1

Anemia o Pregnancy
▪ Increased fibrinogen
- Manifestation of certain conditions and not a disease ▪ Loss of negative charge
- Pale look is due to the redistribution of blood flow o Infections and inflammation
▪ Acute phase reactants;
Lesson 1: Evaluation of RBC Morphology ▪ Increase plasma proteins
o Multiple Myeloma
• Results are flagged when values from the differential or CBC are out of the references range in Hematology ▪ Excess in kappa and lambda chains
automated instrumentation. Agglutination
o RMT must make decision either to perform reflex testing or to pull a peripheral smear for review or - Irregular clumps or red blood cells from antigen-antibody reaction
complete differential to resolve the abnormal result. - Agglutinins react in cold temperature (22oC, cold agglutination) – cold agglutinins
• Proficiency in identifying normal and abnormal red blood cells is a desirable, one that must be practiced as - Warm agglutinins react at 370C directly to RBC antigens
a student and an employee. - AIHA (autoimmune hemolytic anemia)
• Automated cell counting and differential counters have not yet replaced the well-trained eye with respect to
the subtleties of red blood cell morphology. Variations in size are seen as:
• There is no substitute for a well-distributed, well-stained peripheral smear when assessing red blood cell - Microcytes
morphology. - Macrocytes
Normally, RBCs do not clump due to the presence of sialic acid which gives a negative charge.
Microcytic Cells – result from four main clinical conditions:
ARC – Absolute Reticulocyte Count: 1. Iron Deficiency Anemia
- Reticulocytosis – above normal count • Iron is needed for the maturation in the BM
- Reticulocytopenia – below normal count • Results from impaired iron metabolism caused by:
o deficient iron intake
Variations in Red Blood Cell Size – ANISOCYTOSIS
o defective iron absorption
Normocytes o hookworm infection
- Normal red blood cell (discocyte) • Four iron atoms surrounded by the protoporphyrin ring;
- Biconcave, disk-shape • Needs to be incorporated into the four heme structures of each hemoglobin molecule to be
- Approx. 7-8μm, 2.5μm thick absorbed from the bloodstream and transferred, via transferrin, to the Pronormoblasts of the bone
- 90 fL volume w/ 160μm2 average surface area marrow for incorporation into the heme structure.
- Clear area of 1/3 of the cell • Iron-starved red blood cells divide more rapidly than normal red blood cells, searching for iron, and
- MCV: 80-100fL are smaller because of these rapid divisions
- MCHC: 32% to 36% o Rapid divisions lead to decrease in cell size
- Approx. the same size as the nucleus of a small lymphocyte.
- Have a clear area occupying approx. 1/3 (2-3μm) of the cell diameter 2. Thalassemic Conditions (Thalassemia)
• Decreased or absent globin synthesis
Abnormal erythrocyte Arrangements • alpha or beta chains are missing or diminished:
Rouleaux o normal adult hemoglobin is not synthesized
- RBC arranged in stacks of coins or poker chips or flat plates o hemoglobin configuration is impaired
- Abnormal or increased plasma proteins; o leads to microcytic cell
- Loss of negative charge related to zeta potential o increased central pallor – hypochromia
- Can be dispersed by mixing with saline
- Increased erythrocyte sedimentation rate (ESR)
- Associated with:
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3. Iron Overload Disorders

• Show dimorphic blood smear:
o Microcytes mixed with macrocytes
o Red blood cells exhibiting normal hemoglobin levels
o Hypochromia

4. Anemia of Inflammation
• Approx. 10% of individuals who have this disorder arise from renal failure or thyroid dysfunction;
• Microcytic RBC in the peripheral smear;
• Impaired iron delivery to the reticuloendothelial system.

Macrocytic Red Blood Cells

- Diminished life span
- Megaloblastic anemia with MCV of >110fL
Variations in Red Blood Cell Color – Variation in Hb Content – ANISOCHROMASIA
❖ Ineffective Erythropoiesis
• RBC precursors are prematurely destroyed before they are released into the circulation Normochromic Red Cells
❖ Asynchrony • Have hemoglobin concentration that is within the reference range
• Asynchronous development of nuclear structure and cytoplasm Hyperchromic Red Cells
• Nuclear age appears to be out of sync with the cytoplasm development • Have increased concentration of hemoglobin
❖ Megaloblastic Picture: • Hereditary Spherocytosis
• Pancytopenia in CBC • Non-hyperchromic RBC:
• Hypersegmented neutrophils o Related to spherocyte – not biconcave, no central pallor
• Macroovalocytes Polychromasia
❖ Borderline increased MCV, large cells: • Excessive production of red blood cell precursors in response to anemic stress
• Alcoholism • Release reticulocytes, and, sometimes, orthochromic normoblasts (nRBCs)
• Liver disease • Polychromatophilic Macrocytes – reticulocytes:
o Non-megaloblastic o Reticulum can be visualized only when these cells are stained with supravital stain
• High reticulocyte counts o Excellent indicator of bone marrow health
o Polychromatophilic macrocytes (reticulocytes) in the peripheral smear • Observed in the ff. situations:
❖ Nucleated RBC structure o Bone marrow response to anemia
• All immature RBCs are nucleated ▪ Increase reticulocytes;
• Nuclear synthesis depends on Vitamin B12 and folic acid ▪ otherwise, there is bone marrow failure (reticulocytopenia)
o Deficiency of these vitamins will lead to macrocytic cells o Iron deficiency anemia or megaloblastic anemia therapy
• B12 and B9 deficiency: o Bone marrow stimulation due to chronic hematologic conditions (Thalassemia/Sickle cell
o impaired DNA synthesis disease)
o impaired nuclear maturation o Recovery from acute hemorrhage, pregnancy, and in newborns associated with hemolytic
o leads to macrocytic RBC, megaloblastic anemia
o can be caused by D. latum infection
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• Polychromasia Grading: o presents the morphologist with visual clues for what might be the source of the patient’s hematologic
o Slight 1% problems, whether it is decreased RBC production, increased destruction, or defective splenic
o 1+ 3% function
o 2+ 5%
o 3+ 10%
Red Blood Cell Inclusions
o 4+ >11%
- cytoplasm of all normal red blood cells is free of debris, granules, or other structures
• Reference Range for Reticulocytes: - result of distinctive conditions
o 0.5 – 1.5% - adults
o 2 – 6% - newborns
Lesson 2 – Introduction to Anemia
Hypochromic Red Blood Cells
• Exhibit a larger than normal area of central pallor (>3μm) – MCHC of 32% ANEMIA
• Usually seen in conditions in impaired hemoglobin synthesis • Greek origin of term “anaimia” without blood
• Development of hypochromia is usually a gradual process • Anemia is not a disease but an expression/manifestation of an underlying disease or deficiency
• Seen on the peripheral smear as a delicately shaded area of hemoglobin within RBC structure
o Not all hypochromic cells are microcytic, but all microcytic cells are hypochromic Operational Definition:
• Qualitative Grading of Hypochromia: - Reduction in Hb content of the blood caused by decrease RBCs, Hb and Hct
o Normal – central pallor is 1/3 of RBC o Below the lower limit of the 95% reference interval for the individual’s age, sex, race under similar
o 1+ (slight) – central pallor is >1/2 of RBC, pale outer rim of Hb environmental conditions
o 2-3+ (moderate) – central pallor is >2/3 of the cell, pale outer rim of Hb Functional Definition
o 4+ (marked) – only thin rim of pink cytoplasm remains - Decrease in oxygen-carrying capacity of the blood
o Insufficient hemoglobin
o Impaired function of hemoglobin

