INTRODUCTION TO HEMATOLOGY ferr- iron

hemo-/hemato- blood
Hematology hypo- beneath, under, deficient, decreased
• “hema” means blood hyper- above, beyond, extreme
• study of blood cells by staining, counting analyzing and iso- equal, alike, same
recording the appearance, phenotype and genotype of all leuko- white
types of cells macro- large, long
• to predict, detect and diagnose blood diseases and many mega- large, giant
systemic diseases that affect blood cells meta- after, next, change
• to select and monitor therapy for diseases micro- small
• What do we do in RBC and WBC or in hematology lab? myel-/myelo- from BM or spinal cord
1. count (quantitative): anything that is beyond or pan- all, overall, all-inclusive
insufficient the normal value, it is associated with a phleb- vein
certain disease; CBC (complete blood count) procedure phago- eat, ingest
poikilo- varied, irregular
2. morphology (qualitative): looking at the size, shape,
poly- many
presence of inclusion bodies, appearance; PBS
schis- split
(peripheral blood smear) procedure
scler- hard
splen- spleen
❖ anything that is abnormal with its count and morphology
thromb- clot, thrombus
indicates abnormality and disease /thrombo-
❖ hemoglobin carries oxygen inside the RBC; also the xanth- yellow
protein portion of RBC Suffix Meaning
-cyte cell
Blood -emia blood
• Functions: -itis inflammation
▪ transports O2 from lungs to tissues -lysis destruction, dissolving
▪ clears CO2 from tissue to lungs then release to the -oma swelling, tumor
environment -opathy disease
▪ transports biomolecules (proteins, glucose, fats) -penia deficiency
▪ delivers waste to liver and kidneys -phil/-philic attracted to, affinity for
▪ provides coagulation enzymes -plasia/-plastic cell production or repair
▪ protect vessels from trauma and hemostasis -poiesis cell production, formation & development
• average of 5L -poietin stimulates production
• composition
▪ plasma Hematologic Procedures
o transports and nourishes blood cells to different
organs I. Complete Blood Count
▪ cells • can be performed by automation and manual method
o RBC (erythrocytes) • enumeration of cellular elements, quantitation of hemoglobin
o WBC (leukocytes) and describes cell appearance
o platelets (thrombocytes) RBC parameters WBC parameters Platelet
parameters
History RBC count WBC count Platelet count
• Athanasius Kircher (1657) Hemoglobin Neutrophil MPV
▪ described “worm” in blood Hematocrit Lymphocyte
• Anton van Leeuwenhoek (1674) MCV Monocyte
▪ gave account to RBC MCH Eosinophil
• Giulio Bizzozero (1880’s) MCHC Basophil
▪ described platelets as “petites plaques” RDW
Reticulocyte
• James Homer Wright (1902)
▪ developed Wright stain (Wright’s Romanowsky-type
❖ MCV, MCH, MCHC are RBC index/indices; can be comouted
stain)
by using RBC count, Hemoglobin, and Hematocrit values
o polychromatic, mixture of acidic and basic dyes
❖ Reticulocyte: immature RBC; can be seen in BM (majority
o foundation of blood cell identification
can be found and peripheral circulation (few/minor)
▪ scientific term for cell appearance is “morphology”
❖ Erythropoiesis: RBC production; from immature (bone
marrow) to mature (peripheral circulation)
Terminologies
❖ Differential count: performed in PBS; counting WBC, you
will count 100 cells and out of 100, how many neutrophils,
Prefix Meaning
lymphocytes, monocytes, eosinophils, and basophils
a-/an- lack, without, absent, decreased
❖ Neutrophils: 60-70% (normal value)
aniso- unequal, dissimilar
cyte- cell ❖ Lymphocyte: 30-40% (normal value)
dys- abnormal, difficult, bad ❖ Monocyte: 5-10% (normal value)
erythro- red ❖ Eosinophils & Basophils: 0-3% (normal value)
❖ High neutrophils: bacterial infection order to form separate portions (bottom: packed
❖ High lymphocytes: viral infection RBC; top: plasma; middle: buffy coat)
❖ MPV: index of platelets ❖ to find the value of hematocrit, measure the height of
the packed RBC (A), and the whole blood (from
Red Blood Cells plasma to packed RBC) (B), then divide, then multiply
• anucleate, biconcave, discoid cells filled with a reddish it by 100.
protein called hemoglobin 𝑝𝑎𝑐𝑘𝑒𝑑 𝑅𝐵𝐶 (𝐴)
× 100 = ℎ𝑒𝑚𝑎𝑡𝑜𝑐𝑟𝑖𝑡 %
• shape: biconcave 𝑤ℎ𝑜𝑙𝑒 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑 (𝐵)
• central pallor/zone of pallor: inner circle in the RBC; will ❖ WBC and platelets will be found in buffy coat
determine the hemoglobin concentration;
❖ size of central pallor: • Buffy coat
o indirectly proportional to the Hgb concentration: ↑ size ▪ light colored layer between the RBC and plasma
of central pallor = ↓ Hgb concentration ▪ contains the WBC’s and platelets
• staining intensity is directly proportional to Hgb ▪ excluded in Hct determination
concentration:
o ↑ staining (dark color) = ↑ Hgb concentration • RBC indices
o ↓ stain (light color) = ↓ Hgb concentration ▪ MCV (Mean Cell Volume)
• nucleated: immature; anucleated: mature o reflects RBC diameter
• site of production: Bone Marrow o expressed in fL
• appear pink to red cell that measure 6 to 8um in diameter o to compute, you need: RBC count, Hgb, Hct values
with a zone of pallor which occupies 1/3 of their center ▪ MCHC (Mean Cell Hemoglobin Concentration)
reflecting its biconcavity o reflects RBC staining intensity and the amount of
• measurement of volume detects the presence of anemia or central pallor
polycythemia o expressed in g/dL
▪ Anemia: decreased RBC count = ↓ Hgb ▪ MCH (Mean Cell Hemoglobin)
▪ Polycythemia: increased RBC count; hyper viscosity o reflects the mass of hemoglobin
because of too much RBC o expressed in pg
• RBC count in cells per microliter (uL, mcL, mm3, cc, L) ▪ RDW (RBC distribution width)
• first visual RBC counting (1900) but inaccurate o expressed the degree of variation in RBC volume to
tell whether the patient has:
• first to made automated method are Joseph and Wallace
➔ Anisocytosis: variation in the RBC size,
Coulter of Chicago, Illinois (1953)
▪ Coulter principle: cells will be counted via electrical diameter, and volume
impedance
White Blood Cells
• Hemoglobin • leukocytes (granulocytes and agranulocytes)
▪ in oxyhemoglobin, use reagents potassium cyanide and ❖ agranulated doesn’t mean that granules are absent.
potassium ferricyanide to form a much stable form called They are still present but, not evident or visible
cyanmethemoglobin. When you already have • cells for protection from infection and injury
cyanmethemoglobin, then measure it by using • site of production: bone marrow or lymphoid tissues (ex.
spectrophotometer at 540 nm wavelength. The intensity spleen, thymus, liver, lymph nodes)
of solution, measured in the spectrophotometer, is the • colorless in unstained cell suspension, hence “white blood
equivalent of the hemoglobin concentration cell”
▪ protein inside the RBC • normal count: 4,500-11,500 cells/uL of blood
▪ reagents: potassium cyanide and potassium ferricyanide • conditions:
▪ hgb - stable cyanmethemoglobin ▪ Leukopenia: low WBC count
▪ color intensity of the solution is measured in a ▪ Leukocytosis: increased WBC count
spectrophotometer at 540nm ▪ Leukemia: abnormalities in WBC; uncontrolled
▪ compared with a known standard and mathematically proliferation or production of WBC
converted to hemoglobin concentration • Granulocytes (BEN - Basophils, Eosinophils, Neutrophils)
▪ replace it with ionic surfactant sodium lauryl sulfate 1. Neutrophil
▪ neut’s, segmented neutrophil,
• Hematocrit segs, PMN, polymorphonuclear
▪ ratio of the volume of packed RBC’s to the volume of neutrophils
whole blood ▪ granules: purple; fine
▪ also known as PCV (packed cell volume) determined by ▪ phagocytic cells which engulf and
transferring blood to a graduated plastic tube with a destroy microorganisms and foreign materials
uniform bore ▪ “segmented” refers to the multilobed nuclei
▪ after centrifugation, measure the column of RBC’s and ▪ condition:
dividing by the total length of the column of RBC’s plus o Neutropenia: low neutrophils; due to viral
plasma. infection (because of high lymphocyte count),
taking medication that affects the WBC count
❖ in packed RBC, add blood in the capillary tube then o Neutrophilia: increased neutrophils; due to
seal the bottom part using gel. Next, centrifuge in bacterial infection
 ▪ “bands” o Lymphocytopenia: low lymphocytes due to
o immature neutrophil medication or immunodeficient
o found in bone marrow; bone o Leukemia: associated to all cells as long as the
marrow has maturation pool morphology is abnormal due to uncontrolled
and storage pool (reserved proliferation
neutrophils)
o no formed segmentation 2. Monocyte
(continuous nucleus) ▪ mono’s
o cytoplasm of the cell contains submicroscopic, ▪ immature macrophage passing
pink or lavender-staining granules (bacterial through blood from its site of
secretions) production
o left shift: many bands in peripheral circulation ▪ production:
o immature neutrophils can still be released if o BM → PC (transient) → tissues
needed or if the patient has severe bacterial ▪ comprise the most abundant cell type of the body
infection ▪ comprise minor component of peripheral blood
o many bands in peripheral circulation means WBCs because they are transient
severely bacterial infected ▪ comprise minor component of peripheral blood
WBCs
2. Eosinophil ▪ phagocytosed foreign particle
▪ eo’s ▪ assist in assembly and presentation of immunogenic
▪ granules: bright orange epitopes to the lymphocytes
▪ cells with bright orange-red, regular ▪ condition:
cytoplasmic granules o Monocytosis: increased monocytes; non-specific;
▪ condition: possible infection (check other WBC count)
o Eosinophilia: increased eosinophils due to o Monocytopenia: low monocytes theoretical
parasitic infection (not all parasite can increase
the eosinophil count), allergic reaction ❖ monocyte: largest cell in peripheral circulation
o Eosinopenia: low eosinophils (theoretical//very ❖ megakaryocyte: largest cell in the bone marrow;
low) releases platelets
❖ monocyte/macrophage: most abundant cell in the
❖ in differential counting, it is normal to not find body
eosinophils because they have very low quantity in ❖ neutrophil: most abundant cell in the peripheral
the blood circulation (also in bone marrow)
❖ monocyte: located in peripheral circulation
3. Basophil ❖ macrophages: located in tissues
▪ baso’s
▪ granules: dark blue or blue or Platelets
dark purple • “thrombocytes”
▪ cells with dark purple, irregular • fragments from megakaryocyte
cytoplasmic granules which • true blood cells that maintain blood
obscure nucleus vessel integrity by initiating vessel
▪ granules contain histamine and various proteins repair
▪ condition: • adhere to the damaged surfaces,
o Basosophilia: increased basophils due to form aggregates to plug the BV and secrete proteins and
allergic reaction (because of histamine in the small molecules that trigger clot formation or thrombosis
granules) • control hemostasis
o Basopenia: low basophils (theoretical) • 2-4 um in diameter, round or oval, anucleated and slightly
granular
• Agranulocytes • released in megakaryocyte
1. Lymphocyte • condition:
▪ “lymph’s” ▪ Thrombocytosis: increased platelets
▪ complex system of cells that ▪ Thrombocytopenia: decreased platelets
provide host immunity ▪ Essential thrombocythemia: malignant condition;
▪ host immunity: uncontrolled production of platelets
o humoral: B-cells → became
plasma cells, which produces antibodies II. Blood film examination
o cellular: T-cells
• “wedge prep” blood film on a glass
▪ part of humoral and cell-mediated responses
microscope slide
▪ cell: round, slightly larger than RBCs
• morphology of the cells are being
▪ nuclei: round featureless and has a thin rim of non-
examined
granular cytoplasm
• lows it to dry, fixes and stains it with
▪ conditions:
Wright or Wright-Giemsa
o Lymphocytosis: high lymphocytes due to viral
infection
• examines for abnormalities in shape, diameter, color or • coagulant of choice for cell counting and sizing
inclusion bodies using OIO • less shrinkage of RBCs and less of an increase in cell
• estimate WBC count and platelet count for comparison with volume on standing
automation
• cannot give you an accurate count Sodium Citrate
• RBC cannot be counted • EDTA + calcium= insoluble calcium
• WBC differential count salt
• 3.2% sodium citrate
III. Other Procedures • 3.8% sodium citrate
• Coagulation • anticoagulant for aPTT, PT testing
• BM examination and Westergren ESR
• Flow cytometry immunophenotyping • due to dilution of anticoagulant to blood, sodium citrate is
• Cytogenetic analysis generally unacceptable for most other hematology tests
• Molecular diagnosis assay
Specimen Collection Heparin
• inactivates the blood-clotting factor
Equipment for Venipuncture thrombin & factor Xa
• in vitro and in vivo anticoagulant
1. Tourniquet • coat capillary blood collection tubes
• barrier against venous blood flow to locate a vein • inappropriate anticoagulant for many
• disposable elastic strap, Velco strap, blood pressure cuff hematology tests (Wright-stained
• 3-4 in above the venipuncture site blood smears)
• left no longer than 1 min before venipuncture is performed
Oxalate
• distorts the cell morphology
• RBCs become crenated
• vacuoles appear in the granulocytes
2. Collection Tubes • bizarre forms of lymphocytes and monocytes appear
• plastic or glass (OSHA recommends plastic tubes whenever rapidly
possible)
• plastic tubes are covered with silicone (help decrease c) Antiglycolytic agent
possibility of hemolysis and prevent blood from adhering to • inhibits metabolism of glucose by
the sides) blood cells
• additives • sodium fluoride
✓ Clot activators
✓ Anticoagulants ❖ anticoagulated plasma can be
✓ Antiglycolytic agent immediately centrifuged to obtain
✓ Separator Gel plasma

a) Clot activators d) Separator Gel

• blood specimens for serum testing: allowed to clot for 30- • inert material that
60 minutes prior to centrifugation and removal of serum undergoes a temporary
• accelerates clotting process and decreases specimen change in viscosity during
preparation time centrifugation process
• glass or silica particles • separation barrier between
• thrombin liquid and cells
b) Anticoagulants • cannot be used with certain instruments or for blood bank
• prevents blood from clotting procedures
• EDTA, sodium citrate, heparin
• Heparin 3. Needles
▪ binds to antithrombin in • sterile and available in variety Gauge Hub Color
plasma and inhibiting thrombin of lengths and gauges
16G White
and activated factor X • inserted into the vein and has a
18G Pink
point with slanted side
EDTA • gauge number is inversely 19G Cream
• preferred collective tube because it related to the bore diameter - 20G Yellow
can preserve the morphology of the 19-23 gauge: bore for blood 21G Green
cell extraction 22G Black
• EDTA + calcium= insoluble calcium • 21 gauge with 1 in length: most
23G Blue
salt common needle size for adults
24G Medium Purple
• dry additives (K2 EDTA or Na2 • 1 in length: provides better
EDTA) control during venipuncture 25G Orange
• liquid additive (K3 EDTA) 26G Brown
 Green Heparin
4. Needle holders Light blue or clear Buffered Sodiu, citrate (0.105M in
• designed to comply with OSHA’s revised (Hemogard glass, 0.109M in plastics)
• Occupational Exposure to closure) Citrate, theophyline, adenosine,
Bloodborne Pathogens Standards dipyridamole (CTAD)
• needle holders are made to fit White K2 EDTA with gel
Red/light gray or None (plastic)
specific manufacturer’s needle
clear (Hemogard
and should not be interchanged
closure)
• Vacutainer Eclipse Blood Red Silicone coated (glass)
Collection System (BD Medical, Clot activator, Silicone coated (plastic)
Franklin Lakes, NY) with BD
Eclipse needle Order Closure Color Type of Tube
• Jelco multi-sample blood collection needle with Mix by Inverting
Venipuncture Needle-Pro Device (Smith Medical ASD, 1 Yellow Blood cultures-SPS-aerobic
Norwell, MA) and anaerobic 8-10x
• Greiner Bio-One (Monroe, NC VACUETTE QUICK SHIELD 2 Light blue Citrate tube 3-4x
with VACUETTE Visio PLUS multi-sample needle 3 Gold or red/gray BD Vacutainer SST gel
separator Tube 5x
5. Winged Blood Collection Set (Butterfly) Red Serum tube (plastic) 5x
• short needle with Red Serum tube (glass)
plastic wings connected Orange BD vacutainer rapid serum
to thin tubing tube (RST) 5-6x
4 Light green or BC vacutainer PST
• other end of the tubing
Green/gray Gel separator tube with
can be connected to a
heparin 8-10x
needle holder for an
Green Heparin 8-10x
evacuated tube, syringe or a blood culture bottle
5 Lavender EDTA 8-10x
• useful for children or other patient from whom it is difficult to 6 White BD vacutainer PPT separator
collect blood tube K2 EDTA with gel 8-10x
7 Gray Fluoride (glucose) Tube 8-10x
6. Syringes
• consist of barrel, graduated in mm • factors affecting quality of tubes:
and a plunger a) ambient temperature
• have a point at one end and an ▪ low:
open hub at the other end that ▪ high:
attaches to the barrel b) altitude
• pediatric, geriatric and other ▪ high (>5,000 ft)
patients with fragile, tiny or rolling • factors affecting quality of tubes
veins (that would not be able to withstand the vacuum a) humidity
pressure from evacuated tubes ▪ low: hasten the escape of water vapor from a tube
containing a wet additive
Blood Collection Method ▪ high: migration of water vapor inside a tube
b) light
1. Evacuated Tube System ▪ CTAD
• most commonly used
• vacutainer two-way needle, plastic tube holder, evacuated 2. Syringe Method
tube • used less often, still method of choice for some instances
• vacuum tube set should be assembled before applying • barrel and plunger
tourniquet • small, fragile and damaged vein (may collapse under
• provides closed sterile system for specimen collection vacuum pressure)
• follows “order of draw” recommended by CLSI to avoid
cross-contamination 3. Butterfly Infusion Set
• needle with plastic wings, plastic tubing, adapter
• useful for children or other patient from whom it is difficult to
collect blood
• costly

Color Anticoagulant
Lavender K2 EDTA (spray-coated plastic tube)
K2 EDTA (liquid in glass tube)
Pink K2 EDTA (spray-coated plastic tube)
4. Capillary blood • when run is rejected, the cause should be found and
• microhematocrit tubes corrected
• used for hematocrit determination
• small tube may be heparinized or plain ❖ 3 controls: (these specimens are pooled blood means
• mylar layer mixed specimens from different people that has high
▪ keep the pieces intact and safely values and parameters)
contained ▪ high: when the pooled blood is run, all parameters
▪ not interfere with the accuracy for the are high
user’s visual inspection of the sample ▪ normal: when the pooled blood is run, all parameters
are normal
❖ red – with anticoagulant; heparinized ▪ low: when the pooled blood is run, all parameters are
❖ blue – without additives; non-heparinized low
❖ These 3 controls are important to know whether the
Quality Control and Quality Assurance machines are working properly

Quality control Calibration verification

• implies the ability to provide accurate, reproducible/ reliable • detects systematic errors caused by deterioration of the
assay results that offer clinically useful information calibrator or a change in the analytical process
• WHO: internal personnel
• WHEN: daily, every start of duty Steps to correct out-of-control run
Step Description
Quality assurance 1. Reassay When a limit of ±2SDs is
• broader concept used, 5% of expected assay
results fall above or below the
• preanalytical, analytical, and postanalytical variables
limit
• all aspects in the laboratory should be working properly
2. Prepare new control and Controls may deteriorate over
• it makes sure that all the variables and factors in the reassay time when exposed to
laboratory will give the patient a quality type of services, adverse temperatures or
results, etc. subjected to conditions
• WHO: external; organization causing evaporation
• WHEN: yearly 3. Prepare fresh reagents Reagents may have
and reassay evaporated or become
Examples of Components of Quality Assurance contaminated
1. Preanalytical variables: assay selection based on patient 4. Recalibrate instrument Instrument may require repair
indication; implementation of assay selection; patient
identification and preparation; specimen collection Delta check
equipment and technique; specimen transport, preparation, • compares a current analyte result with the result from the
and storage; monitoring of specimen condition most recent previous analysis for the same patient 20%
▪ preparation of: specimen, patient, transport of specimen, deviation
equipment, assays, etc. ▪ investigated for an intervention
2. Analytical variables: laboratory staff competence; assay ▪ analytical error or mislabeled specimen
and instrument selection, assay and instrument validation, • MCV, red cell distribution width (RDW), hemoglobin (HGB),
including linearity, accuracy, precision, analytical platelet count (PLT), PT, INR, and PTT
measurement range (AMR), and specificity; internal quality • when you are checking the previous and present result of the
control; external quality assessment (EQAS) patient
▪ laboratory tests are being done; assay • >20% deviation: investigate if there’s a possible error in
3. Postanalytical variables: accurate transcription and filing machine, reagent, processing, or analytical error
of results, content and format of laboratory report, narrative
report, reference interval (RI) and therapeutic range; External Quality Assessment
timeliness in communicating critical values; patient and • validates the accuracy of hematology and hemostasis
physician satisfaction; turnaround time; cost analysis; assays by comparing results from identical aliquots of
physician application of laboratory results; patient outcome specimens distributed at regular intervals among
▪ file the results, make a narrative report of the results, look laboratories nationwide or worldwide
at the format of the laboratory result, look at the reference • survey or proficiency testing specimens
interval, range, critical values, etc. ▪ aliquots
• target values for the test specimens are established in-house
Internal Quality Control by their manufacturer or distributor
Controls • then further validated by preliminary distribution to a handful
• prepared or purchased assay control of “expert” laboratories
• provide known values and are sampled alongside patient
specimens to accomplish within-run assay validation NEQAS (National External Quality Assessment)
• normal, low or high • requiring laboratories to participate as a condition of
• run at least once per shift licensure.
• within +/-2 SD. • in the Philippines (EQAS in other countries)
 National Reference Laboratory (NRL)
1. Research Institute for Tropical Medicine
a) Parasitology
b) Transfusion Transmissible Infection
c) Bacteriology
2. STD-AIDS Cooperative Central Laboratory
3. East Avenue Medical Center
a) Drinking Water Analysis
b) Drug Testing Laboratory
4. Lung Center of the Philippines for Clinical Chemistry
5. National Kidney and Transplant Institute for Hematology

