Class Review

 This lecture is not all-inclusive

Copyright ©2019 F.A. Davis Company

 Introduction to Hematology
 Hematology is about relationships
• Bone marrow to the systemic circulation
• Plasma environment
to the red blood
cell life span
• Hemoglobin to
the red blood cell

Copyright ©2019 F.A. Davis Company

 Introduction to Hematology
 Blood
• sticky, opaque, metallic taste
• bright red to dark red (oxygenation level)
• pH 7.35-7.45
• 38oC
• 4-6 L of blood
 Study of blood and related
disorders
 Staining, counting, analysis of components

Copyright ©2019 F.A. Davis Company

 Red Blood Cells
 Anucleate
 Biconcave
 Reddish protein (Hgb)
 Transports oxygen and carbon dioxide
 For years, cells were counted to detect
anemia and polycythemia

Copyright ©2019 F.A. Davis Company

 White Blood Cells
 Leukocytes
 Protect from infection
 Heal injuries
 Counted in similar fashion to RBCs
 1:20 dilution with acid
 lyses RBCs
 Seen in peripheral blood, bone marrow,
body fluids, cerebral spinal fluid

Copyright ©2019 F.A. Davis Company

 Platelets
 Thrombocytes
 Repair damaged vessels – thrombosis
 “Cell fragments”
 Small element but responsible for major
conditions

Copyright ©2019 F.A. Davis Company

 Complete Blood Count
 CBC and CBCD
 automated analyzers should give differential
 Blood film examination by technologist not
always required
 Manual differential not
always required either

Copyright ©2019 F.A. Davis Company

 Coagulation
 Platelet function and plasma coagulation
 Coagulation is a complex cascade of a series
of proteins, compounds and elements
 Responsible for clot formation after platelet
plug initiation
 Tests include: Prothrombin Time (INR),
Activated Partial Thromboplastin Time, D-
Dimer, Fibrinogen

Copyright ©2019 F.A. Davis Company

 Coagulation
 Prothrombin Time (PT)
• monitor oral anticoagulation
• Warfarin/Coumadin
• PT in seconds converted to INR
 Activated Partial Thromboplastin Time
• APTT, aPTT, PTT
• monitors IV anticoagulation (usually admitted)
• Heparin
Can also evaluate factor deficiencies and
screen for inhibitors
Copyright ©2019 F.A. Davis Company
 Hematology Department
 Other parts of the department will vary
depending on level of lab complexity and
location… but can include:
• Flow cytometry (Immunophenotyping cells)
• Bone marrow (specimen prep, special stains, etc.)
• Coagulation (thrombin time, clotting factors, etc.)
• Platelet studies (functional testing)
• Erythrocyte Sedimentation Rate (ESRs)
• Reticulocyte count

Copyright ©2019 F.A. Davis Company

 Hematology Department
 Other parts of the department will vary
depending on lab and location… but can
include:
• Manual testing (malarial parasites, infectious
mononucleosis, sickle cell disease, osmotic
fragility
• Body fluid analysis: CSF, synovial fluid, pericardial
fluid, pleural fluid, peritoneal fluid
 Many of these tests are found in higher level
facilities and require specialized/advanced
training and technologists
Copyright ©2019 F.A. Davis Company
 Sample Collection
 Phlebotomy safety must always be followed
 Physical variables can potentially affect some
(time of collection must always be accurately
recorded on sample)
 Ethylenediaminetetraacetic acid
 Sodium Citrate (2 different volumes)
 Order of Draw is crucial. Why?

Copyright ©2019 F.A. Davis Company

 Sample Collection
 Most hematology analyzers use whole blood
 Rare chemistry test uses whole blood
 EDTA prevents clotting giving plasma.
How?
 Sample will separate upon standing
therefore mixing required before analysis
 Blood smear should be made ASAP when
required (morphology can change)

Copyright ©2019 F.A. Davis Company

 Sample Collection
 Plasma descriptions
• normal plasma is light yellow – dark yellow
• icteric often dark yellow – brown

• Lipemic appears as cloudy

• Hemolyzed is pink- red

Copyright ©2019 F.A. Davis Company

 The Hematopoietic Theory
 Consists of
• Production
• Development
• Differentiation
• Maturation

 Highly regulated sequence that the bone

marrow follows to supply the body with
blood cells

Copyright ©2019 F.A. Davis Company

 The Hematopoietic Theory
 Production occurs:
• Intramedullary
‒ within the bone marrow

• Extramedullary
‒ outside of the bone marrow
‒ liver
‒ spleen
‒ etc.

