Enclonar, Kimberly / MLS 3A

Uses:
Intro to Hema o Anticoagulant of choice for hematology cell
October 15, 2020 counts and cell morphology
Glenn Charls Buelis, RMT, MLS (ASPIi) o May be used in preparation of blood smears
(<2 hours)
Physical Characteristics of the Blood o Preferred anticoagulants for platelet counting
Liquid in vivo; may clot in vitro Disadvantages
Red in color hemoglobin o If used in excess:
o Hgb is the oxygen carrying protein of the blood
Shrinkage of RBCs
pH: 7.35-7.45 Degenerative changes in WBCs
SG: 1.055 (1.045-1.065) Platelet swelling
o Because of the solutes o Platelet satellitism
3.5-4.5 times thicker than H2O Adherence of platelets to PMNs
Taste is somehow salty (polymorphonuclear cells)
Induce low platelet count.
Composition of Blood To correct, recollect new
specimen and use citrate.
Multiply result with 1.1 (platelet
multiplying factor)

Citrate
Usually present in light blue and black top
tubes.Mode of action: binds to calcium (Ca2+) to form
soluble complexes
Concentrations:
o 3.2% (0.109M), 1:9 AC:Blood ratio (light blue)
o 3.8% (0.129M), 1:4 AC:Blood ratio (black)
Uses:
o Coagulation studies (Factor V and VIII are
preserved) - light blue
PT and APTT
o Platelet studies (platelet aggregation) - light
blue
Platelet Aggregometry
o Erythrocyte Sedimentation Rate (Westergren) -
Overview of Red Blood Cells black
Also known as erythrocytes Disadvantages:
Anucleate, biconcave, discoid o In excess, prolongs PT and APTT results.
Contains hemoglobin
Pink to red, 6-8 um in diameter Oxalate
Zone of pallor that occupies 1/3 of the diameter Usually present in gray top tubes, can also be present
Major function: Oxygen delivery in black.
Mode of action: binds to calcium to form insoluble
Overview of White Blood Cells complexes.
Also known as leukocytes Optimum concentration: 1-2mg/mL
Major function: Protection Forms:
Examples: o Potassium oxalate - shrinks cells
o Neutrophils o Ammonium oxalate - swells cells
o Eosinophils o Double/Balanced oxalate = ratio of 2:3 (K:NH4)
o Basophils Uses:
o Lymphocytes o Erythrocyte Sedimentation Rate (Wintrobe) -
o Monocytes - precursor of macrophage black
o Glucose testing - gray
Anticoagulants Disadvantages:
Added to the blood to preserve its liquid state. o Distorts cellular morphology
Examples of additives. Crenation of RBCs
Inhibit the coagulation mechanism of the blood in Vacuolation of WBCs
vitro (heparin) Bizarre forms of lymphocytes and
Allows blood specimens to become suitable for monocytes
laboratory testing.
Important in generate accurate and reliable results. Heparin
Other name: mucoitin polysulfuric acid
EDTA (Ethylenediaminetetraacetic Acid) Usually present in green top tubes
Usually present in lavender top tubes. Mode of action: inactivates thrombin by accelerating
Mode of action: chelates calcium (Ca2+) action of antithrombin III
Optimum concentration = 1.5 mg/mL Naturally occurring anticoagulant in the human body
Forms: Optimum concentration: 15-20 U/mL
o K2 EDTA - aka VERSENE; plastic, spray dried Forms:
o K3 EDTA - aka SEQUESTRENE; glass, liquid o Lithium heparin
 Enclonar, Kimberly / MLS 3A
o Sodium heparin - injectable form used in
anticoagulant therapy
Uses:
o Osmotic fragility testing
o Blood gas analysis
o Potassium and Sodium Tests
Disadvantages:
o Causes bluish coloration in blood films when
stained
Acidic blood will attract blue basic dye.
o Not used in coagulation studies - inactivates all
stages of coagulation

Fluoride
Only an additive
Usually present in gray top tubes.
Mode of action: inhibits glycolysis by inactivating
enolase (removes Mg2+)
Fluoride only - serum
Fluoride + oxalate - plasma
Uses:
o Glucose testing (preserves glucose up to 72
hours)

Other Additives
1. Clot activator
o Glass or silica particles - used in STAT serum
determinations
o Thrombin - usually present in orange top tubes
2. Gel separator
o Aka thixotropic gel
o Usually present in gold top tube

Order of Draw
1. Yellow Blood culture tubes 8
1. Light blue Coagulation studies citrate 3-4
tube
1. Red/Gold Serum tubes with or 5 - with clot
without clot activator or gel activator
separator 0 - no clot
activator
1. Green Heparin tubes 8
1. Lavender EDTA tubes 8
1. Gray Glycolytic inhibitor fluoride 8
tubes
 Enclonar, Kimberly / MLS 3A
o Blood pressure cuff (60mmHg)
Blood Collection Applied 3-4 inches above venipuncture site
October 20, 2020 o 7.5-10cm
Glenn Charls Buelis, RMT, MLS (ASPIi) Left for: <1min
Safety Collection tubes
Follow standard precautions (SP) Common: evacuated tube system
o SP includes hand hygiene, universal precaution
Evacuated tube: glass or plastic
does not Has a needle and adapter
Treat specimens as potentially infectious
Can be a potential source of pathogens: hepatitis C, Additives
hepatitis B, hepatitis D, syphilis, malaria, and human Clot activators
immunodeficiency virus (HIV) o Serum testing: allow to clot 30-60 minutes
Accidental puncture (needlestick) may happen at any before centrifugation & removal of serum
time. o
Indirect Transmission: touches a contaminated (STAT)
surface or object & touches the mouth o Ex: glass or silica (activates factor XII or
Handwashing: the most important practice to prevent thrombin)
the spread of infectious diseases. Anticoagulants
o 20s o Prevents blood from clotting
o 1 minute - entire handwashing procedure o Examples:
Wash with soap in between patients, in between
procedures, & when gloves are removed.
Gloves must be worn during blood collection o Invert immediately
procedures. Antiglycolytic
o Inhibits metabolism of glucose by blood cells
designated puncture-resistant containers. o Most common: sodium fluoride
Containers must have the biohazard sign. o serum
o plasma
Role of Phlebo in Infection Control Gel separator
Observance of infection control and isolation policies o Thixotropic gel
Once policies are o Inert material
o Changes viscosity during centrifugation
hygiene o Barrier between liquid & cells
Follow SP at all times. o Also based on the specific gravity.
RBC = 1.05
Physiologic Factors Affecting Test Results Gel separator = 1.04
Physiologic variables under the control of the patient Plasma/Serum = 1.02-1.03
or the phlebotomist (causes preanalytical variation)
Variable Facts Needles
Posture Sterile, length and gauge variety
water from intravascular to interstitial Evacuated: screwed into the adapter, and double
space. pointed
protein, With rubber sleeve
cholesterol, and iron inversely related to the bore size
Blood collection: 19-23
Diurnal Body fluid fluctuations o Adult (common) = 21
Rhythm cortisol, TSH & iron o Children = 23
WBC count, & eosinophil Needle length size: 1.0-1.5 inch
Exercise
AST, WBC count, & HDL cholesterol
Factors: Intensity, duration & frequency
Stress Anxiety & excessive crying =
count
Diet Fasting; either 8-12 hours
Recent
(lipemia)
Smoking

