OLFU Introduction to Hematology

CLINICAL HEMATOLOGY 1 LEC 1 2021 – 2022

1st Semester
RMT 2023 Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
TRANS 1 HEMA311
Date: September 16, 2021 LEC

Outline Table 1.02: Common abbreviations

At the end of the session, the student must be able to learn:
I. Introduction to Hematology Abbreviations Meaning
A. History FBC Full blood count
II. Basic Hematology Terms fL Femtoliter
III. Blood Characteristics and Functions Hb/ hgb Hemoglobin concentration
A. Blood Composition Hct Hematocrit
B. General Characteristics of Blood MCH Mean cell hemoglobin
C. Functions of Blood MCV Mean cell volume
IV. Basic Hematologic Examination MCHC Mean cell hemoglobin
V. Anticoagulants Used in Hematology concentration
VI. Quality Assessment and Quality Control in Hematology CBC Complete blood count
Laboratory pg Picogram
A. Quality Assessment in Hematology Laboratory
B. Quality Control III. BLOOD CHARACTERISTICS AND FUNCTIONS
I. HEMATOLOGY A. Blood Composition
❖ Haemas: Blood
❖ Logos: Study
➢ Study of the cellular elements of the blood
➢ Three formed elements: RBC, WBC, Platelets
➢ Concept of coagulation mechanism (clotting system in the
body)
Liquid Components
A. History

❖ 1657
➢ Describe worms in the blood by Athanasius Kircher
❖ 1658
➢ Discovery of erythrocytes by Swammerdam
❖ 1674
➢ Human erythrocytes described by Anton Van Leuwenhoek
❖ 1842
➢ Platelets were described ❖ Liquid Components
❖ 1846 ➢ 95% Water
➢ PMN (polymorphonuclear neutrophils) distinguished from ➢ 5% (proteins, carbohydrates, electrolytes, enzymes, etc.)
other leukocytes by Wharton Jones ❖ EDTA
❖ 1879 ➢ Plasma (contains fibrinogen)
➢ First complete classification of leukocytes by Ehrlich ❖ RED
❖ 1902 ➢ Serum
➢ Development of Wright’s stain by James Homer Wright
❖ 1920 B. General Characteristics of Blood
➢ Hematology was considered a separate science from clinical
pathology ❖ Blood Volume:
➢ Normal Adult: 5 – 6 liters
II. BASIC HEMATOLOGY TERMS ➢ Newborns: 250mL – 350mL
➢ Women: 4 – 5 liters
Table 1.01: Terms ➢ Hypovolemia: decrease in blood
❖ Viscosity:
Terms Definitions ➢ Thicker than water because of its components (liquid in
a Without nature)
blast Youngest/ nucleated/ immature ❖ Color:
➢ Venous Blood: Dark Red in appearance (unoxygenated)
chomic Color
➢ Arterial Blood: Bright Red (oxygenated)
Cyte Cell
❖ In vivo and in vitro appearance:
Dys Abnormal
➢ In vivo: liquid in state (easily circulate within the vessels)
aemia In the blood ➢ In vitro: from liquid to solid or clot (without anticoagulants)
Ferro/ ferric/ sidero Iron ❖ pH:
Hyper Increased ➢ Neutral level (7.35 – 7.45)
Hypo Decreased ➢ Lungs and Kidneys (bicarbonates) important organs in
Iso Equal maintaining pH level
Macro Large ❖ Specific Gravity:
Micro Small ➢ Whole Blood: 1.045 – 1.066 heavy (cells, chemical
Myelo Marrow constituents)
Normo Normal ➢ Plasma: 1.025 – 1.029
Oid Like ➢ Serum: 1.024

Surell, R. – TRANSCRIBER 1
[HEMA311] 1.01 Introduction to Hematology I Prof. Antonio C. Pascua, RMT, MSMT
Table 2.0: Examples of Potential Preanalytical, Analytical, and
C. Functions of Blood Postanalytical Errors
Preanalytical Analytical Postanalytical
❖ Respiratory Specimen obtained Oversight of Verbal reporting of
❖ Nutritional (delivery of different nutrients) from the wrong instrument flags results
❖ Excretory (removing any unwanted materials) patient
➢ Urine: Filtered formed of blood Specimen procured Out-of-control QC Instrument: (LIS)
❖ Buffering Action (balance pH) at the wrong time results incompatibility error
❖ Maintenance of Constant Body Temperature Specimen collected Wrong assay Confusion about
❖ Transportation of Hormones and other Endocrine Secretion that in the wrong tube or performed reference ranges
Regulates Cell Function container
❖ Body Defense Mechanism Blood specimens Failure to report
collected in the critical values
IV. BASIC HEMATOLOGICAL METHODS OF EXAMINATION wrong order immediately
Incorrect labeling of
❖ Complete Blood Count (screening test) specimen
➢ Red Blood Cell Count Improper processing
➢ White Blood Cell Count of specimen
➢ Platelet Count
➢ Differential Count
➢ Hemoglobin 1. Major Activities
➢ Hematocrit
➢ Blood Indices ❖ Preventive
❖ Reticulocyte Count (bone marrow) ➢ Activities done in prior to the examination of the specimen or
❖ Erythrocyte Sedimentation Rate (inflammation) sample that is intended to establish system conducive
accuracy testing
V. COMMON ANTICOAGULANTS USED IN HEMATOLOGY ❖ Assessment
➢ Activities done during testing to determine whether test
❖ EDTA (Ethylene Diamine Tetraacetic Acid) Violet systems are performing correctly
➢ Action: Inhibits calcium ❖ Corrective
➢ Forms: ➢ Done when error is detected to correct the system
▪ Versene (disodium salts) ➢ Recalibration of the instrument/ machine
▪ Sequestrene (tripotassium salts)
➢ Optimum Concentration: 2. Quality Assessment Programs
▪ 1.5mg/mL of blood
▪ E.g., 3mL of blood, 4.5mg of anticoagulant
❖ Test request procedures
➢ Use:
❖ Patient identification
▪ CBC, Blood Smear
❖ Specimen procurement
▪ Preserves morphology of the cells
❖ Specimen labeling
❖ Citrate
❖ Specimen transportation and processing procedures
➢ Blue: 1:9, Coagulation studies (preserve all clotting factors)
❖ Laboratory personnel performance
➢ Black: 1:4, ESR
❖ Laboratory instrumentation, reagents, and analytical (examination)
➢ Buffered Sodium Citrate: 3.8%
test procedures
➢ Action: Inhibits Calcium
❖ Turnaround times
➢ Use:
❖ Accuracy of the final result
▪ Platelet Count (if there is satellitism)
❖ Heparin (Green)
➢ Action: Inhibits thrombin (thrombin: allows formation of clot) 3. Non-Analytical Factors in Quality Assessment
➢ Optimum Concentration: 15 – 20u/mL of blood
➢ Use: ❖ Qualified personnel
▪ Blood gas analysis, Osmotic fragility test, Platelet ❖ Laboratory Policies
retention test ❖ Laboratory procedure manual
▪ Do not use in blood smear (background will appear ❖ Test requisitioning
bluish) ❖ Patient identification and specimen procurement and labeling
❖ Oxalate (Gray/ Black) ❖ Specimen collection, transport, processing and storage
➢ Action: Inhibits calcium ❖ Preventive maintenance of equipment
➢ Optimum Concentration: 1.2mg/mL of blood ❖ Appropriate methodology
➢ Use: ❖ Accuracy in reporting results and documentation
▪ ESR Testing
B. Quality Control
VI. QUALITY ASSESSMENT AND QUALITY CONTROL
❖ A system of ensuring accuracy and precision in the laboratory
A. Quality Assessment in Hematology Laboratory ❖ Involves process of monitoring the characteristics of the analytical
processes and detects analytical errors during the test
❖ Pre-Analytical ❖ To check the stability of the machines and reagents
➢ Everything before the testing (blood collection, labelling, ❖ “Quality Control” a form of specimen. It should mimic human
proper use of anticoagulants, storage, transport, etc.) specimen
❖ Analytical
➢ Actual testing of the specimen 1. Basic Terminologies
➢ Quality control (just part of quality assurance) takes place
❖ Post Analytical
❖ Sensitivity: Quality of being sensitive
➢ Patient care, results
❖ Specificity: Specific to analyte of interest
❖ Accuracy: Close to the target value
❖ Precision: Quality of being exact and accurate

Surell, R. – TRANSCRIBER 1
[HEMA311] 1.01 Introduction to Hematology I Prof. Antonio C. Pascua, RMT, MSMT

2. Kinds of Quality Control

❖ Intralab
➢ Involves the analyses of control samples together with the
patient specimen
➢ Internal quality control
❖ Interlab
➢ For maintaining long-term accuracy of the analytical methods
➢ External quality control involves proficiency testing

3. Quality Control Material

❖ Resembles human sample

❖ Inexpensive
❖ No communicable disease
❖ No matrix effect
❖ Known analyte concentration
❖ Convenient packaging

