalamin form) as a
cofactor and is catalyzed by the
enzyme methylmalonyl CoA mutase
• The second reaction is the transfer of a
methyl group from 5-
methyltetrahydrofolate (5-methyl THF) to
homocysteine, which thereby generates
methionine.
• This reaction is catalyzed by the enzyme
methionine synthase and uses vitamin B12
(in the methylcobalamin form) as a
coenzyme
•Folate is the general term used for any
form of the vitamin folic acid. Folic acid
is the synthetic form in supplements
and fortified food.
•Folates consist of a pteridine ring
attached to paraaminobenzoate with
one or more glutamate residues
•function of folate is to transfer
carbon units in the form of methyl
groups from donors to receptors.
•Folate deficiency = impaired cell
replication and other metabolic
alterations.
Defect in Megaloblastic
Anemia
• When either folate or vitamin B12 is
deficient, thymidine nucleotide production
for DNA synthesis is impaired
• Folate deficiency has the more direct
effect, ultimately preventing the
methylation of dUMP. The effect of vitamin
B12 deficiency is more indirect, preventing
the production of THF from 5-methyl THF.
Anemia
•when either folate or vitamin B12
is deficient, homocysteine
accumulates because methionine
synthase is unable to convert it to
methionine without vitamin B12 as
a cofactor
Anemia
• In this state of diminished thymidine
availability, uridine is incorporated into DNA.
The DNA repair process can remove the
uridine, but without available thymidine to
replace it, the repair process is unsuccessful.
Although the DNA can unwind and replication
can begin, at any point where a thymidine
nucleotide is needed, there is essentially an
empty space in the replicated DNA sequence,
which results in many single-strand breaks.
Anemia
•Repeated DNA strand breaks lead
to fragmentation of the DNA strand
•Non-functional DNA
•DNA replication is incomplete
•Cell division is stopped = cell is lysed
or apoptosis occurs
•Ineffective hematopoiesis
Anemia
•Erythroid precursors are larger
than normal
•Immature nuclei
•Open finely stippled reticular
pattern of normoblasts
•RNA is unaffected by vit B12 or
folate = normal cytoplasm
Anemia
•Pancytopenia
•Nuclear-cytoplasmic
asynchrony
•Pathognomonic sign of
megaloblastic anemia
Other Causes of
Megaloblastosis
•Congenital dyserythropoietic anemia
(CDA) types I and III
•Myelodysplastic syndromes (MDS)
•Delayed cytoplasm and nuclear
maturation
•congenital dyserythropoietic anemia
(CDA) types I and III
Other Causes of
Megaloblastosis
•FAB M6
•RT inhibitors (vs HIV) = but
also vs DNA production
Systemic Manifestations
of Deficiency
•patients may experience
general symptoms related to
anemia (fatigue, weakness,
and shortness of breath) and
symptoms related to the
alimentary tract
of Deficiency
•Loss of epithelium on the
tongue results in a smooth
surface and soreness
(glossitis). Loss of epithelium
along the gastrointestinal
tract can result in gastritis,
nausea, or constipation
of Deficiency
•After dietary deficiency or
malabsorption begins, it takes
a few years to develop a
vitamin B12 deficiency but only
a few months to develop a
folate deficiency, reflecting the
storage capacity of each
vitamin in the body
of Deficiency
•memory loss, numbness and
tingling in toes and fingers,
loss of balance, and further
impairment of walking by
loss of vibratory sense,
especially in the lower limbs
of Deficiency
•Cardiovascular risk =
homocysteine levels
•Folate levels appear to
influence the effectiveness
of treatments for depression
of Deficiency
•Folate deficiency during
pregnancy can result in impaired
formation of the fetal nervous
system, resulting in neural tube
defects such as spina bifida,
despite the fact that the fetus
accumulates folate at the expense
of the mother.
Causes of Vitamin
Deficiencies
Folate Deficiency
Inadequate intake
•Low folate levels in diet
Increased need
•Pregnancy and lactation
Folate Deficiency
Impaired Absorption
• Food folates must be
hydrolyzed in the gut before
absorption in the small
intestine
•Folate transporter proteins
Folate Deficiency
Impaired Absorption
• Intestinal disease
•Sprue and Celiac disease
•Resectioned small intestine
•Inflammatory bowel disease
Folate Deficiency
Impaired Use of Folate
•Drugs
•Antineoplastic, antibacterial,
