Hematology 1-complete

Hematology Comprehensive Reviewer
K. Molbog & P. Villangca
3J-MT

HEMA 1A: Clinical Hematology I
Chapter Four: Hematopoiesis
 Process of:
1. Blood cell production
2. Blood cell differentiation
3. Blood cell development
 Bone marrow, liver, spleen, lymph nodes, thymus
ORIGIN OF BLOOD CELLS
 Hematopoietic Stem Cells (HSCs)
Types of Human Stem Cells
A. Totipotential Stem Cells
 Present in first few hours after ovum fertilization
 Most versatile = develop into any human cell type
 Development of embryo into fetus
B. Pluripotential Stem Cells
 Several days after fertilization
 Develop into any cell type
 Cannot develop into a fetus
C. Multipotential Stem Cells
 Derived from pluripotent stem cells
 Found in adults
 Limited to specific cell types
 Bone marrow stem cells = RBCs, WBCs, bone cartilage,
adipocytes
 Stem cells maintain cell populations in tissues with high cell turnover
 HSCs = self-renewal + multipotential differentiation
Early Development of Blood Cells
 Mesoderm = middle embryonic germ layer
 Gives rise to mesenchymal tissue, which develop into embryonic
blood cells (except lymphocytes)
 Intraembryonic region = mesenchymally derived; produce HSCs or
progenitor cells before appearing in the yolk sac
 Paraaortic splanchnopleure = earlier stage; produces
multipotent progenitor cells
 Aorta-gonad-mesonephros region = later; produces potent HSCs
 Mammalian embryo = at least 2 spatially separated sources of
hematopoietic cells
 Anatomical sites of blood cell dev’t::
1. Yolk sac
 After gastrulation and mesoderm formation
 Where first HSCs are produced
 Erythroblasts = primitive RBCs; first 2-8 weeks
2. Liver and spleen (hepatic)
 Mesenchymal stem/progenitor cells and HSCs circulate
in peripheral blood (1st trimester) to the secondary
ontogenic sites of hematopoiesis (liver + bone marrow)
 Gradually becomes site of blood cell development
 2nd month of gestation = major hematopoietic site; initial
appearance of granulocytes
 2-5 months
3. Bone marrow (myeloid)
 Hematopoiesis begins in 4th month of gestation
 Primary site of hematopoiesis after 5th month
 Medullary hematopoiesis
BONE MARROW SITES AND FUNCTION
 In cavities of all bones
 Yellow marrow = inactive; adipose tissue
 Red marrow = active production of blood cells
 One of the largest organs (3.5%-6% of body weight; 1500 g in adults
 Composition:
 Hematopoietic cells (erythroid, myeloid, lymphoid, and
megakaryocyte)
 Adipose tissue
 Osteoblasts and osteoclasts
 Stroma
 Hematopoietic cells
 Colonies = compartmentalized in the cords
 When matured in the hematopoietic cords = cells cross walls of
the sinuses, specialized vascular spaces, and enter the
circulating blood
 First few years of life = all bone marrow is red and cellular
 Both appendicular and axial skeleton in young persons
 Confined to axial skeleton and proximal ends of long bones in
adults
 Age 18 = red marrow found only in the vertebrae, ribs, sternum
skull bones, pelvis, and proximal epiphyses of femur and humerus
 Extramedullary hematopoiesis
 Spleen, liver, lymph nodes = produce immature blood cells
 Splenomegaly + hepatomegaly
 Undifferentiated primitive blood cells are present in spleen and
liver (can proliferate)
 Dysfunctional bone marrow (aplastic anemia, infiltration by
malignant cells, overproliferation of a cell line [leukemia])
 Hemolytic anemias (bone marrow insufficient)
CELLULAR ELEMENTS OF BONE MARROW
Progenitor Blood Cells
 Multipotential hematopoietic stem cell
 Progenitor of all blood cells
 Derived from pluripotent stem cell
 Generate mature blood cells over an organism’s lifetime
 Progenitor of 2 major cell lines: lymphocytic and nonlymphocytic
cells
 Stem cells = capable of self-renewal, proliferation, and differentiation into
progenitor cells
 Stem cell plasticity = ability of stem cells to differentiate into an assortment
of unrelated cells
 Multipotent adult progenitor cells (MAPCs)
 Master cells (in bone marrow; rebuild any type of tissue)
 Produce telomerase (keeps cells from aging)

 Become muscle, cartilage, bone, liver, or different types of brain
cells
 Three phases of hematopoietic cells (accdng. to maturity)
a. Primitive, multipotential cells = most immature; self-renew and
differentiate into all blood cell lines
b. Intermediate cells = committed progenitor cells; develop distinct
cell lines
c. Mature cells = most developed; specific functions
 Lymphoid stem cell
 Precursor of mature T cells or B cells
 Nonlymphocytic (myeloid) stem cell
 Progresses into the progenitor CFU-GEMM (granulo, erythro,
mono, megakaryo)
 CFU-GEMM = lead to formation of CFU-GM (granulo, mono), CFU-
Eo, CFU-B, CFU-Meg
 CFU-GEMM to BFU-E (burst-forming unit-erythroid) through
erythropoiesis
 Formation and development of BCs from bone marrow multipotential stem
cell = controlled by growth factors and inhibitors; microenvironment
influences behavior and controls proliferation
 Bone = most appropriate for proliferation and maturation of cells
 HPCs
 Mobilized from bone marrow to blood by hematopoietic growth
factors and chemokines
 Cytokines = may be lineage specific or can regulate multiple
lineages
 Stromal cells + extracellular matrix (fibronectin, collagens,
proteoglycans)
 Umbilical cord blood (UCB) = also contain HPCsa
Erythropoiesis
 Occur in erythropoietic islands
 Niches where erythroid precursors proliferate, differentiate, and
enucleate
 Each island = macrophage surrounded by a cluster of
erythroblasts
 Cell-cell, cell-extracellular matrix adhesion, + and – regulatory
feedback, and central macrophage function
 Erythroid cells = 5%-38% of nucleated cells in normal bone
Granulopoiesis
 Myeloid cells = 23%-85% of nucleated cells in normal bone marrow
 Maturational
 Early cells = located in the cords and around bone trabeculae
 Neutrophils = reside in proliferating pool and the maturational storage pool
 Maturing cells = 3-6 days in the proliferating pool
 Cells can exit storage pool and enter circulation (lifespan = 6-10 hrs.)
Lymphopoiesis
 Lymphoid follicles = where lymphocytes and plasma cells are produced
 Lymphocytes = randomly dispersed in cords
 Plasma cells = in vascular walls
 1%-5% of nucleated cells in bone marrow
Megakaryopoiesis
 Occurs adjacent to the sinus endothelium
 Megakaryocytes = protrude through vascular wall as small cytoplasmic
processes to deliver platelets into the sinusoidal blood
 Develop into platelets in ~ 5 days
Marrow Stromal Cells
 Reticulum cells, histiocytes, adipocytes, and endothelial cells
 Where hematopoietic cells are suspended (semifluid)
 Produce matrix of collagen and proteins (glycoproteins and
proteoglycans)
 Bone marrow cell renewal and differentiation
Mast Cells
 Mesenchymal origin
 Blue-purple granules that obscure the round or oval reticular nucleus =
contain heparin, histamine, serotonin, proteolytic enzymes
 Increased mast cell number = chronic lymphoproliferative disorders or
chronic infections
Macrophages
 Reticulum cells of histiocytes
 Large cells
 Siderophages = macrophages containing iron-rich hemosiderin and
ferritin)
 Gaucher cells = macrophages with uncatabolized glugocerebrosides
Bone Cells
 Osteoblasts = matrix-secreting; resemble plasma cells; increased in
metabolic disease
 Osteoclasts = resemble megakaryocytes; bone-remodeling
INTERLEUKINS
 Proteins that work with hematopoietic growth factors to stimulate
proliferation and differentiation of specific cell lines
 Cytokines; work independently or with other ILs = encourage
hematopoietic growth
 Cell signaling
 Immune cross-talk and communication (messengers of the immune
system)
 35 known
 Inflammatory stimuli and cytokines = limited role in hematopoiesis but
major role in host responses to infection or antigenic challenge
 Pause cell proliferation, cell activation, inflammation, physiology
changes (fever and pain), and allergies (histamine release), and
growth
HEMATOPOIETIC GROWTH FACTORS
 Each is encoded by 1 gene
 Regulate proliferation and differentiation of HPCs + regulate survival and
function of mature BCs
 Bind to receptors on target cells
 Mobilize HPCs from bone marrow to peripheral blood circulation
 Happens after M phase of cell cycle

 Adhesive interactions bet. HPCs and bone marrow extracellular
matrix
 HGFs, chemotherapy, chemokines
A. Erythropoietin
 Located on chromosome 7
 Source = renal peritubular cells; Kupffer cells
 Progenitor cell target = CFU-E, late BFU-E, CFU-Meg
 Mature cell target = none
B. Interleukin-3 (IL-3)
 Long arm of chromosome 5
 Source = activated T lymphocytes
 Progenitor cell target = CFU-blast, CFU-GEMM, CFU-GM, CFU-G,
CFU-M, CFU-Eo, CFU-Meg, CFU-Baso, BFU-E
 Mature cell targets = eosinophils, monocytes
C. G-CSF (Granulocyte colony-stimulating factor)
 Chromosome 17
 Sources = monocytes, fibroblasts, endothelial cells
 Progenitor cell target = CFU-G
 Mature cell target = granulocytes
D. M-CSF (Monocyte colony-stimulating factor)
 Sources = monocytes, fibroblasts, endothelial cells
 Progenitor cell target = CFU-M
 Mature cell target = monocytes
E. GM-CSF (Granulocyte-Macrophage colony-stimulating factor)
 Sources = T lymphocytes, monocytes, eosinophils, monocytes,
fibroblasts, endothelial cells
 Progenitor cell target = CFU-blast, CFU-GEMM, CFU-GM, CFU-G,
CFU-M, CFU-Eo, CFU-Meg, BFU-E
 Mature cell target = granulocytes
EXAMINATION OF MATURING BLOOD CELLS
 CBC (complete blood cell count) = examination of a peripheral blood
smear is important
 Identify normal cells and recognize immature cells that may indicate
disorders
GENERAL CELLULAR CHARACTERISTICS
Overall Cell Size
 Relative to a mature RBC
 RBCs and WBCs = decrease in size as they get more mature
Nuclear-Cytoplasmic Ratio
 Amount of space occupied by the nucleus in relationship to the space
occupied by the cytoplasm
 Nucleus size decreases as cell matures
 Blast forms = high (4:1) N:C ratio
 As cells mature, ratio is reduced to 2:1 or 1:1 (except in thrombocytes,
mature RBCs, and lymphocytes)
 Thrombocytes and erythrocytes = anuclear
 Mature lymphocytes = retain 4:1 to 3:1 N:C ratio
NUCLEAR CHARACTERISTICS
Chromatin Patterns
 Loose-looking to a more clumped pattern as cell matures
 Lymphocytes = smooth/homogeneous chromatin pattern throughout
development until maturation, where heterochromatin is obvious
 Granulocytes = fine to highly clumped pattern
 Monocytes = lacy; becomes finer as cell matures
 Erythrocytes = clumped pattern as maturation progresses, until extremely
dense (pyknotic) nucleus is lost (extruded) from the mature cell)
Nuclear Shape
 Lymphocytes = round/oval; some have small clefts
 Monocytes = kidney-bean; horseshoe shapes
 Neutrophils, eosinophils, basophils = segmented nuclei attached by
filaments; 2-5 lobes
Presence of Nucleoli
 Erythrocytes, leukocytes, megakaryocytes = nucleoli in earliest cell stages
 As cell matures = nucleoli become less visible
 Lymphoblasts = 1-2 nucleoli
 Myeloblasts = 1-5 nucleoli
 Monoblasts = 1-2 nucleoli but sometimes 3-4
 Erythroblasts = 0-2 nucleoli (darker staining)
 Megakaryoblasts = 1-5 nucleoli
CYTOPLASMIC CHARACTERISTICS
Staining Color and Intensity
 Wright-stained
 Darker blue (active protein synthesis) in young cells to lighter blue or pink
in mature cells
 Early cells = medium-blue cytoplasm
 Immature erythrocytes = dark-blue cytoplasm; becomes paler and gray
as hemoglobin is made
 Mature lymphocytes = pale sky-blue cytoplasm
Granulation
 No granules to nonspecific granulation to specific granulation
 Earliest blast forms of leukocytes and megakaryocytes = no granules
 RBCs = never exhibit granules
 Fine to coarse (size)
 Red (azurophilic), blue (basophilic), and orange (eosinophilic)
 Amount of granulation
Cytoplasmic Shape
 Variations in blast forms, monocytes, and megakaryocytes
 Pseudopods = in mature monocytes and in some leukocyte blast forms
 Megakaryocytes (outline becomes more irregular as cell matures)
Quantity of Cytoplasm
 Quantity increases with age
 Megakaryocyte = develops extensive quantities of cytoplasm
 Leukocyte abnormalities = xssive amount of cytoplasm
Vacuolization
 Monocytes = vacuoles throughout life cycle