Copyright ©2019 F.A. Davis Company

 The Hematopoietic Theory
 Site of cell production changes throughout
develop
 Mesoblastic stage
 Hepatic stage
 Medullary stage

Copyright ©2019 F.A. Davis Company

 Liver
 major site of development of blood cells in
fetus
 adults – metabolism of products and
synthesize proteins
 located beneath diaphragm
 vascular
 Kupffer cells = remove debris and damaged
cells
 Porphyrias, hemolytic anemias, other liver
diseases
Copyright ©2019 F.A. Davis Company
 Spleen
 large lymphoid organ
 major filter of blood
 white pulp and marginal zone:
• lymphocytes, macrophages, dendritic cells
 red pulp:
• vascular
• reduced blood flow – remove damaged/old RBCs
• can remove entire RBC (phagocytosis) or partial
removing of RBC (fragments)

Copyright ©2019 F.A. Davis Company

 Spleen
 major storage location of platelets, WBCs
 hematopoiesis in fetal life
 splenomegaly, splenectomy,
autosplenectomy
 post splenectomy – cell counts increase
• increased risk of infections
‒ H. influenzae, S. pneumoniae, N. miningitidis
• abnormal RBC morphology seen on smear
 *4 functions of the spleen Box 2.1

Copyright ©2019 F.A. Davis Company

 The Hematopoietic Theory
 Medullary stage ( 7 months+)
 Bones start to produce cells (entire skeleton)
 clavicle is one of the first
 numerous stages and cell lines
 can measure erythropoietin, Granolocyte
colony stimulating factor (G-CSF)
• Hgb A
• Hgb A2
• Hgb F

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Marrow:
• Children ~1.5 litres
• Adults ~4 litres (half active)
 at birth all bones are active
 Retrogression eventually happens and active
marrow is replaced with adipocytes
 Adult sites: Scapulae, pelvis, sternum,
vertebrae, ribs, skull, proximal portion of
long bones

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Myeloid:Erythroid Ratio
• Myeloid cells: WBCs
• Erythroid cells: RBCs
• normal ratio 3:1/4:1
• Ratio can change in disease
and under stress
 Hematopoietic Stem Cell 
Committed Progenitor Cells

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
Pluripotent Stem cell

Myeloid progenitor Lymphoid progenitor

Colony Forming Unit Pre-B and Pre-T cells

Granulocyte, Erythroid,
Monocyte, Megakaryocyte
(CFU-GEMM) B lymphoblast* T lymphoblast*

Myeloblast*/Monoblast* Pronormoblast* Megakaryoblast* … …

B cell, Plasma cell T cell
… … …
Neutrophil, Monocyte, Erythrocyte Platelets
Eosinophil, Basophil,
Macropage
Copyright ©2019 F.A. Davis Company
Copyright ©2019 F.A. Davis Company
 Hematopoiesis
 Cytokines
• soluble proteins/glycoproteins (cell
communication)
• produced by many different lineages of cells
• the same cytokine may have differing affects
depending on the cell type it interacts with
• can work synergistically with other cytokines
• act throughout develop and maturation

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
Cytokine Cell
IL-2 T/B Cells, NK Cells
IL-6 Stem cells, B cells
IL-11 Megakaryocytes
GM-CSF Granulocytes, macrophages, etc.
EPO RBC progenitor cells

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Review of general morphological features of
cellular maturation
• 1. N:C
• 2. Nucleoli
• 3. Granules (primary/secondary)
• 4. Size
• 5. Cytoplasm

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Leukopoiesis
• Blasts and later can be identified on peripheral
blood smears

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Myeloblast

This image was originally published in ASH Image Bank. Teresa Scordino. Myeloblast with
auer rod. ASH Image Bank. 2016; i#60924. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Promyelocyte

This image was originally published in ASH Image Bank. Teresa Scordino.
Promyelocyte. ASH Image Bank. 2016; i#60399. © the American Society of
Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Myelocyte

This image was originally published in ASH Image Bank. Teresa Scordino. Myelocyt
ASH Image Bank. 2016; i#60507. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Metamyelocyte

This image was originally published in ASH Image Bank. Teresa Scordino.
Metamyelocyte. ASH Image Bank. 2016; i#60398. © the American Society of
Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Band

This image was originally published in ASH Image Bank. Teresa Scordino. Band
Neutrophil. ASH Image Bank. 2016; i#60397. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Neutrophil

This image was originally published in ASH Image Bank. Teresa Scordino. Segmented
Neutrophil. ASH Image Bank. 2016; i#60395. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Hypersegmented Neutrophil

This image was originally published in ASH Image Bank. Teresa Kraus. Segmented
Neutrophil. ASH Image Bank. 2015; i#60047. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Hyposegmented
Neutrophil

This image was originally published in ASH Image Bank. Girish Venkataraman, MD,
MBBS. Segmented Neutrophil. ASH Image Bank. 2016; i#60787. © the American
Society of Hematology.