❖ Every year, laboratories acquire LTO (License to Operate)

from DOH.
INTRODUCTION TO HEMATOPOIESIS AND RBC o erythroblasts oxygenate the embryonic cells
PRODUCTION o primitive erythroblasts already have hemoglobin for
the delivery of oxygen to embryonic tissues (Gower-
Hematopoiesis 1, Gower-2, Portland)
• blood production ▪ surround cavity of yolk sac
• when we talk about blood cells, there’s only 2 steps: o angioblasts are produced and it helps in the formation
▪ production of blood cells of blood vessel
▪ destruction of blood cells o angioblasts are found in the surrounding cavity of yolk
• each of the blood cells in our system has only a certain sac
lifespan: ▪ form blood vessels
▪ RBC: 120 days • primitive but transient yolk sac erythroblasts
• in the production process, there are 4 subsets: ▪ important in early embryogenesis for hemoglobin
• Continuous and regulated process of cell production production
▪ cell renewal formation, development and • occurs intravascularly or it happens inside the blood vessels
▪ cell proliferation specialization of functional (because organs are not yet present)
▪ cell differentiation blood cell (after functional, it • cells from mesoderm also migrate to AGM region
▪ cell maturation will be destroyed)
▪ HSCs (Hematopoietic Stem Cells)
• blood cell kinetics: 4 subsets + destruction of blood cells o produced in the AGM region
• this is happening in the Bone Marrow o pluripotential, totipotent, multipotent (stem cells
▪ all immature cells are found in BM where blood cells originate)
▪ all mature cells are found in peripheral circulation
• Yolk sac → AGM region → fetal liver → Bone Marrow 2. Hepatic Phase
• primary site of production of blood cells: • blood production occurring in the liver
▪ adult: bone marrow • 2nd to 3rd month of fetal development
▪ embryonic stage: Aorta-gonad mesonephros (and yolk • 5-7 week of gestation
sac) • 6th month – will peak
HEMATOPOEISIS (BLOOD CELL PRODUCTION) • 1-2 weeks after birth - will decline
• cells present in this phase:
▪ developing erythroblasts
▪ granulocytes
▪ monocytes
▪ lymphoid cells/lymphocytes ( B cells and T cells)
• hemoglobin type present:
▪ hemoglobin F (predominant)
▪ hemoglobin A (detectable amounts)
• occurs extravascularly (because organs are now present)
• Production of megakaryocytes
• Lymphoid organs:
▪ Thymus
o 1st fully developed organ in fetus
o T cells are produced
▪ Kidney & spleen
o Produce B cells
o B cells are produced
▪ Spleen
1. each cell came from ONE STEM CELL o later part
2. it will be differentiated into committed cells o Decline granulocytic production;
3. maturation / maturing cells; 2 groups: o (replaced by) increases lymphocytic production
▪ immature cells/progenitor/precursor • predominant Hb F and detectable Hgb A
o monocyte, neutrophils, eosinophils, basophils,
• early part: lymphocytes are few, granulocytes are
platelets, RBC, T cell, and B cell
predominant
▪ mature cells
• later part: granulocytes decrease, lymphocytes are now
predominant
Hematopoietic Development
1. Mesoblastic Phase • AGM and yolk sac will disappear during this stage
• when the blood production is occurring in the yolk sac and
3. Myeloid Phase
AGM region
• bone marrow: primary site of cell production
• yolk sac: major site of ell production in the embryonic stage
• starts before the 5th month of fetal development
• 19th day of embryonic development after fertilization
• 24th week of gestation: became the primary site
• primitive erythroblast: blood cell in mesoblastic phase
• BM cavity
• mesoderm to yolk sac
• also known as Medullary hematopoiesis because it happens
▪ central cavity of yolk sac
in the bone marrow cavity
o primitive erythroblasts are produced in the yolk sac
• HCSs and mesenchymal cells migrate into the core of the • y-axis: cellularity/ number of cells
BM • below: development
• bone marrow has Mesenchymal cells 1. in the first few weeks of gestation (in the yolk sac), it will
▪ Type of embryonic tissue decline
▪ usually assisting the bone marrow in production of red 2. production: liver and spleen
cells 3. it will peak in bone marrow (before birth)
o stromal cells: ▪ bone marrows involved:
1. endothelial cells o tibia: infant
2. reticulo-adventitial cells o femur
▪ Differentiate into structural elements that support o rib
developing blood cells o sternum major sites of blood
o vertebrae production: 50, 60 years old
4. Medullary Phase 4. maturation stages
• blood production in the bone
marrow (adult) Blood Cells
• primary site: 24th week of gestation, Stem Cell Theory
wherein the bm become the primary • cell stages of development
site of hematopoiesis ▪ stem cell
• Myeloid-to-erythroid ratio: 4:1 or 1. will actually come from stem
3:1 cell
▪ myeloid: WBC & Platelet 2. stem cell will differentiate
precursors (immature) 3. formation of committed stem
▪ erythroid: RBC precursors cells
• End of the 24 weeks of gestation 4. maturation into maturing cells
▪ BM becomes the 1° of (immature and mature)
hematopoiesis 5. mature cells functions

❖ young: not producing blood cells; 1-5 years old all bones
that has bm are capable of producing blood cells
❖ adult: restraint into several areas

• primary site of bone marrow/medullary phase: Flat bones Hematopoietic stem cells (HSCs)
▪ tibia (infant) • foundation of the adult hematopoietic system
▪ pelvic area, sternum, vertebra (adult) • embryo produces the first adult repopulating HSCs
• Hb F and Hb A (Hb F < Hb A)
• EPO (erythropoietin) Types of Human Stem Cells
regulators of hematopoiesis
• G-CSF, GM-CSF 1. Totipotential stem cells
▪ Granulocyte-colony stimulating factor • first stem cell that is developed upon fertilization
▪ Granulocyte-macrophage-colony stimulating factor • presence: 1st few hours after an ovum is fertilized
• most versatile type of stem cell
❖ regulators are also theoretically seen in hepatic phase, but • develop into any human cell type (from embryo into fetus)
they are not detectable (few amount) • produces pluripotential that will later on help them in
❖ regulators are detected in the medullary phase producing other cells
• stem cells in young individual
Cell Population in the Bone Marrow
Myeloid Lymphoid 2. Pluripotential stem cells
RBC precursors Lymphocytic precursors • presence : days after fertilization
WBC precursors • develop into any cell type, EXCEPT cannot develop into a
Platelet precursors fetus
(megakaryocyte) • usually, it can only produce blood cells
HEMATOPOIETIC DEVELOPMENT • stem cells in young individual

3. Multipotential stem cells

• major stem cell responsible for the formation of blood cells
and other cells
• from pluripotent stem cells
• found in adults, but limited to specific types of cells to form
tissues
• bone marrow stem cells can produce all types of blood cells,
bone cartilage, and adipose (fat) cells
• a fetus
• stem cells in adults: totipotential, pluripotential, multipotential
Bone Marrow ▪ decreases with age
• where all blood cells are produced ▪ red will be decreased and converted into yellow
• found within the cavities of all bones due to resorption of
cartilage and endosteal bone
• trabeculae
▪ radiate out from the bone cortex into the central space
▪ provide structural support for the developing blood cells
▪ supports red marrow or hematopoietic tissue (active
tissue that produces blood cells)
▪ found in spongy bone; mesh
• 2 forms:
▪ Yellow marrow
o normally inactive and composed mostly of fat tissue
o with undifferentiated mesenchymal cells and
macrophages
o full of adipocytes
o can still be activated even in adults if there’s a
demand (ex: if you have anemia)
▪ Red marrow
o normally active in the production of developing blood
cells and their progenitors
o hematopoietic tissue (active tissue that produces • how does the mature cells release from the bone marrow into
blood cells) the peripheral circulation? (refer to the illustration above)
o then later on, active marrow will become yellow ▪ from the cords, it will cross through the sinuses going to
marrow the circulation
o also has adipocytes but in minimal amount ▪ in the bone marrow (trabeculae: mesh-like), one area will
be enlarged
▪ once it is enlarged, you will see that it is surrounded by
hematopoietic cord or tissue (color red)
▪ inside of it, there is sinus, then at the center, there is
artery
▪ in order for the cell to reach the peripheral circulation, it
first needs to reach the central artery
▪ the hematopoietic tissue also contains adipose tissue
▪ the cells inside are in different population (immature
RBC, WBC, megakaryocyte, lymphoid tissues)
▪ there are 2 kinds of cells in between the sinus and
hematopoietic tissue
o adventitial cell: also called as stromal cells
o endothelial cell
▪ hematopoietic cells will pass through the adventitial and
• one of the body’s largest organs endothelial cells
• approx. 3.5% to 6% of total body weight ▪ once the cells reached the sinus, they will now go to the
• average around 1,500 g/1.5 kg in adults central artery
• consists of hematopoietic cells ▪ once they reach the central artery, they will finally reach
▪ erythroid, myeloid, lymphoid, and megakaryocyte the peripheral circulation
▪ fat (adipose) tissue
▪ osteoblasts and osteoclasts
Bone Marrow
▪ stromal (mesenchymal cells – assists blood production) (immature cells/precursor/progenitors)
• hematopoietic cell colonies are compartmentalized in red
marrow
Hematopoietic Tissue
• cords → cross the walls of sinuses → circulation (immature cells/precursor/progenitors)
• First years of life: red and cellular
• 5 and 7 years of age: yellow Central Sinus
marrow (retrogression –
conversion of red marrow to
Adventitial and Endothelial Cells
yellow marrow)
▪ Active marrow restricted in:
o Proximal ends of long Central Artery
bones
o Vertebrae, ribs, sternum, Peripheral Circulation
skull bones, pelvis (mature cells)
• marrow cellularity
▪ ratio of red to yellow
Stromal Cells
• endothelial cells, adipocytes macrophages and
lymphocytes, osteoblasts, osteoclasts, and reticular
adventitial cells

endothelial cells
• broad, flat cells
• where blood cells pass through inside or outside the
hematopoietic tissue (papasok: nutrients; lalabas: mature
cells)
• regulate the flow of particles entering and leaving
hematopoietic spaces in the vascular sinuses
• secrete cytokines (regulator)

adipocytes
• large cells with a single fat vacuole
• regulating the volume of active marrow
• secrete cytokines or growth factors
• once the red marrow becomes yellow marrow, adipocytes LOCATIONS OF DEVELOPING CELLS:
will multiply or increase 1. Erythroblasts
• purpose: to control the volume of red marrow • small clusters throughout the red marrow
• decreased when it is needed by red marrow • mature forms: adjacent to the outer surfaces of the vascular
• increased when it is not needed by red marrow because it sinuses
will now turn into yellow marrow • locations of developing cells:
• release regulators such as cytokines and growth factors
2. Megakaryocytes
• yellow marrow can still be activated even in adults if there’s • adjacent to the outer surfaces of the vascular sinuses
a demand (ex: if you have anemia), adipocytes will decrease • release of platelets into the lumen of the sinus
to have space for hematopoietic tissue • located beside the adventitial cells, near the outer surface
because it will just release the platelets
macrophages
• phagocytosis 3. Immature myeloid cells
• secrete cytokines • metamyelocyte
• found deep within the cords but will also be released once
osteoblasts they mature
• bone-forming cells
Extramedullary hematopoiesis
osteoclasts • adult individual; not normal to produce blood cells in spleen,
• bone-resorbing cells liver, and lymph nodes
• Abnormal
reticular adventitial cells • When spleen, liver and lymph nodes revert back to produce
• secretes cytokines immature blood cells
• incomplete layer of cells in vascular sinuses • enlargement of the spleen and liver
• form a framework or supporting lattice for the developing ▪ they get enlarge because they don’t usually produce
hematopoietic cells blood cells, not their function
• endothelial cells will be the passageway going outside the ▪ they compensate by producing cells
hematopoietic tissue
Stem Cell Theory of Hematopoiesis
regulation of hematopoietic stem and progenitor cell • All cells are derived from a pool of stem cells that are self-
survival and differentiation renewing
• Pluripotential & multipotential stem cells give rise to
Red marrow committed stem cells for each cell line
• compose of developing cells in extravascular cords • Committed stem cells have receptors for specific growth
▪ located in spaces between vascular sinuses factors
▪ supported by trabeculae • Respond to stimulation by division & maturation (precursor
▪ separated from the lumen of the vascular sinuses by cell stages) into end-stage cells
endothelial and reticular adventitial cells
**fx: function; mature cells perform the function

Theories describing the origin of HSCs:

1. Monophyletic theory
• cells are derived from a single progenitor stem cell called a
pluripotent hematopoietic stem cell
• isa lang ang pinanggalingan
• we adapt this theory
• this theory is usually followed because the cells are from a
single stem cell, which is the pluripotent, multipotent, or
totipotent (any of them)
• from a single stem cell, it will be differentiated either myeloid
or lymphoid

2. Polyphyletic theory
• each of the blood cell lineages is derived from its own unique
stem cell
• marami ang pinanggalingan

Theories describing the fate of HSCs:

1. Stochastic model of hematopoiesis
• random process whereby the HSC randomly commits to self-
renewal (continuous production of stem cell) or
differentiation (choice: to renew or to differentiate)
• we adapt this theory

2. Instructive model of hematopoiesis

• microenvironment in the bone marrow determines whether
the HSC will self-renew or differentiate
• we adapt this theory
❖ there are different regulators in every lineage
3 phases of HSC according to cell maturity:
1. Primitive, multipotential/pluripotential cells Regulators
• most immature group capable of self-renewal and • it helps to know whether a cell will go to lymphoid or myeloid
differentiation into all blood cell lines stem cell
• influences the differentiation and maturation
2. Intermediate cells/Committed cells ▪ ex: EPO (erythropoietin) is the regulator for the erythroid
• consists of committed progenitor cells destined to develop lineage, that’s why we have RBCs because EPO
into distinct cell lines influences the stem cells to become red cell
▪ without EPO, there’s no red cell
3. Mature cells • Cytokines & Growth factors
• most developed group with specific functions ▪ regulate the proliferation, differentiation, and maturation
of hematopoietic precursor cells
Stem Cell Theory of Hematopoiesis
Cytokines
• soluble proteins that have direct and indirect effects on
hematopoietic cells
• include interleukins (ILs), lymphokines, monokines,
interferons, chemokines, and colony-stimulating factors
(CSFs)
• stimulation (+); KIT ligand, FLT3 ligand, GM-CSF, IL-1, IL-3,
IL-6, and IL-11) or inhibition (-); transforming growth factor-
beta, TNF-alpha, & interferons ) of production,
 differentiation, and trafficking of mature blood cells and their
precursors Name Source Target Blood Function
• positive (+): promoter Cell
IL-1 B cells, monocytes, T helper cells Costimulation
• negative (-): inhibitor dendritic cells B cells Maturation and
• process is controlled Appears to influence proliferation
different progenitor Activation
Growth factors cells indirectly in NK cells Inflammation
hematopoiesis. It Macrophages
• regulating the proliferation and differentiation of HSCs may act in synergy
• regulating the survival and function of mature blood cells with IL-3, M-CSF,
• specific binding to receptors on the surface of target cells G-CSF, and GM-
• Chromosome 7 (responsible for production of:) CSF to stimulate
cells
▪ Gene for EPO IL-2 Th1 cells Activated T The influences
• Long arm of chromosome 5 (responsible for production of:) cells, and B and regulation of
▪ GM-CSF cells, NK T cells, B cells,
▪ IL-3 cells, NK cells, and
macrophages monocytes. It
▪ M-CSF
acts on activated
• Chromosome 17 (responsible for production of:) B cells as a
▪ G-CSF (Granulocyte Colony Stimulating Factor) growth and
differentiation
REGULATORS: factor
IL-3 Activated Th3 cells, Hematopoietic Promotes the
Growth Cellular Progenitor Mature Cell
mast cells, NK cells, stem cells growth of early
Factor Source Cell Target Target
endothelium, hematopoietic
Erythropoietin Peritubular CFU-E, late None
eosinophils cell lines (e.g.,
cells of the BFU-E, CFU-
proliferation of
kidney, Kupffer Meg
CFU-GEMM,
cells
CFU-M, CFU-
IL-3 Activated T CFU-blast, Eosinophils, Meg, CFU-Eo,
lymphocytes CFU-GEMM, monocytes and CFU-Bs
CFU-GM, CFU- colonies from
G, CFU-M, bone marrow,
CFU-Eo, CFU- IL-3 acts with M-
Meg, CFU- CSF to stimulate
Baso, CFU-E proliferation of
G-CSF Monocytes, CFU-G Granulocytes monocytes and
fibroblasts, macrophages. It
endothelial also stimulates
cells granulocyte,
M-CSF Monocytes, CFU-M Monocytes monocyte,
fibroblasts, eosinophil, and
endothelial mast cell
cells production.
GM-CSF T lymphocytes, CFU-blast, granulocytes Mast cells Growth and
monocytes, CFU-GEMM, histamine
eosinophils, CFU-GM, CFU- release
monocytes, G, CFU-M, IL-4 Th2 cells, just Activated B Proliferation and
fibroblasts, CFU-Eo, CFU- activated naive CD4 cells differentiation,
endothelial Meg, CFU-E cell, memory CD4 IgG1 and IgE
cells cells, mast cells, synthesis
macrophages.
Colony-Stimulating Factors Interacts with G-
• high specificity for their target cells CSF to proliferate
myeloid progenitor
• active at low concentration cells
• names indicate the predominant cell lines that respond to T cells Proliferation
their presence IL-5 Th2 cells, mast Eosinophils Stimulates
• G-CSF: granulocytic cell line cells, eosinophils eosinophil
colony
• GM-CSF: granulocytic-monocytic cell line production and
• GM-CSF + IL3: megakaryocyte cell line interacts with
GM-CSF and IL-
Interleukins 3 in eosinophil
induction
• protein molecules work in conjunction w/ hematopoietic
B cells Differentiation,
growth factors IgA production
• to stimulate proliferation and differentiation of specific cell IL-6 Macrophages, Th2 Activated B Differentiation
lines cells, B cells, cells into plasma cells
• cytokines that act independently or in conjunction with other astrocytes and
endothelium
interleukins to encourage hematopoietic growth Plasma cells Antibody
• cell signaling molecules secretion
• first described as signals for communication between HSCs Differentiation
(inter—between) white blood cells (leuk— from leukocytes)
 T cells, others Induces acute IL-15 Mononuclear T cells, Induces
phase reaction, phagocytes (and activated B production of NK
hematopoiesis, some other cells), cells cells
differentiation, especially
inflammation macrophages
IL-7 Bone marrow Pre/pro-B cell, Differentiation following infection
stromal cells and pre/pro-T cell, and proliferation by viruses
thymus stromal cells NK cells of lymphoid IL-16 Lymphocytes, CD4 T cells CD4
progenitor cells, epithelial cells, (Th cells) chemoattractant,
involved in B, T, eosinophils, CD8 T increases the
and NK cell cells mobility of CD4
survival, T cells
development, IL-17 T helper 17 cells Epithelium, ↑ Inflammatory
and (Th17) endothelium, cytokines
homeostasis, ↑ other
proinflammatory IL-18 Macrophages acts Th1 cells, NK Induces
cytokines as a synergist with cells production of
IL-8 Macrophages, Neutrophils, An inflammatory IL-12 in some of its IFN-γ, ↑NK cell
lymphocytes, basophils, cytokine that is effects activity
epithelial cells, lymphocytes chemotactic for IL-19 - Regulates the
endothelial cells both neutrophils function of
and T cells. It is macrophages,
a potent suppresses the
stimulator of activities of Th1
neutrophils, and and Th2
it activates the IL-20 Biological activities Regulates
respiratory burst similar to IL-10 proliferation and
and the release differentiation of
of both specific keratinocytes
and azurophilic IL-21 Activated T helper All Co-stimulates
granular cells, NKT cells lymphocytes, activation and
contents dendritic cells proliferation of
IL-9 Th2 cells, T cells, B cells Acts as a potent CD8 T cells;
specifically by CD4 CD4 T augments NK
helper cells lymphocyte cytotoxicity;
growth factor. In augments
addition, it has CD40-driven B
been cell proliferation,
demonstrated to differentiation,
support growth and isotype
of BFU-E switching
IL-10 Monocytes, Th2 Macrophages Cytokine promotes
cells, CD8, T cells, production differentiation of
mast cells, B cells Activation Th17 cells
macrophages, B cell Mast cells IL-22 Similar to IL-10 Activates STAT1
subset Th1 cells inhibits Th1 and STAT3 and
cytokine increases
production (IIFN- production of
γ, TNF-β, IL-2) acute phase
Stimulation proteins such as
Th2 cells serum amyloid
IL-11 Bone marrow Bone marrow Multifunctional A, Alpha 1-
stroma stroma regulator of antichymotrypsin
hematopoiesis and haptoglobin
IL-12 Dendritic cells, B Activated T Differentiation in hepatoma cell
cells, T cells, cells into cytotoxic T lines
macrophages cells with IL-2, ↑ IL-23 - Increases
IFN-γ, TNF-α, ↓ angiogenesis
IL-10 but reduces CD8
NK cells ↑ IFN-γ, TNF-α T cell infiltration
IL-13 Activated Th2 cells, Th2 cells, B Stimulates Acts as a
mast cells, NK cells cells, growth and stimulant on
macrophages differentiation of particular
B cells (IgE), populations of
inhibits Th1 cells memory T cells
and the IL-24 - Pays important
production of roles in tumor
macrophage suppression,
inflammatory wound healing,
cytokines (e.g., and psoriasis by
IL-1, IL-6), ↓ IL- influencing cell
8, IL-10, IL-12 survival
IL-14 T cells and certain Activated B Induces growth IL-25 - Supports
malignant B cells cells and proliferation proliferation of
of B cells, cells in the
inhibits Ig lymphoid lineage
secretion
 Induces the
production of IL-
4, IL-5, and IL-
13, which RBC Stages of maturation
stimulate
eosinophil
expansion
IL-26 - Enhances
secretion of IL-
10 and IL-8 and
cell surface 2. Nuclear-cytoplasmic ratio
expression of
CD54 on
• amount of space occupied by the nucleus in relationship to
epithelial cells the space occupied by the cytoplasm
IL-27 - Regulates the • size of nucleus generally decreases as a cell matures
activity of B • N:C ratio decreases
lymphocytes and 𝑁𝑢𝑐𝑙𝑒𝑎𝑟 𝑠𝑝𝑎𝑐𝑒
T lymphocytes • = volume or ratio will be decreased
𝐶𝑦𝑡𝑜𝑝𝑙𝑎𝑠𝑚𝑖𝑐 𝑠𝑝𝑎𝑐𝑒
IL-28 - Plays a role in ▪ decreased because the denominator increases than the
immune defense
against viruses
numerator
IL-29 - Plays a role in • as the cell matures, nuclear space decreases and
host defenses cytoplasmic space increases
against
microbes Features for Nuclear ID
IL-30 - Forms one chain
of IL-27 1. chromatin pattern
IL-31 - May play a role 2. nuclear shape
in inflammation 3. presence of nucleoli
of the skin
IL-32 - Induces Features for Cytoplasmic ID
monocytes and
macrophages to
1. staining color and intensity
secrete TNF-α, 2. granulation
IL-8, and CXCL2 3. shape
IL-33 - Induces helper T 4. quantity of cytoplasm
cells to produce 5. vacuolization
type 2 cytokine when present, it is abnormal
6. inclusion bodies
IL-35 Regulatory T cells Suppression of
T helper cell
activation Features for Nuclear ID
1. chromatin pattern
MARROW DIFFERENTIAL: • most distinctive nuclear feature of a cell in terms of maturity
Cell Type Range (%) Hierarchy and cell type recognition
Erythroblasts 18-24 2nd • Lymphocytes
Myeloblasts Type I 0-1 7th
▪ smooth or homogeneous pattern of chromatin throughout
Myeloblasts Type I 0-2 6th
Promyelocytes 1-4 4th development
PMN’s and precursors 53-63 1st • Granulocytes
Monocytes 0-2 6th ▪ fine to a highly clumped pattern
Eosinophils and 1-3 5th • Monocytes
precursors ▪ lacy pattern, which becomes finer as the cell matures
Basophils and 0-1 7th
precursors • Erythrocytes
Lymphocytes 8-12 3rd ▪ more clumped pattern as maturation progresses, until the
Plasma cells 0-2 6th extremely dense nucleus is lost from the mature cell

• most to least number in peripheral blood differential: 2. nuclear shape

1. neutrophil • round or oval and slightly folded nuclear shape
2. lymphocyte • specific to every cell
3. monocyte • Lymphocytes
4. eosinophil ▪ continue to have a round or oval nucleus
5. basophil • Monocytes
▪ kidney bean–shaped nucleus, but folded or horseshoe
General Cell Characteristics shapes are common
1. Overall cell size • Neutrophils, eosinophils, and basophils (granulocytes)
• compared with the size of a mature erythrocyte ▪ segmented nuclei attached to one another by fine
• erythrocytes and leukocytes filaments with 2-5 depending on the cell type.
▪ as the cell matures, the cell size decreases
• megakaryocytes 3. presence of nucleoli
▪ as the cell matures, the cell size increases • as the cell matures, nucleoli disappear
• erythrocytes, leukocytes, and megakaryocytes
 ▪ all have nucleoli in the earliest cell stages o megakaryocytes
▪ as cells mature, nucleoli are usually not visible
• changes in appearance of nucleoli are proportional to the 4. quantity of cytoplasm
rate of synthesis of ribosomal RNA • actual quantity of cytoplasm increases with age
▪ means that when the nucleoli decrease, RNA synthesis • megakaryocyte
decreases as well ▪ extensive quantities of cytoplasm
▪ as the cell matures (if the nucleoli disappear), RNA ▪ has most cytoplasmic space
synthesis will decrease because they are being replaced • lymphocytes
by protein synthesis ▪ abnormalities frequently
• number of nucleoli varies depending on the cell type: ▪ has least cytoplasmic space
• Lymphoblasts
▪ 1 or 2 nucleoli 5. vacuolization
• Myeloblasts • it is abnormal or not normal to have vacuolization in cells, but
▪ 1 to 5 nucleoli there is an exemption: monocytes, cells are exposed to
• Monoblasts anticoagulants for a longer-than-acceptable period, certain
▪ 1 or 2 nucleoli but occasionally may have 3 or 4 conditions, and older cells
• Erythroblasts • Monocytes
▪ not have any nucleoli or may have up to 2 nucleoli that ▪ having vacuoles throughout their life cycle and under
may stain darker than in other types of blast cells normal conditions
• Megakaryoblasts ▪ commonly seen in older cells and in abnormal conditions.
▪ typically have 1 to 5 nucleoli • Anticoagulants
▪ produce vacuoles as artifacts if the blood is stored for a
Features for Cytoplasmic ID longer-than-acceptable period
1. staining color and intensity ▪ blood cells are >10 days stored in the anticoagulant
• staining color: dark to light as the cell matures • Severe bacterial infections, viral infections and malignancies
• the darker the color/stain, it is an indicative of active RNA ▪ produce a remarkable number of vacuoles in various
synthesis and protein synthesis leukocyte types
• in a Wright-stained blood smear
• vary with cell maturity and type 6. inclusion bodies
• from darker blue in younger cells to lighter blue or pink in • still abnormal
mature cells • indicative of specific diseases
• Immature erythrocytes • monocytes have Auer bodies or Auer rods, specifically the
▪ very distinctive dark-blue cytoplasm that becomes paler monoblast (but it is NORMAL)
and gray looking as the cell synthesizes hemoglobin • myelocytic or monocytic blast forms
• Lymphocytes • erythrocytic inclusions and leukocytic inclusions
▪ pale sky-blue cytoplasmic color • Wright-stained blood smear and special stains