antiseizure
Folate Deficiency
Excessive Loss of Folate
•Kidneys
•Renal dialysis
Vitamin B12 Deficiency
Inadequate Intake
•Strict vegetarians
•Plants cannot synthesize
vitamin B12
Increased Need
•Pregnancy, lactation and
growth
Impaired Absorption
The absorption of vitamin B12 can be impaired by:
(1)failure to separate vitamin B12 from food proteins
in the stomach,
(2)Failure to separate vitamin B12 from haptocorrin
in the intestine,
(3)Lack of intrinsic factor,
(4)malabsorption, and
(5)competition for available vitamin B12
Failure to separate vitamin B12 from
food proteins
•Food-cobalamin malabsorption
• Hypochlorhydria
• Atrophic gastritis
• Gastric bypass surgery
• Long-term use of histamine blockers
and proton-pump inhibitors
Failure to separate vitamin B12 from
Haptocorrin
• Lack of gastric acidity
• Lack of trypsin
• Pancreatic disease
Lack of intrinsic factor
• Pernicious anemia
• Autoimmune disorder
• IF deficiency
• Increased risk for gastric tumors
• autoimmune lymphocyte-mediated destruction
of gastric parietal cells
• loss of H1 production in the stomach constitutes
achlorhydria
• Schilling test
Lack of intrinsic factor
• Pernicious anemia
• production of antibodies to intrinsic
factor and gastric parietal cells that are
detectable in serum
• Other causes of lack of IF
• H. pylori infxn
• Hereditary IF deficiency
Malabsorption
• celiac disease, tropical sprue, and
inflammatory bowel disease
Inherited errors of vitamin B12 absorption
and transport
• Imerslund-Gräsbeck syndrome is a rare
autosomal recessive condition caused by
mutations in the genes for either cubilin or
amnionless.
Competition for vitamin B12
• Blind loops, portions of the intestines
that are stenotic as a result of surgery or
inflammation, can become overgrown
with intestinal bacteria that compete
effectively with the host for available
vitamin 12.
• D. latum
• Splits VitB12 from IF
Laboratory Diagnosis
Screening:
•CBC
•Retic
•WBC differential
•Bilirubin
•Lactate dehydrogenase
•CBC
•Hgb = less than 7
•Hct = less than 20%
•MCV = 100 to 150 fL
•MCH = increased
•MCHC = within reference interval
•RDW = elevated
• CBC & reticulocyte count
• Hypersegmented neutrophils
• Low absolute reticulocyte count
• Dacryocytes
• RBC fragments
• Microspherocytes
• Schistocytes
• Nucleated RBCs, Howell-Jolly bodies,
basophilic stippling, and Cabot rings
• Bilirubin and Lactate Dehydrogenase
• Increased total and indirect bilirubin
• Increased lactate dehydrogenase
• Bone Marrow
• Bone Marrow
Aplastic Anemia
& Bone Marrow
Failure Anemias
Bone Marrow Failure
Anemias
•Reduction or cessation of
blood cell production
•Affects one or more cell lines
•Pancytopenia
Aplastic Anemia
•Failure of the Bone Marrow
•Rare but potentially deadly
•Can be genetic or acquired
•Laboratory findings
•Decreased hgb, hct, retics
•No response to EPO
Aplastic Anemia
•Elevated EPO, TPO, and CSFs
•No splenomegaly
•Pancytopenia
•Normocytic, Normochromic
•Hypocellular BM
•Increased Iron
Aplastic Anemia
•Monosomy 7 and Trisomy 8
Aplastic Anemia
•Acquired
•Approximately 80 to 85% of
aplastic anemia cases
•Two categories:
•Idiopathic
•Secondary acquired aplastic
anemia
Aplastic Anemia
•Acquired
•Two categories:
•Secondary acquired aplastic
anemia
•Chemicals (insecticides,
benzene)
•Viruses (EBV)
•Drugs (Chloramphenicol)
Aplastic Anemia
•Inherited
• ~15% to 20% of aplastic anemia cases
1. Dyskeratosis congenita (DC)
2. Shwachman-Bodian-Diamond (SBDS)
Syndrome
3. Fanconi Anemia (FA)
Aplastic Anemia
1. Dyskeratosis congenita (DC)
•Mucocutaneous abnormalities
•BM failure, Pancytopenia
•Abnormal skin presentation
•Dystrophic nails
•Oral Leukoplakia
Aplastic Anemia
2. Shwachman-Bodian-Diamond (SBDS)
Syndrome
• Pancreatic insufficiency
• Cytopenia
• Skeletal abnormalities
• Predisposition for hematologic
abnormalities
Aplastic Anemia
• Most common inherited Aplastic anemia
• Chromosome instability
• Increased risk for cancer
• Chromosome breakage analysis =
diagnostic test
• Physical abnormalities:
• Skeletal (Thumb malformations,
microcephaly, scoliosis)
Aplastic Anemia
• Physical abnormalities:
• Skin pigmentation (hyperpigmentation,
hypopigmentation, café-au-lait lesions)
• Short stature
• Abnormalities of the eyes, kidneys, and
genitals
• Fanconi Anemia vs Fanconi Syndrome