Hypochromic microcytic anemia

Variations in Red Blood Cell Shape – POIKILOCYTOSIS

- Link to a red blood cell pathophysiology

- Abnormal red blood cell morphology
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General Signs & Symptoms of an Anemic Individual: Types of Anemia

1. Due to decrease in oxygen transport Relative

▪ Fatigue • Normal RBC mass
▪ Syncope • Increased plasma volume
▪ Dyspnea • Conditions that result to hemodilution
▪ Angina pectoris o Pregnancy
▪ Systemic organ failure o Volume overload
2. Due to decrease in blood volume Absolute
▪ Pallor • Decreased RBC mass
▪ Postural hypotension • Normal plasma volume
3. Due to increased cardiac output • Mechanisms involved:
▪ Palpitation o Decreased delivery of RBCs into circulation
▪ Congestive heart failure ▪ Impaired or defective production
Physiologic Response to Anemia ▪ Failure of BM to respond (reticulocytopenia)
o Increased loss of RBCs from the circulation
Sudden loss of blood volume – minute to hours ▪ Acute bleeding, accelerated destruction
• Selective redistribution of blood flow ▪ Respondent BM (reticulocytosis)
• Increased heart and respiratory rate and cardiac output
Erythron - rate of production = rate of destruction
Slow loss of blood – days to weeks
• Shift to the right and increased 2,3-DPG Disturbances on the Erythron
• Increased EPO production in the kidneys
1. Normal red cell destruction but decreased red cell production
Mechanism of Anemia 2. Increased red cell destruction but normal red cell production
3. Increased red cell destruction and decreased red cell production
Effective erythropoiesis
4. Normal red cell destruction and normal red cell production
• red cells that are produced in the bone marrow reaches the circulation and reaches its maturity
• production of functional RBCs Classification of Anemia
• supply peripheral circulation with adequate number of cells
1. Etiologic classification
Ineffective erythropoiesis
2. Morphologic classification
• Red cells that are produced in the bone marrow does not reach the circulation and its lifespan
3. Physiologic classification
• Production of defective erythroid precursors
4. Combined morphologic & physiologic classification
• Undergo apoptosis
• Low blood hemoglobin ETIOLOGIC CLASSIFICATION
• Decreased normal circulating RBCs 1. Decreased production of RBC
Insufficient erythropoiesis • Marrow damage/marrow infiltration
• Decreased erythroid precursors in the BM, decreased RBC production o Hematopoietic stem cell failure
• Iron deficiency o Functional impairment of erythroid precursors
• Deficiency of EPO ▪ Leukemia
• Loss of erythroid precursors ▪ Leukoerythroblastosis
• Suppression of erythroid precursors ▪ aplastic anemia
• Malignancy and infection ▪ pure red cell aplasia
▪ myeloplastic anemia
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• Decreased EPO o Microcytic - <80 fL

o Inflammatory process (ACD) o Macrocytic - >100 fL
o Anemia of liver disease • Classification of RBCs based on the MCHC
o Anemia of endocrine disorder o Normochromic – 32-36g/dL
• Iron deficiency o Hypochromic - <32g/dL
o Hyperchromic - >36g/dL
2. Nuclear maturation abnormality – disturbance of DNA synthesis • Normocytic, normochromic
• Megaloblastic anemia • Microcytic, hypochromic
o Vitamin B12 deficiency • Microcytic, normochromic
o Folic acid deficiency • Macrocytic, normochromic
o Pernicious anemia
• Refractory macrocytic anemia Microcytic Anemia:
• Drugs affecting DNA synthesis 1. Iron deficiency
• Inherited disorders of DNA synthesis 2. Iron-transport deficiency
3. Sideroblastic anemia
3. Cytoplasmic maturation abnormality – disturbance of hemoglobin synthesis 4. Lead poisoning
a. Hemolytic 5. Anemia of infection
b. Acute 6. B^ deficiency
c. Chronic 7. Thalassemia
• Iron deficiency anemia 8. Shahide-Nathan-Diamond syndrome
• Thalassemia
• Sideroblastic anemia Normocytic Anemia:
• Anemia of chronic inflammation 1. Anemia of renal disease
4. Hemolysis – increased RBC destruction or loss 2. Anemia of endocrine disorder
• Intrinsic abnormality (Congenital) 3. Aplastic anemia
o Red cell membrane defect 4. Anemia of blood loss
o Hemoglobinopathies 5. Myelophthisic anemia
o Enzyme defects 6. Anemia of chronic disorders
• Extrinsic abnormality (Acquired) 7. Anemia of pregnancy
o Immune cause 8. Anemia of liver disease
▪ AIHA
▪ Paroxysmal Nocturnal Hemoglobinuria PHYSIOLOGIC CLASSIFICATION
▪ Alloimmune hemolytic anemia • Reticulocyte Production Index (RPI) is <2.0: ineffective erythropoiesis
o Non-immune red blood cell injury o Anemia is due to:
▪ Microangiopathic hemolytic anemia ▪ Hypoproliferative anemia
▪ Macroangiopathic hemolytic anemia ▪ Maturation disorders
▪ Infectious agents • Reticulocyte Production Index (RPI) is >3.0: effective erythropoiesis
▪ Other injury o Anemia is due to:
▪ Hemolysis
MORPHOLOGIC CLASSIFICATION ▪ Blood loss anemia
• Classification of RBCs based on the MCV
o Normocytic – 80-100 fL
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9. Red cell survival time (51Cr)

10. Stool analysis
11. Cytogenic and molecular analysis

Laboratory Diagnosis
1. Efficient use & interpretation of lab measurements
2. Careful history and physical examination
3. Determine whether the Hb, Hct, or erythrocyte count lies below:
• The reference intervals for age and sex, or
• The patient’s previous values
• Underlying cause or mechanism for the anemia
4. Increased numbers of polychromatic macrocytes
• Increased erythropoiesis due to hemorrhage or hemolysis
o History – blood loss
o Physical examination – jaundice, splenomegaly
5. Findings suggestive of hemolysis
• Poikilocytosis: sickle cells, irregularly contracted forms, schistocytes, spherocytes
6. Target cells
• Hemoglobinopathies – Hb C, Hb D, & Hb E, & Thalassemia
• Present in small numbers in any hypochromic anemia
• Without microcytosis – liver disease, absence of the spleen or splenic function
7. Fine basophilic stippling
• Associated with a significant increase in the generation of erythrocytes
o Blood loss
o Hemolysis
8. Course basophilic stippling
• Megaloblastic anemia
• Thalassemia
• Refractory anemias
• Some red cell enzyme deficiencies
• Unstable hemoglobins
• Lead poisoning
(refer to the book, page 293)
9. Oval macrocytes and hypersegmented neutrophils
Laboratory Evaluation of Anemia • Indicates the very likely existence of megaloblastic anemia
1. Complete blood count – Hb, hematocrit, RBC count, blood indices 10. Macrocytic Anemia
2. Reticulocyte count & RPI • Normochromic
3. Blood smear examination • Bone marrow aspiration
4. Osmotic Fragility Test (OFT) • Megaloblastic Marrow
5. Bone marrow aspiration and biopsy o Characteristic changes in both RBC & WBC precursors
6. Serum and marrow iron studies (ferritin, serum iron, TIBC) o Due to folate or cobalamin deficiency
7. Serum bilirubin • Non-megaloblastic Marrow
8. Fecal and urine urobilinogen o Liver disease, hemolytic anemia, hypothyroidism
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o Excessive alcohol intake, hypoplastic anemias • Some of the unstable hemoglobinopathies

