1. Toxicology of the Erythron
Dr R B Cope BVSc BSc(Hon 1) PhD cGLPCP DABT ERT
2. Learning Objectives
• To understand the key basic functional concepts of the
erythron;
• To understand the fundamentals of erythrokinetics;
• To understand and accurately interpret changes in erythrocyte
physical parameters (PCV/hematocrit, MCH, MCHC, MCV);
• To understand and accurately interpret changes to
erythrocyte morphology;
• To understand and accurately recognize common abnormal
changes in the erythron.
3. Basic Concepts of Erythrocyte Function
• The erythron:
– Consists of circulating erythrocytes + bone marrow
precursors + bone marrow progenitor centers + stem cells;
– Key function is oxygen transport mediated by hemoglobin;
4. Basic Concepts of Erythrocyte Function: Heme Synthesis
Disruption of heme synthesis will result
in prophyrias due to the accumulation
of heme precursors or catabolites.
Porphyrias are most commonly genetic
(heritable) disorders of
metabolism, however xenobiotic-
induced porphyrias (or exacerbation of
inherited porphyrias) may also occur
(common: e.g. lead inhibits
ferrochetalase).
Porphyrias may or may not be
associated with anemia depending on
which part of the heme cycle is
disrupted.
The most common cause (in humans)
of reduced heme synthesis is iron
deficiencies.
5. Basic Concepts of Erythrocyte Function: Globin Synthesis
The switch from fetal to adult Hb varies considerably across species. In normal humans, fetal
Hb is present until about 8 months of life. In normal adult humans ~ 97% of Hb is HbA, ~ 3% is
HbA2. The types of Hb present in adults varies across species
6. Note: Fe is in the Fe2+ in the majority of Hb under normal conditions
7. Porphyrias
• Supplementary reading:
– http://emedicine.medscape.com/article/1389981-
overview
– Xenobiotic-induced porphyria cutanea tarda is the only
one I have personally come across:
http://emedicine.medscape.com/article/1103643-overview
8. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Fetal Hb has a higher affinity for O2 than normal adult Hb – lower oxygen delivery to the
tissues compared with adults
10. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
2,3-DPG = 2,3-diphosphoglycerate
• Remember:
• Left shift = less oxygen to the tissues = tissue hypoxia
• Right shift = more oxygen to the tissues
11. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Common and important toxicological situations affecting the
O2Hb dissociation curve include:
– Oxidative attack on erythrocytes and
methemoglobinemias
– Formation of sulfhemaglobin
– Carbon monoxide
12. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Methemoglobinemia
– Common cause of toxicological insult to the erythron
– The presence of Fe in the Fe2+ reduction state is critical for
normal O2 carrying capacity
– Any oxidizing agent has the capacity to produce metHb by
oxidizing Hb Fe2+ to Hb Fe3+ (ferrous ferric)
13. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Methemoglobinemia
– The critical effect of metHb is decreased tissue oxygen
supply (i.e. tissue hypoxia)
– MetHb cannot bind oxygen
– The presence of metHb results in a left shift of the O2Hb
dissociation curve, further exacerbating tissue hypoxia
14. • Blood and mucous membranes are discolored brown (“muddy”)
• Blood color change does not reverse (or reverses slowly) with exposure to
oxygen (i.e. bubbling oxygen through tube of blood)
15.
16.
17. Hemolysis and other signs of oxidative erythrocyte injury are
commonly present (e.g. Heinz bodies, intravascular hemolysis)
18. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Methemoglobinemia
– Note: Rodents are notoriously resistant to oxidative
damage to the erythron and the formation of metHb
– Note: Routine rodent studies alone may not detect the
propensity for metHb formation and/or oxidative damage
to the erythron
– Note: Children under 4 years of age are more susceptible
to oxidative damage to the erythron
– Note: Cats are notoriously susceptible to oxidative damage
to the erythron
19. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Methemoglobinemia
– Note: in some human subpopulations glucose-6-
phosphate dehydrogenase deficiency common (typically
people of African, Middle Eastern and South Asian descent
– protective adaptation to endemic malaria). These
subpopulations are highly susceptible to oxidative assault
on the erythron
20.
21. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Methemoglobinemia
– Note: Persons with pyruvate kinase deficiency are highly
susceptible to oxidative damage to the erythron (lower
energy generation lower capacity to recycle GSH)
• Also increases 2,3-bisphosphoglycerate (2,3 BPG) right shift of
the O2Hb dissociation curve increased tissue oxygenation
individuals with PK deficiency may have a greater capacity for
physical activity than others with similarly low hemoglobin levels.