4. Tools of Quality Assurance and Quality Control

❖ Standard Solution
❖ Control Solution
❖ Blank

5. CBC Quality Control

❖ Commercial Controls
➢ 3 levels (low, normal, high)
➢ Values stored in instrument computer
➢ Lewey-Jennings graph generated and stored for each
parameter
❖ Mode to Mode QC
➢ Most automated hematology instruments have a primary and
secondary mode of sample aspiration
➢ Primary: Automated or Closed
➢ Secondary: Manual or Open
❖ Delta Checks
➢ When the laboratory information system (LIS) and the
instrument are interfaced (connected) delta checks are
conducted by the LIS on select parameters
➢ Comparing of current results to previous results

6. Functions of Quality Control Program

❖ Providing a guide to the functioning of equipment reagents, and

individual technique
❖ Confirming the accuracy of testing when compared with reference
values
❖ Detecting an increase in the frequency of both high and low
minimally acceptable values (dispersion)
❖ Detecting any progressive drift or values to one side of the average
value for at least 3 days (trends)
❖ Demonstrating an abrupt shift or change from the established
average value for 3 days in a row (shift)

Surell, R. – TRANSCRIBER 1
 Hematopoiesis 2021-2022
OLFU CLINICAL HEMATOLOGY 1 LEC 2 1ST Semester

RMT 2023 Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT TRANS 2 HEMA311
DATE: September 24, 2021 LEC

Outline II. STAGES OF HEMATOPOIESIS

At the end of the session, the student must be able to learn: A. Mesoblastic Phase
I. Defining Hematopoiesis’ B. Hepatic Phase
II. Stages of Hematopoiesis C. Medullary Phase
A. Mesoblastic Phase
B. Hepatic Phase A. MESOBLASTIC PHASE
C. Medullary Phase  Chief site (responsible for the production of cells):
III. Adult Hematopoietic Tissue Embryonic Yolk Sac
A. Bone Marrow o it is early in the embryonic development where cells
o Red Bone Marrow from the Mesoderm, migrate to the Yolk sac
o Yellow Bone Marrow  among the cells that migrated, some of these
B. Hematopoietic Microenvironment form Primitive (young cells) erythroblasts,
C. Stromal Cells which are present at the Central cavity of the
D. Extracellular Matrix of the Marrow Yolk sac
IV. Myeloid to Erythroid Ratio (M:E)  Important feature (what makes this phase different from the
A. Abnormal M:E Ratio next stages that occurs in the fetuses & adults):
B. Marrow Differential o this phase occurs INTRAVASCULARLY, wherein it is
V. Other Normal Marrow Cells within the developing blood vessels
VI. Marrow Specimens  unlike the Hepatic & Medullar phases that are
VII. Extra medullary Hematopoiesis extravascular synthesis of cells, this
A. Liver Mesoblastic phase is rather more of
B. Spleen Intravascular synthesis of cells
C. Lymph Nodes o the yolk sac could remain active up to the 8th or the
D. Thymus 12th week of gestation
VIII. Stem Cell Theory  which can later on, discontinue its role because
A. Monophyletic Theory of the continuous development
B. Polyphyletic Theory  after it discontinues its role, there will be a
C. Hematopoietic Stem Cells Transition & Replacement, since
IX. The Different Cytokines & Growth Factors Hematopoiesis never stops (because if it stops,
A. Stem Cell Cycle Kinetics it will also stop the production of the blood cells
B. Stem Cells that might result to complications that can lead
C. Regulation of Hematopoiesis to death)
 in this phase, there are 3 Embryonic Hemoglobins that are
present only during the Embryonic life or during this phase,
! SIDE NOTES GUIDELINES THAT MIGHT HELP ! which are the ff:
Color of side notes: Blue 1. Gower I
 This is a sample info  globin contents present:
o Further discussion o 2 Epsilon chains
of the info above this o 2 Zeta chains
 Discussion of ← side notes indicators
* their indentation may vary 2. Gower II
subtopic above  globin contents present:
this according to the topic it
supports/describes * o 2 Alpha chains
> This is the only bullet used o 2 Epsilon chains
for examples 3. Portland Hemoglobin
 globin contents present:
o 2 Zeta chains
I. DEFINING ‘HEMATOPOIESIS’ o 2 Gamma chains
 Hematopoiesis
 is a continuous & regulated process of blood cell ABOUT THIS PHASE:
production  this phase begins as early as the 19th day of gestation
o this process results in formation, o roughly 3 weeks of pregnancy, the cells being formed
development, & specialization of all functional are Erythroblasts (immature cells), that are important
blood cells (RBCs, WBCs, & Platelets) in the Early Embryogenesis, that produces
 as these blood cells are produced & Hemoglobins
developed, later on, they are  Hemoglobin
released from the Bone Marrow &
 is responsible in the
are present in the Peripheral
delivery/transportation of oxygen
circulation (Blood)
 it is a mixture of Heme & Globin
 it deals with the ff:
 for every hemoglobin molecule, there
o Cell renewal
are 4 units of Heme, paired with 4 units
o Proliferation
of Globin
o Differentiation
 in order to know how to
o Maturation
classify/differentiate these types of
 among humans, Hematopoiesis can be characterized
hemoglobin, you need to check their
as a select distribution of embryonic cells in specific
“-globin content”
sites that rapidly change during development
o during the 19th day, the cells formed are more of a
mass of tissue that needs a lot of oxygen & nutrients in
order for the embryo/tissue to fully mature
 it is confined with Erythropoiesis, since there are primitive
erythroblasts

Bisenio, J. — TRANSCRIBER
[HEMA311] 2.01 Hematopoiesis | Prof. Antonio C. Pascua, Jr., RMT, MSMT
B. HEPATIC PHASE C. MEDULLARY PHASE
 Chief site (responsible for the production of cells): Liver  Chief site (responsible for the production of cells): Bone
o the clusters of cells colonizes the Fetal liver & other Marrow
Reticuloendothelial System (RES) organs (e.g.  Measurable levels of Growth factors: are present during this
Thymus, Spleen, Placenta, Bone Marrow) phase & are needed to make sure that the Stem cells will commit
 Other active sites: Spleen, Kidneys, Thymus themselves in the production of a specific cell
 Important feature (what makes this phase different from the
next stages that occurs in the fetuses & adults): ABOUT THIS PHASE:
o this phase occurs EXTRAVASCULARLY  this phase also referred to as ‘Myeloid stage’
 Hemoglobin production:  this phase begins at the 5th-6th month of gestation
o since there is a production of Red Blood cells (RBCs) o prior to the 5th month of pregnancy, Hematopoiesis
here, there will always be Hemoglobin, which makes then starts to begin in the Bone marrow cavity
the RBCs special & functional o when reaching the 5th month, this transition is called
o the hemoglobins present in this phase are the ff: as, ‘Medullary Hematopoiesis,’ because it occurs in
1. Hemoglobin F or Fetal Hemoglobin (HbF) the medulla or the inner part of the bone
 the predominant hemoglobin for this  by the end of the 24th week of gestation, the Bone marrow is
stage (while the fetus is still inside the then considered as the ‘main site for Hematopoiesis’ & will
womb) persist all throughout our life
 once the fetus is born, there will be a  according to studies, in the Bone marrow during this phase,
decrease in the production of HbF Hematopoietic Stem Cells (HSCs) become present, along with
 globin contents present: Mesenchymal cells, & migrate into the core of the body
o 2 Alpha chains o they are later on responsible for the development &
o 2 Gamma chains production of the blood cells
2. Hemoglobin A or Adult Hemoglobin (HbA)  this phase occurs generally in most of the bones, wherein
 the most abundant hemoglobin among adults, the principal source production are the Flat
among adults bones (e.g. Sternum, Ribs, Pelvis)
 once there is a decrease in the
production of HbF, the production of
HbA will then start to rise Figure 1.01: Sites of Hematopoiesis by Age (source: Rodak’s
 among adults, the ff. are the Hematology 5th edition)
hemoglobins present in our bodies:
2a. Hemoglobin A1 (HbA1)
 predominant HbA
 roughly 95% of our
hemoglobin, is HbA1
 globin contents
present:
o 2 Alpha
chains
o 2 Beta
chains
2b. Hemoglobin A2 (HbA2)
 roughly 2-3% of our
hemoglobin, is HbA2
 globin contents
present: III. ADULT HEMATOPOIETIC TISSUE
o 2 Alpha  Hematopoietic Tissue
chains  is responsible for the synthesis or production of blood
o 2 Delta cells
chains  it is where Lymphoid Development occurs
2c. Little portion of HbF o particularly, having the ff. lymphoid tissues:
 roughly 1-2% of our A. Primary Lymphoid tissue
hemoglobin, is HbF  refers to the Bone Marrow
(produces B-cells) & Thymus
ABOUT THIS PHASE: (produces T-cells)
 this phase begins at 5-7 gestational weeks B. Secondary Lymphoid tissue
o it is in the transition phase from the Mesoblastic to the  where lymphoid cells
Hepatic phase respond to foreign bodies &
o it could reach its peak/highest production on the 3rd antigens
month of pregnancy (roughly 12 weeks)  it is comprised of Spleen,
 it is where the yolk sac has totally discontinued Lymph nodes, & other
its role in Hematopoiesis Lymphoid tissues (e.g.
 this phase ends or it will gradually decline after the 6th month Mucosa-associated lymphoid
period of gestation, where there will be a transition & tissue or MALT)
replacement once again  located in the Reticuloendothelial System (RES):
 it is characterized by recognizable clusters of developing o Bone Marrow
Erythroblasts, Granulocytes, & Monocytes  when you reach the Medullary stage, it
o as well as the presence of Lymphoid cells & the will then further contain the developing
evidences of Megakaryopoiesis cells (RBCs, WBCs, Megakaryocytes
 as you reach this point, aside from Erythroblasts, all other for platelet production, & Lymphoid
hematopoietic cells may be produced, such as WBCs cells for lymphocyte production)
(Granulocytes, Monocytes, Lymphocytes) & Platelets o Lymph nodes
o Spleen
o Liver
o Thymus
Bisenio, J. — TRANSCRIBER
[HEMA311] 2.01 Hematopoiesis | Prof. Antonio C. Pascua, Jr., RMT, MSMT
A. BONE MARROW Figures 2.01 & 2.02: LEFT: Fixed & stained Bone marrow biopsy
 main responsible organ/site for Hematopoiesis specimen (H&E stain, x100). RIGHT: Illustration of arrangement of a
 develops in the embryo by the hollowing-out of the skeletal Hematopoietic cord & vascular sinus in Bone marrow (source: Rodak’s
bones, forming a Central Cavity Hematology 5th edition)
o take note: it is not the bone itself, but it is the soft tissue
inside the bone cavity (central cavity)
 it contains Hematopoietic cells, Stromal cells, & Blood
vessels (e.g. Arteries, Veins, Vascular sinuses)
o Hematopoietic cells— gives rise to the primitive &
undifferentiated cell, or the Hematopoietic Stem Cells
(HSCs)
 Main Function:
o the proliferation & production of blood cells
 since ALL blood-formed elements are
ultimately developed from the HSCs, that are
generally present in the Bone Marrow
 According to Rodak’s:
o at the age of 5-7 years old, it is when Adipocytes become
more present or dominant, & later then occupies the
spaces in our Long bones
o at the age of 20’s, our bone marrows are at around 60-
70% active, which means that we are capable of
producing enough cells in our Flat bones