 Common in older cells in abnormal conditions (except monocytes)
 Artifacts = vacuoles induced by anticoagulants (if blood is stored too long)
 Bacterial and viral infections, malignancies = xssive vacuolization
Inclusion Bodies
 Auer bodies or rods = in myelocytic or monocytic blast forms or ingested
particles
 Erythrocytic inclusions
 Lymphocytic inclusions
 Iron inclusions require special staining, whereas others can be seen by
Wright stain
MATURE BLOOD CELLS IN PERIPHERAL BLOOD
 See Table 4.5

Chapter Five: Erythrocyte Maturation, Physiology, and Life Cycle
ERYTHROPOIESIS
 Mature erythrocyte
 Biconcave disc
 1/3 central pallor
 Hemoglobin performs O2-CO2 transport
 120-day lifespan
 Pliable; moves through capillaries
 Cytoplasmic enzymes are catabolized as the cell ages =
membrane rigidity + phagocytosis + destruction
 Erythrocyte production
 HSC to erythrocyte
 Shed organelles; produce more HgB
 Regulated by ILs + transcription factors
 Molecular chaperones = influence normal cell function
 Hemoglobin (HgB)
 Heme pigment = O2-CO2 transport
 Substances needed for normal erythrocyre and HgB production:
 Amino acids
 Iron (> 20 mg for 200 billion RBCs produced every day; recycle
senescent RBCs; ingest 1-2 mg)
 Vitamin B12
 Vitamin B6
 Folic acid (vitamin B2 complex)
 Co and Ni
 Deficiencies in these substances = abnormal erythropoiesis
 “blast” cells = immature; not supposed to be in peripheral blood
 Nucleoli are found in more immature cells
 Fine to coarse chromatin as cell matures (bluish to reddish)
 Overall cell size becomes smaller as cell matures
ERYTHROPOIETIN (EPO)
 Produced by the kidneys (peritubular cells)
 Also produced in the liver (primary source in the unborn)
 Glycoprotein hormone; stimulates erythropoiesis
 Hematopoietic growth factor
 Chromosome 7
 Low tissue oxygenation (hypoxia) = kidneys produce more EPO
 Number of RBCs increases = O2-carrying capacity increases =
normal O2 level is restored
 Predominant effect on CFU-E (committed erythroid cells) = promote
proliferation and differentiation into erythroblasts
 Prevents erythroid cell apoptosis
 Also promotes production of megakaryocytes
 Produces an increase in RNA followed by an increase in DNA activity and
protein synthesis
GENERAL CHARACTERISTICS OF MATURATION AND DEVELOPMENT
 Once stem cell differentiates into erythroid cell line = cell matures through
the nucleated cell stages in 4-5 days
 Bone marrow reticulocytes mature in 2.5 days
 Young reticulocytes enter circulating blood = remain in the reticulocyte
stage for 1 day
 0.5%-1.5% of circulating erythrocytes
DEVELOPMENTAL STAGES (4-5 days)
Early Cells
 Pluripotent progenitor HSC
 Common myeloid progenitor (non-lymphoid multipotential stem cell)
 CFU-GEMM (colony-forming unit granulocyte-erythrocyte-
monocyte-megakaryocyte)
 CFU-GEMM differentiates into BFU-E (burst-forming unit-erythroid)
 BFU-E = earliest cell in the erythrocyte series
 BFU-E differentiates into CFU-E
 Actively proliferating
 S phase
 Influenced by EPO to undergo cell divisions and cell maturations
to form the mature erythrocyte
Pronormoblast (Rubriblast) or Proerythroblast
 12-19 µm (larger cells = more immature)
 N:C ratio = 4:1 (high N:C ratios indicate more immature cells)
 Large, round (slightly oval) nucleus: dark-appearing
 Nucleoli: 0-2 (more nucleoli = less mature)
 Chromatin: fine pattern
 Cytoplasm: dark distinctive blue (basophilic) color with Wright stain
 No granules
 Blue color = increased RNA activity (hemoglobin synthesis);
immature; basophilic
 Prominent Golgi
 Iron intake
Basophilic Normoblast (Prorubricyte)
 12-17 µm (slightly smaller than Pronormoblast)
 N:C ratio = 4:1
 Nucleus: round to slightly oval
 Chromatin: more clumped (slightly condensed)
 Nucleoli: 0-1 (usually not apparent)
 Cytoplasm: dark blue (super basophilic) with a Wright stain
 No evidence of pink color
Polychromatic Normoblast (Rubricyte)
 Hemoglobin appears for the first time
 11-15 µm (slightly decreased from prorubricyte stage)
 N:C ratio = 1:1
 Chromatin = more clumped (quite condensed)
 Nucleoli: 0 (none)
 Cytoplasm: gray-blue or muddy/light gray(pink staining + basophilia);
evidence of hemoglobinization
Orthochromic Normoblast (Metarubricyte)
 8-12 µm
 Nucleated RBC (NRBC)

 Chromatin: tightly condensed; pyknotic (dense or compact)
 Nucleus: round (later extruded from the cell)
 Nucleoli: 0
 Cytoplasm: more pink or salmon than blue (acidophilic reddish pink); large
quantities of hemoglobin
 3 mitoses from rubriblast to metarubricyte (2/3 occurs in rubricyte stage)
 Cell can no longer undergo mitosis after this stage
Reticulocyte (Polychromatic Erythrocyte)
 Reticulocyte = supravital stain is used
 NMB (new methylene blue) or BCB (brilliant cresyl blue)
 Precipitated ribosome material = deep-blue, mesh-like network
 Polychromatic erythrocyte = Wright stain is used
 Blue appearance = polychromatophilia
 Slightly more blue or purple than mature erythrocyte
 Part of this stage occurs in bone marrow, and the later part occurs in
circulating blood
 Reticular appearance = remaining RNA (supravital stain)
 7-10 µm
 Nucleus: 0 (anuclear)
 Diffuse reticulum
 Lipoxygenase = organelle degradation
 Bcl-x = anti-apoptotic
 Mature in 2.5 days
Mature Erythrocyte
 6-8 µm
 No nucleus
 Cytoplasm: salmon with 1/3 central pallor
 Predominant cell type in peripheral blood
RETICULOCYTES
 Immature erythrocyte
 Formed after metarubricyte (orthochromic normoblast) loses its nucleus
 Remains in bone marrow for 2-3 days before entering circulation
 No nucleus
 With organelles (mitochondria + ribosomes)
 Loss of ribosomes and mitochondria + full hemoglobinization =
mature into erythrocyte
 Quantity in bone marrow = quantity in blood
 Stress or shift reticulocytes = prematurely released due to acute bleeding
Reticulocyte Count
 Wright stain = slight blue tint in some RBCs = polychromatophilia or
polychromasia
 Supravital stain = reticulocytes
 Indicates rate of RBC production
 Expressed as % of total erythrocytes
 Normal range (adults) = 0.5% - 2%
 Normal range (newborns) = 2.5%-6.0%
 Total RBC mass = new RBCs produced x 120
 Decreased RBC mass = decreased RBC production or a
shortened life span
 High reticulocyte count = shortened RBC survival
 Reticulocytosis = body tries to maintain homeostasis; release more
reticulocytes to compensate for short RBC lifespan
Corrected Reticulocyte Count
 Made by correcting the observed reticulocyte count to a normal packed
RBC volume
𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 = 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 𝑥
𝑃𝐶𝑉 (ℎ𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡)
𝑛𝑜𝑟𝑚𝑎𝑙 ℎ𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 𝑏𝑎𝑠𝑒𝑑 𝑜𝑛 𝑎𝑔𝑒 𝑎𝑛𝑑 𝑔𝑒𝑛𝑑𝑒𝑟
 Normal = 0.5%-2.0%
Reticulocyte Production Index
 Percentage calculation = does not account for the fact that prematurely
released reticulocytes require from 0.5-1.5 days longer in the circulating
blood to mature and lose their net-like reticulum
 RPI = measures erythropoietic activity when stress reticulocytes are present
𝑅𝑃𝐼 =
𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡 𝑖𝑛 %
𝑚𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒 𝑖𝑛 𝑑𝑎𝑦𝑠
 Normal = 1
 Hemolytic anemia = 3-7 times higher
 Bone marrow damage, erythropoietin suppression, deficiency of vitamin
B12, folic acid, or iron = 2 or less
DISORDERS RELATED TO ERYTHROCYTE MATURATION AND PRODUCTION
Disorders of Erythropoietin
 Polycythemia = increased concentration of erythrocytes (erythrocytosis) in
the circulating blood that is above normal for gender and age
 Secondary or absolute polycythemias = increase in EPO; not confused with
polycythemia vera or relative polycythemias
 Results from tissue hypoxia (renal neoplasms)
 Smoking
 May result from neoplasms (renal)
 Renal disorders that produce local hypoxia in the kidney
 Familial polycythemia
 Autosomal
 Defect in EPO regulation
Red Cell Increases
 Relative polycythemias
 Increase in RBCs not related to increased EPO
 RBC mass is not increased
 Decreased plasma volume = increased PCV or erythrocyte count
 Causes by loss of body fluids (diarrhea or burns)
Defective Nuclear Maturation
 Megaloblastic maturation
 Vitamin B12 or folate deficiency anemia
 Nuclear maturation lags behind cytoplasmic maturation
 Impaired DNA synthesis
 Asynchronous (interphase and mitosis are prolonged)
 Increased amount of erythrocytic cellular cytoplasm and
increased overall RBC size

CHARACTERISTICS AND BIOSYNTHESIS OF HEMOGLOBIN
Genetic Inheritance
 Adult hemoglobin A
 Defects = amino acid substitutions or diminished production of
polypeptide chains
 Hemoglobinopathies
Chemical Composition and Configuration of Hb
 4 heme groups + 4 polypeptide chains
 574 amino acids
 141 for each of the two α-chains
 146 for each of the two β-chains
 Each chain = attached heme group; 8 helices
 Egg-shaped with central cavity
 Binding of oxygen to 1 heme group induces oxygen binding to the other 3
 Hb denaturation = cannot carry oxygen
2,3-Diphosphoglycerate
 Binds to reduced hemoglobin (deoxyhemoglobin)
 Combines with β-chains = decrease affinity to oxygen
 β-chains are pulled apart when unloading oxygen, 2,3-DPG enters and
forms salt bridges between the chains = lower affinity to oxygen
 Bridges are broken when oxygen uptake happens in the lungs = increase
affinity
 Tissue hypoxia
 Increased deoxyHb
 2,3-DPG binds = reduced affinity = more O2 released
Oxygen Dissociation
 Oxygen content vs. partial pressure of oxygen
 P50 value when oxyHb = deoxyHb: 26.52 mmHg/torr
 Increased oxygen affinity
 Shift to the left
 Higher affinity; decreased release
 Decreased oxygen affinity
 Shift to right
 Lower affinity; increased release
 Tissue hypoxia
 Decrease in pH (Bohr effect)
 Increase in temperature
 Fetal hemoglobin = increased affinity
Carbon Dioxide Transport
 Indirect and direct (erythrocytes) and transport in solution in plasma
A. Indirect (most common; 3/4)
 CO2 enters cell
 Converted to carbonic acid via carbonic anhydrase
 Carbonic acid dissociates into hydrogen and bicarbonate
 Bicarbonate gets out of the cell and a chloride ion enters
(chloride shift); hydrogen ion is accepted by the alkaline
deoxyHb
 Bicarbonate is transported to the lungs in plasma
 In capillaries = bicarbonate is converted into CO2 and water
B. Direct (1/4)
 CO2 (- carbamate groups) binds with deoxyHb (+) = carbamino
hemoglobin
C. Plasma
 5%
 Carried in solution to the lungs
Hemoglobin Biosynthesis
 RBC maturation process
 Most Hb is synthesized before nucleus is extruded
 Heme + globin
Heme Formation from Porphyrin
 Red bone marrow and liver
 Begins in mitochondrion
 Succinyl-CoA + glycine = delta-5-aminolevulinic acid
 Catalyst = ALA synthetase
 Co-factors = vitamin B6 (pyridoxal phosphate; PLP); EPO
 Continues in cytoplasm
 Coproporphyrinogen III = enters mitochondrion and gets
converted into protoporphyrinogen III
 Protoporphyrinogen III is converted into protoporphyrin IX
 Iron is added into protoporphyrin IX (heme synthetase ferrochelatase) to
form heme
 Tetrapyrrole compound
 Rings connected by methene bridges
Role of Iron in Hb Synthesis
 Duodenum
 Iron is converted into ferrous state
 Taken by enterocytes with the help of divalent metal transporter
1
 Stored as ferritin
 Exported into the circulation by ferroportin 1
 Ferroportin
 Last step in Fe absorption
 Allows macrophages to recycle iron
 Unregulated = hemochromatosis (iron overload)
 Transferrin
 Binds to ferric ion
 Transports iron to membrane of immature cell; released back to
the plasma after transport
 Ferrireductase reduces iron
 Iron proceeds to the mitochondrion where it gets incorporated
into protoporphyrin to form heme
Hepcidin
 From liver; peptide hormone
 Regulate iron metabolism (absorption + mobilization from stores
 Interacts with plasma iron transporter and ferroportin to regulate iron use
and storage
 Influences iron available for erythropoiesis
 Dietary Fe absorption is influenced by:
 Hypoxia-inducible factor

