MODULE 5 – ERYTHROCYTE DISORDERS

MODULE OUTCOMES
At the end of this module, the intern is able to:
1. discuss comprehensively the etiology and pathophysiology of the different erythrocyte disorders.
2. apply systematically the procedural steps in common and special diagnostic laboratory examination of
erythrocyte disorders.
3. interpret correctly laboratory results employed for the diagnosis of erythrocytic disorders.

INTRODUCTION
There are many disorders involving red blood cells. RBC disorders may be classified quantitatively – anemia,
a condition where the number of red blood cells is decreased, and erythrocytosis, where the red cells are increased.
There are many types of anemia, with some having more severe consequences than others. RBC disorders may be
diagnosed by taking the medical history, observing for characteristic signs and symptoms, and performing laboratory
examinations.

KEY CONCEPTS
I. CLASSIFICATION OF RBC DISORDERS
A. Anemia
1. Term used to denote conditions associated with decreased to below normal Hb concentration,
erythrocyte count, or hematocrit (Hct).
2. Best defined in reference to a decreased Hb level
3. ↓ amount of Hb/RBC = ↓ O2 reaching the tissues/organs of the body
4. Not a disease but a manifestation of an underlying disease or deficiency
5. Can be classified according to:
a. Etiology/cause
b. Morphology – uses RBC indices (MCV, MCH, MCHC)
c. Physiology
B. Erythrocytosis and Polycythemia
1. Increased RBCs in the circulation
2. Conditions associated with anemia and erythrocytosis can be subdivided into two:
a. Absolute – true increase or decrease in red cell mass
b. Relative – secondary to a change in plasma volume

II. ANEMIA
A. Definition
1. Functional definition: decrease in the oxygen carrying capacity of the blood
2. Operational definition: reduction in the hemoglobin content of blood that can be caused by a decrease in
RBCs, hemoglobin, and hematocrit below the reference interval
a. Moderate anemias – Hb 7 to 10 g/dL
b. Severe anemias – Hb <7 g/dL
3. Not a disease but a manifestation of an underlying disease or deficiency
B. Diagnosis of Anemia
1. Medical History
a. Some anemias are racial/ethnic in origin (inherited)
i. Hemoglobinopathies – black patients with Hb-S, SE Asian with Hb-E
ii. Thalassemias – patients with Mediterranean heritage
iii. RBC enzyme deficiencies – Pennsylvania Amish with PK deficiency, blacks with G-6-PD
deficiency)
b. Exposure to toxic chemicals and medications
c. Travel background (e.g. malaria, babesiosis)
d. Diet
e. Presence of underlying disease (e.g. chronic inflammatory disease, leukemia/lymphoma, endocrine
disorders)
2. Signs and Symptoms
a. Fatigability and dyspnea on exertion, faintness, vertigo, palpitations and headache
b. Pallor, low blood pressure, light fever, rapid bounding pulse, some dependent edema and systolic
murmurs
c. Diagnosis of anemia or of high Hb based on estimation of the color of the skin and of visible mucous
membranes is highly unreliable
d. Physical findings:
i. Skin – pallor (nonhemolytic anemia); jaundice (hemolysis); leg ulcers (sickle cell anemia)
ii. Eyes – “sausage link” veins (Waldenstrom’s macroglobulinemia); tortuous vessels (Hb-SC
disease)
iii. Mouth – “lead lines” on gums (lead poisoning); glossitis or smooth tongue (P.A.); gum hypertropy
(dilantin therapy of AMoL)
iv. Lymph nodes – leukemia/lymphoma; infectious disease
 v. Heart – murmurs; enlargement
vi. Spleen – Mild enlargement (IDA, hemolytic anemia); moderate enlargement (chronic liver
disease); massive enlargement (myeloproliferative disorders)
vii. Liver – alcoholism, myelophthisic anemias
viii. Bone – sternal pain (leukemia); back pain (myeloma)
3. Laboratory Tests
a. Hematologic (Initial)
i. RBC parameters: Hb and Hct (most widely used), RBC count
ii. RBC indices (MCV, MCH, MCHC, RDW)
iii. Examination of peripheral blood smear (RBC morphology)
iv. Reticulocyte count, RPI
v. White cell and platelet counts
b. Additional tests as indicated
i. Bone marrow examination
ii. Urine – protein, bilirubin/urobilinogen
iii. Stool – occult blood, parasites
iv. Serum – LD (↑ in megaloblastic and hemolytic anemia); iron; ferritin (evaluates body iron stores,
proportional to amount of storage iron); TIBC (↑ in IDA); Zinc erythrocyte protoporphyrin test
(determine amount of protoporphyrin not used for Hb synthesis, ↑ in IDA); Plasma iron turn over
59 59
(radioactive iron Fe is IV injected, if there is ↓RBC production Fe stays in the blood longer);
51
RBC life span (uses Cr); folic acid; vitamin B12; bilirubin; protein electrophoresis, etc.
C. General Classification of Anemia
1. Absolute Anemia
a. RBC mass decreased, plasma volume normal
b. Mechanisms:
i. Decreased delivery of RBC into circulation: caused by impaired or defective production, bone
marrow fails to respond (reticulocytopenia)
ii. Increased loss of RBCs from circulation: caused by acute bleeding or accelerated destruction
(haemolytic), bone marrow can respond (reticulocytosis)
2. Relative (Pseudo) Anemia
a. RBC mass normal, plasma volume increased
b. Reticulocyte normal
c. Causes include conditions that result in hemodilution (pregnancy, hyperproteinemia, IV fluids)
D. Etiologic Classification of Anemia
1. Impaired or Defective Production of RBC
2. Increased RBC Destruction (Hemolytic Anemias)
a. Intrinsic (intracorpuscular) defects of RBC – inherited (e.g. membrane defects, hemoglobinopathies,
enzyme defects, thalassemia) or acquired (e.g. PNH)
b. . Extrinsic (extracorpuscular) causes – non immune (e.g. chemicals, toxins, venoms, physical
trauma, infection) or immune (involvement of antibodies)
c. Miscellaneous – Abnormal Hbs, porphyrias
3. Blood Loss
a. Can be acute or chronic
E. Morphologic Classification of Anemia
1. Macrocytic, Normochromic
a. Megaloblastic anemias (vitamin B12 and Folic acid deficiency)
b. Diseases of the liver
2. Normocytic, Normochromic
a. Defective formation of RBC or tumor in the BM – aplastic anemia , multiple myeloma, leukemia,
metastatic cancer, Hodgkins, renal/endocrine diseases, leukoerythroblastosis, chronic inflammatory
diseases
b. Abnormal Hb or ↑ RBC destruction – antibody destruction, enzyme deficiency, hemoglobinopathies,
RBC membrane defect
3. Microcytic, Hypochromic
a. Chronic blood loss
b. Lead poisoning
c. IDA
d. Thalassemia
e. Sideroblastic anemia
F. Physiologic Classification of Anemia
1. Ineffective erythropoiesis (RPI < 2.0)
a. Production of erythroid precursor cells that are defective
b. Defective precursors often undergo apoptosis in the bone marrow
c. Megaloblastic anemia, thalassemia, sideroblastic anemia, hypoproliferative anemias, maturation
disorders
2. Effective erythropoiesis (RPI > 3.0)
a. Bone marrow can respond to anemia
b. Hemolytic anemia, blood loss anemia

III. IMPAIRED PRODUCTION ANEMIA

A. Anemia of abnormal iron/heme metabolism:
 1. Deficiency of the raw material – iron deficiency anemia (IDA)
2. Defective release of stored iron from macrophages – Anemia of chronic disease (ACD)
3. Defective utilization of iron within the erythroblasts – sideroblastic anemia
B. Anemia of abnormal nuclear development: megaloblastic anemias - vit. B12, folate deficiency)
C. Anemias of BM failure: aplastic anemia, pure red cell aplasia (PRCA), congenital dyserythropoietic anemia
(CDA), myelophthisic anemia, anemia of chronic renal disease
D. Hemoglobinopathies
E. Thalassemias

IV. ANEMIA OF ABNORMAL IRON/HEME METABOLISM

A. Laboratory Tests used to Differentiate Disorders of Iron Metabolism
1. Serum Ferritin
a. Good indicator of iron storage status
b. 1 ug/L ferritin = 8mg of tissue iron storage
c. 1st laboratory test to become abnormal in IDA
d. Usually decreased only in IDA
e. Measured using Radioimmunoassays (RIA)
2. Free Erythrocyte Porphyrin (FEP)
a. When iron is deficient, protoporphyrin levels build up in RBCs to several times the normal level.
b. May be measured by a hematofluorometer or by an extraction and fluorescence method
3. Serum iron
a. Samples should be drawn in the morning (S. iron 20% lower in the evening)
b. 12 hours fasting
c. No intake of iron-containing medication for 12-24 hours before the test
4. Total Iron Binding Capacity (TIBC)
a. Indirect measure of the protein transferrin
b. Measures the maximum amount of iron that can bind to serum transferrin
c. Specimen requirement same for serum iron; except, TIBC not time dependent
5. Transferrin Saturation
a. Calculated value the % of transferrin saturated with iron
b. % Transferrin Saturation = [Serum Iron (ug/dL)/ TIBC (ug/dL)] ×100
B. Reference Range
Conventional units Factor SI units
Ferritin (male) 15–200 ng/mL 1 15–200 µg/L
Ferritin (female) 12–150 ng/mL 1 15–150 µg/L
Iron 60–150 µg/dL 0.1791 10.7–26.9 µmol/L
Iron-binding capacity 250–400 µg/dL 0.1791 44.8–71.6 µmol/L
Iron saturation 20–55% 0.01 0.20–0.55
Erythrocyte protoporphyrin 15–50 mg/dL 0.01777 0.27–0.89 µmol/L