2 Types of Bone Marrow:

 Normocellular  Nutrient & Periosteal
1. Yellow Bone Marrow
 Normal bone marrow Arteries
 ‘cellular’
in a stained smear  they supply the
 hematopoietically-wise, it is inactive, which meant that it
is not capable of producing out blood cells  there is 30-70% nutrient & oxygen
 as we grow older, there will be an Hematopoietic cells requirement of the
accumulation of fats, which is (including the mature & bone marrow
then considered to be the yellow immature forms)
bone marrow  Nutrient Artery
 there are special  Hypercellular (Hyperplasia)  supplies blood ONLY
processes/mechanisms wherein  abnormal INCREASE for the bone marrow
this bone marrow may become in the production of
active again  Periosteal Artery
cells
 provides nutrients
2. Red Bone Marrow  there is more than 70%
BOTH for the bone &
 ‘active’ Hematopoietic cells
for the bone marrow
 it consists of the developing cells &
progenitors, that produces the  Hypocellular (Hypoplasia)
mature ones  abnormal SUMMARY:
 it is composed of Hematopoietic DECREASE in the A. Nutrient & Periosteal
cells & Macrophages that are production of cells provide blood & nutrients
arranged in Extravascular cords  there is less than 30% to the bone marrow
o Hematopoietic cells— B. Blood then exits the bone
Hematopoietic cells
develop in specific marrow by the longitudinal
niches within the cords  Aplastic (Aplasia) veins, which runs the end
of the bone marrow
 there is very few or a of the bone marrow
 Main functions:
A. Production of Blood total absence of C. Hematopoietic cells that
cells Hematopoietic cells are located at the
B. Iron storage— especially when RBCs are o due to this, there Endosteal bed, receive
destroyed will be less their nutrients at the
C. B-cell development RBCs to provide Nutrient artery
 According to literature, Mature blood cells of the bone oxygen, less
marrow enter the peripheral circulation by processes that
WBCs to protect
are not clearly understood by far
o What is known so far: Once they mature the body from
within the bone marrow, they will start to leave unwanted
the marrow & go to the peripheral circulation organisms, &
 usually at birth, the bone marrow is 100% red bone less Platelets to
marrow help the body to
form clots

B. HEMATOPOIETIC MICROENVIRONMENT
 it is sometimes referred to as ‘Hematopoietic Inductive
Microenvironment (niches)’
 it is needed to nurture & protect the Hematopoietic stem cells
(HSCs)
 it is responsible for supplying Semifluid matrix (Stroma)

Bisenio, J. — TRANSCRIBER
[HEMA311] 2.01 Hematopoiesis | Prof. Antonio C. Pascua, Jr., RMT, MSMT
oStroma IV. MYELOID TO ERYTHROID RATIO (M:E)
 serves as an anchor for the developing  it is a numerical expression of comparing the relative number of
hematopoietic cells Granulocyte precursors (Myeloid) to the relative number of
 they form an extracellular matrix on the Erythrocyte precursors (Erythroid)
niches, in order to promote cell adhesion, to  when examining the bone marrow, this M:E ratio should also
regulate HSCs, & for the maintenance of be taken in consideration
proteins needed by the maturing cells
 it is used in order to balance in quiescence (inactive/dormant Table 1.01: Normal M:E Ration & its Explanation
cells), and the self-renewal & differentiation of HSCs Normal M:E Ratio Explanation of the Ratio
 as the site of hematopoiesis transitions from the Yolk sac, to
In the Bone marrow, there is more
the Liver, and later on to the Bone marrow, the
Granulocyte Precursor (Neutrophils,
Microenvironment niches for the hematopoietic stem cells
Eosinophils, Basophils) cells as
should also be adjusting
compared to Red blood cells (Immature
o there should be a proper supply of nutrients needed by
cells)
the hematopoietic stem cells, so that they will be able
o this is because of the survival of
to provide mature cells
the Mature cells
 Red cells survive in the
Stromal cells & its Types: Peripheral circulation for
 Stromal cells 120 days
 they play a critical role in the regulation of HSCs & 2:1 up to 4:1 o if Red cells survive, it will last in
progenitor cells, so they can survive & easily the blood for roughly only 1-2
differentiated/distinguish as to their cell line (RBCs, days, because they
WBCs, Platelets) marginate/leave the peripheral
 the key stromal cells that support the HSCs in the blood & go to the tissues
bone marrow niches are the ff: o since there is a lower number of
1. Endothelial cells WBCs compared to the RBCs in
2. Adipocytes the blood, it sends the bone
3. Macrophages marrow a signal to produce more
4. Osteoblasts WBCs, serving as a
5. Osteoclasts Compensation mechanism
6. Reticular cells  Lymphocytes & Monocyte are excluded in this M:E ratio

1. Endothelial cells A. ABNORMAL M:E RATIO

1. Infection
 they regulate the flow of particles, entering & leaving the
hematopoietic spaces  if there is infection, the more you need WBCs
 from 2:1 up to 4:1, it could go as high as 6:1, since more
2. Adipocytes (Fat cells) WBC precursors are present
2. Leukemia
 they are essential for they secrete various steroids that  from 2:1 up to 4:1, it could go as high as 25:1
influence erythropoiesis, maintain bone integrity, & regulate 3. Myeloid Hyperplasia
the volume of bone marrow  here is an elevation of Myeloid precursors
 from 2:1 up to 4:1, it could go as high as 20:1
3. Macrophages
4. Myeloid Hypoplasia
 is important for phagocytosis & the removal of unwanted 5. Erythroid Hyperplasia
materials 6. Erythroid Hypoplasia
 is essential for they secrete Cytokines, which later on
regulate hematopoiesis  When do we test/examine bone marrow specimen:
o it is usually conducted in cases of Hematologic disorders
4. Osteoblasts affecting the cells
> Ex: Anemia, Leukemia, Tumor, Infections
 are referred to as the bone-forming cells
 are sometimes mistaken as plasma cells B. MARROW DIFFERENTIAL
5. Osteoclasts Table 2.01: The Usual percentage (%) of a Particular cell or
Hematopoietic cell in the Bone marrow
 are referred to as the bone-resorbing cells or bone-destroying
cells

6. Reticular cells
Predominant:
 they support the vascular sinuses & hematopoietic cells Granulocyte
precursors,
Extracellular Matrix of the Bone Marrow particularly
the
 Stromal cells secrete semifluid extracellular matrix in order to Neutrophilic
anchor & protect the developing HSCs in the bone cavity precursors
 this matrix is made up of the ff:
1. Fibronectin (FN)
2. Collagen
3. Laminin  Medtechs, Pathologists, & Hematologists usually count 500 cells in
4. Thrombospondin (TSPs) a bone marrow specimen, that will eventually be identified &
5. Tenascin differentiated
6. Proteoglycans o preferably, you need to count 1000 cells, but since it is
> Ex: Chondroitin sulfate, Heparin sulfate, too many, they only count at least 500 cells
Hyaluronate, etc.