Pure Red Cell Aplasia
•Selective and severe decrease in
erythrocyte precursors
•Severe anemia and reticulocytopenia
with normal WBCs and PLTs
•Acquired: Transient
Erythroblastopenia of Childhood (TEC)
•Congenital: Diamond-Blackfan
Anemia
Congenital Dyserythropoietic Anemia
• Hypercellular BM but ineffective erythropoiesis

• Types
• CDA-1 = Spongy heterochromatin with Swiss
cheese appearance
• CDA-2 = Most common subtype; HEMPAS
(Hereditary Erythroblastic Multinuclearity with
Positive Acidified Serum)
• CDA-3 = Least common; BM has megaloblastic
changes; Giant erythroblasts with up to 12
nuclei
Myelophthisic anemia
• Marrow replacement anemia
• Myeloid metaplasia
• Hypoproliferative anemia caused by replacement of
bone marrow hematopoietic cells by malignant cells
or fibrotic tissue (hypercellular BM)
• Associated with cancers
• Dacryocytes
• Normocytic, Normochromic
• Immature RBC and WBC (Leukoerythroblastic blood
picture)
Anemia of Chronic Kidney Disease
•Complication of renal disease

•Inadequate renal production of
EPO
•Uremia inhibits erythropoiesis and
increases RBC fragility
•Burr cells
Blood Loss Anemia
Blood loss anemia
Acute vs Chronic
•Acute blood loss anemia
•Rapid onset
•May be due to trauma or injury
•Clinical symptoms: hypovolemia,
rapid pulse, low BP, pallor
Blood loss anemia
Acute vs Chronic
•Acute blood loss anemia
•Laboratory diagnosis
• Normo, normo
• Initially Retics is normal then
increase in 3-5 days
• N hgb an hct within the first hours
then Inc PLT and WBCs with a
drop in hgb, hct and RBC
Blood loss anemia
Acute vs Chronic
• Chronic blood loss anemia
• Gradual loss
• Often caused by gastrointestinal bleeding
• Laboratory findings:
• Initially normo-normo, that causes a
decrease in hgb and hct
• Gradual loss of iron – micro-hypo
• Ultimately leads to IDA
Hemolytic Anemia
Hemolytic Anemias
•RBC destruction > RBC production
•Hemolysis of mature RBCs is the
primary cause of anemia
•Acute vs Chronic
•Inherited vs Acquired
•Intrinsic vs Extrinsic
•Intravascular vs Extravascular
Hemolytic Anemias
Acute vs Chronic
• Acute
• Rapid onset, episodic; Paroxysmal (PNH,
PCH, HTR)
• Appears and disappears between
episodes
• Chronic blood loss anemia