2. granulation Erythropoiesis
• presence, size, and color of granules are important in cellular • process of erythrocyte production
identification 1. stem cell
• (1) no granules---(2) nonspecific granulation---(3) specific ▪ pluripotent stem cell
granulation ▪ multipotent stem cell
• blast forms of leukocytes and megakaryocytes 2. committed
• Erythrocytes ▪ myeloid stem cell
▪ never exhibit granulation throughout life cycle ▪ CFU-GEMM
• Granulocytes ▪ BFU-E
▪ distinctive granulation ▪ CFU-E
▪ Variation 3. maturing
a. In size: fine, coarse ▪ rubriblast
b. In color: red (azurophilic) ; blue (basophilic) ; orange ▪ prorubricyte immature cells
(eosinophilic) ▪ rubricyte
c. In the amount of granulation per cell ▪ metarubricyte
immature cells
▪ reticulocyte
3. cytoplasmic shape ▪ mature erythrocyte
• all cell has regular outline (round) EXCEPT megakaryocyte **stem cell to committed are unrecognizable under the
and monocyte (irregular outlines) microscope (cannot be differentiated one-by-one; they are
similar to each other)
• useful in cellular identification
**maturing has recognizable stage
• Pseudopods
▪ mature monocytes and in some leukocyte blast forms
• differentiation from the HSC through the mature erythrocyte
• Megakaryocyte
• potential to differentiate into lymphoid or other hematopoietic
▪ develops a more irregular outline as the cell matures
cell types is restricted
o blast forms
• Site according to maturational stage:
o monocytes
 ▪ Yolk sac (mesoblastic)—extramedullary organs (liver)— • the number of cells at each stage before the
red bone marrow (medullary) polychromatophilic erythroblast stage > at each preceding
• erythropoiesis: stage
▪ production—peripheral circulation (for 120 days)— ▪ it means that while the cell matures, our cell number
destruction—erythropoiesis (again) increases because these cells proliferate until
• mature erythrocyte metarubricyte
▪ biconcave disc with a central pallor ▪ kung ano yung number ng metarubricyte, yun din yung
• Hemoglobin roughly number ng magma-mature na erythrocyte
▪ Respiratory protein ▪ rubriblast > prorubricyte > rubricyte > metarubricyte
▪ Heme protein o mas marami si metarubricyte kaysa rubricyte dahil
• lifespan: 120 days nagdi-divide
• nutrients needed by erythropoiesis: amino acids, iron, ▪ the succeeding cells (erythrocyte, reticulocyte) has the
vitamin B12, vitamin B6, folic acid and the trace minerals same quantity
(cobalt, nickel) • after polychromatophilic erythroblast stage, erythroid cells
• 200 billion erythrocytes = >20 mg of elemental iron is needed do not divide
▪ 2 sources of iron: • undergo specialized maturation
o majority will come from the recycled iron from • increased erythrocyte production
old/senescent RBCs • hemoglobin synthesis
o minor will come from the diet (absorbed by intestine)
; we need 1-2 mg/day (from the diet) Early Cells/Committed

Erythropoietin (EPO)
• regulator of erythropoiesis
• EPO acts once we need RBCs
• Site of production: peritubular cells of kidneys
▪ Liver – can also produce 10-15% of EPO ; primary site
for EPO production of infants/unborn
• Characteristics:
▪ Can cross the placenta
▪ 1st human hematopoietic growth factor
▪ Detected in myeloid phase • BFU-E
▪ EPO blood level is inversely proportional to the tissue ▪ earliest cell in erythrocyte series
oxygenation (low oxygen level = high EPO ; high O2 level ▪ first to be committed in the erythroid lineage
= low EPO) ▪ gives rise to large colonies of cells (thousands)
▪ 20 mU/mL /day (can be up to 20,000 mU/mL in case of o those colonies of cells become CFU-E. But they do
anemia) not actively proliferate that’s why when they
• heme protein proliferate, they make many quantity (the reason why
▪ involved in the oxygen-sensing mechanism the produced colonies are large)
• it is working with committed erythroid cells (means from ▪ becomes CFU-E within 1 week
CFU, BFU-E, CFU-E, until the development of maturing cells • CFU-E
; they undergo mitosis until metarubricyte) ▪ second
• predominant effect on the committed erythroid cells ▪ actively proliferating
• promote proliferation and differentiation of CFU-E ▪ give rise to small colonies of cells (100 cells)
▪ usually found in the S-phase of the cell cycle (because
• stimulate the differentiation of BFU-E to becoming CFU
they are actively proliferating)
• prevents erythroid cell apoptosis or cell death
• 6-7 days: maturation period from pronormoblast/rubriblast to
• basic components to prevent apoptosis:
erythrocyte
▪ Lipoxygenase
• 18-21 days: maturation period from BFU-E to erythrocyte
o important in regulating the degradation of internal
organelles
Erythrocyte Maturation & Development
▪ Bcl-x
• after CFU-E, pronormoblast follows
o anti-apoptotic protein
• EPO interacts with IL-3, GMCSF, IL-1 and MEG-CSF
• there is an increase in the production of several types of RNA
▪ means that EPO synthesizes organelles in order to
differentiate every cell from one another
▪ there are some changes in cell characteristics
• increase in DNA activity
• protein synthesis
• increased RNA and DNA activity = increased protein
synthesis = increased cellular organelles
▪ when cellular organelle increases, there is differentiation
of every cell
• erythrocyte matures rapidly ▪ acidophilic

1. Rubriblast 5. Reticulocyte
• earliest recognizable cell in • no nucleus
erythroid lineage • BM = PC
• largest (that’s why it is also called • Reticular appearance caused by
as “mother cell”) remaining RNA
• 12 to 19 um • remnants of RNA can be viewed by
• N:C ratio is 4:1 supravital stain
• Nucleus • (+) Supravital stain
▪ Large, round and contains 0- ▪ new methylene blue
2 nucleoli • Polychromatophilia
▪ dark appearing has fine ▪ Retics with high amount of
chromatin pattern RNA residual
• Cytoplasm ▪ blue appearance in Wright’s stain remaining RNA
▪ color: blue (it means that the RNA activity is high which • 7 to 10 um
is needed in order for us to produce Hb (protein)) • Anuclear
• most iron for hemoglobin synthesis is taken into the cell • Mitochondria and ribosomes are still present
• cytoplasm is pink because it already has Hgb
2. Prorubricyte • Reticulocytes
• 12 to 17 um ▪ Bone marrow: in BM, it will become mature erythrocytes
• N:C ratio is 4:1 for 2.5 days
• Nucleus ▪ Peripheral circulation: in PM, it will mature to
▪ chromatin becomes more erythrocyte within 1 day
clumped • Transition/Marker from reticulocyte to erythrocyte
• Cytoplasm 1. loss of mitochondria and ribosome complete
▪ has lighter stain 2. full hemoglobinization transition
▪ stains a distinctive blue color in
Wright stain **complete transition means you already have mature
• no evidence of the pink color erythrocyte
• RNA activity is still continuing
• smaller than rubriblast 6. Mature Erythrocyte
• still no Hb • 6-8 um
• Radioactice chromium (51Cr)
3. Rubricyte ▪ Determine survivavility
• 11 to 15 um • lifespan: 120 days
• N:C ratio is 1:1 • central pallor of 1 to 3 μm (1/3)
• Nucleus • no remnants
▪ increasingly clumped • has hemoglobin
• Cytoplasm
▪ variable amounts of pink
coloration mixed with basophilia
▪ muddy, light gray appearance
• Hb development starts
• Hgb appears for the first time

4. Metarubricyte
• also known as “nucleated RBC”
▪ because this is the last stage
where nucleus is still present
• last stage capable of mitosis (mitosis
is from BFU-E to metarubricyte)
• limited mitosis (up to 3 mitosis)
• 8 to 12 um
• N:C ratio is 1:1
• Nucleus
▪ chromatin pattern is tightly
condensed
▪ nucleus will be extruded from the cell
• last cell capable of proliferation
• cooling cell that is capable to divide
• Cytoplasm
RBC STRUCTURE, PHYSIOLOGY, METABOLISM AND exocytosis and endocytosis
DESTRUCTION oxidative metabolism
capability to metabolize fatty acids and amino acids
Erythron model
▪ from production to destruction Mature Red Blood Cells
▪ organs involved in terms of production and destruction • no nucleus or cytoplasmic organelles (ribosome and
mitochondria)
❖ RBCs do not have mitochondria, means that they don’t have
oxidative metabolism (refers to: in order for us to generate • limited in metabolic activity
ATP/energy) ▪ metabolism of FA and AA (due to loss of ribosome)
❖ RBCs are deformable because of phospholipids ▪ oxidative metabolism (due to loss of mitochondria)
❖ how do RBC produce energy? • Erythrocyte glycolysis (Anaerobic glycolysis)
▪ it will generate ATP by the process of EMP (it is ▪ source of energy through breakdown of glucose
glycolysis) ▪ we use glucose as source of energy
o glucose is converted into lactate to have 2 molecules
The Life Cycle of a Red Blood Cell of ATP (from EMP pathway)
a. Kidneys respond to a lower-than-normal oxygen ▪ major pathway: EMP (Embden-Meyerhof Pathway)
concentration in the blood by releasing the hormone ▪ other supplementary pathways: (bypass)
erythropoietin. o HMS (Hexose Monophosphate Shunt)
b. Erythropoietin travels to the red bone marrow and stimulates o MHR (Methemoglobin Reductase Pathway)
an increase in the production of red blood cells (RBCs). o LRP (Luebering-Rapoport Pathway)
c. The red bone marrow manufactures RBCs from stem cells • Hemoglobin
that live inside the marrow. ▪ Main cell component
d. RBCs squeeze through blood vessel membranes to enter • Membrane
the circulation. ▪ survival for 120 days in circulation
e. The heart and lungs work to supply continuous movement
• diameter: 6-8 µm
and oxygenation of RBCs.
• volume of RBC: 80-100 fL
f. Damaged or old RBCs are destroyed primarily by the spleen.
• average surface area: 140 µm
• Total volume of blood in the body: 5 L
Reticulocyte
• no nucleus
I. Shape
• they are produced in the erythroblastic island
Biconcave
▪ erythroblastic island are small clusters
❖ RBCs are widely distributed in the bone marrow in • facilitates O2-CO2 transport function
small clusters. Those RBCs are surrounding a central o biconcave shape is easier to be deformed because not
macrophage. Every cluster is called erythroblastic all blood vessels in our body has the same size (some
island. blood vessel has 2 µm, usually the capillaries)
❖ seen in the bone marrow, peripheral circulation • maximize the ratio of the surface area to volume
• Membrane Composition and Characteristics: • allows cell flexibility (RBC deformability)
▪ in erythroblastic island surrounding a central • allows cell to adjust to small vessels and still maintain cell
macrophage viability
▪ BM (bone marrow) and PC (peripheral circulation) • Alteration in ratio is NOT POSSIBLE due to surface area to
▪ reticulocytes have tubulin and actin volume ratio. If there is an alteration in ratio:
o important during terminal erythroid differentiation in ▪ RBCs will be less deformable → prone to lysis and
terms of cell division and cell motility fragmentation
o tubulin and actin are very important for terminal ▪ no deformation happens when: spheroid shape
differentiation for its motility and change its organelles o if it undergoes deformation, the surface area and
▪ Changes: volume will decrease
o Increase in shear resistance of RBCs because of o due to:
membrane that was developed by erythrocytes ➢ membrane loss due to fragmentation →
o Loss of surface area due to loss of membrane decreased surface area
lipid ; erythrocyte became smaller ➢ increased uptake of cations and H2O →
o Acquisition of a biconcave shape; biconcave increased volume
shape is also contributing to the shear resistance of ➔ ex. Na+ K+ : potassium inside, sodium outside
RBCs (PISO)
o Loss of mitochondria and ribosome that’s why ➔ water will go to sodium (partner)
there is a decrease in the metabolic activity; ➔ cation uptake increases means that sodium
➢ mitochondria: loss of oxidative metabolism goes inside as well as the water hence,
(aerobic process) volume increases hence, there’s alteration in
➢ ribosome: loss of capability to metabolize fatty ratio of surface area to volume; volume
acids and amino acids increases
o Loss of tubulin and actin that’s why erythrocytes ➔ altered ratio means less deformable and once
don’t have capabilities to endocytosis and exocytosis it is less deformable, there will be lysis

RBCs cannot do:

II. Membrane Composition and Structure ❖ Eryptosis: RBC death
• composition:
▪ proteins: 50-52% ▪ distribution is energy dependent
▪ lipids: 40% o flippase, floppase, scramblase (membrane-
▪ carbohydrates: 8-10% (glycoprotein, glycolipids) associated enzymes)
➢ these enzyme needs energy
❖ each composition has their own function: ➢ in the membrane of RBC, wherever the most
➔ carbohydrates appear as glycoproteins enzymes are located, you will also see there
(carbohydrate + protein) and glycolipids the energy distribution because enzymes
(carbohydrate + lipids) need energy
▪ disrupted distribution
• allows to function o conditions (thalassemia or sickle cell anemia)
▪ separate IC fluid env. of cytoplasm from the EC fluid env. o aging RBCs
of the plasma
o membrane will separate intracellular to extracellular 2. Cholesterol
barrier ▪ proportion: 40%
▪ allow nutrient and ion passage selectively into and out of ▪ has two types:
the cell o esterified: 10% proportion
o responsible for passage of ions and nutrients (from o unsterified: 30% proportion
outside to inside and vice versa) ▪ responsible for elasticity and fluidity
▪ allow the cell to deform when required ▪ it will not be destroyed even if it gets deformed
• Lipids and Proteins ▪ regulates membrane fluidity
▪ asymmetric arrangement (uneven distribution) from ▪ regulates membrane permeability to electrolytes and
cytoplasmic side to membrane interior or from plasma non-electrolytes
side (reason why the passage is selective; not all can ▪ it also maintains the surface area to volume ratio
come inside and outside) ▪ confers tensile strength (elasticity) to the lipid bilayer
▪ allows selective passage of mol. into and out of the cell ▪ 98% of membrane: unesterified
▪ 70% of plasma: esterified
A. Lipids
• equal proportions of phospholipids & unesterified cholesterol B. Proteins
1. Phospholipids • Bound to lipids throughout the membrane
▪ proportion: 60% • acts as receptor for various molecules & skeletal structure
▪ responsible for barrier, fluidity or flexibility 1. Peripheral protein
▪ arranged in bilayer lipid ▪ also known as cytoskeletal protein
o phospholipid polar groups ▪ important for the skeletal structure of RBC
allows ▪ the reason why it goes back to its normal size and
➢ faces toward water content
membrane
➢ hydrophilic shape (from deformation)
to act as
➢ head liquid
▪ located underneath or underlying the lipid bilayer of
o non polar fatty acids sealer RBC (inside the cell particularly cytoplasm)
➢ not facing towards aqueous environment ▪ it also regulates the shape and deformability
➢ hydrophobic ▪ similar with cholesterol, it provides elastic property of
➢ tail RBC
▪ fluid because FA are free to move laterally w/in the ▪ contain 2 skeletal proteins
membrane, allowing lipids to interact with other lipids o spectrin
• underlie the lipid bilayer
and membrane proteins ➢ band 1 or α on cytoplasmic side
o maintains extreme differences in in osmotic ➢ band 2 or β • regulate the membrane
pressure, cation concentrations, and gas o actin shape and deformability
concentrations between plasma and cytoplasm ➢ band 5 • provide viscoelastic
properties
▪ asymmetrically distributed
o outer: phosphatidylcholine and sphingomyelin (c) portion of membrane
o inner: phosphatidylserine (PS) and
phosphatidylethanolamine

❖ phosphatidylserine is the only negatively charged

phospholipid
❖ phosphatidylserine is important when the RBC
has condition (thalassemia, sickle cell anemia, old
RBC), phosphatidylserine is flipped in the outer
layer because this is where splenic macrophages
attach
❖ phosphatidylserine flips outside so that the
splenic macrophages will attach to the cell, and
the splenic macrophages will remove the
damaged RBC from the circulation
 ▪ also known as transmembrane because they are
attached to phospholipid bilayer
▪ band 3
o most integral protein
▪ functions:
o transport sites
actin junctional
➢ where proteins are attached that enters inside
ankyrin the cells
complex
complex
o adhesions sites
➢ contain sialic acid
lipid ➔ sialic acid gives RBC a negative charge
bilayer
➔ zeta potential
❖ negativity between cells
fibrous ❖ causes cells to repel one another
polypeptide
(protein) ❖ decreased zeta potential: due to
altered plasma proteins
o signaling receptors
❖ fibrous polypeptide is loosely walled
❖ spectrin is bound to phospholipid bilayer
❖ Why do RBCs not aggregate in the peripheral circulation?
Why do RBCs coagulate once they are removed from the
❖ ankyrin complex
peripheral circulation?
▪ is the major connecting protein
➢ because the negative charge is high in the blood vessels,
▪ it connects band 3
so they repel from one another
❖ band 3 is attach to ankyrin via multimer; other than that, 4.2
➢ due to they contain sialic acid, negatively charged, they
stabilizes the linkage between the band 3, ankyrin, and
repel one another
spectrin
❖ when plasma proteins are altered, membrane proteins are
❖ band 3 and protein 4.2 is attached to spectrin via ankyrin
also altered
❖ band 3 is the most abundant integral protein
❖ band 3 multimer needs attachment proteins (ankyrin) (many)
TRANSMEMBRANE PROTEINS:
Transmembrane
❖ actin junctional or 4.1 complex Band Function
Protein
❖ dimer of band 3 is directly attached to spectrin (because they
Aquaporin 1 Water transporter,
are few) Colton antigen
❖ 4.1 protein attaches lipid bilayer, specifically glycophorin C Band 3 (anion Anion transporter, location of
3 ABH antigens
and D, to spectrin via 4.1 protein exhanger, AE1)
❖ additional proteins that link spectrin to complex Ca2+ ATPase Ca2+ transporter
▪ Tropomyosin Duffy G protein-coupled receptor,
▪ Tropomodulin chemokine receptor, Duffy
antigens, receptor for malarial
▪ Actin parasites
▪ Adducin: protein or transmembrane or integral protein Glut-1 Glucose transporter, location of
4.5
ABH blood group antigens
❖ spectrin: contain the most peripheral proteins Glycophorin A PAS-1
Sialic acid transporter; location of
MN blood group antigens
PERIPHERAL PROTEINS: Glycophorin B PAS-4
Sialic acid transporter; location of
Ss blood group antigens
Skeletal Protein Band Function Sialic acid transporter; location of
Glycophorin C PAS-2
α-spectrin 1 Filamentous antiparallel heterodimer, Gerbich blood group antigens
primary cytoskeletal proteins
ICAM-4 Integrin adhesion
β-spectrin 2 cytoskeletal proteins
K+-Cl- Transports K+ , Cl-
Adducin 2.9 Caps actin filament, binds Ca2+ /
calmodulin contransporter
Ankyrin 2.1 Anchors band 3, protein 4,2, and Kell Zn2+ -binding endopeptidase, Kell
other proteins in the ankyrin complex antigens
to spectrin Kidd Urea transporter, Kidd (Jk)
Dematin 4.9 Actin bundling protein antigens
β-actin 5 Binds β-spectrin Na+, K+ -ATPase Na+, K+ transporter
G3PD 6 Carbohydrate metabolism, Na+ -K+ -2Cl- Na+ -K+ -2Cl- transporter
phosphorylates G3P cotransporter
Protein 4.1 4.1 Anchors the actin junctional complex Rh D and CcEe antigens;
to spectrin tetramers, RBC stabilizes band 3 and Rh
cytoskeleton shape
macrocomplexes
Protein 4.2 4.2 Part of ankyrin complex, ATP binding
(protein kinase) protein RhAG D and CcEe antigen
Tropomodulin 5 Caps actin filament component; CO2, cation, and
Tropomyosin 7 Stabilized and regulates actin ammonium transporter
polymerization

2. Integral protein
 • abnormal Hgb leads to lysis
• Normal RBC Hgb concentration has low viscosity and is fluid
• Causes of deformable RBC’s:
▪ Loss of water
▪ Precipitated Hgb
o Heinz bodies
▪ Polymerized Hgb
o Hgb S
▪ Crystalized Hgb
o Hgb C

IV. Energy Metabolism

• Energy is required to:
▪ maintain components of RBC
▪ preserving membrane shape
▪ perform enzymatic reactions
▪ movement of Ca2+, Na+, K+
▪ reduce oxidized proteins
o Hgb must be maintained in reduced state
▪ Hgb site prone to oxidation:
o Iron atom in the heme ring
3. Membrane enzyme ➢ problem in Hgb
▪ Na+, K+- ATPase ➢ you need ferrous (Fe2+) because it has the
o movement of substrates and cofactors in/out of capability to carry oxygen; when oxidized it will
the cell have more oxygen leads to ferric (Fe3+)
o needs energy for the movement of ions in/out of ➔ ferric is incapable to carry oxygen
the cell ➢ Methemoglobin
o controls active transport of Na and K ➔ contains ferric
o increase Na+ w/o loss of K+ : gain water results ➔ formed from oxidation of normal Fe2+ state to
lysis Fe3+ state
o increase K+ : cell shrinkage ✓ normally occurs daily
accumulation of
➢ sodium and water go out methemoglobin ✓ hereditary unstable Hgb
▪ Ca+2, Mg+2 – ATPase happens when: ✓ methemoglobin reductase deficiency
o Moves calcium out of the cell to the plasma ✓ Exposure to oxidant drugs
against high concentration gradient o Sulfhydryl (-SH) groups on the globin chains
o increased intracellular calcium in cell will become ➢ Heinz bodies (formed when there’s oxidation of
rigid (cell) resulting to lysis and it will block the sulfhydryl group)
passage of blood vessels (not flexible) ➔ Heinz bodies: inclusion bodies; abnormal
o calmodulin hemoglobin
➢ binds calcium ➢ problem in oxygen-carrying capacity of RBC
➢ sequesters calcium in place of mitochondria ➢ problem in Hgb
and endoplasmic reticulum, which are absent
in RBC SOURCES OF ENERGY:
➢ RBC contains calmodulin Glucose
o calcium • principal source of energy
➢ involved in regulating and stabilizing • quickly pass-through membrane without expense of energy
membrane phospholipid structure • EMP: conversion of glucose to lactate

C. Carbohydrates Galactose, Fructose and Mannose

1. Glycolipids
▪ responsible for attachments Pentose and Disaccharides
▪ sugar bearing lipids • Not metabolized and cannot pass through the membrane
▪ associate in clumps or rafts • lactose, sucrose, maltose
▪ support carbohydrate side chains that extend into the
aqueous plasma to anchor the glycocalyx Glycogen
o layer of carbohydrates • can be used but not normally used
o net (-) charge prevents microbial attack &
• used when there’s no other carbohydrate that can be a
mechanical damage caused by adhesion to
source of energy
neighboring RBCs or to the endothelium
▪ bears antigens of the ABH and the Lewis blood group
Pathways for Energy Metabolism
systems
1. Embden-Meyerhof Pathway
III. Hemoglobin Viscosity
• metabolize 90-95% of glucose used by the cell (anaerobic)
• normal component of RBC
 • 90-95% of erythrocyte is used for ATP generation • defective: decreased reduced glutathione to neutralize
• glucose catabolized to lactate oxidants
• same as other EMP except for ▪ Reduced glutathione (GSH)
• decrease O2 tension, 2,3-DPG binds to deoxyhemoglobin o principal reducing agent in the cell
form o reduced oxidized sulfhydryl groups in Hgb and
▪ causes Hgb to resist deoxygenation → decrease O2 other proteins
affinity and increase release and delivery of oxygen o yields reduced sulfhydryl groups and oxidized
• ATP and nicotinamide adenine dinucleotide (NAD+ & glutathione (GSSG)
NADH)
• 2-3 DPG ▪ Glutathione reductase & NADPH
▪ responsible to decrease the oxygen affinity and increase o reduce GSSG to GSH
release and delivery of oxygen
▪ formed in bypass Rapoport-Luebering shuttle ❖ we normally have glutathione in our body
▪ generated by Rapoport-Luebering shuttle ❖ RBC is also responsible for the formation of reduced
o bypass pathway in the EMP glutathione
o 1,3-DGP can be catabolized to 3-PG ❖ Reduced glutathione: inhibits the formation of Heinz
o or directly by path w/c high energy ATP is generated bodies and methemoglobin
o you will go bypass when oxygen tension is low ❖ The whitening caused by glutathione is just a side effect
because you need 2-3 DPG ❖ (reduced) glutathione is an antioxidant