Bisenio, J. — TRANSCRIBER
[HEMA311] 2.01 Hematopoiesis | Prof. Antonio C. Pascua, Jr., RMT, MSMT
V. OTHER NORMAL MARROW CELLS 2. Red pulp— contains macrophages & specialized
1. Macrophages macrophages
2. Mast cells 3. Marginal zone— surrounds the white pulp & contains
3. Osteoblasts blood vessels, macrophages, and some B-cells & T-
4. Osteoclasts cells
 Functions:
VI. MARROW SPECIMENS o Sequesters/stores platelets
 stores them in cases of emergency
 Collection site/s:
o Removal of unwanted/abnormal cells in blood
1. Preferred site:
o Posterior Iliac crest  it removes old & damaged blood cells; that is
2. Occasionally preferred sites: why organ is often referred to as the
o Anterior Iliac crest ‘graveyard of the body’
o Spinal processes  there is a constant rate of blood received by
o Vertebral bodies the Spleen, which is around 350 mL/minute
o Sternum
3. Among newborns: C. LYMPH NODES
o Upper end of Tibia  are bean-shaped structures located along the lymphatic
capillaries
 Needle used for Trephine Biopsy:  removes foreign blood contaminants & later on helps in the
o Jamshidi (gauge size: 11) production of blood cells
 Regions:
1. Cortex (Outer layer)— contains B-cells/B-
lymphocytes
 Needle used for Aspiration: 2. Medulla (Inner layer)— contains T-cells/T-
o University of Illinois Sternal lymphocytes
Needle  Functions:
o Lymphocyte proliferation
o Initiation of specific immune response against
VII. EXTRAMEDULLARY HEMATOPOIESIS foreign materials
 it occurs whenever Hyperplasia of the bone marrow cannot meet o Filter unwanted substances (e.g. cellular debris,
the physiologic needs of the body tissue debris, microorganisms/bacteria)
o even if this takes place, there should still be a
production of cells D. THYMUS
o it is where other Reticuloendothelial organs steps in:  it originates from the Endodermal & Mesenchymal tissue
A. Liver  it is populated initially by primitive lymphoid cells from the Yolk
B. Spleen sac & the Liver
C. Lymph nodes  is located in the upper part of the Anterior Mediastinum, at the
D. Thymus level of the great vessels of the Heart
 Lobules:
A. LIVER 1. Cortex (Outer layer)— referred to as the Peripheral
 Main function: provides the cells the different necessary zone
proteins & essential minerals that are needed in the synthesis of 2. Medulla (Inner layer)— referred to as the Central
the different substances for hematopoiesis, such as: zone
1. DNA  Functions:
2. RNA o Normal development of T-cells
 is the major site of cell production in the second trimester of fetal o Conditioning of Lymphocytes
development
 is often involved in blood-related diseases VIII. STEM CELL THEORIES OF HEMATOPOIESIS
> Ex: Porphyria— a defect in the enzymes involved in  All cells are derived from a pool of stem cells that are self-
the synthesis of Heme, which is needed for renewing
Hemoglobin  Pluripotent & Multipotent stem cells give rise to committed stem
 Functions: cells for each stem cell line
o Protein synthesis & degradation o Committed stem cells
o CHO & Lipid metabolism  have receptors for specific Growth factors
o Drug & toxin clearance
 it has committed itself in the production of a
o Iron recycling & storage
specific cell
o Hemoglobin degradation
Figure 3.01: Stem Cell Theory
B. SPLEEN
 is important for phagocytosis & lymphopoiesis
 is the largest lymphoid organ in the body
 is located beneath the Diaphragm, behind the Stomach, in the
upper left quadrant of our Abdomen
 when Spleen is enlarged, more platelets are more
sequestered/stored; even the young & healthy cells may also be
affected & damaged  Hematopoietic stem cells (HSCs)
o this enlargement is caused by an increased need for o are Self-renewing
the removal of unwanted cells o are Pluripotent
o sometimes, if the Spleen has been damaged, there is  based on the Monophyletic theory
a need for it to be removed, which is called as o Apoptosis
Splenectomy  they are able to reconstitute the
 there are 3 Splenic tissues: hematopoietic system of irradiated host
1. White pulp— contains the lymphocytes &
macrophages

Bisenio, J. — TRANSCRIBER
[HEMA311] 2.01 Hematopoiesis | Prof. Antonio C. Pascua, Jr., RMT, MSMT
2 STEM CELL THEORIES: > Ex: if you formed Colonies, and it is because of a Growth
1. Monophyletic theory factor for Erythrocytes, it is referred to as Colony-
 it suggests that all blood cells are derived from a single forming unit Erythroid (CFU-E)
progenitor stem cell, which is Pluripotential  the CFUs promote the proliferation & differentiation of stem cells,
Hematopoietic Stem cell (PHSC) which will allow its further characterization
o these stem cells differentiate into another
type of Committed/progenitor cells IX. THE DIFFERENT CYTOKINES & GROWTH FACTORS
o if you are this PHSC, it is still considered as A. STEM CELL CYCLE KINETICS
primitive or “undifferentiated,” for it is still  DAILY (in a normal adult) & Per kilogram of weight, there are:
confused whether it’ll be RBC, WBC, A. 2.5 billion Erythrocytes
Producing platelet, etc. B. 2.5 billion Platelets
 is the most widely accepted theory among experimental C. 1 billion Granulocytes
hematologies as of today  The determining factor that is controlling the rate of
2. Polyphyletic theory production of these blood cells, is DEPENDENT to the
 it suggests that each blood cell lineage is derived from physiologic needs of the body
its own unique stem cell  1:1000 Nucleated Blood cells
 there is 1 hematopoietic stem cell in every 1000
Nucleated blood cells
Figure 3.02: Stem Cell Theory of Hematopoiesis Process o they are capable of Cell/Mitotic Division when
stimulated with Growth Factors/ Cytokines

B. STEM CELLS
 expression of the chromosomes will help identify the
commitment of a particular progenitor to a cell
 the earliest identifiable human hematopoietic stem cells capable
of initiating long-term cultures express the ff:
CD34+, CD38-, HLA-DRlow, Thy1low, & Lin-

1. CD38 & HLA-DR

 loss of “stemness” in producing out the cells
2. CD33 & CD38
 committed Myeloid progenitors
3. CD10 & CD38
 committed Lymphoid progenitors
4. CD7
 T-lymphoid progenitor cells & natural-killer cells
5. CD19
 B-lymphoid progenitor cells
1. The Pluripotent stem cell, gives rise to Progenitor cells, the
C. REGULATION OF HEMATOPOIESIS
Lymphoid & Myeloid stem cells
2. Lymphoid stem cell  Cytokines
 will produce lymphocytes, B-cell or a T-cell  are group of proteins that have a direct & indirect effects
3. Myeloid stem cell on the hematopoietic stem cells
 will produce mature cells, aside from the lymphocytes  this, along with Growth factors regulate the
proliferation, differentiation, & maturation of
 will commit itself in the production of RBCs, if there will
Hematopoietic precursor cells
be specific Growth factors (Stimulants), that stimulate
the stem cells to make sure that this stem cell become  Functions:
specific/committed o Prevent hematopoietic precursor cells from
4. The Maturing cells cannot be produced all at the same time, dying (by inhibiting Apoptosis)
because a single Myeloid stem cell needs to commit itself to o Stimulate stem cells to divide
o Regulate cell differentiation into different cell
the production of only one
lineages
 but ALL Maturing cells are derived from the Myeloid stem
cell  Cytokines with NEGATIVE INFLUENCES on
hematopoiesis:
Table 3.01: Culture-derived Colony-forming units (CFUs) (source: o Transforming Growth Factors- β
o Tumor Necrosis Factor-α
Rodak’s Hematology 5th edition)
o Interferons
 Colony-stimulating Factor (CSF)
 produced by various cells
 regulates the production of WBCs

Types of Cytokines:
1. Erythropoietin (EPO)
 derived from the Kidneys
 it will help the Myeloid stem cell to produce out RBCs
2. Thrombopoietin (TPO)
 derived from the Liver & Kidneys
 is needed for the production of Megakaryocytes, that
will produce the platelets
3. Granulocyte CSF (G-CSF)
 Based on studies, the Hematopoietic precursor cells give rise to 4. Granulocyte-Macrophage CSF (GM-CSF)
Colonies (CFUs) that can survive from 5-8 weeks, based on  if it’s in the blood, it is Monocyte
laboratory experiments
Bisenio, J. — TRANSCRIBER
[HEMA311] 2.01 Hematopoiesis | Prof. Antonio C. Pascua, Jr., RMT, MSMT
5. Interleukins
 before, they are present/released only by leukocytes
 later on, the studies reveal that other cells also produce
these types of Cytokines
 are signaling mechanisms/molecules that help the
progenitor cells to be committed
 are numbered based on the time they were discovered

Figure 4.01: Derivation of Hematopoietic cells (source: Rodak’s

Hematology 5th edition)

 CYTOKINES WITH POSITIVE INFLUENCES:

o cytokines that help increase the proliferation of
hematopoietic cells
> Interleukins 1, 3, 6, 7, 9, 11
> KIT Ligands (KITLG)
> GM-CSF
> etc.