Venipuncture
Equipment
Tourniquet
Provides barrier vs venous blood flow to help locate
the vein
Types: Deep/light
o Velco strap
 Enclonar, Kimberly / MLS 3A
Needle holders EDTA tube Lavender or pink
In compliance with OSHA
Do not interchange needles (must be according to Sodium fluoride Gray stopper
manufacturer type)
Disposable: discard (single use) Coagulation Testing
-
Winged Blood collection set additive tube or another light blue stopper to clear
Also known as butterfly needle the dead air space in the tubing before the collecting
Short needle with plastic wings tube.
End needle - connect with evacuated tube, syringe, or
blood culture Veni in Children
Ideal: difficult draw Excellent interpersonal skills are needed to deal with
distraught parents and with crying, screaming, or
Syringe frightened
Has a barrel with graduation (mL) Child's arm should be immobilized as much as
possible.
Important: proper attachment (to prevent air)
Ideal: pediatric, geriatric, or other patients with tiny, Complications Encountered in Venipuncture
fragile, or rolling veins Complications Facts
Ecchymosis (bruise) Most common complication
Skin antiseptic techniques
Most common: 70% isopropyl alcohol in Leakage of small amount of
commercially prepared pad blood in the tissue around the
Circular motion - from center then outward puncture site
Allow to dry Apply direct pressure (use
Blood alcohol level test - non-alcohol based antiseptic gauze pad)
(benzalkonium chloride) Hematoma Results when leakage of a
large amount of blood around
Blood culture collection the puncture site.
Two steps: Rapidly swell
1. 30-60 seconds rub with 70% alcohol Remove the needle
2. Cleanse w/ 1-10% povidone-iodine pads (others: immediately and apply
tincture of iodine, chlorhexidine) pressure to the site with a
gauze pad for at least 2
Vein Selection minutes.
Common site: superficial veins of the antecubital
Fainting (syncope) Phlebotomist should always
fossa
Angle: 15-30 degrees ask the patient whether he or
Two anatomical patterns: she has had any prior
o H pattern episodes of fainting during or
Median cubital vein after blood collection.
Cephalic vein Ammonia inhalants: to revive
Basilic the patient
o M pattern If patients begin to faint:
Median vein remove and discard needle
Accessory cephalic vein immediately, apply pressure
Basilic vein - last choice to the site with a gauze pad,
If necessary: patient should make a fist after lower patient's head, and
tourniquet application. loosen any constrictive
Do not pump fist. clothing.
Palpate using index finger: Hemoconcentration Increased concentration of
o Depth cells, large molecules, and
o Direction
o Diameter in water and balance
If not palpable: examine dorsal veins of the hand. Tourniquet should not remain
Veins of the feet should not be used without on the arm for longer than 1
physician's permission. minute.
Endorse to another phlebotomist to attempt to
palpate a vein before using the veins of the feet. remove for 2 minutes and
AVOID THE INNER WRIST reapplied before the
Most crucial step: patient ID venipuncture is performed.

Order of Draw for Venipuncture Hemolysis RBC with consequent escape

of Hgb
Blood culture tube Yellow
Needle is too small during a
Coagulation tube Light blue difficult draw; drew the blood
Serum tube w/ or w/o Red, gold, red-gray marbled, through an existing
activator orange, or yellow-gray stopper hematoma

Heparin tube Green or light green


Pulled back to quickly on the
plunger of a syringe
Forced blood into a tube from
a syringe by pushing the
plunger
Mixing to vigorously
Contaminated with alcohol
Petechiae Small red spots indicating that
small amounts of blood have
escaped into the skin.
Associated with hemostasis
abnormality and should alert
the phlebotomist to be aware.
Allergies Due to antiseptic substances
and adhesive bandages and
tape.
Use hypoallergenic tape or
apply pressure manually until
the bleeding has stopped
completely
Nerve damage No blind probing
If nerve damage: shooting or
sharp pain, tingling, or
numbness in the arm
Remove and the needle. Apply
pressure with a gauze pad
Collect blood from other arm
Seizures Remove and discard needle
Apply pressure with a gauze
pad
Notify nurse or designated
first aid
Vomiting Provide appropriate container
and tissues
Notify nurse or designated
first aid providers.
Special Situations
Complications Facts
Edema Avoid edematous sites
Contaminated with tissue
fluids
Obesity Veins are difficult to palpate
Blood pressure cuff: aids in
locating a vein (not
<40mmHg)
Do not probe blindly
Burned, Damaged, Avoid
Scarred, and No continuous flow of blood
Occluded veins
Intravenous therapy Avoid if possible
If no alternative: draw
below the IV site
(tourniquet should be
placed here as well)
Stop infusion for 2 minutes
Mastectomy Physician consultation
Can lead to pain or
lymphostasis
Enclonar, Kimberly / MLS 3A
Inability to Obtain a Specimen
Case Facts
Failure to Missed veins during collection
draw blood Reposition needle by slightly
withdrawing or advancing the
needle
tube
Patient Patient has the right to refuse
Refusal Do not force patient; notify nurse
or physician
Document!
unsuccessful
Skin Puncture
Ideal for pediatric and newborn patients
therapeutic purposes, extremely obese, geriatric
fluids
Phlebo should note that the sample is from skin
puncture.
Collection sites:
o <1 year old: heel (lateral, medial, plantar)
o >1 year old and adults: palmar surface (3rd and
4th finger)
Perpendicular to finger print lines
Warm the site with commercial heel warmer (not >
42C, not longer than 3-5 minutes)
Clean the skin: 70% alcohol
Allow to dry
Do not use povidone-iodine solution (contamination
risk)
Increases:
o Potassium
o Uric Acid
PUP
o Phosphorus
Wipe first drop (prevent tissue fluid contamination)
Equipment
Sterile Lancets or Blades
Spring loaded in the device
Devices can have varied depths depending on age
Depth of puncture:
o <2mm (infants)
o 2.0-2.5mm (adults) 2- 3 ?
Capillary tubes
With or without heparin
Recommended: plastic or Mylar-coated glass tubes
Red ring - with heparin
Capillary tube length = 70mm or 7cm
Bore size = 1mm or 0.1 cm
Tube should be filled:
o 3/4 (Rodak's)
o At least 50mm (Henry's)
Microcollection Tubes
With or without additives
Same color-coding system with evacuated tubes
Same labelling guidelines
Different order of draw
Fill EDTA first
 Enclonar, Kimberly / MLS 3A
Order of Draw for Skin Puncture Legal Issues
1. Tube for blood gas analysis Two areas of Concern
2. Slides, unless made from specimen in EDTA Breach of patient confidentiality
microcollection tube Patient misidentification
3. EDTA microcollection tube
4. Other microcollection tubes with anticoagulants
5. Serum microcollection tubes

Quality Assurance
Technical Competence
Phlebotomist: trained properly in all phases of blood
collection
Certification is needed
Continuing education is needed
Assess & document competency

Collection Procedures
Review collection SOPs
Proper preparation and patient identification are
crucial

Anticoagulants and Other Additives

Follow manufact
microlots and ensure specimen integrity

accuracy

Requirements for a Quality Specimen

Patient must be properly identified
Patients must properly prepare for draw
Correct order and labelled correctly
Correct anticoagulants and other additives used
Properly mixed by inversion
Not hemolyzed
Should not be contaminated with IV
Not lipemic
Fasting in a timely manner
Timed specimens are drawn at the correct time