• May not be evident but can cause crisis
over time
Hemolytic Anemias
Inherited vs Acquired
•Inherited
• Abetalipoproteinemia – impaired
absorption of fats; deficiency of fat
soluble vitamins
• LCAT deficiency
Hemolytic Anemias
Inherited vs Acquired
•Acquired
•PNH (Paroxysmal Nocturnal
Hemoglobinuria)
• Deficiency of Complement
regulatory proteins (DAF and
MIRL)
Hemolytic Anemias
Intrinsic vs Extrinsic
•Intrinsic
• Abnormal RBCs (cannot donate blood,
only receive)
• Most are acquired
• Abnormality of the:
1. RBC membrane
2. Enzymatic pathways
3. Hemoglobin
Hemolytic Anemias
Intrinsic vs Extrinsic
•Extrinsic
• Caused by environment
• Cannot receive blood, only donate
• Most are acquired
• Substances affecting circulatory system
• May either be:
1. Immunohemolytic
2. Non-immune
Hemolytic Anemias
Intravascular vs Extravascular
•Intravascular
•Happens in the blood vessels
•Fragmentation hemolysis
•Mechanical hemolysis
Hemolytic Anemias
Intravascular vs Extravascular
•Extravascular hemolysis
• Outside blood vessels
• Macrophage-mediated hemolysis
Hemolytic Anemias
Hemolysis
• Detection of hemolysis depends partly on
detection of RBC breakdown products (e.g.
bilirubin)
• Haptoglobin-hemopexin and Methemalbumin
system salvages hemoglobin iron
Hemolytic Anemias
Hemolysis
• Hemoglobin = Heme + globin
• Globin is recycled
• Heme → (via heme oxygenase) →
Biliverdin+iron
• Iron is recycled
• Biliverdin → (via biliverdin reductase) →
Bilirubin
• Bilirubin bound by albumin to liver
• Conjugated bilirubin → intestine →
urobilinogen
Hemolytic Anemias
Hemolysis
• Hemosiderosis and hemoglobinuria is
present if system is overloaded
• Macrophage mediated hemolysis occurs in
the spleen
• Senescent cells or cells with inclusion bodies
or poikilocytosis is digested by macrophages
Hemolytic Anemias Intravascular Extravascular
Haptoglobin DEC SL. DEC

Hemolysis
Hemopexin DEC SL. DEC
Free hgb INC SL. INC
Urine free hgb PRESENT ABSENT
Urine methemoglobin PRESENT ABSENT
Urine sediment PRESENT ABSENT
Schistocytes PRESENT ABSENT
Spherocytes ABSENT PRESENT