 Iron-regulatory proteins in enterocytes
 Hepcidin
 Inhibits iron export by ferroportin
 Increased hepcidin = decrease in [plasma iron]
 Iron is retained in macrophages; lower intestinal absorption
 Deficiencies
 Iron overload syndromes (hemochromatosis)
 Overexpression
 Microcytic anemia
 Bone morphologenetic protein (BMP)
 Activates hepcidin transcription
 Hepcidin expression inhibition
 Hypoxia
 High erythropoietic activity
 Iron deficiency
Globin Structure and Synthesis
 Genetic control (aa sequence is inherited)
Alpha Globin Locus
 Chromosome 16
 2 alpha-globin genes (identical)
 Each cell = 2 chromosomes 16 = 4 alpha-globin genes
 Zeta genes = embryonic; substitute in early development
 α-globin protein
Beta Globin Locus
 epsilon, gamma, delta, and beta
 2 copies of gamma genes in each chromosome 11
 Others = single copies
 2 beta-globin genes = Hb A
DISORDERS RELATED TO HB BIOSYNTHESIS
Disorders of Heme (Porphyrin) Synthesis
 Inherited
 Autosomal (congenital erythropoietic porphyria)
 Acquired
 Pb poisoning (inhibits heme synthesis)
 Inhibits heme synthetase
 Porphyria
 Disease of heme metabolism
 Abnormality = xssive accumulation and excretion of porphyrins
by the biliary and/or renal route
 Neurologic or skin problems
o Acute neurologic
o Non-acute cutaneous
 Purple-red pigment (wine-red color of urine)
 Mild anemia
 Mitochondria = encrusted with iron
 Seen in nucleated RBC with Prussian blue stain
 Cells are called sideroblasts
Disorders of Iron Metabolism
Genetic Defect of Iron
 Transmembrane protein serine 6 (TMPRSS6) gene = enzyme that promotes
Fe absorption and recycling (inhibits hepcidin)
Iron Overload
 Too much iron in hereditary hemochromatosis, porphyria cutanea tarda,
iron-loading anemias (hemolytic dyserythropoietic, myelodysplatic,
aplastic)
 Primary overload
 Increased Fe absorption
 Secondary overload
 Chronic disorders
 Hemolytic anemias
 Sideroblastic anemia
Sideroblastic Anemia
 Mitochondrial iron loading
 Causes
a. Congenital defects (sex-linked [males]; autosomal)
b. Acquired defect = primary (myelodysplastic); may evolve into
acute myelogenous leukemia
c. Malignant marrow disorders (acute myelogenous leukemia,
polycythemia vera, myeloma, myelodysplastic syndromes)
d. Secondary to drugs (isoniazid, chloramphenicol); after
chemotherapy
e. Toxins (alcohol, chronic Pb poisoning)
 Microcytic + hypochromic
 Increased serum iron and serum ferritin (SF)
 Ringed sideroblasts on iron stain of bone marrow aspirate
 Pyridoxine = treatment (hereditary)
 Adequate iron; iron not incorporated in Hb synthesis
 Accumulates in the perinuclear mitochondria of metarubricytes
(non-ferritin iron)
 Decreased activity of ALA-synthetase
 Prussian blue stain = blue iron deposits that surround nucleus
 Laboratory characteristics
 Ringed sideroblasts (iron granules encircling nuclei of
metarubricytes)
 Increased erythropoietic activity (hypercellular marrow)
 Hypochromic + microcytic/normocytic RBCs
Hereditary Hemochromatosis
 Genetic error of metabolism = increased GI absorption of iron
 Xss + abnormal distribution
A. Type 1 (HFE-gene-related)
B. Type 2 (juvenile hemochromatosis)
 HJV
 Caused by hemojuvelin mutations (protein; modulates hepcidin;
deficiency in hepcidin)
C. Type 3
 Midlife
 Transferrin receptor 2
D. Type 4
 SLC40A1 gene

 Ferroportin disease
 Therapeutic phlebotomy or iron chelation therapy
 Remove 1 unit of blood (450 mL) once or twice weekly = most effective
 Genetic hemochromatosis/hemosiderosis
 Amino acid substitution
 Cys to Tyr
 His to Asp
 Acquired
 > 10 units of whole blood
 transfusions
Disorders of Globulin Synthesis
 Globulin synthesis = coordinated with porphyrin synthesis
 Impaired globulin synthesis = protoporphyrin synthesis is reduced
 Impaired porphyrin synthesis = xss globin is not produced
 Deficient globin production = iron accumulation in cytoplasm of cells as
ferritin aggregates
 Thalassemias
 α-thalassemia = defect in alpha-chain synthesis
 β-thalassemia = defect in beta-chain production
Ontogeny of Hb
 several Hb types
A. Embryonic hemoglobins
 Primitive; formed by immature RBCs in the yolk sac
 Gower I, Gower II, Portland types
 Found in human embryo
 Persist until 12 weeks of gestation
 Gower I = 2 zeta, 2 epsilon
 Gower II = 2 alpha, 2 epsilon
 Portland-1 = 2 zeta, 2 gamma
B. Fetal Hb
 Hb F
 Predominant in fetus and newborn
 2 alpha, 2 gamma chains (gamma = 146 aa)
 Gamma chain = either Ala or Gly in 136th position
 5th week of gestation
 Persists for several months after birth
 Hepatic erythropoiesis
C. Hb A
 Adult
 2 alpha, 2 beta
 Hemoglobin A2 = 2 alpha, 2 delta
D. Glycosylated Hb (Hb A1)
 A1a, A1b, A1c
 Formed during erythrocyte maturation
 Hb glycosylation in hyperglycemic persons
 Concentration reflects blood glucose
 Stable
 Addition of carbohydrate group to terminal valine of beta chain
 Diabetes
 3%-6% in normal persons
 6%-12% in both Type 1 and Type 2 diabetes
Variant Forms of Normal Hemoglobin
Carboxyhemoglobin
 Hb binds to CO (210 times greater affinity to CO than O2)
 carboxyHb displaces oxygen = tissue hypoxia
 CO poisoning = cherry-red skin color
Sulfhemoglobin
 Contains sulfur
 Greenish derivative
 Hb precipitates as Heinz bodies
 Cannot transport oxygen
 Can combine with CO to form carboxysulfHb
 Phenacetin and sulfonamides (Clostridium welchii bacteremia)
 Elevated concentrations = cyanosis
Methemoglobin
 Iron in ferric state
 Cannot combine with oxygen
 Metabolic defect OR abnormal Hb structure
 > 10% = cyanosis
 > 60% = hypoxia
Abnormal Hemoglobin Molecules
 Sickle cell anemia
 Due to mutant, codominant genes
 Sickle cell anemia
 Hemoglobin S
 6th position of beta globin chain = Glu residue
 In SCE = Glu is replaced by Val
 Hemoglobin C
 Glu residue at 6th position of beta globin chain = replaced by Lys
residue
ANALYSIS OF HEMOGLOBIN
 Electrophoresis
Alkaline Electrophoresis
 Cellulose acetate electrophoresis
 Hb A, F, S, and C are separated based on negative charges
 Fast hemoglobins = greater electrophoretic mobility than Hb A at pH 8.6;
Bart Hb, Hb H and Hb I (fastest)
 Slowest = Hb C
Citrate Agar Electrophoresis
 Acid pH
 Separate Hbs on the basis of a complex interaction between Hb, agar,
and citrate buffer ions
Denaturation Procedures
 Kleihauer-Betke = differentiate fetal blood (Hb F) from maternal blood (Hb
A) after delivery
 Hb F = resist acid denaturation; stain deep red
 Hb A = denatured by acid; pale pink ghosts
Chromatography
 Quantitation (cation exchange minicolumn chromatography)

 HPLC
MEMBRANE CHARACTERISTICS AND METABOLIC ACTIVITIES OF ERYTHROCYTES
METABOLISM
 Limited metabolic activity
 No organelles or nucleus
 No mitochondria
 Glucose breakdown = main energy source
Erythrocyte Glycolysis
A. Embden-Meyerhof glycolytic pathway
 Anaerobic
 Uses glucose to produce ATP
 Maintain pyridine nucleotides in a reduced state
B. Oxidative pathway/Hexose-Monophosphate shunt/Pentose phosphate
pathway
 Prevents denaturation of globin by oxidation
 Reduce NADP to NADPH
 NADPH is reduced to form glutathione
 Defects = oxidants are not neutralized
 Heinz bodies = globin denaturation due to oxidants
C. Methemoglobin reductase pathway
 Prevents oxidation of heme iron (methemoglobin reductase and
NADH)
 Methemoglobinemia = increased metHb concentration
D. Luebering-Rapaport pathway
 Regulates oxygen affinity of Hb
 2,3-DPG synthesis
 Acid-base balance
 Acidosis = reduced RBC glycolysis; available O2 is increased, 2.3-
DPG falls to normalize tension
 Acidosis = reverse
MEMBRANE CHARACTERISTICS
Reticulocyte Membrane
 Change in protein content and membrane organization
 Become discoid
 Tubulin + actin = differentiation
 Endocytosis and excocytosis
 With organelles
 Changes:
 Increase in shear resistance
 Loss of surface area due to loss of membrane lipid
 Acquisition of biconcave shape
Mature RBC Membrane
 Soft and pliable
 Biconcave shape = increase surface area
 Lipid bilayer + transbilayer proteins
 Cytoskeleton = maintain shape
a. α-spectrin
b. β-spectrin
 Ankyrin = protein; fixes spectrin tetramers to the membrane
 Band 3 = transmembrane protein attached to ankyrin
 Also called anion exchanger protein
 Band 4.2 = stabilize link between ankyrin and band 3
 Glycophorin C = linked to spectrin by the band 4.1 protein
 Band 4.1 R = stabilizes association of spectrin with actin
 Disorders = altered shape; deformability; caused by protein
phosphorylation
 Blood group antigens = carried by transmembrane proteins;
oligosaccharides and glycoproteins
 CD44
 Lutheran blood antigens
 ICAM-4 (intercellular adhesion molecule-4)
 Transporter/pump adhesion proteins:
 Aquaporin 1 (water transporter)
 GLOTI1 and GLUT4 (glucose transporters)
 Sodium/hydrogen exchanger 1
 Na-K ATPase
Aging RBC Membrane
 Age markers
1. PMCA (Plasma membrane calcium) = decreases as RBC ages;
RBC membrane becomes denser
2. HbA1c (glycosylated Hb) = increased as RBC ages
CYTOPLASMIC CHARACTERISTICS
 Hb, glucose, K+
 Enzymes (ferrireductase, Na-K ATPase, etc.)
 Reduced glutathione = cell is not protected from oxidative damage (G6PD
deficiency)
 Pyruvate kinase deficiency = most common
 Decreases phosphofructokinase activity in infants
 G6PD deficiency = limits NADPH regeneration = oxidative damage of Hb
RBC CATABOLISM
 Occurs after 120 days
 Premature infants = 35-50 days
 Fetuses = 60-70 days
 Changes:
a. Less flexible membrane
b. Hb concentration increases
c. Glycolysis diminishes
 Spleen = phagocytosis of aged RBCs
 Splenic venous sinusoids = cell pliability is tested; expose
defective RBCs
Extravascular Catabolism
1. RBC is phagocytized and digested by macrophages of the
reiticuloendothelial system
2. Hb molecule is disassembled:
- Iron
- Protoporphyrin
- Globin
a. Iron = transported in the plasma by transferrin
- Recycled by red bone marrow
b. Globin = catabolized by liver into constituent amino acids and enters
the circulating amino acid pool