Table 4.01: Summary of Growth factors & Progenitor cells that will give
a Specific Cell lines

Bisenio, J. — TRANSCRIBER
 OLFU Lineage Specific Hematopoiesis
CLINICAL HEMATOLOGY 1 LEC 3 2021 – 2022
1st Semester
RMT 2023 Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT
TRANS 3 HEMA311
Date: September 30, 2021 LEC

Outline Table 1.0 Three Erythroid Precursor Nomenclature Systems

At the end of the session, the student must be able to learn: Normoblastic Rubriblastic Erythroblastic
I. Red Cell Maturation Series Pronormoblast Rubriblast Proerythroblast
A. Maturation Process Basophilic Prorubricyte Basophilic
A. Erythroid Progenitor normoblast erythroblast
B. Erythroid Precursors Polychromatic Rubricyte Polychromatic
B. Criteria Used in Identification of Erythroid Precursors (polychromatophilic) (polychromatophilic)
C. Maturation Sequence normoblast erythroblast
A. Pronormoblast Orthochromic Metarubricyte Orthochromic
B. Basophilic Normoblast (Prorubricyte) normoblast erythroblast
C. Polychromatic Normoblast Polychromatic Polychromatic Polychromatic
D. Orthochromic Normoblast (polychromatophilic) (polychromatophilic) (polychromatophilic)
E. Polychromatic Erythrocyte or Reticulocyte erythrocyte erythrocyte erythrocyte
F. Erythrocyte Erythrocyte Erythrocyte Erythrocyte
II. White Cell Maturation Series
A. Granulocyte A. Maturation Process
A. Neutrophils and Neutrophil Development
1. Myeloblasts
2. Promyelocyte A. Erythroid Progenitors
3. Myelocytes
4. Metamyelocytes ❖ Two Progenitors:
5. Band Cell 1. Burst-Forming Unit Erythroid (BFU-E)
6. Segmented Neutrophils ▪ Gives rise to large colonies because they are capable of
7. Neutrophil Kinetics and Functions multi-subunit colonies (bursts)
B. Eosinophil 2. Colony-Forming Unit Erythroid (CFU-E)
1. Eosinophil Development ▪ Gives rise to smaller colonies
2. Eosinophil Kinetics ▪ Cell completes three to five divisions before maturing
3. Eosinophil Functions further
C. Basophils ➢ These progenitors are named for their ability to form colonies
1. Basophil Development on semisolid media in culture experiments that enable the
2. Basophil Kinetics study of their characteristics and development
3. Basophil Functions ➢ BFU-E takes 1 week to mature to the CFU-E and another
D. Mast Cells week for the CFU-E to become a pronormoblast
B. Mononuclear Cells ➢ It takes approximately 6 to 7 days for the precursors to
A. Monocyte become mature enough to enter circulation, approximately 18
1. Monocyte Development to 21 days are required to produce a mature RBC from the
2. Monocyte/Macrophage Kinetics BFU-E
3. Monocyte/Macrophage Functions
B. Lymphocytes
B. Erythroid Precursors
1. Lymphocyte Development
2. Lymphocyte Functions
III. Megakaryopoiesis ❖ Normoblastic proliferation
A. Megakaryocyte Differentiation and Progenitors ➢ is a process encompassing replication to increase cell
B. Thrombocytopoiesis (Platelet Shedding) numbers and development from immature to mature cell
stages
❖ Pronormoblast
I. RED CELL MATURATION SERIES ➢ Earliest morphologically recognizable erythrocyte precursor
and it is derived via the BFU-E and CFU-E from pluripotent
❖ Red Blood Cell/ Erythrocyte hematopoietic stem cells
➢ Carry oxygen from the lung to the tissues, where the oxygen ➢ Able to divide, with each daughter cell maturing to the next
is released stage of development, the basophilic normoblast
➢ Attachment of the oxygen to hemoglobin, the major ➢ From a single normoblast, therefore, 8 to 32 mature RBCs
cytoplasmic component of mature RBCs usually result
➢ Erythroblasts
▪ Nucleated RBC precursors, normally restricted to the B. Criteria Used in Identification of Erythroid Precursors
bone marrow
❖ Three Nomenclatures Erythroid Precursors ❖ Romanowsky stain
➢ Normoblastic ➢ Wright or Wright-Giemsa is commonly used
▪ Commonly used in the United States and is descriptive ❖ The stage of maturation of any blood cell is determined by careful
of the appearance of the cells examination of the nucleus and the cytoplasm
➢ Rubriblast ❖ The most important features in the identification of RBCs:
▪ Parallels the nomenclature used for granulocyte ➢ Nuclear chromatin pattern (texture, density, homogeneity)
development ➢ Nuclear diameter
➢ Erythroblast ➢ Nucleus to cytoplasm (N:C) ratio
▪ Used primarily in Europe ➢ Presence or absence of nucleoli
➢ Cytoplasmic color

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT
A. Pronormoblast
❖ General Trends affect appearance:
➢ Overall diameter of the cell decreases
❖ Nucleus:
➢ Diameter of the nucleus decreases more rapidly than does the
➢ Takes up much of the cell (N:C ratio = 8:1)
diameter of the cell. As a result, N:C ratio also decreases
➢ Round to oval, containing one or two nucleoli
➢ Nuclear chromatin pattern becomes coarser, clumped, and
➢ Purple Red Chromatin is open and contains few, if any, fine
condensed
clumps
▪ The nuclear chromatin of erythroid precursors is
❖ Cytoplasm:
inherently coarser than that of myeloid precursors
➢ Dark blue because of the concentration of ribosomes and
▪ It becomes even coarser and more clumped as the cell
RNA
matures, developing a raspberry-like appearance, in
➢ Golgi complex may be visible next to the nucleus as a pale,
which the dark staining of the chromatin is distinct from
unstained area
the almost white appearance of the parachromatin
❖ Division:
▪ The nucleus becomes quite condensed, with no
➢ Undergoes mitosis and gives rise to two daughter
parachromatin evident at all, and the nucleus is said to
pronormoblasts
be pyknotic
➢ More than one division is possible before maturation into
➢ Nucleoli disappear
basophilic normoblasts
▪ Nucleoli represent areas where the ribosomes are
❖ Location:
formed and are seen early in cell development as cells
➢ Bone marrow
begin actively synthesizing proteins
❖ Cellular Activity:
▪ As erythroid precursors mature, nucleoli disappear which
➢ Begins to accumulate the components necessary for
precedes the ultimate cessation of protein synthesis
hemoglobin production. The proteins and enzymes necessary
➢ Cytoplasm changes from blue to gray-blue to salmon pink
for iron uptake and protoporphyrin synthesis are produced
▪ Blueness or basophilia is due to acidic components
➢ Globin production begins
that attract basic stains such as methylene blue
❖ Length of time in this Stage:
▪ The degree of cytoplasmic basophilia correlates with the
➢ Slightly more than 24 hours
amount of ribosomal RNA
▪ Pinkness, Eosinophilia or Acidophilia is due to
accumulation of more basic components that attract acid
stains, such as eosin

Bluish: Lot of ribosome and RNA

No hemoglobin

B. Basophilic Normoblast (Prorubricyte)

❖ Nucleus:
➢ The chromatin begins to condense, revealing clumps along
the periphery of the nuclear membrane and a few in the
interior
➢ Chromatin condenses, parachromatin areas become larger
and sharper, and N:C ratio decreases to about 6:1
➢ Chromatin stains deep pruple-red. Nucleoli may be present
early in the stage but disappear later
❖ Cytoplasm:
C. Maturation Sequence
➢ Cytoplasm may be a deeper, richer blue than in the
pronormoblast
❖ Erythropoiesis ❖ Division:
➢ Regulated process for maintaining adequate numbers of red ➢ Undergoes mitosis, giving rise to two daughter cells
blood cells in the peripheral blood ➢ More than one division is possible before the daughter cells
➢ A process by which erythroid precursor cells differentiates to mature into polychromatic normoblasts
become mature ❖ Location:
➢ Production of red blood cells ➢ Present in bone marrow
➢ Formation of mature RBC from immature form ❖ Cellular Activity:
➢ Main Regulator: EPO (from kidneys) ➢ Detectable hemoglobin synthesis occurs, but the many
▪ Releases if there is hypoxia cytoplasmic organelles, including ribosomes and a substantial
❖ Note: Immature Cells are present in bone marrow amount of messenger ribonucleic acid
: Mature Cells present in blood ❖ Length of Time in this Stage:
➢ Slightly more than 24 hours