Blood culture specimens

Monitor culture contamination (rate should be lower
than 3%)

results
Reduce by proper venipuncture technique

QC and Preventive Maintenance

annually

Specimen Handling
Start with the initiation of the test request & ends
when the specimen is tested

Transport in upright position

Exposure to light
Child in ice water bath
o Ammonia
o Lactic acid
PLAG
o PTH
o Gastrin

Separate serum within 2 hours

o Glucose
o Potassium
o LDH
 yolk -

liver -
BM
Enclonar, Kimberly / MLS 3A
Hematopoiesis Phases of Hematopoiesis
Mesoblastic
October 22, 2020
Begins around the 19th day of embryonic
Glenn Charls Buelis, RMT, MLS (ASPIi)
development
Sie: yolk sac
Hematopoiesis
Mesodermal cells migrate to the yolk sac:
Continuous, regulated process of blood cell formation
o Central cavity - primitive erythroblasts
that includes:
First primitive cell to be produced (2-8
o Cell renewal
weeks)
o Cell proliferation
Important in early embryogenesis to
o Cell differentiation
produce intrauterine hemoglobin
o Cell maturation
Gower-1
Gower-2
Types of Hematopoiesis
Portland
Synchronous Asynchronous Surrounding cavity - angioblasts (blood vessels)
The nucleus and cytoplasm The nucleus and cytoplasm Occurs intravascularly
develop together (at the does not develop together. Not a definitive hematopoiesis
same rate) Mesodermal cells also migrate to aorta-gonad-
mesonephros (AGM) region
Medullary Extramedullary o Gives rise to hematopoietic stem cells for
Blood production occurs in Blood production occurs definitive or permanent adult hematopoiesis
the inner part of the bone outside the bone marrow.
marrow. Hepatic
Begins at 5-7 gestational weeks
Theories on Blood Formation Peaks by the 3rd month of fetal development
Monophyletic Declines after the 6th month
Unitarian theory Minimal activity is maintained until 1-2 weeks after
Suggests that all blood cells are derive from a single birth
progenitor stem cell called pluripotent hematopoietic Characterized by recognizable clusters of developing
stem cell. erythroblasts, granulocytes, and monocytes
Site: LIVER, thymus, spleen, placenta
Polyphyletic Hepatic hematopoiesis occurs extravascularly
Dualistic theory Hematopoiesis in yolk sac and AGM region disappears
Suggest that each of the blood cell lineages is derived on this stage
from its own unique stem cell. Thymus - first fully developed organ, produces T-cell
Developing kidney and spleen - produces B-cell
Lymphoid cells begin to appear
o Myeloblast Production of megakaryocyte begin during this phase
Basophil Erythroblasts produce HbF, HbA1, HbA2
Eosinophil Most predominant: Hemoglobin F
Neutrophil
o Immature monocyte Medullary
Monocyte Also known as "myeloid" phase
o Megakaryocyte Hematopoiesis begins in the bone marrow cavity prior
Platelets to the 5th month of development
o Pronormoblast Site: bone marrow cavity (medulla)
Erythrocyte o Red bone marrow - hematopoietically active
o Yellow bone marrow - inactive; adipocytes
By the end of 24 weeks (6 months) gestation, the
bone marrow becomes the primary site of
hematopoiesis
-0
Myeloid-to-erythroid ratio 3:1
Measurable levels of erythropoietin, G-CSF, GM-CSF,
Hemoglobin A and F are detected.
Cells at various stages of maturation can be seen in all
blood cell lineages.
Infancy and early childhood:
o All bones contain red marrow.
Adults:
o Red marrow is restricted to:
Sternum
Vertebrae
Scapulae
Pelvis
Ribs
Skull
Proximal ends of long bones
Retrogression - red marrow transformed to yellow
marrow.
red
> -

yellow
 Enclonar, Kimberly / MLS 3A
Bone marrow aspiration
o Child - tibia Stem Cell Phenotype
o Adult - posterior iliac crest Membrane has surface markers or antigens which is
used for blood cell identity which is assigned to as the
cluster of differentiation (CD)
CD38 and HLA-DR Associated with loss of "stemness"
CD33 and CD38 Seen on committed myeloid
progenitors
CD10 and CD38 Seen on committed lymphoid
progenitors
CD7 T-cells and NKCs
CD19 B-cell
CD34 Hematopoietic stem cells (flow
cytometry)

Cytokines and Growth Factors

Stem Cell Theory
Hematopoietic stem cells are:
o Capable of self-renewal
o Pluripotent - capable of giving rise to different
cell types
o Able to reconstitute the hematopoietic system
of the host
Hematopoietic stem cells are directed to one of three
possible fates:
o Self-renewal
o Differentiation
o Apoptosis
2 models
o Stochastic - random
o Instructive - depends on the requirements of
body
o Can be both.
Undifferentiated HSCs can differentiate into
progenitor cells committed to either lymphoid or
myeloid lineages (Monophyletic theory)
o Lymphoid - common lymphoid progenitor
T-cell, B-cell, and natural killer
lymphocyte and dendritic cell lineages
o Myeloid - common myeloid progenitor
Granulocytic, erythrocytic, monocytic,
and megakaryocytic lineage
Hematopoietic growth factors or cytokines - a group
of specific glycoproteins that
o Regulate the proliferation, differentiation, and
maturation of hematopoietic precursor cells
o Responsible for stimulation or inhibition of
production, differentiation, and trafficking of
mature blood cells and their precursors
o Include interleukins, lymphokines, monokines,
interferons, chemokines, and colony-
stimulating factors (CSFs)
Positive influence - stimulating production,
differentiation, trafficking
o KIT ligand, FLT3 ligand, GM-CSF, IL1, IL3, IL6,
IL11
Negative influence - inhibit production,
downregulates differentiates and trafficking
o Transforming growth factor, tumor necrosis
factor, interferons
Colony Stimulating Factors
Substances that have high specificity for their target
cells and are active at low concentrations
Names of the individual factors indicate the
predominant cell lines that respond to their presence
o G-CSF - primarily target is the granulocytic cell
line
o GM-CSF - targets granulocytic-monocytic cell
line
0
o With IL-3, megakaryocyte colony formation
o M-CSF - monocytic cell line

Lineage-Specific Hematopoiesis
Erythropoiesis
o CFU- - -
Pronormoblast
Myelopoiesis
Neutrophils GM-CSF
G-CSF
Monocytes GM-CSF
M-CSF
Eosinophils GM-CSF
IL-5
IL-3
Basophils IL-3
KIT Ligand
Lymphopoiesis
o IL-2, IL-7, IL-12 and IL-15 and to some extent IL-
4, IL-10, IL-13, IL-14 and IL-16

217112,15
 Enclonar, Kimberly / MLS 3A
Megakaryopoiesis
o GM-CSF, IL-3, IL-6, IL-11, KIT ligand and TPO
(thrombopoietin)
o TPO and IL-11 controls the production and
release of platelets

Principles of Normal Cell Maturation

Important characteristics to observe initially in cell
identification
Overall Cell Size N:C ratio
The amount of space occupied
overall size as by the nucleus in relation to the
maturation progresses. space occupied by the
cytoplasm.