C. Differentiating Anemia of Iron Metabolism

Transferrin
Serum ferritin Serum iron TIBC FEP
saturation
IDA ↓ ↓ ↑ ↓ ↑
Thalassemia ↑/N ↑/N N ↑ N
ACD ↑ ↓ ↓ N/↓ ↑
Sideroblastic anemia ↑ ↑ ↓/N ↑ Mixed
Lead Poisoning N–↑ (adults)
N N ↑ ↑↑
N–↓ (children)
D. Iron Deficiency Anemia (IDA)
1. Most common form of anemia (affecting at least one third of the world’s population)
2. Occurs when the iron stores of the body have been depleted
3. Etiology:
a. Excessive loss – GIT or uterine bleeding, excessive menstrual loss, hookworm infection
b. Inadequate intake – milk-fed infants, elderly with poor diets, fad diets
c. Increased requirements – infants and children, pregnancy
d. Decreased absorption – Gastric, celiac or Crohn’s disease
4. Development of IDA
Stage 1 Stage 2 Stage 3
Characteristic Storage iron depletion Transport iron depletion Functional iron depletion
Exhaustion of storage iron;
Loss of storage iron Iron deficient Anemia
iron deficient erythropoiesis
Normocytic,
Blood picture Normocytic, normochromic Microcytic, hypochromic
normochromic
Hemoglobin N N ↓
Serum iron N ↓ ↓
TIBC N ↑ ↑
Ferritin ↓ ↓ ↓
Symptoms None Nonspecific
 5. Laboratory:
a. Microcytic, hypochromic anemia
b. Mostly all lab. tests are ↓ ( Hb, hct, RBC, MCV, MCHC, serum Iron, Ferritin, transferrin saturation)
c. TIBC, FEP and RDW are ↑
d. Smear shows ovalocytes/pencil forms
e. Retic count normal, RPI <2
6. Clinical symptoms:
a. Nonspecific symptoms: fatigue, weakness, dizziness
b. Pica – persistent, compulsive desire to eat a single food or nonfood item such as starch or something
crunchy (ice, clay, plaster, or crunchy foods)
c. Stomatitis – cracks in the corner of the mouth
d. Glossitis – sore tongue
e. Koilonychias – spooning of the nails
E. Anemia of Chronic Inflammation (ACI)
1. Aka Anemia of Chronic Disease (ACD)
2. Present in chronic disorders such as SLE, RA and cancer
3. Most common form of anemia in hospitalized patients
nd
4. 2 to IDA as the most common form of anemia
5. Shows no improvement with iron therapy (only improves with the correction of primary disorder)
6. Etiology:
a. Hepcidin (an APR) decreases intestinal iron absorption and release of iron from macrophages
b. IL 1 secreted by macrophages during inflammation inhibits erythropoiesis and stimulates neutrophils
to release lactoferrin (binds iron)
c. Ferritin (an APR) binds iron
7. Laboratory:
a. Mild to moderate anemia
b. Normocytic, normochromic
c. Normal reticulocyte count; RPI <2
d. Decreased serum iron, TIBC, and transferrin saturation
e. Elevated ferritin and FEP
f. Normal soluble transferrin receptors (sTfRs); sTfRs increased in IDA
F. Sideroblastic Anemia
1. Caused by impaired production of protoporphyrin
2. Characterized by iron loading and the accumulation of ferric iron in mitochondria of normoblasts due to
a defect in heme synthesis
3. Diagnostic laboratory findings are ringed sideroblast in the BM and siderocytes in peripheral blood
(sideroblasts are found in 20-60% of nRBC in BM, but siderocytes are not normally present in the
peripheral blood)
4. 3 Types of sideroblasts:
a. Type 1 – as many as 4 ferritin aggregates per RBC
b. Type 2 – >6 aggregates per cell
c. Type 3 – ringed sideroblast (larger granules situated in a ring/collar around the nucleus); ringed
sideroblasts are the hallmark of the sideroblastic anemias
5. Etiology:
a. Hereditary (genetic) – ↓ALA synthase; most are sex-linked, few autosomal recessive
b. Acquired
i. Primary (idiopathic): AML, MDS, myeloma
ii. Secondary: alcoholism (most common cause of sideroblastic anemia), drug-induced: TB drugs
(isoniazid, cycloserine, pyranizamide), chloramphenicol, chronic transfusions, lead poisoning
6. Laboratory:
a. Microcytic, hypochromic anemia
b. Ringed sideroblast in the BM and siderocytes in peripheral blood (best stained with Prussian blue).
For a diagnosis of sideroblastic anemia, at least 15% of the normoblasts must be type III ringed
sideroblasts.
c. Increased ferritin and serum iron; TIBC is decreased
d. Dimorphic RBC population – microcytic, hypochromic cells together with normal or macrocytic red
cells
G. Lead Poisoning
1. Also Lead Intoxication
2. Occurs in both children and adults
3. Seen mostly in children exposed to lead-based paint (eating chips of flaking lead-containing paint,
chewing painted articles)
4. Also associated with use of improperly glazed pottery for cooking or eating
5. Acceptable Lead levels in children: 15-40 ug/dL of WB (treatment as medical emergency if >80 ug/dL)
6. Pathophysiology
a. Multiple blocks in the protoporphyrin pathway affect heme synthesis. Lead inhibits ALA dehydratase
or PBG synthase (most sensitive) and heme synthase.
b. Lead also injure the RBC membrane (inhibits ATPase)
7. Clinical Symptoms: abdominal pain, constipation, vomiting, muscle weakness, gum lead line (blue/black
deposits of lead sulphate near the teeth), impaired mental development
 8. Laboratory
a. Mild to moderate anemia
b. Hypochromia, microcytosis
c. RBC morphology: basophilic stippling
d. BM: erythroid hyperplasia
e. Increased FEP (much greater extent than in IDA)
f. Urinary ALA and coproporphyrins are increased
g. Normal serum ferritin, marrow iron, sideroblasts
h. Transferrin saturation elevated
i. Urinary excretion of >1 ug of lead per mg of EDTA administered establishes the diagnosis of lead
intoxication
H. Porphyrias
1. Inherited/acquired disorder caused by specific enzyme defects necessary for synthesis of protoporphyrin;
decreased hemoglobin is the result
2. Abnormal protoporphyrin precursors (porphyrins) are excreted in the urine (wine colored) or may
accumulate in the BM/liver
3. Clinical symptoms: dermal photosensitivity and/or neurologic disorders (original werewolf was probably
a person with porphyria)
4. Key Features of the Major Porphyrias
PORPHYRIA INHERITANCE ENZYME DEFICIENCY EXCESS METABOLITES
Acute intermittent Autosomal PBG deaminase PBG and ALA (U)
Porphyria (AIP) dominant Darkened urine on standing
Congenital Autosomal UPG I UP and CP (U)
erythropoietic recessive synthase and/or UPG UP (E)
Porphyria (CEP) III cosynthase Red, fluorescent pigment in urine
Hereditary Autosomal Coproporphyrinogen CP III (F, U)
coproporphyria dominant oxidase PBG and ALA (U)
(HCP) Red, fluorescent pigment in urine
Variegate Autosomal Protoporphyrinogen PBG and ALA (U)
Porphyria (VP) dominant oxidase UP and CP (U, F)
Red, fluorescent pigment in urine
Porphyria cutanea Autosomal Uroporphyrinogen UP I and UP III (U)
tarda (PCT) dominant Decarboxylase Acid induces pink fluorescence
Acquired
Protoporphyria Autosomal Ferrochelatase Free erythrocyte protoporphyrin (FEP)
dominant
U – urine; E – erythrocyte

5. Clinical features of the major porphyrias

a. AIP – Abdominal pain; psychiatric symptoms
b. CEP (aka Gunther's disease) – Photosensitivity; red urine, teeth; hemolysis (anemia); patients rarely
survive past middle age
c. HCP – Photosensitivity
d. VP (aka South african porphyria) – Photosensitivity
e. PCT – Photosensitivity
6. Laboratory:
a. Testing of urine, red blood cells, and feces for various porphyrins (HPLC, fluorometry)
b. Watson-Schwartz test – screening test
c. Hematologic findings are insignificant
I. Iron Overload
1. Iron acquisition exceeds the rate of loss
a. Hemochromatosis – excess accumulation of iron in vital organs such as the liver, spleen and
pancreas causing injury to the tissues
b. Hemosiderosis – iron accumulation that causes little injury; may progress to hemochromatosis
2. Pathogenesis: free iron (ferrous) initiates the generation of superoxide and other free radicals which
damages the membrane lipids (cell, mitochondrial, nuclear, lysosomal membranes)
3. Referred to as “Bronze diabetes” (patients have bronze colored skin and later develops type I DM)
4. Etiology
a. Hereditary: Hereditary hemochromatosis (HFE) protein mutation
b. Acquired: repeated transfusions, dietary intake, alcoholism
5. Laboratory Diagnosis
a. Screening tests: elevated transferrin saturation and ferritin
b. Abnormal liver function tests
c. Genetic testing
d. Liver biopsy with iron staining
6. Treatment
a. Phlebotomy (every 3 months) in hereditary hemochromatosis
b. Iron-chelating drugs (Desferrioxamine) in transfusion-related hemochromatosis

