Midterm Topics Hema 1 Lec

HEM 311: CLINICAL HEMATOLOGY 1
RMT LECTURE 1: OVERVIEW OF CLINICAL HEMATOLOGY

Transcribed by: Surell, Riyoma
2023 INSTRUCTOR: PROF. PAMELA S. SENGSON, RMT

SUMMER TERM, A.Y. 2021 – 2022
Date: June 29, 2022
OUTLINE BASIC HEMATOLOGY TERM AND ABBREVIATION

 Overview of Clinical Hematology
o History of Clinical Hematology TERMS DEFINITION
o Basic Hematology Term and Abbreviation a Without
o Blood Composition -blast Youngest/nucleated
o General Characteristics of Blood -chromic Color
 Overview of Formed Elements -cyte Cell
o Red blood cells Dys Abnormal
o White blood cells -emia In the blood
o Neutrophils Ferro Iron
o Basophil Hyper Increased
o Eosinophil Hypo Decreased
o Lymphocyte Iso Equal
o Monocyte Macro Large
o Platelets Mega Very large/ huge
 Overview of Complete Blood Count Micro Small
Myelo Marrow
Normo Normal
OVERVIEW OF CLINICAL HEMATOLOGY
-oid Like
 Study of the disorder and abnormalities related or
associated with the quality and quantity of the cellular
Abbreviation
elements of the blood FBC Full blood count
 Study of the laboratory procedure and techniques Fl Femtoliter
being used to examine the quality and quantity of the Hb Hemoglobin concentration
cellular elements of the blood Hct Hematocrit
 Average Blood: 5 liters MCH Mean Cell (Corpuscular) Hemoglobin
 Blood Transports Oxygen from Lungs to tissues: MCV Mean Cell Volume
o Clear tissues (carbon dioxide) MCHC Mean Cell Hemoglobin Concentration
o Transports (glucose, proteins and lipids) CBC Complete Blood Count
o Moves wastes (to the liver and kidneys) Pg Picogram
 Plasma: liquid portion of unclotted blood
o Coagulation enzymes that protect vessels
from trauma BLOOD COMPOSITION
o Transports and nourishes blood cells 1. Formed Elements
o maintains circulation - Erythrocytes, leukocytes and
 Three Categories of Blood: platelets
o RBCs: erythrocytes 2. Liquid Portion
o WBCs: leukocytes - Plasma: liquid portion of unclotted
o Platelets: thrombocytes blood (90% H2O – proteins,
HISTORY OF CLINICAL HEMATOLOGY enzymes hormones and lipids –
appears hazy – pale yellow)
 1657 Athanasius Kircher: described “worms” - Serum: is the fluid that remains after coagulation has
 1674 Anton van Leeuwenhoek: gave an account of occurred and clot has formed
RBCs
Serum Plasma
 1842 Giulio Bizzozero: described platelets as “petite
Liquid portion of clotted blood Liquid portion of unclotted
plaques” – French (cell fragment)
blood
 1902 James Homer Wright: wright stain Pale yellow, clear and Pale yellow or straw
 1658 – discovery of erythrocytes by Swammerdam transparent colored/ hazy
 1846 – PMN distinguished from other leukocytes by Has F1, FV, VIII:C, FXIII
Wharton Jones completely consumed in the
 1879 – first complete classification of leukocytes by coagulation process
Ehrlich FII – not completely consumed
 1920 – hematology was considered a separate science >80% consumed
from clinical pathology <20% remains in serum and
called Residual Prothrombin
Overview of Clinical Hematology I Page 1 of 4
GENERAL CHARACTERISTICS OF BLOOD o Increased RBC, RBC Mass: polycythemia
(hyperviscocity)
 In vivo, blood is in fluid; in vitro it coagulates in 5 to 10
minutes Hematocrit, Hemoglobin and Blood Cell Indices
 Thick and viscous 3.5 – 4.5 to 5 times thicker than
1. Haemoglobin: transports oxygen and carbon dioxide
water
2. Haematocrit: ratio of packed RBCs to the volume of
 Makes up 6-8% of the total body component or 60-
whole blood
80ml/kg
3. RBC Indices: internal quality control of automated
 Total Blood volume (TBV)
blood cell analyzers
o Adult Male: 5-6L
a. MCV: RBC diameter, fL
o Adult Female: 4-5L
b. MCH: mass of haemoglobin, pg
o Newborn: 250-350mL
c. MCHC: RBC staining intensity and amount of
 pH of blood
central pallor g/dL
o pH is a measure of the acidity or basicity of an
d. RBC Distribution Width (RDW): degree of
aqueous solution
variation in terms of RBC volume
o it is the negative decimal logarithm of
**Anisocytosis: extreme RBC volume
hydrogen concentration
variability
o Normal: 7.35 – 7.45 (average of 7.40)
o Venous: 7.35 Reticulocytes
o Arterial: 7.45
o pH <7.3  acidic  Polychromatic erythrocytes
o pH >7.5  alkaline  Slightly blue-gray
o maintained by: excretion of CO2 and  Indicate the ability of the bone marrow to increase RBC
excretion of H+ or OH production in anemia
 action of buffered system  Nucleic acid stains or vital/ supravital stains (e.g., new
(carbonate, phosphate, protein and methylene blue and brilliant cresyl blue)
hemoglobin)
 Specific Gravity: depends on RBCs and plasma White Blood Cells
proteins
 Protects its host from infection or injury
o The ratio of the density of a substance to the
 Nearly colorless, unstained
density of a reference substance
o Number of solutes dissolved in the blood  Source: bone marrow or lymphoid tissue
o Whole Blood: 1.045 – 1.066  Leukopenia: decreased WBC
o Serum: 1.024 – 1.028  Leucocytosis: increased WBC
o Plasma: 1.025 – 1.029  Types: neutrophils, eosinophils, basophils,
o Depends: RBCs and Plasma Proteins lymphocytes and monocytes
 Color 1. Neutrophils
o Arterial: bright red  Phagocytic cells (segmented neutrophils or
o Venous: dark purplish red PMN  multilobed nucleus/ nuclei)
o Pulmonary Arteries and Veins Hb with O2:  Major Purpose: engulf and destroy
purple microorganisms and foreign material
o Hb w/o O2: blue  Cytoplasm: pink-lavender staining granules
 Osmolality: concentration of solutes dissolved in the (bactericidal substances)
blood  Neutrophilia: increased neutrophils
o uses osmometer for measurement (bacterial infections)
o Reference Range: 281 – 297 milli-osmoles  Neutropenia: decreased neutrophils
per kilogram (mOsms) (medications or viral infections)
 Bands
OVERVIEW OF FORMED ELEMENTS o Less mature neutrophils with a non-
segmented nucleus
o U or S shape
Red Blood Cells o “left shift”
o Increased: bacterial infection
 Anucleate, biconcave, discoid cells 2. Eosinophils
 Filled with hemoglobin (protein transports O2 and CO2)  Round, bright orange-red cytoplasmic
 Appear salmon pink, 7 – 8um in diameter granules filled with proteins involved in
 w/ a zone of pallor (lightly staining in center) immune system regulation
o before 1900s, physicians and professionals  Eosinophilia: elevated eosinophil count
used RBC count to detect anemia or (allergy, parasitic infection)
polycythemia 3. Basophils
o Low RBC or RBC Hb: anemia  Dark purple, irregular cytoplasmic granules
that obscure the nucleus
 Granules: histamines and proteins Blood Film Examination
 Basophilia: increased basophils (rare,
hematologic conditions)  “wedge-prep”
4. Lymphocytes  WBC Differential Count = 100 WBCs – percent
 For host immunity distribution
 Cell-mediated response
 Nearly round, are slightly larger than RBCs
 Have round featureless nuclei and a thin rim
of nongranular cytoplasm
 Lymphocytosis: increased lymphocytes
(viral infections)
 Lymphopenia/ Lymphocytopenia:
decreased lymphocyte (drug therapy or
immunodeficiency)
5. Monocytes
 Immature macrophage
 Identifies and phagocytize foreign particles
 Assist the lymphocytes in mounting an immune
response (assembly and presentation of antigen
epitopes)
 Have a slightly larger diameter than other WBCs
 Blue-gray cytoplasm with fine azure granules
 Nucleus that is usually indented (horse-shoe
shaped) or folded
 Monocytosis: increased monocytes
Platelets
 Maintain blood vessel integrity by initiating cell wall

repairs
 Platelets rapidly adhere to the surfaces of damaged
blood vessels and formed aggregates with
neighbouring platelets to plug the vessels
 Secrete proteins and small molecules that trigger
thrombosis or clot formation
 Major cells that control hemostasis
o Able to seal wounds and repair blood vessels
wall and maintain vascular latency
(uninterrupted blood flow)
 2 to 4um in diameter
 Round or oval, anucleated
 Slightly granular
 Thrombocytosis: increased platelet count
(inflammation or trauma)
 Thrombocytopenia: decreased platelet count
(consequence of drug treatment and life threatening)
OVERVIEW OF COMPLETE BLOOD COUNT
Basic CBC Measurements by Automated Blood Cell

Analyzer
RBC Parameters WBC Parameters Plt. Parameters
RBC count WBC Count Platelet count
Hgb, Hct, NEUT Count MPV
MCV, MCH, Lymph count
MCHC
RDW, Retic Mono count
EO and BASO
counts

RMT LECTURE 2: HEMATOPOIESIS

SUMER TERM, A.Y. 2021 – 2022
Date: June 29, 2022
OUTLINE  The developing erythroblasts signal the beginning of