This image was originally published in ASH Image Bank. John Lazarchick. Segmented
Neutrophil. ASH Image Bank. 2004; i#2698. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Eosinophil

This image was originally published in ASH Image Bank. Teresa Scordino. Segmented
Neutrophil. ASH Image Bank. 2016; i#60933. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Basophil

This image was originally published in ASH Image Bank. Teresa Scordino. Segmented
Neutrophil. ASH Image Bank. 2016; i#60504. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Review all cell lines and relevant information
 Consider the general morphological
alterations that occur for each cell

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Lymphopoiesis
• 2nd most numerous WBC in peripheral This image was
originally published in
ASH Image Bank.
blood Teresa Scordino.
Lymphocyte. ASH

• usually smaller mature cell

Image Bank. 2016;
i#60510. © the
American Society of
Hematology.

• nucleus can be round - indented

• cytoplasm is light blue to dark
• cytoplasm/margin can be indented by
neighboring RBCs This image was originally
published in ASH Image

• nucleus is generally similar size to RBC Bank. Peter Maslak. Large

Granular Lymphocyte – 1.
ASH Image Bank. 2004;

• differentiate into T and B cells i#62785. © the American

Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Monopoiesis
• larger of the mature cells
• abundant cytoplasm – ground
glass appearance
• possible vacuoles
• Nuclear convolutions, delicate
cytoplasm, dull gray-blue This image was originally published in ASH Image
Bank. Teresa Scordino.. Monocytes. ASH Image
Bank. 2016; i#60935. © the American Society of

cytoplasm, blunt Hematology.

projections/pseudopods

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Lymphocyte vs. Monocyte
• monocyte mistaken for large
lymphocyte
• observe nuclear chromatin,
cytoplasm and shape
• lymphocyte – clumped, larger
granules
• monocyte – linear/lacy,
numerous fine granules

Copyright ©2019 F.A. Davis Company

 Hematopoiesis
 Megakaryopoiesis
• largest cell in BM
• multiple nuclear divisions
• Cytoplasm fragments to release platelets
• platelets approx. 1-4um in diameter
• Thrombopoietin
• endomitosis

This image was originally published in ASH Image Bank. John Lazarchick.
Acute Megakaryoblastic Leukemia – 1. ASH Image Bank. 2011; i#1399. © the
American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Complete Blood Count (CBC)
 Complete blood count includes
• White blood count (WBC)
• Red blood count (RBC)
• Hemoglobin (Hgb)
• Hematocrit (Hct)
• Mean corpuscular volume (MCV)
• Mean corpuscular hemoglobin (MCH)
• Mean corpuscular hemoglobin content (MCHC)
• Platelet count (PLT)
• Red cell distribution width (RDW)
Copyright ©2019 F.A. Davis Company
 Complete Blood Count (CBC)
 RBC – Red Blood cell Count
• reported as 1012/L
• normal range will change depending on age/sex
• adult male = 4.7 to 6.1 X 1012/L
• adult female = 4.2 to 5.4 X 1012/L

Copyright ©2019 F.A. Davis Company

 Complete Blood Count (CBC)
 WBC – White Blood cell Count
• reported as 109/L
• normal range changes depending on age
• Adult = 4.8 to 10.8 X109/L
• leukocytosis
• leukocytopenia
• Absolute differential = WBC

Copyright ©2019 F.A. Davis Company

 Complete Blood Count (CBC)
 PLT – Platelet count
• reported as x109/L
• normal range = 150 – 400 x109/L
• thrombocytosis
• thrombocytopenia

Copyright ©2019 F.A. Davis Company

 Complete Blood Count (CBC)
 HGB – Hemoglobin
• reported as g/L
• hemoglobin found in RBCs
• relates to carrying-capacity of body
• can relate to iron concentration
 aliquot mixed with Drabkin solution
 Hgb  cyanmethemoglobin
 530-540nm
 Normal Range = M: 140 to 180 g/L F: 120 to 160 g/L
Copyright ©2019 F.A. Davis Company
 Complete Blood Count (CBC)
 HCT – Hematocrit
• ratio of volume of packed red cells to volume of
blood
• can be done manually as well in plastic tube and
centrifuged/measured
• reported as 36% or .360
• Adult male = 0.400 – 0.500 L/L
• Adult female = 0.350 = 0.450 L/L