o Refractory anemias w/ hyperplastic bone marrow w/c include the myelodysplastic syndromes • Pyruvic kinase deficiency
11. Microcytic & Hypochromic Anemia 8. Bone marrow and stem cell transplantation
• Reflect a quantitative defect in Hb synthesis • Leukemia
o Iron-deficiency anemias – increased requirement or blood loss not balanced by intake • Lymphoma
o ACD • Hodgkin’s disease,
▪ infection, neoplasia, collagen disease • Multiple myeloma
▪ may be normochromic & normocytic • Myelofibrosis
▪ long-standing: often hypochromic & microcytic • Aplastic disease
o Thalassemia
▪ genetically determined impairment in the rate of globin synthesis Lesson 3 – Anemias of Nuclear Maturation Abnormality
12. Normocytic & Normochromic Anemia
• RPI is the simplest measure of effective erythropoiesis Nuclear Maturation Abnormality – disturbance of DNA synthesis
• Optimal Marrow Response: absolute reticulocyte count, RPI • Megaloblastic Anemia
o marrow has reached an optimal response o Vitamin b12 deficiency
o acute blood loss or hemolysis o Folic acid deficiency
o Pernicious anemia
Therapy • Refractory macrocytic anemia
1. Transfusion of packed RBCs • Drugs affecting DNA synthesis
• For patients who are actively bleeding • Inherited disorders of DNA synthesis
• With a severe & symptomatic anemia
• Palliative and should not be used as a substitute for specific therapy MEGALOBLASTIC ANEMIA
2. Anemia of chronic disorders • Heterogenous group of disorders characterized by diminished capacity for DNA synthesis
• Erythropoietin to reduce transfusion • RBCs with larger size than normal RBCs and have higher N:C ratio compared to normoblastic cells
3. Blood loss or iron deficiency • Maturation of nuclei is delayed, while cytoplasmic development is normal (asynchronous development)
• Correction of the underlying condition
• Oral administration of ferrous sulfate until the anemia is corrected and for several months afterward Causes of Megaloblastic Anemia
to ensure that body stores are replete with iron 1. Gastrectomy (lack of intrinsic factor)
4. Nutritional therapy 2. Dietary deficiency:
• Iron deficiency • Vitamin B12 deficiency (especially in strict vegetarians)
• Vitamin B12 deficiency • Folate is destroyed by prolonged exposure to heat during cooking
• Folic acid deficiency • Alcoholism (alcohol damages the liver and poort nutrition is prevalent among alcoholics)
• Pyridoxine (Vitamin B6) – sideroblastic anemia 3. Disease of the small intestine
5. Corticosteroids – autoimmune hemolytic anemia • Sprue (malabsorption syndrome)
6. Aplastic disorders • celiac disease, stricture formation, jejunal diverticula
• Removal of the offending agent • bacterial overgrowth (blin loop syndrome)
• Supportive therapy for the anemia and thrombocytopenia • Crohns’s disease (an inflammatory bowel disease),
• Prompt treatment of infection • Zollinger-Ellison syndrome (disease of the stomach with gastrointestinal mucosal ulceration)
• Immunosuppressive therapy (e.g., antithymocyte globulin & cyclosporin) • Imerslund-Grasbeck syndrome – hereditary malabsorption of vit. B12
7. Splenectomy • Diphyllobothrium latum – fish tapeworm causes B12 deficiency
• Sufficient improvement for patients w/ hypoplastic, but not totally aplastic marrow • Decreased production of transcobalamin II
• Hereditary spherocytosis and hereditary elliptocytosis
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• Achlorhydia – absent or decreased secretion of HCl in the stomach which is needed for
absorption
• Zinc deficiency – zinc is needed for absorption
4. Increased requirement – pregnancy, infants, lactating mothers, malignancy
5. Liver disease
6. Drugs
• Antifolates, purine analogs, pyrimidine analogs
• Ribonucleic reductase inhibitors
• Anti-convulsants
• Drugs that can depress folates
• Methotrexate
• Drugs that affect cobalamin metabolism
7. Cytotoxic agents
8. Patients undergoing hemodialysis (folate dialyzable)

Vitamin B12 Deficiency

• Cobalamin – necessary for DNA synthesis and maturation
of cells
o organometallic substance containing a corrin ring and
a centrally located cobalt atom
• Associated with neurologic manifestations
o B12 is necessary for fatty acid synthesis of the myelin sheath covering the axons of nerve fibers
• Only vitamin exclusively synthesized by microorganisms found in soil, water, intestine or rumen such as
Propionibacterium shermanii, Streptomyces griseus & Streptomyces aerofaciens
• Sources: meat & milk;
o live, kidneys, oysters, clams, fish, eggs
o cheese and other dairy products
• Individual at risk:
o Breastfeed infants of vegetarian mothers
o Strict vegetarians
▪ body stores of cobalamin usually exceed 1000ug and daily requirements is about 1ug
▪ strict adherence to a vegetarian diet for more than 5 years
• Defects:
o Impaired development;
o Mental retardation and macrocytic anemia;
o Methymalonic aciduria and homocystinuria
• Inherited disorders of cobalamin metabolism include:
o absence of intrinsic factor, abnormal Cbl intestinal adsorption, abnormal transcobalamin II
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• 2 vitB12 coenzymes catalyzing 2 types of reactions: ▪ Blocking antibodies – block the binding of cobalamin to IF
o Adenosylcobalamin – conversion of L-methymalonyl coenzymes A (CoA) to succinyl CoA ▪ Binding antibodies – bind to the cobalamin IF complex and prevents the complex from
o Methycobalamin – methylation, acts as methyltransferase for conversion of homocysteine to binding to the receptors
methionine
• Effect of deficiency:
o Thymidine synthase function is impaired
o Megaloblastic changes because DNA synthesis is diminished
o Prevents the conversion of THF from 5-methyl THF;
▪ as a result, folate becomes metabolically trapped as 5-methyl THF.
▪ This constitutes the “folate trap”

Folic Acid Deficiency

• Folate or pteroylmonoglutamic acid – necessary for DNA synthesis like B12
o composed of a pterin ring connected to p-aminobenzoic acid (PABA) and conjugated with one or
more glutamate residues
• Sources: plants & animal sources
o green leafy vegetables
o broccoli, fruit, whole grains
o meat and liver
o humans do not generate folate endogenously
• 500-20,000ug of folate in body store
o to absorb approx. 50-100ug of folate per day in order to replenish the daily degradation and loss
through urine and bile
o signs and symptoms of deficiency can manifest after 4 months