Hemolytic Anemias
Due to Intrinsic
Defects
Hemolytic Anemias
(intrinsic defects)
1. Hereditary spherocytosis
• Defect in membrane skeletal proteins (spectrin and
ankyrin)
• Autosomal dominant
• Abnormalities cause RBCs to lose unsupported lipid
membrane
• Laboratory findings:
• Spherocytes
• MCHC > 37 g/dL
• Increased osmotic fragility
• Increased serum bilirubin
Hemolytic Anemias
(intrinsic defects)
1. Hereditary spherocytosis
• Disrupted vertical linkages
• 3 key clinical manifestations: Anemia,
Jaundice, Splenomegaly
• Splenectomy for moderate to severe cases.
• Some of assays include OFT, EMA binding test,
SDS-PAGE, RIA, Ektacytometer, Acidified
glycerol lysis test, autohemolysis test,
Hypertonic cryohemolysis test, DAT
Hemolytic Anemias
(intrinsic defects)
2. Hereditary Elliptocytosis (Ovalocytosis)
• Membrane defects caused by polarization of
cholesterol at the ends of the cell rather than
around the pallor
• Disrupted horizontal linkages
• Extravascular hemolysis and anemia but usually
asymptomatic
• Px with Leach phenotype lack the Gerbich antigens
and glycoprotein C
• Hereditary pyropoikilocytosis = Severe form; RBCs
fragment at 41-45 deg C
Hemolytic Anemias
(intrinsic defects)
3. Hereditary Ovalocytosis (South East Asian
ovalocytosis)
• Gene mutation that results in a rigidity of
the membrane
• Resistance to Malaria
• Malaria belt in SEA
Hemolytic Anemias
(intrinsic defects)
4. Overhydrate Hereditary Stomatocytosis
• RBC membrane is excessively permeable
to sodium and potassium at 37 deg C
• RBCs swell
5. Dehydrated Hereditary Stomatocytosis
• Hereditary Xerocytosis
• Most common form of Stomatocytosis
• Potassium leaks out of the cell
Hemolytic Anemias
(intrinsic defects)
6. Hereditary Acanthocytosis
(Abetalipoproteinemia)
• Increased cholesterol:lecithin ratio in the
membrane due to abnormal lipid
concentrations
• Absence of serum beta-lipoprotein needed
for transport
Hemolytic Anemias
(intrinsic defects)
7. Paroxysmal Nocturnal
Hemoglobinuria
• Increased sensitivity for complement binding
• Cells are sensitive to complement lysis
• Flow cytometry
Hemolytic Anemias
(intrinsic defects)
7. Paroxysmal Nocturnal
Hemoglobinuria
• Increased sensitivity for complement binding
• Cells are sensitive to complement lysis
• Lack of GPI linkage
• Flow cytometry
• CD 55 DAF
• CD59 MIRL
• At night, blood is acidic
Hemolytic Anemias
(intrinsic defects)
Other defects:
•Stomatocytosis = Familial
pseudohyperkalemia;
cryohydrocytosis; Rh deficiency
syndrome
•Acanthocytes = Neuroacanthocytosis
(McLeod Syndrome, Chorea
acanthocytosis); spur cell anemia
Hemolytic Anemias
(Enymopathies)
G6PD deficiency
•Vulnerable to oxidative damage and
subsequent hemolysis
•Most common RBC enzyme defect
•VS P. falciparum and vivax in males
•Heinz body anemia
•HMP
Hemolytic Anemias
(Enymopathies)
G6PD deficiency
•5 classes, type 1 is the worst
•Bite cells
•Drugs (primaquine – malarial drug)
•Mothballs, fava beans
Hemolytic Anemias
(Enymopathies)
PK deficiency
•EMP
•Lack of ATP = poor cation pump
(Na,K)
•Decreased RBC deformability
•Reduced lifespan
Hemolytic Anemias (Extrinsic
non-immune defects)
•Condition outside RBCs causing
premature hemolysis
•Not immune related
•Physical or mechanical injury to
RBCs
non-immune defects)
•Microangiopathic Hemolytic
Anemia (MAHA)
•Potentially life threatening
•RBC fragmentation (schistocytes)
•Thrombocytopenia
•Helmet cells and schistocytes
non-immune defects)
Anemia (MAHA)
•Thrombotic Thrombocytopenic
Purpura
•Anemia, severe
thrombocytopenia, markedly
elevated serum LD activity
non-immune defects)
Anemia (MAHA)
Purpura
•Neurologic dysfunction, fever,
renal failure
•Deficiency of vWf-cleaving
protease (ADAMTS-13)
non-immune defects)
Anemia (MAHA)
Purpura
•Platelets-vWF complex accumulate
and blocks small blood vessels
➔Thrombocytopenia,
ischemia, hemolytic anemia
non-immune defects)
Anemia (MAHA)
•Hemolytic Uremic Syndrome (HUS)
• Most often in children after E. coli
infection
• Clots forming renal damage
non-immune defects)
Anemia (MAHA)
•HELLP
• Hemolysis, Elevated Liver enzymes, Low
PLT
• Pregnant women (pre eclampsia)
non-immune defects)
Anemia (MAHA)
•Disseminated Intravascular
Coagulation (DIC)
• Widespread activation of the
hemostatic system
• Systemic clotting triggered by pro
coagulants
• Thrombocytopenia
non-immune defects)
Anemia (MAHA)
•Disseminated Intravascular
Coagulation (DIC)
• Fibrin is deposited in small vessels
causing RBCs to fragment
• Keratocyte+schistocyte
non-immune defects)
Other causes:
• March Hemoglobinuria
• Infectious agents (e.g. malaria; clostridium)
• Mechanical trauma (e.g. prosthetic heart
valves)
• Thermal burns
immune defects)
•RBCs with bound Ab or C’ are
prematurely removed from the
circulation extravascularly by
macrophages
•Complement-mediated hemolysis
removes RBCs intravascularly
immune defects)
•Spherocytes are present
•To ascertain if it is of immune
causes direct antihuman
globulin test is done.
immune defects)
•Classified into three:
1. Autoimmune (WAIHA, CAD, PCH,
Mixed-type)
2. Alloimmune (HTR and HDFN)
3. Drug-induced
immune defects)
WAIHA CAD PCH Mixed
Immunoglobulin IgG IgM IgG IgG and IgM
Temp 37 deg C 4 deg C - 30 deg C 4 deg C 4 deg C - 37 deg C