Copyright ©2019 F.A. Davis Company

 Complete Blood Count (CBC)
 Correlation checks
• Significant part of quality assurance
• “Rule of Three”
• RBC multiplied by 3 = Hemoglobin multiplied by 3
= Hematocrit ± 3

Copyright ©2019 F.A. Davis Company

 Mean Corpuscular Value (MCV)
 One of the most stable  Shift in MCV caused by
parameters in the CBC • Cold agglutinins
 Evaluates preanalytical • Transfusion therapy
• Reticulocytosis
and analytical specimen
integrity  Preanalytical changes in
MCV caused by
 MCV = (Hematocrit/
• IV contamination
RBC) multiplied by 1000
• Specimens from
• Reference range = 80 to 100 hyperglycemic patients
femtoliters (fL) • Patients on some
• red cell size of 6 to 8 chemotherapy drugs
micrometers

Copyright ©2019 F.A. Davis Company

 MCH and MCHC
 Mean Corpuscular Hemoglobin (MCH) and
Mean Corpuscular Hemoglobin Content
(MCHC) provide information concerning
red cell hemoglobinization
 MCH = Hemoglobin/RBC
• Average weight of hemoglobin
• Reference range = 27.5 to 33.0 picograms (pg)

Copyright ©2019 F.A. Davis Company

 MCH and MCHC
 MCHC = Hemoglobin/hematocrit
• reported in g/L
• Amount of hemoglobin per red cell as a percentage
• Reference range = 305 g/L to 360 g/L

 Values <360 g/L

 Values >360 g/L

Copyright ©2019 F.A. Davis Company

 Red Cell Distribution Width
 Red Cell Distribution Width (RDW)
• Mathematical calculation done by instrument
• Anisocytosis (variation in size) and poikilocytosis
(variation in shape) in a peripheral smear
 RDW = Standard Deviation of RBC
volume/mean MCV multiplied by 100
• Reference range = 11.5% to 14.5%

Copyright ©2019 F.A. Davis Company

 CBC
 Retic Count
• represents erythropoietic activity of BM
• last immature stage of RBC development
• contains remnant RNA in cytoplasm (viewed with
new methylene blue supravital stain)
• seen as polychromatic macrocytes on peripheral
smear
• can do manual count as well
• varies with age

Copyright ©2019 F.A. Davis Company

 CBC + Differential
 CBCD
• differential can be relative or absolute
• relative reported as % or decimal
• absolute reported x109/L
• calculated: relative x WBC
• normal ranges depend on age

Copyright ©2019 F.A. Davis Company

 Bone Marrow
 Anatomy and physiology
• One of the largest organs of the body
• Composed of yellow and red marrow
• Intricate supply of nutrients and blood vessels
• Technologists act as assistants (determines if sample adequate)
• Smears made bedside, staining occurs in lab
• Paired with CBC results = diagnose hematologic disorders:
leukemias, cytopenias, etc.
• Site of choice: Posterior Superior Iliac Crest
• Aspirate and Core biopsy

Copyright ©2019 F.A. Davis Company

 Peripheral Smear
 Proper smear prep is essential to accurate evaluation of peripheral
blood
 Drop of blood applied at one end
 Carried across slide with slide/glass
 Thick  Thin
 Does not touch sides of slide or reach the end
 Smooth macroscopic appearance
 No streaks, approx. 2/3 of slide length
 feathered edge = rainbow appearance
 Video on Moodle

Copyright ©2019 F.A. Davis Company

 Making a Push Prep/Wedge Smear

https://www.marketlab.com/diff-safe/p/Diff-Safe/

https://www.fishersci.com/shop/products/alpha-scientific-
diff-safe-blood-dispenser-blood-smears-3/p-177774

*Troubleshooting Poorly made smears

Copyright ©2019 F.A. Davis Company
 Staining Peripheral Smears

 Done ASAP after smear is dried

 Romanowsky stain (Polychrome stain)
 Eosin and methylene blue/Giemsa
 Methanol fixes slides
 Buffer required
 Manual and automated procedures
 Well-Stained Smears
 Examining of Peripheral Smear:

Copyright ©2019 F.A. Davis Company

 Erythrocyte Sedimentation Rate
 Screening test
 Non-specific test used to detect illness related to
• infection
• inflammation
• tissue necrosis
• tissue infarction
 looks at the settling of RBCs in whole blood over a specific
period of time
 measured in mm/hr
 Rouleaux increases rate
 Must know other factors that inc or dec ESR