Other Defects of Nucleoprotein Synthesis

• Congenital defects
o Orotic aciduria – an autosomal recessive condition in which enzymes for pyrimidine synthesis are
absent; increased in urine
o Lesch-Nyhan Syndrome – are rare X-linked disorder of purine metabolism
• Synthetic inhibitors
Pernicious Anemia • Refractory anemia
• Conditioned nutritional deficiency of cobalamin • Congenital dyserythropoietic anemia
o Failure of the gastric mucosa to secrete intrinsic factor (IF) • Erythroleukemia (M6)
o Production of autoantibodies attacking the parietal cells of the stomach or the intrinsic factor itself. • Prolonged nitrous oxide anethesia
• Antibodies of PA: o destroys methylcobalamin;
o Anti-parietal cell antibodies – react w/ gastric parietal cells o revealed an erythroblastic hyperplasia w/ megaloblastic normoblast
o Anti-intrinsic factors antibodies
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Lesson 4 – Anemias of Cytoplasmic Maturation Abnormality 2. Transport iron-depletion (latent iron deficiency)
a. Exhaustion of the storage pool of iron
Cytoplasmic Maturation Abnormality – disturbance in hemoglobin synthesis b. Increased red cell protoporphyrin
• Iron deficiency anemia c. Prussian blue stain of the bone marrow shows essentially no stored iron
• Thalassemia 3. Functional iron depletion (iron deficiency anemia)
• Sideroblastic anemia a. Depletion of storage iron
• Anemia of chronic inflammation b. Diminished levels of transport iron
• Porphyria c. Prevent normal development of RBC precursors
• hemochromatosis d. Primarily normocytic, normochromic;
▪ gradually becomes microcytic, hypochromic
IRON-RELATED DISORDERS
• Iron-restricted anemias
o Iron is the limiting factor – IDA & ACI
o Inadequate production of protoporphyrin – sideroblastic
o Blockage of protoporphyrin production, leads to accumulation of various porphyrins – porphyrias
o Excess accumulations of iron, usually without anemia – hemochromatosis
• Iron-loading anemias
o Chronic erythroid hyperplasia
▪ Hemoglobinopathies & Thalassemia
o Heritable mutations affecting globin chain
structure or their production

IRON-DEFICIENCY ANEMIA
• Most common nutritional deficiency in the world
o Susceptible infants between the aged 6-24
months – milk anemia of infancy
o Adolescent girls and women of childbearing age –
susceptible due to blood loss
o Adult male – if no iron intake, body iron stores of
100 mg would last for 3-4 years before he would
even become iron-deficient
▪ All causes of IDA in adult males would be
due to chronic blood loss

Stages of Iron-deficiency

1. Storage iron depletion (latent iron deficiency)

a. Progressive loss of iron storage
b. Blood loss exceeds absorption – iron mobilized from the stores
c. Storage iron decreases;
▪ plasma ferritin decreases
▪ iron absorption increases
▪ plasma iron-binding capacity (transferrin) increases
 11

o basophilic stippling of erythrocytes

o due to inhibition of the enzyme 5-pyrimidine nucleotidase;
▪ responsible for degradation of RNA material in reticulocytes
• treatment involves removal of lead using a chelating agent such as EDTA

Therapy for IDA

• Ferrous iron – given orally about 200mg/day in 3 doses between meals
• Reticulocytes are expected to increase
• After the patient’s hemoglobin has returned to normal, iron therapy should be continued for at least 2
months to replenish storage iron in the bone marrow

SIDEROBLASTIC ANEMIA
• Defect in heme synthesis particularly involving the incorporation of iron into the heme molecule Anemia of Chronic Inflammation/Infection/Disease
• Congenital enzyme defects • Rheumatoid arthritis, severe trauma
o Delta-aminolevulinic acid synthase • Heart disease
o Heme synthase or ferrochelatase • Diabetes mellitus
• Infection or malignant disease of a long-standing nature
Causes of Sideroblastic Anemia • Infections can cause:
• Hereditary sideroblastic anemia o Impaired mobilization of iron, impaired ferrokinase
o X-linked, common in males; o Impaired marrow response to anemia, diminished erythropoiesis
o defect or a point mutation in the gene for delta aminolevulinic synthase (ALAS2) enzymes; o Shortening red cell survival
▪ needed for heme synthesis • High level of cytokines and interleukins:
• Acquired sideroblastic anemia o Cause the destruction of RBC precursors
o Primary/idiopathic sideroblastic anemia o Decrease the number of erythropoietin receptors on progenitor cell
o Secondary sideroblastic anemia: o Alter iron metabolism
▪ TB drugs (isoniazid, cyclosporine) o Inhibit hematopoiesis
▪ Alcohol (ethanol-induced anemia; most common & reversible) o Decrease EPO secretion
▪ Chloramphenicol (inhibits mitochondrial protein synthesis) o Decrease red cell survival
▪ Copper deficiency
▪ Lead poisoning inhibits several enzymes involved in heme synthesis (delta-aminolevulinate
dehydrate, coproporphyrin oxidase, ferrochelatase)

Lead Poisoning or “plumbism”

• Bluish-black lead line resulting from lead precipitate in the gingiva
• Basophilic stippling in the peripheral blood smear
• Punctate basophilia
 12

o Serum iron, transferrin saturation, and serum ferritin are increased.

Classification of Hemochromatosis
• Hereditary hemochromatosis
o Caused by genetic mutations in the ff, proteins:
▪ HFE
▪ Transferrin receptor protein 2 (TfR2)
▪ Ferroportin
▪ Hemojuvelin (HJV) genes
• Secondary hemochromatosis
o Associated w/ conditions that have an ineffective
erythropoietic component
o increased iron absorption:
Laboratory findings in ACD
▪ B-thalassemia
• Normocytic and normochromic erythrocytes (microcytic, hypochromic in some cases)
▪ congenital dyserythropoietic anemia
• Hypoproliferation of RBCs ▪ sideroblastic anemia
• Low serum iron despite adequate iron stores
• Decreased or normal TIBC (in contrast to IDA which is increased)
• % saturation is decreased Iron overload may also be seen in liver diseases like viral hepatitis and
• Increased erythrocyte protoporphyrin alcoholism.
• Elevated serum ferritin In cases of chronic transfusion, the body recycles the iron from
• Leukocytosis or thrombocytosis (or both) transfused RBCs, in addition to its own senescent RBCs.

Porphyria
• Group of inherited disorders characterized by a block in porphyrin synthesis due to a defect in one or
more enzymes in the heme synthesis pathway leading to the accumulation of porphyrin precursors

Clinical Features of Porphyria

1. Common in intermarrying European monarchies of past centuries;
• Psychosis is a prominent clinical feature
2. Photosensitivity with severe burns on exposure to sunlight
3. Fluorescence of developing teeth and bones
4. Bone marrow specimen the erythroblasts
• will be bright red under a fluorescent microscope
5. Port wine colored urine

Iron Overload
• Hemochromatosis
o Clinical disorder that results in parenchymal tissue damage from progressive iron overload
associated with an iron disorder, but does not involve anemia
o Increased iron stores (absorption greater than loss) and is stored as ferritin and hemosiderin
o Excess iron is often stored around organs (heart, liver, and pancreas)
 13