– Note: Persons with cytochrome b5 reductase deficiency
are highly susceptible to oxidative damage to the erythron
(reduced capacity to reverse metHb)
22. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Methemoglobinemia
– Note: Any organo-amine compound should be suspected
of being a cause of oxidative damage to the erythron until
proven otherwise
– Note: any nitrite or nitrate compound (or compounds that
are metabolized to a nitrite or a nitrate) are similarly
suspect
– Note: chromates are also similarly suspect
– Note: Halogenated benzenes are also similarly suspect
23. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Sulfhemoglobinemia
– Due to the formation of ferric sulfide in Hb
– Cannot be reversed metabolically – requires the
replacement of affected erythrocytes
– Produces a right shift of the O2Hb dissociation curve
• Question: does this make tissue oxygenation better or
worse?
24. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• Sulfhemoglobinemia
– Usually drug induced
• Drugs associated with sulfhemoglobinemia include
acetanilid, phenacetin, nitrates, trinitrotoluene and
sulfur compounds (mainly
sulphonamides, sulfasalazine)
– Occupational exposure to sulfur compounds is another
potential cause
– > 0.5 g% sulfHb is abnormal
25.
26.
27. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• CO-Hb; carbon monoxide poisoning; carboxy-Hb
– Can occur due to either inhalation of CO (notably products
of incomplete combustion) or xenobiotics that are
metabolized to CO (common example is methylene
chloride)
– Hemoglobin binds to carbon monoxide preferentially
compared to oxygen (approx 240:1)
– COHb will not release the carbon monoxide, and therefore
hemoglobin will not be available to transport oxygen from
the lungs to the rest of the body
28. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• CO-Hb; carbon monoxide poisoning; carboxy-Hb
– COHb has a half-life in the blood of 4 to 6 hours
– T½ reduced by exposure to oxygen
– COHb increases the risk of infarction (increased incidence
of ischemic disease in users of methylene chloride)
– Produces a left shift of the O2Hb dissociation curve
• Question: does this make tissue oxygenation better or
worse?
29. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• CO-Hb; carbon monoxide poisoning; carboxy-Hb
– Produces a distinctive “cherry red” discoloration of
blood, tissues, skin
– Skeletal muscle is notably bright red due to the formation
of carboxymyoglobin (used commercially to keep meat red
and more attractive)
– Persons who have died from CO poisoning are described as
looking rosy cheeked and healthy (cherry red means dead)
30. Conditions Affecting Oxygen Carrying Capacity:
Oxygen Hb Dissociation Curves
• CO-Hb; carbon monoxide poisoning; carboxy-Hb
– Important issues:
• Samples must be collected in air-tight tubes
• Samples must be analyzed quickly otherwise falsely low
levels of COHb may be detected
• “Cherry red” discoloration of blood, skin, tissues etc.
fades with time – important diagnostically
• Bubbling oxygen through a tube of blood will hasten
the loss of discoloration – used diagnostically
31.
32.
33.
34.
35. Basic Concepts of Erythrocyte Function: Iron Metabolism
• Except for external blood loss, there is no normal mechanism for the excretion of iron;
• Provided the enterothelium remains intact, iron absorption is highly regulated;
• Under normal circumstances, there is little free iron present in plasma (total iron binding
capacity is not saturated)
• Transferrin is a gamma-globulin
36. Basic Concepts of Erythrocyte Function: Iron Metabolism
• A wide variety of divalent metals compete with Fe2+ for uptake by DMT-1 (notably
Cd2+, Pb2+);
• The pH of the small intestine favors the formation of Fe3+ (mostly insoluble) which must
be reduced by Dcytb to Fe2+ (soluble) for absorption to occur.
37. Basic Concepts of Erythrocyte Function: Iron Metabolism
• Hepcidin is the key hormone regulating Fe uptake from the GI;
• Increased hepcidin occurs in a wide variety of chronic disease states (carcinogenesis) or
inflammatory diseases leading to “anemia of chronic disease.”
• Anemia of chronic disease is a common finding in chronic toxicology studies.
38. Measures of Iron Status: Serum Iron
• Measures the amount of iron bound to transferrin in the
serum
• Unreliable as a measure of total body iron stores
• Can be reported as a % of total iron binding capacity (see
later)
39. Measures of Iron Status: Serum Iron
• Conditions associated with low SI
– Iron deficiency
– Acute and chronic inflammatory processes/disease
(anemia of chronic disease, anemia of inflammatory
disease)
– Hypoproteinemia (notably advanced liver disease)s
– Hypothyroidism
– Renal disease
40. Measures of Iron Status: Serum Iron
• Conditions associated with high SI
– Hemolytic anemia (due to release of Fe from destroyed
erythrocytes)
– Sampling error resulting in hemolysis
– Excessive glucocorticoids in some species (e.g. Cushing’s
disease)
– Iron overload
• Acquired – Fe overload (Fe poisoning, excessive
transfusions etc.)