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT

C. Polychromatic (Polychromatophilic) Normoblast

❖ Nucleus:
➢ The chromatin pattern varies during this stage of
development, showing some openness early in the stage but
becoming condensed by the end
➢ The condensation of chromatin reduces the diameter of the
nucleus considerably, N:C ratio decreases from 4:1 to about
1:1 by the end of stage
❖ Cytoplasm:
➢ This is the first stage in which the pink color associated with
stained hemoglobin can be seen
➢ The color produced is a mixture of pink and blue, resulting in E. Polychromatic Erythrocyte or Reticulocyte
a murky gray-blue
➢ The stage’s name refers to this combination of multiple colors,
❖ Nucleus:
because polychromatophilic means “many color loving”
➢ Beginning at the polychromatic erythrocyte stage, there is no
❖ Division:
nucleus
➢ This is the last stage in which the cell is capable of undergoing
❖ Cytoplasm:
mitosis, although likely only early in the stage
➢ The cytoplasm can be compared with that of the late
➢ Mitosis, producing daughter cells that mature and develop into
orthochromic normoblast in that the predominant color is that
orthochromic normoblasts
of hemoglobin yet with a bluish tinge due to some residual
❖ Location:
ribosomes and RNA
➢ Polychromatic normoblast is present only in the bone marrow
➢ By the end, the cell is the same color as a mature RBC,
in healthy states
salmon pink
❖ Cellular Activity:
❖ Division:
➢ Hemoglobin synthesis increases, and the accumulation
➢ Lacking a nucleus, the polychromatic erythrocyte cannot
begins to be visible as a pinkish color in the cytoplasm
divide
➢ Cellular RNA and organelles are still present, particularly
❖ Location:
ribosomes, which contribute a blue color to the cytoplasm
➢ The polychromatic erythrocyte resides in the bone marrow for
❖ Length of Time:
about 1 to 2 days and then moves into the peripheral blood for
➢ Last approximately 30 hours
about 1 day before reaching maturity
➢ During the first several days after exiting the marrow, the
polychromatic erythrocyte is retained in the spleen for pitting
of inclusions and membrane polishing by splenic
macrophages, which results in the biconcave discoid mature
RBC
❖ Cellular Activity:
➢ The polychromatic erythrocyte completes production of
hemoglobin from a small amount of residual messenger RNA
using the remaining ribosomes
➢ The acidic components that attract the basophilic stain decline
during this stage to the point that the polychromatophilia is
only slightly evident in the polychromatic erythrocytes on a
peripheral blood film stained with wright stain
D. Orthochromic Normoblast ❖ Length of Time:
➢ The cell typically remains a polychromatic erythrocyte for
❖ Nucleus: about 3 days:
➢ The nucleus is completely condensed or nearly so. As a ▪ First 2 days spent in the bone marrow
result, the N:C ratio is low or approximately 1:2 ▪ Third spent in the peripheral blood, although possibly
❖ Cytoplasm: sequestered in the spleen
➢ Increase in the salmon pink color of the cytoplasm reflects
nearly complete hemoglobin production
❖ Division:
➢ Orthochromic normoblast is not capable of division because
of the condensation of the chromatin
❖ Location:
➢ Bone marrow
❖ Cellular Activity:
➢ Hemoglobin production continues on the remaining
ribosomes using messenger RNA produced earlier
➢ Late in this stage, the nucleus is ejected from the cell. The
nucleus moves to the cell membrane and into a pseudopod-
like projection
➢ Loss of vimentin, a protein responsible for holding organelles
in proper location in the cytoplasm, is probably important in
the movement of the nucleus to the cell periphery
❖ Length of Time:
➢ Lasts approximately 48 hours

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT
❖ Neutrophils
F. Erythrocyte ➢ Granules that react with both acid and basic stains, which
gives them a pink to lavender color
❖ Mononuclear Cells
❖ Nucleus:
➢ Monocytes and lymphocytes
➢ No nucleus is present in mature RBCs
➢ Have nuclei that are not segmented but are round, oval,
❖ Cytoplasm:
intended or folded
➢ The mature circulating erythrocyte is a biconcave disc
❖ Kinetics
measuring 7 to 8 micrometer in diameter, with a thickness of
➢ Refers to the movement of cells through developmental
about 1.5 to 2.5 micrometer
stages, into the circulation, and from the circulation to the
➢ It appears as a salmon-pin stained cell with a central pale area
tissues and includes the time spent in each phase of the cell’s
that corresponds to the concavity
life
❖ Division:
➢ The erythrocyte cannot divide
❖ Location and Length of Time:
➢ RBCs remain active in the circulation for approximately 120
days
❖ Cellular Activity
➢ The mature erythrocyte delivers oxygen to tissues, releases it
and returns to the lung to be reoxygenated
➢ The interior of the erythrocyte contains mostly hemoglobin, A. Granulocytes
the oxygen-carrying component
➢ If the cell was spherical, it would have hemoglobin at the
A. Neutrophils and Neutrophil Development
center of the cell that would be relatively distant from the
membrane and would not be readily oxygenated and
deoxygenated ❖ Neutrophils
➢ Are present in the peripheral blood in two forms according to
whether the nucleus is segmented or still in a band shape
➢ Segmented neutrophil make up the vast majority of
circulating leukocytes
➢ It occurs in the bone marrow
❖ Granulocyte-Monocyte Progenitor (GMP)
➢ It shares a common progenitor with monocytes and distinct
from eosinophils and basophils
❖ Granulocyte Colony-Stimulating Factor (G-CSF)
➢ Major cytokine responsible for the stimulation of neutrophil
production
❖ 3 Pools of Developing Neutrophils in Bone Marrow:
1. Stem Cell Pool
Table 1.1 Normoblastic Series: Summary of Stage Morphology ➢ Consists of HSCs that are capable of self-renewal and
Cell/Stage Diameter N:C Nucleoli % NC BM differentiation
in BC transit 2. Proliferation Pool
time ➢ Consists of cells that are dividing and includes common
Pronormoblast 12-20 8:1 1-2 1% 24hr myeloid progenitors (CMPs) also known as colony-forming
micrometer
units-granulocyte, erythrocyte, monocyte and megakaryocyte
Basophilic 10-15 8:1 0-1 1-4% 24hr
Normoblast micrometer (CFU-GEMMs), granulocyte-monocyte progenitors (GMPs),
Polychromatic 10-12 4:1 0 10- 30hr myeloblasts, promyelocytes, and myelocytes
Normoblast micrometer 20% 3. Maturation Pool
Orthochromic 8-10 1:2 0 5-10% 48hr ➢ Consists of cells undergoing nuclear maturation that form the
Normoblast micrometer marrow reserve and are available for release:
Bone Marrow 8-10 No 0 1% 24-48hr metamyelocytes, band neutrophils and segmented
Polychromatic micrometer nucleus neutrophils
Erythrocyte ❖ HSCs, CMPs, and GMPs are not distinguishable with the light
microscope and Romanowsky staining and may resemble early
II. WHITE CELL MATURATION SITE type I myeloblasts or lymphoid cells
➢ Flow cytometry for surface antigen detection
❖ Leukocytes (White Blood Cells) Table 2.0 Neutrophil Granules
➢ Relatively colorless compared to red blood cells Primary Granules Secondary Granules
➢ Number of different leukocytes varies depending on whether Myeloperoxidase B2-Microglobulin
they are being viewed with a light microscope after staining
Acid B-glycerophosphatase Collagenase
with a Romanowsky stain (5 or 6 types) or are identified
Cathepsins Gelatinase
according to their surface antigens using flow cytometry
➢ The overall function of leukocytes is in mediating immunity Defensins Lactoferrin
either innate (nonspecific), as in phagocytosis by neutrophils, Elastase Neutrophil gelatinase-
or specific (adaptive), as in the production of antibodies by associated lipocalin
lymphocytes and plasma cells Proteinase-3 Transcobalamin 1
❖ Granulocytes Others Others
➢ Are a group of leukocytes whose cytoplasm is filled with Tertiary Granules Secretory Granules
granules with differing staining characteristics and whose Gelatinase CD11b/CD18
nuclei are segmented or lobulated Collagenase Alkaline phosphatase
❖ Eosinophils Lysozyme Vesicle-associated membrane-2
➢ Granules containing basic proteins that stain with acid stains acetlytransferase CD10, CE13, CE14, CD16
such as eosin B2 – macroglobulin Cytoplasm 1q receptor
❖ Basophils Development 1q receptor
➢ Granules are acidic and stain with basic stains such as Complement receptor-1
methylene blue Cytochrome b558

