Medt 16

MEDT 16 HEMATOLOGY 1
INTRODUCTION TO HEMATOLOGY
INTRODUCTION HEMATOLOGY
 Hematology is a branch of science concerned with  It is the branch of Internal Medical Science concerned
the study of the characteristic of the blood, its with the study or analysis of blood (various blood
component and their functions. Different mechanism components and blood forming organs).
of blood is essential to maintain homeostasis of the  It also concerned with the study of causes,
body. prevention, prognosis and treatment of blood related
 The works of many scientists like Anton van diseases.
Leeuwenhoek has a significant contribution to the  It refers to the study of the quantity and morphology
advancement of hematology. of the cellular elements found in blood and use these
results to the diagnosis and monitoring of disease.
BRIEF HISTORY OF HEMATOLOGY

DATE SCIENTIST HISTORICAL CONTRIBUTION
 Found out that the disease/plague is caused by worms in blood
1657 Athanasius Kircher
 One of the first scientist/microscopist that investigates the cause of the disease by using microscope
1647 Anton van Leeuwenhoek  Credited as the discoverer of RBCs (also bacteria and Protozoan)
 Coined the term “platelets,” described as “petite plaques”
1800 Guilo Bizzozero
 Identified the function of platelets in coagulation
 American pathologist
 Study various methods of staining tissues and developed a modification of the Romanowsky
James Wright polychrome staining method
 Published an article (A Rapid Method for the Differential Staining of Blood Films and Malarial Parasites
 Identified platelets as enucleate cell fragments and normal blood components
BLOOD – Venous Blood: 7.35

 Blood is the only liquid connective tissue in our body – Arterial Blood: 7.45
 Circulates through our body and delivers essential  Viscosity – measurement of thickness of blood
substances (oxygen) to the body’s cells – 3-4 times viscous than water
 Transport metabolic waste products away from the  Osmolality – measurement of the concentration of
tissues (carbon dioxide) solutes dissolved in the blood; uses osmometer for
measurement
General Characteristic – Reference range: 281-297 milliosmoles/kg (mOsms)
 Color  Specific Gravity – the number of solutes dissolved in
– Bright red (oxygenated) – hemoglobin is saturated the blood
with oxygen; (sat. HbO2) – Whole blood: 1.045-1.066
– Dark red (deoxygenated) – hemoglobin is not – Serum: 1.024-1.028
saturated with oxygen; (0% HbO2) – Plasma: 1.025-1.029
 Weight of Blood  Salinity
– 7-8% of the total body component or 75-85 mL of  Reflects the concentration of salt (NaCl) dissolved in
blood per kg body weight blood
– Approximately 20 g solid per 100mL blood  It is the saltiness or amount of salt dissolved in a
 Total Volume body of water
– Adult Male: 5-6 L  Salts are compounds like sodium chloride,
– Adult Female: 4-5 L magnesium sulfate, potassium nitrate, and sodium
– Newborns: 250-350 mL bicarbonate
 pH  Temperature
– 7.35-7.45 (average: 7.40)  28˚C; slightly higher than body temp
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Blood Composition
Figure 1. Blood Composition
1. Plasma – 55% of the whole blood a. Platelets

 It is the liquid portion of unclotted blood 2. Middle Layer – non-granulated WBCs
 Pale yellow or straw colored but slightly hazy a. Monocytes, lymphocytes
 Serum 3. Lowermost Layer – granulated WBCs
– It is the fluid that remains after coagulation has a. Neutrophils, Basophils, Eosinophil
occurred and clot has formed. b. Reticulocytes
– Pale yellow; clear & transparent c. Immature RBC precursor cells
2. Formed Elements – 45% of the whole blood
 The formed elements are cells and cell fragments
suspended in the plasma.
 The three classes of formed elements are the
erythrocytes (RBCs), leukocytes (WBCs), and the
thrombocytes (platelets)
a. RBC (40-45% of the blood volume)
 Generated from your bone marrow at a rate of
four to five billion per hour.
 Lifecycle of about 120 days in the body Figure 2. Layers and composition of buffy coat
b. WBC (<1% of our blood)
 Protection against infection (attack foreign Terminologies
bodies)  RBC
 Flows via bloodstream but can extend to the – Erythrocytosis – increased number of RBC in
tissues blood
 Generated from bone marrow and have a life – Erythrocytopenia – decreased number of RBC in
span of 13-20 days blood
c. Platelets (<1% of our blood) – Polycythemia – a cancer, causes BM to produce
 Smallest of blood cells (non-active form); too many RBC
controls bleeding.  WBC
 It has a lifespan of 10 days in the human – Leukocytosis – increased number WBC in blood
circulation – Leukopenia – decreased number or WBC in blood
 They received signals and travel to the site  Platelets
and transform (active form). – Thrombocytosis – increased number of platelets
 It forms a cluster and plug the wound in blood
 Buffy Coat – Thrombocytopenia – decreased number of
1. Upper most Layer – lightest, less dense platelets in blood
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Functions of Blood 5. Transport heat to the skin for heat distribution
1. Transport or Metabolic
2. Protection or Defensive
3. Regulation or Regulatory
Metabolic/Transport Function
1. Transport gases between lungs to the other parts of
the body
2. Transport nutrients from digestive tract and storage
sites to the other parts of the body
3. Transport waste products to be removed or Figure 3. Heat distribution across epidermis (skin)
detoxified. It is done in the kidneys and in the liver
4. Transport hormones from glands
HEAT LOSS HEATING UP

 Blood vessels in the skin dilate (vasodilation) to allow more blood from the warm body  Blood vessels in the skin contract
core to flow close to the surface of the body, so heat can be radiated into the environment. (vasoconstriction) to prevent blood from flowing
 As blood flow to the skin increases, sweat glands in the skin are activated to increase close to the surface of the body.
their output of sweat (diaphoresis).  This reduces heat loss from the surface.
 When the sweat evaporates from the skin surface into the surrounding air, it takes the  As the temperature falls lower, random signals
heat with it. to skeletal muscles are triggered, causing them
 Breathing becomes deeper, and the person may breathe through the mouth instead of the to contract.
nasal passages.  This causes shivering, which generates a small
 This increases heat loss from the lungs. amount of heat.
Defensive/Protective Function  Regulated by kidneys and hypothalamus (detects

1. Leukocytes, or white blood cells, destroy invading level of blood water, creates feeling of thirst if low
microorganisms and cancer cells level of water detected in blood)
2. Antibodies (Abs) and other proteins destroy  Pituitary gland release ADH Kidneys (maintain or
pathogenic substances release water)
3. Platelet factors initiate blood clotting and help
minimize blood loss
Regulatory Function
1. pH by interacting with acids and bases
Figure 5. Blood-water regulation mechanism
3. Regulation of Body Temperature

Figure 4. Acid-base disorders HOMEOSTASIS
 It is the condition in which a system such as the
 Regulates pH thru the help of kidneys it excretes human body is maintained in a more-or-less steady
H+ ions or HCO3 ions and lungs by regulation of CO2 state
 Acid-base disorder and compensatory mechanism
2. Water balance by transferring water to and from
tissues
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Components in Maintaining Homeostasis  Feedback is the data that feeds back to control a
1. Stimulus response
 It is provided by the variable (ex. body Temp) that 1. Negative Feedback
is being regulated.  These mechanisms change the variable back to
 Generally, the stimulus indicates that the value of its original state or “ideal value”.
the variable has moved away from the set point or  It serves to reduce an excessive response and
has left the normal range. keep a variable within the normal range.
 Examples of processes controlled by negative
2. Sensor/Receptor feedback includes body temperature regulation
 Monitors the values of the variable and sends data and control of blood glucose
on it to the control center.
3. Control Center
 The control center determines the appropriate
response and course of action. (i.e Central Nervous
System)
 Matches the data with normal values. If the value is
not at the set point or is outside the normal range,
the control center sends a signal to the effector.
4. Input
 Information travels along the (afferent) pathway to Figure 7. Negative feedback mechanism of body temperature
the control center regulation
 Afferent Pathway
– Carries nerve impulses into the central nervous
2. Positive Feedback
 In a positive feedback system, the output
system. For instance, if you felt scorching heat
on your hand, the message would travel through enhances the original stimulus
 Feedback serves to intensify a response until an
afferent pathways to your central nervous
system. endpoint is reached
 Examples of processes controlled by positive
5. Output
 Information sent from the control center down the
feedback in the human body include blood
(effector) pathway to the effector clotting and childbirth
 Efferent Pathway
– Carries nerve impulses away from the central
nervous system to effectors (muscles, glands).
6. Effector
 It is an organ, gland, muscle, or other structure that
acts on the signal from the control center to move
the variable back toward the set point.
Figure 8. Positive feedback mechanism of blood clotting
Homeostatic Imbalance
 Failure to maintain Homeostasis will lead to
Figure 6. Homeostasis mechanism Imbalance (Homeostatic Imbalance)
 Imbalance may result to accumulation of toxic
Feedback Mechanism chemicals that will lead to disorder or Death
 Feedback is essential in maintaining homeostasis.
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BLOOD COLLECTION, HANDLING AND PRESERVATION
BLOOD PHLEBOTOMY  Avoid bruised and scarred areas
 Phlebotomy comes from a Greek word meaning “To  Large vein are not well anchored In tissue and
Cut a Vein” – a process of making incision in a vein. frequently rolled- secured using thumb
 Blood Collection is the process of obtaining blood
sample from the patient.
 It is crucial to the understanding, diagnosis,
prognosis, treatment and prevention of disease.
 A Phlebotomist is the person who practice
Phlebotomy
 It requires social, clerical and technical skills honed
Figure 1. Common sites of venipuncture. (left) Arm’s preferred site
through a lot of practice. for venipuncture; (middle) dorsal hand’s preferred site for
venipuncture; (right) dorsal foot’s preferred site for venipuncture
PHYSIOLOGIC FACTORS THAT CONTRIBUTES TO
PRE-ANALYTICAL VARIATIONS IN TEST RESULTS
1. Posture
 Changing from supine to sitting or standing
 Protein, cholesterol and iron increase their
concentration in blood
2. Diurnal Rhythm
 Refers to daily body fluid fluctuation occurring to
Figure 2. Critical sites for venipuncture. (left) Jugular site for
the constituents of blood venipuncture; (right) femoral site for venipuncture
 Cortisol, TSH and iron are increased in morning and
decreased in the afternoon EQUIPMENTS
 Eosinophil count are low in the morning and high in 1. Vein-locating Devices
the afternoon  The principle of this equipment is that hemoglobin
3. Exercise in blood within the veins absorbs light, causing the
 Increases creatinine, T. protein, CK, myoglobin, asp. vein to stand out as dark lines.
aminotransferase, WBC, HDL 2. Tourniquet
4. Stress  Applied 3-4 inches above the puncture site no
 Anxiety and crying in children longer than 1 minute
 Increases WBC  It is used to provide a barrier against venous
5. Diet blood flow to easily locate the veins
 Eating increases glucose and lipids 3. Needles
 Over fasting decreases glucose and lipids a. Multi-sample Needles (Evacuated Tube System)
6. Smoking  Used in multiple tube collection
 Increased WBC and cortisol  Components:
 Long-termed smoking decreases pulmonary  Multi-sample needle
function and leads to high hemoglobin level  Tube Holder
 Evacuated tubes
BLOOD COLLECTION METHOD b. Hypodermic Needles (Syringe System)
 Length of needle: 1-1.5 inches
Venipuncture  Bevel angle: <30 degrees
 The most common way of collecting blood sample c. Winged Infusion (Butterfly) Needles
from the adults  Allows more flexibility and precision
 It is obtained from the veins on the front of the elbow  Components:
(antecubital veins)  ½ to ¾ inch stainless needle attached to 5 to
12 inches tubing with Luer attachment for
VEIN SELECTION syringe or multi-sample luer adapter
 Choose the vein that are large and accessible
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 2 types of Luer: Needle Gauges
1. Luer slip – without thread  Needle diameter is inversely proportional to the
2. Luer lock – with thread gauge number
 High needle gauge = smaller diameter of needle
 Note: SESIP (Sharp with Engineered Sharp Injury
Protection) – a needle or non-needle sharp with a
built-in safety features or mechanism
NEEDLE GAUGES
GAUGE HUB COLOR SIZE DIAMETER
16G WHITE 1X½" 1.60 mm
18g PINK 1X½" 1.20 mm
19G CREAM 1X½" 1.10 mm
20G YELLOW 1 ", 1 X ¼ ", 1 X ½ " 0.90 mm
21G GREEN 1 ", 1 X ¼ ", 1 X ½ " 0.80 mm
22G BLACK 1 ", 1 X ¼ ", 1 X ½ " 0.70 mm
23G BLUE 1 ", 1 X ¼ ", 1 X ½ " 0.60 mm
24G MEDIUM PURPLE 1 ", 1 X ¼ ", 1 X ½ " 0.55 mm
25G ORANGE 1 ", 1 X ¼ ", 1 X ½ " 0.55 mm
26G BROWN ½ ", 1 X ½ " 0.45 mm
ORDER OF DRAW
ORDER OF STOPPER
ADDITIVES INVERSION DESCRIPTION
DRAW COLOR
1 Color varies SPS 8-10 Bacteriology
2 Light Blue Sodium citrate 3-4 Coagulation
Chemistry
SST gel
Gold Tiger 5 Serology
separator
Immunology
Chemistry
Glass – none
3 Red Serum tube Serology
Plastic – 5
Blood Bank
Chemistry
Orange RST 5-6 Serology
Immunology
Plasma
Light Green Lithium heparin 8-10
Chemistry
4 Ammonia
Dark Green Sodium heparin 8-10 Plasma
Chemistry
5 Lavender EDTA 8-10 Hematology
PPT separator Molecular
6 White 8-10
tube Testing
Glucose
7 Gray Sodium fluoride 8-10
Determination
DNA
8 Yellow ACD solution 8-10
Paternity
ADDITIVES IN COLLECTION TUBES 2. Antiglycolytic Agent

 Most of the collection tubes contain additives.  It inhibits the use of glucose by blood cells
 These additives could either act as a clot activator  Necessary for delayed blood glucose testing.
that accelerates clotting formation or as an  Gray tubes
anticoagulant that prevents blood from clotting. 3. Separator Gel
 Types of Additives  It is an inert material that undergoes temporary
1. Clot Activators change in viscosity during centrifugation
 Initiate or enhance the clotting mechanism enabling separation between liquid and cells
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 It is not used in blood bank processing as it may  Ammonium heparin
interfere the result of testing  Lithium Heparin
4. Anticoagulant Agent  Sodium heparin
 Prevents blood from clotting
 Tubes with anticoagulant requires Immediate COMPLICATION ENCOUNTERED FROM VENIPUNCTURE
 Inversion of tubes after blood collection  Ecchymosis (Bruise)  Petechiae
a. EDTA (ethylene diamine tetra-acetic acid)  Hematoma  Allergies
 Optimum concentration: 1.5mg/ml of blood  Fainting (Syncope)  Nerve Damage
 Removes calcium by forming insoluble salts  Hemoconcentration  Seizures
 Good at preserving cell morphology  Hemolysis  Vomiting
 Forms of EDTA:
 Powdered dipotassium, K2 (Versene), Arterial Puncture
most soluble, hence, most preferred  Blood samples were obtained from the artery.
 Disodium EDTA  Done primarily to determine arterial blood gases
 Liquid tripotassium, K3 (Sequestrene) examining metabolic, respiratory, acid-base
b. Citrate disorders.
 Used for coagulation studies  Usually takes place within the hospital by trained
 Action: binds calcium in soluble complex phlebotomist to assure safety of the patients for it
 Concentration: 3.2% or 0.109M may cause too much bleeding specially to patients
 Ratio: with coagulopathy.
 9:1 – blood to anticoagulant ratio  Samples can be obtained through:
 4:1 – blood to anticoagulant ratio (black  Catheter placed in the artery
top, westergren)  Syringe which is pre-heparinized to minimized air
c. Oxalate exposure
 Action: Binds Calcium to form INSOLUBLE
calcium oxalate SITES FOR ARTERIAL PUNCTURE
 Forms: 1. Radial artery – first choice
 Lithium oxalate – collecting body 2. Brachial or femoral artery – alternative site
fluids which are bloody
 Sodium oxalate – for blood
coagulation studies
 Double oxalate – most commonly used
– Salts of ammonium and potassium
(NH4K) in 3:2 ratio
When used separately:
– Ammonium oxalate (Wintrobes) –
RBC swelling
– Potassium oxalate (Paul-Hellers) –
shrinkage of RBC
d. Heparin Figure 3. Sites for arterial puncture
Optimum concentration: 15-20 U/ml of blood
Inhibits the conversion of prothrombin to Not recommended for the following reasons:
thrombin  Harder to locate
Preferred tube for immunophenotyping and  Have poor collateral circulation
OFT (Osmotic Fragility Testing) or samples  Surrounded by structure that could be damaged
for defibrinated blood
Used in STAT test (e.g Electrolytes) COMPLICATIONS RELATED TO ARTERIAL SAMPLING
 Arteriospasm – involuntary contraction of the
Not for blood film preparation
Distort WBC and platelet morphology artery
 Hematoma
Produced bluish background
 Nerve damage
Forms of heparin:
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 Fainting (syncope) or vasovagal response ORDER OF DRAW
 Drop in blood pressure 1. Tubes for gas analysis
 Sweating and pallor leading to loss of consciousness 2. Slides-peripheral blood smear – if needed
3. Micro-collecting tubes
Skin Puncture a. EDTA
 A process of taking a very small amount of blood from b. Heparin or Other tubes with anticoagulant
the patient usually at the end of the finger c. Serum
 Method used in point of care testing, phlebotomist is
not required for the procedure. SPECIMEN HANDLING
 It is a technique of choice for the following:  Handle specimen as if it is infectious
 Newborn  Specimen should be in an upright position to reduce
 Infant agitation that may result to hemolysis
 Pediatrics and Geriatrics  Exposure to light can cause false decrease result to
 Burned patients bilirubin, carotene, RBC folate and urine porphyrin
 Patients whose vein are reserved for Therapy  Specimens should be placed with ice water bath to
 Patients with thrombotic tendencies and intense maintain low temperature
fear of needles  Other specimen requires warm temperature
 The blood samples obtain is a mixture of venous and  Specimen should be delivered within 45mins-1 hour
arterial blood, also mixed with Interstitial and (max. 2 hours) after collection
Intracellular fluids
BONE MARROW SAMPLES
SITE FOR SKIN PUNCTURE  Bone marrow is the spongy tissue found inside the
 Punctures should be made not more than 2 mm deep bones
 First drop should be wiped-away to prevent  It contains stem cells that developed into red blood
contamination cells, white blood cells and platelets
 Povidone-iodine is not recommended – causes false  It produces 6 billion cells per kg a day
increase in bilirubin, uric acid, potassium and  Bone marrow at early age – composed of red marrow
phosphorous (active cell production)
th
 Warming of site for 3-5 mins not >42°C increases  Bone marrow at 7 year – composed of adipose
blood flow 7 times (replaces red marrow in the long bones)
Indication of Bone Marrow Examination

 Infection
 Metabolic disorder
 Marrow failure and Cytopenia
 Neoplasia diagnosis and Staging
 Monitoring of treatment
Trephine Biopsy
Figure 4. Sites for skin puncture. (left) Third finger as a common  It is a technique or method of collecting samples from
site for finger prick blood collection; (right) lateral area of the heel
for skin puncture.
the bone marrow.
 It is used to estimate cellularity.
EQUIPMENTS
 It demonstrates bone marrow architecture
1. Lancet or blade spring loaded in the puncture device
2. Containers:
(relationship of hematologic cells to fat, connective
 Capillary Tubes
tissue and bone stroma).
 It is used to evaluate diseases that produce focal
– Red band: with anticoagulant Heparin
– Blue band: No anticoagulant
lesions.
 Needle used:
 Microtainer or micro-collection tubes “bullets”
 Jamshidi Biopsy Needle
– Minimum fill: 250 µl
 Westerman-Jensen needle or Snarecoil
– Maximum fill: 500 µl
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Bone Marrow Aspiration 3. The needle will be passed a few centimeter into the
 It is a procedure involves taking an aspirate or fluid bone and rotated to obtain the samples
from the bone marrow drawn under pressure by 4. After the needle is removed, the site will be checked
puncturing the cavity. to ensure that there is no bleeding.
 It is used to determine cell morphology and
enumeration of marrow cellular elements. BONE MARROW ASPIRATION
 Needle used: Gauge 14-18 (University of Illinois 1. A thin needle will be passed through the number skin
Aspiration Needle) into the pelvic bone
 Volume needed: 1 mL to 1.5 mL 2. A small amount of liquid is withdrawn into the syringe
3. The needle will then be removed and the samples put
Collection Site into the appropriate tubes
1. Posterior superior iliac crest (spine) of the pelvis
2. Anterior superior iliac crest of the pelvis
3. Sternum
4. Anterior medial surface of the tibia
5. Spinous process of the vertebrae, ribs
Figure 5. Common collection site for bone marrow samples Figure 7. Bone marrow aspiration and biopsy procedure
Bone Marrow Specimen

1. Direct Aspiration Smears
 Perform wedge smear same as PBS
 Use 6-8 ethanol washed slides
2. Anticoagulated Aspirate Smears
 Bone marrow samples on K3 EDTA
3. Crush Smears
 Gently press to crush spicules
Figure 6. Bone marrow sample collection procedure
 Used ethanol washed slides
Procedure in Collecting Blood Samples 4. Imprints (Touch Preparations)
 Bone marrow samples are usually taken from the  Placed in 2 slides with proper label
posterior superior iliac crest.  Used when specimen is clotted or dry tap
 The patient was asked to lie on the bed and curled up.  Core biopsy and clotted marrow maybe held in
 The skin will be cleansed with antiseptic which can be forceps and repeatedly touched to a washed glass
cold but doesn’t hurt. or coverslip
 A local anesthetic is injected to the area to make the 5. Concentrate (Buffy Coat) Smears
skin numb.  Used when there is decreased nucleated cells in

direct marrow
BONE MARROW TREPHINE BIOPSY  Centrifuge at 2500 g for 10 mins
1. If required, this is done immediately after the  Place 1.5 ml of K3 EDTA anticoagulated marrow
aspiration specimen to a narrow-bored glass or plastic tube