• Hereditary
– Non-regenerative anemias (decreased Fe utilization)
41. Measures of Iron Status: Serum Total Iron Binding Capacity
• TIBC is an indirect measure of how much Fe the transferring
present in serum will bind
• Under normal conditions, about ⅓ of transferring binding sites
are occupied under normal conditions – serum transferrin is
not saturated under normal conditions
• The difference between TIBC and SI is the amount of available
Fe-binding capacity = unbound iron-binding capacity
42. Measures of Iron Status: Serum Total Iron Binding Capacity
• TIBC is increased in Fe deficiency in most species
• TIBC is reduced (or even saturated [i.e. UIBC = 0]) in:
– Fe poisoning
– Intravascular hemolysis
– Sampling error (hemolysis)
43. Measures of Iron Status: Ferritin
• Ferritin represent the labile (i.e. rapidly available) storage
form of Fe
• Small amounts of ferritin are normally present in the
circulation as part of normal Fe homeostasis
• Serum ferritin is decreased during iron deficiency
44. Measures of Iron Status: Ferritin
• Serum ferritin is increased during:
– Hemolytic anemia
– Iron overload
– Acute and chronic inflammation
– Liver disease
– Neoplasia (notably lymphoma)
– Malnutrition
45. Common Causes of Disruptions of Iron Status in Repeated
Dose Toxicology Studies
• Iron deficiency and/or reduced iron uptake
– More likely to be seen in chronic studies
– Early stages may occasionally be seen in sub-chronic studies
– In rodent studies, the animals are relatively young at the start of
the study and are still rapidly growing (increased iron
requirement + reduce capacity to absorb it from the GI + higher
Hb turnover associated with switch from HbF to HbA)
– Iron deficiencies can also can result when high concentrations of
the test article are used in the diet – dietary dilution of essential
minerals
46. Common Causes of Disruptions of Iron Status in Repeated
Dose Toxicology Studies
• Iron deficiency and/or reduced iron uptake
– Test article either inhibits or is a competitive substrate for
enterothelial DMT-1 (typically other divalent metals) or
Dcytb
– Test article is an inhibitor of or competitor ferroportin
– Test article reduced dietary iron bioavailability (binding in
gut insoluble) e.g. starches, tanins, phytates, vit. C, etc..
47. Common Causes of Disruptions of Iron Status in Repeated
Dose Toxicology Studies
• Chronic disease states, notably neoplasia (increased hepcidin)
• Significant inflammatory diseases (acute or chronic; increased
hepcidin)
• Hemolysis (error or due to toxicological effects)
• Severe liver disease
48. Erythrokinetics
Essential for RBC production:
• Iron
• Vitamin B12 (cobalamin)
• Folic Acid
49.
50. Erythrokinetics
• The cell is released from the bone marrow as a reticulocyte
– Circulating red blood cells are ~1% reticulocytes
– After 1–2 days these ultimately become "erythrocytes" or mature red
blood cells
Question: what does it mean if you have an increased % of
reticulocytes in the peripheral blood?
Question: what does it mean if you have an increased % of
normoblasts or nucleated RBCs in the peripheral blood?
51.
52.
53. Erythrokinetics
• The following characteristics can be seen changing in the
erythrocytes when they are maturing:
– They show a reduction in the cell size;
– The cytoplasmic matrix increases in amount;
– Staining (Wright’s stain) reaction of the cytoplasm changes from blue
to pinkish red (this is because of the decrease in the amount of RNA
and DNA);
– Initially the nucleus was large in size and contained open chromatin.
But with the maturation of RBC's the size of the nucleus decreases and
finally disappears with the condensation of the chromatin material.
Question: what does it mean if you have an increase in the
number of RBCs in the peripheral circulation that stain blue
(technical term = polychromasia or polychromatophilia)?
54.
55. Erythrokinetics
Question: what does it mean if you have an increase in variation
of erythrocyte size in peripheral blood (technical term =
anisocytosis)?
56.