V. ANEMIA OF ABNORMAL NUCLEAR DEVELOPMENT

A. General Information
1. Also referred to as megaloblastic anemia
2. Major subgroup of anemia caused by impaired DNA synthesis leading to reduction in the number of cell
division
3. Nuclear-cytoplasmic asynchrony
4. Nuclear maturation is slower than the cytoplasm: enlarged RBC (macrocytes) and hypersegmented
granulocytes
5. RNA synthesis is normal, so the cytoplasm is not affected
6. Causes of Megaloblastic Anemia:
a. Vitamin B12 deficiency – pernicious anemia, other causes
b. Folate deficiency – usually acquired
c. Disorders of DNA synthesis – Inherited defects, drug-induced
7. Folate deficiency more common than vitamin B 12 deficiency
a. Vitamin B12 deficiency takes 3-6 years to develop because of high body stores.
b. Folic acid has low body stores. Folate Deficiency may occur within 2-4 months if no folate is ingested.
B. Clinical Findings in Megaloblastic Anemias
1. Weakness, fatigue and shortness of breath from anemia
2. Sore tongue (glossitis) – severe cases are very painful; tongue appears “beefy red”
3. Skin: lemon-yellow color (pallor + mild icterus); blotchy, brownish pigmentation with patches of vitiligo
may appear
4. Others: Gastritis, nausea, constipation, weight loss due to anorexia, difficulty walking
5. Neurologic symptoms (due to demyelinization of spinal cord and peripheral nerves) pronounced in
Vitamin B12 deficiency but absent in folate deficiency anemia
a. Paresthesias (prickling, tingling, “pins and needles” sensation in the hands and feet)
b. Memory loss, loss of balance, weakness, clumsiness, spasticity
c. Megaloblastic madness – infrequent; personality changes and psychosis (hallucinations, maniacal
outbursts, paranoia, schizophrenia)
C. Causes of Vitamin B12 (Cobalamin) Deficiency Anemia
1. Deficiency of vitamin B12
a. Rare because of large body stores of vitamin B12
b. Vitamin B12 is found in foods of animal origin (e.g. red meat, fish, poultry, eggs, dairy)
c. Seen in malnutrition and strict vegetarians
2. Increased need for vitamin B12
a. Seen in pregnancy, lactation, growth
3. Impaired absorption of vitamin B12
a. Failure to separate vitamin B12 from food proteins in the stomach - hypochlorydia
b. Failure to separate vitamin B12 from haptocorrin in the intestine – lack of gastric acidity or trypsin
c. Lack of intrinsic factor
i. IF is necessary for the absorption of vitamin B12
ii. Without protection by IF, vitamin B12 is destroyed in the GIT before it can be absorbed in the
ileum
iii. Seen in pernicious anemia, H. pylori infection, gastrectomy, hereditary IF deficiency
d. Pernicious Anemia
i. An autoimmune disorder characterized by inability of the gastric mucosa to secrete intrinsic factor
ii. May be caused by antibodies to IF and gastric parietal cells
iii. Diagnostic test: Schilling’s test
4. Intestinal malabsorption
a. General malabsorption: celiac disease, tropical sprue, IBD
b. Transcobalamin deficiency: ↓ transport CHON of vitamin B12 to BM
c. Imerslund-Gräsbek syndrome: deficiency in vitamin B12 receptor sites (cubilin or amnionless)
d. Drugs: neomycin and ethanol
5. Competition for available vitamin B12
a. Fish tapeworm (Diphyllobothrium latum) infection: worm resides in ileum and takes up vitamin B12
b. Small Bowel Bacterial Overgrowth: ↑ bacteria in ileum competes for vitamin B12; seen in mall bowel
diverticulosis, fistulae, Blind loop syndrome
D. Folate Deficiency Anemia
1. Folate deficiency acquired in many ways; it is rarely inherited
2. Manifestation is similar to vitamin B12 deficiency except that neurologic disorders are absent
3. Caused by:
a. Inadequate Intake (Dietary deficiency)
i. Most common cause of folate deficiency
ii. Folate is found green leafy vegetables, fruits, cereals, liver
iii. Food folate destroyed by excessive heat during steaming and boiling
b. Increased need
i. Pregnancy and lactation
ii. Infants and growing children
c. Impaired absorption
i. Folate transport protein deficiency
iii. Intestinal disease (sprue, celiac disease, IBD)
d. Impaired use due to drugs
 i. Includes antimalarial drugs (pyrimethamine), methotrexate (chemotherapeutic agent),
hydrantoins, contraceptives
ii. Impairs DNA synthesis by inhibiting the enzyme dihydrofolate reductase
iii. Alcohol also interferes with folate metabolism
e. Excessive loss with renal dialysis
E. Laboratory Findings in Megaloblastic Anemia
*Items 1-4 are screening tests, the remaining are specific diagnostic tests
1. Complete blood count
a. Macrocytic, normochromic anemia
b. Increased MCV and RDW, normal MCHC
c. Pancytopenia (low RBC count, neutropenia, thrombocytopenia with giant platelets)
d. RBC morphology: oval macrocytes and teardrops, Howell-Jolly bodies, nRBC’s, basophilic stippling,
Pappenheimer bodies and Cabot rings
2. Low absolute reticulocyte count
3. >3.4 hypersegmented neutrophils per 100 WBCs
4. Elevated LDH, total and indirect bilirubin
5. Bone marrow examination
a. Confirmatory for megaloblastosis (megaloblasts have very large, fine chromatin described as
“particulate”)
b. Characteristic cell in BM is the giant metamyelocyte
c. BM is hypercellular with RBC precursors predominating in response to anemia (1:1 to 1:3 ME ratio)
6. Assays for folate, vit. B12, MMA and Homocysteine
a. Folate and vit. B12 measured by immunoassays
b. In vit. B12 deficiency, serum and urinary MMA is increased
c. Deficiency in vit. B12 and folate results in elevated Homocysteine
d. Reference Ranges and Interpretation
Conventional units Factor SI units
Vitamin B12 160 – 950 pg/mL 0.7378 118 – 701 pmol/L
Folate ≥3.5 ng/mL 2.266 >5 nmol/L

Condition Serum B12 Serum Folate RBC Folate

Vitamin B12 deficiency Marked decrease Normal/increased Moderate decrease
Folate deficiency Normal/decreased Marked decrease Marked decrease
Vit. B12 and folic acid deficiency Decreased Decreased Decreased

7. Serum Microbiologic Assays

a. Most common method before 1970’s
b. Microorganisms that require either B12 (Euglena gracilis or Lactobacillus leichmannii) or folic acid
(Lactobacillus casei) for growth are incubated with patient’s serum for 48-72 hours. Serum turbidity
measured spectrophotometrically.
8. Gastric analysis and serum gastrin
e. Achlorhydria is supportive but not diagnostic for pernicious anemia
f. Serum gastrin markedly elevated in gastric achlorhydria
g. X-ray or endoscopic studies verify gastric atrophy and exclude stomach carcinoma
9. Antibody assays
• Anti-IF highly specific and confirmatory for PA
• Parietal cell antibodies not specific for PA
10. Schilling Test
a. Initial test
Normal: >7% of labeled B12 is excreted in the 24-hour urine
b. If initial test is abnormal, repeat test 2-3 days later. Same procedure, except IF is administered with
57Co-B12.
c. Interpretation:
i. Corrected with IF: PA, Gastric resection, Ingestion of corrosives, inert IF
ii. Not corrected with IF: Small bowel bacteria, fish tapeworm, B12 malabsorption
F. Other Conditions affecting DNA Synthesis
1. Orotic Aciduria
a. Rare, autosomal recessive
b. Disoders of pyrimidine metabolism resulting excessive urinary excretion of orotic acid and
megaloblastic anemia
c. Most patients exhibit and physical retardation
2. Lesch-Nyhan Syndrome
a. Rare X-linked disorder of purine metabolism
b. Deficiency in xanthine-guanine phosphoribosyl transferase
c. Features hyperuricemia, self-mutilation, mental and neurologic defects, and megaloblastic anemia
3. Drugs (chemotherapy): antipurines (6-mercaptopurine); antipyrimidines (5-fluoracil, cytosine
aranbinose); antifolates (methotrexate)

VI. ANEMIA OF BONE MARROW FAILURE AND SYSTEMIC DISORDERS

A. Aplastic Anemia
1. Characterized by peripheral pancytopenia
2. Failure to produce all formed elements of the blood (WBC, RBC, platelets) due to damage of
hematopoietic stem cells
3. BM is severely hypoplastic or aplastic
4. Most commonly affects people around the age of 50 and above. It can occur in children.
5. Usually two of the following three blood parameters are needed to diagnose AA:
a. Hb <100 g/L
9
b. Granulocyte count <1.5 × 10 /L
9
c. Platelet count <50 × 10 /L
6. Diagnostic Criteria
Moderate Severe Very Severe
Bone marrow Hypocellular BM plus at Bone marrow cellularity <25% plus Same as SAA
least two of the following: at least two of the following:
9 9 9
Neutrophils 0.5 – 1.5 ×10 /L 0.2 – 0.5 ×10 /L <0.2 ×10 /L
9 9
Platelets 20 – 50 ×10 /L <20 ×10 /L Same as SAA
9
Other Hb ≤ 10 g/dL plus Reticulocytes <20 ×10 /L or <1% Same as SAA
9
reticulocytes <30 ×10 /L corrected for HCT
7. Patients have a poor prognosis with complications that include bleeding* (thrombocytopenia), infection*
(neutropenia) and iron overload due to frequent transfusion needs. *Most serious clinical problems
8. Prognosis: modern treatment protocols by immunosuppressive therapy and bone marrow
transplantation, long-term survival rates greater than 60% are reported.
9. Etiology:
a. Acquired (80-85% of cases)
i. Idiopathic (70% of cases)
ii. Secondary
• Dose dependent: cytotoxic drugs, benzene, radiation
• Idiosyncratic: drugs (chloramphenicol, 5-40× higher risk), insecticides
• Viruses (EBV, Hepatitis virus, HIV)
• Miscellaneous: PNH, autoimmune disease, pregnancy
b. Inherited (15-20% of cases)
i. Fanconi’s anemia
ii. Dyskeratosis congenita
iii. Shwachman-Bodian-Diamond syndrome
10. Fanconi’s anemia
a. Also congenital aplastic anemia or familial aplastic anemia
b. Rare, but most common of the inherited AA
c. Autosomal recessive
d. Chromosome instability disorder characterized by aplastic anemia, physical abnormalities, and
cancer susceptibility
e. Peripheral blood abnormalities manifests 5-10 years after birth
f. Microencephaly, mental retardation, skin pigmentation, short stature, malformations of the thumb,
abnormality of the eyes, kidneys, and genitals
11. Laboratory findings:
a. Severe normocytic, normochromic anemia (↓Hb/Hct) and reticulocytopenia
b. Anemia no response to EPO
c. Leukopenia with granulocytopenia
d. Severe thrombocytopenia with giant platelets
e. No immature cells in the peripheral blood
f. Special tests: LAP score increased, (+) Ham’s test
g. Elevated serum iron
h. Aplasia of the BM with increase in fat (“dry tap” bone marrow)
12. Treatment of choice is Hematopoietic stem cell transplantation (up to 83% survival rate)
B. Pure Red Cell Aplasia (PRCA)
1. RBC production is suppressed with little or no abnormalities in WBCs or platelets
a. BM: cellular, with decrease only in RBC precursors
b. PBS: decrease in RBC; normal WBC and platelets
c. Low reticulocyte count
2. Classification (etiology):
a. Congenital PRCA
i. aka Diamond Blackfan syndrome or Congenital hypoplastic anemia
ii. Autosomal inheritance
iii. Decreased CFU-E in BM or CFU-E insensitive to EPO
iv. Diagnosis made in infancy (Hb 1.7-9.4 g/dL in NB)
v. Abnormal thumbs, retarded growth, osteoporosis, failure of secondary sexual maturation
vi. Severe macrocytic anemia
vii. Treatment: red cell transfusion, BM transplantation
b. Acquired PRCA
i. Seen primarily in individuals >40 years
 ii. Severe anemia (gradual onset), extreme pallor
iii. Treatment: red cell transfusion
iv. Classification:
• Primary: can be idiopathic or due to immune mechanism (Ig inhibitor to RBC precursors,
EPO inhibitor)
• Secondary: benign thymomas, drugs, chemicals, infections, hemolytic anemias (aplastic
crisis)
C. Congenital Dyserythropoietic Anemia (CDA)
1. Represents a family of inherited refractory anemias characterized by ineffective erythropoiesis
2. RBC survival is normal or slightly increased
3. Characteristics of CDA
a. Anemia with rerticulocytopenia
b. Hypercellular bone marrow (erythroid hyperplasia)
c. Ineffective erythropoiesis
i. Presence of multinuclear erythroid precursors in the marrow
ii. Normoblast in the BM show multinuclearity, karyorrhexis and bizarre malformation as a result of
dyserythropoiesis
d. Iron overload (patients may develop hemosiderosis)
e. Increased serum B1 and fecal urobilinogen
4. Has three (3) subtypes:
a. Type I: macrocytic anemia
b. Type II: normocytic anemia
i. RBC contains “i” Ag or HEMPAS antigen; (HEMPAS: hereditary erythroblast multinuclearity with
positive acidified serum test)
ii. Most common form (although it is also rare)
iii. (+) acid serum test, (-) OFT
c. Type III: normocytic to slightly macrocytic anemia
D. Myelophthisic Anemia
1. Also called Leukoerythroblastosis
2. Presence of circulating nRBC’s and immature leukocytes in the peripheral blood
3. Infiltration of abnormal cells into the bone marrow and subsequent destruction and replacement of normal
hematopoietic cells
4. Caused by space occupying malignant tumors of the BM
5. Most common cause is metastatic carcinoma of the breast, prostate, lungs, adrenal or thyroid gland with
bone metastasis
6. Laboratory findings:
a. Mild to moderate anemia
b. BM biopsy is the best method of demonstrating metastatic tumor
c. Distinguishing characteristic: ↑ nRBC in the PBS
d. Dacryocyte
e. Shift to the left
f. Variable WBC and platelet count
E. Anemia of Systemic Disorders
1. Anemia of Chronic Kidney Disease
a. Pathophysiology:
i. Failure of the kidney to produce erythropoietin (principal cause)
ii. Uremia inhibits erythropoiesis and increases RBC fragility
b. Laboratory findings:
i. Low Hct (0.15-0.30)
ii. Normal RBC morphology, occasional burr cells
iii. Abnormal platelet function tests (waste products coat platelets)
iv. “The higher the creatinine, the lower the Hct”
2. Anemia of Endocrine Disorders
a. Commonly anemia is mild
b. BM: decreased erythroid production, normal myeloid and megakaryocyte production
c. Hormones that stimulates erythropoiesis include vasopressin (ADH), pituitary hormones (TSH,
ACTH, GH, Prolactin), testosterone, thyroxine, and cortisol
d. Mechanisms:
i. Hypothyroidism – need for O2 decrease; patients may have macrocytic anemia
ii. Hypopituitarism – GH, prolactin (and thyroxine) stimulates erythropoiesis independent of EPO
iii. Adrenal abnormalities – hypoadrenalism results in Addison disease leading to ↓cortisol and
anemia
iv. Hypogonadism – deficiency of male sex hormone testosterone; testosterone stimulates renal
EPO production
3. Anemia of Pregnancy
a. Etiology
i. Iron and folate (less common) deficiency because of increase demand
ii. Dilutional effects – physiologic expansion of total blood volume
th nd th
b. Anemia usually develops around the 8 week and increases until 32 -34 week
c. Anemia is moderate (seldom <10 g/dL)
VII. HEMOGLOBINOPATHIES
A. General Information
1. Disease state (opathy) involving the hemoglobin molecule
2. Inherited qualitative (structural) defect in hemoglobin production in the BM, specifically in the globin chain
synthesis
3. Most common genetic disease (affects 7% of the population)
4. Majority results from amino acid substitution in the beta chain; changes in Hb structure affect function
5. Alteration in the AA composition causes a change in the net charge of the Hb molecule. These difference
on the net charge of the Hb molecule can be observed on their migration in electrophoresis
6. Hemoglobinopathy terms:
a. Hemoglobin variant – the abnormal hemoglobin caused by a globin chain structural abnormality (e.g.
Hb S)
b. Hemoglobinopathy – the condition diagnosed when the presence of a hemoglobin variant is
confirmed in the blood by laboratory tests. Example, sickle cell diseases (Hb SS) and sickle cell trait
(Hb AS).
c. Homozygous conditions (disease) are more serious than heterozygous conditions (trait)
7. Target cells are seen!
8. Hemoglobin electrophoresis, isoelectric focusing and/or DNA (PCR) analysis may be used to confirm the
diagnosis
9. Common Names and Scientific Designations of the Well-Known Hemoglobin Variants