Increased space for cytoplasm

 Enclonar, Kimberly / MLS 3A
Change in cytoplasm color: blue to gray blue to
RBCs salmon pink
October 27, 2020 o Basophilia = degree of ribosomal RNA
Glenn Charls Buelis, RMT, MLS (ASPIi)

RBCs
Specialized function: Carry oxygen from the lungs to
the tissues
o Oxygen attaches to hemoglobin molecule (major
cytoplasmic component)
Secondary function: Carry carbon dioxide to the lungs
& buffer pH
Mechanisms are in place to control:
o Development
o Production
o Normal destruction of the RBC
No nucleus in its mature state

Normoblastic Maturation
Erythroblasts: nucleated precursors in the bone
marrow.
Can be called normoblasts: normal developing cells in
the bone marrow
Megaloblasts: abnormal erythroblasts Pronormoblast 1st morphologically
Wbrib last
identifiable stage
Maturation Process Globin production begins
Erythroid progenitors
Basophilic normoblast Rise of Hgb concentration
o BFU-E & CFU-E: committed to the erythroid cell
line prowbniyte Last stage with nucleolus
o BFU- - Polychromatic Last stage capable of mitosis
Pronormoblast normoblast End of DNA synthesis
BFU-E to CFU-E (3-5 cell divisions)
o Pronormoblast: 1st morphologically identifiable
Wbrioyle No nucleolus
Orthochromic End of RNA synthesis
0
stage.
o BFU-E to mature RBC: 21 days normoblast End of DNA content
Stages: melawbnoyti Last stage with nucleus
o Pronormoblast Reticulocyte End of RNA content
o Basophilic normoblasts No nucleus
o Polychromatic normoblasts Last stage of Hgb production
o Orthochromic normoblasts
o Polychromatic erythrocyte (reticulocyte)
o Erythrocyte Stages of RBC Maturation
Pronormoblast/Rubriblast
Pronormoblast Nucleus
o Occupies almost the entire cell
1st morphologically identifiable stage.
o N:C ratio 8:1
Divide and daughter cell matures into the basophilic
o Round to Oval
normoblasts.
o 1-2 nucleoli 8 :|
Daughter cells divide into the next stage:
o Purple red chromatin
polychromatophilic normoblast
Simultaneous maturation of the nucleus and Cytoplasm
o Dark blue
cytoplasm.
o Visible golgi complex (pale, unstained area)
Results to the formation of 8-32 mature RBCs.
o Small tufts of irregular cytoplasm
Criteria for Identification of Precursors Division: Undergoes mitosis, gives rise to 2 daughter
Morphologic identification depends on a well-stained pronormoblasts
peripheral blood smear. Location: bone marrow
Stain: Romanowsky stain: Wright or Wright-Giemsa Cellular activity
o Production of proteins & enzymes needed for
Identification parameters:
o Nuclear chromatin pattern iron uptake & protoporphyrin
o Nuclear diameter o Globin production begins
o N:C ratio Length of time: slightly more than 24 hrs
o (+/-) nucleoli
o Cytoplasmic color Basophilic normoblast
Nucleus
o Start of chromatin condensation (clumps along
Trends that affect RBC Appearance
Overall diameter of the cell decreases. the periphery)
o Larger and sharper parachromatin
Nucleus diameter decreases - decreased N:C ratio.
o N:C ratio 6:1
Nuclear chromatin pattern: coarser, clumped, &
o 0-1 nucleoli
condensed.
Nucleoli disappear.
o Deep blue red chromatin 6 :|
 Enclonar, Kimberly / MLS 3A
Cytoplasm Endoribonuclease - digests the remaining ribosomes
o Deeper, richer blue (basophilia)

:
Length of time: 3 days
Division: Undergoes mitosis o 2 days in bone marrow, 1 day in PB
Location: bone marrow
Cellular activity Erythrocyte
o Detectable but undiscernible Hgb synthesis Nucleus: none
(masked by large amounts of mRNA) Cytoplasm
Length of time: slightly more than 24 hrs o Biconcave
Last stage that has nucleolus o 7-8 um (6-8um)
o Thickness 1.5-2.0um
Polychromatic Normoblast o Salmon pink with central pallor (1/3)
Nucleus Division: unable to divide
o Varied chromatin (open at the start then Survives up to 120 days.
condensed at late stages) Cellular activity
o N:C ratio 4:1 o Delivers oxygen to the tissues, shape allows the
o No nucleoli present Hgb to be concentrated at the periphery, must
Cytoplasm
o Pink color (Hgb production)
4:L squeeze to small blood vessels.
Capable of:
o Pink + blue = murky gray-blue o Deformability - because of the membrane of the
Division: last stage capable of mitosis RBCs
Location: bone marrow o Flexibility
Cellular activity
o Increased Hgb synthesis Erythrokinetics
o Ribosomes are still present Dynamics of RBC production and destruction
Length of time: 30hrs Erythron: collection of all stages of erythrocytes in the
body (unified functional tissue)
Orthochromic Normoblast o Immature RBCs in the bone marrow

①
Nucleus o Circulating RBCs in the peripheral blood
o Completely condensed (pyknotic) o RBCs in the vascular spaces
o N:C 1:2
o Last stage with nucleus i. a RBC Production
Cytoplasm Hypoxia
o Increased in salmon-pink color of the cytoplasm Primary oxygen-sensing system: peritubular
o Nearly complete Hgb production fibroblasts of kidney peninsular fibroblasts
o Slightly blue cytoplasm Erythropoietin - produced by the PTF in cases of
Division: not capable of cell division hypoxia
Location: bone marrow Normally, EPO is released in small amounts.
Cellular activity Increased RBC destruction, bleeding or other
o Hgb production continues conditions that cause diminished oxygen carrying
o Ejection of nucleus from the cell capacity.
o Pinching of the cell membrane
If nucleus is not pinched off an
Howell Jolly bodies
Pyrenocyte - nucleus extruded from the cell
o Vimentin
Responsible in holding the nucleus in the
center of the cell
o Non-muscle myosin
Responsible for the ejection of nucleus
Length of time: 48hrs

Polychromatic Erythrocyte/Reticulocyte
Nucleus: None
Cytoplasm
o Predominant color is due to Hgb
o End stage - salmon pink
o Blue in new methylene blue
Division: not capable
Location
o Resides in the bone marrow (1 day or longer)
o Moves to the PB for 1 day before it matures
Cellular activity
o Complete production of Hgb
Last stage of Hgb synthesis
o Digestion of ribosomes
o RNA can be stained by a vital stain
Residual RNA - mesh of small blue strands
Reticulocyte - if stained with new methylene blue
Diffusely basophilic erythrocyte
Erythropoietin
Thermostable, nondialyzable, glycoprotein hormone
Carbohydrate unit
Terminal sialic acid unit
activity in vivo
True hormone, growth factor, receptor mediated
interaction
Three major effects:
o Early release of reticulocytes (Shift reticulocytes)
increased RBC egress into the sinus
Downregulation of adhesion molecules
o Prevents apoptosis
Removing apoptosis induction signal
Direct EPO rescue from apoptosis - RBC
precursors are prevented from dying
Production of anti-apoptotic molecules