 Hematopoiesis definitive hematopoiesis with a decline in primitive
o Embryonic/Fetal Development Mesoblastic hematopoiesis of the yolk sac
Phase  Hematopoiesis during this phase occurs
o Hepatic Phase extravascularly, with the liver remaining the major
o Medullary Phase site of hematopoiesis during the second trimester of
 Adult Hematopoietic Tissue fetal life
 Post-Natal Development
 The developing spleen, kidney, thymus and lymph
o Bone Marrow
nodes contribute to the hematopoietic process during
o Liver
o Spleen this phase
o Lymph Nodes  Thymus is the first fully developed organ in the fetus
o Thymus o Major site of T cell production
 Hematopoietic Microenvironment  Kidney and spleen produce B cells
 Stem Cells  Detectable levels of Hb (F) and Hb A is present
 Hematopoietic Growth Factors
 Control of Hematopoieisis Fetal Hemoglobin Globin Chain Combination
 3 Possible Actions of HSCs HEMOGLOBIN F 2 alpha, 2 gamma
 Cytokines
MEDULLARY/ MYELOID PHASE
HEMATOPOIETIC DEVELOPMENT
 5th month of fetal development hematopoiesis
 Hematopoiesis begins in the bone marrow cavity
o Continuous, regulated process of blood cell  Hematopoietic activity, especially myeloid activity, is
production that includes: apparent during this stage of development, and the
 Cell renewal proliferation myeloid-to-erythroid ratio gradually approaches 3:1
 Differentiation maturation (adult levels)
 Measurable levels of erythropoietin (EPO), (G-CSF),
EMBRYONIC/ FETAL DEVELOPMENT MESOBLASTIC (GM-CSF), hemoglobins F and A can be detected
PHASE  Cells at various stages of maturation can be seen in all
blood cell lineages
 Begins around the 19th day of embryonic development
after fertilization Adult Hemoglobin Globin Chain Combination
 Primitive erythrocytes are found in the yolk sac arise HEMOGLOBIN A 2 alpha, 2 beta
from the mesodermal cell HEMOGLOBIN A2 2 alpha, 2 delta
 Cells from the mesoderm migrate to the yolk sac
 Transient yolk sac erythroblasts are important in
early embryogenesis to produce hemoglobin (Gower- ADULT HEMATOPOIETIC TISSUE
1, Gower-2 and Portland)
 In adults, hematopoietic tissue is located in the
o Delivery of oxygen to rapidly growing
o Bone marrow: developing erythroid, myeloid
embryonic tissues
megakaryocytic and lymphoid cells
 Alpha globin chain production begins at this phase
o Lymph nodes
Embryonic Hemoglobin Globin Chain Combination o Spleen
GOWER I 2 epsilon, 2 zeta o Liver
GOWER II 2 alpha, 2 epsilon o Thymus
PORTLAND 2 zeta, 2 gamma  Lymphoid development occurs in primary (bone
marrow, thymus  T and B lymphocytes derived) and
secondary lymphoid tissue respond to foreign
HEPATIC PHASE antigens (spleen, lymph nodes and mucosa associated
lymphoid tissue or MALT)
 The hepatic phase of hematopoiesis begins at 5 to 7
gestational weeks
 Characterized by recognizable clusters of developing
erythroblasts, granulocytes and monocytes colonizing
the fetal liver, thymus, spleen and placenta
Hematopoiesis I Page 1 of 4
POST NATAL DEVELOPMENT LIVER
 Hematopoietic tissue is involved in the proliferation and  Liver serves as the major site of blood cell production
maturation of blood cells during the second trimester of fetal development
 Organs involved:  Functions
o Bone marrow o Protein synthesis and degradation
o Liver o Coagulation factor synthesis carbohydrate
o Spleen and lipid metabolism
o Lymph nodes o Drug and toxin clearance
o Thymus o Iron recycling and storage
o Hemoglobin degradation
BONE MARROW  Capable of extramedullary hematopoiesis
 Major function is the proliferation and production of SPLEEN
blood cells
 Largest lymphoid organ in the body
Two Major Components:
 It is located directly beneath the diaphragm behind the
1. Red Marrow fundus of the stomach in the upper left quadrant of the
- Hematopoietically active marrow consisting of the abdomen
developing blood cells and their progenitors  It is vital but not essential for life
- Seen in: flat bones of the skull, clavicle, sternum, ribs,  Functions:
vertebrae, pelvis o Indiscriminate filter of the circulating blood
- 4 Major Functions: o Serves as a storage site for platelets
a. Production of blood cells  3 Regions
b. Destruction of erythrocytes o White Pulp: consists of scattered follicles
c. Iron storage from hemoglobin breakdown with germinal centers that contains
d. Central organ for B lymphocyte development lymphocytes, macrophages and dendritic
cells
2. Yellow Marrow o Red Pulp: vascular sinusoids and sinuses
- Abundant: 5 and 7 years of age, where they occupy separated
the spaces in the long bones previously dominated by o Marginal Zone: surrounds the white pulp and
our red marrow forms reticular measure containing blood
- Hematopoietically inactive marrow composed vessels, macrophages and specialized B cells
primarily of adipocytes and (fat cells), with  2 Methods for Removing Senescent or Abnormal
undifferentiated mesencyhmal cells and macrophages RBCs from Circulation
o Culling: involved action of phagocytosis.
HEMATOPOIETIC MICROENVIRONMENT Cells being phagocytized with subsequent
degradation of the cell organelles. Whole cells
 It is responsible for supplying semifluid matrix
degraded. Removal of old or abnormal
(stroma) that serves as an anchor for the developing
RBCs that is within the cords of billroth
hematopoietic cells o Pitting: splenic macrophages removed
 Composition of Stroma; inclusions or damaged surface membrane
1. Endothelial cells – regulate flow of particles from the circulating RBCs. Removal of the
entering and living in hematopoietic spaces unwanted intracellular elements (e.g.,
2. Adipocytes – secretes various steroids and Heinz bodies, Howell-jolly bodies,
influences erythropoiesis and maintains bone Pappenheimer bodies)
integrity. Regulates the volume of the marrow
3. Macrophages – phagocytosis. Secretes various LYMPH NODES
cytokines that regulate hematopoiesis
4. Osteoblasts – bone forming cells. Termed as  Are members of lymphatic system located along the
“water bug” or “comet appearance” lymphatic capillaries
5. Osteoclasts – bone resorbing cells or destroying  Bean shaped structures, 1-5mm diameter
cells  3 Functions:
6. Reticular cells (fibroblasts) – supports the vascular o Play a role in the formation of new
sinuses and developing hematopoietic cells lymphocytes from germinal centers
 Composition of Extracellular Matrix of Bone o Involved in the processing of specific Ig
Marrow o Involved in the filtration of particulate
1. Collagen - matter, debris and bacteria entering the
2. Fibronectin lymph node via the lymph
3. Thrombospondin  Regions;
4. Laminin o Cortex: outer region. Contains follicles of B
5. Proteoglycans – hyaluronate, chondroitin cells. Proliferation centers termed as
sulphate, heparan sulphate germinal centers
o Paracortex: in between the cortex and CONTROL OF HEMATOPOIESIS
medulla. Contains predominantly T cells and
numerous macrophages  The entry of mature blood cells into the intravascular
o Medulla: inner region that consists of T space relies upon:
lymphcoytes and plasma cells o Multiplication of developing cells
o Gradual maturation
THYMUS o Orderly release of cell from bone marrow
 Responsible in conditioning the T lymphocytes 3 POSSIBLE ACTIONS OF HSCs

 Originates from endodermal and mesenchymal
tissues  Self-renewal
 Populated initially by lymphocytes from the yolk sac  Differentiation
and the liver  Apoptosis (programmed cell death, normal physiologic
 An efficient, well-developed organ at birth that consist process that eliminates unwantend abnormal or
of two lobules each measuring 0.5 to 2cm in diameter harmful cells)
STEM CELLS
 Cells that have extensive proliferative capacity CYTOKINES

 Give rise to loose stem cells and differentiate into any  Group of specific glycoproteins called growth factors
blood cell line that regulates the proliferation differentiation and
 HSC are BMC that are capable of producing all types maturation of hematopoietic precursor cells
of blood cells  Includes:
 They differentiate into one or another type of committed o Interleukins (Ils)
stem cells (progenitor cells) o Lymphokines
HEMATOPOIETIC GROWTH FACTOR o Monokines
o Interferons
 CSF: Colony Stimulating Factors o Colony Stimulating Factors (CSFs)
o Specific for various cell lines o Chemokines
1. GM-CSF
o A pan myeloid (all elements of bone marrow) Positive Influence Negative Influence
IL-1 Transforming Growth Factor-B
growth factor that stimulates granulocyte,
IL-3 Tumor Necrosis Factor-a
monocyte, megakaryocyte and eosinophil
IL-9 Interferons
progenitors
IL-11
o Source: fibroblast, T cells and endothelial
GM-CSF
cells
Kit Ligand
2. G-CSF
o Stimulates granulocytes production and
functional activation
o Source: monocytes and fibroblasts
3. M-CSF
o Stimulates monocytes and macrophages
production activity
o Source: monocytes, fibroblast, endothelial
cells
4. Meg-CSF
o Specific to the megakaryocyte lineage
o Source: monocytes, fibroblast,
megakaryocytes
 Erythropoietin (EPO)
o Stimulates proliferation, growth and
differentiation of erythroid precursors and
may have minor effects on megakaryocytes
o Target cells are pronormoblast and CFU-
erythroid cells
o Source: kidney
 Thrombopoietin
o Regulates production platelets
RMT LECTURE 3: LINEAGE SPECIFIC HEMATOPOIESIS

Date: June 29, 2022
OUTLINE  Cytoplasm
 Lineage-Specific Hematopoiesis o Dark blue (concentration of ribosomes)
 General Characteristic of Blast  Location
 Hematopoietic System o Present only in the bone marrow in healthy
o Erythropoiesis states
o Granulopoiesis  Cellular Activity
o Monocyte Development o Begins to accumulate the components
o Lymphocyte Development
necessary for hemoglobin production
o Megakaryopoiesis
o The proteins and enzymes necessary for iron
uptake and protoporphyrin synthesis are
LINEAGE SPECIFIC HEMATOPOIESIS produced
o Lasts 24 hours
 Principle of Normal Blood Cell Maturation
(Synchronistic Maturation) 2. Basophilic Normoblast (Prorubricyte)
o Cytoplasmic Changes  Nucleus
o Cytoplasmic Granules o Chromatin begins to condense
o Nuclear Changes o N:C ratio decreases to about 6:1
o Reduction in Cell Size o The chromatin stains deep purple-red
 Principle of Abnormal Cell Maturation o Nucleoli may be present early in the stage but
(Asynchronistic Maturation) disappear later
o Abnormal Cytoplasmic Differentiation  Cytoplasm
o Abnormal Nuclear Maturation o When stained it is deeper, richer blue in color
o Abnormal Size than in the pronormoblast
 Location
GENERAL CHARACTERISTIC OF BLAST
o Present only in the bone marrow in healthy
 Size: large cell with high N:C ratio states
 Cytoplasm: very dark blue and small in amount in  Cellular Activity
comparison to the size of the nucleus. No granular is o Detectable hemoglobin synthesis occurs,
present but many cytoplasmic organelles, including
 Nucleus: large in size as compared to the size of ribosomes and a substantial amount of
cytoplasm messenger ribonucleic acid (chiefly for
o Chromatin which is reddish purple and hemoglobin production only), completely
indicates predominance of DNA mask the minute amount of hemoglobin
pigmentation
HEMATOPOIETIC SYSTEM
3. Polychromatic (Polychromatophilic) Normoblast or
 Erythropoiesis Rubricyte
 Granulopoiesis  Division
 Monopoiesis o This is the last stage in which the cell is
 Lymphoiesis capable of undergoing mitosis
 Megakaryopoiesis  Location
ERYTHROPOIESIS states
 Cellular Activity
 A process by which erythroid precursor cells
o Hemoglobin synthesis increases and the
differentiates to become mature
accumulation begins to be visible in the color
 Primary Regulator: Erythropoietin
of the cytoplasm
 Length of Time
1. Pronormoblast (Rubriblast)
o 30 hours
 Nucleus
o Takes up much of the cell (N:C ratio of 8:1)
o Round to oval
o Contains 1 or 2 nucleoli
o Purple red chromatin is open and contains
few, fine clumps
Lineage Specific Hematopoiesis I Page 1 of 5

4. Orthochromic Normoblast (Metarubricyte) Nomenclature for Erythroid Precursors
 Nucleus
o Completely condensed Normoblastic Rubriblastic Erythroblastic
o N:C ratio is low or approximately 1:2 Pronormoblast Rubriblast Proerythroblast
 Cytoplasm Basophilic Prorubricyte Basophilic
normoblast erythroblast
o The increase in the salmon-pink color of the
Polychromatic Rubricyte Polychromic
cytoplasm reflects nearly complete
normoblast erythroblast
hemoglobin production
Orthrochromatic Metarubricyte Orthochromic
 Division normoblast erythroblast
o Not capable of division due to the Reticulocyte Reticulocyte Reticulocyte
condensation of the chromatin Erythrocyte Erythrocyte Erythrocyte
 Location
states Erythrocyte Maturation Sequence
Cell Size N:C Nucleoli %BM Transit time Division
5. Reticulocyte Pro 12-20 8:1 1-2 1 24 Mitosis
normoblast (2)
 No nucleus but has mitochondria and ribosomes Basophilic 10-15 6:1 0-1 1-4 24 Mitosis
 Last stage to synthesize hemoglobin Normoblast (4)
Polychromic 10-12 4:1 0 10-20 24 Mitosis
 Last stage in bone marrow before release to the blood Normoblast (16)
 Location Orthrochromic
Normoblast
8-10 1:2 0 5-10 48 -
o Resides in the bone marrow for 1 day or Reticulocyte 8-10 - 0 1 48 -