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT
▪ Patches of grainy pale pink cytoplasm representing
1. Myeloblasts secondary granules begin to be evident in the area of the
Golgi apparatus
➢ Make up 0% to 3% of the nucleated cells in the bone marrow
and measure 14 to 20 micrometer in diameter ➢ Secondary neutrophilic granules slowly spread through the
❖ 3 Types of Myeloblasts cell until its cytoplasm is more lavender-pink than blue
1. Type I Myeloblast ➢ As the cell divides, number of primary granules per cell is
▪ High nucleus to cytoplasm (N:C) ratio of 8:1 to 4:1 (the decreased and membrane chemistry changes so that they are
nucleus occupies most of the cell, with very little much less visible
cytoplasm) ❖ Late Myelocyte
▪ Cytoplasm: Slightly basophilic ➢ Smaller than promyelocytes (15 to 18 micrometer)
▪ Nuclear Chromatin: Fine ➢ Nucleus has considerably more heterochromatin
▪ Nucleoli: 2 to 4 ➢ Nucleoli are difficult to see by light microscopy
▪ Granules: no visible granules under light microscopy
with Romanowsky stains
2. Type II Myeloblast
▪ Cytoplasm: Presence of dispersed primary (azurophilic)
granules
▪ Granules: does not exceed 20 per cell

3. Type III Myeloblast

▪ Rare normal in bone marrows, but they can be seen in
certain types of acute myeloid leukemias 4. Metamyelocytes
▪ Cytoplasm: more purple
▪ Chromatin: darker ➢ Constitute 3% to 20% of nucleated marrow cells
▪ Granules: more than 20 that do not obscure the nucleus ➢ Synthesis of tertiary granules (also known as gelatinase
granules) may begin during this stage
❖ Nucleus
➢ Intended (kidney bean shaped or peanut shaped)
➢ Shape of the nucleus: Cells are no longer capable of division
and the major morphologic change
➢ Nucleoli: absent
❖ Chromatin
➢ Increasingly clumped
❖ Cytoplasm
2. Promyelocyte ➢ Contains very little residual ribonucleic acid (RNA) and
therefore little or no basophilia
➢ Comprise 1% to 5% of nucleated cells in the bone marrow ❖ Size
➢ Larger than myeloblast cells and measure 16 to 25 ➢ Slightly smaller than that of the myelocyte (14 to 16
micrometers in diameter micrometer)
❖ Nucleus: round to oval and often eccentric
➢ 1 to 3 nucleoli can be seen but may be obscured by the
granules
❖ Paranuclear Halo (“hof”)
➢ Usually seen in normal promyelocytes but not in the malignant
promyelocytes of acute promyelocytic leukemia
❖ Cytoplasm: evenly basophilic and full of primary (azurophilic)
granules, first in a series of granules to be produced during
neutrophil maturation
❖ Chromatin: chromatin pumping (heterochromatin) may be visible,
especially around the edges of the nucleus 5. Band Cell

➢ All evidence of RNA (cytoplasmic basophilia) is absent, and

tertiary granules continue to be formed during this stage
➢ Secretory granules (secretory vesicles) may begin to be
formed during this stage
❖ Nucleated Marrow Cells: 9% to 32%
❖ Nucleated Peripheral Blood Cells: 0% to 5%
❖ Nucleus
➢ Highly clumped, and the nuclear indentation that began in the
metamyelocyte stage now exceeds one half the diameter of
3. Myelocytes the nucleus, but actual segmentation has not yet occurred
❖ CLSI (Clinical and Laboratory Standards Institute)
➢ Recommends that bands should be included within the
➢ Make up 6% to 17% of nucleated cells in the bone marrow neutrophil count and not reported as a separate category
and are the final stage in which cell division (mitosis) occurs because of the difficulty in reliably distinguishing bands from
➢ Production of primary granules ceases and cell begins to segmented neutrophils
manufacture secondary (specific) neutrophil granules
➢ Sometimes divided into early and late myelocytes
❖ Early Myelocytes
➢ May look very similar to the promyelocytes in size and
nuclear characteristics
➢ Dawn of Neutrophilia

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT
2. Neutrophil Functions
6. Segmented Neutrophils
➢ Part of the innate immune system (innate immunity:
➢ Make up 7% to 30% of nucleated cells in the bone marrow destruction of foreign organisms that is not antigen specific;
➢ Secretory granules continue to be formed during this stage no protection against re-exposure to the same pathogen,
➢ Present in the highest numbers in the peripheral blood of reliance on the barriers provided by skin and mucous
adults (50% to 70% of leukocytes in relative numbers and membranes
2.3 to 8.1x109/L in absolute terms) ➢ Major function is phagocytosis and destruction of foreign
❖ Pediatric Values material and microorganisms
➢ relative percentages can be as low as 18% of leukocytes in ❖ Complement System
the first few months of life and do not begin to climb to adult ➢ Inclusion of Humoral component
values until after 4 to 7 years of age ❖ Neutrophil extravasation
❖ Morphologic Difference ➢ Involves rolling, adhesion, crawling and transmigration
➢ Presence of between two and five nuclear lobes connected by ➢ Neutrophil recruitment to an inflammatory site begins when
thread-like filaments chemotactic agents bind to neutrophil receptors
❖ Chemotactic agents
➢ Produced by microorganisms, damaged cells or other
leukocytes such as lymphocytes or other phagocytes
❖ First Neutrophil response is to roll along endothelial cells of the
blood vessels using stronger adhesive molecules than those used
by non-stimulated marginated neutrophils
❖ Rolling consists of transient adhesive contacts between neutrophil
selectins and adhesive molecules on the surface of endothelial
cells (P selectins and E selectins)
❖ Activation is facilitated by the rolling of neutrophils on endothelium
7. Neutrophil Kinetics and Functions surfaces by chemokines
❖ One adhesion occurs, neutrophil scans the region while tightly
attached and does not always transmigrate at the location of
1. Neutrophil Kinetics adhesion
❖ Active Crawling depends on signaling between intercellular
➢ Involves the movement of neutrophils and neutrophil
adhesion molecule-1 (ICAM-1: expressed by endothelial cells) and
precursors between the different pools in the bone marrow,
macrophage-1 antigen (MAC-1: expressed by neutrophils)
the peripheral blood and tissues ❖ Transmigrate neutrophils in a directional manner either between
➢ Granulocyte release from the bone marrow is stimulated by
endothelial cells (paracellular) or through endothelial cells
G-CSF
(transcellular) toward the area of greatest concentration of
❖ Neutrophil Production chemotactic agents
➢ between 0.9 and 1.0x109 cells/kg per day
❖ At the Site of infection, neutrophils begin the process of
❖ Proliferative Pool
phagocytosis
➢ 2.1 x 109 cells/kg
❖ Maturation Pool
❖ Second Function of Neutrophils
➢ 5.6 x 109 cells/ kg or a 5-day supply
➢ Generation of neutrophil extracellular traps or NETs
❖ Transit Time Myeloblast – Myelocyte: 6 days ➢ NETs
❖ Transit Time Maturation Pool: 4 to 6 days
▪ Extracellular thread-like structures believed to represent
❖ Peripheral Blood:
chains of nucleosomes from unfolded nuclear chromatin
➢ Circulating neutrophil pool (CNP) material (DNA)
➢ Marginated neutrophil pool (MNP)
▪ These structures have enzymes from neutrophil
▪ Neutrophils are loosely localized to the walls of
granules attached to them and have been shown to be
capillaries in tissues such as the liver, spleen and lung able to trap and kill gram-positive and gram-negative
➢ Ratio of two pools is roughly equal overall, however,
bacteria as well as fungi
marginated neutrophils in the capillaries of the lungs make up
▪ Generated at the time that neutrophils die as a result of
a considerably larger portion of peripheral neutrophils antibacterial activity
❖ Half-life of Neutrophils: 7 hours in blood
➢ NETosis
❖ Diapedesis
▪ Used to describe the unique form of neutrophil cell death
➢ Process wherein integrins and selectins are of significant that results in the release of NETs
importance in allowing neutrophils to marginate as well as exit
❖ Third and Final Function of Neutrophils
the blood and enter the tissues
➢ Secretory function
❖ Apoptosis ➢ Transcobalamin I or R binder protein
➢ Those neutrophils that do not migrate into the tissues
▪ necessary for the proper absorption of vitamin B12
eventually undergo programmed cell death and are removed
▪ source of a variety of cytokines
by macrophages in the spleen, bone marrow and liver
❖ Once neutrophils are in the tissues, their life span is variable
depending on whether or not they are responding to infectious or B. Eosinophil
inflammatory agents
➢ In the absence of infectious or inflammatory agents, the ❖ Eosinophils arise from the CMP
neutrophil’s life span is measured in hours ❖ Eosinophil lineage
❖ Spontaneous Neutrophil Apoptosis ➢ Established through the interaction between the cytokines
➢ Regulated by pro- and antiapoptotic members of the Bcl-2 interleukin-3 (IL-3), IL-5 (induced by IL-33), and GM-CSF and
family 3 transcription factors (GATA-1 hematopoietic transcription
❖ Products of Inflammation and Infection factor, PU.1, and c/EBP)
➢ Mcl-1 and Myeloperoxidase (MPO) tend to prolong the ➢ IL-5 and IL-3 are critical for eosinophil growth and survival
neutrophil’s life span through antiapoptotic signals ❖ Eosinophilic Promyelocytes
➢ MAC-1 trigger the death and phagocytosis of neutrophils ➢ Can be identified cytochemically because of the presence of
Charcot-Leyden crystal protein in their primary granules
❖ Early Myelocyte
➢ First maturation phase that can be identified as eosinophilic
using light microscopy and Romanowsky staining