o Reduction of maturation time (marrow transit
time)
Increase the rate of surviving precursors
enter the circulation
Two ways:
=÷
Increased rate of cellular processes
Accelerated Hgb production
Accelerated bone marrow egress
Accelerated cessation of division
Decreased cell cycle time
Erythropoietin Measurement
Specimen: Plasma & Other body fluids
Methods: Chemiluminescence
Therapeutic use: For anemias due to CKD and
chemotherapy
Bone Marrow Microenvironment
Erythropoiesis occurs in erythroid islands
(macrophages surrounded by erythroid precursors)
Former fact: Macrophages deliver iron to the
developing RBCs (suckling pig phenomenon)
Recent fact: Macrophages release cytokines
necessary for maturation
Erythrocytes are "anchored" to the bone marrow
before entering the circulation
RBC Destruction
Normal RBCs produces enzymes that allows RBCs to
survive up to 120 days
Produces ATP thru anaerobic glycolysis - lack of
enzymes result to senescence (killed by macrophages)
o Senescence - old
Macrophage Mediated (Extravascular) Hemolysis
Happens in the spleen - hostile environment
o Depleted glucose
o Low pH - promoting iron oxidation
Aged RBCs - decreased ATP production
o Oxidation of membrane lipids and proteins
o Imbalance in intracellular potassium and sodium
- cell becomes spherical due to water
Decreased membran
splenic sieve macrophages
Eryptosis - erythrocyte death
When RBCs are lysed within the macrophage
o Iron is removed from heme and stored as ferritin
o Globin from Hgb is degraded to amino acids
o Heme is degraded to bilirubin
Mechanical (Intravascular) Hemolysis
Happens in the blood vessels
Turbulence occurring in the chambers of the heart or
at bifurcation of vessels.
Small breaks in blood vessels & clots - trapping RBCs
Hemoglobin Salvage
When RBCs burst, Hgb is released.
Iron must be salvaged.
Hgb can cause oxidative damage to cells.
Mechanisms to salvage Hgb/iron:
Enclonar, Kimberly / MLS 3A
o Haptoglobin
Hgb binds to haptoglobin in plasma -
avoiding urinary loss of iron
Haptoglobin-hemoglobin complex is taken
up by macrophages in the liver, spleen,
bone marrow, and lungs.
Macrophages express CD163 - haptoglobin
receptor
Inside the macrophage:
Iron is salvaged.
Globin is catabolized.
Protoporphyrin is converted to
unconjugated bilirubin.
Haptoglobin will not survive.
Decreased in intravascular hemolysis.
o Hemopexin
Iron in free plasma Hgb rapidly becomes
oxidized - forming methemoglobin
Metheme binds to the hemopexin in
plasma
Hemopexin-metheme binds to CD91
receptor in hepatocytes
Hemopexin is recycled.
o Methemealbumin (Albumin)
Albumin acts as carriers for different
molecules.
Metheme-albumin binding is just
temporary.
Metheme is rapidly transferred to
hemopexin.
o Reabsorption of iron in the proximal tubular cells
Presence of hemosiderin in urine.
Ferroportin -transports iron the plasma.
RBC Energy Metabolism
RBC relies on anaerobic glycolysis for its ATP supply
Oxygen delivery - does not require ATP
RBC metabolic process - require ATP
Energy (ATP) is important to:
o Perform RBC function efficiently
o Maintain RBC membrane integrity
o Live the entire life span of RBCs
Embden-Meyerhof Pathway
o Produces 90% of energy used by RBCs
Glycolysis Diversion Pathways
Hexose Monophosphate
Also known as Pentose phosphate pathway
Functions:
o Prevents oxidation of hemoglobin
o Protects Hgb from oxidative radicals by
generating NADPH
Free radicals/oxidative radicals
o Group of atoms that are formed when oxygen
interacts with certain molecules
Chief danger: It is when free radicals react with
important cellular elements such as DNA, or the cell
membrane
 Enclonar, Kimberly / MLS 3A
Solution: Production of antioxidants that interacts
with the free radicals and terminate the chain of
reaction.
G6PD provides the only means of generating NADPH
for glutathione reduction, and in its absence,
erythrocytes are particularly vulnerable to oxidative
damage.
Normal G6PD activity is able to detoxify oxidative
compounds and safeguard hemoglobin, sulfhydryl-
containing enzymes, and membrane thiols, allowing
normally functioning RBCs to carry enormous
Rapoport-Leubering
quantities of oxygen safely.
Function: Generation of 2,3-biphosphoglycerate (2,3-
Deficiency: Leads to oxidation of hemoglobin.
BPG) or 2,3-diphosphoglycerate (2,3-DPG)
2,3-DPG binds to Hgb and regulates oxygen delivery
to tissues by competing for the oxygen-binding site of
hemoglobin
When 2,3-DPG binds heme, oxygen is released.
-DPG =

Methemoglobin reductase
Functional Iron: Ferrous (Fe2+)
Oxidative form: Ferric (Fe3+) - Methemoglobin
Functions:
o Prevents oxidation of iron
o Maintains iron in ferrous state
o Reduces methemoglobin to ferrous state.
Methemoglobin reductase or cytochrome B5
reductase.
NADPH may help but slowly.
 Enclonar, Kimberly / MLS 3A
Hemoglobin Metabolism
November 5, 2020
Glenn Charls Buelis, RMT, MLS (ASPIi)

Hemoglobin
Comprises 95% of the cytoplasmic content of RBCs
Concentration within RBCs: 34g/dL
MW: 64,000 daltons
1 gram of Hgb
o 1.34 mL O2
o 3.47 mg Iron
Main function: transport gases; contributes to the
acid-base balance; transports nitric oxide
Globular protein made of 2 different pairs of Iron Kinetics
polypeptide chains Dietary Iron, Bioavailability and Demand
4 heme groups with 1 heme group in each of the 4 Only source of iron for the body is from the diet
polypeptide chains (exogenous Fe3+)
Iron must be in ferrous state to be absorbed into the
Heme Structure intestine (duodenum).
Consists of a ring of C, H, and N called protoporphyrin In the enterocyte is where the absorption of iron
IX takes place in the body from the diet.
Ferrous iron (Fe2+) reversibly binds to one O2 o Conversion of Fe3+ to Fe2+ also happens.
molecule o By: Vitamin C ferrireductase
Ferric iron (Fe3+) oxidized; no longer binds to oxygen; Most dietary iron is in the ferric state (Fe3+)
called methemoglobin Reduction in the ferrous form are enhance by gastric
Heme = protoporphyrin IX + iron acid, acidic food, and duodenal cytochrome b (DcytB),
found in the enterocyte.
Globin Structure Nearly 65% of iron in the body is held within Hgb
Consist of two identical pairs of unlike polypeptide About 25% is in storage, mostly within macrophages
chains and hepatocytes
Variations in amino acid sequences give rise to Remaining 10% is divided among muscles, plasma,
different types of polypeptide chains and cytochromes of cells
Designated by Greek Letter Menstrual bleeding is the main cause of anemia.
Women require 50% more iron than men.