57. Erythrokinetics
• Lifespan of an RBC in peripheral blood is species specific – in
the range of 70 – 150 days in mammals depending on species;
35 days or so in birds
• Eryptosis (RBC programmed cell death)
– Form of apoptosis
– Process makes the RBC prone to the extravascular pathway
of erythrocyte destruction (phagocytosis by the
reticuloendothelial systems of the liver, spleen and bone
marrow [predominantly the red pulp of the spleen])
– Normally eryptosis and erthyropoesis exist in balance
58. Erythrokinetics
• Two pathways for RBC destruction:
– Extravascular pathway (predominantly in the spleen, but also bone
marrow and liver)
– Intravascular pathway (i.e. lyse within the circulation)
59. The anatomic structure of the spleen is ideal for testing the metabolic machinery and
pliability of the red blood cells. Within the splenic pulp, red blood cells are concentrated
and their intracellular metabolic pathways stressed. Following this, red blood cells must
pass through 2- to 5-µm pores to enter the sinusoidal system. Unusually rigid cells or a
cell containing inclusion bodies will be unable to pass this test and will be destroyed by
sinusoidal reticuloendothelial cells.
66. Evaluation of the Erythron:
Indicators of the Circulating RBC Mass
• Endpoints that are commonly used are:
– Hematocrit (= packed cell volume = PCV)
– Hemoglobin concentration of peripheral blood
– RBC count
68. Evaluation of the Erythron: Hematocrit
(=HCT=Packed Cell Volume = PCV)
• HCT measured by centrifugation is a very accurate measure (±
1%)
• Most commonly HCT is a calculated value from automated cell
counters where HCT% = RBC/ul x mean corpuscular volume
(fL)
– Calculated HCT has a higher level of error
– Cell counter MUST be calibrated to the species being
examined
– Birds have eliptical nucleated RBCs which interferes with
this method
69. Evaluation of the Erythron: Hematocrit
(=HCT=Packed Cell Volume = PCV)
• Typically plasma protein concentration by refractometry is
measured at the same time
• Typically the plasma color is examined (red for
hemolysis, yellow of icterus/jaundice, lipidemia = pink and
opaque)
70.
71. Evaluation of the Erythron: Hb Concentration
• Provides the most direct measurement of the oxygen
transport capacity of blood
• Value should be ~ ⅓ of the HCT if RBCs are of normal size
• Typically a colorimetric measurement
• Must be corrected if Heinz bodies, hemolysis, or lipidemia is
present
• Major advantage over HCT is it allows calculation of the MCH
and MCHC
72. Evaluation of the Erythron: RBC Count
• Automated machine counting is reasonably accurate if
appropriately calibrated (except for birds)
• Manual hemocytometer counts have a large error and are of
little value except in birds
• Major value of RBC count is that it allows calculation of the
MCV and MCH (see below)
73. Evaluation of the Erythron: RBC Count
• Low values occur in anemia
• High values occur in polycythemia (or blood doping)
• Spuriously high values occur with dehydration and excitement
in species with a contractile spleen (notably horses)
74. Evaluation of the Erythron:
Factors Affecting HCT, Hb, RBC
• Low values occur in anemia
• High values occur in polycythemia (or blood doping)
• Spuriously high values occur with dehydration and excitement
in species with a contractile spleen (notably horses)
• Overhydration will result in spuriously low values
• Fluid shifts (e.g. shock) can cause spurious values
75. Evaluation of the Erythron:
Factors Affecting HCT, Hb, RBC
• The hydration/intravascular volume status of the animal must be
determined in order to accurately interpret HCT, Hb and RBC!
• How do you do this?
– Total serum or plasma protein (serum or plasma specific gravity)
in combination with the albumen:globulin ratio (A:G) is a useful
quick guide
– If the TP and plasma albumen are increased (in proportion) and
the A:G ratio remains within the normal range, this is an
indicator of dehydration (particularly if combined with an
increased HCT HB & RBC)
Why?
76. Evaluation of the Erythron:
Factors Affecting HCT, Hb, RBC
• If both the TP and A:G are within the normal range and the
HCT Hb & RBC are low, what does this suggest?
• If the TP and A:G ratio are within the normal range and the
HCT Hb & RBC are high, what does this suggest?
77. Evaluation of the Erythron:
RBC Indices Used in the Classification of Anemia
• Mean corpuscular volume (MCV) = average RBC volume in fL
• Mean corpuscular hemoglobin (MCH) = average mass of Hb
present per RBC in pg
• Mean corpuscular hemoglobin concentration (MCHC) =
average concentration of Hb in RBC in g/100 mL of RBCs
• Red cell distribution width = coefficient of variation of the RBC
volume distribution
78. Evaluation of the Erythron:
Mean Corpuscular Volume (MCV)
• Can be a calculated value or determined by an automated cell
counter
– Calculate by dividing the HCT by the RBC count and
converting to fL
79. Evaluation of the Erythron:
Mean Corpuscular Volume (MCV)
• Common causes of increased MCV (macrocytosis)
– Reticulocytosis – Why?