2. A different needle is inserted into the site where 6. Histologic Sections (Cell Block)
aspiration needle was
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HEMATOPOIESIS
HEMATOPOIESIS Hematopoiesis: Adult
 It is defined as the cellular formation, proliferation, 1. Bone marrow – developing erythroid, myeloid,
differentiation and maturation of the blood cells megakaryocytic and lymphoid cells
2. Primary lymphoid organs: thymus and bone marrow –
Blood Cell Formation Theories T-cells and B-cells derived
1. Monophyletic Theory – all blood cells come from 3. Secondary lymphoid organs: spleen, lymph node, and
one origin stem cells GALT – lymphoid cells become competent
2. Polyphyletic Theory
 Different groups of blood cells originate from Bone Marrow Cellularity
different stem cells  Bone marrow can produce 2.5 billion RBC and platelets
 Hemohistioblasts: WBC, RBC and platelets and 1 billion WBC /kg/body weight/day.
 Tissue hemohistioblasts: monocytes, lymphocytes  Cellularity is the ratio of Marrow cells to fat (red
and plasma cells marrow to yellow marrow)
Stages of Hematopoiesis: Intrauterine Normocellular Marrow for Adult

1. Mesoblastic Stage 1. Fat (yellow marrow) 10-50% - composed of adipocytes
 Location: Yolk sac to AGM (aorta-gonad (fat cells), undifferentiated mesenchymal cells and
mesonephros) macrphages
 Begins at the 19th day of embryonic development 2. Hematopoietic cells (red marrow) 40-60% (ave: 50-%)
after fertilization  Consist of developing blood cells and their
 Blood island in the yolk sac remains active for 8-12 progenitors
weeks (1st trimester)  Child below 2 years old has 100% red marrow
 Hematopoietic activity is limited to RBC production
 Products produced: HSCs (hematopoietic stem cells), Hematopoietic Stem Cell (HSC)
primitive erythroblast, and embryonic hemoglobin
2. Hepatic Stage CHARACTERISTICS OF STEM CELLS
 Location: Fetal liver  Capable of self-renewal
th th th
 Begins at the 5 -7 gestational weeks (6 week)  Give rise to differentiated progeny
 Cluster of erythroblast, granulocytes and  Able to reconstitute the hematopoietic system of a
monocytes are developing lethally irradiated host
 Production of RBC and WBC
 At the end of 4th month, primitive cells disappear TYPES OF HUMAN STEM CELLS
 Increased production of definitive erythroblasts, 1. Totipotential Stem Cells
granulocytes, and megakaryocytes  These cells are present in the first few hours after
3. Medullary (Myeloid) Phase an ovum is fertilized.
 Begins at the 5th month of the blood cell  Most versatile type of stem cell and can develop
development into any human cell type including development
 It occurs in the medulla of the bone marrow from embryo into fetus.
 Marrow-primitive stem cells and committed 2. Pluripotential Stem Cells
progenitor cells are confined  These cells are present several days after
 Spleen and lymph nodes – secondary lymphoid fertilization.
tissue for lymphocyte development and  Can developed into any cell type except fetus
differentiation 3. Multipotential Stem Cells
 Primary bones where hematopoiesis occurs: flat  Derived from pluripotent stem cells
bones of sternum (principal source for adults), ribs,  Found in adults
vertebrae, skull, and pelvis  Mature into limited specific cell types to form
tissues
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Lineage Specific Markers CD41, CD61 Megakaryocytes
 These are proteins on the surface that are unique to CD10, CD19 B-lymphoid
specific cells and certain maturation periods CD3 , CD7 T-lymphoid
CD33, CD34 CFU-GEMM (undifferentiated)
CD33, CD 38 Committed myeloid progenitor
MARKERS LINEAGE/CELLS CD10, CD38 Committed lymphoid progenitor
CD33, CD11b, CD14 Myeloid CD15 Granulocyte
CD71, Glycophorin A (GPA) Erythroid
PROGENITOR CELLS GROWTH FACTORS/INTERLEUKINS/CYTOKINES MATURE CELLS

CFU-MEG Thrombopoietin, GM-CSF Thrombocytes
CFU-GM CFU-M GM-CSF, M-CSF, IL-3 Monocytes
CFU-GM CFU-G GM-CSF, G-CSF, IL-3 Neutrophils
BFU-E CFU-E Erythropoietin, GM-CSF, IL-3 Erythrocytes
CFU-Eo GM-CSF, IL-3, IL-5 Eosinophils
CFU-Ba IL-3, IL-4 Basophils
MYELOID-ERYTHROID RATIO (M:E RATIO)

 It is the ratio of granulocytes and their precursors to
nucleated erythroid precursors.
 A normal ratio is between 2:1 and 4:1 (average: 3:1)
 Granulocytes are more numerous due to their short
survival (1-2 days) compared to erythrocytes (120d) Figure 1. Normal myeloid-erythroid ratio
GRANULOPOIESIS
CELL NUCLEUS CYTOPLASM
CELL SIZE (µm) 15-20
Round to oval N:C RATIO 4:1
Nucleoli: 2-5 Moderately basophilic
Chromatin: Granules: absent or up to 20 BONE MARROW 0-2%
immature, fine
PERIPHERAL BLOOD 0%
MYELOBLAST
Round to oval CELL SIZE (µm) 14-24
Nucleoli: 1-3 or
Basophilic N:C RATIO 3:1
more, less prominent
Granules:
Chromatin:
 Primary: >20, red to purple/burgundy BONE MARROW 2-5%
immature, fine, but
 Secondary: none
slightly coarse than
myeloblast PERIPHERAL BLOOD 0%
PROMYELOCYTE
Round to oval, slightly CELL SIZE (µm) 12-18
eccentric
Slightly basophilic to cream colored
Nucleoli: usually not N:C RATIO 2:1
Granules:
visible
 Primary: few to moderate BONE MARROW 5-19%
Chromatin: coarse
 Secondary: variable
and more condensed
than promyelocyte PERIPHERAL BLOOD 0%
MYELOCYTE
NEUTROPHILS
Indented, kidney Pale pink, cream colored to colorless CELL SIZE (µm) 10-15
bean-shaped Granules:
Nucleoli: not visible  Specific granules (rose pink): lysozyme, N:C RATIO 1:5:1
Chromatin: lactoferrin, collagenase, plasminogen
moderately clumped activator, aminopeptidase BONE MARROW 13-22%
Hof/Perinuclear  Primary: few
clearing: common  Secondary: many PERIPHERAL BLOOD 0%
METAMYELOCYTE
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Constricted w/o
Pale pink to colorless cytoplasm
threadlike filaments N:C RATIO
Granules: predominates
Nucleoli: not visible
 Primary: rare
Chromatin: coarse, BONE MARROW 3-11%
 Secondary: abundant
clumped, mature
BAND PERIPHERAL BLOOD 50-70%
2-5 lobes connected Pale pink, cream colored to colorless CELL SIZE (µm) 10-15
by thin filaments w/o Granules: contains acid phosphatase, acid cytoplasm
visible chromatin hydrolase, peroxidase, muramidase, lactoferrin, N:C RATIO
predominates
Nucleoli: not visible oxidants and defensins
BONE MARROW 3-11%
Chromatin: coarse,  Primary: rare
clumped  Secondary: abundant
PERIPHERAL BLOOD 50-70%
SEGMENTED
NEUTROPHIL * Function: phagocytic function of cell
EOSINOPHILS
Colorless to pink
Granules:
Round to oval
 Specific granules (orange red): major basic
Nucleoli: usually not N:C RATIO 2:1-1:1
protein, acid hydrolase, phospholipase,
visible
cathepsin eosinophilic cationic protein,
Chromatin: coarse
eosinophilic-derived neurotoxin, eosinophil BONE MARROW 0-2%
and more condensed
protein X, arylsulfatase, acid phosphatase
than promyelocyte
MYELOCYTE  Primary: few to moderate
PERIPHERAL BLOOD 0%
 Secondary: variable, pale to dark orange

Indented, kidney
Colorless, cream colored N:C RATIO 1:5:1
bean-shaped
Granules:
Nucleoli: not visible
 Primary: few BONE MARROW 0-2%
Chromatin: coarse,
 Secondary: many, pale orange to dark orange
clumped
PERIPHERAL BLOOD 0%
METAMYELOCYTE
Constricted w/o Colorless, cream colored
threadlike filaments Granules: N:C RATIO 1:5:1
Nucleoli: not visible  Primary: few
Chromatin: coarse,  Secondary: abundant, pale orange to dark BONE MARROW 0-2%
clumped orange
PERIPHERAL BLOOD 0%
BAND
2-3 lobes connected Cream colored, may have irregular borders CELL SIZE (µm) 12-17
by thin filaments w/o Granules: contains peroxidase, acid cytoplasm
visible chromatin phosphatase proteolytic enzymes N:C RATIO
predominates
Nucleoli: not visible  Primary: rare
BONE MARROW 0-3%
Chromatin: coarse,  Secondary: abundant, pale orange to dark
clumped orange PERIPHERAL BLOOD 0-5%
EOSINOPHIL * Function: allergy, drug reactions, parasitic infections
BASOPHILS
Moderately cytoplasmic purple black granules of CELL SIZE (µm) 2-23
varying size and shape
Nucleoli: no distinct
Granules: N:C RATIO 50%
nucleolus
 Specific granules (dark purple/blue black):
Chromatin:
histamine, heparin, peroxidase, eosinophilic
moderately BONE MARROW 0-2%
chemotactic factor A
condensed
 Primary: absent
MYELOCYTE  Secondary: absent PERIPHERAL BLOOD 0%
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Pale cytoplasm
Small, indented N:C RATIO 40%
Granules:
Nucleoli: absent
 Specific granules: large, uniform
Chromatin: BONE MARROW 0-1%
 Primary: absent
condensed
 Secondary: absent
PERIPHERAL BLOOD 0%
METAMYELOCYTE

2 lobes usually, Lavender to colorless
connected by thin Granules: contains histamine, heparin, cytoplasm
N:C RATIO
filaments w/o visible chondroitin sulfates predominates
chromatin  Primary: rare
Nucleoli: not visible  Secondary: large, variable in number
BONE MARROW <1%
Chromatin: coarse, w/uneven distribution, may obscure nucleus,
clumped deep purple to black
PERIPHERAL BLOOD 0-1%
BASOPHIL * Function: immediate hypersensitivity
MONOPOIESIS
Round to oval, may be irregular N:C RATIO 4:1

Light blue to gray
Nucleoli: 1-2, prominent
Granules: absent BONE MARROW 0-3%
Chromatin: fine
MONOBLAST PERIPHERAL BLOOD 0%

Irregularly shaped, folded, may
Light blue to gray N:C RATIO 2-3:1
have brain-like convolutions
Granules: fine azurophilic
Nucleoli: may/may not be visible BONE MARROW <1%
(burgundy)
Chromatin: fine to lacy
PROMONOCYTE PERIPHERAL BLOOD 0%
Variable, may be round/ horse shoe/ CELL SIZE (µm) 12-20

kidney bean-shaped, often has folds Blue-gray N:C RATIO 4:1
w/ brain-like convolutions Granules: many, fine
Nucleoli: not visible Vacuoles: absent to numerous BONE MARROW 2%
Chromatin: lacy PERIPHERAL BLOOD 3-11%
MONOCYTE
Eccentric, kidney/ egg-shaped, Abundant w/irregular borders,
indented, elongated may contain ingested material N:C RATIO —
Nucleoli: 1-2 Granules: many course
Chromatin: fine dispersed azurophilic BONE MARROW —
Vacuoles: may be present
PERIPHERAL BLOOD —
MACROPHAGES
(HISTIOCYTES) * Location: resides in tissues (bone marrow, spleen, liver, lungs, etc.)
TYPES OF MACROPHAGES
TYPE OF MACROPHAGE LOCATION FUNCTION
Phagocytosis of small particles, dead cells or bacteria
Alveolar macrophage Lung alveoli
Initiation and control of immunity to respiratory pathogens
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Kupffer cells Liver Initiate immune responses and hepatic tissue remodeling
Microglia CNS Elimination of old or dead neurons and control of immunity in the brain
Splenic macrophages (marginal zone, Spleen marginal zone, red
Elimination of dysfunctional or old RBCs
metallophilic and red pulp macrophages) and white pulp
LYMPHOEISIS
Round to oval Scant, slightly to moderately N:C RATIO 7:1-4:1

Nucleoli: 1 or more basophilic
Chromatin: fine Granules: absent BONE MARROW 0-2%
PERIPHERAL BLOOD 0%
LYMPHOBLAST
Round, indented N:C RATIO 3-4:1
Nucleoli: 0-1, usually single Light blue
prominent large nucleolus Granules: absent BONE MARROW 0-1%
Chromatin: slightly clumped
PERIPHERAL BLOOD 0%
PROLYMPOCYTE
Round to oval, may be slightly Scant to moderate sky blue, vacuoles
N:C RATIO 5:1-2:1
indented may be present
Nucleoli: occasional Granules: none in small lymph, few BONE MARROW 5-15%
Chromatin: condensed azurophilic in larger lymph
LYMPHOCYTE PERIPHERAL BLOOD —

Round/oval, eccentric N:C RATIO 2:1-1:1
Deeply basophilic, often w/
Nucleoli: absent
perinuclear zone (hof)
Chromatin: coarse BONE MARROW 0-1%
Granules: absent
PERIPHERAL BLOOD 0%
PLASMA CELL
TYPES OF LYMPHOCYTES
1. B cell (10-20%) 2. T cell (60-80%)
 Short-lived (3-4 days)  Long-lived (4-10 years)
 Mostly large lymphocytes  Mostly small lymphocytes found in the thymus
 Found in the bone marrow  Direct killing from cell to cell
 Needs to be mature in order to become plasma cell
that will produce an antibody (Ab)
MEGAKARYOPOIESIS
Round N:C RATIO 3:1
Nucleoli: 2-6 Basophilic
Chromatin: homogenous, Granules: absent BONE MARROW 20%
loosely organized
PERIPHERAL BLOOD 0%
MEGAKARYOBLAST (MK-I)
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Indented N:C RATIO 1:2

Basophilic
Nucleoli: variable
Granules: present BONE MARROW 25%
Chromatin: condensed
PERIPHERAL BLOOD 0%
PROMEGAKARYOCYTE (MK-II)
2-32 lobes Blue to pink, abundant N:C RATIO variable

Nucleoli: — Granules: reddish blue,
5-10/100
Chromatin: — few to abundant BONE MARROW
magnification
MEGAKARYOCYTE (MK-III) PERIPHERAL BLOOD 0%
CELL SIZE (µm) 2-4
Light blue to colorless N:C RATIO NA

Absent Granules: red to violet,
abundant BONE MARROW NA
7-25/100 x OIO (x
PERIPHERAL BLOOD
PLATELET/THROMOBOCYTE 1000 magnification)
ERYTHROPOIESIS  Hypoxia – serves as the stimulus for red blood

 It is a process by which erythroid precursor cells production
differentiate to become mature RBC
 Typically occurs in the erythroid island Functions of Erythropoiesis
 Erythopoietin – primary regulator 1. Promotes early release of reticulocytes from the Bone
 3-5 days – differentiation process from Marrow
pronormoblast to reticulocyte 2. Prevents apoptotic cell Death
3. Reduces maturation time
ERYTHROPOIESIS
Round to slightly oval Scanty light blue, may have N:C RATIO 8:1
Nucleoli: 1-2 prominent Golgi body
Chromatin: fine, immature Granules: absent BONE MARROW 1%
PERIPHERAL BLOOD 0%
PRONORMOBLAST/ RUBRIBLAST
Round to slightly oval N:C RATIO 6:1
Nucleoli: 0-1 Dark/deep blue
Chromatin: slightly Granules: absent BONE MARROW 1-4%
condensed, immature
BASOPHILIC NORMOBLAST/ PERIPHERAL BLOOD 0%
RUBRICYTE
Round CELL SIZE (µm) 10-12

Nucleoli: absent Gray-blue
N:C RATIO 4:1
Chromatin: quite Granules: absent
condensed, mature BONE MARROW 10-20%
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POLYCHROMATIC NORMOBLAST/
PERIPHERAL BLOOD 0%
RUBRICYTE
Round N:C RATIO 0.5:1

More pink or salmon
Nucleoli: absent
Granules: absent
Chromatin: fully condensed BONE MARROW 5-10%
ORTHOCHROMATIC PERIPHERAL BLOOD 0%
NORMOBLAST/ METARUBRICYTE
CELL SIZE (µm) 8-8.5
Slightly more blue/purple N:C RATIO NA

Absent than mature erythrocyte
Granules: absent BONE MARROW 1%
POLYCHROMATIC ERYTHROCYTE/
RETICULOCYTE PERIPHERAL BLOOD 0.5-2%
CELL SIZE (µm) 7-8

Salmon w/ central pallor of N:C RATIO NA
Absent about 1/3 the diameter of
cell
Granules: absent BONE MARROW NA
ORTHOCHROMATIC predominant
PERIPHERAL BLOOD
NORMOBLAST/ METARUBRICYTE cell type
ROUTINE HEMATOLOGIC EXAMINATION

HEMOGLOBIN TEST 2. In patients with symptoms of polycythemia
 It measures levels of hemoglobin in your blood 3. Monitor treatment response to anemia and
 Hemoglobin is an iron containing protein found in RBC polycythemia
 It is a protein that carries oxygen from the lungs to 4. Before and after blood transfusion
the rest of the body 5. In patient with a family history of blood disorder
 Purpose: 6. Excessive blood loss from injury or surgical
1. This test is done to check anemia procedure
INDICATIONS OF THE VARIATIONS IN THE LEVELS OF Hgb

HIGH Hgb LEVELS LOW Hgb LEVELS
 Lung disease
 Heart disease  Different types of anemia (low levels of RBCs and globulin)
 Polycythemia vera  Thalassemia
 Kidney tumors  Iron deficiency
 Dehydration  Liver Disease
 Hypoxia  Cancer and Other Diseases
 Carbon monoxide exposure
ROUTINE HEMOGLOBIN Techniques/Methods

TESTS
 Automated Hemoglobin REAGENT-BASED METHODS
Analyzer – enable fast, 1. Hemoglobin Cyanide (HiCN) Method
accurate and reliable results  Using the principle of hemoglobin conversion to
cyanmethemoglobin by adding ferricyanide and
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potassium cyanide, this method of hemoglobin  The absorption of light provides a measurement of
measurement can proceed hemoglobin concentration.
SAHLI’S METHOD
 Sahli’s hemoglobinometer is a manual device that
contains a hemoglobin tube, pipette, and stirrer, as
well as a comparator
 Hydrochloric acid converts hemoglobin to acid
hematin, which is then diluted until the color of the
solution matches that of the comparator block.
Figure 2. Hemoglobin conversion to cyanmethemoglobin
 Advantage:
 The availability of an Internationally accepted
reference standard calibrator or HiCN solution
 Disadvantages:
 Turbidity due to proteins, lipids and cellular matter
is a potential problem with spectrophotometric
estimation
 Heavily lipemic samples and those containing very
high numbers of white cells could raise Figure 3. Sahli’s Method for hemoglobin measurement
concentration of hemoglobin
 Procedure:
2. Vanzetti’s Azide Methemoglobin
 Hemoglobin conversion via potassium ferricyanide
1. Using a dropper, fill in with N10 HCL the
to the colored, stable azide methemoglobin form hemoglobinometer tube up to its lowest marking
2. Put it in the comparator box
that has an almost identical absorbance spectrum
as HiCN 3. Blood is drawn up to 20μl mark of hemoglobin
 A similar reagent used in the HiCN reference pipette exactly
method is used, except a sodium azide is 4. Draw the blood in hemoglobin tube and mix
substituted for the potassium cyanide thoroughly
5. Place the tube at room temperature for 10 minutes
REAGENT-LESS METHOD for complete conversion of hemoglobin into acid
 It is a new technology that measures hemoglobin hematin.
without a reagent based on broad spectrum 6. Add drops of distilled water until the color matches
photometry the color of the comparator box
 This device quantifies absorbance of oxygenated and 7. Once the color is matched with the standard brown
deoxygenated hemoglobin, while turbidity is glass, lift the stirrer up and note down the reading
measured and compensated for at 880 nm. in Sahli’s hgb tube by taking the lower meniscus in
consideration.
NON-INVASIVE METHOD  Advantage:
 Pulse Oxymetry  It is the simple and easy method
 It works by the principle of Spectrophotometry.  Disadvantages:
 When the light rays pass through the finger,  Visual intensity may be different for different
glucose and hemoglobin molecules in the blood individuals
absorbs the light source.  This method estimates only oxy Hemoglobin.
 And then the machine could able to measure the  The endpoint disappears soon
hemoglobin  The Proper stable standard is not available
 Occlusion Spectroscopy  The resulting solution is not a clear solution but a
 Ring-shaped sensor is attached to the subject’s suspension
finger.
 The sensor temporarily ceases blood flow
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Hematology Analyzer LIGHT SCATTER
 An automated hematology or hemoglobin analyzer is  Each cell flows in a single line through a flow cell.
commonly used for providing high throughputs to  As the laser light beam strikes a cell it is scattered in
analyze a variety of red and white blood cells as well various directions.
as hematocrit and hemoglobin levels from the blood  One detector captures the forward scatter light that is
sample proportional to cell size
 Principles:  A second detector captures side scatter light (90°) that
1. Electrical Impedance corresponds to the nuclear complexity and granularity
2. Light Scatter of cytoplasm.
3. Fluorescence
4. Light Absorption
5. Electrical Conductivity
ELECTRICAL IMPEDANCE
 Whole blood is passed between two electrodes
through an aperture so narrow that only one cell can
pass through at a time.
 The impedance changes as a cell passes through. Figure 6. Light scattering method for cell detection and
 The change in impedance is proportional to cell measurement
volume, resulting in a cell count and measure of cell
volume. FLUORESCENCE
 Fluorescence is a type of luminescence caused by
photons exciting a molecule, raising it to an electronic
excited state.
 It is brought about by absorption of photons in the
singlet ground state promoted to a singlet-excited
state.
 As the excited molecule returns to ground state, it
emits a photon of lower energy, which corresponds to
a longer wavelength, than the absorbed photon and
releasing their stored energy in an emitted photon
Figure 4. Electrical impedance principle
 Coulter Electrical Impedance Plot

– These results are plotted using the Frequency
Distribution Graph or Volume Distribution
Histogram
– Y-axis: Relative number of cells Figure 7. Model of fluorescence method through absorption and
– X-axis: Size/volume of the cell emitting of light as the excited molecule return to its ground state
LIGHT ABSORPTION
 The spectrophotometer technique is to measure light
intensity as a function of wavelength.
 It does this by diffracting the light beam into a
spectrum of wavelengths, detecting the intensities
with a charge-coupled device, and displaying the
results as a graph on the detector and then on the
display device.
Figure 5. Plotted results from analyzer using electrical impedance
principle
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 Erythrocytes are lysed and the released
hemoglobin is reduced by sodium dithionite in a
concentrated Phosphate buffer.
 The Reduced HbS is characterized by its very low
solubility and the formation of crystals
 Procedure:
Figure 8. Light absorption application in Spectrophotometer; the 1. The Blood is drawn into a tube that prevents the
absorbed light by the sample is measured
blood from clotting
2. Sodium dithionite is added to the blood
ELECTRICAL CONDUCTIVITY
 Hemoglobin S is insoluble when combined with a
 It is used to determine physical and chemical
composition of leucocytes for their classification. reducing agent (sodium dithionite).
 RBCs “lyse” or break open, releasing the
 Some analyzers use conductivity of High frequency
current to do these leucocyte classification hemoglobin from inside the red blood cells into
the blood plasma.
Other Methods for Hemoglobin Examination  Result:
1. Hgb S if present will crystallize and give a turbid
HEMOGLOBIN F (KLEIHAUER-BETKE METHOD) appearance to the solution.