❖ example: Hgb contains oxygen, oxygen tension crash ▪ R-5-P (Ribose-5-phosphate)

down means there’s hypoxia. 2-3 DPG will now attach to o generated by HMP (Hexose Monophosphate
Hgb so that oxygen can be released and deliver faster. Shunt)
That oxygen will go to the area where oxygen is needed. o used by nucleated cells during nucleic acid
metabolism
❖ phosphofructokinase is the enzyme that we see at the o it is recycled back to EMP
beginning of EMP o major reactant in the conversion of glyceraldehyde-
❖ rate of glycolysis is dependent on the pH level and the 3-phosphate to 1,3-Biphosphoglyceraldehyde
amount of ATP phosphate
❖ pH level: o glyceraldehyde-3-phosphate & fructose-6-
➔ alkaline: increased glycolysis phosphate
➔ acid: decreased glycolysis ➢ Common reactants in both pathways
❖ ATP:
➔ increased ATP = decreased glycolysis (because it is
not needed; ATP is already increased/high)
➔ decreased ATP = increased glycolysis (because it is
needed)

o increased during hypoxia due to alkaline

intracellular pH
o Glycolysis is also affected by ATP level
➢ increase ATP: decrease glycolysis
➢ decrease ATP: increase glycolysis
▪ Pyruvate kinase deficiency

3. Methemoglobin Reductase Pathway

• prevent oxidation of the heme iron
• by reducing peroxide
• NAD is being reduced into NADH, that will be used as
coenzyme of methemoglobin reductase, so that the
methemoglobin will become hemoglobin

2. Hexose Monophosphate Shunt

• we need to reduce NADP; we need NADPH in order to
reduce glutathione
• metabolize 5-10% of glucose used by the cell (aerobic)
• provide reducing potential for the cell
▪ by generating reduced NADPH
• G6P is catabolized (G6PD) to 6-PG instead of passing
Erythrokinetics
EMP
• Erythropoiesis
• increase activity: increase reduced glutathione (reduction
▪ RBC production 3 phases of
of NADP+ to yield NAPH) erythrocyte life
 • Release from BM to PC ❖ when RBCs are aged, it is located in the spleen
• Destruction and death ❖ when RBCs are damaged or has premature lysis (like
▪ spleen and liver problem in membrane, Hgb problem), it happens in the liver
• Requirements for balanced erythron model (balanced
production and destruction): ❖ Liver:
▪ Normally functioning BM ➔ greater blood flow
▪ Normal EPO level (which influences the production of ➔ more active role in the removal of severely damaged
RBCs) cells
▪ Adequate nutrients: Iron, folate, Vit B12 ➔ hemolytic anemia: the membrane is damaged
• Anemia
▪ destruction exceeds production Principal ❖ Spleen:
disturbances in ➔ principal sites of RBC phagocytosis by tissue
• Erythrocytosis erythron model
▪ production exceeds destruction macrophages after damage by normal aging
DIFFERENT ORGANS RESPONSIBLE FOR MAINTAINING
ERYTHROPOIESIS ❖ What happens to RBCs when they are aged?
➔ membrane loss leads to less deformable
➔ low glycolysis leads to decreased ATP levels
➔ enzyme system is already damaged leads to influx of
calcium resulting the cells to be rigid

Extravascular RBC Destruction

• destruction happens in organs (liver or spleen)
• majority of destruction are occurring in the spleen and liver
• 90% destruction of cells
• feces because no stercobilin reached in the intestine

❖ Kidney
➔ produces EPO (erythropoietin) (for adults)
❖ Liver
➔ storage of iron and other nutrients such as, protein, vit.
B12, and folic acid
➔ synthesizes globin (globin is a component of Hgb)
➔ participates in the production of EPO
➔ production: majority in fetus; 10% in adults
❖ Bone Marrow
➔ site of RBC production

Erythrocyte Destruction
• Life span in Infants: 35-50 days
• Life span in Fetus: 60-70 days Intravascular RBC Destruction
• Life span in Adult: 120 days • destruction happens in the blood vessel or peripheral
• As RBC ages: circulation
▪ membrane becomes less flexible (membrane loses lipid, • 10% destruction of cells
protein, carbohydrate)
▪ concentration of cellular Hb increases (Hgb inside PROCESS:
decreases) LIVER
▪ enzyme activity (glycolysis) diminishes; ATP production • you have RBC destruction, then Hgb in the plasma will be
also diminishes released
• Spleen: most active site of phagocytosis of aged cells • this Hgb will be split into alpha and beta dimers (what we call
• Aging RBC globin)
▪ Loss of sialic acid and lipids • This globin, came from Hgb, will bind to a protein called
▪ Decreased ATP levels Haptoglobin, that’s why you will form haptoglobin-
▪ Increased calcium levels hemoglobin complex which will lead to the liver
• RES
▪ Intravascular and extravascular hemolysis all of it cannot be done in the liver especially if it’s too many,
▪ Phagocytic cells (histiocytes, monocytes & that’s why some of it happens in the kidney.
macrophages)
KIDNEY
• we avoid the Hgb in reaching the kidney because it is not
normal for a Hgb to be present in the kidney

• Haptoglobin: protein in the liver that is responsible to bind

with the Hgb
• Hemosiderin: produced in the kidney
• what happens in the plasma? you form methemoglobin
• heme binds to hemopexin
• excess heme binds with albumin

EPO Production and Regulation

Regulation
2 hormones that regulates EPO:
• Prostaglandins
▪ Helps regulate EPO
▪ Enhance effect of EPO in CFU-E
• Estrogen
▪ Inhibit EPO production
Embden-Meyerhof Pathway (Anaerobic Pathway of Glucose Metabolism

NADP will become NADPH,

used for us to convert Oxidized
glutathione (GSSG) to
Reduced glutathione (GSH),
used to maintain the reduced
state of different molecules.

1
2
(bypass) NADH is reduced in NAD+ in
(bypass)
pyruvate to lactate; it became
coenzyme of lactate
will produce dehydrogenase

NAD+ is reduced in NADH as

coenzyme by Glyceraldehyde
3-phosphate dehydrogenase
in conversion of
Glyceraldehyde 3-phosphate
to 1,3-Biphosphoglycerate

NADH is used as coenzyme by

methemoglobin reductase so
that the methemoglobin will go
back to Hgb (Methemoglobin
reductase pathway)
HEMOGLOBIN ▪ the iron and protoporphyrin will combine together forming
a Ferroprotoporphyrin IX or heme
Hemoglobin ▪ purpose of heme: where O2 binds
• is the protein component of RBC
• this is where O2 and CO2 attaches 4. 2,3-Diphosphoglycerate
• Single most common complex organic molecule in • Produced from anaerobic glycolysis
vertebrates ▪ specifically in Rapoport-Luebering shunt (bypass of
• > 1% of total body weight Embden-Meyerhof Pathway)
• Synthesized w/in the maturing nucleated erythrocytes in BM • Hb + 2,3-DPG = decreased O2 affinity ; increased release
• Deliver O2 to tissues and transport CO2 away from lungs • Adequate supply to encourage release of O2to tissues
• Components: • presence of 2,3-DPG in Hgb α O2 affinity in Hgb
▪ Globin chains • adequate tissue oxygenation requires adequate supply of
o 4 polypeptide chains 2,3-DPG
o means it has amino acid sequences
o those 4 polypeptide chains are identical in pair Hemoglobin Variants
o total of 4 individual polypeptide; 2 pairs
▪ Protoporphyrin IX: Proportions (%)
Protoporphyrin + Stages of Hemoglobin
o 4 mol nitrogenous substance Iron = Heme Chains
▪ Iron: Development (Normal) Newborn Adult
o 4 atoms in ferrous state (Fe+2) heme carries O2
Portland γ 0 0
▪ 2,3-DPG ζ
Embryonic Gower-1 0 0
o 1 mol ε
Gower-2 0 0
o may or may not be present
o present: means oxygen affinity (attachment) will Fetal γ 80 <1
Newborn & α
decrease; means it will kick out the O2 resulting to A1 β 20 97
Adult
increase delivery or release A2 δ <0.5 2.5
o absent: O2 affinity increases; decrease O2 delivery
or release ❖ fetal = Hgb F
❖ A1 and A2 = Hgb A
• the hemoglobin concentration within RBC is equivalent to ❖ per Hgb variant, there’s 2 pair of globin chains (ex. Portland:
34g/dL 2 ζ, 2γ ; Gower-1: 2 ζ, 2 ε
• molecular weight of Hgb: 64,000 daltons ❖ Mesoblastic phase: Portland, Gower-1, Gower-2 can be
seen (1st 3 months of Embryonic Development)
Components of Hemoglobin ❖ Hepatic phase: Hgb F is seen (peak at birth; but produced
1. Globin Chains at 4th month of Embryonic Development)
• different types of polypeptides: α, β, γ, δ, ε, ζ, 𝝑 ❖ Hgb A1 and A2 are predominant in adults (1 year of age)
• 4 polypeptide/Hgb; 2 in pairs (depends on the combination) ❖ α and ζ are encoded in chromosome16
❖ β, γ, δ, ε are encoded in chromosome 11
• α and ζ: 141 amino acids, but different components
❖ γ has 146 amino acids; they differ in AA 136 position
• β, γ, δ, ε, 𝝑: 146 amino acids
Hemoglobin Structure
what are the differences of globin chains?
• differences in sequence and number of amino acids
Oxygenated “relaxed” / R Non-oxygenated / “tense” /
• Sequence of AA: polypeptide chains form T form
• Proportions of these chains undergo a series of changes
globin globin
during fetal and early infant life
• After birth, retain proportions throughout life

2. Protoporphyrin IX
• Nitrogenous substance synthesized in mitochondria and
globin globin
cytoplasm of nucleated RBC
• we have 4 protoporphyrin
• we only release and synthesize protoporphyrin in nucleated
RBCs or immature RBC (in mitochondria or cytoplasm)

3. Iron strong
weak bonds anionic salt
• Added at the center of protoporphyrin IX between 2 dimers bridges bonds
• Protoporphyrin IX + Fe+2 = Ferroprotoporphyrin IX or heme between
alpha
• 90% is recycled from extravascular hemolysis and beta
(senescent/old RBC) ❖ H = heme
• 10% came from the diet ❖ heme pocket: where O2 attaches
▪ destruction of RBCs ❖ maximum of 4 mol: Total O2 molecule that can be attached
o 90% extravascular iron came from
❖ 4 globin chains / 2 dimers = 4 heme mol
o 10% intravascular both (90% of iron)
❖ only 2 mol or 1 mol or 3 mol can be attached, depends on • Quaternary structure
the oxygenation ▪ tetromer or also known as complete Hgb molecule
❖ Globular: shape of Hb
❖ Oxygenated doesn’t have 2,3-DPG ❖ 4 O2 mol can be carried by Hgb because you have 4 heme
❖ Hgb A1 :have both alpha and beta (O2 attaches on heme)
❖ dimer: fastened 2 alpha and 2 beta ❖ heme attaches on globin
❖ in between 2 alpha and 2 beta, we have weak bonds
❖ weak bonds means that it’s easily dissociate or detach ; 2,3-
DPG will enter
❖ in between alpha and beta, you have strong bonds
❖ 2,3-DPG will either attach to 2 alpha or 2 beta by the use of
anionic salt bridges
❖ it becomes non-oxygenated because of the presence of 2,3-
DPG

Globin Chain Structure

• Primary structure
▪ referring to amino acid or polypeptide sequence
▪ α and ζ chain: 1 (N-terminal beginning of chain) to 141
(C-terminal) Hemoglobin Synthesis
▪ γ, β, δ and ε chain : 1-146 • occurs in most body cells except erythrocyte/mature RBC. ;
• Secondary structure because it doesn’t have mitochondria and ribosome
▪ helices and non-helices can be seen • red BM and liver
▪ 8 helical segments (A to H) • Heme produced in the erythroid precursors is chemically
o maintains the rigid structure of globin chain identical to that in the cytochromes and
▪ 7 nonhelical segments myoglobin.
o provides flexibility; allow bending to form tertiary
structure
• (NA, AB, CD, EF, FG, GH, HC)

❖ hemoglobin synthesis first happens in mitochondria and

cytoplasm
❖ first mainly happens in mitochondria
• 65% of Hgb: synthesized before the nucleus is extruded ❖ in mitochondria, there is a condensation of succinyl CoA with
▪ rubriblast, prorubricyte, metarubricyte glycine, producing Delta-aminolevulinic acid (delta ALA);
• 35% of Hgb: synthesized in the early reticulocyte ALA synthase + cofactor Vit B6 forms Delta-ALA
▪ nothing happens in erythrocyte because it has no ❖ uroporphyrinogen will then be coproporphyrinogen (I or III)
mitochondria and ribosome; no capability to produce ❖ coproporphyrinogen III will be transferred to mitochondria
protein and becomes protoporphyrin
• HEME + GLOBIN = HEMOGLOBIN ❖ once you have protoporphyrin, it will now combine with iron
• 2,3-DPG can or cannot be seen, depends on hemoglobin (ferrous / Fe2+)
(oxygenated or not) ❖ by the use of heme synthetase, you will now produce heme
• Heme Synthesis
▪ protoporphyrin IX + iron What happens in mitochondria?
▪ Tetramethenetetrapyrole 1. condensation of Succinyl CoA + glycine = Delta-ALA
o protoporphyrin IX 2. formation of coproporphyrinogen to protoporphyrin
➢ protoporphyrin is an isomer or derivative of 3. protoporphyrin + iron
porphyrin
➢ 9th isomer ALA synthase is influenced by:
o cyclic compound consisting of 4 pyrrole rings ❖ Vit B6 or pyridoxal phosphate
connected w/ methene (=CH-) bridges at C 2 & 4 in ❖ EPO production (increased EPO & Vit B6, increased rate of
the pyrrole ring condensation of succinyl CoA and glycine)
o Last compound produced in formation of heme
• Tertiary structure ❖ rate of protoporphyrin synthesis should be equal to rate of
▪ pretzel-like structure globin synthesis
 • Transferrin-iron membrane receptor (developing erythrocyte
heme + globin = hemoglobin precursor)
❖ globin forms in liver (hepatocytes) ▪ Binding, membrane invaginates & vacuole formation
❖ heme forms in cells except erythrocyte ▪ Iron delivered to:
❖ globin is a protein o Mitochondria for synthesis of heme
o For storage as crystalline aggregates of ferritin
❖ protophyrinogen is not needed in the formation of • Sideroblast & siderocytes
protoporphyrin / not needed in heme ▪ happens when you have excess iron
❖ coproporphyrinogen I is the one that formed into ▪ sideroblast: nucleated RBC with stored ferritin
protophyrinogen ▪ siderocytes: mature RBC with stored ferritin

Heme Synthesis Remnants Assembly of Hemoglobin

• In mitochondria • Iron + Globin chain + protoporphyrin IX + 2,3-DPG
▪ Free erythrocyte protoporphyrin (FEP)
▪ Excess porphyrin complexed with zinc 1. Ferric (from ferritin) reduced (Fe+2) ferrous
▪ Elevated FEP in RBC occurs when iron supply is low 2. Insertion of Fe+2 at the center of protoporphyrin IX
(ferrochelatase) = heme
❖ ex. excess protoporphyrin in mitochondria = low iron; excess 3. formation of Globin chain completed in ribosome released in
protoporphyrin will then bind to zinc forming FEP found in cytoplasm (coming from liver)
mitochondria 4. Formation of αβ dimers
❖ low iron happens when you have IDA (Iron deficiency 5. 2 heme mol bind the crevice (E & F helices)
anemia) 6. 2 dimers form α2β2 Hb tetramers (quaternary); this is the
❖ in IDA, FEP is increased complete Hgb molecule (depends if it needs to add 2,3-DPG)
7. one 2,3-DPG inserted in central cavity of Hb structure
• In cytoplasm
▪ Ferritin aggregates Locations of Hemoglobin
▪ Prussian blue • BM except during fetal life
▪ Storage iron that was not used in heme synthesis • Prorubricyte---shortly after release in PC
• During degradation
Iron Metabolism ▪ Phagocytic cells in RES
Location of iron ▪ Spleen, liver and BM
• Throughout the body ▪ Release of iron + globin + biliverdin
• Stored iron intracellular space of liver and bone marrow
• heme synthesis happens in immature cells of erythrocyte Functions of Hemoglobin
production (bone marrow (adult) or liver (fetus)); specifically • Transport of molecular O2 : oxygen saturation per minute:
in cell (cytoplasm) 250 mL of O2 per minute
▪ Ferritin ▪ Plasma: 0.08 mmol of O2 per 1 L WB passes capillary
o all cells except mature RBC bed
o Iron + apoferritin ▪ Hb of 16.0 g/dL: 2.41 mmol of O2
▪ Hemosiderin ▪ Hb of 8.0 g/dL: 1.22 mmol of O2
o hemosiderin forms in the kidney (tubular cells) • Transport of CO2
o if apoferritin is unavailable • Buffering of the blood to prevent changes in pH incompatible
to life
Sources of iron • Oxygen transport
• 90% will be from the senescent or old RBC • Heme-heme interaction
• 10% from the diet
• Dietary iron (Fe+2 & Fe+3) ❖ ([saturation or amount of O2] >O2 bound by Hb, the greater
▪ Only Fe+2 is absorbed the Hb affinity; the more the oxygen increases in Hb, the
▪ ferric (Fe+3) is converted into ferrous (Fe+2) more Hb affinity increases)
o ferric is reduced in acid pH of stomach to produce ❖ ex. Hb with 2 O2 vs Hb with 4 O2 : the latter has greater
ferrous (Fe+2) affinity
o Reduced by acid pH of the stomach ❖ increase affinity, 2,3-DPG will be expelled ; opposite
▪ absorbed in: Small amount in stomach; most in relationship
duodenum and jejunum (intestine)
Oxyhemoglobin Dissociation curve
Transport of iron • relationship between oxygen saturation and oxygen affinity
• Transferrin • high oxygen saturation, high oxygen affinity
▪ iron delivered in the bone marrow (immature RBC; • saturation means the amount of oxygen in Hb;
specifically mitochondria) by transferrin • (percentage of saturation) and partial
▪ Transport Fe+3 released form mucosal cell (ferric- • pressure of oxygen (PO2)
transferrin)
▪ Delivers iron for Hb, myoglobin, cytochrome to iron • Normal curve (different saturation)
storage sites ▪ pO2 < 20 mmHg
 o low O2 affinity • Acute poisoning (beginning 15%-20%) >40% saturation:
o Hb has a very low affinity to O2 sudden loss of consciousness and rapid death
o easily or faster • Treatment: supplemental oxygen
released of O2
o sometimes no O2 2. Sulfhemoglobin
attached • contains sulfur
▪ pO2 is between 20- • in the presence of oxygen, hemoglobin reacts with hydrogen
60mmHg sulfide
o often found in tissues • irreversible change in the polypeptide chains of the
o faster release of O2 / hemoglobin molecule due to oxidant stress
O2 can be released • result in denaturation and the precipitation of hemoglobin
rapidly • cannot transport oxygen = decreased delivery
o Hb affinity to O2
• Carboxysulfhemoglobin
increased
• formed by oxidizing drugs such as phenacetin and
▪ pO2 > 60 mmHg
sulfonamides in cases of bacteremia caused by Clostridium
o 100% saturation =
welchii
low release of O2
• < 1% and seldom exceed 10%
o high saturation and
affinity, slow release and delivery of O2
3. Methemoglobin
o Complete saturation of Hb w/ O2
o often found in lungs because there is where Hb • with iron in the ferric state
oxygenates and the Hb saturation level is high in • incapable of combining with oxygen = decreased delivery
lungs • result from a metabolic defect
• Abnormal structure of Hb mol due to ADT
• Shift to the left • hemoglobin M disorders: asymptomatic cyanosis
▪ increased affinity for O2 • 2% methemoglobin each day (average: 1-3%)
▪ decreased body temperature (cold), no 2,3-DPG, • >10%: cyanosis
decreased CO2, increased blood pH (alkaline), • >60%: hypoxia
decreased delivery • Infants are more susceptible to methemoglobin production
because hemoglobin F, high nitrite quantities in food, water,
• Shift to the right or drugs
▪ decreased affinity for O2 • reversible (in Methemoglobin Reductase pathway)
▪ increased body temperature (hot), with 2,3-DPG,
increased CO2, decreased blood pH (acid) Abnormal Hemoglobin Molecule
• abnormal Hb S
Factors Affecting Hemoglobin Affinity for O2 • sickle gene may occur with Hb C, E, or D
1. Blood (body) temperature • sickled when deoxygenated Hb S polymerizes and forms
▪ increased temperature cause Hb to release O2 rapidly intracellular
2. Blood pH (Bohr effect) aggregates that deform the cell and molecules of
▪ relation between blood pH and O2 affinity of Hb hemoglobin that are almost insoluble on deoxygenation
3. 2,3-DPG
▪ anticoagulants cause WB to lose 2,3-DPG; Hb may not
release O2
4. Carbon dioxide
▪ Haldane effect
o relation between blood CO2 and O2 affinity of Hb
5. Fetal hemoglobin no capability to bind
6. Abnormal hemoglobin variants with O2; no release

Variants of Hemoglobin (Abnormal) Analysis of Hemoglobin

1. Carboxyhemoglobin Alkaline Electrophoresis
• capacity to combine with carbon monoxide in the same • Screening procedure for separation of Hb fractions
proportion as with oxygen • Principle: hemoglobin mol in an alkaline solution have a net
• affinity is 210 times greater = low release (-) charge and move toward the anode in an electrophoretic
system
• even if the concentration of carbon monoxide is extremely
low • hemoglobins A, F, S, and C
• useless for oxygen transport • Fast Hb
▪ mobility than Hb A at pH 8.6
• displaces oxygen and leads to tissue hypoxia
▪ Bart Hb, Hb and Hb I
• normal carboxyhemoglobin level : 1% to 3%
• Media: paper, cellulose acetate, starch block
• Ave. 5% per pack smoked per day
• chronically increased levels: increased erythrocyte
production
• 20% to 30%: dizziness, nausea, headache, and muscular
weakness
Citrate Agar Electrophoresis
• Pakes place at an acid pH
• Separation of Hb is on the basis of a complex interaction
between hemoglobin, agar, and citrate buffer ions
• Principle: separates hemoglobin fractions that migrate
together on cellulose acetate
• Hb S, D, G, C, E, and O
• All hemoglobin specimens that show an abnormal
electrophoretic pattern in alkaline media should undergo
electrophoresis on acid citrate agar

Denaturation Procedures
• Kleihauer-Betke
• determine the amount of fetal blood that has mixed with
maternal blood following deliver
• involves acid denaturation of Hb
• Fetal hemoglobin and adult hemoglobin

Chromatography
• Quantitation of hemoglobin A1
▪ by cation exchange minicolumn chromatography
▪ affected by several types of hemoglobin in addition to
hemoglobin A1
▪ In conjunction, cellulose acetate and citrate agar
electrophoresis to eliminate the possibility of interference
by hemoglobin variants
▪ HPLC and colorimetric methods
RED BLOOD CELL ABNORMALITIES ❖ you need to have PBS for you to observe the morphology of
RBCs
Qualitative: morphology ❖ 3 Portions/Locations of Peripheral Blood Smear
➢ size, shape, color, distribution in the peripheral 1. thick area
blood 2. thin area
➢ anything or any defect or abnormality with this 3. feathery edge
characteristic that leads to disorder of diseases
❖ we normally observe the characteristic of RBCs in thin smear
Quantitative: count
➢ low: anemia
➢ high” polycythemia Abnormal Distribution
1. Rouleaux
RBC Abnormalities • not separated at the usual
• by specific chemical, physical or cellular causes observation area
• Characteristics of Mature Red Blood Cell: • appear as short or long
1. distribution stacks resembling coins
2. size: 6-8 µm or flat plates
3. shape: biconcave or discocytes • entire outline of each cell
4. no inclusions is not visible
5. lack nucleus • first sign of protein
abnormality with increased ESR
▪ thickness: 2.5 µm ▪ ESR is a non-specific indicator of inflammation
▪ volume: average: 90 fL ; range: 80-100 µm • spherocytes: cannot form rouleaux
▪ Surface area: 140 µm
2. Agglutination
❖ any abnormality of defect with these characteristics will • aggregate into random clusters or masses when exposed to
result to a disorder RBC Ab
• entire outline of each cell
• Variations of RBC Abnormalities is not visible
1. distribution on blood film • Autoagglutination
▪ presence of rouleaux formation or agglutination ▪ agglutination occurs
2. size in one’s own plasma
3. shape or serum that contains
4. color no specific agglutinins
5. inclusions ▪ when you centrifuged
the blood of patient, there is a possibility of it to form
Normal Distribution autoagglutination (normal if few agglutination)
• even distribution of RBC (thin portion adjacent to feathery ▪ seen in anticoagulated blood rotated at room
end) temperature
• Characteristics: ▪ increased MCV (Mean Corpuscular Volume)
▪ slightly separated o means that your RBCs are large, not necessarily
▪ barely touching & without overlapping large RBC because agglutination is counted (by the
• thicker portion machine) as one single cell
▪ overlapping cells o aggregation of RBCs makes it look like large cell
▪ unsuitable for evaluation (due to overlapping of cells) • associated with:
• thin area ▪ normal individuals
▪ represent at least 1/3 of the entire film ▪ hemolytic anemias
▪ area of the usual observation of morphology takes place ▪ atypical pneumonia
▪ distribution of RBC is slightly separated or even ▪ staphylococcal infections
distribution ▪ trypanosomiasis
▪ normal site of observation ▪ cold agglutinin disease
• feathery end ❖ side, top: manner of adherence
▪ distribution is irregular with artifactual ❖ cause: aggregation or interaction of the antigen and RBC
▪ artifacts: shapes, color and size distortions antibody
❖ abnormal if they are present in numerous amount
❖ normal if few agglutinations (1-2 agglutination per field)
❖ abnormal if >5 agglutination each field
❖ no antigen aggregates with the antibody, only
autoagglutination of RBCs