BM=1day
longer and then moves into the peripheral PB=1day
blood for about 1 day before reaching RBC D:6-8 - 0 0 BM=0 -
T: 1.5- PB=120days
maturity 2.5
 Reference Range: 0.5 to 1.5%
 Newborn: 2 to 6%
GRANULOPOIESIS
 Reticulocyte Count best indicators of bone marrow
functions NEUTROPHIL DEVELOPMENT
o Supravital stain is used
o Also known as: 1. Myeloblasts
 Polychromatophilic erythrocytes  Make up 0% to 3% of the nucleated cells in the bone
 Diffusely basophilic erythrocytes marrow and measure 14 to 20um in diameter
 Polychromatophilic macrocytes  Cytoplasm
o Clear blue, more heavily colored at its border
6. Erythrocytes o It is non-granular or may have a few
 Nucleus azurophilic granules, depending on the stage
o No nucleus is present in mature RBC of development
 Cytoplasm  Nucleus
o The mature circulating erythrocyte is a o Large, round or oval and occupies about four
biconcave disc measuring 7 to 8um in fifths of the total cell area
diameter, with a thickness of about 1.5 to o It has a very fine chromatic meshwork
2.5mm o Round/ oval nucleus with fine reddish purple
o On a stained blood film, it appears as a staining
salmon pink-staining cell with a central pale o Nucleoli: 2 to 5
area
o The central pallor is about 1/3 the diameter of 2. Promyelocytes
the cell  Comprise 1% to 5% of the nucleated cells in the bone
 Division marrow
o Erythrocyte cannot divide  16 to 25um in diameter
 Length of Time  Nucleus
o Approximately 120 days in circulation o Round to oval and is often eccentric
 Aging of RBCs leads to removal by the spleen o Occupies half or more of the cell
 Cellular Activity o Nucleoli: 1 to 3
o Deliver oxygen to the tissues and releases it  Cytoplasm
and returns to the lung to be oxygenated o Evenly basophilic
o Full of primary granules (azurophilic
granules  first in a series of granules to be
produced during neutrophilic maturation)

 Primary (Azurophilic) Granules Contains: 3. Eosinophil Metamyelocytes and Bands
o Myeloperoxidase  Resemble their neutrophil counterparts with respect to
o Acid b-glycerophosphatase their nuclear shape
o Cathepsins  Secondary granules increase in number and a third
o Defensins type of granule is generated
o Elastase 4. Mature Eosinophils
o Proteinase-3  Nucleus
o Usually display a bilobed nucleus
3. Neutrophil Myelocytes  Cytoplasm
 Make up 6% to 17% of the nucleated cells in the bone o Contains characteristic refractile, orange-red
marrow and are the final stage in which cell division secondary granules
(mitosis) occurs  Circulating Half life
 During this stage, the production of primary o Roughly 18 hours
granules ceases, and the cell begins to manufacture
secondary neutrophil granules Eosinophil Granules
 Primary Granules
4. Neutrophil Metamyelocytes
o Charcot-Leyden Crystal Protein
 Constitute 3% to 20% of nucleated marrow cells
 Secondary Granules
 The cells are no longer capable of division, and the
o Major basic protein (core)
major morphologic change is in the shape of the
o Eosinophil cationic protein (matrix)
nucleus
o Eosinophil-derived neurotoxin (matrix)
 Nucleus
o Eosinophil peroxidase (matrix)
o Indented  kidney bean or peanut shaped
o Lysozyme (matrix)
o Chromatin is increasingly clumped
o Catalase (core and matrix)
o Nucleoli: absent
o b-Glucoronidase (core and matrix)
o Cathepsin D (core and matrix)
5. Neutrophil Bands
o Interleukins 2,4 and 5 (core)
 Make up 9% to 32% of nucleated marrow cells and
o Interleukin-6 (matrix)
0% to 5% of the nucleated peripheral blood cells
o Granulocyte-Macrophage Colony Stimualting
 All evidence of RNA is absent Factor (core)
 Tertiary granules continue to be formed during this  Small Lysosomal Granules
stage o Acid phosphatase
 Secretory granules (secretory vesicles) may begin to o Arylsulfatase B
be formed during this stage o Catalase
 Nucleus o Cytochrome b558
o Highly clumped o Elastase
o Nuclear indentation that began in the o Eosinophil cationic protein
metamyelocyte stage now exceeds one half  Lipid Bodies
the diameter of the nucleus o Cyclooxygenase
o 5-lipoxygenase
6. Segmented Neutrophils o 15-lipoxygenase
 Make up 7% to 30% of nucleated cells in the bone o Leukotriene C4 synthase
marrow o Eosinophil peroxidase
 Secretory granules continue to be formed during this o Esterase
stage  Storage Vesicles
 only morphologic difference between segmented o Carry proteins from secondary granules to be
neutrophils and bands is the presence of between 2 to released into the extracellular matrix
5 nuclear lobes that are connected by a thread-like
filaments BASOPHIL DEVELOPMENT
EOSINOPHIL DEVELOPMENT 1. Immature Basophils

 Nuclei
1. Eosinophilic Promyelocytes o Have round to lobulated nuclei with only
 Can be identified cytochemcally due to the presence of slightly condensed chromatin
Charcot Leyden Crystals protein in their primary o Nucleoli major may not be apparent
granules  Cytoplasm
o The cytoplasm is blue and contains large
2. Eosinophil Myelocytes blue-black secondary granules
 Characterized by the presence of large pale, reddish o Primary azure granules may or may not be
orange secondary granules, along with azure seen
granules in blue cytoplasm

 Basophil Granules o Chromatin pattern is looser than in the other
o Water soluble, therefore may be dissolved if leukocytes and has sometimes been
the blood film is washed too much during described as lacelike or stringy
staining process o Nucleoli: generally not seen with the light
microscope
2. Mature Basophils  Cytoplasm
 Nucleus o Blue gray containing fine azure granules.
o Contain a lobulated nucleus that is often Often referred to as “azure dust”, or
obscured by its granules “ground-glass appearance”
o The chromatin pattern, if visible, is clumped o Small cytoplasmic pseudopods or blebs may
o Actual nuclear segmentation with visible be seen
filaments occurs rarely o In the cytoplasmic and nuclear vacuoles may
 Cytoplasm also be present
o Colorless and contains large numbers of the
characteristic large blue-black granules LYMPHOCYTE DEVELOPMENT
o In the event that granules have been 1. Lymphoblast
dissolved during staining process, they often  Size: 10 to 18um
leave a reddish purple rim surrounding what
 Nucleus
appears to be a vacuole
o Chromatin pattern is coarse
o Round oval in shape
o It contains one or two nucleoli
 Cytoplasm
o No granules present
o Moderate to dark blue
 N:C Ratio: 4:1
2. Prolymphocyte
 Size: same with lymphoblast or smaller
 Nucleus
o Round oval chromatin pattern is more
clumped
 Cytoplasm
o Moderate to dark blue and usually non-
granular
MONOCYTE DEVELOPMENT  N:C Ration: 4:1
1. Monoblast 3. Mature Lymphocyte

 Size: 12 to 20um  Found in the peripheral blood occurring in varying sizes
 Cytoplasm: moderately basophilic to blue or gray;  Size: 8 to 16um in diameter
non-granular  3 Categories:
 Chromatin: fine, lacey o Small
 Nucleoli: 1 to 2  Size: 8 to 10um in diameter
 N:C ratio: 4:1 to 3:1  Cytoplasm:
 usually forms a thin rim
2. Promonocytes around the nucleus
 Size: 12 to 18um in diameter  Moderate blue to dark blue
 Nucleus  Nucleus
o Slightly indented or folded  Round oval in shape
o Chromatin pattern is delicate slightly indented
o At least one nucleolus is apparent  Nucleoli: no visible
 Cytoplasm o Medium
o Blue and contains scattered azure granules  Size: 10 to 12um in diameter
that are fewer and smaller than those seen in  Cytoplasm
promyelocytes  More abundant pale to
moderate blue
3. Monocytes  Nucleus
 Size: larger than neutrophils (diameter of 15 to 20um)  Round or oval in shape
 Nucleus may be slightly indented,
o May be round, oval, or kidney shaped, but no nucleolus is visible
more frequently is deeply indented (horse-
shoe shaped) or folded on itself
o Large  Cytoplasm
 Size: 12 to 16um in diameter o Light blue to purple
 Cytoplasm
 Abundant, clear, very pale
blue may not contain few
non-specific azurophilic
granules
 Nucleus
 Round oval in shape and
may be slightly indented
 Nucleolus: no visible
MEGAKARYOPOIESIS
1. Megakaryoblast (Stage I)
 Size: 14 to 18um in diameter
 Cytoplasm
o Varying shades of blue, may have small blunt
pseudopods
o Narrow band around the nucleus
o As the cell matures the amount of cytoplasm
increases
 Nucleus
o Round oval kidney shaped
 N:C Ratio: 3:1
2. Promegakaryoblast (Stage II)

 Cytoplasm
o More abundant than in previous stage;
granules begin to form in the golgi region
 Nucleus
o Chromatin becomes more coarse
o Multiple nucleoli are visible
 N:C Ratio: 4:1 to 7:1
3. Megakaryocyte (Stage III)

 Cytoplasm
o Contains coarse clumps of granules
aggregating into little bundles which bud off
from the periphery  (platelets)
 Nucleus
o Multiple nuclei are present or multilobed
 N:C Ratio: less than 1:4
4. Platelets

RMT LECTURE 4: ERYTHROCYTES
Date: June 29, 2022
OUTLINE STRUCTURE OF AN RBC MEMBRANE

 Lineage-Specific Hematopoiesis
 General Characteristic of Blast  Membrane Lipids
 Hematopoietic System o Outer Layer
o Erythropoiesis  Phosphatidyl choline
o Granulopoiesis  Sphingomyelin
o Monocyte Development o Inner Layer
o Lymphocyte Development  Phosphatidyl ethanolamine
o Megakaryopoiesis  Phosphatidyl serine
 Membrane Proteins
o Integral Protein
ERYTHROCYTES
 Band 3 (anionexchanger protein)
 Mature RBCs Size: 7 to 8um average 7.5um in  Glycophorin
diameter  Aquaporin
 Has no nucleus nor organelles  Peripheral Protein
 Exists in blood circulation for 120 days o Spectrin
 Limited activity to metabolize fatty acids and amino o Actin
acids o Protein 4.1
o Pallidin (band 4.2)
 Metabolic processes are maintained through different
o Ankyrin
metabolic pathways to produce energy
o Adducing
RBC MEMBRANE CHARACTERISTICS o Tropomycin
o Tropomodulin
 Reticulocyte Membrane
o Young reticulocytes are less stable than those
of mature cells
o Possesses a significant amount of tubulin and
actin
o Changes:
 Increase in shear resistance
 Loss of surface area (about 20%) 
due to the loss of lipid membrane
 Acquisition of a biconcave shape 
if mature erythrocytes
 Loss of cytoplasmic organelles
 Undergoes active endocytosis and
exocytosis which does not occur in 1. Band 3
mature RBCs
 Anion transport
 Mature Red Blood Cell Membrane
o Exchanges bicarbonate or chloride
o Constantly changes as it moves through the o Linkage of lipid bilayer to the underlying
circulation membrane skeleton
o Soft and pliable (flexible) o Important for prevention of surface loss
o Biconcave shape
o Consists of a membrane skeleton (40% lipids 2. Glycophorin
 phospholipids and cholesterol, 8%
 Imparts a negative charge to the cell
carbohydrate  linked to lipid or proteins,
 Glycophorin A – carries MN, Gerbich blood group
52% glycoproteins  >50 transmembrane
antigens
proteins and >half-blood group antigens)
 Glycophorin C, Glycophorin A – important for
protein lattice and lipid bilayer
Plasmodium falciparum invasion and development in
o Deformable and tolerant against mechanical
the RBCs
stress and various pH and salt concentrations
in vivo and in vitro
3. Aquaporin 1
o Cell shape changes reversibly
 Selective pores for water transport
 Allows RBC to remain in osmotic equilibrium with
extracellular fluid
Erythrocytes I Page 1 of 4
 Red Cell Membrane Skeleton o Deformability can be reduced by increases in
o Hexagonal lattice with 6 spectrin molecules associations between skeletal proteins or
o Each are linked to multiple spectrin tetramers between skeletal and integral proteins
o Composition:
 Spectrin CYTOPLASMIC CHARACTERISTICS
 Actin 4.1
 Contents:
o Ankyrin
o Potassium ions
 Links the lipid bilayer to the
o Sodium ions
membrane via interaction with band
o Glucose  utilized by EMB pathway. 90%
3
total RBC glucose has been utilized
 Actin o Intermediate products of glycolysis
o Short, uniform filaments o Enzymes
o Length is modulated by tropomyosin/  Efficiency of cellular metabolism
tropomodulin mainly depends on enzymes
o Approximately 6 spectrin ends interface with
1 actin filament that is stabilize by protein 4.1
 Other Peripheral Proteins METABOLIC ACTIVITY