2. Negative result indicates transparent suspension
 It is used to detect the presence of fetal cells in the
3. The test will not differentiate homozygous from
Maternal circulation during problem pregnancies
 Detect fetal-maternal hemorrhage
heterozygous conditions containing Hemoglobin “S”
4. Follow up a positive solubility test with hemoglobin
 It differentiates hereditary persistence of fetal
hemoglobin from other conditions associated with electrophoresis.
high hgb F levels.
 It determines the required dose of Rh immune-
globulin
 Normal newborns Hgb F levels: 70-90%
 Normal adults Hb F cells are <0.01%
Figure 10. Solubility test for hemoglobin “S”
HEMOGLOBIN ELECTROPHORESIS
 Identification of normal and abnormal hemoglobins.
 Based on net negative charges
– Hemoglobins to migrate from the negative
(cathode) region toward the positive (anode) region.
Figure 9. Kleihauer-Betke method; showing fetal cells and  The distance a particular hemoglobin molecule
maternal ghost cells
migrates is due to its net electrical charge.
 Two types of electrophoresis:
 Procedure:
1. Cellulose acetate at pH 8.6 and
1. Maternal blood is Fixed on a slide with 80%
2. Citrate agar at pH 6.2.
ethanol, treated with Citrate phosphate buffer
2. After staining with Hematoxylin and Eosin, the fetal
cells will be stained while the adult cells appear
like Ghost cells
3. Count lots of cells and report percentage of cells
that are fetal (count the number of fetal blood cells
per 50 low power fields)
Figure 11. Hemoglobin electrophoresis method using cellulose
SOLUBILITY TEST FOR HEMOGLOBIN S (SICKLE CELL acetate and citrate agar gel media
PREPARATION)
 Principle:
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HEMATOCRIT TEST  New Born: up to 60%
 It is the ratio of the volume of Packed Red Blood Cells  The determination of hematocrit by means of
to the total blood volume centrifugation
 Greek words hemato (Greek Haima) = blood; crit  The calculation of hematocrit from the complete
(Krinein) = to seprate blood cell count (CBC)
 It is reported as percentage (%)  The determination of hematocrit by conductivity
 Normal range:  The calculation of hematocrit from ctHb
 Adult: 40-48 %
INDICATIONS OF THE VARIATIONS IN THE LEVELS OF Hct

HEMATOCRIT LEVELS DESCRIPTION CAUSES
 A high hematocrit may reflect an Severe dehydration
absolute increase in the number of Erythrocytosis
High Hct Level
erythrocytes, or a decrease in Polycythemia vera
plasma volume Hemachromatosis
 A low hematocrit (anemia) reflects Internal or external hemorrhage
a low number of circulating red Complication of chronic renal failure
Low Hct Level blood cells and is an indicator of a Pernicious anemia
decrease in the oxygen-carrying Hemolysis
capacity or of overhydration. Autoimmune Disease and Bone Marrow failures
ROUTINE HEMATOCRIT TEST  The hematocrit is determined indirectly from the

 Microhematocrit average size and number of RBCs.
 The reference method recommended by NCCLS of  The reference method is the Coulter impedance
determining hematocrit or packed cell volume (PCV) principle
is through centrifugation.  Calculation of Hematocrit
 Measured by placing whole blood in the capillary  In automated analyzer, the hematocrit is usually
tubes and then centrifuging to read the packed cell calculated by the measured MCV and RBC count by
volume using this formula
 Procedure: 𝑀𝐶𝑉 (𝑓𝑙) × 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑥1012 /𝑙)
𝐻𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 =
1. A minimum of two capillaries is required to 1000
 The “Rule of 3”
ensure balance in the centrifuge. It is important
that the tubes are sealed thoroughly.
2. After five minutes of centrifugation the
hematocrit can be measured while the tubes are
still kept in a horizontal position.
3. A distinct column of packed erythrocytes is
visible in one end of the capillary tube RBC Test
4. The packed erythrocytes are followed by first a
 A blood test to find out how many red blood cells
small turbid layer – the buffy coat layer – and (RBCs) you have. It's also known as an erythrocyte
then a clear column of plasma. count.
5. Hematocrit is estimated by calculating the ratio
 The test is important because RBCs contain
of the column of packed erythrocytes to the total hemoglobin, which carries oxygen to your body's
length of the sample in the capillary tube, tissues
measured with a graphic reading device.  Normal range:
6. The measurement should be performed within 10
 Men: 4.7-6.1 million cells per microliter
minutes to avoid merging of the layers.  Women: 4.2-5.4 million cells per microliter
 Complete Blood Count (CBC)
 Children: 4.0 -5.5 million cells per microliter
 In hematology laboratories, automatic cell count
analyzers measuring multiple parameters are the
most commonly used.
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INDICATIONS OF THE VARIATIONS IN THE LEVELS OF RBC
RBC LEVEL CAUSES SYMPTOMS
Cigarette Smoking
Joint pain
Congenital Heart disease
Tenderness in the palms of the hands or soles
Dehydration
High RBC Level of the feet
Renal cell carcinoma (a type of Kidney cancer)
Itching particularly after a shower or bath
Pulmonary fibrosis
Sleep disturbance
Polycythemia vera
Anemia
Fatigue
Bone marrow failure
Shortness of breath
Erythropoietin Deficiency
Dizziness, weakness, or Light headedness
Low RBC Level Hemolysis, or RBC destruction caused by transfusions and blood vessel injury
Increased heart rate
Internal or external bleeding
Headaches
Leukemia
Pale skin
Malnutrition
ROUTINE RBC COUNT 3. Platelets

4. Hemoglobin (Hgb)
Methods 5. Hematocrit (Hct)
COMPLETE BLOOD COUNT MANUAL RBC COUNTING USING HEMACYTOMETER

 Done via hematology analyzer thru the application of  Manual counting of RBC using Hemocytometer or
Electrical Impedance Neubauer Chamber
 A complete blood count or CBC, measures several  It’s not only the RBC that can be counted in this
component of your blood chamber but also other type of blood cells such as
 A complete blood count may also include WBC’s
measurements of chemicals and other substances in  Very large numbers of Red Blood Cells are present in
your blood. the Blood Specimen.
 These results can give important information about  Practically, counting this amount of Red cells directly
your overall health and risk for certain diseases. under the microscope is highly impossible.
 Different Blood Component included in CBC  So, we have this special type of chamber (Neubauer
1. Red Blood Cells (RBCs) counting chamber) for counting this type of blood
2. White Blood Cells (WBCs) cells.
 A CBC test also includes the classification of
white cells in your blood.
 There are five major types of white blood cells:
a. Neutrophils
b. Eosinophils
c. Basophils
d. Lymphocytes
e. Monocytes
METHODS DILUENT PROCEDURE COUNTING METHOD

1. Fill the RBC pipette up to the 0.5 mark with the blood 1. 3-5mins after charging into the
specimen and wipe out the pipette externally to avoid chamber ,Now we can start
false high results. counting
2. Fill the same pipette with the RBC diluting fluid up to 2. Focus the ruling using the 10x
the mark 101. Objective lens
Hayem’s fluid or 3. Mix the Blood and Diluting fluid in the pipette by 3. Then using the 40x Objective
Micro-dilution Method
Distilled water rotating the pipette (horizontally) between your palms. lens.
4. Placed the cover slip over the grooved area of 4. Count the RBCs in 5 small
hemocytometer squares of the large central
5. Discard 1-2 drops from the pipette before charging the square
chamber 5. The counting is Zigzag, starting
6. Allow a small amount of fluid from the pipette to fill From upper left
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into the chamber 6. Follow the “L-rule” in counting
7. Wait for 3-5 mins before the RBC counting cells
1. Take 3.98 ml of RBC diluting fluid in a Clean, Dry and  Cells touching the Left and
Grease free Test tube. Top lines are counted
2. Add 0.02 ml or 20 µl of Blood Specimen to the tube  Cells touching the Right and
containing diluting fluid Bottom lines are not counted
3. Mix well for few minutes and ready your
Hemocytometer / Neubauer’s Chamber.
Macro-dilution Method 4. Placed the cover slip over the grooved area of
hemocytometer
5. Discard 1-2 drops from the pipette before charging the
chamber
6. Allow a small amount of fluid from the pipette to fill
into the chamber
7. Wait for 3-5 mins before the RBC counting
 Calculation  Out of these 25 squares, the RBCs are counted in

– The No. of RBC in 5 squares of the central square is 5 squares so the Area of 5 small squares is 5/25
considered as ‘N’ = no. of cells. i.e. 1/5
– The Volume of the fluid inside the chamber is the – The depth of the Hemocytometer is 0.1 mm
product of Area and depth of the Hemocytometer. – Now Apply the Following formula to get the Total
 The central area is the 1 sq. mm which is divided Red Blood Cell Count
into 25 parts so the area is 25 squares = 1 sq.  Total RBC Count = N × Dilution / Area × Depth
mm  N × 200 (or 100 as the dilution is made) / (1/5 ×
0.1)
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 × 5
𝑇𝑜𝑡𝑎𝑙 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 =
𝑣𝑜𝑙𝑢𝑚𝑒 (0.1)
𝑇𝑜𝑡𝑎𝑙 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 = 𝑁 × 10,000/𝑚𝑚3
FLOW CYTOMETRY COUNTER RBC Indices

 Principle applied is Light Scattering Principle.  RBC indices are calculated parameters that are part of
 Red blood cells are sphered in a diluent and then an automated blood count report
passed through a laser light detector.  Uses:
 The cells scatter light (at different angles) which is 1. To assist with the differentiation of Anemias
detected by the flow cytometry instrument 2. Serves as quality checks
 The laser detects the number of cells, cell volume and  Includes:
internal content. 1. Mean cell/corpuscular volume (MCV)
2. Mean cell hemoglobin volume (MCH)
3. Mean cell hemoglobin concentration (MCHC)
MEAN CELL/CORPUSCULAR VOLUME (MCV)

 Refers to the average size of the RBCs constituting the
sample
 Reporting units is femtoliters (fL)
 Normal value: 80-100 fL
𝐻𝑐𝑡(%) × 10
𝑀𝐶𝑉 =
𝑅𝐵𝐶 (× 1012 /𝐿)
42 × 10
𝑀𝐶𝑉 + = 100 𝑓𝐿
4.2
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INDICATIONS OF THE VARIATIONS IN THE LEVELS OF MCV
High MCV Low MCV Normal MCV
 Sudden and significant blood loss
 Prosthetic heart valve
 Vitamin B-12 deficiency  Iron deficiency
 Tumor
 Folate deficiency  Thalassemia
 Chronic disease, such as a kidney disorder or endocrine disorder
 Chemotherapy  Lead poisoning
 Plastic anemia
 Pre-leukemias  Chronic diseases
 Blood infection
 Mean cell hemoglobin or mean corpuscular hemoglobin
MEAN CELL HEMOGLOBIN (MCH)  Refers to the amount of hemoglobin relative to the
 Refers to the average weight of hemoglobin in the size of the cell or hemoglobin concentration per red
RBCs in the sample blood cell
 Reporting unit is picogram  Reporting unit is grams per deciliter
 Normal value: 26-32 pg  Normal value: 32-36 g/dL
𝐻𝑏 (𝑔/𝑑𝐿) × 10 𝐻𝑏 (𝑔/𝑑𝐿) × 100
𝑀𝐶𝐻 = 𝑀𝐶𝐻𝐶 =
𝑅𝐵𝐶 (× 1012 /𝐿) 𝐻𝑐𝑡
12.5 × 10 12.5 × 100
𝑀𝐶𝐻 = = 30.5 𝑀𝐶𝐻 = = 34
4.1 37
MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)
INDICATIONS OF THE VARIATIONS IN THE LEVELS OF MCHC

HIGH MCHC LOW MCHC
 Iron deficiency
 Hereditary spherocytosis
 Chronic disease
 Sickle cell disease
 Thalassemia
 Homozygous hemoglobin c disease
 Lead poisoning
ERYTHROCYTE SEDIMENTATION RATE (ESR)  Female: 0-20 mm/hr

 Indirectly measures the degree of Inflammation
present in the body.
 It actually measures the rate of fall of erythrocytes in
a sample of blood that has been placed into a tall,
thin, vertical tube.
 Results are reported as the millimeters of clear fluid
(plasma) that are present at the top portion of the
tube after one hour
 Normal range:
 Male: 0.10 mm/hr
INDICATIONS OF THE VARIATIONS IN THE LEVELS OF ESR

ESR LEVEL CAUSES SOURCES
 Chronic inflammation:
– Rheumatoid arthritis
– Pregnancy
– Bacterial infection
Increased ESR
– Malignancy, tissue damage
– Multiple myeloma
– Waldenstrom macroglobulinemia
– Severe anemia
 Polycythemia
 Clotted sample
 Sickle cell anemia
Decreased ESR  Excess anticoagulant
 Spherocytosis
 “Old blood” (spherocyte form)
 Other condition with poikilocytosis
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Methods
WINTROBE AND LANSBERG

 Tube length 11.5 cm (115mm), bore diameter 3.0 mm
 Uses EDTA/double oxalate as anticoagulant
WHITE BLOOD CELL (WBC) TEST

 The white cell count (WBC) is the total number of
leukocytes in a volume of blood, expressed as
thousands/µL.
 WBC count can be done by manual methods or by
automated cell counters.
ROUTINE WBC TEST
Techniques
 Normal Values: 1. Manual counting using hemacytometer
 Female: 0-20 mm/hr 2. Flow cytometry
 Male: 0-9 mm/hr 3. Differential counting
 Children: 0-10 mm/hr
MANUAL COUNTING USING HEMACYTOMETER
STANDARD/ORIGINAL WESTERGREN  Principle:
 Tube length 30 cm (300 mm ±5 mm, tube bore 2.65  Whole blood collected in EDTA is diluted according
±0.15 mm) to the type of cell count obtained.
 Uses Black top tube with NaCitrate (Sodium Citrate)  The diluted blood suspension is then placed in a
chamber and the cell counted
 The count is multiplied by dilution factor and
reported as number of cells per microliter (µL) of
whole blood
 Procedure:
1. Put the cover slip or glass slip on the top of grid
area in the Chamber (use air tight technique)
2. Dilute you sample: 1: 20 for WBC count using WBC
pipette
 Normal range:
 Female: <50 y/o – 0-20 mm/hr
>50 y/o – 0-30 mm/hr
 Male: <50 y/o – 0-15 mm/hr
>50 y/o – 0-20 mm/hr
 Children: 0-10 mm/hr
MODIFIED WESTERGREN
 It requires dilution of EDTA Blood either using
NaCitrate or NSS 3. To prepare 1:20 dilution, aspirate blood up to 0.5
 2ml of well mixed EDTA blood is diluted with 0.5 ml of
mark then diluting fluid up-to 11mark
3.8% sodium citrate or 85% of Sodium Chloride. 4. Load your sample into the loading area in the
chamber
5. Count the cells in the 4 large squares for WBC
6. Calculate the number of cells counted /µL
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1. Prepare two thin films of blood at conclusion of
venipuncture or from EDTA tube. Label slide in
pencil with patient's full name and date. Allow
slides to adequately air dry.
2. Dip slide in stain for 10 seconds.
3. Dip slide in distilled water for 20 seconds or more
for darker staining.
4. Observe slide under low dry for overall impression
and general appearance of blood cells. Check for
even distribution of WBCs and correct staining of
cells.
5. Observe slide under high dry lens after smearing
6. Estimate WBC count by noting number of white
cells per HPF x1000 in 10 fields.
 10 fields are examined in a vertical direction from
bottom to top or vice versa
 Procedure is repeated until 100 WBCs have been
Counted
 Diluent: Acetic acid (CH3COOH) or Distilled H2O

 Calculation: Using 1:20 dilution
𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 × 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑇𝑜𝑡𝑎𝑙 𝑐𝑜𝑢𝑛𝑡 =
𝑎𝑟𝑒𝑎 (𝑚𝑚2 ) × 𝑑𝑒𝑝𝑡ℎ (0.1)
𝑊𝐵𝐶 = 𝑁 × 50
DIFFERENTIAL COUNTING
 It determines the number of each type of white blood
cell, present in the blood.
 Expressed as a percentage (relative numbers of each
type of WBC in relationship to the total WBC) or as an
absolute value (percentage x total WBC)
 May be performed after the WBC blood count has been
determined by the automated 3 part differential, and
may be used as a double check on the WBC count.
 The differential staining allows one to identify the
types of white blood cells on the smear.
 Procedure:
TYPES OF WBC FOUND IN DIFFERENTIAL COUNTING

REFERENCE RANGE
OLDER
WBC TYPE MALE FEMALE PREGNANT INFANT 8 Y/O
CHILDREN
mm3 % mm3 % mm3 % mm3 mm3 mm3
Neutrophils
 Most common of the WBCs
 Serve as the primary defense
against infection 3,000- 1,800- 3,800- 1,000- 1,500-
50-60 50-60 50-60
 The typical response to 7,000 7,700 10,000 8500 8000
infection or serious injury is an
increased production of
neutrophils.
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Band cells
 Early in the response to
infection, immature forms of
neutrophils will be seen
 Called Stab or Band cells
 The presence of these 0-3 0-3 0-3
immature cells is called a
"shift to the left" and can be
the earliest sign of a WBC
response, even before the WBC
becomes elevated.
Lymphocytes
 Play both an immediate and 1,000- 1,000- 1,300- 1,000-
20-40 20-50 20-50 1,000-7,000
delayed role in response to 4,000 4,800 5,200 9,000
infection or inflammation
Monocytes
 Respond to inflammation,
infection and foreign bodies by 100-600 2-6 0-800 0-8 0-800 0-8
ingesting and digesting the
foreign material.
Eosinophils
 Play a role in allergic disorders
50-250 1-4 0-450 0-4 0-450 0-4
and in combating parasitic
infections
Basophils
 Digest bacteria and other
0.5- 0.5- 0.5-
foreign bodies (phagocytosis) 25-100 25-100 25-100
1.0 1.0 1.0
and also have some role in
allergic reactions
 Lymphocytes – Some cancers

 Increased numbers of lymphocytes are seen in:  Decreased monocyte counts are associated with:
– Most viral infections – HIV infection
– Some bacterial infections – Rheumatoid arthritis
– Some cancers – Steroid exposure
– Graves’ disease – Some cancers
 Decreased numbers of lymphocytes are seen in:  Eosinophils
– Steroid exposure  Increased eosinophil counts are associated with:
– Some cancers – Allergic reactions
– Immunodeficiency – Parasite infections
– Renal failure – Chronic skin infections
– Lupus – Some cancers
 Monocytes  Decreased eosinophil counts are associated with:
 Increased monocyte counts are associated with: – Stress
– Recovery from an acute infection – Steroid exposure
– Viral illness – Anything that may suppress WBC production
– Parasitic infections generally
– Collagen disease
PERIPHERAL BLOOD SMEAR AND STAINS

INTRODUCTION cells but also in identifying cells that are abnormal
 Stain – aids the medical technologist not only in and could be an indication of a disorder or cancer
identifying inclusion bodies found inside the blood
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PERIPHERAL BLOOD SMEAR  If the sample came from EDTA, it should be made
 Purpose within 1h to avoid distortion of cell morphology
1. Evaluates blood-related problems in red blood cells
(RBCs), white blood cells (WBCs) and platelet CHARACTERISTIC OF A GOOD SMEAR
2. Distinguish viral infection from bacterial infection 1. Good smear is a tongue shaped with smooth tail
(cellularity is observed)  The tail of the smear is where examining of blood
3. Evaluating anemias cells/differential counting should be performed
4. Looking for inclusions bodies like basophilic 2. Does not cover the entire area of the slide
stiplling, Howell-jolly bodies and Cabot rings 3. Does not contain any lines or holes (caused by fat,
5. Diagnose blood-borne parasites grease or air bubbles)
 Methods of Peripheral Blood Smear 4. Has both thick and thin areas with gradual transition
Preparation
1. Wedge Technique THICKNESS OF SMEAR
2. Cover Slip Technique  Angle of the spreader (32-45º): the greater the angle,
3. Automated Slide Making and Staining the thicker and shorter the smear
4. Spin Method  Size of the blood drop: more blood will increase the
thickness of smear
Wedge Technique  Speed of spreading: not too slow and not too fast,
 PBS could be made from EDTA (recommended) moderate speed only
 It can also be made directly from finger prick to the
slides METHOD OF COUNTING AND EVALUATING A BLOOD SMEAR
– Disadvantage: No reserved blood if ever 1. Battlement method – used in differential counting;
examination should be repeated most preferred method
2. Longitudinal method – used in manual counting using
SMEAR PREPARATION Neubauer
1. Place a drop of blood, about 2-3 mm in diameter 3. Step-technique
approximately 1 cm from one end of slide.
2. Place the slide on a flat surface and hold the other
end between your left thumb and forefinger.
3. With your right hand, place the smooth clean edge of
a second (spreader) slide on the specimen slide.
4. Hold the spreader slide at a 30-45° angle
(recommended) and draw it back against the drop of
blood.
5. Allow the blood to spread almost to the edges of the
slide.
6. Push the spread forward with one light, smooth, Figure 1. Techniques on counting of cells from a blood smear.
moderate speed (a thin film of blood in the shape of
tongue). CAUSES OF POOR BLOOD SMEAR
7. Label one edge with patient name, lab ID and date 1. Cold agglutinin
8. The slides should be rapidly air dried by waving the  RBCs are clumped together
slides or using an electrical fan.  Solution: Warm the blood at 37°C for 5min then
 If put under the sun, the smear might crack due to remake the smear
the temperature 2. Lipemia (F)
 Holes will appear in the smear
 Sample are obtained either from EDTA from 3. Rouleaux
venipuncture or through direct collection of finger  RBCs will form into stacks resembling coins
prick
 Sample from venipuncture is more ideal when the
lab technician will use multiple smear sample
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3. The slide is dried, loaded into cassette and moved to
the staining position, where stain and then buffer and
rinse are added at designated times.
4. After staining, the slide is moved to a dry position,
then to a collection area where it can be picked up for
microscopic evaluation.
THICKNESS OF THE SPREAD