Variation in Size
• Normocytic
▪ normal diameter of RBC: 6-8 µm
▪ volume: 80-100 fL ; average: 90 fL
▪ normal MCV
• Macrocytic • cases of variant hemoglobin types
▪ >100 fL ▪ abnormal hemoglobin
• Microcytic ▪ abnormal hemoglobin means impaired globulin synthesis
▪ <80 fL • hemoglobinopathies
• Anisocytosis ▪ β-thalassemia
▪ means that there is variation in size ▪ hemoglobinopathies = impaired globulin synthesis
o means in one field, you will see a macrocyte,
normocyte, and microcyte ❖ problem with heme synthesis may result to microcytosis
▪ prominent in severe anemia
▪ chemical or physiological basis Variation in Shape
▪ increased RDW (a lot of cells are variable in sizes) • Poikilocytosis
▪ variation in shape
▪ assume many shapes
▪ chemical or physical alteration
o cellular membrane
o physical contents of the
cell

❖ anisocytosis: variation in size

❖ anisochromia: variation in color

1. Macrocytosis Greek Terminology Common Terms

• size: >8.5-9.0 µm Acanthocyte Acanthocyte
• MCV: >100 fL Echinocyte Burr cell
• increased heme synthesis (hemoglobin synthesis) Crenated RBC
• Reasons why there are macrocytes Elliptocyte Elliptocyte
1. defect in nuclear maturation Ovalocyte
▪ appear as mature, enlarged erythrocytes in PC Schizocyte Helmet cell
Schistocyte
2. defect in stimulated erythropoiesis Discocyte Normal erythrocyte
▪ increases the synthesis of hemoglobin in
Megalocyte Oval macrocyte
developing cells
Drepanocyte, meniscocyte Sickle cell
o more hemoglobin = large cells are produced
Spherocyte Spherocyte
▪ causes a premature release of reticulocytes into PC
Stomatocyte Stomatocyte
▪ (reticulocytes) appear also as basophilic and
Codocyte Target cell
slightly hypochromic on PBS
▪ uncontrolled proliferation of RBC Dacrocyte Teardrop cell
o why do we need to produce more RBC?
o example: when a person has hemolytic anemia, 1. Developmental Macrocytosis
o the bone marrow is normal and released RBCs 1. Oval macrocytes
are normal, but it doesn’t last for 120 days in the • also known as megalocytes
circulation • oval or egg-like appearance
o there’s premature death of RBC • similar in appearance to
o bone marrow will now continuously release cells elliptocytes
until the point that normal and mature cells are ▪ normal cell size volume
no longer available • larger than normal cell-size
o due to this, bone marrow will release immature volume
cells to compensate for the normal ones • macrocytic and have a fuller and rounder appearance
o immature cells are large (macrocytic) • Associated with:
▪ vitamin B12 and folate deficiency
2. Microcytosis o decreased nutrient
• size: <6.2 µm o disruption of mitotic division
• MCV: <80 fL ▪ observed in erythrocytes that are in the reticulocyte stage
• decreased heme synthesis o problem in erythropoiesis
• hemoglobin is affected o release of immature cells in peripheral circulation
• associated with decrease hemoglobin synthesis
▪ produced by a deficiency of iron 2. Membrane Abnormalities
▪ impaired globulin synthesis 1. Spherocytes
▪ mitochondrial abnormality
• lost their normal biconcave
• malabsorption syndrome shape
▪ malabsorption of iron (diet)
• extremely compact, round
▪ we release iron daily, so it needs to be replenished by
shape
increasing it in our diet
• < 6 mm and has an intense
▪ may iron na nawawala sa urine at menstruation
orange-red color
• IDA (Iron Deficiency Anemia)
• Spherocyte-like erythrocytes: artifacts (thin end of a PBS) ❖ in normal state, burr cells have counterpart called Crenated
• loss of cell membrane leads to decreased surface area but RBC
the volume will remain the same at first (80-100 Fl) \ ❖ in crenated RBC, imbalance in osmotic pressure occurs
▪ surface area is decreased so the volume ratio will also (sodium is released)
decrease, which leads to membrane instability, when it ➢ the water in RBC will then be released
reaches small blood vessels it will not deform, which ➢ kasama lagi ni Na+ si H2O kaya nagkaroon ng crenation
leads to premature cell destruction or lysis ❖ crenated RBC has no associated condition

4. Acanthocytes
• known as spiculated RBC
▪ has irregular projections
• irregularly distributed multiple thorny,
spike-like projections
• few spicules.
• spherocytes: shape of RBCs if hemolytic anemia is present • abetalipoproteinemia and spur cell
• Microspherocytes are associated with: anemia
▪ ABO hemolytic disease of the fetus and newborn (HDN) ▪ imbalance between erythrocyte and plasma lipids
▪ storage phenomenon that produces microspherocytes in ▪ inability to absorb lipids in intestine
the recipient of a blood transfusion leads to:

2. Elliptocytes
• also known as ovalocytes
• narrower & more elongated than
megalocytes
• rod, cigar, or sausage shape
• membrane defect: loss of integrity ; end
point: lysis • Liver cirrhosis with hemolytic anemia
• Associated with: • heparin administration
▪ hereditary elliptocytosis • hepatic hemangioma
▪ anemias associated with malignancy • neonatal hepatitis
▪ Hb C disease • post-splenectomy
o crystalized hemoglobin
▪ hemolytic anemias 5. Stomatocytes
▪ IDA • have slitlike opening that resembles a mouth on one side of
▪ pernicious anemia the cell
▪ sickle cell trait • result from increased Na+ ion and decreased potassium
▪ thalassemia (K+) ion
▪ osmotic imbalance
❖ in elliptocytes, you form this cell because there is a problem ▪ Na+ should be outside (PISO: potassium
in the membrane inside, sodium outside) but here, Na+
❖ smaller than megalocyte but more elongated enters inside, which leads to the
increase of Na+ that’s why K+ has been kicked-out
3. Burr cells • different from Created RBC (osmotic imbalance)
• also known as echinocyte ▪ Crenated RBC: lumabas ang
• known as spiculated RBC Na+
▪ has regular projections ▪ Stomatocytes: pumasok ang
• one or more spiny projections (uniformly Na+
shaped) • Associated with
• elongated cell or assume quarter moon ▪ acute alcoholism
shape ▪ alcoholic cirrhosis
• less spherical than acanthocytes ▪ glutathione def.
• produced as artifacts in vitro ▪ hereditary spherocytosis
• decreased deformability ▪ IM
▪ depends on loss of membrane: ▪ lead poisoning
o decreased surface area:volume ratio ▪ Malignancies
o abnormal hemoglobin ▪ thalassemia minor
o decreased lipid in plasma membrane ▪ hereditary stomatocytosis and Rh null disease (means
❖ increased red cell rigidity leads to less deformable. Less no Rh antigen)
deformable leads to premature destruction and lysis (end
point)
❖ 2 kinds of lipid (cholesterol and phospholipid) ; when these
are decreased, it can lead to the formation of burr cell
6. Target Cells 3. Teardrop cells
• also known as codocytes • smaller than normal erythrocytes
• resemble a shooting target • resemble tears
• central red bull’s-eye is surrounded by a • Associated with:
clear ring & outer red ring ▪ homozygous beta-thalassemia
• cells are thinner than normal ▪ myeloproliferative syndromes
• excessive membrane lipid: volume--- ▪ pernicious anemia
decreased volume: membrane surface ratio ▪ severe anemias
▪ surface area:volume ratio is affected (decreased
because the volume increases) 4. Semilunar Bodies
▪ thalassemia • hemoglobin has been released
• maldistribution of hemoglobin ▪ nawawala yung hemoglobin at
▪ abnormal hemoglobin natitira nalang ay membrane
• enzyme defects: increased cholesterol and ▪ ghost cell dahil nawala yung
phosphatidylcholine incorporated into the membrane lipid hemoglobin
• associated with: • Large, pale-pink staining ghost of
▪ hemoglobinopathies the red cell: the membrane remaining after the contents have
▪ hemolytic anemias been released
▪ hepatic disease with or without • Large as leukocytes
jaundice • Associated with:
▪ IDA ▪ Malaria
▪ after a splenectomy ▪ Conditions causing overt hemolysis
▪ can occur as an artifact
4. Abnormal Hemoglobin Content
3. Trauma 1. Sickle Cells
1. Schistocytes • resemble a crescent
• also known as schizocytes • two pointed ends or at least one of
• small and irregularly shaped the ends of the cell must be pointed
fragments of RBC • membrane is smooth
• result of the breaking apart of an • stains uniformly throughout
erythrocyte • result from the gelation of
▪ half the size of a normal polymerized deoxygenated Hb S
erythrocyte Hb S forms/happens when:
▪ deeper red appearance ▪ lowered oxygen levels (hypoxia)
• Increased numbers associate with: ▪ decreased blood pH (acidic pH of blood)
▪ hemolytic anemias related to • influx of sodium ions (osmotic imbalance)
burns (exposed to heat kaya nag- • increased level of intracellular calcium ions
rupture yung blister cells) and • Associated with sickle cell anemia
prosthetic implants (foreign
materials in cases of implants) as Other Poikylocytes
well as renal transplant rejections
1. Blister Cell
• containing one or more vacuoles
2. Helmet Cells
that resemble a blister
• also known as dacrocyte or teardrop, holly leaf or
• has thinned area at the periphery
drepanocyte, and keratocyte or helmet cell
or outer border
• larger scooped out part of the cell that remains after the
• vacuoles may rupture
rupturing of a blister cell
• Associated with:
• from physical process of fragmentation
▪ damage to the membrane
▪ formed in the spleen and intravascular fibrin clots
▪ traumatic interaction of blood
• distorted cell from blister cells (because the blister cell has
vessels and circulating blood
ruptured due to trauma)
• increased numbers:
▪ result of pulmonary emboli
o sickle cell anemia
o microangiopathic hemolytic anemia (MAHA)
dacrocyte; drepanocyte; keratocyte;
teardrop holly leaf helmet cell
➢ other terms depending on the morphology of the
cell (can be formed after rupture)
❖ pale vacuole and homogenous stain of RBC
❖ the erythrocyte has formed vacuole
❖ when the vacuole is exposed to trauma, it is ruptured
❖ that’s why you will now form 2 cells (schistocytes and helmet
cells) 3. Hypochromia
• central pallor exceeds one third (1/3) of the cell’s diameter,
2. Knizocytes which means Hgb synthesis is decreased
• resemble a pinched bottle • pale overall appearance
• Associated with: • inadequate iron stores = decrease in hemoglobin synthesis
▪ hemolytic anemias • seen in IDA (Iron Deficiency Anemia)
▪ hereditary spherocytosis
4. Polychromatophilia
• seen in reticulocytes
3. Leptocytes • reflect a state of cell immaturity
• resemble target cells but the ▪ the greater polychromatophilia, greater immaturity (>
inner, central portion is not polychromatophilia = > immaturity)
completely detached from the ▪ intense polychromatophilia = decrease immaturity of
outer membrane RBC
• Associated with: • seen in nonnucleated erythrocyte has a faintly blue-orange
▪ hepatic disorders color
▪ IDA blue-orange color is present means:
▪ thalassemia ▪ lacks the full amount of hemoglobin
▪ diffusely distributed residual RNA in the cytoplasm
• polychromatophilic erythrocyte
4. Pyknocytes ▪ basophilic erythrocyte
• similar to blister cells ▪ supravital stain: Prussian blue (+)
• distorted, contracted RBC
• Associated with: Inclusions
▪ acute, severe hemolytic anemia • Causes:
▪ G6PD def. 1. developmental organelles
▪ hereditary lipoprotein def. 2. abnormal hemoglobin precipitation
▪ seen in small numbers during the 1st 2 to 3 months of life
as infantile pyknocytes 1. Developmental Organelles
1. Howell-Jolly bodies
5. Spiculated erythrocytes • one inclusion per cell
• either burr cells or acanthocytes • 1 to 2 mm in size
• spiculated erythrocytes is the general term. We use to refer • round, solids staining, dark-blue to
to burr cells or acanthocytes purple inclusions
• irregularly contracted erythrocytes • Seen in mature cells (RBCs) than
• may also be referred to as burr cells, crenated cells, immature erythrocytes
pyknocytes, spur cells, acanthocytes, and echinocytes • not seen in normal erythrocytes
• are formed by nuclear remnants
REVIEW:
predominantly composed of DNA
• there are 4 possible reasons why you will have variation in
• usually seen when or develop in
the shape
accelerated or abnormal erythropoiesis
• Variation in the shape
• stain: supravital stain (+)
1. developmental macrocytosis
2. membrane abnormalities
❖ spleen is important in the destruction of RBCs
3. trauma
❖ if RBCs are not destroyed, there will be continuous formation
4. abnormal hemoglobin
of inclusion bodies in the cytoplasm
Alteration in Color
2. Basophilic stippling
• color is directly proportional to Hgb concentration
• tiny, round, solid-staining, dark-blue
▪ pale color = low Hb content ; intense color = high Hb
granules
content
• inclusion bodies all throughout the RBC
• central pallor is indirectly proportional to Hgb
• evenly distributed throughout the
▪ large central pallor = low Hgb content
cytoplasm of the cell
• pinkish-red appearance with a lighter-colored center
▪ Coarse basophilic stippling
▪ reflects the amount of hemoglobin present
o punctate stippling
o larger than in the fine form
1. Normochromic
o more serious in terms of pathological significance
• central pallor is normal
• Central pallor does not exceed 1/3 diameter of the cell

2. Anisochromia
• variation in the normal coloration
• 2 types of basophilic stippling: ▪ hemolytic anemias secondary to drugs such as
1. fine basophilic stippling phenacetin
2. coarse basophilic stippling ▪ some hemoglobinopathies
• granules composed of precipitated
ribosomes and RNA during the 2. Crystals
process of staining • hemoglobinopathies
• Associated with: • Hb C crystals
▪ disturbed erythropoiesis ▪ crystalized Hgb
▪ lead poisoning ▪ rod-like or angular opaque structures
▪ Heavy metal poisoning; specifically lead ▪ Associated with Hb C disease
▪ Thalassemia • Hb H bodies
▪ Pyrimidine-5-nucleotidase deficiency ▪ brilliant cresyl blue stain
• stain: supravital stain (+) ▪ blue globules
▪ represent polymers of the beta chains of HB A
3. Pappenheimer bodies/ Siderotic granules
• associated with excessive iron 3. Parasitic inclusions
• also known as siderotic granules Plasmodium sp. Inclusions
• Wright-stained smears as purple P. vivax
dots Schüffner dots
P. ovale
• infrequently seen in peripheral P. falciparum Maurer dots
blood smears
• Siderotic granules P. malariae Ziemann stippling
▪ Stain: iron stains (+)
▪ dark-staining particles of iron in the erythrocyte
▪ appear as blue dots and represent ferric (Fe3+) ions
• Pappenheimer bodies
▪ aggregates of mitochondria, ribosomes, and iron
particles
• Associated with:
▪ iron-loading anemias
▪ Hyposplenism
▪ hemolytic anemias

4. Cabot rings
• found in the periphery of the
cell
• ring-shaped, figure-eight, or
loop-shaped structures
• formed of either double or
multiple rings
• bell or tall hat shape on scanning electron microscope
• stain a red or reddish-purple color and no internal structure
• represent remnants of microtubules from the mitotic spindle
• represent nuclear remnants or abnormal histone
biosynthesis
• Associated with:
▪ lead poisoning
▪ pernicious anemia
▪ megaloblastic anemia

2. Abnormal Hemoglobin Precipitation

1. Heinz bodies
• Hgb is exposed to oxidation
• 0.2 to 2.0 mm
• stain: supravital stain (+)
▪ crystal violet or brilliant cresyl
blue
• represent precipitated, denatured hemoglobin
• Associated with:
▪ congenital hemolytic anemia
▪ G6PD deficiency
RBC DISORDERS • Relative Erythrocytosis
▪ decrease plasma volume
RBC Dirorders ▪ normal RCM
• if there is a defect either in the structure, morphology, and ▪ dehydration
count, that is an indicator of a disorder ▪ the fluid will transfer from the blood vessel to the tissue
• quantitative in cases of dehydration
• Physiologic State ▪ hemoglobin is the indicator of RCM ; (RCM is also
▪ Erythrocytosis = RCM equivalent to hemoglobin concentration)
▪ at constant level regulated by erythropoiesis ▪ in cases of relative erythrocytosis, RBC count is high but
▪ erythrocytosis or erythropoiesis or the production of RBC the hemoglobin is normal
is equivalent to RCM (Red Cell Mass) ▪ hematocrit is the indicator of erythrocytosis
▪ decreased RBC production, decreased RCM ▪ hemoglobin is not used as indicator in erythrocytosis
▪ increased RBC production, increased RCM because RCM is normal
• Anemia
▪ Decreased RBC in circulation Laboratory Profile
• Polycythemia or Erythrocytosis • Anemia
▪ Excess in circulation ▪ hemoglobin is the indicator
▪ high blood cell production ▪ decreased hemoglobin
o decreased O2 carrying capacity (hypoxia)
Relative Count vs Absolute Count • Polycythemia
• Absolute ▪ hematocrit is the indicator
▪ true decrease / increase in RCM ▪ increased hematocrit
▪ Absolute anemia or Polycythemia o hypervolemia
▪ referring to true count o hyperviscosity
▪ in here, when RBC is low, it really means that the RBC
count is low Physiologic Response to Anemia
• Relative Anemia 1. shift to the right
▪ fluid shift from extravascular to intravascular ▪ increased release of oxygen
compartment ▪ decreased O2 affinity in hemoglobin
▪ Relative polycythemia 2. increased 2,3-DPG
▪ there is secondary reason or disorder on why the RBC ▪ when 2,3-DPG attaches to the Hgb, O2 is released
count is low ▪ 2,3-DPG and O2 doesn’t combine
▪ reason on why falsely-decreased RBC in the peripheral 3. selective redistribution of blood flow to areas of
circulation highest O2 demand
▪ we have blood vessel (intravascular) and tissue ▪ normal response of our body is that when a certain
(extravascular), when the fluid transfer from extra to organ is in need, the same area has high blood flow
intra, hemodilution occurs (solute dilutes in the blood because that area needs O2
vessel) ▪ ex. lower limbs need O2, the blood flow in that area is
▪ meaning to say, when hemodilution occurs, when you high in order to provide O2
collect a blood sample from that specific blood vessel, 4. increased cardiac output
the count decreases because it dilutes ▪ in anemia, the pump of heart is fast
▪ expanding plasma volume and diluting RCM ▪ when cardiac output or pumping is high, delivery of
▪ causes why blood volume is expanding or why the fluid blood is much faster and blood flow increases
transfer from extravascular to intravascular:
▪ in cases of pregnancy and hyperproteinemia Hematologic Response to Anemia
▪ pregnant women have imbalance in hormone so • increased red cell production
osmotic pressure is also affected ▪ shift reticulocytes increased RPI
▪ in hyperproteinemia, the blood vessel has many o 6-8 fold increase (indication that BM is responding to
solutes because proteins are solute. anemia)
Hyperproteinemia means your plasma protein level in o 1 week to manifest
blood is high • slower type of response
• this can manifest 1 week after the O2 has decreased
❖ in both scenario, solutes in the blood vessel needs to be • slower but more effective because the management or effect
diluted is long-term
❖ the physiologic response of our body when the plasma of • in this type of response, the red cell production increases
our blood is highly viscous is it needs to be added some • increased reticulocytes (released first because it is the
fluid. The fluid needs to be transferred from extra to intra. immediate precursor or mature RBCs)
When this happens, dilution occurs that will also result to • to measure the presence of reticulocytes is by the
hemodilution measurement of RPI (Reticulocyte Production Index)
▪ RPI
▪ when you have anemia, the hemoglobin is low because o when RPI increases, bone marrow is effective
RCM is low because your bone marrow is responding (to anemia
• 3x > the normal value: reticulocytes released in cases of
anemia
 • when RPI doesn’t increase, ▪ decreased production of erythropoietin: anemia of
▪ ex. in anemia, low Hgb, bone marrow doesn’t respond renal disease
• Failure in Bone Marrow:
1. intrinsic disease in BM Anemia Caused by Increased Red Blood Cell Destruction or
2. lack of growth factors Loss
3. failure of mechanism • Intrinsic RBC abnormality:
▪ membrane defects: hereditary spherocytosis,
Signs and Symptoms hereditary elliptocytosis or pyropoikilocytosis,
• Mild anemia paroxysmal nocturnal hemoglobinuria
▪ No symptoms because of physiologic response ▪ enzyme deficiencies: glucose-6-phosphate
▪ Palpitations and dyspnea (exercise) dehydrogenase deficiency, pyruvate kinase deficiency
• Increasing severity ▪ globin abnormalities: sickle cell anemia, other
▪ Tachycardia hemoglobinopathies
▪ Shortness of breath • Extrinsic RBC abnormality:
▪ Headaches ▪ immune causes: warm-type autoimmune hemolytic
▪ Pallor (dermal vasoconstriction & blood redistribution) anemia, cold agglutinin disease, paroxysmal cold
▪ Leg cramps, dizziness, fatigue, insomnia hemoglobinuria, hemolytic transfusion reaction,
▪ in this case, the O2 is decreased hemolytic disease of the fetus and newborn
• Severe ▪ nonimmune red blood cell injuries: microangiopathic
▪ Coma hemolytic anemia (thrombotic thrombocytopenic
▪ Death purpura, hemolytic uremic syndrome, HELLP syndrome,
▪ no compensation disseminated intravascular coagulation),
▪ organs are shutting down because there are no longer macroangiopathic hemolytic anemia (traumatic cardiac
source of O2 hemolysis), infectious agents (malaria, babesiosis,
2 Classification of Anemia bartonellosis, clostridial sepsis), other injury (chemicals,
• Pathophysiologic drugs, venoms, extensive burns)
▪ mechanisms • Blood loss: acute blood loss anemia
• Morphologic
▪ morphology or structure Ineffective Erythropoiesis RPI < 2.0
(ineffective BM)
Pathophysiologic Classification of Anemia Hypoproliferative anemias Maturation disorders
I. Hypoproliferative Effective Erythropoiesis RPI > 3.0
▪ decreased production of RBC (effective BM)
II. Maturation disorders Hemolytic Anemias Blood loss anemia
▪ defective DNA synthesis
▪ defective Hgb synthesis Morphologic Classification of Anemia
▪ decreased erythropoietin production RBC indices & Stained PBS
III. Hemolytic disorders • MCV
▪ intrinsic RBC abnormality ▪ RBC index used to indicate and measure the size of RBC
o problem of the RBC itself ▪ volume/size
o membrane defect ▪ normocytic: 80-100 fL (normal MCV)
o globin abnormalities ▪ microcytic: <80 fL
o enzyme deficiencies ▪ macrocytic: >100 fL
▪ extrinsic RBC abnormality • RDW:
o immune ▪ to measure the anisocytosis
➢ antibody-antigen reaction leads to lysis of RBC ▪ to determine it the sizes of RBC are different from each
o non-immune other
➢ chemical causes • 4 categories based on RBC indices:
➢ physical causes 1. Normocytic, normochromic
➢ trauma ▪ normal size, normal Hgb
IV. Blood loss 2. Microcytic, hypochromic
▪ small RBC size, low Hgb
❖ bone marrow transplantation is the most effective 3. Microcytic, normochromic
management for anemia ▪ small RBC size, normal Hgb
Anemia Caused by Decreased Production of Red Blood 4. Macrocytic, normocytic
Cells ▪ large RBC size, normal Hgb
• Bone marrow failure: acquired and congenital aplastic
anemia, pure red cell aplasia, anemia associated with ❖ cell size and hemoglobin content can be observed in the
marrow infiltration (myelophthisic) PBS
• Impairment of erythroid development: ❖ look for the central pallor and the staining intensity to
▪ disorders of DNA synthesis: myeloblastic anemia estimate the hemoglobin content
▪ disorders of hemoglobin synthesis: iron deficiency ➢ lighter = low Hgb
anemia, thalassemia, sideroblastic anemia, anemia of ➢ red/pink = normal Hgb
chronic inflammation ➢ increased central pallor = decreased Hgb content
 ➢ bread, junk foods/chips, and other foods that
Microcytic Macrocytic Normocytic children usually eat
Common Common Common o children need iron supplement
IDA Folic acid def. Hypoproliferative o increase demand and low reserve
Myelophthistic
anemia
▪ pregnancy
Her. SA Refractory o 2 individuals need iron
dysmyelophthistic o pregnant women also need iron supplement
Occasional Occasional HA o pregnant woman is advised to take ferrous
ACD Hypoproliferative Hemoglobinopathies sulfate and mycolic acid
Hemoglobinopathies Refractory anemia BLA o pregnant women are prone to anemia
Liver dis. ACD
o the iron in the milk in pregnant woman is high
HA Acq. SA
Blood loss anemia Occasional because they need it and they need it to provide
Early IDA for the baby
Refractory anemia ▪ functional iron deficiency
o iron stores are adequate but the iron is not
MCV Low MCV Normal MCV High available to support normal erythropoiesis
Microcytic Normocytic Macrocytic ▪ too much demand but low storage
RDW Normal
-----------Homogenous----------- ▪ usual demand of iron/day: 30-75 mg dietary
Microcytic Normocytic Macrocytic
RDW High iron/day to compensate the iron from the diet
-----------Heterogenous-----------