1. Protein 4.1
o Stabilizes actin-spectrin interactions
2. Adducin
o Also stabilizes interaction of spectrin with
actin
o Promotes interaction and influenced by
calmodulin (a calcium binding protein)
3. Ankyrin
o Interacts with band 3 and spectrin to achieve
linkage between lipid bilayer and skeleton and
later on augmented by protein 4.2
 Red Cell Mechanics
o Influenced by:
 Cell shape (ratio of cell surface area
to cell volume) EMBDEN-MEYERHOF PATHWAY
 Cytoplasmic viscosity (regulated by
MCHC)  Also known as anaerobic glycolysis
 Membrane deformability and  Glucose undergoes glycolysis to form ATPs
stability (important property of RBC  Maintains pyridine nucleotides in reduced state to
function) permit their function in oxidation-reduction reactions
 Cell Shape within the cell
o Creates an advantageous surface area over  Deficiencies to production of ATP can be exhibited by:
volume relationship o Premature cell death due to inherited defects
o Facilitates deformation while maintaining in glycolysis
constant surface area o Loss of viability during the storage of blood for
o Progressive loss of intracellular and transfusion
membrane components results in biconcave
shape and improved deformability
o SA/V ratio alterations result in more spherical
shape with less redundant surface area
o Wherever there is membrane loss, that will
lead to reduced surface area
o Increased cell water content, will lead to
increased volume
 Membrane Deformability/ Stability
o During pressure upon RBC
 Spectrin molecules they undergo
reversible change, some of those
become uncoiled and extended,
others appears compressed
o During extreme or sustained pressure
 Membrane exhibit permanent plastic
deformation
HEXOSE MONOPHOSPHATE SHUNT LEUBERING-RAPOPORT PATHWAY
 also known as oxidative pathway  This mechanism is low in energy consumption

 Oxidative catabolism of glucose with reduction of  Important in oxygen carrying capacities of the RBCs
NADP to NADPH  Capable of regulating oxygen transport even with
o Required to reduced glutathione hypoxia and acid-base disorders
 If the pathway is defective:  Permits the accumulation of 2,3 DPG
o Amount of reduced glutathione becomes  Increased deoxyhemoglobin results to binding of 2,3
insufficient to neutralize oxidants DPG
o Stimulates glycolysis
METHEMOGLOBIN REDUCTASE PATHWAY Metabolic Pathways in the Erythrocyte
 Depends on EMB pathway because it needs the Metabolic Pathway Function

reduced pyridine nucleotides that keeps haemoglobin Embden Meyerhof Maintains cellular energy by
generating ATP
in a reduced state
Oxidative or Hexose- Prevents denaturation of
 Prevents the oxidation of heme iron Monophosphate Shunt globin of the hemoglobin
 Requires the reducing action of NADH and the enzyme molecule by oxidation
methemoglobin reductase Methemoglobin Reductase Prevents oxidation of heme
iron
Leubering-Rapoport Regulates oxygen affinity of
hemoglobin
ERYTHROKINETICS
 Term describing the dynamics of RBC production and

destruction
 Erythron o 10% of RBC undergo destruction
o Name given to the collection of all stages of intravascularly
erythrocytes throughout the body, developing
precursor in the bone marrow and the
circulating RBC in the peripheral blood
ERYTHROPOIETIN
 A hormone produced in the kidney in response to

tissue hypoxia
 Specific Action:
o Induces committed progenitor cells in the
bone marrow to differentiate and proliferation
into pronormoblast
o Shortens the generation time of
pronormoblast
o Promotes the early of reticulocytes to the
o Causes:
peripheral blood
 ABO mismatched blood transfusion
 Increased: cases of erythroid hyperplasia,
 Cold agglutinin disease
polycythemia, hemorrhages and increased RBC
 Paroxysmal cold hemoglobinuria
destruction
 Burns
 Decreased: cases of Anemia (e.g., caused by kidney
 Snake bites
damage, the kidneys will produce less EPO  a
 Bacterial – C. perfringens
hormone that signals your bone marrow to make RBC
 Parasitic Infections – P. malaria
 lesser EPO, fewer RBCs, less oxygen delivered in
 Mechanical heart valves
organs and tissues)
 Paroxysmal nocturnal
hemoglobinuria
MECHANISM OF RED CELL DESTRUCTION
 Fragmentation  Extravascular
o Loss of a portion of the erythrocytes o Lysis of erythrocytes outside the circulation
membrane  Helps by reticuloendothelial system
o Accompanied by a loss of cellular contenets of the cell which are the liver and the
including Hgb spleen
 Osmotic Lysis  Usually happens through
o Passing of water into the red cell eventually phagocytosis and about 90% aged
burst the cell RBCs is destroyed extravascularly
 Erythrophagocytosis o Little or no hemoglobin escapes into the
o Ingestion of the whole RBCs by a circulating circulation
monocytes or neutrophils or by macrophages o Decreased haptoglobin
(mononuclear phagocyte system) o Normal plasma hemoglobin
o Causes:
 Complement Induced Cytolysis
 Bacterial/ viral infections
o Complement attach itself to the cells and
 Drug induced
induce lysis
 Autoimmune
 Hemoglobin Denaturation
 Microangiopathy
o When Hgb is exposed to oxidant stress 
 Hemoglobinopathies
later on denature the Hgb
 Membrane defects
o Happens when mechanism to protect the cell
 Metabolic defects – G6PD
after exposure to stress fails to work and
deficiency
denatured Hgb later on precipitates forming
inclusion bodies (Heinz Bodies)
2 TYPES OF DESTRUCTION
 Intravascular
o Usual outcome of sensitization of
erythrocytes with complement
o Lysis of erythrocytes which occurs within the
circulation through the classic pathway
RMT LECTURE 5: HEMOGLOBIN

Date: July 04, 2022
OUTLINE
 Lineage-Specific Hematopoiesis
 General Characteristic of Blast
 Hematopoietic System
o Erythropoiesis
o Granulopoiesis
o Monocyte Development
o Lymphocyte Development
o Megakaryopoiesis
HEMOGLOBIN
 One of the most studied proteins in the body because

of the ability to be easily isolated
 Consists of:
o 95% of cytoplasmic content of RBCs
 Oxygen Transport (from lungs to the tissues)
 Carbon Dioxide Transport (from the tissues to the lungs
for exhalation)
 Contributes to the acid base balance by releasing
hydrogen ions
 Nitric Oxide Transport
o Regulator of the vascular tone (contractile
activity of vascular smooth muscle cells in the
walls of smooth arteries and arterioles 
major determinant of blood flow throughout
the circulation)
COMPONENTS
 Globin
o Occurs in ribosomes
o Varied sequence of amino acids
o Difference in globin chains designation
relates both to the sequence and number of
amino acids
HgbA  4 heme groups and 4 polypeptide chains (574: total
Symbol Name # Amino Acids amino acid of the entire chain)
α Alpha 141
β Beta 146  Heme Production
γA Gamma A 146 (position 136: alanine) o Requires the formation of protoporphyrin IX
γG Gamma B 146 (position 136: glycine) and the availability of iron
𝛿 Delta 146 o The synthesis of heme begins in the
ε Epsilon 146 mitochondria with the formation of D-ALA
ζ Zeta 141 (Delta Aminolevunilic acid) from glycine and
θ Theta Unknown succinyl coenzyme A
Hemoglobin I Page 1 of 4
o Immature Red Cell with Ferritin:
sideroblasts
o Mature Red Cell: siderocytes
o Stain with: Prussian blue
Iron Compartments in Normal Humans

Compartment Form & Site % Total Typical Iron
Body Iron Content (g)
Functional Hemoglobin iron in blood 68 2.400
Myoglobin iron in muscles 10 0.360
Peroxidase, catalase, cytochromes, 3 0.120
riboflavin enzymes in all cells
Storage Ferritin and Hemosiderin mostly in 18 0.667
macrophages and hepatocytes; small
amounts in all cells except mature
RBC
Transport Transferrin in plasma <1 0.001
 Protoporphyrin IX  The connection of heme and globin through chemical

o PIX = nitrogenous substance synthesized bonds forms the basis of hemoglobin molecule
partly in mitochondria and partly in the HEMOGLOBIN STRUCTURE
cytoplasm of a nucleated red cell
 PRIMARY: specified sequence of amino acid residues
Role of Iron in Heme Synthesis
 SECONDARY: dividing the chain into 8 separate
helical segments (A to H)
 TERTIARY: arrangement of the helices into a pretzel-
like configuration
 QUARTERNARY: describes the complete hemoglobin
molecule
 Iron
o Most abundant transition metal in the body
o Iron uptake = controlled  maintain iron
balance
 E.g., Duodenum dietary 3 iron is
reduced to ferrous iron and taken up
from the intestinal lumen into the Normal Hemoglobin Variants
enterocytes. DMT1 (a protein called
divalent metal transporter 1 
primarily responsible for transporting
ferrous iron to the enterocytes).
DMT1 is instrumental in the uptake
of iron  stored in ferritin
(enterocytes)  or exported into the
circulation by another transport
protein which is Ferroportin
o In the ferrous form is required to convert
protoporphyrin to become heme
o Doesn’t have mechanism of active excretion
o Body adjust iron levels by intestinal
absorption depending on our body needs
o Sources: dietary, recycled from senescent
erythrocytes
o Storage: ferritin and hemosiderin
o Transport: transferrin and ferroportin
Hemoglonin I Page 2 of 4
NORMAL HEMOGLOBIN FORMS
 Oxyhemoglobin
o Hgb with ferrous iron and oxygen which is
seen in arterial circulation
 Deoxyhemoglobin
o Hemoglobin with ferrous iron but no oxygen
which is seen in venous circulation
 Regulator: phosphate  regulating oxygen affinity
 2,3-DPG (diphosphoglycerate) combines with the
beta chains of deoxyhemoglobin
REGULATION OF HEMOGLOBIN PRODUCTION
 Heme Regulation
o HEME: inhibits transcription of the ALA
synthase gene, ALA dehydrase and PBG
deaminase (negative feedback mechanism 
stops the synthesis)
 Globin Regulation
o Highly regulated  balance of heme and
globin HEMOGLOBIN DERIVATIVES
o Controlled at the transcription level
 Initiators:  Dyshemoglobins  dysfunctional  no oxygen
 Promoter DNA sequence transport  formed and may accumulate to toxic levels
 Key transcription factor (KLF-1 or after exposure to certain drugs, chemicals or gases
Kruppel-like factor 1) o Most are acquired
 Other transcription factors o Methemoglobinemia: small fraction are
(GATA-1, Ikaros, TAL-1, p45-NF- hereditary
E2 and LDB1)  Carboxyhemoglobin
 Enhancer region of DNAse 1 o Carbon monoxide will bind with oxygen (240
hypersensitive nucleic acid times)
sequences (Locus Control o Shift to the left
Region) o Cannot bind and carry oxygen
o KLF1, GATA1, Ikaros, TAL1, p45-NF-E2, and o Gasoline motors, gas heaters, defective
LDB1 stoves, smoking
o Locus Control Region o It is also known as “silent killer” because it is
odorless and colorless
OXYHEMOGLOBIN DISSOCIATION CURVE o Patient may quickly become hypoxic
 Methemoglobin
 Readily bind oxygen molecules to the lung o Reversible oxidation of heme iron to ferric
 Efficiently unload oxygen to the tissues (requires low state
oxygen affinity) o Decreased delivery of oxygen to tissue
 During oxygenation each of the 4 heme iron atoms can o Levels of Methemoglobin:
reversibly bind oxygen (1.35mL of O2 = 1g of Hgb)  <25% asymptomatic
 Hemoglobin has the ability to bind large quantities of  >30% cyanosis (bluish discoloration
O2, however, hemoglobin must be willing to release O2 of the skin and mucous membranes)
when needed and symptoms of hypoxia (dyspnea,
 Shift to the left: lesser oxygen being released to the headache, vertigo and change in
tissue mental health status)
 Shift to the right: oxygen given more to the tissues  >50% coma and death
o Derivative of hemoglobin in which the ferrous
Shift to the Left Shift to the Right ions is oxidized to ferric state
DECREASED: INCREASED: o Chocolate brown discoloration of blood,
pCO2 pCO2
cyanosis and functional anemia
Hydrogen Ions Hydrogen Ions
2,3-DPG 2,3-DPG  Sulfhemoglobin
Temperature Temperature o Mainly caused by:
INCREASED: DECREASED:  drugs (sulfanilamides, phenacitin,
pH (alkalosis) pH (acidosis) nitrites and phenyl hydrogen)
Presence of Hgb F/HbF  exposure to chemicals (industrial
**remember yung kwento ng pangangaliwa and environmental setting)
 exposure to sulfur
o Addition of sulfur atom to pyrrote ring of heme
(It has GREENISH pigment)
o Ineffective for oxygen transport
o Patients with elevated sulfhemoglobin may
exhibit cyanosis
o Mixture of oxidized, partially denatured forms
of hemoglobin that form during oxidative
hemolysis
o Associated with sufonamides administration,
severe constipation, Clostridium perfringens,
enterogenous cyanosis
o Cannot be reduced back to hemoglobin
RMT LECTURE 6: RED BLOOD CELL ABNORMALITIES