 Increased hematocrit: the angle of the spreader slide
Figure 2. Unacceptable type of smears. should be decreased
 Decreased hematocrit: the angle of the spreader slide
Cover Slip Technique should be increased
 Used for bone marrow aspirate smears
 2 coverslips are used
 Disadvantage: Labelling is difficult due to the size of
the coverslip
PROCEDURE
1. Take a clean cover glass.
2. Touch the cover glass to the drop of blood.
Figure 4. Making peripheral blood smears. Adjustment on the
3. Place another similar cover glass over the other in
angle of spreader to produce a good smear.
crosswise direction with the side containing drop of
blood facing down. Spin Method
4. Pull the cover glass quickly.  This method uses special centrifuge to make a smear
5. Dry it and stain it.  It also an automated method but only until smearing,
6. Mount it with a mountant, film side down on a clean staining is done manually
glass side.
PROCEDURE
1. Place a drop of blood in the center of a glass slide.
2. Spin at a high speed in a special centrifuge called
Cytospin.
3. Blood spread uniformly.
4. Dry then stain it.
Figure 3. Making a smear using coverslip.
Automated Slide Making and Staining

 Aside from PBS, CBC can also be done using this
 Depending on the hematocrit reading, the system
adjust, depends on the following:
 Size of the drop of blood to be used
Figure 5. Making a smear using spin method. A drop of blood
 Angle and speed of the spreader slide in making a
sample was placed on the slide and subjected to centrifugation.
wedge preparation
 After each blood film is prepared, the spreader slide
STAINS
is automatically cleansed.  Purpose: to easily recognize the blood cells on the
prepared smear and identify cells and make an
PROCEDURE evaluation/assessment regarding the morphology of
1. Blood films are produced approximately every 30sec.
the cells including the presence of cells’ inclusion
2. The detail of the specimen is printed on the slide.
bodies (might be normal but some has a significance
to some disorders/abnormalities).
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 A well-stained smear aids to the identification of May-Grunwald-Giemsa (MGG) Stain
blood cells and its main purpose is to differentiate the  Used for staining of blood, bone marrow smear, and
normal from abnormal blood cells. clinical cytological specimens
 Also used in morphological inspection and differential
ROMANOWSKY STAIN counting of blood cells
 Two Main Ingredients: methylene blue (alkaline  Contains alkaline methylene blue, related azures and
staining acids), eosin Y (acidic staining bases) acidic eosin.
 Giemsa staining makes the effect of azure more
COLOR RESPONSE OF BLOOD COMPONENT TO prominent staining stains all cellular components.
ROMANOWSKY STAINING  Basic Dyes:
CELLULAR COMPONENT COLOR – Carry net (+) charges, and stains nuclei (because of
Nuclei Chromatin purple the [–] charges of phosphate groups of DNA and
Nucleoli Light blue RNA molecules), granules of basophil granulocytes,
Cytoplasm
Erythroblast Dark blue
and RNA molecules of the cytoplasm of WBCs.
 Acid Dyes:
Erythrocyte Dark pink
Reticulocyte Grey-blue – Carries net (–) charge and stains red blood cells
Lymphocyte Blue and granules of eosinophil granulocytes.
Metamelocyte Pink – The pH is a very critical factor in staining, so any
Monocyte Grey-blue change will lead to wrong staining reaction.
Myelocyte Pink – The limits of the most suitable pH are between 6.5
Neutrophil Pink / orange
Promyelocyte Blue
and 6.8.
Basophil Blue
Granules PREPARATION OF STOCK SOLUTION
Promyelocyte (primary granules) Red or purple 1. Stock solution is prepared by mixing 0.15g of Giemsa
Basophil Purple black powder in 12.5 ml of glycerin and 12.5 ml of methyl
Eosinophil Red-orange alcohol.
Neutrophil Purple 2. Before use, dissolve 1 volume of stock solution in 9
Toxic granules Dark blue
Platelet Purple
volumes of buffered water (1:9 dilution)
Other inclusions
Auer body Purple PROCEDURE
Cabot ring Purple 1. Prepare thin smear of blood sample and air dry so
Howell-Jolly body Purple that it will not be washed out.
Döhle body Light blue 2. Fix smears for 3min with methanol or with cytofix
*Parasites Dark purple / dark blue fixative (Cat. No. 207030410050).
 Methanol binds blood cells to the slide so it won’t
 Factors that influence smear staining method be washed out when rinsing of the PBS is done.
1. Concentration of the stain
3. Stain the smear in May-Grunwald stain diluted with an
 Low concentration: produce pale colored cells
equal volume of distilled water for 5mins.
(under stained) 4. Put the smears without washing in 1:3 solution of
 High concentration: produce dark stained smear
Giemsa stain diluted with distilled water for 8-10mins.
(over stained) 5. Wash the smears in distilled water and let them dry.
2. Time of exposure
6. Observe under microscope, 40x and 100x under oil
 Too long: overstaining
immersion lens.
 Too short: understaining
 Types: INTERPRETATION OF RESULTS USING MAY-GRUNWALD-
1. May-Grunwald-Giemsa (MGG) Stain
GIEMSA STAIN
2. Wright’s Stain
 Erythrocytes: light pink to brown
3. Jenner’s Stain
 Lymphocytes: blue with purple nucleus
4. Leishman’s Stain
 Eosinophil: blue violet
5. Field’s Stain
 Eosinophilic granules: deep red
 Neutrophil: deep blue to blue violet
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 Neutrophil granules: red 5. Allow the film to air dry.
 Basophil: blue violet 6. Dip air dried blood film in undiluted stain for 15-
30secs.
7. Decolorized the stained smears by immersion in
distilled or deionized water.
8. Let air dry in a vertical position.
INTERPRETATION OF RESULTS USING WRIGHT’S STAIN

 Erythrocytes: yellowish red
 Polymorphonuclears: dark purple nucleus, reddish
iliac granules, pale pink cytoplasm
Figure 6. May-Grunwald-Giemsa stain. Showing different blood  Eosinophils: blue nuclei, red to orange-red granules,
cells as the end-product of MGG staining. blue cytoplasm
 Basophils: purple to dark blue nucleus, dark purple
Wright’s Stain granules
 It is named after James Homer wright who devised  Lymphocytes: dark purple nuclei, sky blue cytoplasm
the stain in 1902 based on the modification of the  Platelets: violet to purple granules
Romanowsky stain.
 It is used to differentiate nuclear and cytoplasmic
morphology of platelets, RBC, and WBC.
 It can also be used to stain blood smears in the
detection of blood parasites.
 The stain distinguishes easily between blood cells and
became widely used for performing differential WBC
count which are routinely ordered when infections are
expected.
PROCEDURE Figure 7. The unique features of each blood cells using Wright’s
 Thin blood film (Dip Method) stain
1. Dip air dried film in undiluted stain for 15-30secs.
2. Decolorize the stained smears by immersion in Jenner’s Stain
distilled or deionized water.  a.k.a. methylene blue-eosinate
3. Air-dry in vertical position.  It is a mixture of several thiazin dyes (basic) in a
 Thin blood film (Rack Method) – ideal for multiple methanol solvent
smears  It is similar to wright stain but differ by not using
1. Air dried the slides on the racks and flood with polychromed methylene blue
stain for 10-15secs.  The staining solution has ionic and cationic properties
2. Add an equal volume of deionized/distilled water – The (–) charged phosphoric acid groups of DNA
and stain for 10secs. attract the purple polychromatic cationic dyes to the
3. Rinse the slide by dipping in deionized/distilled nuclei
water for 30secs.  The stain is dark blue and results in very observable
4. The slide may also be rinsed by swishing or by just clearly stained nuclei
washing with deionized/distilled water.
 Thick blood film IMMERSION STAINING PROTOCOL (Procedure)
1. Allow film to air dry thoroughly for several hours 1. After the smearing, we thoroughly dry the blood or
or overnight. bone marrow smears.

2. Lake the thick film by immersing in distilled or 2. Fix smears in absolute methanol for 15-5min.
deionized water for 10mins. 3. Stain smears in Jenner’s stain solution for 2min.
3. Allow the film to air dry thoroughly. 4. Stain in mixture of 50ml of Jenner’s stain solution,
4. Fix-air-dried film in absolute methanol for 30secs in 75ml of pH 6.6 phosphate buffer solution and 175ml
a couplin jar. deionized water for 5min.
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5. Rinse in standing deionized water for 1.5mins or rinse PREPARATION
in running deionized water 1. Dissolved 0.2g of powdered Leishman’s dye in 100 ml
6. Air drying of acetone-free methyl alcohol in a conical flask.
7. Examine under microscope 2. Warm it to 50°C for 30min with occasional shaking.
3. Cool it then filter it.
HORIZONTAL STAINING PROTOCOL (Procedure)
1. Place a slide thoroughly dried film in a horizontal PROCEDURE FOR STAINING
staining rack. 1. Pour Leishman’s stain drop-wise (counting the drops)
2. Flood smear with absolute methanol for 15-30sec and on the slide and wait for 2min.
then drain. 2. Add double quantity of buffered water dropwise over
– Methanol is applied as fixative to prevent the the slide.
smears being washed-out. 3. Mix by rocking for 8min.
3. Flood smear with 1ml Jenner’s solution and let stand 4. Wash in water for 1-2min.
for 3mins. 5. Air dry and examine under oil immersion lens of the
4. Add 1ml of pH 6.6 phosphate buffer solution and 1ml microscope.
deionized water to smear and let stand for 45 secs.
5. Rinse briefly with running deionized water. INTERPRETATION OF RESULTS
6. Air dry then examine the smear under microscope.  RBC: red to yellowish red
 Neutrophils: dark purple nuclei, pale pink cytoplasm,
INTERPRETATION OF RESULTS (Jenner’s Stain) reddish-lilac small granules
 Erythrocytes: pale pink  Eosinophils: blue nuclei, pale pink cytoplasm, red to
 Eosinophilic granules: reddish orange orange-red large granules
 Leukocyte nuclei: purple  Basophils: purple to dark blue nucleus, dark purple
 Cytoplasm: bluish purple cytoplasm, dark granules
 Neutrophilic granules: light purple  Lymphocytes: dark purple to deep bluish purple
nuclei, sky blue cytoplasm
 Platelets: violet
 Parasites (e.g. Malaria): dark blue
Figure 8. Jenner’s staining. Showing neutrophils and lymphocytes.
Leishman’s Stain Figure 9. The result image of the blood cells including Leishman’s
 Neutral stain for blood smear, devised by a British parasite after the staining procedure.
surgeon B.B. Leishman (1865–1926)
 It is a differential stain that stain the various Field’s Stain
components of the cells.  Field’s stain is a rapid stain used primarily on thin
 Used to study the adherence of pathogenic organisms films for malarial parasites and other parasites.
(parasites, bacteria) to the human cell.  Composition:
 Component:  Field’s Stain A: methylene blue and azure 1
 Mixture of eosin (acidic stain) dissolved in phosphate buffer solution
 Methylene blue (basic stain) in methyl alcohol and  Field’s Stain B: eosin Y in buffer solution
is usually diluted and buffered during the staining
procedure THIN FILM (Procedure)
1. Fix thin film with methanol.
2. Dry microscopic slide on filter paper.
3. Immerse slide in field stain B (eosin) for 5secs.
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4. Immediately wash with water. 4. Differentiate between leukemoid reaction and
5. Immerse slide in field’s stain A (methylene blue) for leukemia (i.e. chronic myeloid leukemia [CML]).
10 secs. – Both conditions show elevated levels of WBC
6. Immediately wash with water. – Leukemoid – increased neutrophils due to
7. Dry thin films. bacteria
– Leukemia – impaired, destroyed, abnormal,
THICK FILM (Procedure) immature WBCs (blasts)
1. Immerse thick film in field’s stain A (methylene blue) 5. Confirm diagnosis of hairy cell leukemia
for 3secs. (overproduction of hairy B-cell lymphocytes).
2. Rinse immediately with tap water. 6. Detects cytoplasmic abnormalities and enzymes
3. Immerse thick film in field’s stain B (eosin) for 3secs. deficiency in myeloid disorder.
4. Then rinse immediately with tap water. 7. Differentiate type of acute myeloid leukemia (AML)
5. Let the slide dry. and acute lymphoblastic anemia (ALL).
8. Classify leukemia.
 Types of Cytochemical Stains
1. Enzymatic Cytochemical Stain
a. Myeloperoxidase
b. Phosphatase
i. Neutrophil/Leucocyte Alkaline Phosphate
Figure 10. Field’s staining procedure. (NAP/LAP)
ii. Acid Phosphatase Reaction
INTERPRETATION OF RESULTS (Field’s Stain) c. Esterase
 Red cells: pink i. Specific
 Nuclei: blue ii. Non specific
 Neutrophilic granules: lilac 2. Non-Enzymatic Cytochemical Stain
 Eosinophilic granules: orange a. Sudan Black B (SBB)
 Parasite (trypanosoma, plasmodium): dark blue b. Periodic Acid Schiff (PAS)
c. Toluidine Blue
d. Perl’s Iron Stain (Prussian Blue Reaction)
Myeloperoxidase (MPO)
 It is an enzyme present in primary and secondary
granules of neutrophils and their precursors.
 It is also present in eosinophil granules and in
Figure 11. Field’s stained smear showing blood cells and parasite. azurophillic granules of monocytes.
 Purpose:
CYTOCHEMICAL STAINS 1. It produces hypochlorous acids to carry out their
 It is a technique used to identify diagnostically useful antimicrobial activity.

enzymes or other substances in the cytoplasm of – Responsible for destroying bacteria.
hemapoeitic cells, usually by development of color 2. Differentiate between myelogenous and monocytic
reactions leukemia from ALL.

 Elements: enzymatic, non-enzymatic 3. Diagnose congenital deficiency of neutrophil MPO.
 Purpose:  Principle:
1. To characterize the blast in acute leukemia as – MPO splits H2O2 in the presence of chromogenic
myeloid (cancer originated from blood forming cells electron donor (e.g. diaminobenzidine [DAB]) and
in the bone marrow) or non-myeloid (bone marrow forms an insoluble reaction product.
is not involved). – The reaction product is stable, insoluble and non-
2. Identify early granulocytic and monocytic cells in diffusible.

acute leukemia.  Method: Pseudo peroxidase or Lepehre reaction
3. Identify unusual cell lineages.
TPMEB 2020
 Reagents:  Stock substrate solution: dissolve 30mg of napthol AS
– Fixative: Buffered formal acetone (BFE) in 0.5ml dimethylformamide and make final volume
 Acetone: 40ml up to 100ml with 0.2M Tris buffer.
 Buffer: 30ml  Coupling azo-dye: fast blue BB salt
 Formalin: 25ml  Counterstain: 0.02% aqueous neutral red
– Substrate: 3,3’-DAB
– Buffer: Sorensen’s phosphate buffer, pH 7.3 PROCEDURE
– Hydrogen peroxide (H2O2, 30% w/v) 1. Fix air dried blood films for 30sec in cold 4% formalin
– Counterstain: Aqueous hematoxylin methanol.
2. Rinse with tap water and air dry.
PROCEDURE 3. Incubate slides with working substrate solution for
1. Fix air dried smear in cold buffered acetone for 30sec. 15mins
2. Rinse in running tap water and dry.  Working substrate solution: 40ml stock solution add
3. Incubate for 10min in working substrate solution. 24mg of fast blue BB
 Working substrate solution: 30mg of DAB in 60ml 4. Wash in tap water and air dry.
buffer, and add 120μl H2O2 just before use. 5. Counterstain for 3mins in 0.02% aqueous neutral red,
4. Wash, counter stain with hematoxylin for 3-5min. rinse with water and air dry.
5. Rinse in running tap water and air dry.
RESULTS
RESULTS  Reaction product: blue and granular
 Reaction product: brown and granular  Intensity of reaction: (–) to strongly (+)
 Nuclei: blue
 (+) reaction: red-brown precipitate GRADING
 0 = negative, or no granules
 +1 = occasional granules
 +2 = moderate number of granules
 +3 = numerous number of granules
 +4 = heavy positivity, numerous granules crowding
cytoplasm overlying nucleus
Figure 12. Blood smear stained with myeloperoxidase (MPO).  Count 100 neutrophil and score them 0 to +4 then
calculate the final score by adding the total scores.
 MPO stains reveal a strong granular cytoplasmic  Normal NAP/LAP score: 38-178
staining in many leukemic blasts
 MPO (+) Auer rods are present (intensified stain).
Neutrophil Alkaline Phosphate (NAP)
 Also called leucocyte alkaline phosphatase (LAP).
 Alkaline phosphatase activity is found predominantly
in mature neutrophil and metamyelocyte
 Purpose: To differentiate between leukamoid
reaction and chronic myeloid leukemia
 Principle: The enzyme activity is associated with a
poorly characterized intra-cytoplasmic membranous
component distinct from primary and secondary
granules
 Reagents: Figure 13. NAP/LAP scoring used to differentiate leukamoid
 Fixative: 4% formalin methanol reaction and chronic myeloid leukemia
 Substrate: napthol AS phosphate
 Buffer: 0.2M Tris buffer, pH 9.0
TPMEB 2020
INTERPRETATION OF RESULTS Esterase
NAP/LAP SCORE NAP/LAP SCORE  Group of enzymes that hydrolyses acyl and chloro-
 Leukamoid reaction acyl esters of α-naphthol or naphthol AS.
 Newborn babies  Purpose:
 Pregnant women
1. Useful in differentiating myelocytic series from
 Aplastic anemia
 In CML monocytic series
 Pernicious anemia
 Hereditary hypophosphatasia
 Neutrophilia of infection 2. To distinguish between normal and leukemic cells
 Paroxysmal Nocturnal
 Polycythemia vera of both series
Hemoglobin (PNH)
 Hodgkin lymphoma  Principle: Esterase enzyme present in leukocyte
 Myelofibrosis
 Essential thrombocytosis hydrolyses the substrate esters and the product
 Multiple myeloma formed react with a diazonium salt to give a brightly
 Obstructive jaundice formed-colored compound
 Classification:
Acid Phosphatase (ACP) Reaction 1. Specific Esterase: Isoenzymes 1, 2, 7, 8, and 9;
 Ubiquitous in hemapoietic cells stained by NASD CAE
 Purpose: For the diagnosis of T-cell ALL and hairy 2. Non-specific Esterase: Isoenzymes 3, 4, 5, and 6;
cell (B-cell) leukemia which is tartrate resistant inhibited by sodium fluoride (NaF); stained by ANBE
 Principle: and ANAE
– ACP enzyme is present in myelocytic, lymphocytes,
monocytic, plasma cell, and platelets.  Isoenzyme – have different amino acid sequence
– ACP in the presence of L-tartrate produces no color but has the same function
while hairy cell ACP will give a (+) color reaction.
 Types:
RESULTS 1. Naphthol AS-D Chloroacetate Esterase (NASD CAE)
 Reaction product: red with mixture of granular and 2. α-Naphthyl Butyrate Esterase (ANBE)
diffuse positivity 3. α-Naphthyl Acetate Esterase (ANAE)
INTERPRETATION NAPHTHOL AS-D CHLOROACETATE ESTERASE
 ACP without tartaric acid  stain for specific esterase
– All acute and chronic T-lineage leukemia show  Useful as a marker of cytoplasmic maturation in
strong positivity (polar positivity). myeloid leukemia.
– Granulocytes are strongly (+).  Reagents:
– Monocytes, eosinophils and platelets show variable  Fixative: Buffered formal acetone
positivity.  Buffer: 66mmol/L phosphate buffer, pH 7.4
– In bone marrow macrophages, plasma cells and  Substrate: Naphthol AS-D chloroacetate in buffer
megakaryocytes are strongly (+)  Coupling Reagent: Hexazotized new fuchsin in 4%
 ACP with tartaric acid sodium nitrate solution
– In hairy cell leukemia the majority of leukemic cells  Counterstain: Aqueous hematoxylin
show positivity in the presence of tartaric acid.  Procedure:
1. Fix air dried smears in cold buffered formal acetone
for 30secs.
2. 2. Rinse gently in running tap water and air dry.
3. 3. Treat the slides with substrate solution for 5-
10mins.
4. 4. Rinse in running tap water and air dry.
5. 5. Counter-stain with aqueous hematoxylin for
1min.
Figure 14. Acid phosphatase reaction with tartrate resistance. 6. 6. Rinse with running tap water and air dry.
Seen are granular staining in the lymphocytes.  Results:
 Reaction product: bright red
TPMEB 2020
 T-lymphocytes show focal dot like positivity (not
seen)
 Erythroblast show focal or diffuse positivity
Figure 15. Naphthol AS-D chloroacetate esterase stain. Seen are

myelocytes (bright red)
α-NAPHTHYL BUTYRATE ESTERASE

 Stain for non-specific esterase
 Useful to identify monocytic component in AML
Figure 17. α-naphthyl acetate esterase stain. Seen was a smear
 Substrate: α–naphthyl butyrate with acute monoblastic leukemia (AML)
 Coupling Reagent: Fast garnet GBC
 Results:  Combined ANAE and CAE – to avoid the need to
 Reaction product: brown and granular compare results from the spate slide; to detect
 Interpretation: myelomonocytic leukemia
 The majority of monocytes (>80%) stain strongly  Combined CAE and NABE – to identify monocytic
 Granulocytes and platelets are negative and granulocytic component in AML
 B-lymphocytes are negative
 In bone marrow monocytes, monocytes precursors Sudan Black B (SBB)
and macrophages stain strongly  Sudan black B is a lipophilic dye that stains intra-
cellular phospholipids and other lipids.
 Purpose:
1. Differentiate between ALL and AML.
2. Done in old smears in which MPO cannot be
performed.
 Principle: A lipophilic dye binds irreversibly to an
unidentified/undefined granule in granulocytes and
eosinophils
 Method: Sheehan and Storey
 Reagents:
 Fixative: 40% formaldehyde vapors
 Stain: 0.3% SBB in absolute alcohol
Figure 16. α-naphthyl butyrate esterase stain. Seen are monocyte  Phenolic buffer: 16gm crystalline phenol in 30ml of
stained (+) (arrow) suggesting that it is an abnormal/neoplastic absolute ethanol and final volume up to 100ml with
cell. buffer
 Working stain solution: add 40ml phenolic buffer to
α-NAPHTHYL ACETATE ESTERASE 60ml SBB solution
 Stains non-specific esterase  Counter stain: Leishman stain
 Substrate: α-naphthyl acetate
 Coupling Reagent: Hexazotized pararosaniline in PROCEDURE
4% sodium nitrite solution 1. Fix air dried smear in in formalin vapors for 5-10mins.
 Results: 2. Then air wash for 15mins.
 Reaction product: diffuse red/brown 3. Now stain with working solution of SBB stain solution
 Interpretation: for 1h.
 Leukemic and normal monocyte stain strongly 4. Now give 3 washings of ethanol for 30secs each.
 Normal granulocytes are negative 5. Wash with water and air dry.
6. Now counter stain with Leishman stain.
TPMEB 2020
RESULTS
 Reaction product: black and granular
 Nuclei: blue
Figure 19. Periodic acid Schiff (PAS) stained smear
INTERPRETATION
 Granular precursors show diffuse weak positivity,
Figure 3.3.18. Sudan black B stained smear. (+) stain in AML

with neutrophils showing intense granular positivity
patients. and act as internal positive controls.
 Monocytes and their precursors show variable diffuse
INTERPRETATION positivity.
 The results are similar to MPO staining both in normal  Normal erythroid precursors and RBCs are negative
and leukemic cells. but dysplastic erythroblasts are positive.
 Difference: eosinophil granules are SBB (–)  Megakaryocytes and platelets are positive.
 In rare cases (1-2%) of ALL shows no- granular  Lymphoblast shows PAS blocks (i.e. block positivity).
smudgy positivity not seen in MPO staining
Toluidine Blue
Periodic Acid Schiff (PAS)  It is a basic thiazine metachromatic dye with high
 Use to detect the glycogen present in tissues. affinity to acidic tissue components.
 Purpose:  Purpose: useful for the enumeration of basophils and
1. Differentiate between AML and ALL. mast cells
2. Useful in AML and MDS to identify abnormal  Reagents: 1% toluidine blue in methanol (w/v)
erythroblast and dysplastic megakaryocytes.
3. Confirm diagnosis of acute promyelocytic leukemia. PROCEDURE
 Principle: 1. Place air dried blood smears on staining rack and
 Periodic acid specifically oxidizes 1-2 glycol groups flood with toluidine blue solution and incubate for 5
of carbohydrates to produce stable di-aldehydes. min.
 These di-aldehydes give a red reaction product 2. Rinse in running tap water until clear and air dry.
when exposed to Schiff’s reagent.
 Reagents: RESULTS AND INTERPREATATION
 Fixative: Methanol  Granules of basophils and mast cells: bright red and
 1% periodic acid (HIO4) Schiff’s reagent purple
 Counter stain: Aqueous hematoxylin  Nuclei: blue
 Cells with abundant RNA may show blue tint to the
PROCEDURE cytoplasm
1. Fix films for 15mins in methanol.
3. Treat slides with 1% periodic acid for 10mins.
4. Rinse in running tap water for 10mins and air dry.
5. Now treat with Schiff’s reagent for 30mins.
7. Counterstain with aqueous hematoxylin for 5mins
then wash and air dry.
RESULTS Figure 20. Toluidine blue (+) basophils in chronic myelogenous