3. Porphyrin = broken at the alpha methene bridge by heme oxidase to form
CO and tetrapyrrole (bilirubin)
4. Alpha carbon leaves as carbon monoxide exhaled thru the respiratory s.
5. Tetrapyrrole (unconjugated bilirubin) is carried by plasma albumin to the
liver
6. Plasma albumin-bound tetrapyrrole (bilirubin) is conjugated to
glucuronide and excreted in the bile
7. Bilirubin-glucuronide = excreted into the gut and converted by bacterial
into stercobilinogen, which is excreted in the feces
8. Small amount of urobilinogen is reabsorbed into the blood circulation and
excreted in the urine
Intravascular Catabolism
1. RBC is destroyed intravascularly, releasing Hb
2. Hb dissociates into alpha and beta dimers
3. Haptoglobin (plasma globulin) binds the dimers
 Binding prevents urinary excretion of plasma Hb
4. Complex is removed from the circulation by hepatocytes
 Catabolized in liver
5. Unbound alpha and beta dimers in blood are filtered by glomeruli and
reabsorbed by renal tubular cells and converted into hemosiderin
 Limited capacity for renal tubular uptake = free Hb and metHb
appear in urine
 Hb not bound by haptoglobin nor excreted in urine = oxidized to
metHb
6. Heme from metHb is released and bound by hemopexin (transport
protein)
7. Hempoxin-heme complex is removed from the circulation by the low-
density lipoprotein receptor-related protein to eliminate potentially toxic
free heme
8. Heme groups in xss of hemopexin-binding capacity combine with albumin
to form methemalbumin until more hemopexin is available
MEASUREMENT OF ERYTHROCYTES
Mean Corpuscular Volume
𝑀𝐶𝑉 =
𝑃𝑎𝑡𝑖𝑒𝑛𝑡′
𝑠 𝑃𝐶𝑉 (
𝐿
𝐿
)
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (
𝑥1012
𝐿
)
= 𝑓𝐿
 Average RBC volume
 Reference values = 80-96 fL
Mean Corpuscular Hemoglobin
𝑀𝐶𝐻 =
ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 (
𝑥10𝑔
𝑑𝐿
)
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (
𝑥1012
𝐿
)
= 𝑝𝑔
 Average weight of Hb in an average RBC
 Directly proportional to amount of Hb and RBC size
 Reference values = 27.5-33.2 pg
Mean Corpuscular Hb Concentration
𝑀𝐶𝐻𝐶 =
ℎ𝑒𝑚𝑜𝑔𝑙𝑜𝑏𝑖𝑛 (
𝑔
𝑑𝐿
)
𝑃𝐶𝑉 (
𝐿
𝐿
)
= 𝑔/𝑑𝐿
 Average concentration of Hb per unit volume of RBCs
 Ratio of the weight of Hb to the volume of RBCs
 Reference values = 32-36 g/dL

Chapter Six: Erythrocyte Morphology and Inclusions
ERYTHROCYTES: NORMAL AND ABNORMAL
 Discoytes = normal mature RBCs
 Biconcave disc
 Anuclear
 6.2-8.2 µm in diameter
 Variations:
 Size
 Shape
 Color
 Inclusions
 Alterations in RBC distribution on a PBS
VARIATIONS IN SIZE (anisocytosis; severe anemias)
a. Normocytic
 6.2-8.2 µm
 Normal
b. Macrocytic
 > 8.2 µm
 Macrocytosis -= defect in either 1) nuclear maturation or 2)
stimulated erythropoiesis
 Maturation defect = B12 or folate deficiency = disrupt mitotic
activity in bone marrow
 EPO stimulation = increase Hb synthesis = premature release of
reticulocytes
c. Microcytic
 < 6.2 µm
 Microcytosis = decrease in Hb synthesis
 Iron deficiency
 Impaired globulin synthesis
 Abnormal mitochondria (impaired heme synthesis)
VARIATIONS IN SHAPE (poikilocytosis)
 Chemical/physical alteration of cell membrane or contents of the cell
a. Acanthocytes
 Thorny, spine-like projections around membrane
 Disorders:
1. Abetalipoproteinemia
o Imbalance between RBC and plasma lipids
(impaired lipid absorption) = membrane defect
2. Spur cell anemia
3. Liver cirrhosis + hemolytic anemia
4. Following heparin administration
5. Hepatic hemangioma
6. Neonatal hepatitis
7. Post-splenectomy
b. Blister cells
 1 or more vacuoles that resemble blisters on skin
 Thinned area at periphery of cell membrane
 Ruptured vacuoles = keratocytes + schistocytes are produced
 Disorders:
1. Pulmonary emboli in sickle cell anemia
2. Microangiopathic hemolytic anemia
c. Burr cells (echinocytes)
 1 or more spiny projections of cellular membrane; uniform
 No spicules (acanthocytes have spicules and more spherical)
 Quarter moon-shaped
 Increased cell rigidity = premature destruction
 Disorders:
1. Anemias
2. Bleeding gastric ulcers
3. Gastric carcinoma
4. Peptic ulcers
5. Renal insufficiency
6. Pyruvate-kinase deficiency
7. Uremia
d. Crenated RBCs (echinocytes)
 Short, scalloped, or spike-like projections; distributed regularly
 Loss of intracorpuscular water (osmotic imbalance)
e. Elliptocytes
 Narrower; more elongated than megalocytes (oval macrocytes)
 Rod/cigar/sausage shape
 Membrane defect = loss in integrity
 Disorders:
1. Hb C disease
2. Ellipsocytosis
3. Anemias associated with malignancies
4. Hemolytic anemias
5. Fe-deficiency anemia
6. Pernicious anemia
7. Sickle cell trait
8. Thalassemia
f. Helmet cells (schizocytes)
 Larger scooped out part of cell that remains after blister cell
rupture
 Cell fragments
 Formed in the spleen and IV fibrin clots
 Hemolytic anemias related to burns or prosthetic implants
g. Keratocytes
 Partially deformed; not cut
 Spicules (horn-like) = ruptured vacuole
 Half-moon or spindle
 Disseminated IV coagulation
h. Knizocytes
 Pinched bottle
 Hemolytic anemias (spherocytosis)
i. Leptocytes
 Resemble target cells
 Inner central portion is not completely detached from outer
membrane
 Hepatic disorders, IDA, and thalassemia

j. Oval macrocytes (megalocytes)
 Oval or egg-like
 Fuller and rounder than elliptocytes
 Vitamin B12 and folate deficiencies
 RBCs in reticulocyte stage
k. Pyknocytes
 Distorted, contracted erythrocytes; similar to burr cells
 Disorders:
1. Acute, severe hemolytic anemia
2. G6PD deficiency
3. Hereditary lipoprotein deficiency
l. Schistocytes (schizocytes)
 RBC fragments
 ½ the size of an RBC; deeper red color
m. Sickle cells (drepanocytes)
 Crescent-shaped; smooth membrane
 Gelation of polymerized deoxygenated Hb S
 Sodium ion influx = increased intracellular Ca2+ = membrane
rigidity
 Sickle cell anemia
n. Spherocytes
 RBCs that have lost biconcave shape
 Compact, round shape
 Orange-red color
 Membrane instability = decreased deformability = premature
destruction
 Genetic defect or physical trauma (heat or chemical)
 Associated with hemolytic disease of the newborn
(microspherocytes)
 Splenomegaly
 Disorders:
1. Acquired hemolytic anemia
2. Blood transfusion reactions
3. Congenital spherocytosis
4. DIC
o. Spiculated RBCs
 Irregularly contracted
 Also called burr cells, crenated cells, pyknocytes, spur cells,
acanthocytes, and echinocytes
p. Stomatocytes
 Slit-like opening that resembles a mouth
 Increased Na+ and decreased K+ within cytoplasm
 Acute alcoholism, alcoholic cirrhosis, glutathione deficiency,
hereditary spherocytosis, infectious mononucleosis, Pb poisoning,
malignancies, thalassemia minor, transiently accompanying
hemolytic anemia
 Rh null disease
q. Target cells (codocytes)
 Resemble a shooting target
 Central red bull’s eye surrounded by a clear ring and then an
outer red ring
 Thinner than normal
 Hb maldistribution
 Cholesterol and phosphatidylcholine incorporated into cell
membrane lipids
 Hemoglobinopathies:
1. Hb C disease
2. S-C and S-S disease
3. Sickle cell thalassemia
4. Thalassemia
5. Hemolytic anemias
6. Hepatic disease with or without jaundice
7. IDA after splenectomy
r. Teardrop cells (dacryocytes)
 Smaller than normal
 Resemble tears
 Disorders:
1. Homozygous beta-thalassemia
2. Myeloproliferative syndromes
3. Pernicious anemia
4. Severe anemias
ALTERATIONS IN COLOR (anisochromia)
 Normochromic = pinkish-red; central pallor (1/3)
 Hypochromia = central pallor exceeds 1/3 of cell’s diameter
 Inadequate iron stores = decreased Hb synthesis
 IDA
 Polychromatophilia
 Non-nucleated RBC has a faintly blue color
 Lacks full amount of Hb; blue color = diffused residual RNA in
cytoplasm
 Polychromatic erythrocyte (larger)
 Reticulocyte = supravital stain (thread-like netting)
 Basophilic erythrocyte = if the cell stained an intense blue or blue-
gray color without a pink cast
 Increased polychromatic RBCs = rapid blood regeneration + increased
bone marrow activity
VARIETIES OF INCLUSIONS
a. Basophilic stippling (fine)
 Tiny, round, solid-staining, dark-blue granules
 Punctate stippling = coarse basophilic stippling
 RNA and ribosome granules precipitated during blood smear
prep
 Disorders:
1. Disturbed erythropoiesis (defective or accelerated
heme synthesis)
2. Pb poisoning
3. Severe anemias
b. Cabot rings
 Ring-shaped, figure-eight, or loop-shaped
 Double or multiple rings
 Red/reddish-purple
 Remnants of microtubules from mitotic spindle
 May be nuclear remnants or abnormal histone biosynthesis

 Pb poisoning, pernicious anemia
c. Crystals
 Hb C crystals = rod-like or angular opaque structures within RBCs
(Hb C disease)
 Hb H bodies = brilliant cresyl blue stain
o Blue globules
d. Heinz bodies
 Stain with crystal violet or brilliant cresyl blue
 Precipitated, denatured hemoglobin
 Congenital hemolytic anemia, G6PD deficiency, hemolytic
anemias (phenacetin), hemoglobinopathies
e. Howell-Jolly bodies
 Round, solid-staining, dark-blue to purple inclusions
 1 or 2 per cell
 Most frequently seen in mature RBCs that lack a nucleus
 Not seen in normal RBCs
 Nuclear remnants composed of DNA
 Develop in periods of accelerated or abnormal erythropoiesis
(spleen cannot keep up with pitting these remnants from the cell)
 Hemolytic anemias, pernicious anemia
 Post-splenectomy, physiological atrophy of spleen
f. Pappenheimer bodies (siderotic granules)
 Wright-stained smears; purple dots
 Dark-staining particles of iron in the RBC visible with Prussian blue
o Blue dots (Fe3+ ions)
 Aggregates of mitochondria, ribosomes, and iron particles
 Iron-loading anemias, hyposplenism, and hemolytic anemias
ALTERATIONS IN RBC DISTRIBUTION
a. Agglutination
 Erythrocyte clumping
 True agglutination = produced by the presence of antibodies
reacting with antigens on the RBC
b. Rouleaux formation
 Stack of coins arrangement
 Thick portions of blood smears
 If seen in thin areas of blood smear = pathogenic rouleaux
 Associated with the presence of cyroglobulins
PARASITIC INCLUSIONS IN ERYTHROCYTES
Malaria
a. Plasmodium vivax
 Accolé forms = trophozoites; crescent-shaped masses at RBC
periphery
 Early trophozoites = minute blue discs with a red nucleus within
the pink cytoplasm of RBC
 Infected RBCs are large and pale-staining
 Schüffner dots/granules = fined red or pink;
 Gametocytes = fill cell almost completely
b. Plasmodium falciparum
 RBCs retain original size
 Young trophozoites = minute rings
 Gametocyte = elongated crescentic or sausage-shaped
 Maurer dots = irregular dark red- or pink-staining, rod- or wedge-
shaped granules
 RBCs are sequestered intravascularly
c. P. malariae
 Ziemann stippling = dust-fine, pale-pink dots in RBC cytoplasm
d. P. ovale
 Schüffner dots

Chapter Seven: Classification and Laboratory Assessment of Anemias
CAUSES OF ANEMIA
 Anemia = Hb concentration or PCV is below the lower limit if the 95%
reference interval
 Decrease in the oxygen-carrying capacity of blood
 Reference interval: 12-16 mg/dL
 Affected by age, sex, race
 Neonates are polycythemic (>16 mg/dL)
 Males = higher Hb due to higher body mass
 Females = lower Hb
 Causes:
 Blood loss
 Impaired red cell production
 Accelerated red cell destruction (hemolysis in excess of the
ability of the marrow to replace these losses)
 Not a disease; rather = manifestation of underlying disease or deficiency
CLINICAL SIGNS AND SYMPTOMS
 Diminished O2 delivery to tissues due to lowered Hb concentration
 Fatigue (easy fatigability)
 Dyspnea on exertion
 Pallor (pale palpebral conjunctiva and nail beds)
 Fainting and vertigo
 Congestive heart failure (strain on the heart)
 Jaundice (hemolytic anemias)
 Petechiae, mouth mucosal bleeding, sternal tenderness,
lymphadenopathy, cardiac murmurs, splenomegaly, and hepatomegaly
 Classic symptoms: fatigue and shortness of breath
CLASSIFICATION OF ANEMIAS
Based on Etiology/Pathophysiology
A. Hemolytic (RBC destruction) = accelerated destruction
 Inherited
 Acquired
B. Impaired Production = decreased rate of formation
 Aplastic
 Iron deficiency
 Sideroblastic
 Anemia of chronic disease
 Megaloblastic
C. Anemia of blood loss = acute or chronic
 Acute = normocytic and normochromic
 Chronic = microcytic and hypochromic (hookworm, peptic ulcer
disease, gynecologic bleeding)
Based on Morphology (based on MCV; Wintrobe’s Indices)
A. Microcytic (maturation defect)
 IDA
 Thalassemia
 Sideroblastic anemia
 Anemia of chronic inflammation
B. Macrocytic (maturation defect)
 Megaloblastic
 Vitamin B12 deficiency
 Folate deficiency
 Myelodysplasia and erythroleukemia
 Non-megaloblastic
 Newborns
 Liver disease
 Aplastic anemia
 Alcoholism
 Spherocytosis
 Hypothyroidism
C. Normocytic = use retic. count; hypoproliferation
 High Retic
 Hemolytic anemia
o Intrinsic: membrane defects,
hemoglobinopathies, enzyme deficiencies
o Extrinsic: immune and non-immune tissue
injury
 Normal Retic
 Aplastic anemia
LABORATORY ASSESSMENT
Quantitative Measurement
A. Decreased hemoglobin concentration
B. Reduced PCV
C. Decreased RBC concentration
D. RBC indices (MCV, MCH, MCHC)
E. Red cell histogram
F. RDW or RCMI (red cell morphology index)
Semiquantitative Grading of Erythrocyte Morphology
 Direct observation of a peripheral blood smear for abnormalities in RBC
morphology
Grading for RBC Morphology
0 Normal or slight variation
1+ Small population
showing abnormality;
slightly increased or few
2+ More than occasional
numbers of abnormal
RBCs; moderately
increased
3+ Severe increase in
abnormal RBCs in each
microscopic field; many