– Anything that inhibits nucleic acid synthesis and inhibits cell
division i.e. reduced number of cell divisions during RBC
ontogeny in the bone marrow. Commonly B12 deficiency
• B12 deficiency is classically macrocytic and hypochromic (see later)
– Anything that delays RBC maturation – Why?
– Genetics – must make sure you are using the correct normal
control values for the given strain
– Spurious – RBC agglutination will result in falsely high MCV
values
80. Evaluation of the Erythron:
Mean Corpuscular Volume (MCV)
• Common causes of increased MCV (macrocytosis)
– Megaloblastic anemia, the most common cause of macrocytic
anemia, is due to a deficiency of either vitamin B12, folic acid, or both
– Pernicious anemia is caused by a lack of intrinsic factor, which is
required to absorb vitamin B12 from food. A lack of intrinsic factor
may arise from an autoimmune condition targeting the parietal cells
(atrophic gastritis) that produce intrinsic factor or against intrinsic
factor itself. These lead to poor absorption of vitamin B12. Alcoholism
is a major cause of B12 malabsorption
– Macrocytic anemia can also be caused by damage to the stomach
(reduced B12 and folate absorption)
– Hypothyroidism
– Methotrexate, zidovudine, and other drugs may inhibit DNA
replication.
81. Evaluation of the Erythron:
Mean Corpuscular Volume (MCV)
• Common causes of increased MCV (macrocytosis)
– Macrocytic anemia can be further divided into
"megaloblastic anemia" or "nonmegaloblastic macrocytic
anemia".
– The cause of megaloblastic anemia is primarily a failure of
DNA synthesis with preserved RNA synthesis, which results
in restricted cell division of the progenitor cells. The
megaloblastic anemias often present with neutrophil
hypersegmentation (six to 10 lobes).
– The nonmegaloblastic macrocytic anemias have different
etiologies (i.e. unimpaired DNA globin synthesis,) which
occur, for example, in alcoholism.
82. Evaluation of the Erythron:
Mean Corpuscular Volume (MCV)
• Immature animals of most species have small RBCs and lower
normal MCV values – must use the correct normal range for
the species, strain and age group!
• Causes of reduced MCV (microcytosis)
• Iron deficiency is a classical cause of microcytosis – due
to an additional round of cell division required to reach
the critical cytoplasmic concentration of Hb necessary
to stop further DNA synthesis and cell division of RBC
precursors. Typically Fe deficiency anemias are
microcytic hypochromic anemais
83. Evaluation of the Erythron:
Mean Corpuscular Volume (MCV)
• Causes of reduced MCV (microcytosis)
– Heme synthesis defect
• Iron deficiency anemia
• Anemia of chronic disease (more commonly presenting
as normocytic anemia)
– Globin synthesis defect
• Alpha-, and beta-thalassemia
• HbE syndrome
• HbC syndrome
• Various other unstable hemoglobin diseases
84. Evaluation of the Erythron:
Mean Corpuscular Volume (MCV)
• Normocytic anemias
– Normocytic anemia occurs when the overall hemoglobin
levels are decreased, but the red blood cell size (mean
corpuscular volume) remains normal.
– Causes include:
• Acute blood loss
• Anemia of chronic disease
• Aplastic anemia (bone marrow failure)
• Hemolytic anemia
85.