Mean Cell Volume 2. Inadequate intake

Decreased Normal Increased ▪ 1 mg/day of iron is lost
• α- or β- • Anemia of • Aplastic anemia ▪ iron-deficient diet
Thalassemia chronic • Chronic liver
trait inflammation disease
▪ the iron in our body decreases everyday
• Anemia of • Anemia of renal • Alcoholism o we release and excrete cells and those cells
RDW chronic disease • Chemotherapy have mitochondria. Iron is stored in the
Normal inflammation • Acute
• Hb E hemorrhage
mitochondria
disease/trait • Hereditary o ex. excrete cells → squamous cells →
• low MCV , spherocytosis desquamated in the urine and feces
normal RDW
• Iron • Early iron, • Folate or
o dry skin or old skin cells also contains iron, when
deficiency folate, or vitamin 12 it is removed in our body, iron decreases
vitamin B12 deficiency ▪ red meat has more iron than vegetables
• Sickle cell-β- deficiency • Myelodysplastic
thalassemia • Mixed syndromes
RDW
deficiency of • Cold agglutinin 3. Inadequate absorption
iron + vitamin disease ▪ malabsorption caused by celiac disease
Increased
B12 or folate • Chronic liver
• Sickle cell disease ▪ inherited mutations of iron regulatory proteins
anemia • Chemotherapy o increased hepcidin (preventing iron absorption)
• Hb SC disease
production
• Myelodysplastic
syndromes ▪ decrease stomach acidity: achlorohydria

Microcytic Normochromic/ Hypochromic Anemia 4. Chronic Blood loos

1. Iron Deficiency Anemia ▪ menstrual flow or uterine malignancies
▪ low iron ▪ GI bleeding, chronic bleeding, tumor, parasitosis,
▪ low raw iron ulcerative colitis
▪ raw material is defective ▪ regular blood donation
2. Chronic Disease ▪ Chronic intravascular hemoglobinuria (PNH)
▪ defective release of iron store from macrophages Paroxysmal Nocturnal Hemoglobinuria
3. Sideroblastic Anemia o increase Hgb and iron release in the urine
▪ defective utilization of iron o excess loss in urine in the form hemoglobinuria
▪ excessive iron store (7.8 mg/dL)
4. Spherocytic
▪ membrane defect LABORATORY DIAGNOSIS:
5. Thalassemia • Chemical Tests
▪ globin defect (Hgb) 1. Serum ferritin
1. Iron Deficiency Anemia ▪ tissue iron store ; good indicator of iron storage
• Causes: status
1. Increased physiologic demand o high serum ferritin = high iron storage
▪ rapid growth: infants & children ▪ 1st lab test to become abnormal when iron store
o in children or infant growth, mas mabilis sila decreases
lumaki, hindi nakakasabay yung iron store and ▪ decreases before RBC morphology shows
iron intake na meron sila kaya merong mga iron ▪ principle: radioimmunoassays
supplement ang mga bata kasi di enough yung ▪ RR: M: 15-200 ug/L F: 12-150 ug/L
milk (especially in infants) and the diet they eat ▪ iron store is increased in menopausal women
o in fortification (foods), iron is included
 o 1 ug/L ferritin = 8 mg of tissue store = 120 ug o excess/unbound iron removed
iron/kg o sample is analyzed for the remaining iron
▪ content
o iron measured = capacity of iron to bind
❖ when we lack in iron, the storage is the first that will ➢ we measure the capability so that when the
compensate and help in releasing iron TIBC value is high, iron is also high (this iron
❖ ex. kapag nabawasan ng iron, mangangailangan yung is added to saturate transferrin in the
RBC ng iron galing sa storage sample)
❖ iron is lower in female because of menstruation ➢ ex. level of transferrin is high, real iron level
is low
2. Free Erythrocyte Protoporphyrin (FEP) ▪ specimen requirements:
▪ protoporphyrin + zinc = FEP o sample: non-hemolyzed serum
▪ protoporphyrin combines with zinc when the iron is o collection: fasting sample
low or decreased ▪ RR: 250-450ug/dL
▪ iron +protoporphyrin = heme ▪ relationship of TIBC to iron: indirect
▪ compound in w/c ferrous iron is added to form heme o decreased iron = increased TIBC
▪ increased in both IDA and ACD ▪
▪ mixed in sideroblastic anemia
▪ normal in thalassemia 5. Transferrin Saturation
▪ FEP cannot differentiate IDA and ACD because ▪ calculated from serum iron and TIBC values
both have increased FEP in this kind of disorder ▪ principle: % transferrin saturation =
𝑠𝑒𝑟𝑢𝑚 𝑖𝑟𝑜𝑛
× 100
𝑇𝐼𝐵𝐶
▪ principle: measuring using hematofluorometer or
▪ RR: 22-55% saturation
extraction & fluorescence
▪ Reference Range: < 50 ug/dL of RBC Iron Stage 1 Stage 2 (Iron Stage 3
Replete (Iron Deficient (IDA)
3. Serum Iron (Normal) Deplete) Erythropoiesis)
Serum ↓ <12
❖ iron in plasma: iron binds in transferrin ferritin
>12 ↓ <12 ↓ <12
❖ to measure the iron, you need to remove transferrin Marrow iron 2-3 ↓ 0-1 ↓0 ↓0
first by adding 0.05 N HCL then add chromogenic TIBC 300-360 360 ↑ 390 ↑ 410
reagent in order to reduce the form. Reduced Serum iron 65-165 115 ↓ <60 ↓ <40
ferrous will undergo spectrophotometer at 562 nm. Transferrin
20-50 30 ↓ <15 ↓ <10
sat
That will now be equivalent to serum iron FEP <50 <50 ↑ 100 ↑ 200
❖ low serum iron = low iron in the body Marrow ↓ <10
40-60 40-60 ↓ <10
▪ principle: serum ferric iron removed from transferrin sideroblasts
Hgb, MCV, ↓ Microcytic,
(0.05 N HCL) RBC NORMAL NORMAL NORMAL Normo /
o iron + chromogenic reagent (acid-ferrozine) = morphology hypochromic
reduced ferrous ❖ stage 1
o spectrophotometer at 562 nm ➢ store is the first to manifest (first to decrease)
▪ specimen requirements: ➢ storage is deficient but other factors/tests are still normal
o sample: non-hemolyzed serum ❖ stage 2
o collection: morning & fasting sample ➢ TIBC up to marrow sideroblasts are now defective/ value
▪ RR: 50-150ug/dL; M: 125 F: 100 is abnormal
➢ all are defective EXCEPT morphology
❖ hemolyzed = false increase because when the ❖ stage 3
sample is hemolyzed, Hgb releases. When Hgb ➢ even the morphology is defective
releases, iron also releases. It will now be added to
the measurement in the serum iron value • Laboratory Diagnosis
❖ anticoagulated plasma (EDTA or Heparin) = ▪ CBC: decreased Hct & Hgb (<8 g/dL)
false decrease. EDTA and Heparin decreases iron ▪ Blood indices: decreased MCV, MCH, MCHC
content because they bind with iron. When ▪ PBS: Microcytic, hypo/normochromic
measured, it will result to falsely decreased ▪ RDW: increased (you will observe elongated cells, tailed
❖ morning sample is collected sample because elliptical cells, microcytes)
evening sample is <25% ▪ Platelet count: Low, Normal, or High
❖ fasting: 12-24 hours no intake of iron medication ▪ RPI: decreased (<2.0)
o BM cannot compensate without sufficient iron stores
4. Total Iron Binding capacity o decreased because this is a maturational disorder,
▪ additional test for diagnosis of iron metabolism meaning you have defective BM
disorder ▪ BM:
▪ principle: indirect measurement of the ability of o Prussian Blue Reactivity is low
serum transferrin to bind with iron ➢ Prussian Blue is used for iron stain
o transferrin is saturated by adding ferric iron to o Prussian blue reactivity will also estimate the iron
serum stores
➢ we are now measuring ferric iron that binds
with transferrin
2. Anemia of Chronic Disorders
• in association with infectious, inflammatory or malignant dis.
>1-months duration (which results to mild to moderate
anemia)
• secondary to type of disorders, it can be infectious,
inflammatory or malignant
• Mild to moderate anemia
• Infectious:
▪ Tb
▪ Pulmonary infection
▪ Pelvic inflammatory disease
▪ Chronic fungal disease ❖
▪ Osteomyelitis
▪ meningitis
• Non-infectious: 2. Shortened erythrocyte survival
▪ Rheumatoid Arthritis (RA) ▪ non-specific stimulation of macrophages causes to
▪ Thermal injury increase activity
▪ SLE ▪ low iron store (macrophage)
▪ Myocardial infection ▪ low RBC survival
• Malignant:
▪ Carcinoma, Hodgkin’s disease 3. Impaired marrow response to shortened red cell
▪ Lymphosarcoma, Leukemia life span
▪ EPO not increased → diminished erythropoiesis
• Causes: ▪ inflammatory cytokines (such as tumor necrosis
1. Decrease iron release from storage macrophage factor-a and interleukin-1 from activated
▪ interleukin-1 / leukocyte endogenous mediator macrophages and interferon-g from activated T
o stimulate synthesis of APR apoferritin cells

❖ increased apoferritin trap iron in macrophage • Laboratory diagnosis

❖ reduced amount of iron circulating to marrow ▪ CBC: Slightly decreased Hct & Hbg (<1-2 g/dL)
sideroblast ▪ PBS: Normocytic, normochromic, hypochromic anemia
❖ decreased serum iron and marrow sideroblast ▪ Blood indices: decreased MCHC
▪ Reticulocyte count: N or slight increased
❖ we can’t release iron in macrophage ▪ RPI: <2.0; hypoproliferative
❖ why is there iron in macrophage then? ▪ BM: Prussian blue stain positive (Prussian blue is an iron
➢ we have lysed RBCs in the circulation, stain)
macrophage is one of the cells that engulfs o Increased serum ferritin (serum ferritin is from
destroyed RBC macrophage)
➢ the iron came from the engulfed RBC o Decreased sideroblasts
❖ some of the macrophages in our body, which are
responsible for engulfing these damaged RBCs 3. Sideroblastic Anemia
contains iron • excess accumulation of iron deposited in mitochondria and
❖ macrophages release the iron, but in the chronic normoblasts
disorder cases, macrophage can’t release the iron • Prussian blue stain: ringed sideroblast
❖ sideroblast are immature RBCs that contains • 3 types:
apoferritin (stored iron) ▪ Type I
❖ kapag kulang tayo ng iron sa katawan, isa sa unang o ferritin aggregates (4) per cell randomly distributed
bumababa ay yung stored iron (located in marrow ▪ Type II weak to Prussian
sideroblast) o ferritin aggregates (6) per cell Blue stain
❖ if you have anemia of chronic disorders, isa sa unang ▪ Type III
mababawasan sa katawan natin ay yung mga o pathologic ringed sideroblasts or sideroblastic
marrow sideroblast kasi nau-used up na yung mga anemia
stored irons natin o larger granules situated in the ring or collar around
❖ in this case, the normal/increased iron storage are for nucleus of normoblast (for dx: >15% of normoblast)
macrophages not for marrow sideroblast
❖ in this case, macrophages can’t release iron • Hereditary
▪ X-linked recessive trait
o ALA-synthase deficiency
▪ Autosomal recessive trait
• Acquired
▪ Idiopathic
o Acute myeloid leukemia
o Myelodysplastic syndromes
 o Myeloma ▪ chronic leg ulcers, gall stones, spherocytes and
▪ Secondary to drugs or toxins stomatocytes in PBS
o Alcoholism • Laboratory Diagnosis
o Lead poisoning ▪ CBC: decreased Hgb > 10 ug/dL
o Chloramphenicol ▪ Increased: OFT, B1, LDH, urobilinogen, MCHC, Retics
o Isoniazid, cyclosporin, Pyrazinamide ▪ Decreased: haptoglobin, MCV
▪ Normal: RDW
• Laboratory Diagnosis (hereditary) ▪ Autohemolysis after 48hrs at 37C
▪ CBC: decreased Hgb (6.0 g/dL) ▪ *Splenectomy may be beneficial
▪ PBS: Microcytic, hypochromic ▪ Inherited disorder
▪ RBC morphology: anisocytosis & poikilocytosis with ▪ Defective membrane protein skeleton structure,
target cells, basophilic stippling elongated elliptical cells
▪ dimorphic blood picture ▪ Variant: hereditary pyropoikilocytosis
▪ high: serum iron, serum ferritin, transferrin saturation
▪ normal TIBC 5. Thalassemia
▪ N/ low: FEP • causes:
▪ BM: ▪ reduced production of globin chains
o erythroid hyperplasia with excessive iron in ▪ formation of structurally abnormal Hb
macrophages
o 40% normoblasts (pathologic ringed sideroblast with
rings in the polychromatophilic or orthochromatophilic
normoblast

• Laboratory Diagnosis (acquired)

▪ CBC: decreased Hgb (7-10 g/dL)
▪ PBS: Normocytic/ sl. macrocytic, hypochromic with
anisocytosis & poikilocytosis
▪ RBC morphology: RBC fragments, target cells,
5.1 ⍺-thalassemia
basophilic stippling
▪ Dimorphic blood picture • Nomenclature:
▪ High: serum iron, serum ferritin, transferrin saturation, ▪ Normal haploid: αα
FEP ▪ Thalassemia: gene deletion
▪ (+) PAS; Pappenheimer bodies o One: -α (α thalassemia 2 or α+thalassemia )
▪ BM: o Two: -- (α thalassemia 1 or α° thalassemia)
o erythroid hyperplasia with M:E ratio 1:1 Genotype Description Disorder
o 95% normoblasts (pathologic ringed sideroblast with αα/αα Normal None
-α/αα Heterozygous Silent carrier
rings in the polychromatophilic or orthochromatophilic
-α/-α Homozygous α-thalassemia
normoblast)
--/αα Heterozygous minor
CS Hb H/ Constant
--/α α Heterozygous
Spring Disease
--/-α Heterozygous Hb H disease
Barts Hydrops
--/-- Homozygous
fetalis

Red Cell Hb
Genotype Disorder
Morphology Electrophoresis
αα/αα None Normal Normal
-α/αα Silent carrier Normal / SI
-α/-α α-thalassemia ↓ MCV, MCH Normal
--/αα minor ++ Hb H incl.
↓ MCV, MCH Hb A, H (2-40%)
--/-α Hb H disease +++ Hb H ± Hb Barts Hb
incl. A2
Barts Hydrops ↓ MCV, MC Hb Barts (80%)
-/--
fetalis ↑ NRBC Hb Portland
4. Hereditary Spherocytosis
• trait in whites
Genotype Demographics Anemia LE
• Causes: Asians, Chinese,
1. defective binding of spectrin to 4.1 Silent carrier None
Filipinos
▪ autosomal dominant Both: Southeast
2. deficient synthesis in spectrin Asians, Chinese,
Filipinos Normal
▪ autosomal recessive α-thalassemia
Homo: Med. None/Mild
minor
Blacks
• Clinical Features: Hetero: rare in
▪ jaundice, splenomegaly, skeletal abnormalities blacks
 Hb H/ CS Orientals
None/Mild
▪ (δβ)°/ (δβ)°
Disease Med. populations o Deletion of δ and β structural genes found in
Southeast Asia, chromosome 11
Hb H disease Med. Islands, Moderate
Middle east
Southeast Asia, Thalassemia minor
Barts HF Med. Islands, Severe Fatal • Genotypes:
Middle east
▪ Hetero β° (β°/ β) or β+ (β + / β)
o High-Hb A2 thalassemia
Hb H- Constant Spring Disease
o Combination of normal β gene + either β+/ β°
• caused by compound hetero inheritance of Hb CS ▪ Hetero δβ: (δβ)°/ β
• and α°thalassemia (--/αCSα) ▪ Hetero Hb Lepore: (δβ) Lepore / β
• Hb CS ▪ Heterozygous βSC
▪ 2 β chains + 1 normal α chain + 1 abnormal α chain (172 Red Cell Hb
Diagnosis RBC Count
aa) Morphology Electrophoresis
• deficit in normal α chain MCV, MCH
Thalassemia ++ stippling Hb F Var. Hb A2
• when inherited with double α gene deletion major +++ NRBC & ± Hb A
▪ Hb H like disorders target
MCV, MCH
Hb H Disease Thalassemia + stippling N/ Hb F V. Hb
intermedia ± NRBC A2 ± Hb A
• caused by deletion of 3 of 4 globin chains (--/-α) ++ target cells
• non deletional forms: (ααT/ ααT) and (ααT/--) Thalassemia
MCV, MCH
N/ Hb F V. Hb
+ stippling &
minor A2 & Hb A
target
Barts Hydrops Fetalis Normal/ Sl.
• caused by deletion of 4 α globin chains (--/--) Thalassemia MCV & MCH,
N N
▪ high affinity to oxygen minima ± stippling &
target
▪ not effective release of oxygen to tissues
▪ fatal
5.3 Hereditary Persistence of Fetal Hemoglobin
• Hb Portland
• increased Hb F in adults in the absence of usual clinical and
▪ survival into 3rdtrimester of fetal life
hematologic features of thalassemia
5.2 β-thalassemia • deletion/ inactivation of δ and β structural gene complex
• lack/reduced production of beta chains, excess of alpha • compensatory persistence of γ chain into adult
chains • Categories:
• massive imbalance: severe erythrocyte dysfunction ▪ Pancellular
o RBCs contain increased levels of Hb F (acid elution
• result: ineffective erythropoiesis
slide test)
• Classifications:
▪ Heterocellular
▪ Thalassemia major
o only subpopulation of RBCs contain inc. levels of Hb
o Severe anemia with iron overload
F
▪ Thalassemia intermedia
o British, Georgia, Swiss, Atlanta, Seattle
o Moderate anemia
▪ Thalassemia minor
o Asymptomatic; may or may not produce mild anemia Laboratory Diagnosis
▪ Thalassemia minima a. Hemoglobin electrophoresis
o No detectable clinical abnormalities • Cellulose acetate (alkaline medium)
▪ Separates Hb variants (screening)
Thalassemia major ▪ Hb Barts, Hb CS, Hb Lepore
• Citrate agar (acid pH)
• Genotypes:
▪ Useful in differentiating abnormal hemoglobins w/c
▪ β°/ β°
migrate together on cellulose acetate (Hb Lepore &
▪ β+/ β+
Hb S)
▪ β°/β+
▪ δβ (Lepore)/δβ (Lepore)
b. Quantitation of Hb F
o Hb Lepore
➢ 2 normal alpha chains + 2 abnormal non-alpha • Significantly increased
chains formed by fusion of N-ter end of δ chain and ▪ Homo β°and β+ (Mediterranean form), δβ
C-ter end of β chain thalassemia, Hb Lepore, pancellular HPFH
➢ Baltimore, Boston, Hollandia • Moderate/ Sl. elevation
▪ Thalassemia minor and Heterocellular forms of
HPFH
Thalassemia intermedia
• Genotypes: c. Brilliant Cresyl Blue Stain for Hb H
▪ β+/ β+
• induce precipitation of intrinsically unstable Hb H
o Americans and African blacks
• Hb H inclusion: denatured beta globin chain
o Less impairment of β chain synthesis than med. form
 ▪ small, multiple, irregular shaped greenish blue o Tuberculosis
bodies with pitted golf ball appearance • Severe:
▪ (+): Hb H disease, α-thalassemia trait, silent α- ▪ BM cellularity
thalassemia o <25% of normal or <50% of normal cellularity w/
<30% hematopoetic cells
d. Acid Elution Slide Test for Hb F • PLUS any 2 of the following:
• differentiate the intracellular distribution of Hb F in ▪ Neutrophil count: <500/uL / <0.5x109/L
thalassemia (non-uniform) with increased Hb F in ▪ Platelet count: <20000/uL / <20x109/L
pancellular HPFP (uniform) ▪ Reticulocyte count: <10000/uL / <1%
• Hb F: bright pink to red (infants) ▪ Treatment
▪ ”ghost cells”: only outer membrane is visible (adult) o BM transplantation
o blood transfusion won’t work because the bone
Normocytic, Normochromic marrow is not responding and even the stem cells are
1. Anemia of Marrow Failure affected
2. Anemia of Chronic Renal Disease
3. Hemolytic Anemia 1.2. Pure Red Cell Aplasia
4. Acute Blood Loss • decreased RPI because BM is not responding
• hypoplasia of erythrocyte precursors only
1. Anemia of Marrow Failure • severe anemia with reticulocytopenia (↓ retics)
1.1. Aplastic Anemia • only RBC precursor decreases
• Pancytopenia • normal cellularity
▪ decrease in all cellular constituents in peripheral blood • BM: absence of erythroid precursors with normal myeloid
and bone marrow (WBC) and platelet elements
• Decreased retics • clinical findings:
• Hypocellular marrow ▪ pallor, splenomegaly, hepatomegaly
▪ thrombocytopenia • associated with:
▪ neutropenia ▪ hemolytic anemia, parvovirus inf., drugs, thymoma
• depletion of hematopoietic stem cells • Diamond-Blackfan anemia
• Acquired
MAA SAA VSAA
▪ Primary
Bone Hypocellular bone Bone marrow Same as SAA
Marrow marrow plus at least cellularity o Idiopathic
two of the following: <25%* plis at ➢ we don’t know the cause
least two of the o Immune mechanism
following: ➢ immunoglobulin inhibitor to RBC precursors
Neutrophils 0.5-1.5 0.2-0.5 <0.2
➢ EPO inhibitor
(x109/L)
Platelets 20-50 <20 Same as SAA ▪ Secondary
(x109L) o Benign thymoma, drugs, chemicals, infections, HA
Other HGB ≤ 10 g/dL plus Reticulocytes Same as SAA (aplastic crisis)
reticulocytes <20x109?L or • Inherited
<30x109L <1% corrected
▪ Diamond-Blackfan anemia
for HCT
**MAA: moderate aplastic anemia ; SAA: severe aplastic anemia o congenital
; VSAA: very severe aplastic anemia
1.3. Myelopthisic Anemia
1.1. Aplastic Anemia • results when BM is replaced with abnormal cells (metastatic
• decreased RPI because BM is not responding carcinoma)
• Primary • infiltration of abnormal cells BM not responding
▪ Congenital Fanconi Anemia (rare) • found in patients with carcinoma
▪ Acquired idiopathic (no known precipitating factors) • decreased RPI because BM is not responding
▪ most common cause: Chlorampphenicol • hypoproliferative anemia
• Secondary • degree of anemia is correlated with tumor burden
▪ Drugs • used interchangeably with leucoerythroblastic reaction
o antibacterial, anti-inflammatory, diuretics, ▪ presence of NRBCs and immature leukocytes in PB
anticonvulsants, antithyroid, oral hypoglycemic, ▪ not synonymous
antimalarial
▪ Chemicals
o Benzene and derivatives, Hydantoins, Sulfonamide, 2. Anemia of Renal Disease
Gold preparations • decreased RPI because BM is not responding
▪ Radiation • hypoproliferative anemia (erythroid)
o Chlordane, chlorerophenothane (DDT), Lindane • occurs inpatients with chronic renal failure
▪ Immune mechanism • failure of the renal production and release of EPO (growth
▪ Infection factor for erythroid maturation)
o Viral • decreased EPO poduction
o Hepatitis C • anemia + increased BUN
 ▪ chronic leg ulcers, gall stones, spherocytes and
3. Hemolytic Anemia stomatocytes in PBS
• shortened red cell survival • Laboratory Diagnosis
• RBC destruction > RBC production ▪ CBC: ↓ Hgb > 10 ug/dL
• intracorpuscular or extracorpuscular defects ▪ Increased: OFT, B1, LDH, urobilinogen, MCHC, Retics
• Classification: ▪ Decreased: haptoglobin, MCV
▪ Intrinsic: defect in RBC (membrane, enzyme [G6PD, ▪ Normal: RDW
pyruvate kinase], hemoglobin [globin]) ▪ Autohemolysis after 48hrs at 37C
▪ Extrinsic: RBC damaged from external forces ▪ *Splenectomy may be beneficial
▪ Intravascular – RBC lysis inside the blood vessels ▪ Inherited disorder
▪ Extravascular – RBC lysis outside the blood vessels ▪ Defective membrane protein skeleton structure,
elongated elliptical cells
Predominantly ▪ Variant: hereditary pyropoikilocytosis
Predominantly
Macrophage-
Fragmentation
Mediated
(Intravascular
(Extravascular 3.1.2 G6PD Deficiency
Hemolysis
Hemolysis) • X-linked disorder
Agents from Outside the RBC
• Immune hemolysis cold • Immune hemolysis
• Inability to neutralize oxidation stress
antibody warm antibody • reduced NADPH not formed
• Microangiopathic • Hemoglobin molecule is unstable, susceptible to hemolysis
hemolysis
• Infectious agents, as in • Rapid intravasular destruction
Extrinsic Acquired
defects
malaria
conditions • Aging cells are more susceptible to destruction
• Thermal injury
• Chemicals/drugs
• Venoms
• Prosthetic heart valve
Membrane Abnormalities
• Spur cell anemia of
severe liver disease
(ED) • Hereditary
membrane defects
• Paroxysmal nocturnal (HD) [spherocytosis,
Intrinsic hemoglobinuria (ID) elliptocytosis] Hereditary
• Clinical Patterns
defects Abnormalities of the RBC Interior conitions ▪ neonatal jaundice
• Enzyme defects such ▪ congenital hemolytic anemia
as G6PD deficiency
• Globin abnormalities
▪ drug-induced hemolysis (primaquine, phenylhydrazine
such as sickle cell etc)
disease, thalassemia ▪ favism (Mediterranean enzyme variant)
• ***May confer resistance to Malaria
3.1 Intrinsic Hemolytic Anemia • Laboratory Diagnosis
• Hereditary defects ▪ CBC: ↓ Hb (3-4 g/dL)
▪ Abnormalities of red cell membrane ▪ Increased
o Spherocytosis, Elliptocyrosis o reticulocytes, plasma Hb, bilirubin, serum LDH,
▪ Inherited RBC enzyme defect uroilinogen
o G6PD def., pyruvate kinase def ▪ PBS: normo/ normo; polychromasia, poikilocytosis,
▪ Disorders of Hgb production some spherocytes & “bite” cells (acute hemolytic
o hemoglobinopathies (sickle cell, Hgb C) episodes)
o thalassemia (alpha, beta) o (+) Heinz bodies:
• Acquired defects: ➢ ↓ reduced glutathione → ↑ oxidative stress
▪ Paroxysmal Nocturnal Hemoglobinuria (PNH) ➢ (+) supravital stan: ex. Romanowsky
o it is a complement-mediated lysis ▪ Hemoglobinuria & hemosiderinuria
▪ Decreased haptoglobin
3.1.1 Hereditary Spherocytosis ▪ Negative Coomb’s test
• defect in RBC membrane o Antiglobulin test
• trait in whites ➢ used to R/O immune related hemolysis
• Causes: ▪ we use G6PD fluorescent spot test: screening test
• defective binding of spectrin to 4.1 ▪ Confirmatory: G6PD Assay
▪ autosomal dominant History Recent infection, Direct Negative
administration of antiglobulin
• deficient synthesis in spectrin drugs associated test result
▪ autosomal recessive with hemolysis,
• NOTE: or ingestion of
fava beans
▪ spectrin is: Clinical Chills, fever Indicators of ↓ Serum haptoglobin
o part of cytoskeleton of RBC manifestations headache, hemolysis (severe)
nausea, back ↑ Serum lactate
o responsible for shape, flexibility of RBC pain, abdominal dehydrogenase
• Clinical Features: pain
▪ jaundice (↑ bilirubin in plasma), splenomegaly (because
Jaundice ↑ Serum indirect
of compensation), skeletal abnormalities bilirubin
 Dark urine ↑ Plasma hemoglobin Amino acid S β 6 A3 Glu → Val
Complete ↓ Hemoglobin Hemoglobinuria substitution
blood count (moderate to Amino acid •C •β •6 • A3 • Glu → Lys
results severe) deletion • D-Los •β • 121 • GH4 • Glu → Gln
↓ Reticulocyte Angeles
count • D-Punjab
Selected ↓ G6PD activity (mild •E •β • 26 • B8 • Glu → Lys
Peripheral additional to severe), may be • O-Arab •β • 121 • GH4 • Glu → Lys
blood film tests falsely normal as a • Gun Hill •β • 91-95 • F7-FG2 • NA
findings result of Amino acid Constant α NA NA NA
Direct reticulocytosis, elongation Spring
anticoagulant leukocytosis, or Globin chain Lepore- δβ NA NA NA
test result thrombocytosis and in fusion Baltimore
individuals with mild
deficiencies