4+ Most severe; prevalent in
each microscopic field;
marked or marked
increase
ANEMIA OF BLOOD LOSS
Acute Blood Loss
 Trauma
 Decrease in blood volume = hypotension
 Heart rate, respiratory rate, and cardiac output are increased
 Redistribution of blood flow from the skin and viscera to heart, brain, and
muscle
 Hb and Hct are initially unchanged (balanced loss of plasma and cells);
Hct and Hb decrease as fluid moves from the extravascular to the
intravascular (dilution)
Chronic Blood Loss
 Asymptomatic
 Strain on the heart = cardiac failure
 Increased EPO secretion
 Examples: hookworm, intermittently bleeding colonic polyp
 Induces iron deficiency
 Low reticulocyte count
MICROCYTIC ANEMIAS
 MCV < 80 fL
Iron-Deficiency Anemia
 Disorder of heme metabolism
 Nutritional deficiency, faulty Fe absorption, increased demand for Fe,
excessive Fe loss
 Tests:
a. Serum iron
b. TIBC (total iron-binding capacity)
c. Hemoglobin
d. Ferritin
 Stages:
a. Stage 1 (pre-latent)
 Decrease in storage iron
 Storage Fe depletion
 Low ferritin
 Normal Hb, serum Fe, and TIBC
b. Stage 2 (latent)
 Decrease in iron available for erythropoiesis
 Low ferritin
 Low serum iron
 High TIBC
 Normal Hb
c. Stage 3 (anemia)
 Decrease in circulating RBC parameters and decrease
in O2 delivery to tissues
 Low Hb
 Low ferritin
 Low serum iron
 High TIBC
Anemia of Chronic Inflammation
 Caused by biochemical changes that occur during inflammation
 Shortened RBC lifespan
 Main defect: hepcidin = decreased Fe absorption from the GI
 Pb poisoning = defective protoporphyrin synthesis
 ALA-synthetase and ferrochelatase are inhibited
 Fe2+ is not incorporated into heme
 Basophilic stippling
 Peppenheimer bodies (siderotic granules)
 May be induced by drugs (chloramphenicol and isoniazid)
 Prussian blue stain = ringed sideroblasts
Thalassemia
 Defect in globin chain synthesis
 α-thalassemia = alpha chain defect
 β-thalassemia = beta chain defect
MACROCYTIC ANEMIAS
 MCV > 100 fL
 RNA in cytoplasm
 DNA in nucleus (require B12 and folate)
Megaloblastic Anemias = conditions that impair DNA synthesis
A. B12 deficiency
 Common in vegetarian elders
 Absorbed in the ileum (Fe = duodenum and jejunum)
 B12 needs to complex with intrinsic factor for it to be absorbed
from the ileum (B12-IF complex)
o IF = produced from gastric parietal cells
 Pernicious anemia = gastric parietal cells do not produce intrinsic
factor
 Symptoms: neurologic disorders (gait and stance)
 Deficiencies are due to:
1. Failure of B12 to separate from food
2. Haptocorrin
3. Ileal disease
 Schilling Test = differentiate the cause of B12 deficiency
 Reflex testing = measure methylmalonic acid when B12 levels are
low
 Treatment: lifelong IV injection of B12
B. Folate deficiency
 Folate = ubiquitous
 Easily destroyed by overcooking
 Lost in dialysis and anti-epileptics
 Neural tube defects = spina bifida
 Inflammatory bowel disease and malabsorption
C. Erythroleukemia
 M6-like (like leukemia) marrow

 Di Guglielmo syndrome
NORMOCYTIC ANEMIAS
 MCV = 80-100 fL
HEMOLYTIC ANEMIA
 Normocytic; high reticulocyte count
 Hyperbilirubinemia = sign of hemolytic anemia
 High B1 = unconjugated; pre-hepatic; hemolytic (HDN or HTR)
 High B2 = conjugated; post-hepatic; obstructive (stones)
 High B1 and B2 = hepatocellular (hepatitis)
 Haptoglobin = binds free Hb
 Intravascular catabolism
 High haptoglobin = more free Hb; hemolysis
 LDH (tetramer) = high isoenzyme 1 (HHHH) = hemolysis
Intrinsic (intracorpuscular; inherent to RBC)
A. Inherited
a. Membrane defects
1. Hereditary spherocytosis
 High MCHC
 Band 3 conformational change = senescent RBCs
become spherocytes
 Lack of spectrin
 Spleen catabolizes RBCs = splenomegaly
2. Hereditary ovalocytosis
3. Hereditary elliptocytosis
4. Hereditary stomatocytosis
5. Acanthocytosis
6. Spur cell
7. Pyropoikilocytosis
8. Xerocytosis
b. Enzyme deficiencies
1. G6PD (glucose-6-phosphate dehydrogenase) deficiency
 Heinz bodies (hemoglobin denaturation)
 Hexose monophosphate pathway
2. PK (pyruvate kinase) deficiency
c. Hemoglobinopathies
1. Hb S disease
 Triggered by hypoxia
 Sickle cell disease
 Microvascular occlusion = sickle cells cannot navigate
 Polymerization causes sickling
 β-chain = 6th amino acid (Glu) is substituted by Val
2. Hb C disease
 β-chain = 6th amino acid (Glu) is substituted by Lys
 Hexagonal crystal of dense Hb protruding from RBC
membrane
3. Hb SC disease
 Finger-like or quartz-like crystals
4. Hb D disease
5. Hb E disease
6. Hb H disease
7. Methemoglobinemia
8. Unstable Hb
B. Acquired
 Paroxysmal nocturnal hemoglobinuria
 On and off
 At night
 Excreting free Hb
 During sleep = pH changes = hemoglobinuria
 Complement-mediated lysis of RBCs
 Diagnosed by Ham test
 Absence of CD55 and CD59
 Due to mutations in PIGA-1 (phosphatidylinositol
phospholipid)
 C56 + C6-C9 = membrane attack complex; rapid osmotic lysis
 CD55 = acts on C5- and C9-convertase
 CD59 = acts on membrane attack complex
 Later on develop myelodysplastic syndrome, then leukemia
Extrinsic (extracorpuscular)
A. Immune causes (antibody-mediated) = Direct Coumb’s Test (direct anti-
globulin test; +DS = red cell sensitization in vivo)
1. Isoimmune Hemolytic Anemias
a. Hemolytic Disease of the Newborn (HDN) or erythroblastosis
fetalis
 Rh-(-) mother and Rh-(+) father = 2nd child dies; give Rhogam
at 27th week of gestation
 High B1 (w/ affinity to fatty tissue; brain) = kernicterus (yellow
brain; brain damage)
 B1 is converted to B2 (water-soluble) using light
b. Hemolytic Transfusion Reactions (HTR) = incompatibilities
2. Autoimmune Hemolytic Anemias
a. Warm agglutinins (antibodies)
b. Cold agglutinins
c. Mixed warm and cold agglutinins
3. Drug-Induced Hemolytic Anemias
B. Non-immune RBC injury
a. Mechanical
 Microangiopathic hemolytic anemia
 Thrombotic thrombocytopenic purpura
 Hemolytic uremic syndrome
 Disseminated intravascular coagulation
 Cardiac valve prosthesis = valves with sharp edges;
replaced due to S. pyogenes infection
 March hemoglobinuria = foot-pounding trauma
b. Infectious agents
 Malaria
 Babesia
c. Chemical and physical agents: drugs, toxins, burns
APLASTIC ANEMIA
 Normocytic; normal or low reticulocyte count
 Pancytopenia (decrease in all cell lines)
 Bone marrow fails to produce HSCs (HSC failure)
 Chloramphenicol, radiation

 Hypocellular (marrow is all adipose)
MYELOMA, MYELOFIBROSIS, OR PRIMARY REFRACTORY ANEMIA
 Hypercellular
 Normocytic; normal or low retic count
NEOPLASM, UREMIA
 Normal cellular
 Normocytic; normal or low retic count

Chapter Eight: Acute and Chronic Blood Loss Anemias
ACUTE BLOOD LOSS ANEMIA
Etiology
 Trauma
 During or after surgery
 Accidents
 Severe injuries
Physiology
 No immediate anemia produced
 Severe hemorrhage (losing more than 20% of circulating blood)
 Shock (other cardiovascular problems)
 Decreased total blood volume = circulatory failure
 Anemia = dilutional effect of extravascular fluid on cells in the circulation
Clinical Features of Acute Hemorrhage
Volume of Blood
Loss (mL)
Blood Volume (%) Symptoms
500-1000 10-20 Few or none
1000-1500 20-30 At rest
(asymptomatic)
Upright position-
light-headedness,
hypotension,
tachycardia
1500-2000 30-40 Thirst, shortness of
breath,
clouding/loss of
consciousness;
decrease: BP,
cardiac output,
venous pressure;
cold, clammy,
and pale
extremities
2000-2500 40-50 Lactic acidosis,
shock; irreversible
shock, death
Laboratory Findings (in order)
1. Transient fall in platelet count
2. Platelet count rises within 1 hour
3. Neutrophilic leukocytosis (10-35 x 109/L)
 Shift to the left (more immature forms)
4. 24 hours = still normochromic and normocytic
 Normal RBC indices
5. After 48-72 hours (2-3 days) = Hg and Hct fall
 Tissue fluids move into the blood circulation to compensate for
the blood lost
 Cells are diluted
6. 3-5 days = transient macrocytosis
 Maximum after 10 days
 Results from increased reticulocytes due to increased
erythropoiesis
7. 2-4 days = total WBC count returns to normal
8. 2 weeks = WBC morphological changes disappear
9. Return of red cell profile to normal takes longer
CHRONIC BLOOD LOSS ANEMIA
Etiology = blood loss of small amounts occurs over an extended period (months)
 GI disorders (bleeding from ulcers)
 Gastritis due to alcohol or aspirin ingestion
 Tumors
 Parasitosis (hookworm)
 Diverticulitis
 Ulcerative colitis
 Hemorrhoids
 Heavy menstruation (menorrhagia)
 Fibroid tumors and uterine malignancies in women
 Urinary tract abnormalities
 Kidney stones, tumors, or chronic infections
Laboratory Findings
 Features seen in acute blood loss are absent
 RBC regeneration is slower
 Normal reticulocyte count (or slightly increased)
 Initially = normochromic and normocytic
 Chronic bleeding results in iron deficiency = microcytic +
hypochromic
 Normal or decreased WBC count
 Increased platelets
 Later decreased due to severe iron-deficiency
Acute (24 hours) Chronic (months)
Etiology Trauma GI tract
Menstruation
Urinary tract
Blood volume
disruption
Yes No
Iron deficiency No Yes
Hct Normal Decreased
WBC count Increased Normal
Platelets Increased Normal
Reticulocytes Normal Increased