Copyright ©2019 F.A. Davis Company

 Microhematocrit

 PCV
 measured after centrifuging aliquot of sample
 erythrocytes are packed at one end of capillary tube
 buffy coat (platelets and WBCs) sit between RBCs
and plasma
 HCT is read below the buffy coat
 helps indicate hydration status, anemia, acute
hemorrhagic conditions

https://eclinpath.com/hemat
ology/tests/hematocrit/

Copyright ©2019 F.A. Davis Company

 RBC

 Main component =
hemoglobin
 RBC morphology:
• nucleus is round (baseball)
• no granules
• cytoplasm is very basophilic (younger cells)
 lavender
 Erythron:
 RBC mass:
 Primary and Secondary roles
Copyright ©2019 F.A. Davis Company
 Red Cell Membrane
 Plasma membrane
• maintains osmotic pressure
• ratio of phospholipids:cholesterol constant
• energy dependent
• disruption results in deformities, sickled cells,
thalassemia, senescent RBCs and signals
macrophages for destruction

Copyright ©2019 F.A. Davis Company

 Red Cell Membrane
 Structure: trilaminar
• Outer: glycolipids and glycoproteins
• Central: cholesterol and phospholipids
• Inner: (cytoskeleton) spectrin, ankyrin, actin,
protein
 Integral and peripheral proteins
 50% protein, 40% lipid, 10% cholesterol

Copyright ©2019 F.A. Davis Company

 Proteins
 Integral proteins
• Transport (active and passive)
• Structure for some antigens
‒ Glycophorin A (M and N antigen),
‒ Glycophorin B (S and s antigen),
‒ Glycophorin C (point of attachment)

Copyright ©2019 F.A. Davis Company

 Proteins
 Peripheral proteins
• flexible rod-like protein
• Cytoskeleton (responsible for deformability and
elasticity of the red cell membrane)
‒ 𝝰 and β Spectrin and ankyrin
• provide lateral and horizontal membrane stability

Copyright ©2019 F.A. Davis Company

 Proteins
 Peripheral proteins
• inherited abnormalities exist
‒ hereditary elliptocytosis
‒ hereditary spherocytosis
• membrane fails to rebound after membrane
deformation
• membrane gets removed in small portions and
causes change of shape

Copyright ©2019 F.A. Davis Company

 Deformability
 flexibility necessary for RBC survival
 travel through microvasculature
 reduced deformability, changes in shape has
been linked to hemolytic anemias
 rigid cells = picked off by spleen
 bite cells and spherocytes

Copyright ©2019 F.A. Davis Company

 Transport Mechanisms
 Diffuse freely through special channels
• Water
• Chloride (Cl-)
• Bicarbonate (HCO3-)
 More highly regulated intra:extracellular
ratio
• Sodium 1:12
• Potassium (K) 25:1
• Calcium

Copyright ©2019 F.A. Davis Company

 Transport Mechanisms
 Cellular metabolic processes require ATP
• maintaining cation gradient
• phospholipid distribution
• protecting cell proteins from denaturing
• Functional ferrous hgb
 Damage to the osmotic balance
= influx of sodium
= swelling of cell
= colloid osmotic hemolysis

Copyright ©2019 F.A. Davis Company

 Red Cell Metabolism
 Embden-Meyerhof pathway
• Provides 90% of ATP
• Anaerobic glycolysis (Glucose  Pyruvate)
• Net gain of 2 ATP
• important in formation of 2-
3Diphosphoglycerate (2-3DPG)

Copyright ©2019 F.A. Davis Company

 Red Cell Metabolism
 Phosphogluconate pathway
• Hexose Monophosphate Shunt
• Provides 5% to 10% A T P
• produces NADPH
• detoxifies peroxide
• extending the lifespan of the RBC

Copyright ©2019 F.A. Davis Company

 Red Cell Metabolism
 Methemoglobin reductase pathway
• Heme iron constantly exposed to oxidizing
agents (oxygen and peroxide)
• Maintains iron in the reduced ferrous state
(Iron2+)
• Helps NADPH reduce Met-Hgb

Copyright ©2019 F.A. Davis Company

 RBC Morphology Evaluation in the
Laboratory
 Sample flagged for review by automation
 Prepare a quality peripheral smear
 Review at least 10 fields (red cells not
touching)
• Morphology in every field?
• Is morphology artificial or pathologic?
 Quantify abnormalities
• 1+ to 4+
• Follow grading guidelines for site

Copyright ©2019 F.A. Davis Company

 RBC Morphology
 *Must choose proper location on slide
 RBCs touching, not overlapping
 Minimal area between cells
 Morphology will be affected if you choose
wrong area

Copyright ©2019 F.A. Davis Company

 RBC Morphology

Copyright ©2019 F.A. Davis Company

 Microcyte
 MCV <80 fL
 <6um in diameter
 Associated with
• Iron deficiency (IDA), chronic
inflammation, thalassemia
minor, lead poisoning,
sideroblastic anemia
 retain central pallor

This image was originally published in ASH Image Bank. Teresa Scordino.
Microcytosis. ASH Image Bank. 2016; i#60999. © the American Society of
Hematology.