MOLECULAR Hemoglobin AA Number Scientific Designation

ABNORMALITY Common Name Affected
Amino acid S 6 β 6 Glu → Val
substitution C 6 β 6 Glu → Lys
D-Los Angeles 121 β 121 Glu → Gln
D-Punjab
E 26 β 2 Glu → Lys
O-Arab 121 β 121 Glu → Lys
AA deletion Gun Hill 91-95 β 91-95 (F7-FG2) Leu His Cys Asp Lys →0
AA elongation Constant Spring NA α +31c (142 Gln)
Globin chain fusion Lepore-Baltimore NA δ (1-50) β (86-146)
10. Structural Mutations of Common Hemoglobinopathies
Hemoglobin Comments
th
S Valine @ 6 position instead of glutamic acid on the beta chain
th
C Lysine on 6 position instead of glutamic acid on the beta chain
st
D Glycine on 121 position instead of glutamic acid on the beta chain
th
E Lysine @ 26 position instead of glutamic acid on the beta chain

B. Hemoglobin S
th
1. Valine @ 6 position instead of glutamic acid on the beta chain
2. Most common hemoglobin variant
3. Occur most commonly in sub-Saharan Africa, Arab-India, the Americas, Eurasia, and SE Asia
4. Autosomal codominant
5. Apparent immunity to Plasmodium falciparum
6. Pathophysiology of sickle cell disease (SCD)
a. When oxygen decreases at the tissue level, RBC undergoes sickling phenomenon
b. Hb polymerizes leading to formation of crystals/tactoids, that grow in length beyond the diameter of
the RBC causes the cells to become rigid
c. Hemolysis is common
i. Extravascular hemolysis
ii. Intravascular hemolysis: RBC unable to traverse the microcirculation
7. Sickle Cell Crises
a. Vassoocclusive crisis (VOC)
i. Hallmark of SCD
ii. Microthrombi, vascular occlusions, and microinfarctions in the joints, extremities and organs
(organ failure)
iii. Sickled cells can’t flow freely through microvasculation of joints and spleen which can cause
plugging up of small vessels furthering the hypoxic environment. This results in tissue
death/necrosis.
iv. All organs are affected, with kidney failure being a common outcome; hyposplenism, leg ulcers,
stroke and joint swelling also occur
b. Infectious crises: primary cause of death in sickle cell anemia (bacterial infection); due to abnormal
splenic function
c. Aplastic crises: thought to be caused by infection and fever
d. Bone, joint and other crises
i. Pain where sickle cells accumulate
ii. Hand-foot syndrome or dactylitis: painful swelling of back of hands and feet
 e. Splenic sequestration crises: spleen enlarges as more RBCs are trapped, leading to hypovolemia,
which may cause shock and even death
8. Two-step process in diagnosis of SCD
a. Screening
i. Demonstrates insolubility of deoxygenated Hb S
ii. Hemoglobin Solubility Test (Dithionite tube test)
iii. Sodium Metabisulfite Method
b. Confirmatory
i. Hemoglobin electrophoresis, HPLC, or capillary electrophoresis
ii. Isoelectric focusing, tandem mass spectrometry, or DNA analysis
C. Sickle Cell Anemia
1. Homozygous inheritance (Hb SS); sickle cell gene inherited from both parents
2. No Hb A is produced
3. RBC contains 80% Hb S and 20% Hb F are seen. HbA2 is variable
4. Sickling occurs when oxygen saturation is <85%
5. Symptoms don’t appear until 6 months of age because of the presence of Hb F
6. Life expectancy of 50 years with proper treatment.
7. Death usually results from infection or congestive heart failure
9. Organs affected by sickle cell anemia
a. Liver – hepatomegaly; jaundice and hyperbilrubinuria
b. Heart – heart failure due to iron deposition (frequent transfusion)
c. Spleen – splenomegaly; hyposplenism
d. Skin – leg ulcers
e. Kidneys – kidney failure because of infarction
f. Lungs
10. Laboratory findings in sickle cell anemia:
a. Severe normocytic, normochromic hemolytic anemia
b. PBS: sickle cells and target cells (hallmark), bronze elliptocytes, nRBCs, Pappenheimer bodies,
Howell-Jolly bodies, anisocytosis
c. Leukocytosis and thrombosis common
d. Special hematologic tests:
i. Positive Hb solubility screening test
ii. Hb electrophoresis – Hb S migrates with Hb D and G on alkaline Hb electrophoresis; can
differentiate using acid electrophoresis
iii. BM erythroid hyperplasia (decreased M:E ratio)
e. Chemistry: increased LD, B1 and urobilinogen; decreased haptoglobin
D. Sickle Cell Trait
1. Heterozygous for Hb S (Hb AS)
6 Glu Val
2. α 2β 1β 2 →

3. Patients inherit a normal B globin gene from one parent and a sickle globin gene from the other parent
4. RBC contains 40% Hb S, 60% Hb A
5. No clinical symptoms as the patient lives a normal life span.
6. Treatment rarely required.
7. Sickling does not occur in normal conditions; sickling may occur in extreme tissue hypoxia (<40% Hb O2
saturation)
8. Deoxygenation of RBCs occur in respiratory infections, dehydration, violent exercise, obstetric delivery,
high altitude, flying in unpressurized aircraft, exposure to extreme cold, administration of anaesthesia
9. Lab. Findings in Sickle Cell Trait
a. Anemia generally not present
b. Normal RBC morphology, few target cells and occasional sickle cells
c. Special tests
i. (+) Sickle solubility test
ii. Hb electrophoresis: Hb A always higher than HbS; normal Hb F
E. Compound Heterozygotes
1. Hb SC
a. Most common compound heterozygous syndrome
b. Inheritance of 2 Hb variants: Hb S and Hb C
c. Less severe than sickle cell anemia, but more severe than Hb C disease
d. Laboratory findings in Hb SC Disease:
i. Normocytic, normochromic anemia (may not be present)
ii. RBC morphology: target cells, Hb SC crystals, pocketbook cells
iii. Hb electrophoresis: No Hb A is produced; 50% Hb S and 50% Hb C
e. Hb SC crystal: have finger-like projections and usually protrude outisde the cell membrane; “Hand in
glove" or "Washington monument" appearance.
2. Hb S-Thalassemia
a. Doubly heterozygous condition
b. Has several forms
i. Hb S- α thalassemia: common, clinically insignificant
ii. Hb S - β thalassemia: same findings as sickle cell anemia, milder
c. Sickle solubility tests are positive
3. Hb SD
 a. Combination of Hb S and Hb D
d. Less severe than sickle cell disease