86. Evaluation of the Erythron:
Mean Corpuscular Hb (MCH)
• MCH = Hb ÷ RBC count
• Generally factors affecting the MCV affect the MCH in a
similar way
– If the MCV is decreased (e.g. Fe deficiency), the MCH
decreases since smaller RBCs contain less Hb
• In general, the MCH is much less value than the other
parameters in the classification of anemias
• Interpretation of the Hb status of RBCs should be based on
the MCHC rather than the MCV because the MCHC corrects
for cell volume
87. Evaluation of the Erythron:
Mean Corpuscular Hb Concentration (MCHC)
• MCHC = Hb ÷ HCT and then adjusted to g/dL
• MCHC is the most accurate of the RBC indices because it does
not require a RBC count
• MCHC is the most useful parameter in classification of
anemias
• A true increase in MCHC does not normally occur
– Increased MCHC is almost always spurious – due to
hemolysis (calculation assumes that all Hb is contained
within RBCs which is not true if hemolysis has occurred)
88. Evaluation of the Erythron:
Mean Corpuscular Hb Concentration (MCHC)
• Reduced MCHC (Hypochromasia = hypochromia)
– Occurs with reticulocytosis – reticulocytes are larger cells
and contain less Hb, accordingly the Hb concentration is
decreased
– Iron deficiency due to reduced synthesis of Hb
– Vitamin B6 deficiency – reduced synthesis of Hb
– Lead exposure (inhibition of synthesis of Hb)
– Drugs or xenobiotics that disrupt Hb synthesis
90. Evaluation of the Erythron:
RBC Distribution Width (RDW)
• Coeficient of variation of the RBC volume distribution
• RDW = SDMCV ÷ MCV x 100
• Index of the variation in size (i.e. aniocytosis) of RBCs
• Anemias which are microcytic or macrocytic will have
increased anioscytosis and an increased RDW
• Significant reticulocytosis will result in an increased RDW and
anisocytosis
93. Peripheral Blood Smear – RBC Morphology
• Stains
– New methylene blue – acidic structures stain blue or
purple (i.e. DNA, RNA, basophil granules)
– Romanowsky stains (e.g. Wright’s, Diff-Quick) – acidic
structures stain blue to purple, basic groups stain red
(protein, eosinophil granules)
94. Peripheral Blood Smear – RBC Morphology
• Rouleaux formation
– RBCs for stacks
– Presence indicates an alteration of RBC surface charge
(zeta potential)
– Interpretation depends on the species
• Horses normally have some Rouleaux formation in
health
– Rouleaux formation correlates with the RBC sedimentation
rate i.e. protein coating of the surface of RBCs (e.g. fibrin
during inflammatory processes, increased
globulins, multiple myeloma) increased ESR + increased
Rouleaux formation
95.
96. Peripheral Blood Smear – RBC Morphology
• Agglutination
– Grape-like congregations of RBCs due to the presence of a
cross-linking antibody (typically anti RBC IgM)
– Important in toxicology because there are numerous
xenobiotics that act as haptens on the surface of RBCs and
generate an autoimmune anemia
97.
98. Peripheral Blood Smear – RBC Morphology
• Spherocytes
– Associated with immune mediated anemias – an important
biomarker of extravascular immune-mediated RBC
destruction
– Result of phagocytosis of antigens on the surface of RBCs
– Can result from phagocytosis of Heinz bodies (areas of
oxidative precipitation of Hb in the erythrocyte –
biomarker of oxidative damage to Hb)
– Rarely, can result of changes to cell membrane compositon
(acquired or hereditary)
99.
100. Peripheral Blood Smear – RBC Morphology
• Polychromatophilia
– Cells have a light blue-gray color when stained with a
Wright’s stain
– Classical feature of increased numbers of immature
peripheral RBCs in the peripheral blood i.e. accelerated
release of RBCs from the bone marrow
– Classical characteristic feature of reticulocytes i.e.
reticulocytosis is described as macrocytic, polychromasia
and polychromatophilic..
101.
102. Peripheral Blood Smear – RBC Morphology
• Poikilocytosis
– General term for abnormally shaped RBCs
– Can be spurious due to storage of sample before analysis
– Can be normal in some species and at some stages of
development e.g. switching from fetal to adult Hb
– Indicate trauma to the RBC cell membrane due to
• Turbulent blood flow
• Intravascular fibrin (microangiopathic effects)
– Specific types of poikilocytes can be associated with
particular diseases e.g. acanthocytes are associated with
particular types of liver diseases
104. Peripheral Blood Smear – RBC Morphology
• Echinocytosis
– Form of poikiloctyes
– Can be due to artifact
– Can be due to xenobiotic exposure, particularly if effected
blood electrolytes or membrane electrolyte transport or
osmotic effects
105.
106. Peripheral Blood Smear – RBC Morphology
• Hemlet cells (keratocytes)
– Biomarker of oxidative damage to RBCs
– Develop due to rupturing of a cell membrane vesicle or
membrane blebs
– Can also be produced by microangiopathic processes (see
schistocytes below)
107. Peripheral Blood Smear – RBC Morphology
• Schistocyte
– Irregular RBC fragments due to cutting of the RBC into
pieces by intravascular fibrin deposits
108.
109. Peripheral Blood Smear – RBC Morphology
• Basophilic stippling
– Due to residual clumps of RNA, or precipitated ribosomes
or mitochondria in RBCs
– A characteristic of lead exposure (rare finding) – typically
there is basophilic stippling but NO polychromasia (why?)
– Can occur during regenerative anemias in conjunction with
polychromasia
– Also seen with severe burns, some xenobiotics, septicemia
110.