 Reaction product: red leukemia.
 Intensity: pink to bright red
 Cytoplasmic positivity: diffuse or granular
TPMEB 2020
Perl’s Iron Stain (Prussian Blue Reaction) – This ferric iron then reacts with dilute potassium
 Principle: ferrocyanide solution to produce an insoluble blue
 Siderotic granules are found in the cytoplasm of compound ferric ferrocyanide (prussian blue)
developing cells in bone marrow in the form of
ferric.
+3
 [Fe ] + Brussian blue (Perl’s reagent)  blue
color
 Reagents: potassium ferricyanide + HCl
– When the tissue is treated with an acid ferrocyanide
solution, it will result in the unmasking of ferric iron
in the hemosiderin, in the form of ferric hydroxide
(FeOH3) via dilute hydrochloric acid
Figure 21. Prussian blue stained smear showing (+) siderotic
granules in nucleated RBC
PERIPHERAL BLOOD SMEAR EVALUATION

INTRODUCTION  Seen in iron deficiency anemia
 As a part of peripheral blood smear (PBS) evaluation,  Thalassaemia
not only the color and morphology are being observed  Hypochromia
and discussed but also the cellularity of blood 3. Hyperchromic
components.  Increase in hgb content in the RBC
 It also explains the causes of these changes and  Red cells stain deeply
discusses the significance of these various changes.  Have less central pallor
 Increase in MCH
VARIOUS CHANGES IN RBC  Seen in megaloblastic anemia
 Hereditary spherocytosis (MCH is normal but
Color MCHC is increased)
 It is determined by hemoglobin content of the red
blood cells (RBC).
 The average amount of hemoglobin (hgb) in the RBC
 Mean corpuscular/cell hgb (MCH): 27-31 pg
 Mean corpuscular/cell hgb concentration (MCHC):
32-35 pg
1. Normochromic – with normal hemoglobin content
per RBC
2. Hypochromic
 Decrease in hgb content of RBC
 Increase in central pallor (>1/3rd) Figure 1. RBCs with different amount of hemoglobin.
 Decrease in MCH and MCHC
COLOR CHANGES/VARIATIONS IN RBC

DEFINITION/DESCRIPTION SEEN IN
Dimorphic
 Sideroblastic anemia
 Some weeks after
iron therapy for iron
Presence of hypochromic cells and normochromic cells in the same
deficiency anemia
film
 Hypochromic anemia
Figure 2. PBS of patients with dimorphic after transfusion with
anemia, seen in combination of normal cells
hypochromic cells and normochromic cells
TPMEB 2020
Polychromatophilia  It is a condition of RBC where there is an appearance of blue grey
tint of red cells due to hgb and RNA (residual) in young cells.
 They are larger than normal and may lack central pallor; implies
 Hemolysis
reticulocytosis
 Acute blood loss
 This phenomenon are also seen in reticulocytes/polychromatic
Figure 3. Polychromatophilia showing
erythrocyte where the RBCs appears in a blue-grey color because
blue-grey tinted RBC of the presence of RNA
Size Changes/Variations in RBC  Iron deficiency anemia

 Thalassemia
ANISOCYTOSIS  Anemia of chronic disease
 Increase variation of size of RBC  Sideroblastic anemia
 Normal: mean corpuscular/cell volume (MCV) is -80- 3. Macrocytic anemia
100 fl  This refers to when RBCs are larger than usual
 Microcytes: MCV <80 fl  Macrocytes – when MCV of RBC is increased
 Macrocytes: MCV >100 fl (>100 fl)
 Macro-ovalocytes are seen in megaloblastic
3 TYPES OF ANEMIA (variation of RBC sizes) anemia
1. Normocytic anemia  Myelodysplastic syndrome
 A blood problem when a person has normal-sized  Round macrocytes seen in alcoholism, liver
RBCs, but there is a low amount of them. disease
2. Microcytic anemia
 This is when the RBCs are smaller than usual. Shape Changes/Variations in RBC
 Microcytes – size of RBC is reduced (<80 fl); seen  Poikilocytosis – abnormal shape of RBCs
when hgb synthesis is defective
SHAPE CHANGES/VARIATIONS IN RBC

DEFINITION/DESCRIPTION SEEN IN
Acanthocytes/ Spur Cells
 Abnormal phospholipid metabolism
Red cells with small number of spicules of  Abetalipoproteinemia
inconstant length, thickness and shape,  Inherited abnormalities of red cell membrane protein
irregularly disposed over the surface.  Splenectomy
 End stage of liver disease
Figure 4. Acanthocytes erythrocytes
Echinocytes
 Also called crenated cells
 Uremia (i.e chronic renal disease)
 There are numerous, short, regular
 Artifact (i.e older renal disease)
projection
 Liver disease
 Commonly occur as an artifact during
 Hyperlipidemia
preparation of film
Figure 5. Crenated erythrocyte on PBS
Schistocytes/ Helmet Cells  Indicative of a mechanical stress microangiopathic

 Typically irregularly shaped, jagged, and hemolytic anemia (MAHA)
have two pointed ends.  MAHA – caused by destruction of erythrocytes due to
 Smaller than normal red cells and of physical shearing as a result of passage through small
varying shape vessels occluded by systemic microthrombi.
 Suggests RBC injury from damaged  Typical for disseminated intravascular coagulopathy (DIC).
Figure 6. Jagged, irregularly-shaped endothelium  Thalassemia
erythrocytes  Direct thermal injury
Keratocytes/ Horn Cells
 Have pairs of “horns” or spicules either
one or two pairs.  Mechanical damage
 Sometimes termed as bite cell or helmet  Removal of Heinz body by pitting action of spleen
cell
Figure 7. Bite cell or helmet cell in PBS
TPMEB 2020
Elliptocytes/ Ovalocytes
 Iron deficiency anemia
 Elliptical in shapes
 Myelofibrosis with myeloid metaplasia
 Most abundant in hereditary
 Megaloblastic anemia
elliptocytosis
Figure 8. Elliptocytes in PBS
Spherocytes
 Nearly spherical
 Hereditary spherocytosis
 Diameter is smaller than normal
 Some cases of autoimmune hemolytic anemia
 Lack central pale area (central pallor) or
 Direct physical or chemical injury
have a smaller, eccentric, pale area
Figure 9. Spherical-shaped erythrocytes
(spherocytes)
Stomatocytes/ Hydrocytes  Contains a mouth-like/slit-like pattern
that replaces the normal central zone of
pallor.
 Hereditary stomatocytosis (HSt) – liver disease due to
 Red cells with central biconcave area
alcoholism
appear slit-like in dried film and cup-
 Myelodysplastic syndromes
shaped in wet film.
Figure 10. Red cells with central  The normal biconcave disc becomes a uni-
biconcave area concave cup RBC
Codocytes/ Target Cell RBC
 Cells in which central round stained area  Common in Thalassaemia
and peripheral rim of cytoplasm  Chronic liver disease
 Have the appearance of a shooting target  Hereditary hypo- betalipoproteinemia .
with a bullseye  Iron deficiency anemia
 Have an excess of cell membrane relative  Hemoglobinopathies (hgb C, hgb H, sickle cell anemia)
Figure 11. Erythrocytes with a shooting to cell volume  Post-splenectomy
target, bull’s eye appearance
Sickle Cell RBC
 Present in film of patient with homozygosity for hgb S.

Cells are sickle (boat shape) or crescent
 Usually absent in neonates and rare in patients with high
shape
hgb F percentage
Figure 12. PBS demonstrating

irreversibly sickled cells
 One side of cells is tapered and other is
Tear Drop Cell/ Dacrocytes blunt.
 It is a result of the removal of an  Thalassemia
inclusion from the cell as it moves  Splenic abnormalities
through the spleen.  Myelofibrosis
 Since red cells are quite flexible and  Frequently associated with infiltration of the bone marrow
usually return to their normal shape by fibrosis, granulomatous inflammation, or hematopoietic
Figure 13. Erythrocytes with an following pitting, it has been theorized or metastatic neoplasms.
appearance of being tapered and blunt on that in this case the membrane may have  Vitamin B12 deficiency and some other forms of anemia
one end been stretched too far and thus cannot
return to its original shape
ARRANGEMENT CHANGES/VARIATIONS IN RBC

DEFINITION/DESCRIPTION ASSOCIATION
Rouleaux Formation
 Paraproteinemia (monoclonal gammopathy)
 Elevated plasma fibrinogen or globulin level
Alignment of red cells one upon another so  Chronic liver disease
that they resemble stacks of coins  Malignant lymphoma
 Multiple myeloma
Figure 14. Rouleaux formation in  Chronic inflammatory disease
peripheral blood smear
TPMEB 2020
Red Cell Agglutinin  Infectious mononucleosis,
 These are autoantibodies that cause RBC
 Mycoplasma pneumoniae
clusters to form and can create anemia
 Other bacterial, viral or parasitic infections.
by accelerating their destruction
 More rarely, with cancers such as
 It may also lead to false low RBC counts
lymphoma, leukemia or multiple myeloma.
when the blood count is analyzed
Figure 15. PBS showing RBC agglutinin  Cold Agglutinin Disease
VARIATIONS OF INCLUSION BODIES IN RBC

DEFINITION/DESCRIPTION ASSOCIATION/SEEN IN
Howell-Jolly Bodies Associated with:
 Single
– Megaloblastic anemia
 Smooth single large round inclusions
– Hemolytic anemia
 These are the remnant of nuclear
– Post-splenectomy
chromatin
 Multiple
Figure 16. Howell-Holly bodies seen in – Megaloblastic anemia
PBS – Abnormal erythropoiesis
 Post-splenectomy
Heinz Bodies  Thalassemia
 Purple, blue, large, single or multiple
 Hemolytic Anemia
inclusions attached to the inner
 Oxidative stress
surface of the red blood cell.
 Glucose-6-phosphate dehydrogenase deficiency
 Represent precipitated normal or
 Glutathione synthetase deficiency
unstable hemoglobins.
Figure 17. Heinz bodies seen in PBS  Drugs & Toxins
 Unstable hemoglobin
Papenheimer Bodies
 It is an abnormal basophilic granules Associated with:
of iron found inside RBC  Sideroblastic anemia
 These are small single or multiple  Thalassemia
peripherally sited angular basophilic  Seen in the peripheral blood following a
Figure 18. Pappenheimer bodies seen in
(almost black) erythrocyte inclusions. splenectomy
erythrocytes
 Fine stippling
– Increased polychromatophilia
Basophilic Stippling – Increased production of red cells
 Coarse stippling
 These are aggregates of Ribosomes
– Lead and heavy metal poisoning
or fragments of ribosomal RNA
– Disturbed erythropoiesis
precipitated throughout the
 Megaloblastic anemia
cytoplasm of circulating erythrocytes
 Thalassemia
 Stain deep blue with Wright’s stain
Figure 19. Basophilic stippling seen in  Infection
erythrocytes  Liver disease
– Unstable Hgb
– Pyrimidine-5’-nucleotidase deficiency
Cabot’s Ring
 These are thin, red-violet, threadlike
strands, Ring shaped, figure of eight
 Observed rarely in – Pernicious anemia
or loop shaped.
 Lead poisoning
 Red or Reddish-purple with Wright’s
Figure 20. Cabot’s ring seen in
stain and have no internal structure
erythrocytes
Malarial Stippling
 These are Plasmodium parasites in
the form of trophozoites found inside
the RBC Associated with malaria
 Gametocytes form of Plasmodium are
found outside the RBC
Figure 21. Malarial stippling seen in PBS
Hemoglobin H Inclusions  It is an unstable hemoglobin which Associated with a-thalassemia
TPMEB 2020
forms precipitates just below the red
blood cell membrane.
 Located just inside the red blood
cells’ membrane and push outward.
 Can be observed with Brilliant Cresyl
Figure 22. Hgb H inclusions seen in PBS
Blue (BCB) Stain
WBC MORPHOLOGY  It indicates a response by the bone marrow to the

Inflammatory or granulopoietic cytokines, which
Neutrophils increases the marrow production
 Shift to the Left
 This Blood shift is an increase in the number of
immature cell types among the blood cells in a
sample of blood
 Usually band cells can be seen in the PBS, but
earlier forms can also be seen
 Reasons
 It is due to the release of bone marrow stores
Figure 23. Shift to the left; blood shift showing increase number of
immature neutrophils
 Myeloid hyperplasia (not always)
WBC GRANULES
DEFINITION/DESCRIPTION ASSOCIATION
Toxic Granulation  Bacterial infection and
other Inflammatory
condition
Dark Blue cytoplasmic granules
 Aplastic anemia
 Myelofibrosis
Figure 24. Toxic granulation seen in neutrophils  Administration of G-CSF
Hypogranulation
It is described as decrease in number or
Myelodysplastic syndrome
complete absence of specific (primary)
(MDS)
granules
Figure 25. Hypogranulation seen in neutrophils
Alder-Reilly Anomally
Granules are Large, coarse, dark purple

Mucopolysaccharidosis
azurophilic granules in the cytoplasm
Figure 26. Granules found with Alder-Reilly anomaly

Chediak-Higashi Sydrome
 Granules are giant, scanty azurophilic
 Neutrophil has a functional defect
 It is a defect in microtubules function Staphylococcus and
that prevents lysosomes from fusing Streptococcus
with phagosomes to produce
Figure 27. Granules in neutrophils associated with phagolysosomes
Chediak-Hiagashi syndrome
Dohle Bodies  These are single or multiple blue
cytoplasmic inclusions that are
remnants of rough endoplasmic  Bacterial infection
reticulum  Inflammation
 Small, round or oval, pale blue-gray  During pregnancy
structure
Figure 28. Dohle bodies seen in neutrophils  Found at the periphery of neutrophil
May-Hegglin Anomaly  Granules resemble Döhle bodies
TPMEB 2020
 Inclusions occur in all types of
leucocytes except lymphocytes
 Contains small basophilic cytoplasmic
granules
Figure 29. Inclusion bodies seen in neutrophils
Vacuoles in Neutrophils
 Severe sepsis
 As an artifact with
prolonged standing
Figure 30. Neutrophil with vacuoles in its cytoplasm
NUCLEI
OTHER
DEFINITION/DESCRIPTION ASSOCIATION/SEEN IN
NOTES
Hypersegmentation
 Uraemia
Presence of 6 or more lobes of more
 Iron deficiency anemia
than 3% of neutrophils
Drugs – Cytotoxic treatment with methotrexate
Figure 31. Hypersegmentation seen in
neutrophils
Pelger-Huet Cells  Neutrophils with dumbbell-shaped,
bilobed nuclei
 Neutrophil fail to segment properly
 Defect of terminal neutrophil
differentiation secondary to
Figure 32. Hyposegmentation seen with
mutations in the lamin B receptor
neutrophils (LBR) gene
Pseudo-Pelger-Huet Cells
 Morphological similar to Pelger-Huet  Myelodysplastic syndrome
anomaly  Acute myeloid leukemia with dysplastic
 Bilobed with more condensed maturation
chromatin Occasionally in Chronic myelogenous leukemia
Figure 33. Hyposegmentation seen in pseudo-
Pelger-Huet anomally
Pyknotic Neutrophil (Apoptosis)
 Dead homogenous Nuclei (pyknotic)  Accelerated phagocytosis in Infections,
 Small numbers of Dead or Dying cells  Poison, burns and Heatstroke
may normally be found in the blood Invitro after standing for 12-18h
Figure 34. Pyknotic neutrophils seen in PBS
 Eosinopenia
– Prolonged steroid administration
 Eosinophilia
– Allergic conditions and asthma
 Severe eosinophilia
Eosinophils
– Reactive eosinophilia
– Eosinophilic leukemia
– Idiopathic hyper-eosinophilic syndrome
– T-cell lymphoma, B-cell lymphoma
Acute lymphoblastic leukemia
 Nucleus segment fold up on each
other resulting compact irregular
Basophils dense nucleus (closed lotus flower) Increased in chronic myeloproliferative disorder
 Granules are large, variable in size,
dark blue or purple often has
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obscure nucleus
 Granules are rich in
– Histamine
– Serotonin
– Heparin
 It is the largest circulating leucocyte
Increased in:
(15-18 um)
 Chronic infections
 Cytoplasm is Gray-blue
 Inflammatory conditions such as:
 Nucleus is Large, curved ( horse-shoe
Monocytes – Tuberculosis and Crohn’s disease
shape)
– CML
 No segmentation
– Acute leukemia with monocytic component
 Chromatin is fine and evenly
Infectious mononucleosis
distributed
 The body doesn't make enough lymphocytes.
 The body makes enough lymphocytes, but
they’re destroyed.
Lymphocytopenia
 The lymphocytes get stuck in the spleen or
 Abnormally low number of
lymph nodes.
lymphocytes in the blood
 Infectious diseases, such as AIDS, viral
hepatitis, tuberculosis, and typhoid fever.
Lymphocytes
Autoimmune disorders, such as lupus.
Lymphocytosis  Had a recent infection (most commonly viral)
 Higher-than-normal amount of  Long-lasting inflammation, like arthritis
lymphocytes  A reaction to a new medication
 A condition that often results from  Trauma
your immune system working to  Had their spleen removed
fight off an infection or other disease Leukemia or Lymphoma
 Thrombocytopenia 3. Increased destruction of platelets
– A condition of having low level of  Pregnancy
platelets in the blood stream  Immune thrombocytopenia
– Processes that causes low  Bacteria in the blood
platelets:  Thrombotic thrombocytopenic purpura
1. Trapping of platelets in the  Hemolytic uremic syndrome
spleen Medications
 Enlarge spleen - can harbor  Thrombocytosis
too many platelets, which – A condition of having high levels of
Platelets decreases the number of platelets in the blood stream
platelets in circulation – Reactive thrombocytosis
2. Decreased platelet production  Chronic inflammatory disorder (i.e
 Leukemia and other cancers Juvenile rheumatoid arthritis)
 Some types of anemia  Acute infection
 Viral infections such as  Iron deficiency anemia
hepatitis C or HIV  Hemolysis, hemorrhage
 Chemotherapy drugs and  Cancer
radiation therapy  Splenectomy or hyposplenism
 Heavy alcohol consumption – Essential thrombocytosis
Giant Platelet
Seen in:
 May-Hegglin anomaly
Platelets as size of RBC  Bernard-Soulier syndrome
 Alprt syndrome
 Storage pool syndrome
Figure 35. Giant platelets seen in PBS
ERYTHROPOIESIS
RED BLOOD CELL DIFFERENTIATION
RBC NUCELUS CYTOPLASM CLINICAL CONDITIONS &
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*OTHER NOTES
Rubriblast/Pronormoblast
(14-22m) Shape: round to slightly oval
Color: deep blue Hemolytic disease of newborn
N/C Ratio: 8:1
Contents: Golgi,
Color: purple-red
mitochondria, producing *Earliest recognizable stage of
Chromatin: fine
light blue perinuclear halo erythropoiesis
Nucleoli: 1-2 prominent with bluish tint
Prorubricyte/Basophilic
Normoblast (12-17m) Shape: round, centered
Color: deep blue
N/C Ratio: 6:1
Contents: Golgi (may
Color: purple interspersed with light
produce light blue area Hemolytic disease of the newborn
areas
near the nucleus),
Chromatin: slightly coarse
mitochondria
Nucleoli: usually not visible
Rubricyte/ Polychromatophilic Color: bluish-pink to grey- Hemolytic disease of the newborn