Date: July 04, 2022
OUTLINE  Variation in Red Cell Color

 Red Blood Cell Abnormalities o Polychromasia
o Variations of RBCs  Variation in hemoglobin content
o Hemoglobin Content showing a slight blue tinge (wright
 Variation in Red Cell Shape stain), gray-blue and larger than
o Developmental Macrocytosis normal; residual RNA
o Membrane Abnormalities o Hypochromasia
o Trauma  Larger than normal central area of
o Abnormal Hemoglobin Content
pallor
 Inclusions
 IDA, thalassemia, anemia of chronic
disease, sideroblastic anemia,
RBC ABNORMALITIES myelodysplastic anemia
 Cell Size = microcytosis, macrocytosis Table 1: Hypochromasia Grading

 Variability in Size = anisocytosis
GRADE DESCRIPTION
 Cell Color = hypochromia 1+ Central pallor is ½ of cell diameter
 Cell Shape = poikilocytosis  cellular inclusions 2+ Central pallor is 2/3 of cell diameter
3+ Central pallor is ¾ of cell diameter
4+ Thin rim of hemoglobin
VARIATIONS OF RED BLOOD CELLS
 Average Size: 7.2um Table 2: Polychromasia Grading

o 6 to 8um in diameter, 6.8 – 7.5 or 7-8um in
GRADE % POLYCHROMATIC RED CELLS
diameter
Slight 1%
 Variation in Size: 1+ 3%
o Normocyte  normal size 2+ 5%
o Microcyte  smaller size (<6) 3+ 10%
 Associated with a decreased 4+ >11%
hemoglobin synthesis
 Deficiency of iron (IDA), impaired
globulin synthesis, mitochondrial HEMOGLOBIN CONTENT
abnormality affecting the heme
synthesis of the heme unit  The concentration of hemoglobin with RBCs is
(mitochondrial abnormality) approximately 34g/dL, and its molecular weight is
 IDA  malabsorption approximately 64,000 Daltons
 Hemoglobinopathies  Normochromic: 32 to 36g/dL
o Macrocyte  larger size (>8)  Hypochromic: <32g/dL
 Result in defect in nuclear o Seen in cases of thalassemia, iron deficiency
maturation  Hyperchromic: >36g/dL (misnomer)
 Deficiency in Vitamin B12 o Spherocytes  full  no central pallor
or folate
VARIATION IN RED CELL SHAPE
 Disruption of regular mitotic
division inside the bone  Poikilocytes secondary to developmental macrocytosis
marrow  Poikilocytes secondary to membrane abnormalities
 Stimulation of erythropoietin (EPO)  Poikilocytes secondary to trauma
 Increased hemoglobin
production (premature DEVELOPMENTAL MACROCYTOSIS
released of reticulocytes in
blood circulation   Ovalocyte
macrocytic and basophilic o Egg-like or oval shaped, wider than
 slightly hypochromic elliptocytes
cells) o Reduction in membrane cholesterol:
 Appear as mature and large  Megaloblastic bone marrow
erythrocytes  myelodysplasia
 Macrocytic and basophilic cells
Red Blood Cells Abnormalities I Page 1 of 5
o Also known as megalocytes (macrocytic and  Codocytes
rounder appearance) o Also known as target cell
o Features: o Bull’s eye appearance
 Markedly increased MCV o The cells are thinner than normal, which may
 Megaloblastic erythropoiesis be because of an excessive ratio of
 No central area of pallor membrane lipid to cell volume
 Megaloblastic anemia o Causes:
 Hemoglobinopathies
Differential Diagnosis of Macrocyte  Thalassemia
 Obstructiver liver disease
 Oval Macrocyte
 Post-splenectomy
o Seen in:
 IDA
 Folic acid deficiency
 Vitamin B12 deficiency
 Pernicious anemia
 Round Hypochromic Macrocyte
o Seen in:
 Alcoholism
 Hypothyroidism
 Liver disease
 Blue-Tinged Macrocyte
o Seen in:
 Neonate response to anemic stress  Spherocyte
 Response to anemic stress o Low surface area to volume ratio
o Defect or loss of membrane:
MEMBRANE ABNORMALITIES  Hereditary spherocytosis
 Isoimmune and autoimmune
 Acanthocytes haemolytic anemia
o Also known as Spurr/thorn cell  Severe burns
o Spheroid with 3-12 irregular  Banked blood stored for a long time
spikes/spicules
o Associated Conditions:
 Alcoholic cirrhosis with haemolytic
anemia
 Malabsorption states
 Post splenectomy,
 Hepatitis of newborn
 Pyruvate kinase
 Abetalipoproteinemia
 Stomatocyte
o Also known as mouth cell or slit like pallor
area (one side of the cell)
o Increase Sodium Ion and Decrease
Potassium Ion
 Echinocytes o Increase permeability of the membrane to
o Also known as Burr cells sodium
o Having one or more spiny projections of o Associated Conditions:
cellular membranes  Hereditary stomatocytosis
o Quarter-moon shape  Rh Null
o Less spherical than acanthocytes  Alcoholism
o Regular 10 – 30 scalloped short  Cirrhosis
projections evenly distributed  Obstructive liver disease
o Causes:
 Uremia
 Pyruvate kinase deficiency
 Anemai with renal insufficiency
 Renal disease

 Elliptocyte ABNORMAL HEMOGLOBIN CONTENT
o Narrower and more elongated than
megalocytes  Drepanocytes
o Rod or cigar shaped o Also known as sickle cells/ meniscocytes
o Defect is considered to be in the cytoskeleton, o Crescent shaped cell
with a decrease in the membrane protein o Membrane: smooth and stays uniformly
band 4.1 throughout
o Associated Clinical Disorders: o Polymerization of deoxygenated hemoglobin
 Hereditary elliptocytosis S (decreased O2 levels and blood pH)
 Anemias associated with o Associated Conditions:
malignancy  Sickle cell anemia
 Hemoglobin C disease  SC disease
 Haemolytic anemias
 Iron deficiency anemia
 Pernicious anemia
 Sickle cell trait
 Thalassemia
 Other Poikilocytes
o Blister Cells
 Red cell with single or multiple
POIKILOCYTES SECONDARY TO TRAUMA vacuoles or markedly thinned areas
at the periphery
 Schistocytes
 Characteristic of Heinz body
o Also known as Schizocyte, Fragmentocyte
mediated hemolysis
and “egg shell”
 Appear to have a partially raised or
o Fragmentation produced by damage of RBC
blistered membrane
by fibrin, altered vessel walls, prosthetic heart
valves
o Schistocyte with Horn-like projections
 Keratocytes/ Helmet cells
o Conditions Associated:
 Disseminated intravascular
coagulation (DIC)
 Thrombotic thrombocytopenic
purpura (TTP)
 Burns
 Microangiopathic haemolytic  Degmacyte (Bite Cell)
anemia o Erythrocytes with irregular membrane results
from splenic macrophage mediated removal
of denatured hemoglobin molecule
o Drug-induced anemias
o G6PD Deficiency
o Thalassemia
o Unstable
o Hemoglobinopathies
 Dacryocyte
o Also known as teardrop cells
o Squeezing and fragmentation during splenic
passage (one side is pointed resembling pear
shape)
 Myeloid dysplasia
 Hypersplenism
 Thalassemia

POIKILOCYTES
 Slight: less than 5%

 Moderate: 5 to 15%
 Marked: more than 15%
Particular Variation:
 Occasional: <1%
 Few: 1 to 5%
 Frequent: 5 to 10%
 Many: >10%
INCLUSIONS
 Heinz Bodies
 Howell-Jolly Bodies
o Deep purple, irregularly shaped inclusions
o Dark blue purple, dense and round granule
o Requirement:
o 1% only
 Supravital stains (crystal violet 
dark or deep purple inclusions that
 Megaloblastic anemia
are attached to inner RBC
 Severe haemolytic process
membrane)
 Thalassemia
 Accelerated erythropoiesis
 Defects in HMP
o Composition:
 G6PD Deficiency
 DNA (nuclear fragments)
 Unstable Hb
 Splenectomy
 Thalassemia
o Composition:
 Precipitated denatured hemoglobin
(can formed in patients with G6PD
deficiency)
 Basophilic Stipplings
o Dark blue granules
o Blueberry bagel
 Lead poisoning
 Pyrimidine-5-nucleotidase
deficiency
 Heavy metal poisoning
 Hb H Inclusions
 Thalassemia
o Inclusions represent precipitated Hb H
o Composition:
o Uses supravital stain (brilliant cresyl blue)
 Precipitated RNA
o Hb H Disease:
 Alpha thalassemia with moderate
hemolytic anemia
o Composition:
 Precipitated B-globin chains of
hemoglobin
 Cabot Ring
o Rings, loops, figure of eight, red to purple
(some are blue)
o Bell or tall hat shape on scanning EM
 Megaloblastic anemia
 Severe anemias
o Composition:
 Mitotic spindle remnants

 Hb CC Crystals
o It can be Rhomboid, Tetragonal or Hexagonal
shaped crystals
o Seen after splenectomy
o Hexagonal with blunt ends and stain darkly
o Homozygous C (Hb CC) disease
 Malaria
o Protozoan transmitted by
mosquitoes
o Maturation Stages:
 Rings
 Trophozoites
 Schizonts
 Hb SC Crystals  Gametocytes
o Finger-like, quartz-like crystal of dense
hemoglobin protruding from the RBC
membrane
o Dark-hued crystals of condensed Hb distort  Babesia
the red cell membrane o Protozoan inclusion, tick
o Crystalline projection is often straight with bite
parallel sides and one blunt, pointed, o “Maltese cross” formation
protruding end
 Others
o Agglutination:
 Clumping of red cells
 Red cell aggregation
o Rouleaux:
 “Stack of coins” pattern of red cells
 Increase plasma globulin
 Ringed Sideroblast
o Nucleated RBC that contains non-heme iron
particles
o Excessive iron overload in mitochondria of
normoblasts
 Sideroblastic anemia
 MDS (Myelodysplastic Syndrome)
RBC MORPHOLOGY GRADING
MORPHOLOGY GRADE
Polychromatophilia 1+ = 1 to 5/field
Helmet cell, dacrocyte 2+ = 6 to 10/field
Spherocyte, acanthocyte, schistocyte 3+ = >10/field
Poikilocytosis 1+ = 3 to 10/field
Codocyte, Burr Cells 2+ = 11 to 20/field
Stomatocyte, Ovalocyte, Elliptocyte 3+ = >20/field
Rouleaux 1+ = 3 to 4 agg.
 Pappenheimer Bodies
2+ = 5 to 10 agg.
o Dark staining iron granules that usually 3+ = numerous
clumped together at periphery of the cell agg.
o Associated Conditions: Sickle cells POSITIVE ONLY
 Sideroblastic anemia Basophilic stippling
 MDS Pappenheimer bodies
 Thalassemia Howell-Jolly
 Haemolytic anemia
 Defective erythropoiesis
o Composition:
 Iron