F. Hemoglobin C
th
1. Lysine on 6 position instead of glutamic acid on the beta chain
nd
2. 2 most common Hb variant
3. Found primarily people of African descent
4. Hemoglobin C Disease
a. Homozygous state (Hb CC)
b. Hb CC tend to crystallize when dehydrated, hence the name “crystal Hb” or “Hb C” (older cells most
vulnerable)
c. Hb C crystals (rod shaped crystals) may be seen in Wright stained smear. Crystals may be
demonstrated by incubating RBC @ 37°C with Na citrate
d. RBCs may fragment in microcirculation or removed by spleen causing a mild hemolytic anemia
e. Patients generally asymptomatic and live a normal life span. Treatment is not required.
f. Mild to moderate normocytic, normochromic anemia
g. Numerous target cells and few spherocytes, occasional Hb C crystals
h. Hb electrophoresis
i. Almost 100% Hb C in RBC (90% Hb C, 2% Hb A2, 7% Hb F)
ii. No Hb A is produced
iii. On cellulose acetate (alkaline pH), Hb C migrates with Hb A2, E and O-Arab. Hb C separated by
citrate agar (acid pH) electrophoresis where it migrates farther toward the anode
5. Hemoglobin C Trait
a. Heterozygous state (Hb AC)
b. No clinical symptoms
c. Laboratory findings:
i. No anemia
ii. Slightly hypochromic RBCs
iii. 40% target cells
iv. 60% Hb A, 40% Hb C
6. Hb-C Harlem
a. Aka Hb C-Georgetown
b. Rare
6 Glu Val; 73 Asp Asn
c. Multiple amino acid substitution (α 2β 2 → →
)
d. Hb SC-Harlem have a clinical picture similar to Hb SC disease
e. Heterozygotes are asymptomatic
G. Hemoglobin D
1. Hb D disease associated with a mild hemolytic anemia
2. Asymptomatic in the heterozygous state
3. Have several varieties with abnormality on both alpha or beta chain
4. 16 β-chain variants and 6 α-chain variants
121 Glu Gln
5. Hb D-Punjab or Hb D-Los Angeles (α2 β2 →
), is the variant with highest frequency
68 Asn Lys
6. Hb G-Philadelphia (α2 →
β 2)
rd
a. 3 most common variant among blacks after Hb S and Hb C
b. Homozygous form not compatible with life
H. Hemoglobin E
th 26Glu Lys
1. Lysine @ 26 position instead of glutamic acid on the beta chain (α2β2 →
)
rd
2. 3 most common hemoglobin variant
3. Common in SE Asian, African and African-American populations
4. Homozygous state (Hb EE) manifests as a mild anemia, Hb E trait is asymptomatic
5. No therapy required
I. Hemoglobin M
3+
1. Hb M has iron in the ferric state (Fe )
2. Has seven variants
3. Hb M-Boston, Hb M-Iwate, and Hb Auckland (α chain variants)
4. Hb Chile, Hb M-Saskatoon, Hb M-Milwaukee-1, and Hb M-Milwaukee-2 (β chain variants)
5. Findings: cyanosis, 30-50% methemoglobin
J. Unstable Hemoglobin Disease
1. Also called Congenital Heinz body hemolytic anemia
2. Disease results to denaturation and precipitation of globin chains
3. Heinz bodies are characteristically present causing ↓ MCHC
4. Over 70 variants identified
5. Occur as a result of AA substitutions, deletions or cross overs
6. Examples are Hb H, Kӧln, Gun Hill, Leiden and Zurich (may cause hemolysis)
K. Hemoglobinopathies Affecting O2 Affinity
1. Hb with increased O2 affinity
a. More than 90 variants discovered
b. Hb Gun Hill and Hb Chesapeake
c. Results in compensatory erythrocytosis
d. Asymptomtic, normal lifespan, no treatment required
2. Hb with decreased O2 affinity
 a. May cause slight anemia
b. Hb Kansas, Seattle, Vancouver and Mobile

VIII. THALASSEMIA
A. General Information
1. Inherited quantitative defect in the production of globin chain synthesis
2. Normal globin structure but rate of formation is decreased or absent
3. Found mainly in the descendants of Mediterranean area ( “thalassic” – greek, great sea), Asia and Africa
4. Named after the globin chain affected: Alpha (α), Beta (β), Gamma (γ), Delta (δ)
5. Hypochromic anemia with normal to high serum iron
6. Pathophysiology of Thalassemia
a. A reduced or absent production of a particular globin chain, which diminishes hemoglobin synthesis
and produces microcytic, hypochromic RBCs
b. An unequal production of the α- or β-globin chains causing an imbalance in the α/β chain ratio; this
leads to a markedly decreased survival of RBCs and their precursors.
c. The α/β chain imbalance is more significant and determines the clinical severity of the thalassemia
7. Laboratory findings in Thalassemia:
a. Microcytic, hypochromic anemia
b. Hct and Hb values vary
c. RBC count usually elevated
d. PBS: anisocytosis, poikilocytosis, polychromasia, target cell, basophilic stippling, nRBC
e. Normal to high serum iron
f. Citrate agar electrophoresis: Hb F usually elevated
8. Lab. Differentiation of IDA and Thalassemia Minor
Thalassemia minor IDA
9 9
RBC count > 5.5 × 10 /L < 5.5 × 10 /L
RDW Normal or slightly increased Increased
S. Ferritin Normal or slightly increased Decreased
S. Iron Normal Decreased
TIBC Normal Increased
Transferrin saturation Normal Decreased
ZnPP/H Normal Increased
IDA and Thalassemia minor are the two most common causes of microcytic, hyphochromic blood
picture
B. Alpha-Thalassemia
1. Reduced or absent alpha chain synthesis affecting the 4 genes for alpha chain synthesis
a. Total of 4 genes is inherited, 2 from each parent
b. Normal: αα/αα
c. Gene deletion is the predominant genetic defect
2. Common in SE Asia
3. Genotypes of α-Thalassemia
Genotype Genotypic Description Disorder
––/–– Homozygous α° thal Bart’s hydrop fetalis
+
––/–α Heterozygous α° thal / α thal Hb H disease
––/αα Heterozygous α° thal α Thalassemia minor
+
–α/–α Homozygous α thal α Thalassemia minor
+
–α/αα Heterozygous α thal Silent carrier
CS
––/α α Heterozygous α° thal / Constant Spring Hb H / Constant Spring disease
O
a. Two Haplotypes: α –thalassemia (deletion of both α-globin genes on chromosome 16 resulting in
+
no α chain production), α –thalassemia (deletional or non-deletional mutation in one of the two a-
globin genes on chromosome 16 results in decreased a chain production from that chromosome)

C. Four Categories of α-Thalassemia

1. Bart’s Hydrop’s Fetalis Syndrome
a. α Thalassemia Major
b. 4 gene deletion (--/--)
c. No production of alpha chain; therefore, no normal hemoglobins produced
d. Excess gamma chains accumulate forming Hb Barts
e. Hb Barts has a high affinity for O2
f. Infants are usually stillborn or survive until birth due to Hb Portland (but they usually die)
g. Fetus is severely anemic, which leads to cardiac failure and edema in the fetal subcutaneous tissues
(hydrops fetalis)
h. Laboratory findings
i. Severe microcytic, hypochromic anemia
ii. Numerous nRBCs in PBS
iii. Marked erythroid hyperplasia
2. Hemoglobin H Disease
a. 3 gene deletion ( – – / – α )
 b. Decrease in alpha chain production leads ↑B chain production
c. Decrease in alpha chain production leads to accumulation of B chain (tetramers) forming Hb H (β4).
Hb H precipitates forming rigid RBCs which are destroyed by the spleen.
d. Mild to severe hemolytic anemia
e. Patient development and life expectancy are usually normal.
f. Some may have physical and intellectual abnormalities
g. Laboratory Findings
i. RBC indices are decreased (MCV, MCH, MCHC)
ii. RDW increased
iii. Reticulocytosis (approx. 5%) but RPI indicates inadequate BM response
iv. Almost all RBCs have Hb H inclusions (golf ball or raspberry appearance)
v. Hb electrophoresis: affected neonates shows 20-40% Hb Barts but is gradually replaced by Hb
H
vi. Hb H Inclusions demonstrated by BCB or NMB stains
3. α-Thalassemia Minor
a. α Thalassemia 1 Trait
b. 2 gene deletion: (– – / α α ) or (– α / – α); both forms are common in SE asians, Chinese and Filipinos
c. Patient are usually asymptomatic or may have mild anemia
d. Microcytosis in the presence of normal Hb A2 and Hb F level is very suggestive
4. Silent Carrier α-Thalassemia
a. α-Thalassemia 2 Trait
T
b. 1 gene deletion (– α / α α); rarely, a non-deletional mutation (α α / α α)
c. Asymptomatic
d. Hematologic picture normal
e. Diagnosed on gene (DNA) analysis
f. Borderline low MCV may be the only sign
D. Beta-Thalassemia
1. Defect in the production of beta chains
2. Most common thalassemia, common in Mediterranean area
3. Lack or reduced production of beta chains
a. Alpha chain synthesis normal
b. Massive imbalance causes excess alpha chain to precipitate in RBC causing dysfunction and
hemolysis (RBC removed by BM and spleen)
c. Minor imbalance causes no problem
4. Peripheral Blood Morphology in Thalassemia:
a. Microcytic, hypochromic RBCs
b. nRBC (major and intermedia only)
c. Anisopoikilocytosis
d. Target cells
e. Basophilic stippling
E. Four Categories of β-Thalassemia
1. β-Thalassemia Major
a. Aka Cooley’s anemia
b. Homozygous state
c. Clinical findings:
i. Severe anemia – patients require regular transfusion
ii. Mongoloid appearance – BM expansion causes marked skeletal deformities: frontal bossing,
cheek and jaw protrusions; distortions of ribs and vertebrae, pathologic fractures of long bones
iii. Retarded growth and sexual development
iv. Without adequate treatment: cardiomegaly, hepatosplenomegaly, gallstones, chronic leg ulcers,
hypersplenism
d. Life expectancy:10-20 years
e. HSC transplantation is the only curative therapy
f. Laboratory findings
a. Severe microcytic, hypochromic anemia
b. Mild reticulocytosis with RPI <3.0
c. Erythroid hyperplasia in the BM (1:20 M:E ratio)
d. Greatly increased Hb-F (30-100%)
e. Increased serum iron and bilirubin
2. β-Thalassemia Intermedia
a. Milder than β-Thalassemia major but more severe than β-Thalassemia minor
i. Similar peripheral blood picture
ii. Moderate anemia
b. Growth and development of children relatively normal.
c. Transfusion independent (does not require regular transfusion)
3. β-Thalassemia Minor
a. Aka Cooley’s trait, β-Thalassemia trait
b. Usually asymptomatic and patients live a normal life span
c. Anemia mild or absent
d. PBS RBC morphology significantly abnormal
 e. Elevated Hb A2 !
4. Silent Carrier State of β-Thalassemia
a. Aka Thalassemia minima
silent
b. Genotype: β /β
c. Silent form of an abnormal β-globin gene
d. No clinical or laboratory abnormality is usually detected
F. Hereditary Persistence of Fetal Hemoglobin (HPFH)
1. Increased levels of Hb F in adults in the absence of clinical and hematologic features of thalassemia
2. Benign suppression of B-chain and δ-chain synthesis with increased production of Hb F
3. Hb F resistant to in vivo sickling of RBC
4. 2 major categories:
a. Pancellular HPFH – all RBCs contain increased Hb F
b. Heterocellular HPFH – only a subpopulation of RBCs conatin increased Hb F