Copyright ©2019 F.A. Davis Company

 Macrocyte
 MCV > 100fL
 >9um in diameter

 Associated with
• Vitamin B12 deficiency, Folic
acid deficiency, Liver disease,
Megaloblastic anemia,
Neonates, Reticulocytosis

Copyright ©2019 F.A. Davis Company

This image was originally published in ASH Image Bank. Teresa Scordino.
Microcytosis. ASH Image Bank. 2016; i#60999. © the American Society of
Hematology.

This image was originally published in ASH Image Bank. Teresa Scordino.
Normocytis rbcs with lymphocyte for comparison. ASH Image Bank. 2016;
i#60304. © the American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Anisocytosis
 Abnormal variation in
cell sizes
 RDW >14.5%
 Associated with:
• Hemolytic anemia,
megaloblastic anemia,
IDA, treatment of anemia

Copyright ©2019 F.A. Davis Company

 Anisocytosis
 2 distinct populations of
RBCs
 Dimorphic Population
 RDW >14.5%
 Associated with:
• Transfusions,
myelodysplastic syndrome,
IDA, sideroblastic anemia,
vitamin B12/folate
deficiency

Copyright ©2019 F.A. Davis Company

 Hypochromic

 Larger central pallor

than normal red cell
• Hemoglobin synthesis is
impaired
• MCHC less than 30.5%
• Associated with:
‒ IDA, thalassemia,
sideroblastic anemia

Copyright ©2019 F.A. Davis Company

 Polychromasia
 Reticulocytes (when
stained with New
Methylene Blue)
• Gray/blue in color
• Round nucleus
• Cell size: 10 to 12
micrometers
 Associated with:
• Hemorrhage, hemolysis,
neonates, treatment for
anemia
Copyright ©2019 F.A. Davis Company
 Spherocytes

 Round, compact, no
central pallor
 Darker colour (reduced
SA:V ratio)
 Associated with:
• Hereditary spherocytosis
(Spectrin abnormality),
Immune hemolytic
anemia, transfused cells,
extensive burns
Copyright ©2019 F.A. Davis Company
 Sickle Cells
 Drepanocyte
 Reversible: more
rounded, half-moon
shaped
 Irreversible: crescent
shaped and pointed
projections
 Associated with:
• Sickle cell disease
(hemoglobin S),
hemoglobin SC disease
Copyright ©2019 F.A. Davis Company
 Ovalocytes and Elliptocytes

 Egg shaped
 Cigar-shaped

 Associated with:
• Hereditary Elliptocytosis
(abnormal spectrin/protein
4.1), thalassemia major, IDA,
megaloblastic anemia

Copyright ©2019 F.A. Davis Company

 Target Cells

 Codocyte
 Bull’s eye–shaped cell
 Increased SA:V
 Increased cholesterol
and decreased Hgb
 Associated with:
• Hemoglobinopathies,
thalassemia, IDA,
splenectomy, obstructive
liver disease
Copyright ©2019 F.A. Davis Company
 Stomatocytes

 Elongated area of
central pallor
 Looks like mouth or
stoma
 Associated with:
• Hereditary
stomatocytosis,
alcoholism, liver disease,
Rh null syndrome,
artifact

Copyright ©2019 F.A. Davis Company

 Acanthocytes

 Spur cell, thorn cell

 Smaller red cell
surrounded by
uneven thorn-like
spicules/spines (3-10)
 Associated with:
• Severe liver disease,
splenectomy,
abetalipoproteinemia
This image was originally published in ASH Image Bank. Teresa Scordino,
MD. Acanthocytes. ASH Image Bank. 2016; i#60266. © the American
Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Echinocyte
 Burr cell
 Short, evenly spaced
projections (10-30)
 Associated with:
• Uremia/Renal Disease,
pyruvate kinase deficiency,
neonates, artifact

This image was originally published in ASH Image Bank. Teresa Scordino,
MD. Burr Cells or Echinocytes. ASH Image Bank. 2016; i#60291 © the
American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Dacrocyte
 Teardrop cell
 Single pointed extension
 Resembles teardrop or
pear
 Associated with:
• Primary myelofibrosis,
myelophthisic anemia,
thalassemia, megaloblastic
anemia