111. Peripheral Blood Smear – RBC Morphology
• Heinz bodies
– A classical biomarker of oxidative damage to the erythron
– Due to denatured Hb
– RBCs prone to hemolysis
112. Peripheral Blood Smear – RBC Morphology
• Metarubricyte
– Term refers to the presence of any form of nucleated RBC
in the peripheral blood
– Indicates highly increased demand for RBCs and may be
appropriate (e.g. response to increased erythropoesis in
responsive anemias) or inappropriate (e.g. due to lead
poisoning, Fe deficiency, Cu deficiency etc.)
113. Bone Marrow Evaluation
• Parameters:
– Cellularity
– Number of megakaryocytes (platelet precursors)
– Myeloid: erythroid ratio
– Morphology
114.
115. Bone Marrow Evaluation
• M:E ratio
– Usually used to determine the efficiency of the response
to anemia
– Should be interpreted with the WBC count i.e. if the WBC
count is normal and the M:E is decreased it is indicative of
an increased production of RBCs and a normal response to
anemia
– A high M:E ratio in the presence of a normal WBC count
and anemia indicates a problem with RBC production
116.
117. Regenerative Vs Non-Regenerative Anemias
• Characteristics of regenerative anemias = evidence of
increased production of RBCs
– Polychromasia – Why?
– Reticulocytosis – Why?
– Anisocytosis + increased RDW – Why?
– Increased MCV + macrocytosis – Why?
– Basophilic stippling – Why?
– Hypercellular bone marrow with a reduced M:E – Why?
– Are all of the above responses appropriate and if so, why?
118. Regenerative Vs Non-Regenerative Anemias
• Characteristics of regenerative anemias = evidence of
increased production of RBCs
– Polychromasia – Why?
– Reticulocytosis – Why?
– Anisocytosis + increased RDW – Why?
– Increased MCV + macrocytosis – Why?
– Basophilic stippling – Why?
– Hypercellular bone marrow with a reduced M:E – Why?
– Are all of the above responses appropriate and if so, why?
119. Regenerative Vs Non-Regenerative Anemias
• Characteristics of regenerative anemias
– Tend to be non-chronic
– Internal blood loss and hemolytic anemias tend to respond
more rapidly than external blood loss anemias (why?)
120. Regenerative Vs Non-Regenerative Anemias
• Characteristics of non-regenerative anemias
– Tend to chronic – why?
– By definition there is an inadequate bone marrow response
which may be due to:
• Not enough time – RBC destruction is faster than RBC
production
• Inadequate precursor materials (e.g. Fe, B12, B6 etc.)
• Increased hepcidin due to inflammatory diseases (decreased
Fe)
• Renal disease – not enough erythropoeitin
• Bone marrow destruction (neoplasia, infections)
121. Case Studya1
• Following data (group mean from treatment group) was
from day 45 of a 90 day study in rats:
Parameter Value High or low relative to normal Range, Pre-
exposure measurements and control Group (all
changes statistically significant)
HCT 13 % Low
Hb 3.9 g/dL Low
RBC 1.59 x 106/ul Low
MCV 81 fL High
MCH 24.5 pg
MCHC 30.0 % Low
Reticulocytes 16.5 % High
264 x 103/ul
Nucleated RBCs 3 per 100 WBC High
Total protein 3.8 g/dL Low
Albumin 1.8 g/dL Low
A:G 0.90 Normal
RBC morphology: anisocytosis,
polychromasia
122. Case Studya1
• Following data (group mean from treatment group) was
from day 45 of a 90 day study in rats:
Parameter Value High or Low Compared With Normal
Range and Control Group
Platelets 653 x 103/uL High
WBC 17.5 x 103/uL High
WBC morphology is normal 1.59 x 106/ul Low
123. Case Study 1
• Answer the following questions
– What is the type of erythron problem present?
– What other problems are present?
– What is the likely mechanism involved?
– Is the problem being reversed/repaired?
– What would you hope to see by day 90 of the study?
– What would you expect to see 30 days following the
cessation of exposure to the test article?
• Why?
• What conditions might have to happen for any change
to occur?
• What conditions might prevent any putative recovery?
124. Case Studya2
• Following data (group mean from treatment group) was
from day 90 of a rat study
Parameter Value High or low relative to normal Range, Pre-
exposure measurements and control Group (all
changes statistically significant)
HCT 15% Low
Hb 4.4 g/dL Low
RBC 1.79 x 106/ul Low
MCV 84 fL High
MCH 24.6 pg
MCHC 29.3 % Low
Reticulocytes 24 % High
430 x 103/ul
Nucleated RBCs 6 per 100 WBC High
Total protein 6.5 g/dL Normal
Albumin 2.9 g/dL Normal
A:G 0.81 Normal
RBC morphology: anisocytosis,
polychromasia, spherocytosis,
125. Case Study 2
• Answer the following questions
– What is the type of erythron problem present?