Chapter Nine: Aplastic and Related Anemias
APLASTIC ANEMIA
 Paul Ehrlich
 Acquired or congenital
 Hypoproliferative disorder (reduced growth/production of blood cells)
 Damage or destruction of hematopoietic tissue in the bone marrow =
deficient blood cell production
 Most cases = immune-mediated destruction of hematopoiesis
ETIOLOGIC CLASSIFICATION
Acquired Aplastic Anemia
A. Idiopathic Aplastic Anemia (70% of cases)
 No established history of chemical or drug exposure or viral
infection
 Unknown cause
B. Secondary (10%-15% of cases)
 Iatrogenic causes = radiation; chemotherapy
o Transient marrow failure
 Benzene
 Intermediate metabolites of some common drugs
Inherited Aplastic Anemia
A. Constitutional Aplastic Anemia
 Congenital (genetic-predisposition)
 Diamond-Blackfan and Fanconi anemias
OR
ANEMIA FROM DIRECT TOXICITY
 Iatrogenic causes (radiation and chemotherapy)
 Ionizing radiation
 Benzene
 Intermediate metabolites of some common drugs
 Injure proliferating and quiescent hematopoietic cells = damaged DNA =
apoptosis
ANEMIA FROM IMMUNE-MEDIATED CAUSES
 Iatrogenic causes (transfusion-associated graft versus host disease)
 Eosinophilic fasciitis
 Hepatitis-associated disease
 Intermediate metabolites of some common drugs
 Idiopathic aplastic anemia
 Iatrogenic agents:
1. Benzene and its derivatives
2. TNT (trinitrotoluene)
3. Insecticides and weed killers
4. Arsenic (inorganic)
5. Antimetabolites (antifolates; purine and pyrimidine analogues)
6. Antibiotics
Benzene
 Metabolized in the liver to form phenolic and open-ring structures:
 Hydroquinones = inhibit maturation and amplification of bone
marrow stem and blast cells
 Metabolites = alter function of marrow stromal cells = cannot support
hematopoiesis
 Anemia takes years to manifest
Drugs (idiosyncratic reactions)
 Chloramphenicol
 Recombinant human EPO (epoetin)
 Tetracyclines, organic arsenicals, phenylbutazone, trimethadione,
methylphenylethylhydantoin
Infections
 Viral
 HBV, HCV
 Measles
 Epstein-Barr virus
 CMV
 Human parvovirus-B19 = infects erythroid replicating cells
 Causes: drug exposure; direct stem cell damage by the virus;
depressed hematopoiesis by the viral genome; virus-induced
autoimmune damage
 Hepatitis-associated aplastic anemia
 Anemia follows an acute attack of hepatitis
 Pancytopenia after 1-2 months of viral hepatitis
PATHOPHYSIOLOGY
 Most common laboratory findings:
 Pancytopenia
 Reticulocytopenia
 Bone marrow hypocellularity
 Depletion of HSCs
 Immune-mediated in most cases (type 1 cytotoxic T cells)
 Cellular immune suppression
 Thymomas = pure red cell aplasia
 Insufficiencies or defects in:
a. Pluripotent stem cells (CFU, stem cells [CFU-S])
b. Committed stem cells (CFU, committed cells [CFU-C])
 Mechanisms:
a. Microenvironment is unable to provide for normal development
of HSCs
b. Stimulators may be absent (cytokines and interleukins)
c. Bone marrow failure = from excessive suppression of
hematopoiesis by T cells or macrophages
o Immunologically-mediated, tissue-specific organ
destruction
o No spontaneous healing = death due to infection or
bleeding within a few years
d. Stem cells inhibiting each other

PHASES OF APLASTIC ANEMIA
A. Onset of Disease
 Immune-mediated destruction of hematopoietic compartment
 Few surviving stem cells = support hematopoiesis for some time,
but eventually, cell counts become very low and symptoms
appear
B. Recovery
 Partial or complete response (no increase in numbers of stem
cells)
 In the minority = primitive cell compartment repopulates over
time (stem cell self-renewal)
C. Late Disease
 Pancytopenia relapse after years (emergence of abnormal
clone of stem cells)
 PNH, MDS, or AML
IMMUNE SYSTEM-HSC INTERACTIONS
 Mononuclear cells = suppress hematopoietic colony formation by normal
marrow cells
 Increased numbers of activated cytotoxic T-lymphocytes
 ATG therapy = anti-thymocyte globulin
 IFN-γ and TNF in marrow microenvironment = suppress proliferation of early
and late HPCs and HSCs
 TNF- α = specific marker for aplastic anemia
 Inhibitory lymphokine
CLINICAL FEATURES
 Total bone marrow failure
 Pancytopenia (reduction in circulating RBCs, WBCs, and
platelets)
 Anemia = fatigue and pallor
 Thrombocytopenia = bleeding and bruising
 Neutropenia = susceptibility to infections
 Acute course (fulminating) = profound pancytopenia and rapid
progression to death
 Chronic course (insidious onset)
 High risk for malignancies or late clonal hematologic diseases:
 PNH (paroxysmal nocturnal hemoglobinuria)
 MDS (myelodysplastic syndrome)
 AML (acute myelogenous leukemia)
 Preleukemic condition
CLINICAL FEATURES
 Pancytopenia (all cell lines are affected)
 Erythrocytic, leukocytic, thrombocytic
 If one cell line is involved = erythrocytic
 Severe aplastic anemia: 2 of 3 blood values fall below critical levels
 Granulocytes less than 0.5 x 109/L
 Platelets less than 20 x 109/L
 Reticulocytes less than 1.0%
 Bone marrow hypocellularity
 Less than 30% of residual hematopoietic cells
 More fat cells
 Very severe aplastic anemia
 Neutrophil: less than 2.0 x 109/L
 Platelets: less than 20 x 109/L
 Reticulocyte: less than 0.6%
 Normocytic and normochromic RBCs
 There may be macrocytosis
 RDW = normal in non-transfused patients
 Leukopenia
 Decrease in granulocytes
 Lymphocytosis
 Thrombocytopenia
 Increased serum iron
 Decreased plasma iron turnover
 Decreased iron use by RBCs
 Decreased effective and total erythropoiesis
 Very few early erythroid and myeloid cells; scanty megakaryocytes
 Primitive progenitor stem cells (1% of marrow cells) = absent
 If radiation is the cause:
 Reticulocyte count falls
 RBCs decline slowly
 Neutrophilic leukocytosis within the first few hours
 Decrease in lymphocytes after the 1st day = early leukopenia
 After 5 days = granulocytes decrease
 Platelets decrease later
 Platelets = last to return to normal in the recovery phase
TREATMENT
 Immunosuppressive therapy
 If no donor for HSCT
 Antithymocyte globulin (or antilymphocyte [ALG or ATG])
 Cyclosporine-A (Cy-A)
 Splenectomy
 Lymphocytapheresis
 Hematopoietic stem cell transplant (allogenic)
 Patients younger than 40 years of age
 Suitable donors: identical twin or an HLA-matched donor
CONGENITAL RED BLOOD CELL-RELATED DISORDERS
 Inherited bone marrow failure syndromes = initially exhibit aplastic anemia
 Fanconi anemia
 Congenital amegakaryocytic thrombocytopenia
 Diamond-Blackfan anemia = rare
Telomeres
 TERC (telomerase RNA component) = gene for RNA component of
telomerase
 Heterozygous mutations = short telomeres in congenital aplastic
anemia
 Mutations = risk factors for marrow failure
 Hexameric repetitive DNA sequences (TTAGGG in the leading strand;
CCCTAA in the lagging strand)
 Capped by proteins at the extremities of linear chromosomes
 Cannot be fully duplicated during cell division = shorten with every cell
cycle

 End-replication problem = telomere shortening after every cell
division
 Critically short telomere = cell proliferation arrest, senescence,
and apoptosis (p53, p21, and PMS2)
 If cells bypass these inhibitory pathways = telomeres continuously
shorten and lose their function = chromosomal instability
 Telomere maintenance = TERT and shelterin
 Telomerase (TERT) = expressed by highly proliferative cells
 Telomerase reverse transcriptase
 Uses RNA molecule (TERC) as the template to elongate 3’ ends of
telomeres
 Tightly regulated; very low levels in stem and progenitor cells =
telomeres eventually shorten with age
 Lymphocytes have shorter telomeres than do granulocytes
 Shelterin = DNA-binding proteins that cover and protect telomeres
 Mutations in genes for telomere repair = repair defects and defects in
protection
 Telomerase complex gene mutations (associated with trisomy 8)
 Telomere-protecting complex (shelterin) gene mutations
 The shorter the telomeres = higher risk for subsequent
development of malignancy (AML)
 Excessive telomere loss = bone marrow failure and malignancy
 Short telomeres = inhibit cell proliferation and predispose to
chromosomal instability
 Techniques in measuring telomeres
a. Southern blot analysis
b. Quantitative PCR
c. Flow-FISH (combination of flow cytometry and fluorescence in
situ hybridization) = most accurate
LABORATORY FINDINGS IN BONE MARROW FAILURE SYNDROMES
 Absent to severe peripheral cytopenia
 Most common = unexplained red cell macrocytosis
 Elevated Hb F levels
 Dysplastic changes in marrow cells:
a. 1 or 2 nuclei in micromegakaryocytes
b. Pseudo-Pelge Huët anomalies
c. Mild Megaloblastic features (nuclear:cytoplasmic dyssynchrony)
d. Multi-nucleated erythroid precursors
PURE RED CELL APLASIA
 Hypoproliferation of erythroid elements
 3 forms:
1. Congenital: Diamond-Blackfan Syndrome
2. Acquired Chronic: idiopathic (thymoma and lymphoma)
3. Acute (transient): parvovirus, other infections, drugs, riboflavin
deficiency
 Disturbed erythropoiesis due to:
 Immune suppression (patients respond to steroid treatment)
o Antibodies against erythroid precursor cells
o Lymphocytes that inhibit erythropoiesis
 Malnutrition
 Neoplasia (thymomas)
 Bone marrow
 Erythroid precursors absent
 Increased serum EPO (erythropoietin)
 Acquired pure red cell aplasia
 Selective failure of RBC production
 Middle-aged adults
 Reticulocytopenia
 Only the most primitive erythroid precursors are present in
marrow
 Normal WBC and platelet production
 Associated with thymoma
 Chronic acquired red cell aplasia
 Drugs, collagen vascular disorders, lymphoproliferative disorders
 Autoimmune cytopenias
 Therapy = corticosteroids and immunosuppressive drugs
Diamond-Blackfan Anemia (DBA)
 Characteristics:
a. Proapoptotic hematopoiesis
b. Bone marrow failure
c. Low stature
d. Birth defects
e. Cancer predisposition
 Other names:
 Blackfan-Diamond syndrome
 Congenital pure red cell aplasia
 Congenital hypoplastic anemia
 Inherited bone marrow failure syndromes (IBMFS)
Pathophysiology
 IBMFS due to a ribosomal disorder
 Gene mutation in RPS19 (most frequently mutated; 77)
 Whole gene deletions
 Translocations
 Truncating mutation
 DBA genes = encode protein components of either the small 40S or large
60S subunits
 Mechanisms:
a. Hypoproliferation of erythroid cells
b. Enhanced sensitivity of hematopoietic progenitors to apoptosis
c. Defective ribosomal subunit maturation = delay globin gene
translation = excess of free heme = erythroid-specific apoptosis
and anemia
Laboratory Manifestations
 Diagnosis in the first year of life
 Symptoms = pallor and lethargy
 Diagnostic criteria:
1. Anemia appearing prior to the 1st birthday
2. Normal or slightly decreased neutrophil count
3. Variable platelet counts (often increased)
4. Macrocytosis
5. Normal bone marrow cellularity with few red cell precursors
 Non-classical DBA: slowly progressive, refractory anemia
 No concurrent leukopenia or thrombocytopenia
 Severe: normochromic and slightly macrocytic

 Low reticulocyte
 Normal leukocytes or slightly decreased
 Platelets are normal or increased
 Reduction in erythroid cells
 Normal granulocytic and megakaryocytic cell lines
 Elevated Hb F
 Fetal membrane antigen “i” is present
 Elevated RBC adenosine deaminase level
Treatment
 Steroids (75% partially respond)
 Steroid treatment may be long-term
Fanconi Anemia
 Best described c ongenital form of aplastic anemia
 13 different subtypes
 Inherited
 Autosomal recessive mode (except FA-B subtype); X-linked
 Chromosomes 9q and 20q
 Chromatid breaks, gaps, rearrangements, reduplications, and
exchanges
 More common in males
Clinical Signs and Symptoms
 Low birth weight (< 2500 g)
 Skin hyperpigmentation (café au lait spots)
 Short stature
 Skeletal disorders (aplasia or hypoplasia of the thumb)
 Renal malformations
 Microcephaly
 Hypogonadism
 Mental retardation
 Strabismus
Laboratory Findings
 Among patients with congenital malformations, 28% are diagnosed
before the onset of hematologic manifestations
 5 years of age = progressive pancytopenia becomes apparent
 Moderate to severe pancytopenia: 5-6 g/dL Hb concentration
 Predisposition to neoplasia and nonhematopoietic developmental
anomalies
 Marker: susceptibility to an immune cytokine
 Gold standard: demonstration of increased chromosomal breakage
following exposure to clastogens (mitocytic C and diepoxybutane)
Therapy
 Bone marrow transplantation
 Prevent infection and hemorrhage
 Treatment of choice for patients with an HLA-identical
unaffected sibling
 Steroids and androgens
 Cryopreserved umbilical cord blood transplantation
 From sibling
 HLA-identical
 High percentage of CD34+ cells (stem cell marker by cluster
designation)
 Recombinant granulocyte-CSF (colony-stimulating factor)
 Reducing neutropenia
 Ineffective in stimulating erythrocyte or thrombocytic cell lines
Familial Aplastic Anemia
 Subset of Fanconi anemia with a low incidence of congenital
abnormalities
 1-77 years
 Children: bleeding manifestations secondary to thrombocytopenia
 Eventually develop pancytopenia and hypocellular bone
marrow
 Skin hyperpigmentation
 Stunted growth
Transient Erythroblastopenia of Childhood (TEC)
 Pure red cell aplasia (children)
 Occurs in previously healthy children (younger than 8 years of age; 1-3
years)
 Self-limited; self-recovery after 1-2 months
 Humoral inhibition of erythropoiesis or decreased stem cells
 Parvovirus is not a cause
 Erythroid marrow recovery = 1-2 weeks after onset
 Moderate to severe normocytic anemia and severe reticulocytopenia
 Normocellular marrow
 Absence of erythroid precursors
Congenital Dyserythropoieitic Anemia (CDA)
 4 types
 Characteristics:
a. Indirect hyperbilirubinemia
b. Ineffective erythropoiesis
c. Peculiarly-shaped multinuclear erythroblasts
d. Refractory anemia
e. Reticulocytopenia
f. Secondary hemosiderosis
Type 1 CDA
 Mildly macrocytic anemia
 Prominent anisocytosis and poikilocytosis
 Apparent at birth; not a threat to life
Type 2 CDA
 Most common type
 Positive acidified serum test
 RBCs similar to those of patients with PNH (red cells in both abnormalities
are susceptible to hemolysis)
 To differentiate: type 2 CDA red cells react strongly with both anti-
i and anti-I
Type 3 CDA
 Similar to type 1
 Giant multinucleated erythroblasts
 Red cells are not susceptible to lysis by acidified normal serum
 Megaloblastic changes not prominent
Type 4 CDA
 Similar to type 2 but serological abnormalities are absent