Thalassemia
• Formerly known as Mediterranean anemia
• Diverse group of genetic disorders characterized by a primary, quantitative reduction in globin chain
synthesis
• Mechanisms:
o A reduced or absent production of a particular globin chain
▪ Diminishes hemoglobin synthesis
▪ Produces microcytic, hypochromic RBC
o An unequal production of the a- or B-globin chains
▪ Imbalance in the a/B chain ration
▪ Markedly decreased survival of RBCs and their precursors
• α-thalassemia – there is complete or partial defect in α-chain synthesis (chromosome 16)
• β-thalassemia - there is complete or partial defect in β-chain synthesis (chromosome 11)
o β-thalassemia major
▪ bone marrow expansion from excessive ineffective erythropoiesis
▪ skeletal deformities with frontal bossing, cheek bone, and jaw protrusions, and distortions of
vertebrae and ribs
▪ dipole of the skull is thickened and perpendicular striations appear (hair on end appearance)
▪ forwarded protrusions of the upper teeth and overbite leads to dental and orthodontic
problems
▪ growth and sexual development are retarded and intercurrent infections are common

Laboratory Findings of Thalassemia

• Anisocytosis and poikilocytosis
• Normal or increased RBC count
• Increased RDW
• Target cells
• Basophilic stippling
• NRBCs in the peripheral blood (in some types only
• Decreased osmotic fragility test
• Erythroid hyperplasia in the bone marrow and extramedullary erythropoiesis
 14

o Mild macrocytosis – stress erythropoiesis

Hereditary Persistence of Fetal Hemoglobin

• Heterogenous group of inherited disorders characterized by increased levels of Hb F in adults in the
absence of the clinical & hematologic features of thalassemia
• It is characterized by either deletion or inactivation of the β and δ gene complex but compensatory
persistence of γ chains

Types of Aplastic Anemia

• Primary aplastic anemia
o Inherited aplastic anemia
▪ Fanconi’s anemia – an autosomal recessive disorder
▪ Dyskeratosis congenita
▪ Shwachman -Diamond Syndrome
▪ Cartilage-hair hypoplasia
▪ Pearson syndrome
▪ Amegakaryocytic thrombocytopenia (thrombocytopenia-absent radius, TAR
Lesson 5 – Anemias of Decrease RBC Production
syndrome)
Mechanism for decrease RBC production leading to anemia include the ff: ▪ Dubowitz syndrome
• Marrow damage/ Marrow infiltration o Idiopathic aplastic anemia
o Hematopoietic stem cell failure • Secondary or acquired aplastic anemia
o Functional impairment of erythroid precursors o Chemical agents or toxic substances that damage bone marrow
▪ Leukemia, Leukoerythroblastosis, aplastic anemia, pure red cell aplasia, ▪ Ionizing radiation
myelophthisic anemia ▪ Benzene
• Decreased EPO ▪ Antineoplastic drugs (busulfan, urethane)
o Inflammatory process (ACD), renal disease, anemia of liver disease, anemia of endocrine ▪ Antimicrobial (salvarsan, chloramphenicol, sulfonamides, chlortetracycline,
disorder streptomycin)
• Iron Deficiency ▪ Anticonvulsants (mephenytoin, trimethadione)
▪ Antimalarials (amodiaquine, chloroquine, mepacrine)
APLASTIC ANEMIA ▪ Insecticides (DDT)
• A syndrome of bone marrow failure characterized by ▪ Hair dyes
peripheral pancytopenia and bone marrow hypoplasia ▪ Bismuth, mercury, and gold
o Bleeding - thrombocytopenia; easy bruising, o Infection and other diseases
bleeding gums and episodic nosebleeds ▪ Viral infection (parvovirus B19, HIV, EBV, Hepatitis virus)
o Lack of palpable spleen and the patient is ▪ Brucellosis
susceptible to bacterial infections ▪ Immunologic disorders (SLE, RA)
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▪ Transfusional GVHD CONGENITAL DYSERYTHROPOIETIC ANEMIA

• A heterogenous group of refractory, congenital anemia characterized by both abnormal and ineffective
PURE RED CELL APLASIA erythropoiesis
• Describes a condition in which there is nearly absent RBC precursors in the BM, but normal
megakaryocytes and WBC precursors Classification of CDA
• Acquired pure red cell aplasia • CDA Type I
o Thymomas (T cell-mediated erythroid rejection) o May cause neonatal jaundice
o Autoimmune diseases o Red cells are slightly macrocytic
▪ RA, SLE, AIHA, chronic active hepatitis, collagen-vascular diseases, and chronic o Anisocytosis and poikilocytosis
lymphocytic leukemia o Basophilic stipplings and cabot rings
▪ Immunoglobulin G (IgG) antibodies suppress the growth of RBC precursor o Binucleated cells and erythrophagocytosis in the BM
• A congenital form of pure red cell aplasia – Diamond-Blackfan in 1938 o Swiss cheese nuclei
• Other causes; Respiratory infections, gastroenteritis, primary atypical pneumonia, infectious, o Thin Feulgen-positive intranuclear chromatin bridge joining 2 normoblasts
mononucleosis, mumps, and viral hepatitis • CDA Type II
o HEMPAS (hereditary erythroblastic multinuclearity with positive acidified serum test)
Types of Pure Red Cell Aplasia o Mos common
• Transitory arrest of erythropoiesis (transient aplastic crises) o Normocytic; anisocytosis, poikilocytosis, and basophilic stippling
o Occur during the course of a hemolytic anemia (often preceded by an infection) o Absence of megaloblastic marrow
o Caused by parvovirus B19 infection o Positive acidified serum test (similar to PNH) – unique HEMPAS antigen found on the red cell
▪ Cross the placenta in infected women and infect and destroy mature erythroid surface
progenitor cells (CFU-E) in the fetus o Negative sugar water test
o Can induce spontaneous abortion • CDA Type III
• Transient erythroblastopenia of childhood (TEC) o Multinuclearity of the normoblasts in the BM
o Temporary condition occurs in healthy children following viral infection o Other normoblasts called gigantoblasts (up to 50-60 um in diameter)
• Congenital red cell aplasia (Diamond-Blackfan Syndromw) o Normocytic to slightly macrocytic
o Rare, congenital disorder with an abnormal gene that codes for ribosomal protein subunit of o Negative acidified serum test
nucleolar localization • CDA Type IV
o Without this gene, BFU-Es and CFU-Es exhibit acceleration in apoptosis o Associated with severe transfusion-dependent anemia
o Marked by erythroid hyperplasia, normoblastic
MYELOPHTHISIC ANEMIA o Slight to moderate increase in nonspecific dyserythropoietic change, including markedly
• Bone marrow replacement – myelophthisis irregular or karyorrhectic nuclei
• Bone marrow space is taken over by cells or material that should not be there o Absence of the marrow abnormalities characteristics of CDA I – III
o Marrow replacement of hematopoietic stem cells by neoplastic cells in metastatic carcinomas, o Absence of precipitated protein within erythroblasts
o multiple myelomas, leukemia, lymphoma, granulomatous disease (seen in military tuberculosis) • CDA V
o myelofibrosis and storage diseases o Normal or near-normal Hgb with normal or slightly increased MCV
• Laboratory Findings: o Markedly normoblastic to mildly or moderately megaloblastic erythroid hyperplasia
o Normocytic and normochromic red cells o Ineffective erythropoiesis with little or no erythroid dysplasia
o Pancytopenia • CDA VI
o Polychromasia and stippled red cells o Normal or near-normal Hgb with marked macrocytosis
o Immature erythroid and myeloid cells in the peripheral blood o Erythroid hyperplasia with cobalamin and folate independent megaloblastic erythropoiesis and
o Teardrop cells are present nonspecific dyserythropoiesis
o Ineffective erythropoiesis
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• CDA VII • Interferon and ribavirin (drugs for treatment of hepatitis C) suppresses erythropoiesis
o Marked normoblastic erythroid hyperplasia with nonspecific dyserythropoietic changes, • Thin macrocytes - in alcoholics
especially irregular nuclear shapes • Target cells – obstructive jaundice
o Markedly ineffective erythropoiesis
o Intraerythroblastic inclusions resembling precipitated globin ANEMIA OF RENAL INSUFFICIENCY
• Occurs in patients with end stage renal disease
ANEMIA IN MALIGNANCY • Primary deficiency of erythropoietin production by the interstitial fibroblast;
• Mechanisms o Type I interstitial cells, thereby leading to anemia
• Direct effects • Failure to produce adequate amounts of EPO – decreased RBC production and thrombocytopenia
o Replacement of marrow by malignant cells (decreased RBC production) (bleeding)
o Acute and chronic blood loss • BUN and creatinine are elevated
• Indirect effects • Burr cells and fragmented RBCs
o Anemia of associated organ failure (renal, hepatic) • Treatment includes
o Malnutrition and vitamin deficiency o recombinant erythropoietin
o Immune hemolytic anemia o renal transplant
• Treatment-associated anemia (chemotherapy or radiation therapy) o dialysis (patients may develop folate deficiency since folate is dialyzable)