Complement
Variable Yes Yes Yes
activation
Hemolysis Extravascular Extravascular Intravascular Both
I (most); i (some); Pr
Blood group Rh P Panreactive
(rare)
immune defects)
•WAIHA
• Warm Autoimmune Hemolytic Anemia
• Most common immune hemolytic anemia
• Antibodies = IgG
• RBCs are coated with complement or
antibodies ➔ Macrophages either
phagocytize RBCs or remove antibody
complement ➔ RBCs hemolyze / have
membrane loss
immune defects)
•CAD
• Cold Agglutinin Disease
• Cold agglutinins = Autoantibodies of the IgM class
• React optimally at 4 deg C
• Acute CAD may be secondary to M. pneumoniae
infection, IM, and other infections
• Blood specimen must be maintained at body temp
after collection
immune defects)
•PCH
•Paroxysmal Cold Hemoglobinuria
•IgG biphasic antibody
•Fixes complement to RBC when cold;
lyses complement-coated RBC when
warmed
immune defects)
•Mixed type
• WAIHA + CAD
immune defects)
•Drug induced HA
• 4 mechanisms:
1. Drug absorption
2. Drug-RBC membrane protein immunogenic
complex
3. RBC autoantibody induction
4. Membrane modification method
immune defects)
•HTR
• Most common cause of AHTR is
accidental transfusion of ABO-
incompatible donor RBCs into a recipient
(IgM)
• DHTR = usually IgG; happens days to
weeks after transfusion as the titer of
alloantibodies increases
immune defects)
•HDFN
• Occurs when an IgG alloantibody
produced by the mother crosses the
placenta into the fetal circulation and
binds to fetal RBCs that are positive for
the corresponding antigen
• IgG-sensitized fetal RBCs are cleared
from circulation by macrophages in the
spleen
Hemoglobinopathies
Hemoglobinopathy
•A disease state involving Hgb
•Most common genetic diseases
•Point mutation
•QUALITATIVE defect
Hemoglobinopathy
•A disease
Hemoglobin S state
6thinvolving Hgb
amino acid of Beta chain; GLUTAMATE is replaced by VALINE
•Most common
Hemoglobin C
genetic diseases
6th amino acid of Beta chain; GLUTAMATE is replaced by LYSINE
•Point mutation
Hemoglobin E 26th amino acid of Beta chain; GLUTAMATE is replaced by LYSINE
Hemoglobin O 121st amino acid of Beta chain; GLUTAMATE is replaced by LYSINE
Hemoglobin D 121st amino acid of Beta chain; GLUTAMATE is replaced by GLYCINE