Chapter Ten: Iron Deficiency Anemia, Anemia of Chronic Inflammation and
IRON DEFICIENCY ANEMIA
 Iron = essential in Hb synthesis
Early Diagnosis
 Prevent cognitive and psychomotor skills decline in young children (< 2
years of age)
 Reduce maternal morbidity
Etiology
a. Decreased Iron Intake (nutritional deficiency)
 Not enough iron is consumed to meet the normal, daily required
amount of iron
 Fad diets; imbalanced vegetarian diet (iron-deficient diets)
b. Increased Iron Utilization (increased physiological demand)
 Pregnancy
 Growth years (post-natal growth spurt & adolescent growth spurt)
 Periods of increased blood regeneration
c. Excessive Loss of Iron (pathological or physiological)
 Physiological = menstruation, pregnancy
 Pathological = GI bleeding, urogenital bleeding, pulmonary
hemosiderosis, intravascular hemolysis, malignancy (colon
cancer)
d. Faulty/Incomplete Iron Absorption (physiological iron deficiency)
 Autoimmune gastritis
 Celiac disease
 H. pylori infection
 Achlorhydria following gastric resection
 Chronic diarrhea
PHYSIOLOGY
 Iron
 35-50 mg per kilogram of body weight
 3.5—5.0 g total iron
 1 mg/day iron loss
o Exfoliation of intestinal epithelial cells
o Exfoliation of skin cells
o Bile and urinary excretion
 Replacement = 1 mg/day of iron from diet
 Operational Iron = iron used for O2-binding and biochemical reactions
 Found in heme of Hb or myoglobin
 Hemoglobin = contains 2/3 of the iron present in the body
Iron Needs in Infants and Children
 Premature infant = lower total body Fe than full-term newborn
 Faster rate of postnatal growth = iron depletion is more rapid than
full-term infants
 Develop by 2-3 months of age
 Supplement 75 mg of Fe (normal levels at birth)
 Diarrhea and cow’s milk = may lead to increased Fe loss
 Cell extrusion from intestinal mucosa, skin, and UT = iron loss
 Lose 20 µg/kg/day
 7-12 month infants = 11 mg of Fe a day
 Babies younger than 1 year = given iron-fortified cereal + breast milk
 0.5-1.0 mg = iron in breast milk and cow’s milk
 Different bioavailability (higher Fe absorption [50%] from breast
milk; cow’s milk: 10%)
Dietary Iron
A. Non-heme iron
 Iron salts from food
 More abundant in infants (low meat diet)
 Enhanced intake:
1. Ascorbic acid
2. Meat
3. Fish
4. Poultry
5. Heme iron
6. Orange juice
 Absorption inhibitors:
1. Bran
2. Polyphenols
3. Oxalates
4. Phytates
5. Vegetable fiber
6. Tannin in tea
7. Phosphates
B. Heme iron
 From hemoglobin and myoglobin of meat
 Well-absorbed
Metabolism
1. Iron intake in ferric state (Fe3+)
2. Reduction to ferrous state (Fe2+) by stomach secretions
 Stomach secretions = reducing agents:
1. Glutathione
2. Ascorbic acid
3. Sulfhydryl groups of proteins and digestion products
 Low pH (gastric juice) = makes Fe available from iron-containing
meat
 Very little Fe absorbed from stomach
3. Iron passes from the stomach to the duodenum and upper jejunum
 Absorption occurs
 GI epithelial cells (brush border microvilli) = absorb just enough
iron; absorb 5-10% of 10-20 mg/day intake
4. Transferrin (plasma protein from liver) = binds absorbed iron
 Transferrin = beta-globulin (glycoprotein)
 Iron is chelated within the intestinal lumen; transferrin shuttles it
into the mucosal cells of the small intestine
5. Cellular iron uptake:
a. Transferrin-iron complex binds to a specific receptor
 Higher number of transferrin receptors in cells of organs
with the highest requirements (erythroid bone marrow
cells & placental trophoblasts)

 Regulation (by transferrin receptor mRNA) = based on
iron content and requirements
 Iron-deficient cells = more receptors
 Iron-replete cells = down-regulated receptors
b. An invagination of the cell membrane forms
c. A vacuole is formed
d. Iron is released intracellularly
e. Transferrin = released to plasma to resume Fe transport
6. Iron use or storage
 Body cells
 Storage of ferritin (heme iron) and hemosiderin (non-heme iron)
in the mononuclear phagocyte system
 Fe binds with apoferritin to form ferritin
 Storage iron = hepatocytes and macrophages; ferritin
 Hemosiderin = when apoferritin levels are low
 Transferrin = can take Fe from storage deposits and
transport them to erythroid precursors
 Mononuclear phagocyte system = 1st-line of iron
supply
 Incorporation into Hb
 Catabolism
7. Iron excretion
 Exfoliation of epithelial cells or bile, urine, feces
 RBC loss
 Menstruation
PATHOPHYSIOLOGY
 Does not develop rapidly
 Stages:
I. Stage I = pre-latent; decrease in storage iron
II. Stage 2 = latent; decrease in iron available for erythropoiesis
(transport)
III. Stage 3 = anemia; decrease in circulating RBC parameters;
decrease in oxygen delivery to tissues (functional)
Stage 1 Stage 2 Stage 3
Bone marrow
iron stores
Decreased Absent Absent
Serum ferritin Decreased < 12 µg/L < 12 µg/L
Transferrin
saturation
Normal < 16% < 16%
Free RBC
protoporphyrin
Normal Increased Increased
Serum
transferrin
receptor
(TIBC)
Normal Increased Increased
Reticulocyte
Hb content
Normal Decreased Decreased
Hb Normal Normal Decreased
MCV Normal Normal Decreased
Clinical Signs
and
Symptoms
Present
 Pallor, fatigue, and weakness
 Papilledema
 Due to abnormal hemodynamics
 Optic nerve swelling = visual loss if left untreated (examine optic
fundi)
 Infants
 Bulging of the fontanelles (reversible)
 Children
 Psychomotor and mental impairment in the first 2 years if life
 Pica = compulsive ingestion of non-nutritive substances (ice, wooden
toothpicks, chalk, dirt, or cornstarch)
 Replace dietary Fe OR inhibit Fe absorption
 50% of IDA patients
LABORATORY CHARACTERISTICS
Hematology Studies
 CBC
 Hb
 Hct
 RBC count
 WBC count
 Platelet count
 Peripheral blood smear examination
 MCV = < 80 fL (microcytic)
 Patients at the early stage of the disease will have normocytic
and normochromic RBCs
 % HYPO = percentage of hypochromic RBCs
 Most sensitive and specific parameter of functional Fe deficiency
 Reticulocyte count
 Normal = 0.5-2.5%
 Reticulocyte hemoglobin content (CHr)
 Early indicator for Fe deficiency
 Infants and toddlers; prevent psychomotor and cognitive
development problems
 Sysmex XE2100 and Bayer ADVIA 2120
 < 27.2 pg Hb content = iron deficiency
 93.3% sensitive; 83.2% specific
 Identify IDA early in RBC hemoglobinization
 Hallmarks:
a. Decreased Hb and Hct (perhaps a normal RBC count)
b. Decreased MCV, MCH, and MCHC
c. Hypochromic and microcytic cells upon full manifestation
d. Normal platelet count (can be increased after blood loss)
e. Normal leukocyte count
f. Reticulocyte count = decreased or normal
g. Bone marrow exam = decrease in stainable iron; erythroid
hyperplasia
Clinical Chemistry Studies

 Iron studies
 Differentiate IDA from sideroblastic anemia and thalassemia
(also microcytic, hypochromic)
 Increased transferrin levels
 Reflects depletion of iron stores
 10-15 g/dL serum iron = iron deficiency
 Low percent saturation (more transferrin is available for less Fe)
 Iron turnover + percentage of serum iron used in RBC production
= increased; get the most possible from the little iron available
 Serum ferritin = detects deficient iron stores
 ≤ 12 µg/L = iron deficiency
 Acute-phase reactant
 Increased serum ferritin = infection, malignancy, iron overload,
inflammation, liver disease
 Low sensitivity (bone marrow biopsy or a trial of iron therapy to
differentiate IDA from other anemias)
 Distinguish between microcytic hypochromic anemias (IDA = low
ferritin; thalassemia and sideroblastic anemia = normal)
 Soluble transferrin receptor (sTfR)
 TfR = membrane-bound protein that captures transferrin with its 2
Fe atoms from ECF; fragment finds its way into serum
 Decreased iron stores = cells produce more TfR = higher serum
concentrations
 Differentiate between IDA and ACDs
 Indicator of erythropoietic activity
 Not an acute-phase reactant
 Exclude iron deficiency when serum ferritin concentrations > 30
µg/L in patients with inflammatory disease
 Distinguish ACD from IDA (TfRs do not increase in ACD)
 Also increased in sideroblastic anemia and thalassemia
ANEMIA OF INFLAMMATION or ANEMIA OF CHRONIC DISORDERS
Etiology
 2nd most prevalent (after IDA)
 Inflammation, infection, malignancy, systemic diseases
 Tuberculosis
 Lung abscess
 Bacterial endocarditis
 Neoplasms
 Rheumatoid arthritis
 Rheumatic fever
 Systemic lupus erythematosus (SLE)
 Uremia
 Chronic liver disease
 Minor hematologic abnormality
Pathophysiology
 Hypoproliferative anemia (underproduction of RBCs)
 Mildly shortened RBC lifespan
 Hepcidin (25-amino acid peptide hormone from the liver or monocytes)
 Gene expression regulated by:
1. Iron availability (hepatocyte signaling through the BMP
receptor complex)
2. IL-6-mediated inflammatory signaling pathway
 Interacts with ferroportin (cellular receptor; iron export channel)
= block release of iron from cells (tissue macrophages and jejunal
enterocytes)
 Hepcidin = iron sequestration and hypoferremia
 Blocks cellular efflux by binding and inducing degradation via
internalization of ferroportin at enterocytes and macrophages of
the MPS
 Excessive Hepcidin = MPS iron blockage = defective iron supply
for erythropoiesis = defective EPO production = anemia
 ACD due to malignancy:
 Inadequate EPO production
 Inadequate response of erythroid marrow to EPO
 Impaired Fe release due to increased Hepcidin production
producing a functional Fe deficiency
 Alterations in production of proinflammatory cytokines
 Decresed RBC production because of direct bone marrow
infiltration by malignant tumor cells or by primary marrow cell
malignancies
 Increased RBC destruction (immune or microangiopathic
hemolytic anemia)
 Acute or chronic blood loss
 Toxic effects of invasive therapy (chemotherapy or radiation
therapy)
 Indirect multiple causes (malignant disease, organ failure,
hemolytic anemias)
 Systemic diseases = release acute-phase reactants
 Elevated CRP, fibrinogen, haptoglobin, and ceruloplasmin
 Initiated by interleukin-1-β = fever, neutrophilia, leukocytosis,
acute-phase protein synthesis, lymphokine production
stimulation, lactoferrin release from granulocytes
 Anemia associated with renal disease
 Decreased EPO production by damaged kidneys
 Chronic liver disease
 Alcohol = toxic to bone marrow HPCs, marrow cellularity, and
RBC morphology
LABORATORY CHARACTERISTICS
 Elevations in:
 Platelet counts
 Total WBC counts
 Acute-phase reactants (CRP, fibrinogen, haptoglobin,
ceruloplasmin)
 CRP = may correlate with Hepcidin levels
Hematology Studies
 Mild hypoprolific anemia (28%-32% Hct range)
 Hb = as low as 5 g/dL in uremia
 Normochromic and normocytic
 Autoimmune disorder or chronic renal disease
 Compensatory erythroid hyperplasia OR reticulocytosis is absent
 Normal or increased bone marrow stores in macrophages
 Malignancy-associated:
 Leukoerythroblastosis = presence of both immature erythrocytes
and leukocytes are seen