IX. INCREASED DESTRUCTION ANEMIA

A. General Information
1. Also referred to as Hemolytic anemia
2. Increased loss of RBCs from circulation due to shortened lifespan of mature RBC’s
3. BM can respond: reticulocytosis is seen
4. Signs of RBC destruction:
a. Extravascular hemolysis – increased indirect bilirubin, increased urine/stool urobilinogen
b. Intravascular hemolysis – increased plasma hemoglobin, hemoglobinuria, decreased plasma
haptoglobin, methemoglobinemia
B. Classification of Hemolytic Anemia
1. Intrinsic Hemolytic Anemia
a. All are associated with a negative direct antiglobulin test (DAT)
b. Etiology:
i. Hereditary: membrane abnormalities, enzymopathies, hemoglobinopathies
ii. Acquired: PNH
c. Three categories:
i. Membrane abnormalities – H. spherocytosis, H. elliptocytosis, etc
ii. Enzymopathies – PK deficiency, G-6-PD Deficiency
iii. Plasma Constituent Abnormalities – abetalipoproteinemia, LCAT deficiency
2. Extrinsic Hemolytic Anemia
a. RBCs are structurally and functionally normal, but a condition outside of the RBCs causes premature
hemolysis
b. Etiology:
i. Nonimmune – presence of a condition that causes physical or mechanical injury to RBCs (e.g.
MAHA, infections)
ii. Immune – mediated by antibodies, complement, or both (e.g. autoimmune, drug-induced,
isoimune hemolytic anemias)
C. Paroxysmal Nocturnal Hemoglobinuria (PNH)
1. Acquired intravascular hemolytic anemia characterized by intermittent (paroxysmal) sleep associated
(nocturnal, acid pH at night) blood in the urine (hemoglobinuria)
2. Hematopoietic stem cell mutation that results in the lack of GPI-anchored proteins
3. RBC (also WBC and platelets) more sensitive to lysis by complement
4. RBC membrane that regulates C’ are missing:
a. CD55 – decay-accelerating factor (DAF); inhibits C3 and C5 convertases in the alternate pathway
b. CD59 – membrane inhibitor of reactive lysis (MIRL); prevents formation of MAC
5. Increased fixation of C3; increased complex C5 through C9; increased ability of complex to penetrate
lipid membrane of cell, which leads to destruction of RBCs, WBCs and platelets
6. Clinical findings: mild to severe HA, thrombosis, BM failure
7. Laboratory findings:
a. Anemia (Hb may drop <6 g/dL)
b. Hemoglobinemia, hemoglobinuria, hemosidenuria
c. Relative reticulocytosis
d. Decreased WBC with low LAP score
e. Thrombocytopenia
f. Positive Ham's test and sugar water test (traditionally used)
g. Flow cytometry to detect deficiencies of CD55 and CD59 (standard test now)

X. RBC MEMBRANE ABNORMALITIES

A. General Information
1. RBC membrane responsible for the discoid shape of RBCs as well as its deformability. Loss of
deformability leads to premature RBC lysis.
2. Most RBC membrane defects are hereditary
3. All hereditary RBC membrane defects are inherited in an autosomal dominant manner except
abetalipoproteinemia (autosomal recessive)
4. Hereditary: H. spherocytosis, H. elliptocytosis, H. ovalocytosis, H. stomatocytosis, Acanthocytosis,
Abetalipoproteinemia
5. Acquired: Acquired stomatocytosis, spur cell anemia, PNH
 6. All are associated with a negative DAT
7. RBC-related proteins
a. Spectrin – framework of the RBC
b. Actin – link together spectrin dimers
c. Protein 4.1 – mediates spectrin-actin linkage
d. Ankyrin – attaches spectrin to protein 3
B. Hereditary Spherocytosis (HS)
1. aka Congenital Hemolytic anemia/jaundice
2. Most common RBC membrane defect
3. 75% autosomal dominant
4. Caused by mutations that disrupt the vertical membrane protein interactions, which results in loss of
membrane and decrease in surface area-to-volume ratio
5. Defective binding of spectrin to protein 4.1
6. Quantitative defect in spectrin (less common)
7. Defects lead to rigid RBCs which are trapped by the spleen
8. RBC survival decrease only when spleen is present (treatment of choice is splenectomy)
9. Increased RBC permeability to sodium will increase the H2O content of RBC leadng to swelling and
hemolysis
10. Clinical findings: anemia, jaundice, splenomegaly
11. Laboratory Findings
a. Numerous microspherocytes in PBS (hallmark)
b. Mild anemia (adult Hb frequently >10 g/dL)
c. Only disease where an MCHC can be truly increased (MCHC >36%)
d. Increased reticulocyte count and RDW
e. Increased OFT (most useful diagnostic test)
f. Increased autohemolysis test, corrected by addition of glucose
C. Hereditary Elliptocytosis (HE)
1. Caused by impaired connection of spectrin or polarization of cholesterol at the ends of the cell rather
than around pallor area
a. Protein 4.1 deficiency
b. Abnormal spectrin dimer-dimer interaction
c. Defective ankyrin-protein 3 interaction
2. Mechanism of hemolysis involve membrane loss, decreased RBC deformability and splenic destruction
3. Most are asymptomatic, 10% display hemolytic anemia and splenomegaly
4. Laboratory findings:
a. Hb >12 g/dL
b. >25% of RBC are elliptocytes
c. Osmotic and autohemolysis test usually normal
D. Hereditary Pyropoikilocytosis (HPP)
1. Extremely rare
2. Thermal sensitive form of HE
3. Spectrin-spectrin association defect (similar to HE)
4. Laboratory findings:
a. Bizarre RBC morphology
b. Most striking feature is extreme microcytosis (25-55 fL MCV)
c. Thermal sensitivity: RBC fragment as low as 45-46°C (normal RBC >49 °C)
d. Extreme anisocytosis and micropoikilocytosis with budding red cells, fragments, spherocytes,
elliptocytes, and other bizarre shaped cells.
E. Hereditary Ovalocytosis (HO)
1. Aka Southeast Asian ovalocytosis (SAO)
2. About 30% oval RBCs
3. Mutation in the gene for band 3 results in increased rigidity of the membrane and resistance to invasion
by malaria
4. Hemolysis is mild or absent
F. Stomatocytosis
1. Overhydrated Hereditary Stomatocytosis
a. Also Hydrocytosis
b. Increased permeability of RBC (↑Na, ↓K) ) causing an influx of H2O
c. 5-50% stomatocytes
d. Moderate to severe HA
2. Dehydrated Hereditary Stomatocytosis
a. Aka H. xerocytosis (HX)
b. Most common form of stomatocytosis
c. RBC membrane permeable to K+ causing it to leak out of RBCs
i. Efflux (outflow) of K+ is greater than influx (inflow) of Na.
ii. Water moves out of the cell
iii. Cells become dry (greek word xeros, dry)
d. <10% stomatocytes
e. Mild to moderate HA
3. Other Hereditary Membrane Defects with Stomatocytes
 a. Familial Pseudohyperkalemia - excessive potassium leaks out of the RBCs at room temperature in
vitro but not in vivo
b. Cryohydrocytosis (CHC) – mild to moderate hemolytic anemia with leakage of sodium and potassium
from RBCs
c. Rh Deficiency Syndrome
4. Acquired Stomatocytosis
a. Stomatocytosis occurs frequently as a drying artifact on Wright-stained peripheral blood films
b. In normal individuals, only 3% to 5% of RBCs may be stomatocytes
c. In true stomatocytosis, such cells should be found in all areas of the blood film
G. Neuroacanthocytosis
1. Abetalipoproteinemia (ABL)
a. Abnormal RBC lipid content – increase in sphingomyelin (rigid layer) over lecithin (fluid layer) causing
decreased deformability; mild anemia
b. Acanthocytes! 50-100% of RBC in PBS
c. Decreased plasma triglycerides and cholesterol
d. Fat malabsorption, ataxia, neuropathy
2. McLeod syndrome (MLS)
a. X-linked disorder, mutations in the KX gene
b. Kx protein, precursor of the Kell blood group antigens
c. Chorea, neuropsychiatric manifestations
3. Chorea acanthocytosis (ChAc)
a. Deficiency of chorein
b. Chorea, neuropsychiatric manifestations
H. LCAT Deficiency
1. LCAT (Lecithin-Cholesterol Acyltransferase) converts cholesterol to cholesterol ester
2. LCAT deficiency leads to a reduction in plasma cholesterol ester and an increase in free plasma
cholesterol
3. Target cells due to increased RBC membrane cholesterol
4. With renal disease and corneal opacities

XI. RBC ENZYMOPATHIES

A. General Information
1. RBC enzyme defects
2. Most commonly encountered enzymopathies are deficiencies of G6PD and pyruvate kinase. Other RBC
enzymopathies are rare.
B. Glucose-6-Phosphate Dehydrogenase (G-6-PD) Deficiency
1. Sex-linked inheritance
2. Most common RBC enzyme abnormality (5%in the global population)
3. G-6-PD is an enzyme in the hexose monophosphate shunt that is responsible for the production of
reduced glutathione to prevent denaturation of Hb
4. G6PD-deficient RBCs cannot generate sufficient NADPH to reduce glutathione and thus cannot
effectively detoxify the hydrogen peroxide produced upon exposure to oxidative stress.
5. Denatured Hb precipitates in RBC (Heinz bodies) after exposure to oxidative substances causing
hemolysis. RBC with precipitates can’t deform to pass through microcirculation and they hemolyze
6. Usually not hemolytic until oxidatively challenged
7. Oxidative substances: anti-malarial drugs (primaquine), sulphonamides, analgesic, antipyretics
(prevention by the avoidance of these drugs)
8. Degree of hemolysis varies from asymptomatic (majority) to life-threatening
9. Lab. findings:
a. Normocytic, normochromic anemia
b. Increased reticulocyte count
c. PBS: “Bite” cells seen often, Heinz bodies
d. G6PD fluorescent spot test – screening test
e. Quantitative spectrophotometric assays – gold standard
f. Methemoglobin reductase test
g. (+) Ascorbate cyanide test – brown color within 2 hours (EDTA specimen) or within 2-4 hours
(heparin, ACD) indicates G6PD deficiency
C. Pyruvate Kinase Deficiency
1. Autosomal recessive
nd
2. 2 most frequent RBC enzyme deficiency
3. Most common enzyme deficiency in the Embden Meyerhof pathway
4. RBC have a ↓ life span due to their inability to utilize glucose leading to lack of ATP production
(impairment of cation pump)
5. RBCs are unable to retain H2O causing a nonspherocytic hemolytic anemia (HNSHA)
6. PK deficiency is the most common cause of HNSHA
7. Most patients have symptoms of hemolysis
8. Laboratory findings:
a. Hb 6-12 g/dL
b. No prominent morphologic features in PBS
c. Burr cells common
d. Fluorescent Spot Test – recommended screening test
 e. Autohemolysis test – screen for PK deficiency and G6PD deficiency; more specific enzyme assays
are now available
f. Quantitative PK Assay
D. Methemoglobin Reductase Deficiency
3+
1. Causes the formation of methemoglobin (iron in Fe state) which is unable to transport O2
2. Primary characteristic: cyanosis and chocolate brown blood