DNA-based mutation
detection usually 1. Sickle Cell Anemia
needed to identify • Two types:
heterozygous
females
▪ homozygous:
o 2 gene inherited (SS): sickle cell disease
Heinz bodies o more severe manifestation
observed on
supravital stain ▪ heterozygous:
o 1 normal and 1 hemoglobin S (SA): sickle cell trait
3.1.3 Pyruvate Kinase Deficiency
• E-M pathway Hb S
• Autosomal recessive disease (rare) • most common abnormal hemoglobin
• effect is more profound in older cells • normal glutamic acid at 6thposition in the β chain is replaced
• failure to generate sufficient ATP by valine
▪ results in defective control of ions (Na+& Ca +enter cell) • Results in:
▪ damage to membrane phospholipid ▪ altered solubility
▪ **reticulocytes - mitochondrial oxidative phosphorylation ▪ altered ability to withstand oxidation
o (+) ATP ▪ instability
• Increase 2,3-DPG ▪ increased propensity for methemoglobin production
▪ because of ↑ ATP ▪ increased or decreased oxygen affinity
▪ shift to the right: ↓ oxygen affinity ↑ oxygen release • Hb A is lacking, Hb S is present
• Jaundice and splenomegaly • Sickling is increased
• Confirmatory test: PK Assay ▪ low oxygen tension
▪ low pH
▪ increased body temp

Homozygous S (SS)
• lifelong, severe, hemolytic anemia
• Sickle cell crisis:
▪ rigid sickle cells increase blood viscocity
▪ aplastic crisis, vaso-occlusive episodes, prone to
infection (pneumococcus), splenic sequestration, bone
and joint pain
▪ organs affected: liver, heart, spleen, skin, lungs, kidney

❖ PK deficiency: Sickle cell trait

➢ mature RBC: (-) ATP because of absence of PK • Heterozygous (AS)
➢ early retics: (+) ATP because of mitochondria • usually with no apparent disease
• protected from Plasmodium falciparum infection
3.1.4 Hemoglobinopathies • normal PBS
1. β-chain substation • (+) sickle cell solubility test because you still have 1 S
• Sickle cell anemia, Sickle cell trait, Hb S Thalassemia (positive basta may S)
• Hb C disease, Hb SC, Hb D, Hb SD, Hb E, Hb O-arab
2. Multiple AA substitution Laboratory Diagnosis
• Hb C-Harlem • PBS:
3. α-chain substitution ▪ target cells, ovalocytes, schistocytes are visible
• Hb G-Philidelphia ▪ presence of inclusions like Basophilic stippling, Howell-
4. AA acid deletion Jolly bodies, and Pappenheimer bands
• Hb Gun Hill ▪ marked poikilocytosis, anisocytosis, inclusion bodies,
Hemoglobin
Amino
Helical sickle cells (6-18%)
Molecular Affected Acid β-chain
Abnormality
Common
Chain Numbers
Numbers
Substitution
▪ increased WBCs, platelets, retics, nRBC
Name Affected
Affected • Bone Marrow:
▪ BM is responding ; RPI: >3.0
 ▪ marked erythroid hyperplasia: M:E ratio 1:1 (myeloid to • Cellulose acetate: migrates with Hb S
erythroid normal ratio is 4:1)
▪ high iron storage 3.2 Extrinsic Hemolytic Anemia
• Blood Chemistry: • Non-Immune
▪ increased B1, serum iron ▪ mechanical
• Hgb S solubility test (+) ▪ microangiopathic
▪ All patients with Hb S genotype, (+) in sickle cell solubility ▪ chemical & toxic agents
screening test ▪ infections
▪ Hb SS, AS, S-thalassemia, SC ▪ hypersplenism
• Hgb electrophoresis shows Hgb S ▪ systemic disease
• Hb D-Los Angeles, G-Philidelphia, Q-India migrate in the • Immune
same position as in cellulose acetate ▪ *Auto – own antibody
• OFT: decreased o primary
• Half-life of RBC with Hb S: 10-20 days o secondary
o drug induced
2. Hemoglobin C Disease o infections
• resembles Hb S but instead of valine, lysine is seen on the ▪ *Allo – antibody from donor
6th position of beta chain o transfusion reactions
• mild hemolytic anemia & splenomegaly
• Hb C crystals and clam shaped cells 3.2.1. Microangiopathic Hemolytic Anemia
▪ Hb CC tend to crystallize when dehydrated • acquired extracorpuscular defect
▪ 2ndmost common after Hb S • fragmentation syndrome
• Hb AC • microangiopathic means disease of small blood vessels
▪ Asymptomatic • caused by:
▪ No anemia ▪ fibrin deposition in blood vessels
▪ severe hypertension
3. Hb SC Disease ▪ vessel abnormalities
• Inherited Hb S and Hb C
3.2.2. Disseminated Intravascular Coagulation
4. Hemoglobin D • occurs after extensive damage to vessels or exposure to
• 16 beta chain variants and a 6 alpha chain variants compounds that initiate clotting
• Migrate in same position as Hb S • fibrin deposition in vessel lumen → RBC fragmentation
• Homozygous (Hb DD) and heterozygous (Hb AD) • no age preference
▪ Hb D-Los Angeles
o Hb D-Punjab 3.2.3. Thrombotic Thrombocytopenia Purpura
o Glycine is substituted for glutamic acid in • seen in young adults
121stposition of beta chain • also known as “Moschkovitz syndrome”
▪ Hb SD
o Hb SD-Los Angeles 3.2.4. Hemolytic Uremic Syndrome
• involves acute intravascular hemolysis and renal failure
5. Hemoglobin E
• seen in young children
• Beta chain in which lysine is substituted for glutamic acid in
the 26th position
3.2.4. Paroxysmal Nocturnal Hemoglobinuria
• Hb EE and Hb AE exist
• complement mediated lysis
• 3rd most common
• acquired intracorpuscular defect
• BM stem cells show abnormal sensitivity to complement in
6. Hb O-Arab
acidified plasma or low ionic strength environment
• Same substitutions as Hb C
• result: complement mediated lysis
• Glutamic acid is replaced by lysine in 121stposition
• all erythrocytes, granulocytes & platelets have defects
• Migrates same as Hb A2, C and E
• Types of sensitivity in erythroid cells:
▪ PNH I - react normally in presence of complement
7. Hemoglobin C-Harlem
• Hb C-Georgetown ▪ PNH II - 2-5X sensitivity
▪ PNH III - 15-25x
• 2 AA acid substitution
• increased susceptibility to complement mediated red cell
• occurs at 6th(same as Hb S) and 73rdposition (aspartic acid;
lysis
asparagine)
• Morphologic:
• Hb C-Harlem, Hb C-Ziguinchor, Hb S-Travis
▪ EM: protuberances on the red cell surface
▪ (+) sickle solubility test
▪ Cellulose acetate: migrates with Hb S • Chemical abnormalities:
▪ deficient acetylcholinesterase activity & abnormally
8. Hb G-Philadelphia constituted glycoproteins
• Replacement of asparagine by lysine in 69thposition
3.2.4. Paroxysmal Nocturnal Hemoglobinuria
• (-) sickle solubility test
• Rare, acquired disorder
• Characterized by proliferation of abnormal clones of • when the red cells are warmed, these antibody caused lysis
hematopoietic cells in the BM in the presence of complement.
• Manifested by hemosiderin in urine after a night’s sleep • Laboratory findings:
• Laboratory Diagnosis ▪ elevated Reticulocyte count
a. Sugar of Sucrose Lysis Test ▪ Increased concentration of Indirect Bilirubin
▪ screening ▪ Hemoglubinuria
▪ excessive hemolysis when exposed to low ionic ▪ Positive Donath-Landsteiner of Rosenbach or
strength solution • Ehrlich or Sanford test
b. Ham’s acid hemolysis test ▪ Positive for methemalbumin
▪ confirmatory
▪ excessive hemolysis when exposed to complement 4. Blood Loss
containing serum at low pH 4.1. Hemorrhagic Anemia
• Hemorrhage
3.2.5 Immune Hemolytic Anemia ▪ body adjusts, increased heart rate
Autoimmune Hemolytic Anemia ▪ expanding circulatory volume
•Warm autoimmune hemolytic anemia (WAIHA) ▪ **hematologic response to acute blood loss
▪Idiopathic ▪ increase plt, circulating granulocytes
▪Secondary
o Lymphoproliferative disorders ▪ increased EPO levels in 6 hours
o Nonlymphoid neoplasms ▪ reticulocytosis in 24 hours
o Collagen-vascular disease
o Immunodeficiency disorders Macrocytic Anemia
o Viral infections
• Megaloblastic Anemia
•Cold agglutinin disease (CAD)
▪Idiopathic ▪ Vit B12 deficiency: Pernicious, Non-pernicious
▪Secondary ▪ Folate deficiency
o Acute: infections (Mycoplasma pneumoniae, infectious • Non-Megaloblastic Anemia
mononucleosis, other viruses) ▪ Anemia of Liver Disease
o Chronic: lymphoproliferative disorders
•Paroxysmal cold hemoglobinuria
▪Idiopathic 1. Megaloblastic Anemia
▪Secondary • Deficiency in:
o Viral infections ▪ Vitamin B12 (Cobalamin)
o Syphilis ▪ Folic acid
•Mixed-type autoimmune hemolytic anemia
Alloimmune Hemolytic Anemias • Defective DNA production, abnormal nuclear and
•Hemolytic transfusion reaction (HTR) cytoplasmic development and the production of large,
•Hemolytic disease of the fetus and newborn (HDFN) dysmature megaloblstic cells
Drug-Induced Immune Hemolytic Anemia • Earliest signs: hypersegmented PMNs, oval macrocytes
•Drug dependent
•Drug independent 2. Vitamin B12 Deficiency
• Intrinsic factor: needed to absorb Vit. B12 in terminal ileum
• Autoimmune hemolytic anemia (AIHA) • Pernicious anemia: most common cause
• Ab’s directed to antigens present in own erythrocytes ▪ Ab’s against intrinsic factor
▪ Warm autoimmune hemolytic anemia • Lack of IF from parietal cells of gastric mucosa
▪ Cold autoantibody hemolytic syndrome ▪ gastrectomy, atophic gastritis
▪ Cold agglutinin disease ▪ Veganism
▪ Paroxysmal cold hemoglobinuria ▪ D. latum infection

3.2.5.1. AIHA associated with cold antibody 3. Folate Deficiency

• due to Ig M cold reactive antibodies • caused by:
▪ react best at temp below 320C ▪ dietary (most common)
▪ occur in association w/ infection, malignancy or ▪ intestinal malabsorption
autoimmune disorders ▪ increased demand
• Laboratory findings: ▪ excess loss
▪ Direct Antiglobulin test is positive / Coomb’s ▪ defective synthesis
▪ Cold agglutinins titer is increased
• Peripheral smears:
Laboratory Diagnosis
▪ polychromatophilia
• CBC
▪ spherocytosis
▪ macroovalocytes
▪ agglutination of red cells
▪ WBCs and platelets are low
o agglutination occurs because of RBC antibodies
▪ hypersegmented PMNs
▪ Low retics
3.2.5.2. Paroxysmal Cold Hemoglobinuria
• BM
• hemolysis occurs when blood is warmed after previous
▪ marked erythroid hyperplasia
exposure to chilling
• Blood Chemistry
• anemia is caused by the presence of an autohemolysin in
▪ increased B1, serum iron and LDH
the plasma that becomes attached to the red cells in the cold
• Pernicious anemia ▪ iron deposits in liver
▪ Schilling test abnormal • Hemosiderosis
▪ Ab’s to gastric cells, IF ▪ secondary iron accumulation.
• Vit B12 ▪ iron deposits in parenchymal cells and Kupffer cells in the
▪ levels are low portal tract
▪ high urine methylmalonate
• Folic acid Lead Poisoning
▪ low levels • Adults-occupational
• Children-PICA
Liver Disease • *It depresses enzymes
• Non- megaloblastic anemia • lacks (↓) delta amino levulinic dehydratase causing ALA to
• Decreased cholesterol synthesis accumulate
• Spurr cells, acanthocytes • heme synthetase causing red cells to accumulate excessive
protoporphyrin
Erythrocytosis • Laboratory Diagnosis:
• Excess production of red cells ▪ Anemia (micro/normo)
• Primary Erythrocytosis ▪ Increased reticulocytes
▪ uncontrolled growth of cells for no apparent purpose ▪ Decreased OFT
• polycythemia vera ▪ RBC changes: _____
▪ Blood lead usually >40 ug/mL
Refractory Anemia ▪ BM: erythroid hyperplasia; ringed sideroblasts
• Abnormal cell production, unresponsive to treatment
• Myelodysplastic – may be pre-leukemia Disorders of red cell formation
• Hallmarks • Deficiency states
▪ ineffective erythropoiesis ▪ IDA, chronic disease, sideroblastic anemia, lead
▪ markedly hypercellular marrow poisoning, megaloblastic
▪ abundant abnormal erythroid precursors (megaloblastic) • Hypoproliferative
▪ aplastic anemia, renal dse, pure red cell aplasia
Differentials of Erythrocytosis • Ineffective erythropoiesis
• Primary Polycythemia: ▪ refractory anemia: uncontrolled proliferation
▪ PV
• Secondary: References:
▪ high altitude • Lotspeich-Steininger e.t al; Clinical hematology principles,
▪ CPD procedures, correlations, Lippincott Company, 1992
▪ cyanotic congenital heart disease • Turgeon, Clinical Hematology: Theory and Procedures 5ht
▪ low cardiac output ed., Lippincott Williams & Wilkins, 2012
▪ hypoventillation syndrome • Keohane et. al, Rodak’s Hematology: Clincal Principles 6th
• Relative erythrocytosis: ed., Elsevier, 2020
▪ dehydration, stress • DOH Dept Circ 2017-0173 Schedule of EQAS Application
▪ high affinity Hgb variants
▪ neoplasms-renal artery stenosis
▪ renal transplant
▪ renal cyst

Porphyrias
• Genetically acquired inborn error of metabolism
• Deficiencies of enzymes involved in porphyrin-heme
biosynthetic pathway
• Excessive build-up of precursor compounds
▪ ↑ D-ALA, porphobilinogen

Genetic Porphyrias
• Manifestations may be neurologic (excruciating pain and
other neurologic symptoms), cutaneous
• *Acute intermittent porphyria (AIP)
• Laboratory Diagnosis:
▪ Spectroscopy and biochemical analysis of blood, urine
and stool
▪ Urine phorphobilinogen is markedly elevated

Syndromes of Iron Overload

• Hemochromatosis
▪ inc. GIT iron absorption and systemic iron overload
Morphologic Classification of Anemia:

has problem with globin

(involved in Hgb synthesis)

• high RPI • low RPI

• effective BM • ineffective BM

has problem with iron

(involved in Hgb synthesis)

maturational disorder

• basis is MCV

Combined Physiologic and Morphologic Classification of Anemia

GRANULOPOIESIS • deep within the cords
• form sinus to peripheral circulation (once they mature)
• both granulocytes and agranulocytes has granules
• granulocyte has more prominent granules which makes it easier to Regulators
bee seen under the microscope Growth Mature Cell
Cellular Source Progenitor Cell Target
• agranulocyte has granules but cannot be seen using the light Factor Target
microscope Erythropoietin Peritubular cells CFU-E, late BFU-E, None
• granules are used for identification (if it’s neutrophil, basophil, of the kidney, CFU-Meg
Kuffer cells
eosinophil)
IL-3 Activated T CFU-blast, CFU-GEMM, Eosinophils,
• lymphocytes CFU-GM, CFU-G, CFU- monocytes
M, CFU-Eo, CFU-Meg,
Hematopoiesis CFU-Baso, BFU-E
• production of blood cells G-CSF Monocytes, CFU-G Granulocytes
• Continuous and regulated process of cell production fibroblasts,
endothelial cells
▪ cell renewal
M-CSF Monocytes, CFU-M Monocytes
▪ cell proliferation fibroblasts,
▪ cell differentiation endothelial cells
▪ cell maturation GM-CSF T lymphocytes, CFU-blast, CFU-GEMM, Granulocytes
• BM → peripheral circulation monocytes, CFU-GM, CFU-G, CFU-
eosinophils, M, CFU-Eo, CFU-Meg,
▪ immature and precursor cells are found in the bone marrow monocytes, BFU-E
▪ from bone marrow going to → peripheral circulation fibroblasts,
endothelial cells
mesoblastic
• hematopoiesis is a controlled process because we have different
• Yolk sac → AGM region (Aorta-gonad mesonephros) → fetal liver growth factors
→ bone marrow • Colony-Stimulating Factors
hepatic ▪ high specificity for their target cells
▪ active at low concentration
medullary ▪ names indicate the predominant cell lines that respond to their
• granulokinetics – production until destruction presence
▪ G-CSF: influences for us to produce granulocytic cell line
▪ GM-CSF: granulocytic-monocytic cell line
▪ GM-CSF + IL3: megakaryocyte cell line

Marrow Differential
Cell Type Range (%)
Erythroblasts 18-24
Myeloblasts Type I 0-1
Myeloblasts Type II 0-2
Promyelocytes 1-4
PMN’s and precursors 53-63
Monocytes 0-2
Eosinophils and precursors 1-3
Basophils and precursors 0-1
Lymphocytes (B cells, T cells) 8-12
Plasma cells 0-2
• Polymorphonuclear leukocytes (PMNs) – mature cells (neutrophil,
eosinophil, basophil)

Granulocyte Maturation & Development

Red Marrow • granulocytes are neutrophil, eosinophil, and basophil
• active in the proliferation of red cells • CFU-GEMM – Colony forming unit: granulocyte, erythrocyte,
• locations of developing cells: megakaryocyte, macrophage
1. Erythroblasts • from myeloblast → promyelocyte → myelocyte → metamyelocyte →
• small clusters throughout the red band → segmented (mature cell)
marrow • myelocyte – is where differentiation starts (eosinophil myelocyte,
• mature forms: adjacent to the outer basophil myelocyte, neutrophil myelocyte)
surfaces of the vascular sinuses • Early cells:
❖ in cross section of BM, sinus is Pluripotent stem cells
where the immature cells are ↓
released if they are ready to myeloid stem cell (committed stem cell)
become mature cell ↓
2. Megakaryocytes CFU-GEMM
• adjacent to the outer surfaces of the vascular sinuses ↓
CFU-GM
• release of platelets into the lumen of the sinus
↓
❖ nasa gilid lang because they release platelets going to the
myeloblast →
sinus (platelet has no capability to travel on its own because
myelocyte
it doesn’t have nucleus and cellular organelles)
❖ naghahatid sa platelets papuntang sinus para ma-release
Granulopoiesis
sa peripheral circulation
• Granulocytic kinetics
3. Immature myeloid cells
▪ development, distribution, and
• metamyelocyte
destruction of NEB
 ▪ production and destruction • Basophils – 8.5 hrs
• BM
▪ 2 compartments found in bone marrow: Granulocyte Maturation & Development
o mitotic pool or proliferative compartment
o maturation storage compartment