 Teardrop RBCs (dacryocytes), schistocytes, helmet cells, fibrosis,
and hypochromia
 Total WBC count = based on degree of infection
 Platelets = normal
 Hemosiderin in bone marrow = increased or normal
 Decreased sideroblasts
 Recticulocyte count = < 2%
Clinical Chemistry Studies
 Decrease in:
 Serum iron levels (impaired recycling of iron from macrophages)
 Transferrin (TIBC)
 Percentage of saturation = very low in inflammation with abundant iron in
the marrow
 Low sTfR = synthesis is impaired by proinflammatory cytokines
 AOI may co-exist with IDA in women of childbearing age
 Hallmarks:
 Low serum iron
 Low TIBC
 Stainable iron in bone marrow
Treatment
 Treat the underlying cause
 Severe anemia = blood transfusion
 Recombinant human EPO (rHu-EPO) = counteract suppressive effects of
cytokines (IL-1)
 Ameliorates anemia in infants with infections
SIDEROBLASTIC ANEMIA
 Impaired production of adequate amounts of protoporphyrin
 Microcytic, hypochromic
 Iron = abundant in the bone marrow
 Prussian blue stain of bone marrow
 Normoblasts with iron deposits in the mitochondria surrounding
the nucleus = ring sideroblasts
 May be hereditary or acquired
a. Hereditary = X-linked, autosomal
b. Acquired
1. Primary sideroblastic anemia (refractory)
2. Secondary sideroblastic anemia caused by drugs and
bone marrow toxins
o Anti-tubercular drugs (isoniazid)
o Chloramphenicol
o Alcohol
o Lead
o Chemotherapeutic agents
Lead Poisoning
 Pb = interferes with porphyrin synthesis in these steps:
 Conversion of D-ALA to porphobilinogen (PBG) by ALA
dehydratase (or PBG synthase) = accumulation of D-ALA
(measurable in urine)
 Incorporation of iron into protoporphyrin IX by ferrochelatase
(also called heme synthetase) = accumulation of iron and
protoporphyrin
 Anemia due to Pb poisoning is normocytic and normochromic
 Chronic = microcytic, hypochromic
 Erythroid hyperplasia in bone marrow
 Basophilic stippling
 Lead inhibits pyrimidine 5’-nucleotidase = enzyme involved in the
breakdown of RNA in reticulocytes
 EDTA = used to chelate lead so it can be excreted in the urine
Porphyrias
 Impaired production of porphyrin component of heme
 Acquired or hereditary
 Single deficiencies of most enzymes in the synthetic pathway
 Products from earlier stages in the pathway accumulate in cells that
produce heme (RBCs and hepatocytes)
 Excess porphyrins = leak from the cells as they age and die
 Products also accumulate in body tissues
 Photosensitivity in skin with severe burns upon exposure to sunlight

Chapter Eleven: Megaloblastic Anemias
MEGALOBLASTIC ANEMIAS
 2 major divisions:
a. Vitamin B12 (cobalamin, Cbl) deficiency
b. Folic acid deficiency
 Altered DNA synthesis = megaloblastic (abnormal RBC precursors)
 Macrocytosis (MCV > 100 fL)
 Can occur even without a megaloblastic process (e.g.,
reticulocytosis)
 Most common causes: acquired
 Cbl deficiency = pernicious anemia; gastrectomy, inflammatory
disorders of the ileum, fish tapeworm (D. latum)
 Folate deficiency = inadequate dietary intake
 Abnormal nuclear maturation of RBCs
 Dyssynchrony or asynchrony (imbalance between nuclear and
cytoplasmic maturation)
 Prolonged premitotic interval = large nucleus, increased
cytoplasmic RNA, and early synthesis of Hb)
 Many cells do not undergo mitosis
 Cells break down in the bone marrow and produce increased
levels of LDH (lactate dehydrogenase)
 Impaired maturation in myelogenous WBCs and
megakaryocytes = leukopenia + neutrophilic
hypersegmentation and thrombocytopenia
 Megakaryocyte fragments + giant platelets = seen on PBS
 Pernicious anemia = also characterized by active intramedullary
hemolysis
 Neurological symptoms are common (neuropsychiatric disturbances)
 Peripheral neuropathy or depression
 Reversible
ETIOLOGY
B12 Deficiency
a. Increased utilization of vitamin B12
 Parasitic infections (D. latum [fish tapeworm])
 Pathogenic bacteria causing diverticulitis and small bowel
stricture
b. Malabsorption syndrome
 Gastric resection
 Gastric carcinoma
 Celiac disease (sprue)
c. Nutritional deficiency or diminished supply of B12
 Primary dietary sources = meat, eggs, milk
 Need for 5-15 µg/day to meet requirements
 Body can store large amounts of Cbl = years are required for
deficiency to develop
d. Pernicious anemia
 Due to chronic atrophic gastritis
 Deficiency in intrinsic factor from gastric parietal cells
Folate Deficiency
a. Abnormal absorption
 Celiac disease (sprue)
b. Increased utilization
 Pregnancy
 Acute leukemias
c. Treatment with antimetabolites
 Antimetabolites = folate antagonists
EPIDEMIOLOGY
 More women are affected than men
 Underlying gastritis = immunologically related to:
 Autoantibody to IF
 Serum IF inhibitor
 Autoantibodies to parietal cells
 Genetic predisposition (clustering of disease and autoantibodies in
families)
 Pernicious anemia = may be associated with autoimmune
endocrinopathies and anti-receptor autoimmune disease:
 Autoimmune thyroiditis (Hashimoto thyroiditis)
 IDDM
 Addison disease
 Primary ovarian failure
 Primary hypoparathyroidism
 Graves disease
 Myasthenia gravis
PHYSIOLOGY
 Megaloblastic dyspoiesis = due to B12 or folate deficiency
 Vitamin B12
 5000 µg in the body (1/3 in the liver)
 Loss per day: 5 µg/day
 Required amount per day: 5 µg/day (to compensate for the lost
amount)
 Amounts in normal diet (meat, eggs, milk) = 5-30 µg
 Folate
 Leafy vegetables; organ meats (liver and kidneys); fruits, dairy
products, cereals
 Low on storage (stores last only for about 3-4 months if diet is
folate-deficient)
 Alcohol = most common pharmacological cause of folate-
deficiency
Vitamin B12 (cobalamin) Transport
 3 binding proteins:
a. Intrinsic factor (IF)
b. Transcobalamin II
c. R proteins
 Intrinsic Factor (glycoprotein)
 Gastric parietal cells (mucosa of fundus of stomach)
 Acidic pH = B12 splits from food and binds with IF to form B12-IF
complex

o Loss of acidity (achlorhydria) = B12 does not split with
food and is not absorbed
 B12-IF complex = highly specific
o Absorbed in the ileum
o Only ileal enterocytes have the receptors to attach to
this complex
 Transcobalamin II (plasma polypeptide)
 From the liver
 Carrier of the vitamin to the liver and other tissues
 Also stimulates Cbl uptake by reticulocytes
 R proteins (Cbl-binding glycoproteins)
 Storage sites
 Eliminate excess cobalamin
 Produced by leukocytes
 TC I and TC III (in plasma, saliva, milk)
 TC I = backup transport system for endogenous cobalamin
(endogenous Cbl = produced in the GI by bacteria; not
absorbed)
Vitamin B12 and Folic Acid Deficiencies
 Vitamin B12 deficiency
 Dietary: malnutrition
 Medications (inhibitors) = H2-receptor antagonists; proton-pump
inhibitor drugs
 Malabsorption: achlorhydria; gastric resection
 Intestinal: microorganism overgrowth (short bowel syndrome);
sprue; D. latum infection
 Impaired utilization: TC II deficiency
 Increased demand: pregnancy
 Folate deficiency
 Inadequate intake
 Increased demand (pregnancy, infancy)
 Malabsorption disorders (sprue)
 Biologic competition for folate (bacterial overgrowth)
 Medications (inhibitors): anticonvulsants, chemotherapy agents,
methotrexate
 Alcohol
 Antibodies to IF or parietal cells
 Pernicious anemia = autoimmune disorder
PATHOPHYSIOLOGY
 Pernicious anemia = associated with chronic atrophic gastritis
Gastric Pathological Findings
 Fundus and body (of stomach) = acid-producing gastric parietal cells +
pepsinogen-secreting zymogenic cells
 Affected in pernicious anemia
 Target of autoantibodies: α and β subunits of the H+/K+-ATPase of
gastric parietal cells
 Early lesions: chronic infiltration of the submucosa with
inflammatory cells (gastritis)
 Intestinal metaplasia: zymogenic and parietal cells are replaced
with cuboidal cells that resemble those of the small intestinal
epithelium
 Autoantibodies = CD4 Th1 T-cells
 Antrum = gastrin-producing cells
 Not affected by pernicious anemia
 Clinical manifestations of autoimmune type A gastritis:
 Autoantibodies to parietal cells
 Autoantibodies to IF
 Achlorhydria
 Low serum pepsinogen (due to destruction of zymogenic cells)
 High serum gastrin
 Insidious onset (acquired megaloblastic anemias) = slow-developing
 Lemon-yellow skin
 Hyperpigmentation (melanin deposition) of:
 Nail beds
 Skin creases
 Periorbital areas
 Angular cheilitis = cracking at the corners of the mouth
 Dyspepsia
 Diarrhea
 Glossitis and painful tongue
 Early graying of the hair
 Tiredness, dyspnea on exertion, vertigo, or tinnitus secondary to anemia
 Congestive heart failure, angina, or palpitations
 B12 deficiency = neurological and cognitive abnormalities
 Parethesias (pins and needles), loss of balance, visual changes,
paraplegia, memory loss, dementia
 Granulocytopenia: infection, bleeding, or bruising
LABORATORY FINDINGS
Hematology
 Hb = severely decreased
 RBC count = decreased
 Hct = severely decreased (variable; since macrocytosis may not reflect the
decrease in RBCs)
 MCV = high (> 130 fL)
 MCH = increased (90% of cases)
 MCHC = normal
 Platelet counts = slightly decreased or normal
 WBC count = low (leukopenia); neutropenia (hypersegmented PMNs; > 4
lobes); eosinophilia
 Anisocytosis and poikilocytosis (macrocytic, ovalocytic)
 Metarubricytes (nRBCs) in PBS
 Inclusions: Howell-Jolly bodies, basophilic stippling, Cabot rings
 Reticulocyte count = < 1%
 Increase up to 25% in 5-8 days
 Pancytopenia = in ADVANCED cases only
 Promegaloblasts and nRBCs = due to extramedullary hematopoiesis
(spleen and lymph nodes)
 RDW = high (reflects degree of anisocytosis)
 Bone marrow examination:
 Hypercellular

 Megaloblastic changes in erythroid line or all cell lines
 Can also be hypocellular
 RBC precursors = enlarged; decreased nuclear-cytoplasmic ratio
(?)
o Erythroblasts = salami-like appearance of nuclear
membrane
 Dysplastic appearance
 Granulocytic precursors = enlarged; giant metamyelocytes with
incompletely-segmented nuclei
o Increased # of mitoses = diminished myeloid:erythroid
ratio
Clinical Chemistry
 Increased Fe stores
 Low serum B12 for B12 deficiency mb
 Low serum folate for folate deficiency mb
 Other assays for B12 deficiency
 Serum methylmalonic acid (high)
 Total homocysteine
 IF-blocking antibody
 Parietal cell antibody
 High gastrin levels
 Low pepsinogen levels
 Achlorhydria (absence of HCl); stomach
 LDH (lactate dehydrogenase) levels = elevated due to
intramedullary hemolysis
 Schilling Test
 Establish cause of B12 deficiency in a patient with no detectable
antibodies
TREATMENT AND MONITORING THERAPY
 B12 deficiency = monthly injections of 100 µg of vitamin B12
 Correct the anemia and also the neurological symptoms (if
present)
 Elderly patients with gastric atrophy
 Tablets with 25 µg to 1 mg vitamin B12 daily
 Treatment timeline (response is seen in the bone marrow 8-12 hours
following treatment)
1. In 2-3 days = reticulocyte count increases; peaks in 5-8 days
2. 1 week = Hct begins to increase
3. 4-8 weeks = Hct normalizes
4. 3-4 days = MCV increases (due to reticulocytosis) then decreases
5. 25-78 days = MCV normalizes
6. 6 months = disappearance of neurological symptoms
7. 24 hours = serum iron levels decrease

Hematology 1-complete

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