XII. NONIMMUNE HEMOLYTIC ANEMIA

A. General Information
1. All cause a normocytic/normochromic anemia caused by trauma to the RBC
2. All are acquired disorders that cause intravascular hemolysis with schistocytes and thrombocytopenia
B. Microangiopathic Hemolytic Anemia (MAHA)
1. Microangiopathic: a disease of small vessels
2. Can be caused by a complication of one of several conditions in which there is a disturbance of the
microvascular environment
3. Thrombotic thrombocytopenic purpura (TTP)
a. Aka Moschkovitz syndrome
th
b. Occurs most often in adults (4 decade)
c. Rare, life-threatening
d. Etiology
i. Idiopathic – autoantibodies to ADAMTS-13 (a disintegrin and metalloprotease with a
thromobospondin type 1 motif, member 13)
ii. ADAMTS 13 is an enzyme responsible for breaking down vWF multimers. When multimers are
not broken, clot forms.
iii. Secondary – infections, pregnancy, inflammation, and disseminated malignancy depressing
ADAMTS-13 synthesis
iv. Inherited – also Upshaw-Schülman syndrome
e. Laboratory findings:
i. Hb typically below 10 g/dL
9
ii. Severe thrombocytopenia (10-30 X 10 /L)
iii. PBS: reticulocytosis, nRBCS, large # of schistocytes, poikilocytosis
iv. Coagulation tests: within reference range
v. Chemistry: increased plasma LDH, Hb and B1; decreased haptoglobin
4. Hemolytic uremic syndrome (HUS)
a. Characterized by MAHA, thrombocytopenia, and acute renal failure
b. Two general types
i. Typical HUS – Shiga toxin-associated HUS (stx-HUS); 90% of cases, occurs most often in
children following a GIT infection (commonly E. coli); syndrome often progress to oliguria or
anuria
ii. Atypical HUS – uncontrolled activation of alternative complement pathway
c. Pathophysiology:
i. Toxin damages the capillary lining
ii. Platelets clump in the area to stop blood loss and initiate healing.
iii. Clots damages RBCs and prevents adequate blood flow to affected organs, resulting in organ
dysfunction or failure
d. Laboratory findings:
i. PBS: severely decreased Hb (4 g/dL), leukocytosis, thrombocytopenia, schistocytes, burr cells
ii. Coagulation tests: within reference range
iii. Chemistry: markedly elevated plasma Hb, decreased haptoglobin
iv. Urinalysis: albuminuria, RBCs, WBCs, casts
5. HELLP Syndrome
a. HELLP: Hemolysis, elevated liver enzymes, low platelet count
b. A serious complication in pregnancy
c. Maternal endothelial cell dysfunction which leads to platelet activation and fibrin deposition
d. Exact pathogenesis unknown
e. Major diagnostic criteria: low APC, increased LDH and AST
6. Disseminated intravascular coagulation (DIC)
a. Widespread activation of the hemostatic system resulting to fibrin thrombi formation in small vessels
b. Fibrin causes RBC fragmentation
c. Multiple organ failure can occur due to clotting and bleeding due to consumption of platelets
d. Basic Causes of DIC
i. Release of tissue thromboplastin-like substances into the circulation: obstetric complications,
acute promyelocytic and monocytic leukemia, intravascular hemolysis, burns, etc
ii. Activation of coagulation proteins by damaged endothelium: infections (viral, bacterial, fungal),
heat stroke, shock
iii. Activation of coagulation proteins in association with intravascular aggregation of platelets: toxins
and drugs, antigen-antibody complexes
e. Laboratory Findings:
i. PBS: schistocytes, thrombocytopenia
ii. Hb and Hct vary
 iii. Prolonged PT and APTT
iv. Decreased fibrinogen
v. Increased D-dimer
vi. Chemistry: increased LD (LD1 and 2), decreased haptoglobin
C. Mechanical Injury to Erythrocytes
1. Schistocytes is a characteristic finding
2. Macroangiopathic Hemolytic Anemia
a. Traumatic Cardiac Hemolytic Anemia
i. Seen in aortic valvular disease such as aortic stenosis or cardiovascular prosthesis
ii. Artificial heart valves (Waring Blender Syndrome)
b. Stress or March Hemoglobinuria
i. Transient hemolytic anemia that occurs after forceful contact of the body with hard surfaces
ii. Seen in marathon runners, soldiers, karate practitioners, tennis players
3. Miscellaneous Extracorpuscular Mechanism of Injury
a. Infections and infestations
i. Intracellular: malaria, Babesiosis
ii. Malaria is the most common infectious disease that causes a hemolytic anemia
iii. Extracellular infections: T. gondii, Clostridium perfringens, Staphylococcus, Streptococcus,
Bartonella
b. Exposure to chemicals and toxins: spiders and snakes (rarely) venom contain enzymes that lyze the
RBC membrane
c. Thermal injury: heat (third degree burns) cause direct damage to RBC membrane causing RBCs to
fragment

XIII. IMMUNE HEMOLYTIC ANEMIA

A. General Information
1. Acquired/extrinsic immune hemolytic anemias
2. Conditions in which RBC survival is shortened due to an antibody-mediated mechanism
3. Classification:
a. Autoimmune – WAIHA, CAD, PCH, mixed-type AIHA
b. Drug induced – drug dependent, drug independent
c. Alloimmune – HTR, HDFN
4. Associated with a positive DAT
5. Pathophysiology of Immune Hemolysis
a. IgM
i. Requires complement
ii. Hemolysis may be extravascular (mainly in the liver) if complement is partially activated to C3b,
or intravascular if complement is fully activated to C9.
b. IgG
i. Occurs with or without complement activation
ii. IgG-sensitized RBCs are removed from the circulation by macrophages in the spleen; partial
phagocytosis produces spherocytes, which are prematurely trapped in the spleen and
phagocytized; IgG- and C3b-sensitized RBCs are removed by macrophages in the spleen and
liver.
B. Autoimmune Hemolytic Anemia (AIHA)
1. Caused by an altered immune response resulting in production of antibodies against the hosts own RBC
2. Warm AIHA
a. Majority are caused by IgG (rarely IgM, IgA) antibodies
b. Antibodies bind optimally @ 37°C
c. Hemolysis usually extravascular (Ag-Ab complexes are cleared by the spleen)
d. Classification/Etiology:
i. Idiopathic – 60% of cases
ii. Secondary to diseases that alter the immune response (lymphoproliferative diseases,
autoimmune disorders, immunodeficoency disorders, viral infections, tumors)
e. Laboratory: spherocytes; ↑ OFT; positive (DAT helpful in differentiating from hereditary
spherocytosis)
3. Cold Agglutinin Disease (CAD)
a. Caused by IgM with optimum reactvity at 4°C and a thermal amplitude >30°C
b. Two type of cold agglutinins
Nonpathologic Cold Agglutinins Pathologic Cold Agglutinins
Polyclonal Monoclonal
Occur in low titers Occur in high titers
No reactivity >30°C Capable of reacting >30°C
c. Ab binds to RBCs at cold temp, at central circulation IgM dissociates, but C3b remains
d. Hemolysis predominantly extravascular
e. Classification:
i. Acute CAD – commonly found as a complication of M. pneumoniae (anti-I), respiratory infections,
IM (anti-i), malignancy, etc
ii. Chronic CAD – rare, seen in middle aged to elderly patients
f. Laboratory: RBC clumping macroscopically and microscopically; positive DAT
 g. If antibody titer is high enough, sample must be warmed to 37°C to obtain accurate RBC and indices
results (Causes falsely increased MCV and decreased RBC count)
4. Paroxysmal Cold Hemoglobinuria (PCH)
a. Caused by a C’ dependent IgG biphasic hemolytic antibody (Donath Landsteiner Antibody/Auto-anti-
P)
b. Antibody attaches to RBC @ cold temperature (<20°C) and binds C’ (C1-C4) and causes lysis @
37°C
c. Etiology: idiopathic or secondary (viral respiratory infection, congenital syphilis)
d. Laboratory Findings:
i. DAT positive
ii. Increased plasma hemoglobin and bilirubin
iii. Decreased haptoglobin
o
iv. Pos-tive Donath-Landsteiner test: Incubate patient’s fresh serum with RBCs at 4 C. Warm the
o
mixture at 37 C for 30 minutes and centrifuge. Hemolysis indicates positive result.
5. Mixed-Type AIHA
a. Patient simultaneously develops an IgG autoantibody with optimum reactivity at 37° C (WAIHA) and
a pathologic IgM autoantibody that reacts optimally at 0° C to 10° C but has a thermal amplitude of
greater than 30° C (CAD)
C. Drug-Induced Immune Hemolytic Anemia (DIIHA)
1. Condition is suspected when there is a sudden decrease in hemoglobin after administration of a drug
with evidence of hemolysis
2. Immediate action is to discontinue the drug
3. Most common drugs implicated in DIIHA in are cefotetan, ceftriaxone, trimethoprim, and piperacillin
4. Mechanisms of Drug Induced Hemolysis
i. Drug adsorption – drug-RBC hapten complex (e.g. penicillin, cephalothin, quinidine)
ii. Immune Complex – drug-antibody complex attaches nonspecifically to RBC and activates C’
iii. Autoantibody formation – methyldopa, levodopa, mefenamic acid
iv. Membrane modification – drug alters membrane causing nonspecific absorption of IgG, IgM, IgA and
C’ (e.g. cephalosporin)
D. Alloimmune Hemolytic Anemia
1. A foreign antigen stimulates the host to produce an alloantibody
2. Hemolytic Transfusion Reactions (HTRs)
a. Recipient has antibodies to antigens on donor RBCs
b. Donor cells are destroyed intravascular and/or extravascular
c. Classification
i. Acute (AHTR) – occur within minutes to hours after transfusion; most common cause is
transfusion of ABO incompatible units
ii. Delayed (DHTR) – occur days to weeks after transfusion; seen in Duffy and Kidd blood groups
d. Physical findings: hypotension, fever, increased respiratory and pulse rates; in severe cases, renal
failure and DIC
e. Laboratory findings:
i. Hemoglobinemia (increased plasma hemoglobin)
ii. Hyperbilirubinemia
iii. Hb and urobilinogen in the urine
iv. Positive DAT
v. In DIC: depletion of fibrinogen, factor VIII and platelets; positive D-dimer test
3. Hemolytic Disease of the fetus and Newborn (HDFN)
a. Occurs when IgG alloantibody from mother crosses the placenta and binds to fetal RBCs positive for
the corresponding antigen
b. Sensitized RBCs are removed by the spleen
c. Also erythroblastosis fetalis (because of increased nRBC in newborns peripheral blood)
d. very high bilirubin levels cause kernicterus
e. May be due to:
i. Rh incompatibility – Rh negative woman is exposed to Rh antigen from fetus and forms IgG
antibodies; these antibodies will cross the placenta and destroy RBCs of the next fetus that is
Rh positive; cause kernicterus
ii. ABO incompatibility – group O woman develops IgG antibody that crosses the placenta and
coats fetal RBCs when fetus is group A or B. The coated RBCs are phagocytised.
f. Rh VS ABO incompatibility
Rh incompatibility ABO incompatibility
Mother Rh (D) negative O
Child Rh (D) positive A or B
Severity of disease Severe Mild
Jaundice Severe Mild
Bilirubin levels Very high Slightly increased
Spherocytes on PBS Rare Usually present
Anemia Severe If present, mild
DAT Positive Negative or weakly positive