Hemoglobinopathy
• Sickle Cell Disease (Hgb SS)
• No Hemoglobin A1 is produced
• Approximately 80% is Hgb S and 20% is
Hgb F
• Hgb A2 is variable
• When deoxygenated, Hgb
crystallizes/polymerizes forming the
classic sickle cell shape
Hemoglobinopathy
•Sickle Cell Trait (Hgb AS)
•Defect is inherited from 1 parent
only
•60% Hgb A1 and 40% Hgb S
•Hgb A2 and Hgb F are normal
Hemoglobinopathy
•Hemoglobin C disease (Hgb CC)
• No Hgb A is formed
• 90% Hgb C; 2% Hgb A2; 7% Hgb F
Hemoglobinopathy
•Hemoglobin SC disease (Hgb SC)
• Double heterozygous condition
• Abnormal sickle cell gene from one
parent and abnormal C gene from other
parent is inherited
• No Hgb A is formed
• 1:1 Hgb S and Hgb C
• Compensatory Hb F may be found
Thalassemias
Thalassemia
•QUANTITATIVE defect
•Reduced synthesis of one or more of
the globin chains of Hgb
•Anemia, hepatosplenomegaly,
mongoloid faces
•Imbalance of A vs B chain ratios
•Thalassemia minor = resistance to
malaria
Thalassemia
•QUANTITATIVE defect
•Reduced synthesis of one or more of
the globin chains of Hgb
•Anemia, hepatosplenomegaly,
mongoloid faces
•Imbalance of A vs B chain ratios
•Thalassemia minor = resistance to
malaria
Thalassemia
•Normal Hemoglobins in an adult
• Hgb A1 = 95-100%
• Hgb A2 = 0 – 3.5%
• Hgb F = 0 – 2%
Normal B-chain = B/B

Normal A-chain = aa/aa
Alpha-thalassemia
Alpha-thalassemia major (--/--)
• All four alpha chains are deleted
• No normal Hgb is produced
• Hydrops fetalis
• 80% Bart’s Hgb (4 gamma chains)
• Cannot carry oxygen
• Incompatible with life 
Alpha-thalassemia
Alpha-thalassemia minor/trait (a-/a-) or
(aa/--)
• Two alpha genes are deleted
• Px are usually asymptomatic and
discovered accidentally
• Up to 6% Hgb Barts in newborns = diag.
• Mild microcytic, hypochromic anemia
• High RBC count
• Target cells
Alpha-thalassemia
Hgb H disease (a-/--)
•3 alpha genes are deleted; excess Beta
chains
•Heinz bodies are formed
•RBCs are rigid and are destroyed in the
spleen
•Moderate microcytic, hypochromic
•70% Hgb A; 30% Hgb H
Beta-thalassemia
Beta-thalassemia major (homozygous)
•Cooley anemia
•Absence of both Beta chains
•Excess in alpha chains
•No Hgb A; compensation is 90% Hgb F
•Requires regular transfusion
• Iron burden = deferoxamine is given
Beta-thalassemia
Beta-thalassemia major
•Clinical findings:
• Sever micro-hypo
• Target cells, teardrop cells, nRBCs,
basophilic stippling, Howell-Jolly bodies,
Pappenheimer bodies, Heinz bodies
• Increased serum iron and bilirubin
Beta-thalassemia
Beta-thalassemia minor (heterozygous)
• Mild; asymptomatic
• One beta chain is normal
• One beta chain is absent or weakly expressed
• Hgb A decreased but Hgb A2 increased to
compensate
• Mild micro-hypo
• Target cells
• Basophilic stippling
NNNNNNNNNNNN

Anemia

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Anemia

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Laboratory

• Fanconi Anemia vs Fanconi Syndrome

• Hypercellular BM but ineffective erythropoiesis

•Complication of renal disease

• Chronic blood loss anemia

Haptoglobin DEC SL. DEC

Free hgb INC SL. INC

Urine free hgb PRESENT ABSENT

Urine methemoglobin PRESENT ABSENT

Urine sediment PRESENT ABSENT

Schistocytes PRESENT ABSENT

Spherocytes ABSENT PRESENT

Immunoglobulin IgG IgM IgG IgG and IgM

Temp 37 deg C 4 deg C - 30 deg C 4 deg C 4 deg C - 37 deg C

Hemoglobin O 121st amino acid of Beta chain; GLUTAMATE is replaced by LYSINE

Hemoglobin D 121st amino acid of Beta chain; GLUTAMATE is replaced by GLYCINE

Normal B-chain = B/B

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