RMT LECTURE 7: WHITE BLOOD CELL ABNORMALITIES

Date: July 05, 2022
OUTLINE CIRCULATING KINETICS AND MORPHOLOGY

 White Cells
o Circulating Kinetics and Morphology  Band Cells/ Stab Cells
 Nuclear Abnormalities o Cell undergoing granulopoiesis, derived
o Pelger-Huet from a metamyelocyte, and leading to a
o Pseudo-Pelger Huet Anomaly mature granulocyte
o Hypersegmentation o Left Shift: increase release of precursors
 Cytoplasmic Abnormalities from the bone marrow
o Alder-Reilly Granules o Right Shift: characterised by the presence of
o Auer Rods hypersegmented polymorphonucleocytes
o Chediak-Higashi  In the Circulating System:
o Dohle Bodies o Stem Cell Pool
o May-Hegglin Anomaly
 Consists of hematopoietic stem cells
o Toxic Granules
o LE and Tart Cell (HSCs) that are capable of cell
 Lymphocyte Abnormalities renewal and differentiation
o Reactive Lymphocytes o Proliferation Pool
o Downey Cells  Also known as mitotic pool
o Basket/ Smudge Cell  Consists of cells that are dividing
o Hairy Cell (common myeloid progenitors or
o Sezary Cell also known as CFUs)
o Flame Cell o Maturation Pool
o Grape Cell  Also known as storage pool
 Cells undergoing nuclear maturation
that formed the marrow reserve that
WHITE CELLS
are available for release
 Major Function:  Neutrophils are believed to survive 2 to 5 days after
o The major function of neutrophils is to entering the tissues
respond rapidly to microbial invasion to kill the o Cells Available for Release:
invaders (phagocytosis)  Metamyelocytes
o Steps:  Band neutrophils
1. Chemotaxis (movement of segmented  Segmented neutrophils
neutrophil)
NUCLEAR ABNORMALITIES
2. Adherence (adherence of phagocytic cell
to bacterial cell wall). It can be enhanced
by opsonisation (uses opsonins  to
tag foreign pathogens) PELGER – HUET
3. Engulfment
4. Phagosome formation (formed when  Also known as true or congenital PHA
phagocyte disrupts a portion of plasma  Autosomal dominant disorder characterized by a
membrane around the particle) decreased nuclear segmentation
5. Fusion (phagosome fuses with 1 or more  Nuclei:
lysosomal granules) o Round oval or bilobed which are spectacle-
6. Digestion (normally results in autolysis or like, pince-nez like, dumbbell or peanut
disruption of the phagocyte)  Chromatin:
o Intense nuclear clumping
 Pelger-Huet Anomaly is also described as a blood
laminopathy associated with the lamin B receptor
o Mutation of lamina B receptor or the LBR
gene on the band 1q42
White Blood Cells Abnormalities I Page 1 of 5

PSEUDO-PELGER HUET ANOMALY Table 1: Mucopolysaccharidoses Disorders
 Acquired anomaly Name Subtype Enzyme Deficiency

 Helpful in diagnosis of myeloproliferative neoplasms MPS I Hurler Syndrome a-I-iduronidase
 Causes: MPS I Scheie Syndrome a-I-iduronidase
o High stress conditions like burns MPS II Hunter Syndrome Iduronate sulfatase
o Drug reactions MPS III A Sanfilippo Syndrome heparan N-sulfatase
o MDS MPS III B Sanfilippo Syndrome a-N-
o Malignancy acetylglucosaminidase
o Myeloproliferative disorder MPS III C Sanfilippo Syndrome Heparan acetyl-CoA:
a-glucosaminidase
N-acetyltransferase
MPS IV A Morquio Syndrome Galactose-6-Sufatase
MPS IV B Morquio Syndrome B-Galactosidase
**Hurler syndrome: most severe form;
Scheie syndrome: lesser severe form
**refer to Table 26.3 (Rodaks) for the complete details about

Mucopolysaccharidoses Disorders
AUER RODS
 Needle-like bodies seen in cytoplasm of myeloblasts

and granulocytes in certain leukemias
HYPERSEGMENTATION
 Pink or red rod shaped structures
 Normally 3 to 5 lobes (>5 lobes)  Found in myeloid and monocytic series
 Sometimes referred to as a myeloid “right shift”  Cytoplasmic inclusions that result from fusion of
 Also called polycytes or macropolycytes primary azurophilic granules
 Associated Condition:  If found it confirm the presence of myeloblasts
o Megaloblastic anemia o Presence of non-lymphocytic leukemia
 Larger than normal (myeloid leukemia)
o Chronic granulocytic leukemia
 Never seen in lymphoblast and it uses as
differentiator between lymphoblastic and myeloblastic
leukemia
 Classified as pathological
CYTOPLASMIC ABNORMALITIES
CHEDIAK-HIGASHI GRANULES
ALDER-REILLY GRANULES
 Giant red, blue to grayish round inclusions
 Rare inherited disorder characterized by granulocytes  These bodies are formed by aggregation and fusion
that are large, darkly staining cytoplasmic granules of the primary and secondary specific granules
 Large purple-black coarse cytoplasmic granules  Complications:
o Reilly Bodies o Neutropenia
 Accumulation of degraded mucopolysaccharides o Thrombocytopenia
 The primary defect is in special granules present in
skin pigment cells and certain white blood cells
 Chediak-Higashi Syndrome is caused by mutations
in the LYST gene (specifically CHS I LYST 
Lysosomal Trafficking Regulator Gene  located in
chromosome 1q42.1 – 2  encodes for a protein that
regulates the morphology and function of the lysosome
related organelles)
o Rare autosomal recessive disease of immune
dysregulation
TOXIC GRANULES
 Manifested by presence of large granules in the

cytoplasm
 Band or segmented neutrophils in the peripheral blood
 Large purple to black (some are red) granules 
azurophilic granules
o Normally present in early myeloid forms only,
not normally seen in band or segmented
 Clinical Manifestation: neutrophils
o Partial albinism o Contains peroxidases and hydrolases
o Paired with a severe recurrent life threatening  Toxic granulation is clinically significant because it
bacterial infections or sepsis appears to reflect a poorer prognosis
 Include giant lysosomal granules in the
 Associated Conditions:
granulocytes, monocytes or lymphocytes o Severe infection  most frequent cause of
DOHLE BODIES toxic granulation
o Rheumatoid arthritis
 Single or multiple blue inclusions o Less frequent but can result to
 Aggregates of free ribosomes of RER autophagocytosis
 Inclusions consisting of remnants of rRNA
 Typically found either in band or segmented
neutrophils
 Can also appear together with toxic granulation
 Confused with May-Hegglin
o If it can be seen in eosinophils, basophils and
monocytes
 Associated Conditions:
o Infection
o Burns LE CELL
o Leukemia
 Lupus Erythematosus
o Chemotherapy
o Intact neutrophil that has an engulf
o Inflammatory states
homogenous mask of degenerated nuclear
material  displaced the normal nucleus
 Neutrophil with large purple homogenous round
inclusion
 Ingested: neutrophil  degenerated nuclear material
MAY-HEGGLIN ANOMALY
 Inherited dominant condition

 Dohle-Body Like Inclusions
o 2 to 5um
o Basophilic  Tart Cell
o Large basophilic inclusions that resemble the o Monocyte/ histiocyte with ingested
Dohle bodies are present lymphocyte
 Pale blue inclusion o Not important and pathologic and has no
 With the presence of giant platelets diagnostic value

LYMPHOCYTE ABNORMALITIES HAIRY CELL
 Nuclei: round to ovoid, lack nucleoli

 Relatively abundant cytoplasm with ragged
REACTIVE LYMPHOCYTE projections
 Also known as Atypical Lymphocyte/ Transformed  Lymphocyte with hair-like cytoplasmic projections
Lymphocyte/ Variant Lymphocyte surrounding the nucleus
 Large and exhibit deep blue to pale gray cytoplasm  Hairy Cell Leukemia
 Seen in benign reactive processes o Diseases of B cell lineage commonly found in
middle-aged men (50 years old)
 Stimulated
o Spleen, blood and bone marrow is the major
 Presence of reactive lymphocytes indicates immune
focus of this disease
stimulation (e.g., virus  Epstein Barr Virus  causes
o Typically presenting Splenomegaly
infectious mononucleosis)
o Cytopenias
 Reactive Lymphocyte
o Preferred term
DOWNEY CELL SEZARY CELL
 Early classification system of certain forms of variant  Mononuclear cell with cerebriform (grooved like or
lymphocytes folded convoluted pattern, surrounds thin rim of
 Type I cytoplasm) nucleus
o Turk’s irritation cell  Cutaneous T cell lymphomas
o With block of chromatin o Leukemic phase is the Sezary syndrome
o Small cells with minimum cytoplasm  Round lymph cell with nucleus that is grooved or
o Nucleus: indented convoluted
 Type II  Sezary Syndrome
o IM cells  Infectious Mononucleosis Cells o Leukemic phase of T cell lymphomas
o Most common type o Characterized by exfoliative erythroderma,
o Large cells with abundant cytoplasm peripheral lymphadenopathy
o Round mass of chromatin o Can be seen in skin, lymph nodes and
o Ballerina skirt appearance peripheral blood
 Type III o Mycosis fungoides
o Vacuolated
o Large moderated basophilic cytoplasm
o Nucleoli: apparent
o Swiss chief or moth eaten appearance
BASKET CELL/ SMUDGE CELL
 Natural artifacts
 Represents the bare nuclei of leukocytes
 Degenerated nucleus
 Pressure in making smear
 Increased number = Chronic Lymphoblastic
Leukemia (CLL)

FLAME CELL
 Cytoplasm: stains bright red to pink

 Increased quantities of glycogen and has intracellular
deposits of amorphous matter
 Plasma cell with red to pink cytoplasm
 Associated with increased Ig
o Multiple myeloma
GRAPE CELL
 Also known as Mott/ Berry Cell

 Cytoplasm: filled with russel bodies (acidophilic,
refractile (bodies are transparent) globules that
represent gamma globulin secretions)
 Plasma cell with vacuoles
 Large protein globules
 Multiple myeloma