ANEMIA OF ENDOCRINE DISEASE

• Pituitary disorders Lesson 6 – Hemolytic Anemias (Part I)
o Deficiencies in hormones;
▪ Normocytic, normochromic anemia and leucopenia Hemolysis – increased RBC destruction or loss
o Correct as normal function is restored by replacement therapy • Intrinsic/intracorpuscular abnormality
• Thyroid disorders o Red cell membrane defect – mostly hereditary poikilocytosis, abnormal lipid membrane
o Hypothyroidism – mild degree of anemia; MCV is usually high/normal composition
▪ Decrease in thyroid hormones results to smaller tissue oxygen requirement –
o Hemoglobinopathies – globin abnormality
decreased production of EPO by the kidneys
o Thyrotoxicosis – mild degree of normochromic anemia in 20% of cases ▪ Sickle cell anemia, other hemoglobinopathies
▪ Erythroid activity is increased but a disproportionate increase in plasma volume o Enzyme defects
means either no change in Hgb concentration or mild anemia ▪ G6PD deficiency, PK deficiency
• Adrenal disorders
o Hypoadrenalism - normochromic, normocytic anemia Hemolysis is the premature destruction of erythrocytes
▪ Corrected by replacement mineralocorticoids • Lead to hemolytic anemia when marrow activity cannot compensate for erythrocyte loss
o Hyperadrenalism (Cushing’s) – erythrocytosis with atypical net increase in Hgb (1-2g/dL) • Associated with increased red cell destruction;
• Parathyroid disorders – anemia from impairment of erythropoietin production, or in some cases from o Red cell production is normal
secondary marrow sclerosis
o Membrane abnormalities, enzyme deficiencies, or other extrinsic/extracorpuscular
• Sex hormones – androgens stimulate erythropoiesis and are occasionally used to stimulate red cell
factors
production in aplastic anemia. The influence of androgens explains the higher Hgb in adult males than
females • Laboratory Findings:
o Hemoglobinemia, hemoglobinuria
ANEMIA IN LIVER DISEASE o Hemosiderinuria
• Red cells may be normocytic to slightly macrocytic o Methemoglobinemia
• Hypoplastic anemia secondary to viral-induced marrow suppression o Increased unconjugated bilitubin
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o Decreased hemopexin and haptoglobin • Mutation in the gene for band # (SLC4A1) that results in increased rigidity of the membrane
o Increased LDH • Large oval RBCs with one to two transverse bars or ridges
o Decreased HbA1c
o Increased urobilinogen in urine and stool HEREDITARY PYROPOIKILOCYTOSIS
o Decreased RBCs but increase reticulocytes – polychromasia • Rare hemolytic disorder characterized by extreme microcytosis (MCV is 25-55 fL)
o Spherocytes, elliptocytes, budding, RBC fragments, and other bizarre shaped cells
o Increased nucleated red cells
• Severe subtype of HE
o Erythroid hyperplasia (M:E Ration = 0.5-1)
• Impaired spectrin tetramer formation and spectrin deficiency or presence of mutation spectrin
o Blood film shows poikilocytosis (schistocytes/spherocytes)
o disruption of membrane skeletal lattice and cell destabilization
• heat sensitive
HEREDITARY SPHEROCYTOSIS
o fragment at temperatures as low as 45°C to 46°C ( normal temp for fragmentation is above
• RBC structural proteins functions in vertical interaction with lipid bilayer 49°C)
• Mutation in genes: • OFT and autohemolysis test are increased
o ANK1 (ankyrin) o Negative DAT (direct antiglobulin test)
o SPTA1 (α-spectrin)
o SPTB (β-spectrin) HEREDITARY OTOMATOCYTOSIS SYNDROMES
o EPB42 (protein 4.2)
• Overhydrated Hereditary Stomatocytosis
• Deficiency in membrane proteins (ankyrin and spectrin) o Permeability and flux of sodium and potassium
o Problems with red cell deformability and permeability o Increased water content and swollen cells – mouth like central pallor
• Loss in RBC membrane o Decreased surface are-to-volume ratio
o Decrease surface-to-volume ration o Autohemolysis and OFT increased
o Resulting to spherocyte • Hereditary Xerocytosis
• Clinical Findings: o Greater efflux of potassium than influx of sodium
o Splenomegaly, gallstone formation, jaundice o Dehydrated hereditary stomatocytosis (DHS)
o Increased reticulocyte count; MCHC increased o Formation of target cells
o Increased OFT, positive autohemolysis test, negative direct antiglobulin test (anti-human o Mutation if PIEZO1 gene (ion channel component protein 1)
globulin test) o Elevated MCHC and decreased OFT

HEREDITARY ELLIPTOCYTOSIS ABNORMAL MEMBRANE LIPID COMPOSITION

• RBC structural proteins T=that mediate horizontal interaction in the RBC cytoskeleton • Spur Cell Anemia
• Deficiency of protein 4.1 o Severe hepatocellular disease such as cirrhosis
o Association of spectrin and actin (Spherocytic HE) o Serum lipoproteins increase – excess of erythrocyte membrane cholesterol
• Defect in spectrin-dimer-dimer interaction and ankyrin-protein 3 interaction – Atypical HE o Decreased membrane fluidity and cell deformability
• OFT is normal to slightly increased o Membrane fragments are lost; acquires irregular spikelike projections typical of spur cells
• Classification of HE: (acanthocytes)
o Common HE (elliptocytes) – impaired spectrin or protein 4.1 o Laboratory Findings:
o Spherocytic HE – spherocytes and fat elliptocytes ▪ Normocytic, normochromic anemia
o Stomatocytic HE – roundish elliptocytes that are stomatocytic ▪ Increased reticulocytes
o Atypical HE ▪ 20-8-% of RBCs are acanthocytes, spherocytes
▪ Presence of echinocytes
HEREDITARY OVALOCYTOSIS • Abetalipoproteinemia (Hereditary Acanthocytosis)
• Southeast Asian ovalocytosis or Melanesian elliptocytosis
 18