Proliferative Compartment
• myeloblasts, promyelocytes, myelocytes
• capable of cell division
• myeloblasts, promyelocytes, myelocytes: continuous cell division
until maximum cell division (50x)
• metamyelocyte
▪ last stage of mitotic division (4-5x of cell division)
• based on picture:
▪ percentage means population in the BM
o progenitor cells 0.1-0.2% - population of cells in the entire
BM
▪ hours mean maturation period
o it takes 15 hours for myeloblasts to become promyelocytes 1. Myeloblast
o promyelocyte to myelocyte = 24 hours • earliest morphologically identifiable cell in granulocytic cell line
o myelocyte to metamyelocyte = 4.3 days • as it matures, nucleus decreases its size while cytoplasm increases
• myeloblast – first morphologically identifiable cell in the granulocytic • 10-18 um
cell line; earliest morphologically identifiable cell in the granulocytic • N:C ratio 4:1
cell line • Nucleus
▪ finely reticular chromatin
▪ 1-5 light-staining nucleoli
• Cytoplasm
proliferative compartment ▪ small rim of basophilic cytoplasm
▪ lacks granules
▪ Auer rods
o inclusion bodies
o aggregates of fused lysosomes
o appear as red, needle-like crystalline cytoplasmic inclusions
o indicator for myeloblast
Maturation Storage Compartment o differentiates myeloblast to promyelocyte
• it is normal to have Auer rods in myeloblast

2. Promyelocyte
maturation stage compartment • presence of prominent granulation
• N:C ratio 3:1
• Granules
▪ cytoplasm has primarily azurophilic (non-specific) granules
▪ contains enzymes myeloperoxidase and chloroacetate esterase
• metamyelocytes and bands are immature cells ▪ storage compartment of enzymes
• segmented cells are mature cells and called marrow reserved ▪ presence of enzymes in phagocytes for digestion of foreign
bodies
• neutrophils: 4-8 days of life reserved cells in bone marrow
• 14 to 20 um
Peripheral Circulation Compartments • Nucleus
• circulating pool and marginating pool ▪ more condensed
▪ nucleoli are present
• in cases of infection, these are the first one that will be consumed
• Cytoplasm
• Circulating pool
▪ pale grayish blue
▪ found in the bloodstream
▪ presence of primarily azurophilic granules
• Marginating pool
▪ lining the endothelium
3. Myelocyte
▪ adhere to the endothelium of the blood vessels
• where neutrophils, eosinophils, and basophils can be differentiated
▪ there are neutrophils in the endothelium and they are the first
cells to attack the foreign body invaders in the area of • appearance of secondary or specific cytoplasmic
inflammation granulation
• nucleoli are no longer visible
• Circulation pool to peripheral tissues • NEB became visibly recognizable
▪ diapedesis: passes, migration, or movement of lag cells to the • N:C ratio 2:1 or 1:1
tissues • 12 to 18 um
▪ Neutrophils act as phagocytes in the site of invasion of foreign • Nucleus
bodies ▪ more oval in appearance
• Segmented neutrophils – 7-10 hrs (lasts in the peripheral circulation) ▪ nucleoli are no longer visible
• Eosinophil – few hrs ▪ Chromatin more clumped
▪ Charcot-Leyden crystals: produced when there is an excessive • Cytoplasm
number of eosinophils due to: ▪ blue-pink (depends on the granules of specific cells)
o infection • Granules:
o damaged eosinophil ▪ Neutrophilic granules
o degenerated eosinophil
 o fine and stain a blue-pink color with Romanowsky stain 2. Promonocyte
(Wright stain and Giemsa stain) • proliferation: 2-3 mitotic divisions in 2-2.5 days
➢ Giemsa stain: for staining of bacterial cells and also • last stage of proliferation
human cells • maturation period until it becomes monocytes: 2-2.5
➢ Wright stain: for staining procedures of blood smears, days
urine samples, and bone marrow aspirates
o enzymes found: 3. Monocytes
➢ lysosomal hydrolase • No large reserve of cells in maturation-storage pool
➢ lysozyme ▪ monocyte becomes macrophage (tissue monocyte) (goes to the
➢ myeloperoxidase tissues)
▪ Eosinophilic granules • Circulating and marginating pool in PC (1:3.5)
o larger than neutrophilic granules, round or oval-shaped • maturation period: 8.5 hours
o orange and have a glassy or semi-opaque texture • Largest mature cells in the peripheral circulation
o two types of granules: ❖ largest cell in the bone marrow: megakaryocyte
➢ small round granules ❖ most abundant cell in the human body: macrophage
• present in few quantities and small sizes ❖ most abundant cell in the peripheral circulation and bone
• content is acid phosphatase marrow: neutrophil
➢ large crystalline granules • Irregular cytoplasmic outline
• elliptical shape • Commonly observed vacuoles
• present in large or greater amounts • blue-gray cytoplasm, with fine granulation resembling
• has myeloperoxidase and acid phosphatase
▪ Basophilic granules Lymphocyte Maturation & Development
o dark blue-black color and dense appearance 1. Lymphoblast
o contains heparin and histamine • 1st morphologically identifiable cell of the
lymphocytic maturational series in BM
4. Metamyelocyte • not entirely from the bone marrow, can be
• nucleus begins to assume an indented or kidney from secondary lymphoid organs such as
bean shape thymus and lymph nodes
• nucleus becomes indented (distinguishing factor) • 15 to 20 um
• Nucleus • N:C ratio of 4:1
▪ chromatin continue to condensed • Nucleus
▪ color of the specific granulation continues to become a major ▪ round or oval
distinguishing feature ▪ 1-2 nucleoli
• Cytoplasm: Pink (Romanowsky stain) • Cytoplasm
▪ chromatin pattern is delicate looking
5. Mature Granulocytes ▪ medium blue or darker-blue border
• Band form (because it still has more RNA content)
▪ immature cell ▪ no granules
o peripheral circulation: lesser quantities
o also present in bone marrow 2. Prolymphocyte
▪ elongated nucleus • from BM, thymus & 2° lymphoid organs
▪ no lobulation • 15 to 20 um
• Segmented form • N:C ratio of 4:1 or 3:1
▪ mature cell
• Nucleus
▪ has lobulation
▪ round or slightly indented
• Mast cells ▪ 0-1 nucleoli
▪ called “tissue basophil” because this is the last developmental
• Cytoplasm
stage of basophil
▪ chromatin pattern is slightly condensed
▪ not observed in the blood of healthy persons.
▪ medium blue with thin, darker-blue rim
▪ appearance similar to that of the blood basophil
▪ few azurophilic granules (differentiates from lymphoblast)
▪ round or oval nucleus
▪ granules of the mast cell do not overlie the nucleus as they do
3. Mature Lymphocyte
in basophils
• resembles nRBCs or nucleated RBCs
• either T lymphocyte (cytotoxic T cells) or B lymphocyte (becomes
Monocytes & Macrophages Maturation & Development
plasma cell)
• CFU-GM
• already present in the peripheral blood
• CFU-M
• plasma cell
• CFU-G
▪ tissue form of lymphocyte
▪ produces antibodies
• same sizes, same N:C ratio, same no granules or granules are not
▪ memory cells that interact with foreign bodies just in case it
easily seen
returns (alam na kung ano yung gagawin kapag bumalik ulit
• granules in cytoplasm are not identifiable
dahil may na-produce nang antibodies)
• nucleus: lazy pattern chromatin and convoluted or twisted shape ▪ cannot be seen in peripheral circulation
▪ can be seen in the bone marrow (<2%)
1. Monoblast
• BM, thymus & 2° lymphoid organs
• 17 to 20 to 6-9 um
• N:C ratio of 2:1 or 4:1 to 3:1
• Nucleus
▪ round or oval or indented
▪ Nucleoli not visible
• Cytoplasm
 ▪ chromatin pattern is dense and appears clumped
▪ light sky blue and very scanty
▪ few azurophilic granules
▪ bluish (differentiates from erythrocytes: pinkish and clean)

4. Mature B cell
• 8 to 20 um
• Nucleus: round or oval and may be eccentrically placed
• Cytoplasm
▪ fine pattern chromatin
▪ nongranular
▪ mottled blue color

Megakaryocytic Maturation & Development

• megakaryoblast and promegakaryocyte are difficult to identify and
differentiate from the cells in other cell lines

Megakaryocytes
• largest cells found in the bone marrow
• easiest to be identified
• has irregular cytoplasm
• Thrombopoietin
• BFU-M
▪ most primitive progenitor cell committed
to megakaryocyte lineage
• CFU-M
• final stage of megakaryocyte development is
the morphologically identifiable
• 160 um
• no nucleoli
• multilobular nucleus
• segmented nucleus
• located near the sinus of bone marrow because it has proplatelet
projections
▪ proplatelet projections becomes platelets, which can be seen in
the peripheral circulation

Mature Platelets
• 2 to 4 um
• younger platelets larger than older ones
• no nucleus (anucleated)
• doesn’t have cellular organelles
• Cytoplasm: light blue, with evenly dispersed, fine red to purple
granules (hard to see because it is too small)
References:
• Lotspeich-Steininger e.t al; Clinical hematology principles,
procedures, correlations, Lippincott Company, 1992
• Turgeon, Clinical Hematology: Theory and Procedures 5th ed.,
Lippincott Williams & Wilkins, 2012
• Keohane et. al, Rodak’s Hematology: Clincal Principles 6th ed.,
Elsevier, 2020
WBC ABNORMALITIES ▪ drugs and hormones

• quantitative: low or high count ❖ high neutrophil indicates leukemia because it is a malignant
• qualitative: morphology and function condition so there will be an uncontrolled proliferation, which leads
• any of the morphology among the morphology, function, and to increased WBC count
whether the count s high or low is a clue for us to diagnose disorders ❖ most abundant WBC is the neutrophil that’s why neutrophil is
or diseases affected when there’s leukemia
❖ non-malignant condition includes inflammatory condition and
Granulocyte Quantitative Abnormalities infection (bacteria)
• Leukocytosis
▪ Neutrophilia Eosinophilia
▪ Eosinophilia • Persistently and significantly numbers of eosinophils
▪ Basophilia
• Causes
▪ Monocytosis
▪ active allergic disorders
• Leukopenia o asthma
▪ Neutropenia o Hay fever
▪ Eosinopenia ▪ dermatoses
▪ Basopenia ▪ nonparasitic infections
▪ Monocytopenia ▪ forms of leukemia
▪ parasitic infections patients with significant
Leukocytosis o filarial worms – most common parasite that causes the
• there is an increase proliferation of WBCs (circulatory) increase of eosinophil count
• high concentration, count, percentage of leukocyte in peripheral • vacuolization and degranulation
circulation ▪ Charcot-Leyden crystals
• ↑ in the conc. or % of any of the leukocytes in the Peripheral Blood o remnants of disintegrated eosinophils
(circulating pool) o found in secretions like tissues, exudates (fluids), sputum,
• Neutrophils or lymphocytes: most common cause and stool
• Causes:
▪ ↑ movement of immature cells out of BM’s proliferative ❖ includes allergic conditions
compartment
o from the proliferative compartment, it goes to the circulating Basophilia
pool • > 0.075 ×109/L
▪ ↑ mobilization of cells from the MSC of BM to PB • number of circulating basophils is not remarkably affected by factors
o from maturation storage compartment to circulating pool such as time of day, age, and physical activity
▪ ↑ movement of mature cells from MP to CP • Causes:
o from marginating pool to circulating pool ▪ hormones
▪ ↓ movement of mature cells from circulation to tissue ▪ ulcerative colitis
o hindi na napupunta sa tissues ▪ hyperlipidemia
o example: monocyte is transient in peripheral circulation it ▪ some viral infections
goes to tissues as macrophage; basophil goes to tissues as o smallpox
mast cell o chickenpox
o if no cell goes to the tissue, there will be an increase amount ▪ chronic sinusitis
of monocyte and basophil in the peripheral circulation, thus, ▪ Chronic Myelocytic Leukemia (CML)
total WBC count increases ▪ polycythemia vera
o increase of all blood cells
❖ in bone marrow there’s 2 compartment, proliferation compartment
and maturation storage Monocytosis
❖ in peripheral circulation, we have marginating and circulating pool • significant absolute ↑ in circulating monocytes
❖ we get specimen in the circulating pool
• Causes:
❖ if there will be an increase in WBCs in the peripheral circulation
▪ Infections
(circulating pool), probability that is because of neutrophilia,
▪ fever of unknown origin
eosinophilia, basophilia, and monocytosis
▪ inflammatory bowel disease
❖ the most common reason why WBC increases are usually because
▪ RA
of the increased neutrophil or increased lymphocyte due to when
▪ hematological disorders
neutrophil and lymphocyte increases, total WBC count will instantly
▪ tuberculosis
increase (they are the majority of population)
▪ bacterial endocarditis
❖ when we say leukocytosis, it means total WBC count
• tissue macrophages
▪ response to foreign antigens
Neutrophilia
• ↑ in the number of neutrophils
Leukopenia
• Causes:
• most common cause are neutrophils
▪ present in some forms of leukemia and nonmalignant conditions
• ↓ neutrophils = ↓ total cell count because neutrophil is the most
▪ physical stimuli
abundant cell in the PC
o heat and cold trauma
• decreased total WBC count
➢ marginating pool decreases leads to release going to
the peripheral circulation
Neutropenia
o surgery
• reduction in the number of circulating neutrophils
o burns
o stressful activities • Causes:
o vigorous exercise ▪ bone marrow injury or infiltration
o nausea o infiltration: ↑ abnormal cells, ↓ precursors of PMN, thus ↓
o vomiting neutrophil count in PC
 o bone marrow injury: not releasing of cells (other populations 1. Toxic granulation
are also affected) • peroxidase (+) azurophilic granules
▪ nutritional deficiencies ▪ peroxidase stain
o ↓ Vitamin B12, folic acid, iron, growth factors (important in ▪ azurophilic means primary/non-specific
the proliferation) granules
▪ cyclic neutropenia • fine or heavy dark granulation in bands and
o hereditary disorder segmented neutrophils or monocytes
o not producing of neutrophil due to easily destruction or the ▪ natatabunan yung cytoplasm
bone marrow has the problem itself (cannot release) • represent the precipitation of ribosomal protein (RNA)
▪ increased destruction or utilization of neutrophils • caused by metabolic toxicity within the cells
o increased destruction than production ▪ that’s why there is a release of precipitation of ribosomal protein
o ↓ production ↑ destruction RNA
▪ entrapment in the spleen • graded on a scale of 1+ to 4+
o a lot of neutrophils are trapped in the spleen means low • causes:
count ▪ severe bacterial infection
▪ starvation ▪ burns
▪ anorexia nervosa ▪ malignant disorders
• Transient causes: ▪ drug therapy
▪ means it can increase after bumagsak yung count (not liefetime • grading is dependent on the coarseness and amount of granulation
mababa yung neutrophil count) in cytoplasm
▪ acquired disorder ▪ grading is subjective
▪ viral infections
▪ inability to release mature granulocytes into the blood 2. Chediak-Higashi syndrome
• Congenital causes: • autosomal recessive trait seen in children and
▪ all throughout the life of the patient, they have low neutrophil young adults
count • large granules, gigantic, peroxidase (+) deposits
▪ congenital agranulocytosis of the Kostmann type
• cause: abnormal lysosomal development in
▪ Myelokathexis
neutrophils, monocytes and lymphocytes.
▪ reticular dysgenesis
• Neutrophils
▪ type IB glycogen storage disease
▪ impaired chemotaxis
▪ transcobalamin-II deficiency
o broken communication channel
▪ delayed killing of ingested bacteria
Eosinopenia
o due to abnormal lysosome
• normal: low count
• Patients suffer from frequent infections
• rare, stress-related condition
▪ not efficient bacteriocidal cells
• Causes:
• appearance is similar to toxic granulation but not fine
▪ glucocorticosteroid hormones
▪ result of acute bacterial or viral inflammation
❖ large granules in the picture are the abnormal lysosomal deposits
❖ lysosome is very important in phagocytosis
Basopenia
➢ bacteria will attach to the phagocyte
• normal: low count ➢ vesicle will enclose the bacteria
• causes: ➢ vesicle will be fused in the lysosome
▪ hormones ➢ lysosome will digest
o Corticotropin ❖
o Progesterone
▪ thyrotoxicosis Syndrome Enzyme deficiency Substance stored
Hurler’s Alpha-L-iduronidase Mucopolysaccaride1
Monocytopenia Hunter’s Iduronidate sulfatase Mucopolysaccaride2
• normal: low count Sanfilippo’s Form A: Heparin N-sulfatase Mucopolysaccaride3
Form B: N-acetyl-a glucosaminidase
Scheie’s Alpha-L-iduronidase Mucopolysaccaride5
Granulocyte Qualitative Abnormalities: Morphology
• Toxic granulation 4. Döhle Body
• Chediak-Higashi syndrome • aggregates of rough endoplasmic reticulum (RNA)
• Alder-Reilly Inclusions • single or multiple, lightblue–staining inclusions
• Döhle Body • near the periphery of the cytoplasm.
• May-Hegglin Anomaly • Neutrophils, monocytes or lymphocytes
• Hypersegmentation • causes:
May-Hegglin anomaly
• Pelger-Huët Anomaly ▪ Infections
• Ehrlichia ▪ Burns
▪ Drug therapy
❖ in memorizing, check first the: • Döhle body–like inclusions:
1. appearance
2. cause 5. May-Hegglin Anomaly
3. condition associated • presence of Döhle body–like inclusions
4. cells affected (is it neutrophil, eosinophil, etc.) • neutrophils, eosinophils, and monocytes
• coexist with
▪ large and poorly granulated platelets
▪ thrombocytopenia
• approximately 50% of patients do not have symptoms
• others have bleeding tendencies
6. LE cell o acute and chronic leukemias, myelodysplastic syndromes,
• Neutrophil with large purple homogenous round Hodgkin disease, and carcinoma
inclusion • Chronic Granulomatous Disease
▪ rare disorder, X-linked trait or autosomal recessive
▪ neutrophils and monocytes ingest, but cannot kill catalase (+)
7. Hypersegmentation org.
• segmented neutrophils with > 5 lobes ▪ leads to recurrent infections by cat. (+) org. on the 1st year of
• causes: life
▪ Vitamin B12 or folic acid def. ▪ do not generate O2−, produce H2O2, or consume O2 at an
• coexists along with: accelerated rate via
• pseudohypersegmentation o respiratory burst is not activated
▪ old segmented neutrophils o free radical forms of reduced O2 are not produced
▪ Causes:
8. Pelger-Huët Anomaly ▪ severe def. or instability of leukocyte G6PD
• autosomal dominant disorder o (-) nitroblue tetrazolium (NBT) screening test
• produces hyposegmentation of mature • Lactoferrin deficiency
neutrophils ▪ specific granules are reduced in quantity
• nuclear shape: resemble a dumbbell or a pair of ▪ devoid of the specific granule protein
eyeglasses ▪ Causes:
• chromatin clumping and cytoplasmic maturation: normal o unresponsiveness to chemotactic signals
o diminished adhesiveness to surfaces of particles
• due to abnormal nucleic acid metabolism
▪ Results:
• Pseudoanomaly
o pyogenic infections
▪ drug induced
• manifested as lipid storage diseases
▪ maturational arrest associated
o with some acute infections • macrophages
▪ prone to accumulate undegraded lipid products
• Function is normal
▪ leads to an expansion of the reticuloendothelial tissue
▪ benign anomaly
• monocytic disorders
9. Ehrlichia ▪ Gaucher disease
▪ Niemann-Pick disease
• Human granulocytic ehrlichiosis (HGE)
• caused by
▪ Ehrlichia chaffeensis, E. ewingii Monocyte-Macrophage: Qualitative Abnormalities
1. Gaucher Disease
▪ bacterium extremely identical to E. phagocytophila
• seen in children
• transmitted by:
▪ lone star tick: Amblyomma americanum • mild: relatively normal life
▪ black-legged tick: Ixodes scapularis • severe: die prematurely
▪ western black-legged tick: I. pacificus • deficiency of β-glucocerebrosidase
• form vacuole-bound colonies known as morulae ▪ splits glucose glucosylceramide
▪ cerebroside accumulates in histiocytes
Granulocyte Qualitative Abnormalities: Function • Gaucher cells
• Defective Locomotion and Chemotaxis ▪ rarely found in the PC
▪ large, with 1-3 eccentric nuclei and wrinkled cytoplasm
• Defects in Microbicidal Activity
▪ RES
▪ CGD
▪ ↓ erythrocytes & leukocytes
▪ MPO deficiency
▪ Infiltration into the BM
1. Defective Locomotion and Chemotaxis
2. Niemann-Pick Disease
• Impaired leukocyte mobility
• similar to Gaucher disease
▪ rheumatoid arthritis
▪ cirrhosis of the liver • seen in infants and children
▪ CGD • def. of the enzyme that cleaves phosphoryl choline from
• Defective locomotion or leukocyte immobility sphingomyelin
▪ Corticosteroids treatment ▪ Sphingomyelin accumulates in the tissue macrophages
▪ Lazy leukocyte syndrome • Pick cell
• Defective chemotaxis ▪ same appearance to the Gaucher cell
▪ diabetes mellitus ▪ cytoplasm of the cell is foamy in appearance
▪ Chédiak-Higashi anomaly
▪ sepsis 3. Tart cell
▪ high levels of antibody IgE • monocyte with ingested lymphocyte
• rough and unevenly stained
2. Defects in Microbicidal Activity • mistaken as LE cell
• Myeloperoxidase Deficiency
▪ Alius-Grignaschi anomaly
▪ autosomal recessive genes Lymphocyte: General Qualitative Abnormalities
▪ absence of MPO enzyme from neutrophils and monocytes, but 1. Variant lymphocytes
not eosinophils • atypical lymphocytes, Downey cells, reactive or transformed
▪ MPO lymphocytes, lymphocytoid or plasmacytoid lymphocytes, and
o mediates oxidative destruction of microbes by H2O2 virocytes
▪ functional abnormality is not severe • healthy persons: 5% or 6%
▪ infections are not usually serious ▪ morphological evidence of a normal immune mechanism
▪ partial def. • numbers:
▪ IM
 ▪ viral pneumonia & viral hepatitis Plasma cell: Qualitative Abnormalities
1. Grape or Mott cells
• cytoplasm is completely filled with
• Plasma cell with vacuoles
• Large protein globules
• Multiple myeloma
• Characteristics:
▪ overall size 2. Flame cells
▪ enlarged nucleus • cytoplasm stains a bright-red color
▪ nuclear shape: lobulated or monocytoid • contains increased quantities of glycogen or intracellular deposits of
▪ chromatin pattern: fine to coarse amorphous matter
▪ 1-3 nucleoli • Associated with:
▪ abundant, foamy and vacuolated cytoplasm ▪ Increased Ig
▪ gray to light blue or intensely blue cytoplasm ▪ Multiple myeloma
▪ presence of granules • viral disorders
• allergic conditions
Lymphocyte: Specific Qualitative Abnormalities • chronic infections
1. Binucleated Lymphocytes • collagen diseases
• seen in viral infections • plasma cell dyscrasias
• > 5% ▪ increased plasma cells or completely infiltrate BM
▪ either lymphocytic leukemia or leukosarcoma o Waldenström macroglobulinemia
o Multiple myeloma
2. Rieder cells
• similar to normal lymphocytes except that the nucleus is notched, References:
lobulated, and cloverleaf-like • Lotspeich-Steininger e.t al; Clinical hematology principles,
• seen in: procedures, correlations, Lippincott Company, 1992
▪ CLL • Turgeon, Clinical Hematology: Theory and Procedures 5th ed.,
▪ artificially produced through blood smear preparation Lippincott Williams & Wilkins, 2012
• Keohane et. al, Rodak’s Hematology: Clincal Principles 6th ed.,
3. Vacuolated lymphocytes Elsevier, 2020
• associated with:
▪ Niemann-Pick disease
▪ Tay-Sachs disease
▪ Hurler syndrome
▪ Burkitt lymphoma
• Vacuoles can also be seen in:
▪ variant lymphocytes
▪ reaction to viral infections, radiation, and chemotherapy

4. Smudge cells/ Basket cells

• natural artifact in the preparation of a blood
smear
• bare nuclei of lymphocytes and neutrophils
• Increased fragility of cells
▪ contributes to the increased percentage of smudge cells
• seen in:
▪ increased proportions in lymphocytosis (CLL)

5. Downey cells
• Type I
▪ Turk’s irritation cell
▪ with block of chromatin
• Type II
▪ IM cells
▪ Round mass of chromatin
▪ Ballerina skirt appearance
• Type III
▪ vacuolated
▪ Swiss chief or moth eaten appearance

6. Sezary cells
• round lymph cell with nucleus that is grooved or convoluted
• sezary syndrome
• mycosis fungoides

7. Hairy cell
• lymphocyte with hair-like cytoplasmic projections
surrounding the nucleus
• Hairy cell leukemia