Iron Compartments

Compartment Form and Anatomical site % of total

body Iron
Function Hemoglobin ~68 (65)
Myoglobin ~10
Peroxidase, catalase, ~3
Complete Hgb Structure cytochromes, riboflavin
Globin chains loop to form a cleft pocket for heme enzymes in cells
Tetrameric in structure
Spherical Storage Ferritin and hemosiderin ~18 (25)
4 heme groups attached to 4 polypeptide chains and mostly in macrophages
carry up to 4 O2 and hepatocytes; small
amounts in all cells except
mature RBCs
Hemoglobin Biosynthesis
Heme Synthesis Transport Transferrin in plasma <1
Occurs in the mitochondria and cytoplasm of bone
marrow erythrocyte precursors
Pronormoblast to reticulocyte in the circulation Ferrous (Fe2+) Functional
Transferrin - carries iron in the ferric form (Fe3+) to Absorbable
developing erythroid cells
Carried by DMT1
Iron is transported into the mitochondria where it is
Carried by ferroportin
reduced to ferrous state and united to
Oxidized by Hephaestin to Fe3+
protoporphyrin IX to form heme, then released to the
cytoplasm Ferric (Fe3+) Non-functional; dietary
Methemoglobin
Circulates in the plasma (bound to
transferrin)
Reduced by Vitamin C ferrireductase to
Fe2+
 Enclonar, Kimberly / MLS 3A
Iron Absorption Cellular Iron Disposition
Body's system to regulate iron is via absorption When iron stores inside the cell are sufficient,
Divalent metal transporter 1 (DMT1) - carries ferrous production of
iron across the luminal side of the enterocyte inside the cell, production of
o Gateway to enterocyte (entrance)
o Responsible for absorption of other metals (Zn, Systemic Body Regulation
Cu, Co) Hepcidin - protein released in the liver that binds to
Ferroportin - carries iron across the enterocyte into ferroportin and inactivates it
the bloodstream o Stimulated by
o Only known protein that exports iron in across IL-6
the cell membrane Iron
ILBH
o Gateway to circulation (exit) Bacterial LPS
HFE protein - main regulator
By HFE gene (hemochromatosis gene)
Iron Transport Interact with other protein to regulate
Iron, as it leaves the enterocyte, is in ferrous state iron
Iron must be converted to the ferric form for Hereditary
transport in the blood hemochromatosis - iron overload
Hephaestin - protein that oxidizes iron as it exits the
enterocyte absorption
Apotransferrin (apoTf) - protein that carries ferric Body iron
form in the plasma; binds 2 molecules of Fe3+ is able to transport iron into the blood
Transferrin - apoTf with Fe3+
75% proceed to bone marrow for erythropoiesis Iron Recycling
10-20% in the liver Littoral cells possess ferroportin in their membranes.
Macrophages (during extravascular hemolysis) serve
as iron transporter
Hepatocytes also possess ferroportin so that salvaged
iron can be exported to transferrin and to other cells

Summary
Vitamin C Converts dietary Fe3+ to Fe2+
ferrireductase
DMT1 Gateway of Fe2+ to enterocyte
Ferroportin 1 (Ireg- Gateway of Fe2+ to circulation
1)
Hephaestin Converts Fe2+ to Fe3+
Decrease iron plasma concentration
Cellular Iron Absorption Apotransferrin Protein without Fe3+
Relies on receptor-mediated endocytosis
Transferrin Protein with 2 molecules of Fe3+
Transferrin receptor 1 - binds to transferrin
Clathrin-coated endosome = 2Fe-Tf + TfR + DMT1 Hepcidin Released in the liver to bind with
Endosome is acidified by H+ (pH 5.5) ferroportin for regulation of Fe2+
o Fe2+ Fe3+ (ferritin) Transferrin Attachment site of transferrin for
mitochondria receptor 1 cellular absorption
o Endosome is released out of the cell
If there is enough iron inside the cell, TfR1 is halted
Iron may bypass the cytoplasmic iron-sensing system Globin Synthesis
and moves directly to the mitochondria P arm of chromosome 16: codes of zeta and alpha
P arm of chromosome 11: codes for epsilon, gamma,
delta, beta
Synthesized in the ribosomes, then released to the
cytoplasm

Hemoglobin Assembly
Each globin chain binds to a heme molecule to form a
heterodimer in the cytoplasm
A-chain has a + charge and has affinity to B-chain (-
charge)

Phase Hgb Present Composition

Mesoblastic Phase Gower 1 2E + 2Z (EZ)
Gower 2 2A + 2E (TAE)
Portland 2Z + 2G (ZG)
 Enclonar, Kimberly / MLS 3A
Hepatic Phase up to F 2A + 2G
before birth (3rd (FAG) Sulfhemoglobin
trimester) Formed by the addition of sulfur atom to the pyrrole
ring of heme
Birth F (60-90%) 2A + 2G Irreversible; persists for the life of the cell
(FAG) Ineffective for oxygen transport
A (10-40%) 2A + 2B (2B) Patient is cyanotic at high levels
Color of the blood: mauve lavender
2 years through Adulthood A1 (>95%) 2A + 2B (2B)
Presence of greenish pigment
(6months) A2 (<3.5%) 2A + 2D (2D) Treatment: avoidance of offending agent
F (1-2%) 2A + 2G Assay:
(FAG) o 630nm
o Differentiated to MetHb when the spectral curve
does not shift when cyanide is added
Hemoglobin Ontogeny
Hgb changes reflect the sequential activation and
Carboxyhemoglobin
inactivation (switching) of the globin genes
Formed by the combination of CO with heme iron
After 6 months after birth, HgbA is the major
CO has 240x affinity that O2
hemoglobin.
Impairs the delivery of oxygen to the tissues
Silent killer - odorless, colorless, victims quickly
become hypoxic
Reversible
<2% formed endogenously
Exogenously derived from exhaust of automobiles,
tobacco smoke, and industrial pollutants
20-30%: Toxic effects; headache, dizziness, and
disorientation
>40%: coma, seizure, hypotension, cardiac arythmias,
pulmonary edema, and death
Diagnosis of CO poisoning:
o COHb is >3% in nonsmokers and >10% in
Dyshemoglobins smokers
Unable to transport oxygen Treatment:
Offending agent modifies the structure of the o Administration of 100% oxygen
hemoglobin molecule, preventing it from binding o Hyperbaric oxygen therapy
oxygen Assay: spectral absorption at 540nm
Most are acquired, some are hereditary Color of blood: cherry red

Methemoglobin
Formed by the reversible oxidation of heme iron to
ferric state
1% is maintained by the methemoglobin reduction
systems
>30%: Cyanosis and hypoxia
>50%: coma or death
Methemoglobinemia
Acquired
o Acquired after exposure to exogenous oxidant,
such as nitrites, primaquine, dapsone, or
benzocaine
Hereditary
o Mutations in the gene for NADH-cytochrome b%
reductase 3 (CYB5R3)
o Mutations in the A, B, G- gene (HgM or M
hemoglobin)
Treatment for Acquired methemoglobinemia
o >30%: Intravenous methylene blue
administration
o Exchange transfusion
No effective treatment for hereditary
methemoglobinemia
Color of blood: chocolate brown
o Does not revert back to the normal red when
oxygenated
Assays:
o Spectral absorption analysis (CO-oximeter)
o Absorption peak at 630nm
o HbM: Electrophoresis; HPLC; DNA mutation
testing
 Enclonar, Kimberly / MLS 3A
Oxygen Dissociation Curve
November 10, 2020
Glenn Charls Buelis, RMT, MLS (ASPIi)
Shift to the Right
Functions of Hemoglobin
50% O2 saturation occurs at a pO2 of >27mmHg
1. Transport of molecular oxygen from lungs to the Hgb has a for O2 and readily releases it to
tissues the tissues
2. Transport of CO2 from tissues to the lungs Hypoxia
3. Buffering of the blood pH Respiratory acidosis ( ) = hypoventilation
4. Transport of nitric oxide High altitude
Congestive heart failure
Oxygen Transport
Severe anemia
Hemoglobin readily binds oxygen molecule in the
lungs which require high oxygen affinity to transport
oxygen (high oxygen tension)
-DPG
To unload oxygen, it requires low oxygen affinity
Oxygen affinity is affected by heme-heme interaction
Transported via
o Plasma <2%
Factors affecting Hgb Affinity for Oxygen
o RBCs
Body temperature
CO2 Transport
(shift to the right)
Dissolved in plasma (10%)
Active tissues release acid and heat which in turn
Carbamino Hgb (20%)
stimulate the release of O2
Bicarbonate ions (by carbonic anhydrase) - exchange
Cl-
pH
Relationship between blood pH and the O2 affinity of
Heme-Heme interaction
Hgb is referred to as the Bohr effect
Binding of one molecule causes an increased affinity
H+ binds oxyhemoglobin and the O2 is released from
of the other heme groups of O2
the Hgb due to the Bohr effect.
Relates to the partial pressure of oxygen (pO2)
CO2
CO2 exerts an effect of O2 uptake and delivery similar
Partial pressure of Oxygen (pO2)
to the Bohr effect
Defined in terms of the amount of oxygen needed to
Non-
saturate 50% of Hgb
The shift in the curve related to the CO2 level is called
Or the p50 value
Haldane effect
Described by the oxygen dissociation curve