Shape: round, centered to eccentric
Normoblast (10-15m) blue Myelodysplastic syndromes (CML)
N/C Ratio: 4:1
Contents: visible Hemolytic anemias
Color: red-purple
Thalassemia major
Chromatin: coarse, condensed; perinuclear halo, hgb
parachromatin distinct, producing a causing the pink-grey color,
*Last subdivision during
“checkerboard” appearance RNA causing the light blue maturation
Nucleoli: none color *The production of hgb starts
Orthochromic Normoblast/ Shape: round, centered to eccentric: may
Metarubricyte (7-12m) Hemolytic disease of the newborn
be fragmented or extruding
Myeloproliferative syndrome (CML)
N/C Ratio: 1:2 Color: pink to orange-pink
Thalassemia major
Color: — Contents: hgb production
Sickle cell disease
Chromatin: condensed and homogenous increased
(pyknotic)
*Extrusion of nucleus stars
Nucleoli: none
Reticulocyte (7-10m)
Color: pink to slightly
Increased erythrocyte production
pinkish gray
Hemolytic anemias
none Contents: remnants of
Membrane disorders
Golgi and mitochondria,
Hemolytic disease of the newborn
residual RNA (reticulum)
Erythrocyte/ Normocyte
(6-8m)
Color: pink, central pallor
None but the cell itself is round, biconcave
about 1/3 of cell *Matured RBC
disk (discocyte)
Contents: no mitochondria
Normoblast to reticulocyte takes 3-5d to develop/form. After the formation of these in the bone marrow it will need 1-2d before it goes out of the
one marrow into the blood circulation. From there it will wait for another day for it to become a mature RBC which is the erythrocyte/normocyte.
ERYTHROCYTE STRUCTURE
Membrane
 Shape of the RBC (biconcave) is crucial to its function
 Allows close to maximum surface-to-volume ratio and
optimal gaseous exchange
Figure 1. RBC shape, (discocyte, biconcave)
 Consists of:
1. Carbohydrates – 10%
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 Internal lipids: phosphatidylethanolamine,
phosphatidylinositol, phosphatidylserine
Figure 2. Part of RBC membrane consists of lipid
2. Protein – 50%
a. Integral Proteins – penetrates the lipid layer
 Glycophorin (A&C), band 3, Rh complex
b. Peripheral Proteins – outside the cell
membrane
 Spectrin, anykyrin, actin, adducing, band 4.1,
band 4.2, tropomyosin
3. Lipid – 40% (20% phospholipid, 20% cholesterol)
 Phospholipid – a type of lipid molecule; main Figure 3. Phospholipid layer structure, forming a double layer
which is the characteristic of a cell membrane
component of the cell membrane; hydrophobic
tail, hydrophilic head
 External lipid: phospholipid, phosphatidylcholine
glycolipid, sphingomyelin
RED CELL MEMBRANE INTEGRAL PROTEINS

PROTEIN FUNCTION
 It is a sialic acid-rich protein
Glycophorin  Red blood cells (RBCs) have a net negative surface charge and this bulk charge is due to ionized sialic acid
 Imparts a (–) charge to the cell, reducing interaction with other cells/endothelium
 a.k.a. Membrane Inhibitor of Reactive Lysis Type II (MIRL II)
Glycophorin A (GPA)
 Complement regulator and is a receptor for bacteria, viruses, and Plasmodium falciparum malarial parasite
Glycophorin C (GPC)  Maintains erythrocyte shape and regulates membrane material properties, through its interaction with protein 4.1
 Major RBC transmembrane protein and major integral protein
 Most abundant membrane protein in human RBC
 a.k.a. Ion exchanger protein; exchange bicarbonate for chloride (chlorine shift)
Band 3
 Catalyze chloride-bicarbonate exchange
 Contains binding sites for Ankyrin, band 4.1, band 4.2, and several glycolytic enzymes
 Plays a role in recognition and removal of normal senescent RBCs
 Form a core complex that is critical to the structure of the erythrocyte membrane, and may play a physiologically
Rh complex Protein role in the sequestration of blood ammonia.
 RBCs which lack Rh antigens (Ag) have an abnormal shape.
RED CELL MEMBRANE PERIPHERAL PROTEINS

PROTEIN FUNCTION
Spectrin  Principal structural element of RBC membrane, which plays a major role in the RBC cytoskeleton membrane organization
Ankyrin  Primary determinant of mechanical coupling of the lipid bilayer to the membrane cytoskeleton
 Abundant protein in the cell membrane
Actin
 Participates more in protein to protein interactions
Adducin  Promotes association of spectrin with F-actin
Dual functions:
Band/
 Promotes high-affinity association between spectrin and F-actin
Protein 4.1
 May link the skeleton to the membrane by virtue of its associations with glycophorin and band 3
Band/  Regulates the association of protein 3 within ankyrin
Protein 4.2  A deficiency of the protein is associated with several types of inherited hemolytic anemias
Tropomyosin  Stabilizing the actin filaments
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Figure 4. Chloride-bicarbonate exchange where the anion Figure 5. Integral and peripheral proteins inside the
exchange is regulated by the band 3 integral protein RBC membrane
 Myosin supports the cytoskeleton/shape of RBC EMBDEN-MEYERHOR PATHWAY (EMP)

 The concavity of the RBC is supported by the  It is the major pathway in tissues lacking
contraction of myosin mitochondria
 It involves sequence of reaction converting glucose or
PHYSIOLOGIC FUNTION glycogen to pyruvate or lactate
1. Maintain cell structure and deformability  The degradation of glucose generates ATP and
2. Maintain osmotic balance between plasma and cell provides intermediates for other synthetic and
cytoplasm metabolic pathways
 Osmosis – diffusion of water from low  The process occurs in the cell cytosol ( the fluid
concentration to high concentration present in the cell membrane) of all tissues
3. Acts as supporting skeletal system for surface antigen  This pathway maintain cation gradients (potassium in,
(Ag) and receptors sodium out)
4. Aid in transportation of essential cellular ions and  Maintain RBC membrane flexibility
gases  Affected by pyruvate kinase deficiency
 Product: 2 pyruvates, ATP, NADH
Nutritional Requirements of an RBC for its
Synthesis
 Amino acid – globin-chain (structure of hgb)
synthesis
 Vitamin B12 (cyanocobalamin) and folic acid –
DNA replication and cell division
 Vitamin B6 (pyridoxine) – iron conversion; low
levels of these causes anemia; ferrous form (Fe++) is
the most functional form of Fe
 Vitamin B2 (riboflavin) – RBC production and
oxygen transportation
 Vitamin B5 (pantothenic acid) Vitamin B3
(nicotinic acid/niacin) – production of RBC
++
 Iron (Fe ) –oxygen transport
Metabolism of RBC
1. Embden-Meyerhof Pathway (EMP)
2. Hexose Monophosphate Shunt (HMPS)/Pentose
Phosphate Pathway (PPP)
3. Rapport-Luebering Pathway (RLP)
4. Methemoglobin Reductase Pathway (MRP)
Figure 6. EMP generating pyruvates, ATP, and NADH

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HEXOSE MONOPHOSPHATE (HMP) SHUNT/PENTOSE
PHOSPHATE PATHWAY (PPP)
 Alternative pathway to glycolysis and TCA cycle for
the oxidation of glucose
 No ATP is directly utilized or produced in this pathway
 It gives protection to RBC from oxidant damage by the
production of reduced glutathione (antioxidant
activity)
 Reduced glutathione – maintain the integrity of the
RBC membrane
 It prevents denaturation of hemoglobin
 Affected with G6PD deficiency
 Product:
 NADPH – for reductive biosynthesis; required to
Figure 8. RLP generating 2,3-BPG
maintain glutathione in reduced state
 Ribose 5-phosphate – nucleic acid biosynthesis
METHEMOGLOBIN REDUCTASE PATHWAY (MRP)
 Hemoglobin is also called methemoglobin (if ferric
 Other sugar phosphates
[Fe+++] form)
++
 Maintains iron in Fe form (functional form) in hgb
 Functions to limit the production of methemoglobin
 In the absence of this enzyme (methemoglobin
reductase), methemoglobin accumulates and it cannot
carry oxygen
+++
 Hemoglobin with iron in Fe state cannot combined
and carry oxygen
 Affected with NADH cytochrome b5 reductase
Figure 7. PPP generating NADH and production of reduced

glutathione
RAPPORT-LUEBERING PATHWAY (RLP)

 It is a supplementary pathway to glycolysis
 It is mainly concerned with the synthesis of 2,3-BPG
(2,3-biphosphoglycerate)
 Regulates oxygen delivery to the tissues Figure 9. MRP maintaining iron in ferrous (Fe++) form
 Affected with phosphofructokinase deficiency
 Increased 2,3-BPG  decrease O2 affinity of hgb
HEMOGLOBIN
 It is a conjugated globular protein that carries oxygen
– Loose hgb, hence O2 will be released from the hgb
 Decreased 2,3-BPG  increase O2 affinity of hgb
then transport it to the tissues
 A complete hemoglobin molecule consists of 4 heme
molecules/subunits.
– Each of which is attached to 1 molecule of the
protein globin; each contains heme and globin
 Molecular weight: 64,458 Dalton
 Hemoglobin in RBC: 34 g/dl
 Oxygen per gram of hemoglobin: 1.34ml
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Figure 10. Hgb structure showing globin chains with iron-containing heme
Heme
++
 Consist of 1 iron (Fe ) atom and 4 pyrrole rings that  Genetic coding for globin chains (loci of chromosome)
are joined to each other  Chromosome 16: alpha and zeta genes
 1 heme = 1 mole of oxygen  Chromosome 11: beta, delta, gamma, epsilon genes
 1 hgb = 4 moles of oxygen
Figure 11. A porphyrin ring structure of heme

Figure 13. Cluster of chromosomes 11 and 16 encoding globin
genes
Globin Molecule
 Has 4 polypeptide chains and each chain is attached to
Main Function of Hemoglobin
1 heme group
1. Its main function is to transport oxygen from the
– 4 Identical heme groups, each consisting of a
lungs to tissue and carbon dioxide from tissue to
protoporphyrin ring and ferrous iron
lungs
– 4 globin polypeptide chains determine the type of
2. It contributes in acid-base balance by binding and
hemoglobin present
releasing hydrogen ion and transport of nitric oxide
(vasodilator)
Figure 14. Nitric oxides enlarge blood vessels and increase blood
Figure 12. 4 globin polypeptide chains of hgb flow
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ACID-BASE BALANCE HEMOGLOBIN SYNTHESIS
 Increasing the concentration of hydrogen ion, causes  65% occurs during nucleated stage of RBC maturation
the pH to fall (acidic)  35% occurs during the reticulocyte stage
 Ex. HCl  hydrogen + chloride ions  Heme synthesis: occurs in the mitochondria
+
 Decreasing the concentration of H ion, causes the pH  Globin synthesis: occurs in the ribosomes
to rise (alkaline)
– +
 Ex. NaOH  donates OH and reacts with H ion Biosynthesis of Hemoglobin
 The synthesis begins in mitochondria with the
OXYGEN-HEMOGLOBIN DISSOCIATION CURVE formation of delta-aminolevulinic acid (d-ALA) from
 Important for the understanding of how blood carries the condensation of glycine and succinyl coenzyme A
and releases oxygen with the participation of ALA synthase.
 It is expressed as P50 values which designates partial – This process is activated by pyridoxal phosphate
oxygen (PO2) (PLP)
– P50 of normal blood is 26-30 mmHg  D-ALA enters cytosol and yields porphobilinogen (PBG)
 An increase in P50 represents decrease in and water molecules with the help of ALA
hemoglobin-oxygen affinity or a shift to the right or dehydratase enzyme
vice versa  4 PBG molecules joined by uroporphyrinogen-I
synthase as linear tetrapyrrole called
Shift to the Left hydroxymethylbilane and ammonia
 Results to an increase in hemoglobin-oxygen affinity  Linear tetrapyrrole forms a ring known as
but a decrease in oxygen delivery to the tissues uroporphyrinogen (UPG) III with the participation of
 RBC are much less efficient in releasing O2 to tissues UPG III synthase
 Occurs in condition such as:  All acetyl groups of UPG-III are converted to methyl
 Alkalosis; decrease in body temperature groups by decarboxylation and generates
 Increased quantities of abnormal hgb coproporphyrinogen (CPG) III and CO2
(methemoglobin and carboxyhemoglobin; increased  CPG-III is converted to protoporphyrinogen by CPG
quantities of HbF) oxidase
 Multiple transfusion of 2,3-DPG-depleted stored  Protopophyrinogen (PPG) is further oxidized to form
blood protoporphyrins
 Iron is incorporated by ferrochelatase to generate
Shift to the Right heme
 Occurs to alleviate a tissue oxygen dissociation curve
such as hypoxia
 Mediated by increased levels of 2,3-DPG, results in a
decrease in the affinity of hgb to oxygen molecule and
an increase in oxygen delivery to the tissues
 RBCs have become more efficient in oxygen delivery
 May occur also in response to acidosis or a rise in
body temperature
Figure 16. Biosynthesis of hgb
COMPOSITION OF HGB FOUND IN NORMAL

HUMAN DEVELOPMENT
GLOBIN CHAINS HGB STAGE OF DEVELOPMENT
22 Gower 1
22 Gower 2 Embryonic
Z2Y2 Portland
Figure 15. Oxygen-hemoglobin dissociation curve
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2AY2 Sulfhemoglobin
F Fetus
2GY2  Formed by irreversible oxidation of hgb by drugs,
22 A1 sulfonilamide and phenacetin
Newborn and adult
22 A2  It is formed by the addition of sulfur atom to the
pyrrole ring of heme – produces greenish pigment
DEVELOPMENTAL EXPRESSION OF HGB  Could result to the formation of Heinz-bodies
DEVELOPMENTAL STAGE HGB  It could combine to carbon monoxide resulting to
1. 1st 2mon of gestation  Gower 1 carboxysulfohemoglobin
2. 2nd month of gestation  Gower 2, Portland  It cannot be converted back to normal hemoglobin A
3. 10wk  Switching to HgF until it breaks down
4. Throughout fetal life  HgF  Wavelenght: same with Hi (sulfhemoglobin curve does
5. Shortly after birth  Regulation of beta chain
not shift when cyanide is added)
6. 6mon to adulthood  HgA1, HgA2
Carboxyhemoglobin (HbCO)
 Derived from the combination of carbon monoxide
with heme iron
 Carbon monoxide is termed as “silent killer”
 It is light sensitive and has a typical color of cherry
Figure 17. Developmental stage of human Red
 Its affinity to hgb is 240x than of oxygen
HEMOGLOBIN DERIVATIVES  Wavelength: 540 nm
1. Methemoglobin (Hi)
2. Sulfhemoglobin
3. Carboxyhemoglobin (HbCO)
Methemoglobin (Hi)
 Derivatives of hgb in which ferrous iron is oxidized to
ferric state
 Associated with the exposure to exogenous oxidants
such as nitrite, primaquine, dapsone and benzocaine
 Hereditary causes linked to mutation in NADH-
cytochrome B5 reductase 3 (CYB5R3)
 Causes chocolate brown discoloration of blood Figure 18. Molecular structure of carboxyhemogloin containing the
 Wavelength: 525nm carbon monoxide
ANEMIA
ANEMIA Causes of Anemia
 Anemia is defined by the decreased in hemoglobin 1. Increased reticulocyte production in the bone marrow
(hgb), hematocrit (hct), and red blood cell (RBC) count (BM)
 It is the inability of the blood to supply adequate 2. RBC loss of accelerated RBC destruction
amount of oxygen (O2) to the tissues 3. BM production is impaired
4. Hereditary or acquired defect
5. Nutritional deficiency
6. Blood loss
a. Blood loss up to 20%
 At rest without clinical signs
 With mild exercise the patient may experience
Figure 1. Comparison of smear w/normal individual vs w/anemia tachycardia (rapid heartbeat)

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b. Blood loss of 30-40% leads to circulatory collapse Reticulocyte Count
and shock  It is the reflection of the amount of effective RBC
c. Blood loss of 50% leads to imminent death production taking place in the BM
 The number of reticulocytes/1000 RBC is counted and
 In slowly developing anemias, a very severe drop reported as a %
in hgb of up to 50% may occur without the threat of  Normal value:
shock or death  At birth: 1.8-8%
 Adult: 0.5-1.5%
Clinical Symptoms
 Pallor  Gastrointestinal symptoms
 Weakness  Pica
 Easily fatigability  Syncope
 Lethargy or malaise  Dizziness
 Exercise dyspnea  Headache
 Palpitation  Tinnitus or vertigo
 Difficulty sleeping  Irritability
Figure 3. Reticulocytes found in PBS
DETECTION OF ANEMIA
Hemoglobin Electrophoresis
1. RBC count
 Can be used to identify the presence of an abnormal
2. Hgb count
3. Hct or packed cell volume (PCV)
hgb (hemoglobinopathies)
 Different hgb will move to different regions of the gel
4. Mean corpuscular volume (MCV)
5. Mean corpuscular hemoglobin concentration (MCHC)
and the type of hgb may be identified by its position
on the gel after electrophoresis
6. Mean corpuscular hemoglobin (MCH)
7. Bone marrow smear (BMS)
8. Peripheral blood smear (PBS)
9. Red cell distribution width (RDW)
10. Reticulocyte count
11. Hgb electrophoresis
12. Antiglobulin test
13. Osmotic fragility test
14. Sucrose hemolysis test
15. Acidified serum test (Ham’s test)
Red Cell Distribution Width (RDW)

 A coefficient of variation in size distribution of RBCs
 Formula:
𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑑𝑒𝑣𝑖𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑀𝐶𝑉 × 100
𝑅𝐷𝑊 =
𝑀𝐶𝑉
 Normal range: 11.5-14.5%
– A value >14.5 means that there is increased
variation in cell size above the normal amount
(anisocytosis)
– A value <11.5 means that the RBC population is
more uniform in size than normal
Figure 4. Hgb electrophoresis for the detection of abnormal hgb in

cellulose acetate and citrate agar
Figure 2. Abnormal variation in size of red cell seen in PBS

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Antiglobulin Testing or with the C3b or C4d component of complement,
 Tests that detect the presence of antibody (Ab) or must be added to the system.
complement on the surface of the RBC and can be used  This will form a “bridge” between the Abs or
to support a diagnosis of an autoimmune hemolytic complement coating the red cells, causing
anemia agglutination.
– These Abs are produced because the immune  Types:
system mistakenly recognizes their own RBCs as 1. Direct Antiglobulin Test (DAT) – detects Abs or
foreign complement coating patient’s red cells in vivo
 Principle: 2. Indirect Antiglobulin Test (IAT)
 Red cells coated with complement or IgG Abs do not  Uses a 37ºC incubation step so Abs in serum can
agglutinate directly when centrifuged. These cells react with antigens (Ags) on cells in vitro
are said to be sensitized with IgG or complement.  After washing the cells antiglobulin reagent is
 In order for agglutination to occur and additional used to detect Ab coating of cells
Ab, which reacts with the Fc portion of the IgG Ab,
Figure 5. Antoglobulin test (direct and indirect Coomb’s test)
Osmotic Fragility Test

 Measures the RBC sensitivity to a hypotonic solution
of saline
 Saline concentrations of 0-0.9% are incubated with
RBCs at room temperature and the percent of
hemolysis is measured
 Persons with increased osmotic fragility have a
limited ability take up water in a hypotonic solution
and will, therefore, lyse at a lower sodium
concentration than will normal RBCs
Figure 6. Osmotic fragility test

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Sucrose Hemolysis Test 2. Absolute Anemia
 It is the confirmatory test for Paroxysmal Nocturnal 3. Microcytic Hypochromic Anemia
Hemoglobinuria (PNH) when the sugar water test is 4. Normocytic Normochromic Anemia
(+) 5. Hemolytic Anemia
 Sucrose provides a low ionic strength that permits 6. Aplastic Anemia
binding of complement to RBCs 7. Blood Loss
 Exposure of RBC to small amount of serum and 8. Lead Poisoning
isotonic, low ionice strength sucrose solution leads to 9. Macrocytic Normochromic Anemia
complement coating of RBC
– Hemolysis is observed in PNH samples due to Relative (Psuedo) Anemia
increase sensitivity of PNH erythrocyte to  RBC mass is normal, but plasma volume is increased
complement  Secondary to an unrelated condition and can be
 Paroxysmal Nocturnal Hemoglobinuria (PNH) – transient in nature
an acquired clonal disorder of bone marrow stem cells  Causes include conditions that result in hemodilution,
which gives rise to blood cells that are unusually such as pregnancy and volume overload
susceptible to activated complement  Reticulocyte count is normal
 Normocytic/normochromic anemia
Absolute Anemia
 RBC mass is decreased, but plasma volume is normal
 Mechanisms involved include:
1. Decreased delivery of red cells into circulation
 Caused by impaired or defective production
 BM fails to respond; reticulocytopenia
2. Increased loss of red cells from the circulation
 Caused by acute bleeding or accelerated
destruction (hemolytic)
Figure 7. Sucrose hemolysis test method
Acidified Serum Test (Ham’s Test) Microcytic Hypochromic Anemia

 It is defined by having low levels of RBC that are both
 It is the definitive diagnostic test for PNH
 In acidified serum, complement is activated by the
smaller and paler than normal
 It includes:
alternate pathway, binds to RBCs, and lyses the
1. Iron Deficiency Anemia
abnormal RBCs found in PNH
2. Thalassemia
 Patient RBCs are placed in mild acid
3. Sideroblastic anemia
 A (+) result (increased RBC fragility) indicates PNH or
4. Anemia of Chronic Disease (ACD)
congenital dyserythropoietic anemia
IRON DEFICIENCY ANEMIA (IDA)
 It occurs when your body doesn’t have enough of the
mineral iron (essential to make hgb)
 Signs and symptoms:
 Fatigue and headache
 Weakness and dizziness
 Pale skin
 Shortness of breath
 Pica
 Tingling sensation in the legs
Figure 8. Ham’s test method  Tongue swelling or soreness
 Old hands and feet
TYPES OF ANEMIA  Tachycardia
1. Relative (Psuedo) Anemia
 Brittle nails
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 Other symptoms:
 Koilonychias or spooning of the nails
 Esophageal webs
 Glossitis
 Blue sclera
 Muscle dysfuction
Figure 9. Sideroblastic cell stained with Prussian blue to show
 Common causes: accumulation of iron in the cell’s cytoplasm
 Heavy menstruation
 Poor diet  Common causes:
 Intestinal disease 1. Inherited SA
 Malabsorption syndromes a. Congenital SA – sex-linked
 Chronic blood loss and pregnancy b. Autosomal Recessive SA
 Hookworm infection 2. Acquired SA
a. Primary/Idiopathic
THALASSEMIA  Irreversible and the blocks are unknown
 It is a group of heterogenous disorder in which one or  i.e myelodysplasia – caused by poorly
more globin chains are reduced or absent formed blood cells or ones that don’t work
 It is a type of anemia that is caused by the mutations properly
in the genes needed for normal hgb production b. Secondary
 These genetic mutations lead to qualitative defects in  Reversible
the amount of globin chain produced  i.e antituberculosis drugs (isoniazid and
 The most affected chains are the α and β genes chloramphenicol)
 The type and severity depends on the globin gene  Lead and alcohol
mutated (α or β) and the number of genes affected
 Clinical manifestation: ANEMIA OF CHRONIC DISEASE (ACD)
1. Diminished hgb synthesis  Anemia of inflammation
 Production of microcytic hypochromic RBCs  Inability of RBC to use available iron for hgb
 Due to the absent or reduced production of a production
particular globin chain  Impaired release of storage iron associated with
2. Decreased survival rate fo RBCs and their precursor increased hepcidin
 Due to unequal production of α and β-globin  Associated with persistent infections, chronic
chains causing imbalance in α/β chain reaction inflammatory disorders (SLE, rheumatoid arthritis,
 Signs and symptoms: Hodgkin lymphoma, cancer)
 Fatigue  Characterized by: mild hypochromic microcytic,
 Weakness inadequate erythrocyte production, low serum iron,
 Pale or yellowish skin low binding capacity (i.e low transferrin)
 Facial bone deformities
 Slow growth Normocytic Normochromic Anemia
 Abdominal swelling  Defined by having low level of RBC with either normal
 Dark urine size and hgb concentration