o Absence of serum β-lipoprotein, low serum cholesterol, low TAG, low phospholipid, increased o Poikilocytosis w/ variable NRBCs
in the ratio of cholesterol to phospholipid o Echinocytes
o Defective synthesis of apoprotein B, a TAG transfer protein or mutations in Band 3 protein o No Heinz bodies
o Transport of fat-soluble vitamins is impaired
o prothrombin time increased due to reduced vit. K stores HEMOGLOBINOPATHY
• Lecithin-cholesterol acyl transferase (LCAT) deficiency • Disease that results from an inherited qualitative abnormality of the structure or synthesis of the globin
o Impaired formation of cholesterol esters from cholesterol portion of the Hb molecule
o Low LDL and HDL
o Elevated levels of VLDL and lipoprotein X
• McLeod Syndrome (MLS)
o X-linked disorder caused by mutation in the KX gene
▪ Membrane precursor of the Kell blood group antigens
▪ Lack KX on RBC - reduced RBC deformability and shortened RBC survival

G6PD DEFICIENCY
• G6PD needed for NADPH and reduced glutathione; protects enzyme hemoglobin against oxidarion by
reducing hydrogen peroxide and free radicals
• Cannot generate sufficient amount of glutathione
• Hb oxidized to metHb
• Globin chain denatures forming Heinz bodies
• Mediterraneans are occasionally associated with severe potentially fatal hemolytic episodes ff. ingestion
of fava beans (favism)
o Contain oxidants like vicine and cinvicine; oxidize glutathione
• Protective against P. falciparum
• Laboratory Findings:
o Decreased Hb w/ normocytic, normochromic RBCs
o Polychromasia, poikilocytosis, and spherocytosis
o Intravascular hemolysis
o Heinz bodies
o Increased autohemolysis test
o Bites cells or helmet cells

PYRUVATE KINASE DEFICIENCY Hemoglobins with tetramers of normal chains:

• Enzyme in the Embden-Meyerhoff pathway – breaks down glucose into lactate accompanied by energy • Hb Bart’s – consists of four gamma chains (γ4) and occurs in newborns or infants
production in the form of ATP • Hb H – consists of four normal beta chains and occurs in older children
• Inability of the cell to maintain normal ATP levels • Laboratory tests for diagnosis of hemoglobinopathies:
o Alteration of erythrocyte membrane causing potassium loss and dehydration o CBC with blood indices
o Decreased membrane deformability o Hb H test
o enhances sequestration in the splenic cord ▪ Supravital staining of the BM with methyl violet
• Laboratory Findings: ▪ Appear as irregularly shaped bodies to the nucleus of normoblasts – multiple
o Decreased Hb w/ normocytic, normochromic RBCs greenish-blue dots, like the pitted pattern on a golf ball
o Polychromasia o Heat denaturation test – unstable Hb precipitates
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o Isopropanol precipitation test – unstable Hb precipitate HEMOGLOBIN S

o Alkali denaturation test for Hb F • Associated with sickle shaped RBCs
o Acid elution slide test fro F cells • Point mutation in the 6th amino acid of the β-chain, in which glutamic acid is replaced by valine
▪ Kleihauer-Betke test (RBC with Hb F stained red cells containing Hb A as pale pink • Found in black population
ghosts) • Mos common variant
o Sickling tests • Polymerization of deoxygenated Hb S
o Ferritin assay o RBC appear:
o Hb electrophoresis – Hb separated based on the interactions among Hb variants, agar, and ▪ Crescent-shaped
buffer ions in addition to te electrical charge of the various hemoglobins at an acid or alkaline ▪ Boat-shaped
pH ▪ Filament-shaped
o Isoelectric focusing ▪ Holly-leaf form
▪ Envelope cells
• Associated w/ resistance, but not immunity to P. falciparum
o Heterozygous inheritance of sickle cell gene (AS) – sickle cell trait
o Homozygous inheritance (SS) – sickle cell anemia

Sickle Cell Trait

• Heterozygous form of sickle cell disease
• Hb A is present in higher percentage
• No symptoms; experience painful crises
• Normal lifespan and treatment are rarely required
• Laboratory Findings:
o Anemia is not present
o Morphology is normal w/ few target cell and occasionally sickle cells
o Positive solubility test

Sickle Cell Disease

• Majority is Hb S
• Clinical symptoms:
o Chronic hemolytic anemia
o Autosplenectomy
o Vasoocculation, vasoocclusive crises
o Susceptibility to bacterial infections
o Acute chest syndrome
o Pulmonary hypertension, myocardial infarction

Sickle Cell Crises

• Painful SCC – occur in persons with SCA due to deoxygenation of RBC; low-grade fever
• Vasoocclusive crises – occur when rigid sickle cell increased blood viscosity
o Cause microthrombi, vascular occlusion
o Microinfarts can cause organ failure
• Infectious crises – depression of immune function
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o S. pneumoniae – major infectious agent among children ▪ TTO, HUS, HELLP, DIC
o Primary cause of death in SCA o Macroangiopathic hemolytic anemia
• Bone joint and other crises – occur ff. accumulation and occlusion of sickle cells in the bone and joints ▪ Traumatic cardiac hemolysis, March hemoglobinuria
• Splenic sequestration crises – sickle cell become trapped in the splenic microcirculation o Infectious agents
o May cause shock due to hypovolemia and death ▪ Malaria, babesiosis, bartonellosis, clostridial sepsis)
• Aplastic crises - occur ff. infection and fever leads to temporary reduction in normoblasts circulating o Other Injury
RBCs, Hgb and Hct values ▪ Chemicals, drugs, venoms, extensive burns

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

• Rare acquired defect of insidious onset which arises in the BM stem cells
• Show abnormal sensitivity to complement in acidified plasma or low ionic strength environment
o Complement-mediated cell lysis
• Absence of a membrane glycolipid (GPI pr glycosyl-phosphatidyl inositol) - defect in the PIG-A gene
o GPI serves as anchor of complement-regulating proteins;
o MIRL (membrane inhibitor of reactive lysis or CD 59)
o DAF (decay accelerating factor or CD 55)
o C8 binding protein (C8bp)
• Proteins cannot bind to RBC, complement action is not regulated resulting to uncontrolled lysis of RBCs
• Laboratory Findings:
o Severe anemia
o Normoblasts and NRBCs are found in the peripheral blood
o Thrombocytopenia
o Leukopenia w/ relative lymphocytosis
o Decreased LAP activity
o Positive sucrose hemolysis test
o Positive Ham’s test