– What other problems are present?
– What is the likely mechanism involved?
– Is the problem being reversed/repaired?
– What would you hope to see by day 90 of the study?
– What would you expect to see 30 days following the
cessation of exposure to the test article?
• Why?
• What conditions might have to happen for any change
to occur?
• What conditions might prevent any putative recovery?
126. Case Study 3
• Following data (group mean from a treatment group) was from day 3 of a 90 day
study in cats. Some deaths have occurred in this exposed group
Parameter Value High or low relative to normal Range, Pre-
exposure measurements and control Group (all
changes statistically significant)
HCT 25.2% Low
Hb 11.2 g/dL Normal
RBC 5.5 x 106/ul Low
MCV 45 fL Normal
MCH 20 pg
MCHC 44 % High
Total protein 8.5 High
Albumin 3.9 g/dL Normal
A:G 0.85 Normal
RBC morphology: excessive Heinz
bodies present
Additional comments: serum is
red tinged, blood was chocolate
colored, urine was dark brown
127. Case Study 3
• Answer the following questions
– What is the type of erythron problem present?
– What other problems are present?
– What is the likely mechanism involved?
– Is the problem being reversed/repaired?
– What other problems might occur with this?
– What would you hope to see by day 90 of the study?
– What would you expect to see 30 days following the
cessation of exposure to the test article?
• Why?
• What conditions might have to happen for any change
to occur?
• What conditions might prevent any putative recovery?
128. Case Studya4
• Following data (group mean from treatment group) was
from day 90 of a 90 day study in rats:
Parameter Value High or low relative to normal Range, Pre-
exposure measurements and control Group (all
changes statistically significant)
HCT 12.8 % Low
Hb 4.0 g/dL Low
RBC 2.25 x 106/ul Low
MCV 56.9 fL High
MCH 17.8 pg Low
MCHC 31.3 % Low
Reticulocytes 0% Low
Nucleated RBCs 0 per 100 WBC High
RBC morphology: hypochromasia
129. Case Study 4
• Answer the following questions
– What is the type of erythron problem present?
– What other problems are present?
– What is the likely mechanism involved?
– Is the problem being reversed/repaired?
– What other problems might occur with this?
– What would you hope to see by day 90 of the study?
– What would you expect to see 30 days following the
cessation of exposure to the test article?
• Why?
• What conditions might have to happen for any change
to occur?
• What conditions might prevent any putative recovery?
130. Case Studya5
• Following data (group mean from treatment group) was
from day 90 of a 90 day study in rats:
Parameter Value High or low relative to normal Range, Pre-
exposure measurements and control Group (all
changes statistically significant)
HCT 26 % Low
Hb 8.8 g/dL Low
RBC 5.23 x 106/ul Low
MCV 49.7 fL Normal
MCH 16.8 pg
MCHC 33.8 % Normal
Reticulocytes 0.1 % Normal
52 x 103/ul
Nucleated RBCs 0 per 100 WBC High
Total protein 9.3 g/dL High
Albumin 1.9 g/dL Low
A:G 0.0.26 low
RBC morphology: Normal
131. Case Study 5
• Answer the following questions
– What is the type of erythron problem present?
– What other problems are present?
– What is the likely mechanism involved?
– Is the problem being reversed/repaired?
– What other problems might occur with this?
– What would you hope to see by day 90 of the study?
– What would you expect to see 30 days following the
cessation of exposure to the test article?
• Why?
• What conditions might have to happen for any change
to occur?
• What conditions might prevent any putative recovery?
132. Case Study 1
• Regenerative blood loss anemia
• Macrocytic hypochromic anemia with polychromasia and
reticulocytosis
• Loss of total protein due to hemorrhage + subsequent fluid
shifts into the vascular compartment
133. Case Study 2
• Macrocytic hypochromic regenerative hemolytic anemia with
evidence of extravascular erythrophagocytosis
• Autoimmune mediated anemia
134. Case Study 3
• Oxidative intravascular hemolytic anemia + met Hb
135. Case Study 4
• Microcytic, hypochromic non-regenerative anemia
• Fe deficiency
136. Case Study 5
• Normocytic, normochromic anemia + hyperproteinemia due
increased globulins + decreased albumen
• Chronic inflammatory disease