 This type of anemia may be cause by:
SIDEROBLASTIC ANEMIA (SA) 1. Hemolytic Anemias
 This condition blocks or interferes in the 2. Aplastic Anemias
protoporphyrin pathway resulting in defective hgb 3. Stem cell-related anemias
synthesis and iron overload 4. blood loss
 The iron inside RBC is inadequately used to make hgb, 5. Lead Poisoning
despite normal amounts of iron 6. Pregnancy
 Excess iron accumulates in the mitochondrial region of 7. Renal disorder

the immature erythrocyte in the BM and encircles the 8. Liver Disease
nucleus; cells are called ringed sideroblast
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Hemolytic Anemia c. Elliptocytosis
 Premature destruction of erythrocytes d. Glucose-6-phosphate Dehydrogenase (G6PD)
 The BM cannot compensate for the erythrocyte loss deficiency
 The severity depends on whether the onset of e. Pyruvate kinase deficiency
hemolysis is gradual or abrupt and extent of 2. Acquired Hemolytic Anemia
erythrocyte destruction a. Viral or bacterial
 Hemolysis could be: b. Drugs (e.g antimalarial, sulfa drugs and etc.)
1. Mild hemolysis – can be asymptomatic c. Blood cancers & tumors
2. Severe hemolysis – can be life-threatening d. Autoimmune disease
 Symptoms: e. Hypersplenism (overactive spleen)
 Paleness f. Mechanical heart valves
 Jaundice g. Severe reaction from Blood transfusion
 Dark-colored urine
 Fever  Also includes:
 Weakness, dizziness and confusion 1. Immune Hemolytic Anemia
 Hepatosplenomegaly a. Autoimmune
 Tachycardia b. Alloimmune
 Two Main Types of Hemolytic Anemia: c. Drug-induced
1. Inherited Hemolytic Anemia 2. Mechanical Hemolytic Anemia
a. Sickle cell anemia & Thalassemia 3. Paroxysmal Nocturnal Hemoglobinuria
b. Spherocytosis
INHERITED HEMOLYTIC ANEMIA

Sickle Cell Anemia
 The body makes abnormal hgb

 The RBC of the patient with this condition has a
sickle or crescent shape
 Sickle-shaped RBC lasts only for 10-20d
Figure 10. Sickled cells in PBS

 It is a type of anemia that is caused by the
Thalassemia mutations in the genes needed for normal hgb
production
Hereditary Spherocytosis  This is on the most common form of hemolytic
anemia
 This condition involves defects in the cell
membrane of the erythrocyte
 Like in sickle cell anemia, the erythrocyte
appears like a ball or has a sphere-shaped
Figure 11. Abnormal red cells (spherocytes) in PBS appearance
Hereditary Elliptocytosis (Ovalocytosis)
 It also has a defect in cell membrane

 That defect results to the abnormal shape of the
erythrocyte
 The RBC becomes elliptical in shape
Figure 12. Abnormal red cells (elliptocytes) in PBS

 G6PD is an enzyme essential in the production of
energy (ATP) inside the RBC
 In this condition, G^PD is missing
Glucose-6-Phosphate Dehydrogenase
 As a result, the RBC burst and die that leads to
(G6PD) Deficiency
anemia
 Hexose-monophosphate shunt or pentose
phosphate pathway
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 In this condition, pyruvate kinase which is
involved in the chemistry inside the RBC is
missing
Pyruvate Kinase
 The deficiency will destroy the RBC in the
circulation
 The Embden-Meyerhof pathway is affected
ACQUIRED HEMOLYTIC ANEMIA

Immune Hemolytic Anemia  The immune system destroys RBC
 In this condition, Abs are produced by the immune system to attack and destroy RBC
 50% of hemolytic anemia are AIHA
 AIHA may developed even after blood transfusion or BM transplant
 Cold-Reactive Antibodies
Autoimmune – Developed in some cases of AIHA
– It is Active in cold temperature
– It is active when the parts of the body is exposed to low temperature (0-10ºC)
 Warm-Antibody
– More common than cold antibody AIHA
 The antibodies attacks the RBC that came from blood transfusion
Alloimmune – It happens if the blood transfused is not compatible with the recipient
– e.g the Rh(–) blood mother having an Rh(+) baby
 There are certain drugs that may react and then develops hemolytic anemia
Drug-induced
 e.g penicillin
 Physical damage to RBC membrane can cause death to erythrocyte
 Damage may be due to:
Mechanical Hemolytic Anemia – Artificial heart valve
– Hemodialysis
– Heart lung bypass, etc
 It is a disorder in which the RBC are impaired due to lack of certain proteins
Paroxysmal Nocturnal Hemoglobinuria  The RBCs are destroyed quickly because of this lacking protein
 PNH has increased risk of blood clots in the vein and low levels of WBC
Aplastic Anemia  This condition leads to pancytopenia (reduction in RBC,

 Occurs when your body stops producing enough new WBC, and platelet in the blood
blood cells  Since BM is defective, BM has no response to
 Characterized by defective function of the BM erythropoietin
Figure 13. BM of individual with aplastic anemia compared to normal
THREE TYPES OF APLASTIC ANEMIA

GENETIC (FANCONI ANEMIA) ACQUIRED (SECONDARY) IDIOPATHIC
 About 30% of acquired aplastic anemias are due to drug
 50-70% of aplastic anemia have
– Antibiotics: chloramphenicol, sulphonamide
no known cause
– Chemicals: benzene, herbicides
 Autosomal recessive trait – Diamond-Blackfan anemia
– Viruses: B19 parvovirus secondary to hepatitis, measles, CMV,
 Dwarfism, renal disease, – True red cell aplasia
Epstein-Barr virus
mental retardation (leukocytes and platelets
– Radiation/chemotherapy
normal in number)
– Myelodysplastic syndromes, leukemia, solid tumors, paroxysmal
– Autosomal inheritance
nocturnal hemoglobinuria
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 Symptoms:
 Fatigue and shortness of breath
 Rapid and irregular heartbeat
 Unexplained/easy bruising
 Nosebleed and bleeding gums
 Prolonged bleeding from injuries
 Headache, dizziness and fever
 Usually, aplastic anemia have no symptoms
 Treatment: medications, blood transfusions, stem

cell therapy, BM/stem cell transplant
Blood Loss
1. Acute Post-Hemorrhagic Anemia
 Sufficient amount of blood is lost over a short
period of time
– Hypovolemia develops after a single episode of
bleeding
– Rapid pulse rate Figure 14. Lead poisoning diagram, inhibiting d-ALA and
– Pallor and low blood pressure ferrochelatase
 Laboratory findings:
– Normal hct/hgb and reticulocyte in few hours Macrocytic Normochromic Anemia
– Increase platelet count and leukocytes  Defined by having a large RBC but contains only a
– Drop in hgb, hct and RBC normal amount of hgb
– Reticulocytosis in 3-5d  MCV: >96fl
2. Chronic Post-Hemorrhagic Anemia  MCHC: normal
 Small amount of blood is lost over an extended  Types:
period of time 1. Megaloblastic Anemia
– Often caused by gastrointestinal bleeding 2. Non-megaloblastic Anemia
 Laboratory findings:
– Decreased in hgb, hct and RBC count MEGALOBLASTIC ANEMIA
– Gradual loss of iron leads to microcytic  Defined by defects in DNA synthesis but normal in
hypochromic anemia RNA synthesis
 Results to the delayed maturation of nucleus to that of
Lead Poisoning the cytoplasm
 Seen mostly in children exposed to lead-based paint  The erythroblast is large and the nuclear maturation
 Mechanism: fails but the hemoglobinization is normal
– There are multiple blocks in the protoporphyrin  The chromatin maintains an open and lacey
pathway that affects the heme synthesis appearance despite normal hgb formation in the
 Clinical symptoms: erythroblast as they mature
 Abdominal pain and weakness  Causes:
 Gum lead line (forms blue-black deposits of lead 1. Vitamin B12 deficiency (cobalamin) – pernicious
sulfate) anemia
 Laboratory findings  Pernicious Anemia
 Basophilic stippling  It is a condition that caused the deficiency of
the intrinsic factor in the parietal cells
 The body’s immune system attacks and
destroys parietal cells that produced intrinsic
factors
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 This intrinsic factors are essential for the  Eggs, meats, etc
absorption of vitamin B12 by the intestine 3. Abnormalities of vitamin B12/folate metabolism
 Other causes: 4. Inherited disorders of DNA synthesis
– Parasitism  Laboratory findings:
– Poor dietary intake  MCV: 120-140fl
– Gastrectomy  Oval shape
– Malabsorption syndrome  Hypersegmentation of neutrophil
– Atrophy and achlorhydria of parietal cells  Elevated bilirubin and iron
 Delayed development: high reserved amount of  Inclusion bodies:
cobalamin in the body  Howell jolly bodies
 Clinical symptoms:  Basophilic stippling
– Fatigue, weakness, headache, chest pain,  Pappenheimer bodies
weight loss, pale skin  Cabot’s ring
– For severe (prolonged) cases: memory loss,
dementia, depression, unsteady gait,
peripheral neuropathy
 Treatment: vitamin B12 injection, oral
supplementation
2. Folic acid deficiency (vitamin B9) – nutritional
megaloblastic anemia
 Folate is a water-soluble vitamins and it is not
restored in the fat cells
 Urinating contributes to the deficiency of folate
 It is associated to pregnancy
Figure 15. Megaloblastic anemia caused by deficiency in vitamin
B12
 Has a similar features of vitamin B12 deficiency
 Other causes:
NON-MEGALOBLASTIC ANEMIA
 Poor Diet
 These are type of anemia with no impairment in the
 Disease
DNA synthesis but has a defective production of
 Genetics
erythrocyte resulting to macrocytic anemia
 Medication side-effects
 Causes:
 Alcohol intoxication
 Liver disease
 Treatment:
 Alcoholism
 Dietary intake of folate
 Conditions that cause accelerated erythropoiesis
 Intake of methylated folate
 Chronic obstructive pulmonary disease (COPD)
 Prevent taking alcohol and Fruits have high
amounts of folate
HEMOGLOBINOPATHIES
INTRODUCTION  It is the most common genetic disease worldwide
 Hemoglobin (hgb) is a very important content or red  In this condition, the hgb molecule has an altered
blood cells (RBCs) as it is responsible for the delivery amino acid sequence (AAS) within the globin chains
of oxygen (O2) to the tissues and carries carbon which changes the structure impairing its function
dioxide (Co2) to be released from out body.  Hemoglobin
 Thalassemia is an inherited disorder that affects the – It is produced by genes that controls the expression
hgb the result to anemia. of the hgb protein
– Defects in genes leads to production of abnormal
HEMOGLOBINOPATHY hgb
 Disease involving hgb
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Types of Gene Mutation
1. Deletion – removal of one or more nucleotides
2. Insertion – addition of one or more nucleotides
3. Fusion – involves joining of one or more of the adult
globin genes
4. Substitution – it is the replacement of one original
nucleotide in the normal gene with different type of
nucleotide
Figure 1. Hgb in RBCs carrying oxygen
Categories of Hemoglobinopathy
1. Qualitative Hemoglobinopathy
 Synthesis of hgb is normal or near-normal rate
 Alteration in the AA within the globin chains of hgb
 The change in AAS change/alter the structure and
causes defect (structure defect) in the hgb structure
and also affects the function (qualitative defect)
2. Quantitative Hemoglobinopathy
 Happens in thalassemia
 Results in reduced rate of hgb synthesis
(quantitative defects)
 Does not affect the structure of hgb or the AAS
 The reduced amount of synthesized hgb due to
the mutation results to anemia.
Figure 2. Types of gene mutation; the base substitution, addition,
 It triggers or stimulates the production of other
and deletion
hgb not affected by the mutation to compensate
for the loss of hgb
QUALITATIVE HEMOGLOBINOPATHY
DESCRIPTION CLINICAL FEATURES LABORATORY DIAGNOSIS
 It is the most common type of hemoglobinopathy
 An inherited variant of normal adult hgb
 Mutation in the 6th position of the beta globin
 Substitution of glutamine to valine
Hemoglobin S
 Polymerizes into long, thin polymers to form sickles when O2
saturation decreases
 It may be reversible (associated with vaso-occlusion) or
 Poikilocytes: drepanocytes
irreversible
and target cells
 a.k.a. homozygous sickle cell disease/sickle cell anemia
 Increased reticulocyte
– Results when gene for hgb S is inherited from both parents
count
– During the 1st few months of life, moderate to severe
 Dithionite solubility (sickle
anemia develops as the amount of fetal hgb decreases and
screen): positive
hgb S increases
 Hemoglobin Electrophoresis
 Homozygous hgb S
– positive for hgb S and no
 Normocytic, normocytic anemia
Hgb A present
Sickle Cell  Infants and children are susceptible with bacterial infection
 Hallmark: vasoocclusive
Disease and splenic sequestration that could lead to death
crisis
 Major threat: bacterial infections (septic shock) from
Staphylococcus aureus, Streptococcus pneumoniae,
Haemophilus influenzae
 Prophylactic oral penicillin and folic acid should be
administered before 3mos up to age six to avoid morbidity
and death
 Observed to be resistant against P. falcifarum
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 a.k.a. hgb S trait
 Heterozygous for hgb S (majority of hgb present is hgb A)
– The gene for hgb S is inherited from one parent and hgb A  Homozygous state (hgb
gene from the other EE)
Sickle Cell – Hgb A and hgb S are present – Manifest mild
Trait  Patients are usually asymptomatic. anemia with
– This carrier state does not result in health problems though microcytic and  Solubility testing: negative
– Microscopic hematuria is found in some patient target cells  Confirm via HPLC or
– Rare manifestation: extreme hypoxia – Low MCV (55-65 fl) electrophoresis
– Sickle cell screen positive – It resembles  No treatment is required
 Mutation at the 6th position of beta globin thalassemia trait
Hemoglobin C  Substitution of glutamine to lysine – P. falciparum
Disease  Polymerizes into thick crystals when O2 saturation decreases multiplies more
 Crystal resembles “bars of gold” or “Washington monument” slowly in
 It was first described in 1954 with a 30% prevalence in homozygous hgb E
southeast Asia (hgb EE)
 Mutation in the 26th position of beta globin  Heterozygous state
Figure 3. Microcytes and target cells
 Substitution of glutamine to lysine – Low MCV (65 fl) in patients with hgb E trait
– Polymerization does not occur because of the position of the – Slight
Hemoglobin E erythrocytosis with
substitution in B-chain
– However, the amino acids substitution at codon 26 inserts a presence of target
cryptic splice site that causes abnormal alternative splicing cells
and decreased transcription of the functional mRNA for the
hgb E globin chain
 It is the most common compound disorder (compound
heterozygous syndrome)
 Combination disorder with inheritance of mutation for both
hgb S and hgb C
– Different AA substitution is found on each 2 globulin chains.
 Glutamic acid is replaced by valine (hgb S on position 6 on 1
beta-Globin chain and by lysine (hgb C) on the other B-globin
chain
– The frequency in west Africa is 25%
– In US, the prevalence is approximately 1-833 births per
 Similar picture to sickle
year cell disease, however,
doesn’t manifest till
teenage years
– Cause all vaso-
occlusive  Mild normocytic
complications of normochromic with many
sickle cell anemia features of sickle cell
 Hemolytic anemia is anemia
moderate • Hemoglobin 11-13 g/dl
Hemoglobin SC
 Many exhibit • Reticulocyte count is 3- 5%
splenomegaly • PBS: sickle cells with target
Figure 4. Substitution of glutamic acid to valine on the 6th position
 Respiratory tract cells and intraerythrocytic
infections with S. crystalline structure
pneumoniae are  Solubility testing positive
common
 Patients with hgb SC
live longer than with
hgb SS and have fewer
painful episodes
Figure 5. Substitution of a single AA (glutamic acid to valine) in the 6th position of

the 146 AA of the beta chain of hgb
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QUANTITATIVE HEMOGLOBINAPATHIES  Whipple called it Thalassic (Greek for “great
sea”) referring to the high incidence of patient of
Thalassemia Mediterranean during that time
 It is a group of heterogenous disorder in which one or  1940s
more globin chains are reduced or absent  Investigators described thalassemia major
 It is a type of anemia that iss caused by the mutations  Thalassemia Major – patient homozygous
in the genes needed for normal hgb production with this disorder shows severe course
 These genetic mutations leads to qualitative defects in  Thalassemia Minor – heterozygotes are
the amount of globin chain produced carrier and has milder form of anemia
 The most affected chains: alpha and beta genes  1950s
 The type and severity depends on the globin gene
mutated (α or β) and the number of genes affected Normal Human Hemoglobin
 Brief History  These are the different types of hgb present on a
 1925 – Cooley and Lee particular developmental stage of human
 First describes four children with anemia,
hepatosplenomegaly and mongoloid facies HEMOGLOBIN STRUCTURAL FORMULA
 Later on, these characteristic becomes typical Hb-A 22 97%
Adult
with untreated beta thalassemia major referred Hb-A2 22 1.5-3.2%
to as “Cooley’s Anemia” Fetal Hb-F 22 0.5-1%
 1932 – Whipple and Bradford
Hb-Gower 1 22
Embryonic Hb-Gower 2 22
 Published a paper outlining the detailed autopsy
Hb-Portland 22
studies of these children with this disorder
DESIGNATION FOR NORMAL  AND  – GLOBIN GENES

DESIGNATION DEFINITION
Normal -globin gene
 Normal amount of beta chains produced
One gene located on each chromosome 11
Normal 1 and 2 globin genes on one chromosome haplotypes 
 Normal amount of  chains produced
Two genes located on each chromosome 16
DESIGNATION FOR MAJOR THALASSEMIC GENES

DESIGNATION DEFINITION
B-globin gene mutation
O
No globin chains are produced
+ Mutation results in 5-30% decreased in -chain production
silent Mutation that results in mildly decreased -chain production
δ -globin gene deletion or non-deletion mutation
δ O No δ or  chains are produced
Accompanied by some increase in γ-gamma chains production
δ -globin gene fusion
Produces small amount of fusion product, hgb Lepore
δ lepore
No δ or  chains are produced
Hereditary Persistence of Fetal Hemoglobin
δ -globin gene deletion or non-deletion mutation in γ-gamma globin gene promoter
HPFH
No δ or  chains are produced
THALASSEMIA  Genetic blood disorder passed down through family

 This disorder leads to the destruction of large number where the body produces abnormal form/inadequate
of RBCs which results to anemia. amount of hgb.
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 Hgb is the protein in RBCs that carries O2
 Thalassemia are named according to the chain with
mutated, reduced or absent synthesis.
 Types of Thalassemia:
1. Alpha thalassemia – genes related to the alpha
globin protein are missing or changed (mutated)
2. Beta Thalassemia – genes defects affect
Figure 8. Anisocytosis. Poikilocytosis and numerous nucleated
production of the beta globin protein RBCs in the blood film of neonate with hgb Bart’s hydrops fetalis
 Forms of Thalassemia:
1. Thalassemia Major – inherit the gene defect HEMOGLOBIN H (_ _/_)
from both parents  It is mild to moderate form of alpha thalassemia
2. Thalassemia Minor – occurs if you receive the  Usually caused by presence of only one gene
faulty gene from only one parent producing alpha chains (_ _/_α)
 People with this form of the disorder are carriers  Appeared in early childhood and may continue to live
 Most of the time, they do not have symptoms into adulthood
 After 6mos, gamma chains  beta chains
Alpha Thalassemia  Accumulation of unpaired beta chains that will form a
 It is caused by mutation or deletion in the alpha tetramers (B4)
globin chains that leads to the formation of excess  This tetramer precipitate and forms an inclusion to
gamma chains (hgb Bart) matured RBC that causes hemolytic anemia
 α-chains is a component of both fetal and adult hgb  Signs and symptoms:
 Mechanism in fetus and newborn:  Hepatosplenomegaly
 Increase production of γ-chain is a result of the  Jaundice
reduced production of α-chains  Overgrowth of upper jaw and prominent forehead
 γ-chain does not precipitate and forms hemoglobin
tetramer (hgb Bart) ALPHA THALASSEMIA MINOR (_/_) or (_ _/)
 After 6mos, γ-chain switch to β  B4 hgb H  Caused by homozygous α+ (_α/_α) or
 It does not have severe ineffective erythropoiesis heterozygous αO (_ _/αα) forms
 Inclusion in mature RBC is observe due to the  Two genes of α-globin genes are deleted
precipitation of the tetramer B4 of hgb H  Clinically similar to B-thalassemia minor but hgb A2 is
not elevated
HEMOGLOBIN BART SYNDROME (_ _/_ _)  Asymptomatic
 Hemoglobin Bart Hydrops Fetalis Syndrome/Alpha
Thalassemia Major (4 gamma chains SILENT CARRIER (_/) or (T/)
 Excess fluid builds up in the body before birth  It is associated with normal RBC and no clinical
 It has a very high affinity to O2 symptom presented
 Signs and Symptoms:  Deletion of one gene of alpha-globin genes
 Anemia  There is a slight decrease in alpha chain production
 Hepatosplenomegaly  Slight excess of y-chains at birth that form tetramers
 Heart defects and abnormalities in urinary system of hgb Bart (y4) in the range of 1-2%
or genitalia +
 A non-deletional a mutation in one globin gene
 In mother: high blood pressure with swelling, (aT/aa) results in silent state
premature delivery and bleeding
COMPARISON OF DIFFERENT FORMS OF ALPHA THALASSEMIA