RMT LECTURE 8: HEMATOLOGIC PROCEDURES

Date: July 05, 2022
OUTLINE Hemacytometer
 Complete Blood Count
o Manual Cell Count  Also known as counting chamber
 Differential Count  Improved Neubauer/ Levy Chamber
o Absolute Count
o Rule of Three
 Red Cell Indices
o MCV
o MCH
o MCHC
 Red Cell Distribution Width
 Reticulocyte Count
o Miller Disc
o Absolute Reticulocyte Count
o Corrected Reticulocyte Count
o Reticulocyte Production Index
o Automated Reticulocyte Count
 Erythrocyte Sedimentation Rate (ESR)
 Osmotic Fragility Test (OFT)
 Point of Care Test
 Automation
o Electronic Impedance Table 1: Manual Cell Counts with Most Common Dilutions,
o Optical Scatter Counting Areas
o Radiofrequency
Cells Diluting Fluid Dilutio Objecti Area
o CBC Parameters and Quality Control Counted n ve Counted
WBC 1% ammonium oxalate or 120 10x 4mm2
3% acetic acid or 1:100 10x 9mm2
COMPLETE BLOOD COUNT 1% hydrochloric acid
RBC Isotonic Saline 1:100 40x 0.2mm2 (5
small
 Cell Counts squares of
o RBC Count center
o Platelet Count  different request from the square)
Platelets 1% ammonium oxalate 1:100 40x 1mm2
physician, not included in CBC phase
o WBC Count **hayems fluid  RBC  much better result than isotonic saline
o Differential Count because it can cause formation of rouleaux
 Hemoglobin
 Hematocrit Calculations
 Blood Cell Indices (MCV, MCH, MCHC)
The general formula for manual cell counts is as follows and can
MANUAL CELL COUNT be used to calculate any type of cell count: (1 chamber only
proceed directly to the computation but if 2 chamber counted 
 Red Blood Cell Count get the average and proceed to the formula below)
 Platelet Count
𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
 White Blood Cell Count Total Count =
𝑎𝑟𝑒𝑎 (𝑚𝑚2)𝑥 𝑑𝑒𝑝𝑡ℎ (0.1)
Materials Needed 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟 𝑥 10∗

Total Count =
𝑎𝑟𝑒𝑎 (𝑚𝑚2)
 Hemacytometer
 Thoma Pipet
 Suction Device RBC HEMACYTOMETER:
 Thick Coverslip
 Cell Counter  25 medium squares  1 square = 0.04mm  5
 Diluting Fluids squares is = 0.2mm2
 16 small squares
Hematologic Procedures I Page 1 of 6

N-RBC
 Falsely counted as WBC

 Not lysed by WBC diluting fluids
 >5 NRBC per 100 WBCs seen in PBS  CORRECT
THE WBC COUNT
 Correct the WBC Count:
𝑢𝑛𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑊𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 𝑥 100
𝑛𝑢𝑚𝑏𝑒𝑟𝑠 𝑜𝑓 𝑛𝑅𝐵𝐶 + 100
 Remarks: “corrected WBC count”
Correct Order of Leukocytes in the Peripheral Blood:
Indirect Platelet Count
1. Segmented Neutrophil (50%)
Platelet Estimate of Report Platelet Estimate as 2. Lymphocyte (34%)
0 – 49,000/uL Markedly Decreased 3. Monocyte (4%)
50,000 – 99,000/uL Moderately Decreased 4. Band Neutrophil (3%)
100,000 – 149,000/uL Slightly Decreased 5. Eosinophil (2.7%)
150,000 – 199,000/uL Low Normal 6. Basophil (0.3%)
200,000 – 400,000/uL Normal
ABSOLUTE COUNT
401,000 – 599,000/uL Slightly Increased
600,000 – 800,000/uL Moderately Increased  More accurate than the relative count
Average 800,000/uL Markedly Increased  Actual number of specific WBC in a liter of blood
 Absolute count = percentage x WBC count
Disposable Blood Cell Count Dilution Systems
Hemoglobin
 Capillary pipette and diluent reservoir systems
 Higher in the morning and lower in the evening
o WBC Count and Platelet Count
 Screen for anemia and may detect RBC breakdown/
o Leuko Check (Biomedical Polymers)
hemolytic anemia
 1,98mL of 1% ammonium oxalate
 Higher in high altitudes
 Blood: 20uL
 Increase in strenuous muscular activity
o EDTA or Skinpuncture
 Reference Values:
o Allow 10 minutes to lysed RBCs
o Male: 13.5 to 18.0g/dL
o First 3 to 4 drops discard, succeeding drops
o Female: 12.0 to 15.0g/dL
can be used in hemacytometer
DIFFERENTIAL COUNT CYANMETHEMOGLOBIN METHOD
 One hundred WBCs are counted and classified  Blood is diluted using Drabkin Solution (potassium
through the use of push-down button counters ferricyanide, potassium cyanide, sodium bicarbonate,
 Results are reported as percentages surfactant)
 Principle: a stained smear is examined to determine Hemoglobin (Fe2+) + K3 Fe (CN)6  methemoglobin (Fe3+) +
the percentage of each type of leukocyte to present KCN  cyanmethemoglobin
and assess the erythrocyte and platelet
morphology 1. Hemoglobin first oxidized to methemoglobin by
 WBC Abnormalities are also reported potassium ferricyanide
o Reporting: 2. Converts methemoglobin to cyanmethemoglobin
 Reactive Lymphocytes (measured by spectrophotometer)
 Separate % as a % of total 3. Absorbance of cyanmethemoglobin will be read at
lymphocytes 540nm  directly proportional to hemoglobin
 Semi-quantitatively: concentration
occasional to many
 Toxic Granulation Hematocrit
 Present  “packed cell volume”
 Semi-quantitatively:  Volume of packed RBCs that
slight to marked/ 1+ to 4+ occupies a given volume of
 Specimen whole blood
o Peripheral blood  Reported: percentage (%) or
o Bone marrow L/L
o Body fluid sediments  Reference Range:
o Female: 36 to 45%
o Male: 41.5 to 50.4%

RULE OF THREE  Normal Value: 32 to 36g/dL
 Applicable if red cells are normocytic-normochromic Table 2: Red Blood Cell Indices, Red Blood Cell Morphology,
 The value of the haematocrit should be three times the and Disease States
value of the hemoglobin plus or minus 3
MCV MCHC RBC Found in
Hgb =12g/dL (fL) (g/dL) Morphology
<80 <32 Microcytic, IDA, Anemia of
HCT = 36% (0.36L/L) Hypochromic inflammation, thalassemia
Hb E disease and trait,
sideroblastic anemia
According to the rule of three,
80 to 32 to 36 Normocytic, haemolytic anemia,
100 Normochromic myelophthisic anemia, bone
Hgb (12) x 3 = HCT (36)
marrow failure, chronic renal
disease
An acceptable range for the haematocrit
>100 32 to 36 Macrocytic, Megaloblastic anemia,
would be 33% to 39%. These values conform Normochromic chronic liver disease, bone
to the rule of three marrow failure,
myelodysplastic syndrome
RED CELL INDICES
 Used to define the size and hemoglobin content of the RED CELL DISTRIBUTION WIDTH
red blood cell
 Aids in diagnosing and differentiating anemia  It reflects the degree of red cell variation in size
o MCV  mean cell volume  Formula:
o MCH  mean cell hemoglobin 𝑆𝐷
o MCHC  mean cell hemoglobin RDW= 𝑥 100
𝑀𝑒𝑎𝑛 𝑆𝑖𝑧𝑒
concentration
 Normal Value: 11.5 to 14.5%
MEAN CELL VOLUME (MCV)  Increased RDW  anisocytosis
 Microcytic/ Macrocytic  normal RDW + dec or inc
 Average volume of red blood cells expressed in MCV
femtoliters (fL)  Anisocyte wih size within reference range = increase
 Formula: RDW + Normal MCV
𝐻𝐶𝑇 (%) 𝑥 10  Anisocyte with size below or above the normal range =
MCV = abnormal MCV and RDW
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑥1012 /𝐿)
For example, if the HCT = 45% and the RBC

count = 5 x1012/L the MCV = 90fL Representative Critical Values
 Normal Value: 80 to 100fL  Hemoglobins: less than 5.0g/dL

 Haematocrit: less than 15%
MEAN CELL HEMOGLOBIN (MCH)  Platelet Count: less than 30,000 per microliter
 Average weight of hemoglobin in a red blood cell  WBC Count: less than 2,500 per microliter and greater
expressed in picograms (pg) than 30,000 per microliter
 Not considered in the classification of Anemias
 Formula:
RETICULOCYTE COUNT
𝐻𝐺𝐵 (𝑔/𝑑𝐿) 𝑥 10
MCH =
𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑥1012 /𝐿)
 An indicator of the rate of erythrocyte production
For example, if the hemoglobin = 16g/dL and  Use of supravital stains (new methylene blue)
the count = 5 x 10/L, the MCH = 32pg o Any non-nucleated RBC that contains 2 or
 Normal Value: 26 to 32pg more particles of the blue stain granulo-
material is defined as Reticulocyte
MEAN CELL HEMOGLOBIN CONCENTRATION (MCHC)  Specimen: whole blood in EDTA
 The count is expressed as a percentage of total
 An average concentration of hemoglobin in each erythrocytes
individual red blood cell expressed in grams per  Formula:
decilitre (g/dL)
 Formula: Reticulocytes (%) =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑥 100
1000 (𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡𝑒𝑑)
𝐻𝐺𝐵 (𝑔/𝑑𝐿) 𝑥 10
MCHC = For example, if 15 reticulocytes are counted,
𝐻𝐶𝑇 (%)
For example, if the HGB = 16g/dL and the 15 𝑥 100

Reticulocytes (%) = = 1.5%
HCT = 48%, the MCHC = 33.3g/dL 1000

 Reference Range:  Calculation (RODAKS)
o Adult: 0.5 to 1.5% CRC (%) = Reticulocyte (%) x
𝑝𝑎𝑡𝑖𝑒𝑛𝑡 𝐻𝐶𝑇 (%)
45
o Newborn: 2.0 to 6.0%
MILLER DISC  Reference Value: 0.5% to 1.5 – 2.0%
 Designated to reduce the labor – intensive process of RETICULOCYTE PRODUCTION INDEX (RPI)
counting reticulocytes
 Measures erythropoietic activity when stress
 Disc is inserted into the eyepiece
reticulocytes are present
of the microscope
 Shift Reticulocytes
 Minimum of 112 cells should be
o Prematurely released reticulocytes from the
counted in the small square
marrow
o Smaller Square (B):
o Shifted from the bone marrow to the
1/9 of the larger square
peripheral blood
o 112 cells = 1,008 red
 Acute bleeding (low levels of RBC
cells
count)
o Larger Square (A): reticulocytes
 Anemia
 Formula:
o Instead of 1 day  2 to 3 days in the
Reticulocytes % =
𝑛𝑢𝑚𝑏𝑒𝑟 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 (𝑙𝑎𝑟𝑔𝑒 𝑠𝑞𝑢𝑎𝑟𝑒)𝑥 100
peripheral blood
𝑛𝑢𝑚𝑏𝑒𝑟 𝑅𝐵𝐶𝑠 𝑖𝑛 𝑠𝑞𝑢𝑎𝑟𝑒 𝐵 (𝑠𝑚𝑎𝑙𝑙 𝑠𝑞𝑢𝑎𝑟𝑒) 𝑥 9 o Larger and bluish color
For example, if 15 reticulocytes are counted in the large  Reference Value:

square and 112 RBCs are counted in the small square, o >3 = adequate bone marrow response
Reticulocytes % =
15 𝑥 100
= 1.5% o <2.1 = inadequate bone marrow response
112 𝑥 9  Formula:
𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 (%)𝑥 (𝐻𝐶𝑇 % 45%)
RPI =
ABSOLUTE RETICULOCYTE COUNT (ARC) 𝑚𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
𝑐𝑜𝑟𝑟𝑒𝑐𝑡𝑒𝑑 𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒 𝑐𝑜𝑢𝑛𝑡
Or RPI =
𝑚𝑎𝑡𝑢𝑟𝑎𝑡𝑖𝑜𝑛 𝑡𝑖𝑚𝑒
 The actual number of reticulocytes in 1 liter (L) or 1
microliter (mL) of blood For example, for a patient with a reticulocyte count of
 Formula: 7.8% and a HCT of 30%, and with polychromasia noted, the
𝑟𝑒𝑡𝑖𝑐𝑢𝑙𝑜𝑐𝑦𝑡𝑒𝑠 (%)𝑥 𝑅𝐵𝐶 𝑐𝑜𝑢𝑛𝑡 (𝑥1012 /𝐿) previous table indicates a maturation time of 2 days. Thus
ARC =
100
7.8 𝑥 (30/45)
For example, if a patient’s reticulocyte count is 2% and RPI =
2
the RBC count is 2.20 x 1012/L, the ARC is calculated as follows
(note that the calculated result has to be converted from 10 12/L RPI = 2.6
to 109/L) Table 3: Hematocrit Value and Maturation Time
2 𝑥 (2.20𝑥1012 /𝐿)
ARC = = 44 𝑥109 /𝐿 Patient’s Hematocrit Value Correction Factor
100
(%) (Maturation Time, Days)
 Reference Range: 20x109/L to 115x109/L 40 – 45 1
35 – 39 1.5
CORRECTED RETICULOCYTE COUNT (CRC) 25 – 34 2
15 – 24 2.5
 In specimens with a low haematocrit, the percentage of <15 3
reticulocytes may be falsely elevated because the
whole blood contains fewer red blood cells
 A correction factor is used, with the average normal AUTOMATED RETICULOCYTE COUNT
haematocrit considered to be 45%
 Analyzers evaluate reticulocytes using optical scatter
 Formula:
or fluorescence after the red blood cells are treated
CRC = TURGEON
with fluorescent dyes or nucleic acid stains to stain
𝑝𝑎𝑡𝑖𝑒𝑛𝑡 ′ 𝑠 𝑝𝑎𝑐𝑘𝑒𝑑 𝑐𝑒𝑙 𝑣𝑜𝑙𝑢𝑚𝑒 (ℎ𝑐𝑡)
Reticulocyte (%) x =% residual RNA in the reticulocytes
𝑛𝑜𝑟𝑚𝑎𝑙 ℎ𝑐𝑡 𝑏𝑎𝑠𝑒𝑑 𝑜𝑛 𝑎𝑔𝑒 & 𝑔𝑒𝑛𝑑𝑒𝑟
Example: if an adult male has a haematocrit of 30%