Copyright ©2019 F.A. Davis Company

 Schistocyte
 Fragmented erythrocyte
(rupture in circulation)
 Numerous sizes and
shapes
 Associated with:
• Microangiopathic
hemolytic anemia
(Hemolytic uremic
syndrome (HUS),
Disseminated intravascular
hemolysis(DIC), TTP),
burns

Copyright ©2019 F.A. Davis Company

 Bite Cell Blister Cell

 Splenic pitting of Heinz

bodies
Copyright ©2019 F.A. Davis Company
 Hemoglobin C crystals

 Hexagonal crystal of
dense hemoglobin
 Stain dark red

 Associated with
Homozygous Hgb C
disease

Copyright ©2019 F.A. Davis Company

 Hemoglobin SC

 1-2 fingerlike
projections
 Composed of
hemoglobin SC

 Associated with Hgb SC

disease

Copyright ©2019 F.A. Davis Company

 Poikilocytosis
 Many abnormally
shaped RBCs

This image was originally published in ASH Image Bank. Teresa Scordino,
MD. Hereditary pyropoikilocytosis. ASH Image Bank. 2016; i#60300 © the
American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 RBC Agglutination
 Clumping of
erythrocytes
 Outline of cells not
usually evident
 Associated with:
• Antibody reactions, cold
agglutinins

Copyright ©2019 F.A. Davis Company

 Rouleaux
 Erythrocytes in rows
 Looks like stacked coins
 Can be associated with
background staining
(high protein conc.)
 Associated with:
• Inflammation, Plasma cell
myeloma, lymphoma

Copyright ©2019 F.A. Davis Company

 Howell-Jolly Bodies

 Dark blue-purple
 Round-oval
 Usually 1 per cell
 Composed of DNA
 Associated with:
• Hyposplenism,
Splenectomy,
megaloblastic anemia,
hemolytic anemia

Copyright ©2019 F.A. Davis Company

 Pappenheimer Bodies

 Siderotic granules
(Prussian Blue Stain)
 Irregular clusters
 Small, light blue
 Often near periphery
of cell
 Composed of Iron
• Associated with
Sideroblastic anemia, hemoglobinopathies, thalassemias,
megaloblastic anemia
Copyright ©2019 F.A. Davis Company
 Basophilic Stippling
 Dark blue-purple granules
 Very fine to coarse
 Numerous granules
throughout cell
 Composed of
RNA/ribosomes
 Associated with:
• lead intoxication,
thalassemia, abnormal
heme synthesis/ alcoholism
Copyright ©2019 F.A. Davis Company
 Heinz Bodies
 Round, dark blue-
purple
 Not visible on Wright
stain (supravital stain)
 Composed of
denatured Hgb
 Associated with
• G6PD deficiency,
unstable hemoglobins

Copyright ©2019 F.A. Davis Company

 Cabot Ring
 Dark blue-purple
 Loop, ring or figure
eight
 Looks like beads on a
string
 Composed of remnants
of the mitotic spindle,
microtubules
 Megaloblastic anemia
 Confused with malaria
Copyright ©2019 F.A. Davis Company
 Malaria

This image was originally published in ASH Image Bank. Peter Maslak.
This image was originally published in ASH Image Bank. Eitan Fleischman,
Malaria-1. ASH Image Bank. 2011; i#3516 © the American Society of
MD. Severe Malaria Blood Smear. ASH Image Bank. 2020; i#63067 © the
Hematology.
American Society of Hematology.

Copyright ©2019 F.A. Davis Company

 Microorganisms
 Malaria (transmitted by mosquitos)
• Plasmodium vivax
• Plasmodium malariae
• Plasmodium ovale
• Plasmodium falciparum
• Plasmodium knowlesi
 Babesia microti
• transmitted by tick bites

Copyright ©2019 F.A. Davis Company

 Microorganisms
 Microfilaria

Copyright ©2019 F.A. Davis Company

 Microorganisms
 Trypanosomes

Copyright ©2019 F.A. Davis Company

 Hemoglobin Synthesis
 The function of the R B C’s is to produce,
package, protect, and transport hemoglobin
among various tissues
 Hemoglobin
• Responsible for carrying oxygen (oxyhemoglobin)
• Pulls C O2 away from tissues
• Keep balanced p H between the blood

Copyright ©2019 F.A. Davis Company

 Red Cell Composition
 Heme
• 4 iron atoms iron2+ (linked through histidine a.a)
• Iron surrounded by protoporphyrin ring (multi-
step enzymatic procedure)
• ferroprotoporphyrin
 Globin
• 2 pairs of globin chains made up of amino acids
• Alpha and beta chains