GENOTYPE Hgb A Hgb Bart Hgb H
Normal 97-98% 0 0
Silent Carrier 96-98% 0-2% 0
Alpha Thalassemia Trait 85-95% 5-10% 0
Hemoglobin H Disease Dec 25-40% 2-40%
Hydrops Fetalis 0 80% (with 20% hgb Protland) 0-20%
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Beta Thalassemia  Clinical signs and symptoms vary because of multiple
 Inherited in an autosomal recessive pattern genotypic presentation
 Caused by mutation or deletion of beta globin chains – Iron overload is present
 Remains asymptomatic till 6mos due to increase hgb F – Cardiac, liver and endocrine complications
 Between 6-24mos after completion of switching  Homozygous silent thalassemia gene mutation
gamma to beta chains, appearance of symptom begins (βsilent/βsilent)
 Mechanism: – It usually presents increased level of hgb F and hgb
 Unpaired and excess α-chains precipitates in A2
developing RBC  oxidative stress and damage to  Compound heterozygous: one gene caused mild
the membrane of RBC apoptosis decrease of b-chain production and one other gene
 Accumulation of iron and inflammatory cytokines in caused marked decrease b-chain production
RBC precursor  apoptosis  Dominantly inherited B-thalassemia (rare)  only one
 Stimulates increase production of EPO that results gene mutated
in massive but ineffective erythroid hyperplasia
 Ineffective erythropoiesis BETA THALASSEMIA MINOR/BETA-THALASSEMIA TRAIT
– It is the premature death of RBC precursor in the  One beta-globin gene is affected
bone marrow (BM)  Mild hemolytic anemia
– BM attempts to produce RBC but is not able to  Hgb A is 92-95% elevated
release viable cells into the circulation  Hgb A2 level is elevated (3.5-7.0%)
 Bone deformities due to BM expansion  Hgb F level 1-5%
– Thinning of the cortex of the bone  increase  No clinical symptoms (hepatosplenomegaly in few
risk of fracture patients)
– Lacy appearance of long bone  Erythroid hyperplasia (minimal ineffective
– Frontal bossing erythropoiesis)
 Extramedullary erythropoiesis  Laboratory diagnosis
– Hepatosplenomegaly  Normal to elevated RBC level with decreased hgb
– Causes jaundice (increase level of bilirubin) and hct
– Increased level of iron  Hgb level : 12-14 (men), 10-12 (women)
– Suppressed hepcidin production in the liver  MCV: <75 fl and MCH <26 pg
 Reticulocyte: slightly increased
COOLEY’S ANEMIA/BETA THALASSEMIA MAJOR  Poikilocytosis (elliptocytes and target cells)
 Severe beta-thalassemia is usually diagnosed
between 6th month and 2y of age after completion of SILENT CARRIER (silent/)
gamma to beta switch  The blood is completely normal
 It has a severe clinical symptoms with  Heterozygous state with NO hematologic
hepatosplenomegaly & distinct bone changes abnormalities or clinical symptoms
 Transfusion dependent  The β-globin gene mutation produced only a small
 Laboratory diagnosis decrease in β-chain production
 Decreased hgb level: 3-4g/dl
 Decreased MCV: 50-70fl COMPARISON OF DIFFERENT FORMS OF
 RC inclusions, basophilic stipplings, Howell-jolly and BETA-THALASSEMIA
pappenheimer bodies GENOTYPE Hgb A Hgb A2 Hgb F
 Reticulocyte is moderately elevated Normal Normal Normal Normal
Silent Carrier Normal Normal Normal
Minor Dec Normal to inc Normal to inc
BETA THALASSEMIA INTERMEDIA (silent/ silent), (δ O/O), Intermedia Dec Normal to inc Usually inc
(O/ δ O) Major Dec Usually inc Usually inc
 It has a severe clinical symptoms and usually not
transfusion dependent
TPMEB 2020
LEUKEMIA
LEUKEMIA Certain abnormalities cause the cell to grow and
 A clonal disease that develops subsequent to the divide more rapidly and to continue living when
malignant transformation of one or more normal normal cells would die.
hematopoietic progenitor cell FORMS OF LEUKEMIA
 A group of malignant disorder affecting the blood and 1. Acute Leukemia – characterized short duration,
blood-forming tissues of the bone marrow, lymph many immature cell forms in the BM and/or peripheral
system and the spleen blood, and an elevated total leukocyte count
 Unregulated proliferation and accumulation of blast 2. Chronic Leukemia – has symptoms of long
cell in the bone marrow (BM) and body tissues duration, mostly mature cell forms in the BM and/or
 Symptoms seen in leukemic cells due to: peripheral blood, and total leukocyte counts that
 Bone marrow failure: anemia, neutropenia, range from extremely elevated to lower than normal
thrombocytopenia 3. Lymphocytic Leukemia – affects the lymphoid
 Infiltration to body organs: liver, spleen, lymph cells (lymphocytes), which form lymphoid/lymphatic
nodes, brain, meninges, skin or testes tissue
 Etiology 4. Myelogenous Leukemia – affects the myeloid
1. Genetic and environmental influences cells; myeloid cells give rise to RBCs, WBCs and
2. Chronic exposure to chemicals such as benzene platelet-producing cells
3. Exposure to radiation
4. Cytotoxic therapy for cancers Incidence
5. Congenital anomaly  Acute leukemia can occur in all age groups
6. Presence of primary immunodeficiency and – Acute Lymphocytic Leukemia (ALL) – children
infection with human T-cell leukemia virus type 1 – Acute Myeloid Leukemia (AML) – adults
 Pathophysiology  Chronic leukemia are usually a disease of adults
 Leukemia is thought to occur when some blood cells – Chronic Lymphocytic Leukemia (CLL) – extremely
acquire mutations in their DNA. rare in children and unusual before the age of 40
– Chronic Myeloid Leukemia (CML) – peak age: 30-50
ACUTE VS CHRONIC LEUKEMIA

VARIABLE ACUTE CHRONIC
Age All ages Adults
Clinical onset Sudden Insidious
Prognosis (untreated) 6mos or less 2-6 years
Cells Immature >30% blasts More mature cells
Anemia Prominent Mild
WBC count Varies Increased
Thrombocytopenia Prominent Mild
Lymphadenopathy Mild Present, often prominent
Splenomegaly Mild Present, often prominent
CLASSIFICATION OF LEUKEMIA 2. ALL: L1, L2, L3

 FAB Leukemia Classification  Laboratory Investigation
1. Myelogenous – Complete blood count (CBC)
2. Lymphocytic  Increase WBC count
3. Monocytic  Lymphoblast present in PBS
 WHO Leukemia Classification  Decreased RBC count
1. Myeloid  Decreased Platelet Count
2. Lymphoid – Bone marrow aspirate – all normal marrow
3. Histiocytic/dendritic cell elements are depressed and replaces with
ACUTE LEUKEMIA abnormal blast cells
1. AML: M0, M1, M2, M3, M4, M5, M6, M7
TPMEB 2020
CHRONIC LEUKEMIA  “Chronic” in chronic myelogenous leukemia indicates
that this cancer tends to progress more slowly than
Chronic Lymphocytic Leukemia acute forms of leukemia
 The disease occurs in older patients (>50y/o)  “Myelogenous” in chronic myelogenous leukemia
 Generalized lymphadenopathy: enlargement of refers to the type of cells affected by this cancer
cervical, axillary or inguinal lymph nodes  It typically affects older adults and rarely occurs in
 Hepato-splenomegaly occurs in 50-60% of cases children, though it can occur at any age
 Features of anemia: spontaneous bruises, pupura,  Symptoms (usually does not have any):
bleeding gums due to thrombocytopenia  Bone pain
 Immunosuppression is a significant problem  Easy bleeding
 Bacterial infections followed by viral and fungal  Feeling full after eating a small amount of food
infections such as herpes zoster are also seen  Feeling run-down or tired
 Fever
Chronic Myelogenous Leukemia  Weight loss without trying
 It is an uncommon type of cancer of the bone marrow  Loss of appetite
 Define by an increased number of WBCs in the blood  Pain or fullness below the ribs on the left side
 Excessive sweating during sleep (night sweats)
DIFFERENTIAL DIAGNOSIS OF AML AND ALL

(+)
NAME STAINED PART INTERPRETATION NOTES
RESULT
Peroxidase produces brown granules in
Myeloperoxidase (MPO) AML Primary granules For fresh specimen only
cytoplasm or granulocytes and monocytes
Dark purple black granules in neutrophil
Sudan Black B (SBB) AML Phospholipids and lipids Parallel with MPO
precursors
Marker for immature
Periodic Acid Schiff (PAS) ALL Glycogen, glycoprotein Bright fuchsia pink
lymphocytes
A-Naphthol Choloroacetate Immature neutrophil and
Granulocytes Bright red Specific esterase
Esterase mast cells
B-Naphthyl Acetate
Monocyte Monocyte, megakaryocyte Red brown Non-specific esterase
Esterase
A-Naphthyl Butyrate Monocyte, promonocyte,
Dark red Specific esterase
Esterase monoblast
Acid Phosphatase Hairy cell Lysosomes Purple to red granules Not stored
Leukocyte Alkaline Decrease in Tertiary granules of 100 cells counted and neutrophils are
Normal value: 35-100
Phosphatase (LAP) AML neutrophils scored 0-4 based on the staining activity
Figure 1. Characteristics of leukemic cells through enzymatic and non-enzymatic staining

TPMEB 2020
ACUTE MYELOGENOUS LEUKEMIA SUBTYPES
DESCRIPTION SIGNS ANS SYMPTOMS LABORATORY DIAGNOSIS
FAB M0 – Undifferentiated
Acute Myeloblastic
Leukemia
 With minimal differentiation
 Blasts are (–) for B and T
lymphoid Ag, platelets; (+) for
CD13, CD33 and CD11b
Figure 2. Leukemic cells with minimal

differentiation
 Rapid or gradual onset that
resemble an acute infection
 Physical examination: bone
tenderness (particularly the ribs
FAB M1 – Acute Myeloid and sternum), ulcerated mucous
Leukemia without membranes, petechiae, and
Maturation purpura
 50% of patients: hepatomegaly,  Anemia and thrombocytopenia
splenomegaly, lymphadenopathy  >1/3 of patients has
 >90% cells are myeloblasts
 Cellular infiltration of organs is leukocytosis
 3% of blasts stain for MPO
less prominent in AML than ALL  Total leukocyte count:
 Localized tumor masses >100,000 L
consisting of myeloblasts may
Figure 3. Leukemic cells of AML without arise in bine or soft tissues
maturation – In these tumors, the presence
of large quantities of the
enzyme MPO produces a green
appearance if the tissue is out
(a tumor called chloroma)
 Anemia and thrombocytopenia
 30-90% are myeloblasts  Leukocytosis: total leukocyte
FAB M2 – Acute Myeloid  Numerous single Auer rods count exceeding 300,000 L
Leukemia with Maturation  Associated with t (8:21)  Hemorrhagic manifestations  PBS: myeloblast predominates;
karyotype which are common initial granulocytic cell line
 Usually occurs in middle-aged symptoms (easy bruising, maturation is observed
people epistaxis, gingival bleeding, – Nuclei: usually round/oval
 The median age of occurrence petechiae) with one/more prominent
is 48y, however, approx. 40%  Hepatomegaly, splenomegaly nucleoli and fine reticular
of cases occur in 60y/o or older and lymphadenopathy are seen chromatin
Figure 4. Leukemic cell of AML with  Male:Female ratio = 1.6:1 infrequently – Cytoplasm: basophilic with a
maturation  The median survival time is variable no. of azurophilic
8.5mos granules
 Auer rods are commonly seen
FAB M3 – Acute
Promyelocytic Leukemia
 Cells contain t (15:17) on 95%
 The most aggressive of acute
of cases and are HLA-DR (–)
leukemia with a sever bleeding
 Laboratory findings similar to
 The median age of occurrence tendency and a fatal course, if
those of the FAB M2
is 38y untreated, of only weeks
 Promyelocytes –
 Median survival of approx.  Hypofibrinogenemia and
predominating cell type
16mos hemorrhage are common
 The promyelocytes may be
 Male:Femal ratio = 2:1  An increased in DIC is common
hypergranular, microgranular,
 Other signs and symptoms are
or hypogranular variations
like M2
Figure 5. Leukemic cells of acute

promyelocytic leukemia
TPMEB 2020
Figure 6. Vairationsof promyelocytes in

PBS, (up to down) hypergranular,
microgranular, hypogranular
 Coarsely granular
promyelocytes with dumbbell-
shaped or bilobed nuclei may
be seen
 The nuclear chromatin is finely
reticular and the cells often
lack a nucleoli
 Cytogenetically, M3 is
characterized by a balanced
reciprocal translocation
between chromosomes 15 and
17, which results in the fusion
between PML gene and retinoic
acid receptor α (RARα) which
inhibits maturation
 Peroxidase stain intensely (+)
 Peroxidase-, Sudan-,
chloroacetate esterase-, and
non-specific esterase-positive
 Symptoms: similar to those of cells
other forms of acute leukemia  Proliferation of granulocytes
 Patients with FAB M4/FAB M5/ALL and monocytes
FAB M4 – Acute  Referred to as Naegeli-type
(predominantly of the T-cell type)  Anemia and thrombocytopenia
Myelomonocytic Leukemia monocytic leukemia.
with hyperleukocytosis are at are present
 Uncommon in children and
risk of leukostasis development.  The total leukocyte count varies
young adults
 Leukostasis – a pathological from leukopenia to
 Highest frequency of
finding of slightly dilated, thin- leukocytosis
occurrence = adults older than
walled vessels filled with  On PBS, early myeloid cells
50 years of age
leukemic cells; the brains and the predominate, approx. 20% of
 Male:Female ratio = 1.4:1
lungs are the most commonly the cellular elements are
Figure 7. Leukemia cells of acute  The average length of survival
myelocytic leukemia involved organs. monocytes
being approx. 8mos
 Symptoms of leukostasis:  Mildly elevated serum and
headache, visual impairment, urine lysozyme
shortness of breath  Blast may have indented,
convoluted nuclei in monocytes
 The number of nucleoli
averages from 3-5
 It is uncommon and comprises  The onset is dramatic, with  Total leukocyte count: 15,000-
FAB M5 – Acute Monocytic
less than 1% of all leukemia headaches and fevers being the 100,000 μl
Leukemia
 Two forms (FAB M5a and FAB chief complaints  Monocytes and Promonocytes
TPMEB 2020
M5b) have been distinguished  Physical examination: gingival constitute 25-75% of the
 WHO – acute monoblastic hyperplasia, as in nucleated cells
leukemia (FAB M5a) and acute myelomonocytic leukemia; pallor;  Blasts frequently have a muddy
monocytic leukemia (FAB M5b) and skin lesions or smoggy gray-blue cytoplasm
 FAB M5a form is most common  Uncommon – enlargement of the containing tiny granules, and
in young adults lymph nodes and spleen pseudopods are common
Figure 8. Leukemic cells of acute  FAB M5b has a peak occurrence  Extramedullary masses may be  The nucleus has a reticular
monocytic leukemia
characteristically during middle seen in about 1/3 of patients granular chromatin pattern and
age  DIC occurs may contain from 1-5 large
nucleoli
 A few immature erythrocytes
may be seen occasionally.
Figure 9. (Top) acute monocytic leukemia

FAB M5a, (bottom) acute monocytic
leukemia M5b
 It is very resistant to therapy

 The life expectancy 5-8mos
depending on the type.
 The WHO synonym for FAB M6a
 Blast cells of erythroid and
and FAB M6b is acute erythroid
myeloid
leukemia
FAB M6 – Erythroleukemia  Erythroblasts have an irregular
 This form of leukemia, also
outline with a high nuclear-
referred to as erythemic
 A common presenting symptom is cytoplasmic ratio
myelosis or Di Guglielmo
bleeding manifestation  Some of the blast exhibit the
syndrome represents a
 Pancytopenia is common at intense blue color associated
proliferation of both immature
diagnosis with rubriblasts
granulocytic and erythrocytic
 Erythroblasts – strongly PAS
cell types
Figure 10. Erythroleukemia blast cells and CD71 (+), express ABH
 This form of leukemia is
blood group antigens, and react
usually acute
with antihgb antibody
 Median age of occurrence: 54y
 Cytopenia is usually present,
particularly thrombocytopenia
 Dysplastic features in the
FAB M7 – Acute  WHO: acute megakaryoblastic neutrophils and platelets may
Megakaryoblastic Leukemia leukemia be present
 50% or more of the blasts are  There is no unique
 Organomegaly is infrequent
of the megakaryocyte lineage chromosomal abnormality
except in children
 Acute megakaryocytic associated w this form of AML.
 Radiographic evidence of bone
leukemia occurs in children and  Immunophenotyping reveals
lytic lesions has been observed in
adults that megakaryoblasts express
children
 This form of leukemia one or more of the platelet
Figure 11. Blast cells of acute comprises approximately 3-5% glycoprotein: CD41 or CD61
megakaryoblastic leukemia in PBS of cases of AML  Blasts are negative with anti-
MPO antibody
 Prognosis is usually poor,
particularly in infants
Other Types of Leukemia
 Rare, it is usually acute and  Chronic cough, pulmonary
Eosinophilic Leukemia
death occurs within 1y infiltration of leukocytes, and
TPMEB 2020
 Tissue infiltration and cardiac CNS involvement
failure is common  Biopsy in skin may show
Charcot-Leyden crystals,
marrow or either of eosinophil
accumulation
Figure 12. Eosinophilic leukemia cells in
PBS
Basophilic Leukemia  This is the rarest form of all
leukemias
 An infiltration on mast cells in
large numbers into affected
skin is observed
 PBS can demonstrate greater
than 50% basophils in this
Figure 13. Basophilic leukemia cells in
PBS disorder
ACUTE LYMPHOCYTIC ANEMIA
CLASSIFICATION OF ALL
FAB TYPE BLASTS SIZE NUCLEAR SHAPE NUCLEOLI CYTOPLASM
L1 Small Indistinct Scant Transparent
L2 Large, heterogeneous Indented, prominent Large, abundant Moderately clefted
L3 Large Regular oval/round Prominent, basophilic Prominent, vacuoles
 Signs and Symptoms – Auer rods are absent

– Lymphadenopathy and hepatomegaly are present – Terminal deoxynucleotidyl transferase (TdT) is an
in 75% of patients intracellular enzyme that catalyzes the non-specific
– Leukemic meningitis and cranial nerve palsies incorporation of nucleotides into DNA that are
caused by nerve infiltration by leukemic blast are present in lymphoblast of patients with ALL of
quite common either T- and B-cell lineage
 Laboratory Diagnosis – The common ALL antigen (cALLA) is found on the
– PBS – predominance of blast cells in about 50% of surface of lymphoblast in 70% of patients with ALL
patients; usually composed of close to 100% – CD20 expression is associated with inferior
lymphoblast, lymphocytes, and smudge cell survival in adults with ALL
TPMEB 2020

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MEDT 16 HEMATOLOGY 1

BRIEF HISTORY OF HEMATOLOGY

BLOOD – Venous Blood: 7.35

Figure 1. Blood Composition

1. Plasma – 55% of the whole blood a. Platelets

HEAT LOSS HEATING UP

Defensive/Protective Function  Regulated by kidneys and hypothalamus (detects

Figure 5. Blood-water regulation mechanism

3. Regulation of Body Temperature

Figure 8. Positive feedback mechanism of blood clotting

ADDITIVES IN COLLECTION TUBES 2. Antiglycolytic Agent

Indication of Bone Marrow Examination

Bone Marrow Specimen

Procedure in Collecting Blood Samples 4. Imprints (Touch Preparations)

skin numb.  Used when there is decreased nucleated cells in

aspiration specimen to a narrow-bored glass or plastic tube

aspiration needle was

Stages of Hematopoiesis: Intrauterine Normocellular Marrow for Adult

PROGENITOR CELLS GROWTH FACTORS/INTERLEUKINS/CYTOKINES MATURE CELLS

MYELOID-ERYTHROID RATIO (M:E RATIO)

CELL SIZE (µm) 10-15

CELL SIZE (µm) 10-14

BASOPHIL * Function: immediate hypersensitivity

Round to oval, may be irregular N:C RATIO 4:1

MONOBLAST PERIPHERAL BLOOD 0%

CELL SIZE (µm) 12-20

Variable, may be round/ horse shoe/ CELL SIZE (µm) 12-20

Round to oval Scant, slightly to moderately N:C RATIO 7:1-4:1

LYMPHOCYTE PERIPHERAL BLOOD —

CELL SIZE (µm) 8-20

Indented N:C RATIO 1:2

2-32 lobes Blue to pink, abundant N:C RATIO variable

MEGAKARYOCYTE (MK-III) PERIPHERAL BLOOD 0%

CELL SIZE (µm) 2-4

Light blue to colorless N:C RATIO NA

ERYTHROPOIESIS  Hypoxia – serves as the stimulus for red blood

Round CELL SIZE (µm) 10-12

Round N:C RATIO 0.5:1

Slightly more blue/purple N:C RATIO NA

CELL SIZE (µm) 7-8

ROUTINE HEMATOLOGIC EXAMINATION

INDICATIONS OF THE VARIATIONS IN THE LEVELS OF Hgb

ROUTINE HEMOGLOBIN Techniques/Methods

Figure 4. Electrical impedance principle

 Coulter Electrical Impedance Plot

HEMOGLOBIN F (KLEIHAUER-BETKE METHOD) appearance to the solution.

Figure 10. Solubility test for hemoglobin “S”

INDICATIONS OF THE VARIATIONS IN THE LEVELS OF Hct

ROUTINE HEMATOCRIT TEST  The hematocrit is determined indirectly from the

ROUTINE RBC COUNT 3. Platelets

COMPLETE BLOOD COUNT MANUAL RBC COUNTING USING HEMACYTOMETER

METHODS DILUENT PROCEDURE COUNTING METHOD

 Calculation  Out of these 25 squares, the RBCs are counted in

FLOW CYTOMETRY COUNTER RBC Indices

MEAN CELL/CORPUSCULAR VOLUME (MCV)

INDICATIONS OF THE VARIATIONS IN THE LEVELS OF MCHC

ERYTHROCYTE SEDIMENTATION RATE (ESR)  Female: 0-20 mm/hr

INDICATIONS OF THE VARIATIONS IN THE LEVELS OF ESR

WINTROBE AND LANSBERG