WARM -ANTIBODY AUTOIMMUNE HEMOLYTIC ANEMIA

Lesson 6 – Hemolytic Anemias (Part II) • Occurs when the patient’s own immune system produces anti-red cell antibodies that react most
effectively at warm temperature (37°C)
• Antibody is usually IgG
EXTRINSIC ABNORMALITY (HEMOLYSIS)
• DAT is positive and the RPI is greater than 3
• Immune Cause:
o Paroxysmal Nocturnal Hemoglobinuria (PNH)
COLD -ANTIBODY AUTOIMMUNE HEMOLYTIC ANEMIA/ COLD HEMAGGLUTININ DISEAS
o Autoimmune hemolytic anemia (AIHA)
• Caused by antibody that react most effectively at cold temperature (0-4°C)
▪ Warm AIHA (WAIHA)
• Antibody is IgM (usually anti-I)
▪ Cold Hemagglutinin Disease (CHAD)
• Can develop secondary to Mycoplasma pneumoniae or Epstein-Barr virus infections
▪ Paroxysmal Cold Hemoglobinuria (PCH)
o Alloimmune hemolytic anemia
▪ Hemolytic transfusion reaction (HTR) COLD PAROXYSMAL HEMOGLOBINURIA
▪ Hemolytic disease of the newborn (HDN) • Rare autoimmune disease similar to cold-antibody AIHA
• Non-immune red cell injury • Caused by binding of the Donath-Landsteiner (DL) autoantibody to the patient’s red cells ff. cold exposure
o Microangiopathic hemolytic anemia o D-L autoantibody is IgG (anti-P)
 21

o Binds to red cells at 0-4°C and hemolysis occurs on warming due to complement activation
• DAT is positive and the Donath-Landsteiner test is positive

HEMOLYTIC TRANSFUSION REACTION

• Due to interaction of foreign (non-self) erythrocyte antigens and plasma Ab
• Ab produced in HTRs cause immunologic destruction of donor cells and not with the RBCs of the person
making the antibody
o Acute hemolytic transfusion reaction
▪ Infused RBCs react with Ab that already exist in the blood
▪ Result of clerical or human error
o Delayed hemolytic transfusion reaction
▪ Anamnestic response whereby the donor erythrocytes contain an antigen to which
the patient has been previously sensitized

HEMOLYTIC DISEASE OF THE NEWBORN

• HDN or erythroblastosis fetalis
• Destruction of the infant’s red cells when maternal antibodies (usually IgG) specific to an antigen on
the infant’s red cells cross the placenta
• Kleihauher-Betke stain allows simple detection of fetal red cells in the maternal circulation
o Test takes advantage of the relative resistance of fetal hemoglobin to alkaline denaturation
o Maternal cells appear as washed-out ghosts while fetal cells stand out ff. staining DRUG-INDUCED RED CELL DESTRUCTION
• Laboratory Findings: • Hapten or drug absorption mechanism
o Reticulocytosis o Hapten drugs: penicillin, streptomycin, and cephalothin attach to serum proteins
o Increased NRBCs ▪ Drug-protein complex adsorbs to red cell surface and induces production of
o Increased indirect bilirubin antibody specific for the drug
o Positive DAT ▪ Antibody is usually IgG
▪ IgG-coated cells are then subject to extravascular destruction in the spleen
▪ DAT is strongly positive due to IgG sensitization, while the eluate is often negative
• Immune complex or bystander mechanism
o Drugs: quinine, stibophen and phenacetin
 22

▪Complex reacts with antibody against the drug while it is still in the plasma and • Intraerythrocytic protozoa (malaria) rupture RBC
attaches to the red cell membrane o Malaria anemia is the major cause of morbidity and mortality of millions of individuals living in
▪ Antibodies either IgM or IgG endemic areas who are infected by the malarial spp., P. falciparum
▪ Have the ability to bind complement which results in intravascular hemolysis • C. perfringens – released hemolytic toxins
▪ DAT is positive, while the eluate is often negative • Extravascular hemolysis can be caused by Bartonella
• RBC autoantibody induction
o Drugs: levodopa, aldomet, and mefenamic acid Burns
▪ Induced the production of autoantibodies specific for the Th antigens on the • 3rd degree burns induce changes on the blood film
red cell surface o Direct action of heat at 49°C denatures spectrin;
▪ Antibody is usually IgG and is produced only after several months of continuous ▪ Budding, fragmentation, microcytes and microspherocytes
therapy o Presence of microspherocytes and microcytes falsely elevates the platelets number
▪ DAT is strongly positive and the eluate is also positive ▪ Need for manual count
• Membrane modification
o Cephalosporins – capable of modifying the erythrocyte membrane Hemolytic transfusion reaction
▪ Normal plasma proteins including immunoglobulins and complement bind to the • Due to interaction of foreign (non-self) erythrocyte antigens and plasma Ab
membrane in a nonimmunologic manner • Ab produced in HTRs cause immunologic destruction of donor cells and not with the RBCs of the person
• Unifying Theory making the antibody
o Drugs bund to erythrocyte membrane
▪ Antibodies produced react with epitopes specifically to the drug, combined drug MICROANGIOPATHIC HEMOLYTIC ANEMIA
and erythrocyte membrane epitopes, or epitopes on the erythrocyte membrane • Small vessel diseas
• From physical damage to red cells as they pass through very small orifices or damaged and
MACROANGIOPATHIC HEMOLYTIC ANEMIA sclerosed vessels
• Chronic intravascular hemolysis • Characterized by RBC fragmentation (Schistocytes) and thrombocytopenia
o Low serum haptoglobin o Occurs intravascularly by the mechanical shearing of RBC membrane
o Hemosiderinuria, reiticulocytosis ▪ Cells rapidly pass through turbulent areas of small blood vessels that are partially
o Red cell abnormalities (schistocytes, irregularly contracted cells) blocked by microthrombi or damaged endothelium
• Mechanical damage of red cells in the turbulent environment of a leaky valve
o After surgical replacement of a diseased heart valve with prosthesis Hemolytic Uremic Syndrome (HUS)
o After surgical repair of a septal defect with a plastic patch • Associated with thrombocytopenia and renal involvement
o March hemoglobinuria o Results of clot forming in the microvasculature of the kidney
▪ Seen in soldiers after a long march and in joggers or athletes ▪ Found I young children with a history of EHEC or Shigella dysenteriae
▪ Chronic intravascular hemolysis may lead to iron deficiency as a result of loss Hb
in the urine

Drugs and Chemicals

• Lead from gasoline, paint, or other industrial products
o Interferes with ATP production and inhibits heme synthesis
• Quinine sensitivity – new, unusual
o In one case in tonic water, was implicated by the finding of quinine-dependent antibodies
reactive with platelets, granulocytes, and erythrocytes in patient serum

Infectious agent
Thrombotic Thrombocytopenia Purpura (TTP)
 23

• Microthrombi composed of platelets

• Unusually large forms of von Willebrand factor that occlude capillaries
• Deficiency on the VWF-cleaving protease;
o Disintegrins and metalloproteinase with a thrombospondin type1 motif, member 13
(ADAMTS-13)

Disseminated Intravascular Coagulation (DIC)

• Initiated by damage to the endothelial lining of vessels
o Damage causes release of thromboplastic substances that activate the coagulation mechanism
in vivo
o Activation of all parts of the hemostatic systems leading to the production of fibrin clots;
▪ Consumption of platelets and coagulation proteins, and degradation fibrin
▪ Clotting and bleeding occur

HELLP Syndrome
• Obstetric complications characterized by hemolysis, elevated liver enzymes, and low platelet count
• Findings similar to microangiopathic conditions
• Pre-eclampsia
o Abnormalities in the development of placental vasculature;
▪ Poor perfusion and hypoxia
o Anti-angiogenic proteins
▪ Released from placenta
▪ Block action of placental growth factors including endothelial growth
o Leads to platelet activation and fibrin deposition in the microvasculature - liver