Copyright ©2019 F.A. Davis Company

 Hemoglobin Molecule

Copyright ©2019 F.A. Davis Company

 Hemoglobin
 Embryonic hemoglobin
• Hemoglobin Gower one (ζ2, ε2)
• Hemoglobin Gower two (α2 ε2)
• Hemoglobin Portland (ζ2 γ2)
 Fetal hemoglobin (3 months of fetal
development)
• Hemoglobin F (α2 γ2) – up to 90%

Copyright ©2019 F.A. Davis Company

 Hemoglobin (continued)
 Adult hemoglobin (3 to 6 months after
delivery)
• Hemoglobin A (α2 β2): 95% to 98%
• Hemoglobin A2 (α2 δ2): 3% to 5%
• Hemoglobin F (α2 γ2): less than 2%

 Table 4.1

Copyright ©2019 F.A. Davis Company

 Oxygen Dissociation (O D) Curve
 OD curve
• Sigmoid shape (“S” shape)
• Partial pressure of O2 (PO2) = P50 value
• ↑ bound oxygen when ↑ PO2
• Lungs: PO2 is 100 millimeters of mercury = 97%
Hemoglobin saturation with oxygen
• Circulation: PO2 is 40 millimeters of mercury =
75% hemoglobin saturation with oxygen

Copyright ©2019 F.A. Davis Company

 Oxygen Dissociation (O D) Curve
(continued)

Copyright ©2019 F.A. Davis Company

 O D Curve Changes
 Shift to right
• Less attraction to oxygen (release)
• At 40 millimeters of mercury, hemoglobin is 50%
more saturated but willing to give up 50% of
oxygen to tissue if needed
• Lower affinity
‒ Anemia
‒ Acidosis (decreased p H)
‒ Increased 2,3-D P G
‒ Elevated temperature (fever)

Copyright ©2019 F.A. Davis Company

 O D Curve Changes (continued)
 Shift to left
• More attraction for oxygen
• At 40 millimeters of mercury, hemoglobin is 75%
more saturated but willing to release only 12% to
tissues
• Higher affinity
‒ Presence of abnormal hemoglobins (high affinity)
‒ Alkalosis (increased p H)
‒ ↓ 2,3-D P G, body temp
‒ Multiple transfusions of stored blood where 2,3-D P G
is depleted

Copyright ©2019 F.A. Davis Company

 Hemolysis
 Extravascular 90%
• available energy stores are reduced
• R E S system: spleen, liver, lymph nodes, bone
marrow
• Releases heme and globin contents to be
recycled
• Gobin, Heme
• Iron  transferrin  BM

Copyright ©2019 F.A. Davis Company

 Figure 4.4

Copyright ©2019 F.A. Davis Company

 Hemolysis
 Intravascular
• Lysed directly into blood vessels
• free hemoglobin = haptoglobin
• during complement activation (ABO transfusion
reactions)

Copyright ©2019 F.A. Davis Company

 Anemia

 decrease in oxygen delivery capacity

 insufficient hemoglobin
 defective hemoglobin function
 history and physical examination is
important for clinical diagnosis
• classic symptoms: fatigue, shortness of breath,
decreased energy levels

Copyright ©2019 F.A. Davis Company

 MCV

 Microcytic anemia
• MCV <80 fL
• diameter <6um
• often seen with hypochromia, increased central
pallor
• hemoglobin synthesis decreased
• Heme: Iron deficiency (IDA), chronic
inflammation, defective protoporphyrin synthesis
• Globin: Thalassemia

Copyright ©2019 F.A. Davis Company

 MCV

 Macrocytic
• MCV >100 fL
• Diameter >8um
• Megaloblastic states: Vit B12, folate deficiency
(oval macrocytes and hypersegmented
neutrophils)
• Non-megaloblastic: membrane disruption of
phospholipid-cholesterol balance, liver
disease/alcoholism

Copyright ©2019 F.A. Davis Company

 MCV

 Normocytic
• MCV in the normal reference range
• destruction of RBCs
• elevated retic counts in hemolytic anemia
• Intrinsic vs Extrinsic defects leading to hemolysis

 Other related classifications: based on absolute

reticulocyte count and red cell distribution width

Copyright ©2019 F.A. Davis Company

 Homework
 Know reference ranges
 Review Chapters in Textbook
 Review Cell Atlas and Morphology (All cells
covered in class)
 Of course… Lab Safety

 It has been a great semester! Good luck with

the exams, next year and your careers!
Copyright ©2019 F.A. Davis Company
 Exam
 Monday April 19, 2021
 830 - 1130
 Multiple Choice
 Short Answer
 Photos
 Everything is testable!

Copyright ©2019 F.A. Davis Company