XIV. ANEMIA OF BLOOD LOSS

 A. General Information
1. Increased loss of RBCs from circulation
2. Blood loss may be internal or external
3. Minor loss (≤500 mL) generally is not serious
4. BM can respond: reticulocytosis is seen
B. Acute Blood Loss Anemia
1. aka Acute Posthemorrhagic Anemia
2. Characterized by a sudden loss of blood from trauma or other severe forms of injury
3. Signs and symptoms related to degree of blood loss
a. 10% blood loss – may be none at rest, lightheadedness, increased pulse on arising
b. 20% blood loss – hypotension, exertional tachycardia
c. 30% blood loss – decreased cardiac output, hypotension, rapid pulse, cold and clammy skin
d. 40-50% blood loss – severe shock and death
4. Laboratory:
a. Immediately after an acute major blood loss. The hemoglobin and hematocrit remain normal
b. 3-4 hours, fluid enter circulation, causes dilution leading to normocytic, normochromic anemia
c. Anemia becomes evident after 12 – 24 hours. increase in WBC count (left shift) and APC
d. Reticulocytosis in 3-5 days
C. Chronic Blood Loss Anemia
1. Characterized by a gradual, long-term loss of blood
2. Often caused by GIT bleeding (e.g. carcinoma of the colon, hookworm infection, etc.)
3. Laboratory:
a. Initially shows a normocytic/normochromic anemia that over time causes a decrease in
hemoglobin/hematocrit
b. Gradual loss of iron causes microcytic/hypochromic anemia
c. Reticulocytes and WBC count is low

XV. ERYTHROCYTOSIS
A. General Information
1. Also referred to as Polycythemia
2. Disorders involving an increased number of circulating red cells and thus an increased RCM
3. Term used to signify an above normal Hb, hct (>50%), RBC
4. Classically defined as an elevated Hct level above the normal range
5. WHO definition of polycythemia:
a. Hb of >18.5 g/dL for men and >16.5 g/dL for women
b. Lower values but with a documented unexplained Hb increase of at least 2 g/dL
c. Hct >99th percentile of the method-specific range

6. Classification of Erythrocytosis
a. Relative erythrocytosis
i. Decreased plasma volume
ii. No true increase in red cell mass (RCM)
b. Absolute erythrocytosis
i. True increase in RCM
ii. Primary Erythrocytosis: polycythemia vera
iii. Secondary Erythrocytosis: appropriate production of EPO or inappropriate production of EPO
B. Relative Polycythemia
1. Refers to an increase in Hct or red cell count as a result of decreased plasma volume
2. Actual # of RBC is not increased, only the # of cells per unit volume is increased
3. Seen in:
a. Acute dehydration – severe diarrhea, burns
b. Patients on diuretic therapy
c. Gaisböck’s syndrome (aka stress/spurious polycythemia) – associated with tobacco smoking,
cardiovascular problem, hypertension and diuretic therapy; seen in middle-aged obese men
4. Laboratory findings:
a. Normocytic-normochromic red blood cells, Normal WBC, RBC and platelet morphology
b. WBC may be normal or slightly elevated in dehydration
c. Normal bone marrow
d. LAP is normal
C. Polycythemia Vera
1. Absolute Primary Erythrocytosis
2. From vera, latin term meaning “true”
3. A chronic myeloproliferative disorder (CMPD)
4. Excessive proliferation of granulocytic and megakaryocytic cell lines but affects primarily the erythroid
series
5. More frequent in men than in women.
6. Usually begins in middle age, often over 60 years of age (but condition seen in all age groups)
7. Criteria for diagnosis (WHO)
a. Major criteria
 i. Elevated Hb >18.5 (males) or 16.5 (females) g/dL, or other evidence of increased red cell
volume.
ii. Presence of Janus 2 kinase (JAK2V617F) mutation or similar mutation such as JAK2 exon 12
mutation.
b. Minor criteria
i. BM biopsy showing panmyelosis with prominent erythroid and megakaryocytic proliferation
ii. Low serum erythropoietin
iii. Endogenous colony formation in vitro.
8. Clinical features
a. Increased blood viscosity (2-3 times normal) and blood pressure, sludging of blood flow, and
thromboses, which lead to poor oxygen delivery (headaches, dizziness, blurred vision, vertigo, MI,
ruddy cyanotic appearance
b. Hepatomegaly (40%) and splenomegaly (70%) – because of extramedullary hematopoiesis
c. Thrombotic or hemorrhagic phenomena occur in about half of patients (upper gastrointestinal
bleeding, often from peptic ulcer, is the most common bleeding problem).
d. Erythromelalgia (condition characterized by episodes of pain, redness, and swelling in various parts
of the body, particularly the hands and feet)
9. Laboratory findings:
12
a. Increased RBC (7-10 × 10 /L), hct, Hb
9
b. Platelets: increased (>1000 × 10 /L); decreased aggregation to ADP and epinephrine
9
c. Neutrophilia (10–30 × 10 /L)
d. Arterial oxygen saturation is normal!
e. Serum and urine erythropoietin are decreased
f. Blood picture: Normocytic-normochromic RBC with moderate anisocytosis
g. Blood viscosity is high, and it may be difficult to prepare good blood films
h. Bone marrow: panhyperplasia/panmyelosis
i. ↑erythroid, granulocytic and megakaryocytic cell lines
ii. obvious features: ↑ megakaryocytes and normoblasts
iii. BM iron stores are decreased or absent
i. LAP is increased (distinguish PV from other erythrocytosis)
10. Course of the disease
a. Latent phase – asymptomatic
b. Proliferative phase – patients may be hypermetabolic or have symptoms of hyperviscosity or
thrombosis
c. Spent phase – anemia, leukopenia, secondary myelofibrosis, increasing liver and spleen size
d. Late complication – acute leukemia or myelodysplastic syndrome (MDS)
e. Patients usually live 10–20 years under good control.
11. Management
a. Phlebotomy – most efficient method
b. Alkylating agents – chlorambucil, Chlorambucil, busulfan, and pipobroman; high rates of iatrogenic
leukemia.
32
c. Radioactive phosphorus ( P) – myelosuppresant
d. Hydroxycarbamide (hydroxyurea) – interferes with DNA synthesis
D. Secondary Erythrocytosis
1. Caused by an increased level of erythropoietin
2. Etiology:
a. Appropriately increased EPO production – normal response to hypoxia
iii. Decreased oxygen loading: arterial O2 unsaturation; seen in high altitude, pulmonary disease,
cyanotic heart disease, carboxyhemoglobinemia, methemoglobinemia, Hb M
iv. Decreased O2 unloading: high O2 affinity hemoglobinopathy ; biphosphoglycerate deficiency
(deficiency of 2,3-DPG mutase)
b. Inappropriate production of EPO – in this case EPO is elevated but O2 saturation is normal and no
conditions of hypoxia present
i. Neoplasms, benign or malignant, express high level of EPO Mrna (Wilms’ tumor, renal
carcinoma, cerebellar hemangioma, hepatoma)
ii. Mass impinging on the kidney induces increased renal production of EPO as a result of increased
pressure or local hypoxia within the kidney (renal cysts, ovarian carcinoma, some hepatomas)
iii. Localized tissue hypoxia (polycystic kidney, renal artery stenosis)
iv. Postrenal transplant recipients
v. Acute hepatitis
3. Laboratory findings:
a. Normocytic-normochromic red blood cells
b. Normal WBC, RBC and platelet morphology
c. No thrombocytosis and leukocytosis!
d. Bone marrow: erythroid hyperplasia
e. Normal LAP!
4. Polycythemia vera VS Secondary Polycythemia
Polycythemia Vera Secondary Polycythemia
 O2 saturation Normal Decreased or Normal
EPO Decreased Increased
LAP Increased Normal
WBC count Increased Normal
Platelet count Increased Normal

E. Genetic Polycythemia
1. Von Hippel–Lindau syndrome
a. Autosomal dominant disorder
b. Mutated VHL gene (These mutations result in increased levels of HIF-1α and increased levels of
EPO)
2. Chuvash polycythemia
a. Autosomal recessive congenital polycythemia
b. Mutated EPO receptor
c. Endemic in the Chuvash population of the Russian federation
d. Associated with a high mortality rate caused by thrombotic and hemorrhagic complications.
3. Primary familial and congenital polycythemia (PFCP)
a. Autosomal dominant disorder
b. Associated with low serum EPO levels and in vitro hypersensitivity of erythroid progenitors to EPO.

FURTHER READING
1. Brown, B. A. (1993). Hematology: Principles and procedures (6th ed.). Estados Unidos: Lea & Febiger.
2. Keohane, E. M., Smith, L. J., Walenga, J. M., Rodak, B. F. (2016). Rodak's hematology: Clinical principles and
applications (5th ed.). St. Louis, MO: Elsevier.
3. Lo, R. W., Liu, E. B., Orillaza, M. A., Faundo, A. C., & J., D. C. (2012). PCQACL'S Standardization and
harmonization of complete blood count in the Philippines (1st ed.). Quezon City: C & E Publishing.
4. McPherson, R. A., & Pincus, M. R. (2011).
5. Henry's Clinical Diagnosis and Management by Laboratory Methods E-Book. Saunders.
6. Stiene-Martin, E. A., Lotspeich-Steininger, C. A., & Koepke, J. A. (1998). Clinical Hematology: Principles,
procedures, correlations. Philadelphia: Lippincott-Raven.
7. Turgeon, M. L. (2012). Clinical hematology: Theory and procedures (5th ed.). Philadelphia, PA: Wolters Kluwer
Health/Lippincott Williams & Wilkins.