Oxygen Dissociation Curve

Carbonic anhydrase to form HCO3
Plots the percent oxygen saturation of hemoglobin
versus the pO2 2,3-biphosphoglycerate
Shape is sigmoidal
-

Involves conformational change in the structure of

o
hemoglobin
o
Oxygenated = relaxed
S curve R conformation
Y-axis reflects the percentage of hemoglobin
Change in conformation of the Hgb tetramer occurs
saturated with oxygen for an Hgb solution at pH 7.4
Disruption of the salt bridges and release of 2,3-BPG
X-axis reflects the partial pressure of oxygen at
15-degree rotation of the A1B1 dimer
equilibrium
Deoxygenated = tensed
pO2 of 27mmHg results in 50% oxygen saturation of
T conformation
the Hgb Stabilized by the binding of 2,3-BPG between the B-
Shape reflects: chains and the formation of salt bridges
o When the pO2 is <20mmHg, Hgb has affinity -

-
for O2
o pO2 of 20-60mmHg, affinity for O2
Fetal Hemoglobin
o pO2 >60mmHg, curve flattens which indicate
Causes shift to the left
almost complete saturation
pO2 should not be <40mmHg
weakened ability to bind 2,3-BPG
Shift to the Left
50% oxygen saturation occurs at a pO2 of <27mmHg
Hgb has an for O2 and does not readily
release it to the tissues ( )
Multiple transfusions due to depleted 2,3-
delivery
Respiratory Alkalosis ( ) = hyperventilation
Presence of hemoglobin variants with high affinity to
O2
 Enclonar, Kimberly / MLS 3A
WBCs Promyelocyte
November 24, 2020 Comprise 1-5% of nucleated cells in the bone marrow
Glenn Charls Buelis, RMT, MLS (ASPIi) 16-25um, relatively larger than myeloblasts
Round to oval, eccentric nucleus
WBC / Leukocytes
Paranuclear halo or "hof"
Relatively colorless compared to red blood cells Evenly basophilic cytoplasm
General function: Defense against infections Cytoplasm is full of primary (azurophilic) granules
Can be grouped to: Primary (non-specific) granules are visible in this stage
o Granulocytes
(Steininger)
o Mononuclear cells
Chromatin clumping may be visible
1-3 nucleoli can be seen but can be obscured by
WBC Development
granules
Stem cell pool: consists of hematopoietic stem cells
capable of self-renewal and differentiation Myelocyte
Proliferation (mitotic) pool: consists of cells that are 6-17% of nucleated cells in the bone marrow
actively dividing 16-24um (Steininger)
Maturation (storage) pool: consists of cells Final stage of mitosis
undergoing nuclear maturation that form the marrow Begins to produce secondary, specific granules
reserve and are available for release
Early myelocyte
o Morphologically similar to promyelocytes in
White Blood Cells
terms of size and nuclear characteristics
Bone Marrow PB o "Dawn of neutrophilia" - patches of grainy pale
Bands Neut 9-32% 0-5% pink cytoplasm which represent secondary
granules in the Golgi area
Neutrophil 7-30% 50-70% Late myelocyte
Lymphocyte 18-24% o Smaller than promyelocyte
o 15-18um
Monocyte 2-11%
o Nucleus has more heterochromatin
Eosinophil 1-3% 1-3%
Basophil <1% 0-2% Metamyelocyte (Juvenile cell)
3-20% of nucleated cells
14-16um
Neutrophil First stage incapable of mitosis
Most common leukocyte in normal peripheral blood Begins to synthesize tertiary granules
Polymorphonuclear Indented nucleus (kidney bean or peanut shaped)
Responds to bacterial infection
2 forms: If present in blood: leukemia
o Segmented - majority
o Band Bands
9-32% of the nucleated cells in the bone marrow
Development 0-5% of nucleated cells in peripheral blood
Share common progenitor cell with monocyte (GMP) Absence of RNA
G-CSF - major cytokine responsible for stimulation of Tertiary granules continue to form
neutrophil production Secretory granules begin to form
HSCs, CMPs, and GMPs are not morphologically Nucleus is highly clumped
distinguishable Nuclear indentation exceeds 1/2 of the nucleus
Neutrophil myeloblasts and promyelocytes are not diameter
morphologically distinguishable from the other WBCs Difficult to distinguish from segmented neutrophil
Last stage before the cell matures
Myeloblast Other names: Stab or Staff Cell
Makes up 0-3% of nucleated cells in the bone marrow
First morphologically identified stage Segmented (Mature)
14-20um in diameter 7-30% in BM
Type I Highest numbers in peripheral blood (50-70%)
o High N:C ratio of 8:1 to 4:1 2-5 nuclear lobes connected by a threadlike filament
o Nucleus occupies most of the cell Pink to tan cytoplasm with violet or lilac granules
o Slightly basophilic cytoplasm
o With fine nuclear chromatin and 2-4 visible
% Size Nucle Granule Others
nucleoli in oli s
o Has no visible granules
BM
Type II
o Presence of dispersed primary (azurophilic) Myeloblast 0- 14- TI: None First
granules in the cytoplasm 3% 20u 2-4 morphological
o <20 granules per cell m ly identifiable
Type III T1: 8:1 or 4:1
o Darker chromatin and more purple cytoplasm TII: Dispersed
o >20 granules that do not obscure the nucleus primary
o Rare in the normal bone marrows granules
o Difficult to distinguish from type II myeloblasts
 Enclonar, Kimberly / MLS 3A
Promyelocyt 1- 16- 1-3 Primary Paranuclear Gelatinase and collagenase breakdown collagen -
e 5% 25u halo or "hof" allowing neutrophils to move through the tissue
m Neutrophils migrate towards the area with highest
concentration of chemotactic agents
Myelocyte 6- 16- Seconda Can be Once at the site of inflammation, neutrophils begin
17 24u ry differentiated the process of phagocytosis.
% m Final stage of Phagocytosis involves the engulfment, killing, and
mitosis digestion of microorganisms and foreign materials.
Early Dawn of Second function: Formation of neutrophil
myelocyte neutrophilia extracellular traps (NETs)
o NETs: threadlike structures with enzymes from
Late 15- Heterochrom
neutrophils granules that trap and kill bacteria
myelocyte 18u atin
and fungi
m
o Release of NETs - when neutrophils die
Metamyeloc 3- 14- 0 Tertiary Kidney bean Third Function: Secretion of substances
yte (Juvenile 20 16u or peanut- o Transcobalamin I or R binder protein - proper
cell) % m shaped absorption of vitamin B12
o Secretion of cytokines
Bands 9- Tertiary 0-5% in
(Stab/Staff 32 Secretor peripheral Eosinophil
cell) % y blood Make up 1-3% of nucleated cells in the bone ma
Indentation: Accounts for 1-3% of PB leukocytes
>1/2 Responds to helminthic infections and mediates
Last stage allergic reactions
before the cell
matures Development
Segmenter 7- 50-70% in Similar to neutrophils - CMP
30 blood Lineage: IL-3, IL-5, GM-CSF, and transcription factors
% Pink to tan IL-5: critical for eosinophil growth and survival