(0.30L/L) and a reticulocyte count of 3%, the corrected
reticulocyte count would be
CRC =
0.30 𝐿/𝐿
0.3 x = 0.02 = 2%
0.45 𝐿/𝐿 (𝑎𝑑𝑢𝑙𝑡 𝑚𝑎𝑙𝑒 𝑛𝑜𝑟𝑚𝑎𝑙 𝑣𝑎𝑙𝑢𝑒)

ERYTHROCYTE SEDIMENTATION RATE (ESR)  Hemoglobin
o Measured by modified hemoglobinometers or
 Non-specific measurement used to detect and monitor by oximeters integrated with a blood gas
an inflammatory response to tissue injury analyser
o Rheumatoid arthritis  E.g., HemoCue (lysing agent and
o Infections reagents  in contact with blood 
o Malignancies hemoglobin azide  570nm and
 It is non-specific because it can also elevated in cases 880nm
of pregnancy, anemia, older age and plasma cell  Cell Counts
myeloma o Traditional cell counting methods
 Not commonly ordered because it is prone to error that o Employs a buffy coat analysis method
can cause false elevated  E.g., QBC Star Q (QBC Diagnostics)
 It is not considered as screening test for infections most  Centrifugation of
especially in asymptomatic individuals specialized capillary tubes
 Distance in millimetres at which the RBCs fall in 1 hour  Expansion of buffy coat
 9 parameters: Hct, Hgb,
Stages of ESR
MCHC, WBC count,
 Initial rouleaux formation Granulocyte, Granulocyte
o First 10 minutes of the procedure (after you %, Lymphocyte,
put blood) Lymphocyte %, Platelet
 Rapid settling of RBCs Count
o Initial sedimentation  Disadvantage: not specific
o Takes 40 minutes
AUTOMATION
 Final sedimentation of RBCs
o Packing Stage  Two General Priciples
o 10 minutes o Electronic Resistance (impedance)
 After the final sedimentation, the cells will fall down  o Optical Scatter
measures rate of fall of packed red cells
ELECTRONIC IMPEDANCE
 Reference Ranges:
o Male (0 to 5yrs old) = 0 to 15mm/hr  Developed by Coulter 1950s
o Female = 0 to 20mm/hr  Most common methodology used
 Based on the detection and measurement of changes
OSMOTIC FRAGILITY TEST (OFT) in electrical resistance
o Mainly produced by the cells that travels to the
 A measure of the ability of red cells to take up fluid small aperture
without lysing o Check for the number of pulses,
 Decreased surface area to volume ratios proportional to the number of cells
 Employed to help diagnose different types of anemia, counted
in which the physical properties of red cells are altered o Height of the voltage is directly
proportional to the volume of the cell
Increased OFT Decreased OFT
 Utilizes non-conductive properties of blood cells
Decrease resistance Increase resistance
o As blood cell passes through orifice of
Hemolytic anemia Splenectomy
aperture it displaces its own volume
Hereditary spherocytosis Liver disease
o RBCs and platelets counted together,
Spherocyte cells Sickle cell anemia
separated by pulse heights
Iron Deficiency Anemia
o Hydrodynamic focusing forces cells to pass
Thalassemia
single file though sensing zone
 Factors Affecting Volume Measurement
POINT OF CARE TEST o Aperture diameter
 Red cell/platelet  smaller than the
 Produced rapid and accurate results to facilitate faster white blood cell aperture (increased
treatment and to decrease length of stay of patients platelet counting sensitivity)
 Diagnostic testing at/or near the site of patient care  White cell
o Bedside of the patient  Protein build-up: decreases
 Hematocrit diameter  slow down flow of cell 
o Centrifuge-based device (microhematocrit low cell counts machine detect
centrifuge) higher volume  falsely elevate cell
 ISTAT 1 (Abbott) volumes
o Conductivity method
 EPOC (Siemens)

o Cell carryover CBC PARAMETERS
 To prevent or minimize  internal
cleaning systems Parameter Unit of Reporting Common Method of
Determination
o Coincident passage loss WBC x109/L Impedance count x calibration
 Falsely decreased cell count  factor
falsely increase volume RBC x1012/L Impedance count x calibration
o Orientation of the cell in the center of the factor
aperture HGB g/dL Colorimetric absorbance in
proportion to hemoglobin
 Decreased hemoglobin content
MCV fL From RBC histogram
o Deformability of the RBC HCT x 10 / RBC
 Decreased hemoglobin content HCT % RBC x MCV / 10
o Recirculation of cells back into the sensing MCH Pg HGB X 10 / RBC
zone MCHC g/dL or % HGB x 100 / HCT
 To prevent  back wash or sweep RDW % Impedance (from histogram)
Platelet x109/L Impedance count x cal factor
flow mechanism
WBC Diff Absolute: x109/L Light scatter, flow cytometry
 Falsely elevated cell count Percent of WBC: %
OPTICAL SCATTER
CBC QUALITY CONTROL
 A hydrodynamically focused sample stream is directed
through a quartz flow cell past a focused light source  Commercial Controls:
(Tungsten-halogen lamp or helium neon laser) o 3 levels (low, normal, high)
 Cells counted as passed through focused beam of light o Values stored in instrument computer
(LASER  monochromatic  light emitted here is at o Levey-Jennings graph generated and stored
single wavelength) for each parameter
 Sum of diffraction, refraction and reflection  Mode to Mode QC:
 Multi angle polarized scatter separation o Most automated hematology instruments
(M.A..P.S.S) have a primary and secondary mode of
o Forward angle light scatter (0) sample aspiration. Controls must be run on
 cell volume BOTH and correlate
o Forward low angle light scatter (2 to 3)  Primary = automated or closed
 cell volume and refractive index  Secondary = manual or open
o Forward high angle 5 to 15
 cell volume, refractive index  Delta Checks
o Orthogonal light scatter (90) o When the laboratory information system (LIS)
 Also known as side scatter and the instrument are interfaced (connected)
 results from refraction and reflection delta checks are conducted by the LIS on
of light from larger structures inside select parameters
the cell
Table 5: Advantages and Disadvantages of Automated
RADIOFREQUENCY Analyzers
 also known as alternating current resistance Advantages Disadvantages
 Used in conjunction with electrical impedance Rapid, objective, statistically Produce cell counts which
 Cell interior density is proportional to pulse height or significant are falsely increased or
change in the RF signal decreased
Not subject to the Some analyzers check only
distributional bias of the the volume and number of
manual count particles
More efficient and cost Platelet clumps may be
effective misclassified as leukocytes
or erythrocytes and
nucleated red blood cells can
be misclassified as
leukocytes, or specifically
lymphocytes
The precision of the
automated differential makes
the absolute leukocyte
counts reliable and
reproducible

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HEM 311: CLINICAL HEMATOLOGY 1

RMT LECTURE 1: OVERVIEW OF CLINICAL HEMATOLOGY

2023 INSTRUCTOR: PROF. PAMELA S. SENGSON, RMT

OUTLINE BASIC HEMATOLOGY TERM AND ABBREVIATION

 Maintain blood vessel integrity by initiating cell wall

OVERVIEW OF COMPLETE BLOOD COUNT

Basic CBC Measurements by Automated Blood Cell

Overview of Clinical Hematology I Page 3 of 4

2023 INSTRUCTOR: PROF. PAMELA S. SENGSON, RMT

OUTLINE  The developing erythroblasts signal the beginning of

 Responsible in conditioning the T lymphocytes 3 POSSIBLE ACTIONS OF HSCs

 Cells that have extensive proliferative capacity CYTOKINES

2023 INSTRUCTOR: PROF. PAMELA S. SENGSON, RMT

Lineage Specific Hematopoiesis I Page 1 of 5

o Resides in the bone marrow for 1 day or Reticulocyte 8-10 - 0 1 48 -

Lineage Specific Hematopoiesis I Page 2 of 5

EOSINOPHIL DEVELOPMENT 1. Immature Basophils

Lineage Specific Hematopoiesis I Page 3 of 5

1. Monoblast 3. Mature Lymphocyte

2. Promegakaryoblast (Stage II)

3. Megakaryocyte (Stage III)

Lineage Specific Hematopoiesis I Page 5 of 5

OUTLINE STRUCTURE OF AN RBC MEMBRANE

 Other Peripheral Proteins METABOLIC ACTIVITY

 also known as oxidative pathway  This mechanism is low in energy consumption

METHEMOGLOBIN REDUCTASE PATHWAY Metabolic Pathways in the Erythrocyte

 Depends on EMB pathway because it needs the Metabolic Pathway Function

 Term describing the dynamics of RBC production and

 A hormone produced in the kidney in response to

MECHANISM OF RED CELL DESTRUCTION

2023 INSTRUCTOR: PROF. PAMELA S. SENGSON, RMT

 One of the most studied proteins in the body because

Iron Compartments in Normal Humans

 Protoporphyrin IX  The connection of heme and globin through chemical

REGULATION OF HEMOGLOBIN PRODUCTION

2023 INSTRUCTOR: PROF. PAMELA S. SENGSON, RMT

OUTLINE  Variation in Red Cell Color

 Cell Size = microcytosis, macrocytosis Table 1: Hypochromasia Grading

 Average Size: 7.2um Table 2: Polychromasia Grading

Red Blood Cells Abnormalities I Page 2 of 5

Red Blood Cells Abnormalities I Page 3 of 5

 Slight: less than 5%

Red Blood Cells Abnormalities I Page 4 of 5

Red Blood Cells Abnormalities I Page 5 of 5

2023 INSTRUCTOR: PROF. PAMELA S. SENGSON, RMT

OUTLINE CIRCULATING KINETICS AND MORPHOLOGY

White Blood Cells Abnormalities I Page 1 of 5

 Acquired anomaly Name Subtype Enzyme Deficiency

**refer to Table 26.3 (Rodaks) for the complete details about

 Needle-like bodies seen in cytoplasm of myeloblasts

 Manifested by presence of large granules in the

 Inherited dominant condition

White Blood Cells Abnormalities I Page 3 of 5

 Nuclei: round to ovoid, lack nucleoli

DOWNEY CELL SEZARY CELL

BASKET CELL/ SMUDGE CELL

White Blood Cells Abnormalities I Page 4 of 5

 Cytoplasm: stains bright red to pink