cytoplasm
with violet or Promyelocyte
lilac granules Charcot-Leyden crystal proteins are present in
primary granules (cytochemical stains)
Indistinguishable from other WBCs
Kinetics
Movement of neutrophil and its precursors between Myelocyte
the different pools in the bone marrow, PB, and Large, pale, reddish-orange secondary granules
tissues
0.9-1.0 x10^9 cells/kg per day Metamyelocyte
Proliferative pool: 2.1x10^9 cells/kg Secondary granules increase in number and become
Maturation pool: 5.6x10^9 cells/kg more distinct
Myeloblast to myelocyte - 6 days Begin to produce tertiary granules
Maturation pool - 4-6 days
Release from BM is stimulated by G-CSF Bands
In the PB, neutrophils are divided into: Similar to neutrophil counterpart
o Circulating neutrophil pool Reddish-orange granules in cytoplasm
o Marginated neutrophil pool
Half life = 7hrs Mature eosinophil
Diapedesis Bilobed nucleus
o Movement of WBCs from the blood to the Refractile, orange-red secondary granules
tissues
o Integrins and selectins Kinetics
Last myelocyte division to emergence of mature
Functions eosinophil from BM - 3.5 days
Major function: Mean turnover: 2.2x10^8 cells/kg per day
o Phagocytosis and destruction of foreign material Large storage pool in bone marrow:
and microorganisms o 9x10^8 cells/kg
Process involves seeking and destruction Half life: 18hrs
In response to chemotactic agents, neutrophils ROLL Survival time in tissues: 2-5 days
along endothelial cells of blood vessels
Rolling is mediated by neutrophil selectins and Functions
adhesive molecules on the surface of endothelial cells Degranulation
Integrins help tighten binding of neutrophils and o Classical exocytosis
endothelial cells o Compound exocytosis - clump before release
Diapedesis - migration of neutrophils between or o Piecemeal degranulation - excreted piece by
through endothelial cells piece
As neutrophils migrate, they release gelatinase and Immune regulation
collagenase Responds to parasitic helminths
o Destruction of tissue-invading helminths
 Enclonar, Kimberly / MLS 3A
Major basic protein (MBP) Monocytes
Eosinophilic cationic protein (ECP) 15-20um
Hallmark of allergic disorders Round, oval, deeply indented (horseshoe shaped)
o Increased when there are allergic reactions nucleus
o Mediates allergic reactions Lacelike or stringy chromatin pattern
o Suggested to have a role in airway modelling Nucleoli are generally not seen
o Regulates basophils Blue-gray cytoplasm with fine azure granules (azure
dust), ground-glass appearance
Basophil
<1% in bone marrow Kinetics
0-2% in PB Promonocyte pool: 6x10^8 cells/kg
Must be differentiated from MAST cells o 7x10^6 monocytes/kg per hour
Basophils are true leukocytes Normally, promonocytes undergo 2 mitotic divisions
Mast cells are tissue effector cells - mature in tissues in 60 hrs giving rise to 4 monocytes
and only found in tissues In increased demands, promonocytes undergo 4
mitotic divisions in 60hrs to give rise to 16 monocytes
Development No storage pool
Immature Basophils Marginal pool of monocytes is 3.5x the number in the
Round to somewhat lobulated nuclei circulating pool
Slighly condensed chromatin Remains in the circulation for 3 days
(+/-) nucleoli Monocytes enter the tissues and may transform into:
Large blue-black secondary granules (water soluble) o Macrophages (40-50um)
Primary granules may not be visible o Osteoclasts
o Dendritic cells
Mature Basophils
Unsegmented or lobulated nucleus, often obscured Functions
by granules Innate immunity
Clumped chromatin o Act as phagocytes
Colorless cytoplasm with large blue-black granules Synthesizes nitric oxide
Adaptive immunity
Kinetics o Antigen presentation - activation of lymphocytes
Poorly understood Dendritic cell - most efficient and potent
Life span: 60hrs antigen-presenting cell
Housekeeping functions (macrophage)
Functions o Removes debris and deal cells
Poorly understood o Destruction of senescent RBCs and maintenance
Initiators of allergic inflammation of iron storage
o Granzyme B o Synthesize various proteins
Release of IL-4 and IL-13 - TH2 cytokines
Synthesis of IgE Lymphocytes
Production of retinoic acid Divided into 3 majors groups:
Angiogenesis - expression of VEGF o T cells - adaptive + cellular
Involved in control of helminthic infections o B cells - adaptive + humoral
o NK cells - adaptive + cellular
Monocyte Different from other leukocytes
2-11% of circulating leukocytes o Lymphocytes are not end cells (resting cells)
o Recirculate from blood to tissues and back to the
Development blood
o B and T cells - capable of rearranging antigen
Similar to neutrophils because both are derived from
receptor gene segments
GMP
o T and NK cells mature outside the bone marrow
Macrophage-CSF: major cytokine responsible for the
growth and differentiation of monocytes
Development
Monoblast For B and T cells, development can be subdivided
Very rare and difficult to distinguish from myeloblasts into:
o Antigen independent
based on morpholoby
Large, round, centrally-placed nucleus with soft, fine- Bone marrow and thymus
stranded chromatin o Antigen dependent
Single, large nucleolus Spleen, lymph nodes, tonsils, etc.

Promonocyte B lymphocytes
12-18um Develop in the bone marrow
Slightly indented or folded nucleus Pro-B, pre-B and immature B cells
Delicate chromatin pattern Immature B cells (hematogones)
At least 1 nucleolus o Antigen naïve
o Migrates to secondary lymphoid organs
Blue cytoplasm with scattered azure granules
o Homogenous nuclear chromatin and extremely
scanty cytoplasm
 Enclonar, Kimberly / MLS 3A
T lymphocytes
Develop in the thymus
Pro-T, pre-T, and immature T cells
Immature T cells migrate to secondary lymphoid
organs

Mature lymphocytes
Types of lymphocytes are indistinguishable
N:C ratio of 5:1 to 2:1
Chromatin is arranged in blocks
Deep purple, round, oval, or indented nucleus
Sky blue or "Robin egg" blue cytoplasm

Small lymphocytes 7-10um

Medium lymphocytes 10-12um
Large lymphocytes 11-25um

B lymphocytes 3-21%
T lymphocytes 51-88%
NK cells 4-29%

Functions
B lymphocytes
o Antibody production
o Antigen presentation
o Production of cytokines regulating T cells and
antigen presenting cell functions
o Plasma cell
T lymphocytes
o CD4 T cells - helper cells
o CD8 T cells - Killer cells (cytotoxic T cells)
Secretes granzyme and perforins
NK lymphocytes
o Part of innate immunity
o Kills tumor cells and virus-infected cells
o Also produce perforins
o Modulation of macrophages and T cells