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT
2. Eosinophil Kinetics
1. Eosinophil Development
❖ Time from the last myelocyte mitotic division to the emergence of
1. Eosinophil Myelocytes mature eosinophils from the marrow is about 3.5 days
❖ The mean turnover of eosinophils approximately 2.2 x 108 cells/kg
❖ Characterized by the presence of large (resolvable at the light per day
microscope level), pale, reddish-orange secondary granules, along ❖ Large storage pool of eosinophils in the marrow consisting of
with azure granules in blue cytoplasm between 9 and 14 x 108 cells/kg
❖ Nucleus ❖ In Circulation:
➢ Similar to that described for neutrophil myelocytes ➢ Eosinophils have a circulating half-life of roughly 18 hours
❖ Transmission Electron Micrographs however, the half-life of eosinophils is prolonged when
➢ Many secondary eosinophil granules contain an electron- eosinophilia occurs
dense crystalline core ❖ Tissue Destination:
➢ Underlying columnar epithelial surfaces in the respiratory,
gastro-intestinal and genitourinary tracts
❖ Survival Time: 2 to 5 days in tissues

3. Eosinophil Functions
❖ Eosinophil granules are full of a large number of previously
synthesized proteins, including cytokines, chemokines, growth
factors and cationic proteins
❖ Inflammation Process Degranulation
➢ Classical Exocytosis
▪ Granules move to the plasma membrane, fuse with the
plasma membrane and empty their contents into the
extracellular space
➢ Compound Exocytosis
2. Eosinophil Metamyelocytes and Bands ▪ Second mechanism in which granules fuse together
within the eosinophil before fusing with the plasma
❖ Resemble neutrophil counterparts with respect to their nuclear membrane
shape ➢ Piecemeal degranulation
❖ Secondary granules increase in number and a third type of ▪ Secretory vesicles remove specific proteins from the
granule is generated called the secretory granule or secretory secondary granules
vesicle ▪ These vesicles then migrate to the plasma membrane
➢ Become more distinct and refractory and fuse to empty the specific proteins into the
❖ Electron Microscopy indicates the presence of two other extracellular space
organelles: lipid bodies and small granules ➢ Cytolysis
▪ When extracellular intact granules are deposited during
cell lysis
❖ Eosinophils
➢ Play important roles in immune regulation
➢ They transmigrate into the thymus of the newborn and are
believed to be involved in the deletion of double-positive
thymocytes
➢ Capable of acting as antigen-presenting cells and
promoting proliferation of effector T cells
➢ Implicated in the initiation of either type 1 or type 2 immune
responses because of their ability to rapidly secrete
performed cytokines in a stimulus-specific manner
➢ Important factors in acute and chronic allograft rejection
3. Mature Eosinophils ➢ Regulate mast cell function through the release of major
basic protein (MBP)
❖ Display a bilobed nucleus ▪ Causes mast cell degranulation as well as cytokine
❖ Cytoplasm: contains characteristic refractile, orange-red production, and they also produce nerve growth factor
secondary granules that promotes mast cell survival and activation
❖ Electron Microscopy ➢ Increased in infection by parasitic helminths, and in vitro
➢ Reveals extensive secretory vesicles, and their number studies have found that the eosinophil is capable of destroying
increases considerably when the eosinophil is stimulated or tissue-invading helminths through the secretion of MBP and
activated eosinophil cationic protein as well as the production of
reactive oxygen species
➢ Number of eosinophils in blood and sputum correlated with
disease severity. This has led to the suggestion that the
eosinophils one of the causes of airway inflammation and
mucosal cell damage through secretion or production of a
combination of basic proteins, lipid mediators, reactive
oxygen species, and cytokines such as IL-5
❖ Eosinophilia
➢ Hallmark of allergic disorders of which asthma has been the
best studied
❖ Anti-IL 5 monoclonal antibody treatment
➢ Reduce exacerbations in certain asthmatic patients
❖ Eosinophils in GI tract
➢ Occurs in allergic disorders such as food allergy, allergic
colitis and inflammatory bowel disease such as Crohn’s
disease and ulcerative colitis

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT
Table 2.01 Eosinophil Granules
Primary Granules Secondary Granules 2. Basophil Kinetics
Charcot-Leyden Crystal Major basic protein (core)
Protein ❖ Lifespan of basophils is relatively longer than that of the other
Eosinophil cationic protein (matrix) granulocytes, 60 hours
Eosinophil-derived neurotoxin ➢ Attributed to the fact that when they are activated by IL-3 and
(matrix) IL-25, antiapoptotic pathways are initiated that prolong the
Eosinophil peroxidase (matrix) basophil life span
Lysozyme (matrix)
Catalase (core and matrix) 3. Basophil Functions
B-Glucuronidase (core and matrix)
Cathepsin D (core and matrix) ❖ Basophils were regarded as “poor relatives” of mast cells and
Interleukin-2, -4, and -5 (core) minor players in allergic inflammation because, like mast cells, they
Interleukin-6 (matrix) have IgE receptor on their surface membranes that, when cross-
Granulocyte-macrophage-colony linked by antigen, result in granule release
stimulating factor (core) ❖ Basophilic functions in both innate and adaptive immunity
Small Lysosomal Granules Lipid Bodies ❖ Basophils are capable of releasing large quantities of subtype 2
Acid phosphatase Cyclooxygenase helper T cell (TH2), cytokines such as IL-4 and IL-3 that regulate
Arylsulfatase B 5-Lipoxygenase the TH2 immune response
Catalase 15-Lipoxygenase ❖ It also induce B cells to synthesize IgE. Initiators of the allergic
inflammation through the release of preformed cytokines
Cytochrome b558 Leukotriene C4 synthase
❖ Basophil activation is not restricted to antigen-specific IgE cross-
Elastase Eosinophil peroxidase
linking, but it can triggered in non-sensitized individuals by growing
Eosinophil cationic protein Esterase list of parasitic antigens, lectins, viral superantigens, cytokines,
chemokines growth factors, proteases, and components of the
C. Basophils complement system
❖ Capable of synthesizing granule proteins based on activation
❖ Basophils signals
➢ Derived from progenitors in the bone marrow and spleen, ➢ E.g., basophils can induced to produce a mediator of allergic
where they differentiate under the influence of a number of inflammation known as granzyme B
cytokines, including IL-3 and TSLP (thymic stromal ➢ Mast cells can induce basophils to produce and release
lymphopoietin) retinoic acid (a regulator of immune and resident cells in
❖ 2 Basophil Populations allergic diseases)
➢ IL-3 elicited basophils: immunoglobulin E (IgE) dependent ❖ Play important role in angiogenesis through expression of
➢ TLS elicited basophils: non-IgE dependent vascular endothelial growth factor (VEGF) and its receptors

1. Basophil Development D. Mast Cells

1. Immature Basophils ❖ Mast cells are not considered to be leukocytes. They are tissue
effector cells of allergic responses and inflammatory reactions
❖ Round to somewhat lobulated nuclei with only slightly condensed ❖ Development and Function
chromatin ➢ Their precursors circulate in the peripheral blood for a brief
❖ Nucleoli: may or may not be apparent period on their way to their tissue destinations
❖ Cytoplasm: blue and contains large blue-black secondary ➢ Mast cells have several phenotypic and functional similarities
granules with both basophils and eosinophils
❖ Primary azure granules may or may not be seen ❖ Mast Cell Progenitors (MCPs)
❖ Basophil granules are water soluble and therefore may be ➢ Originate from the bone marrow and spleen
dissolved if the blood film is washed too much during the staining ➢ Released to the blood before finally reaching tissues such as
process the intestine and lung, where they mediate their actions
❖ KIT ligand (stem cell factor)
➢ Major cytokine responsible for mast cell maturation and
differentiation
❖ Once the MCP reaches its tissue destination, complete maturation
into mature mast cells occurs under the control of the local
microenvironment
❖ It function as effector cells in allergic reactions through the release
2. Mature Basophils of a wide variety of lipid mediators, proteases, proteoglycans, and
cytokines as a results of cross-linking of IgE on the mast cells
❖ Nucleus: lobulated, often obscured by granules surface by specific allergens
❖ Chromatin pattern: if visible, clumped ❖ It can function as antigen-presenting cells to induce the
❖ Actual nuclear segmentation with visible filaments occurs rarely differentiation of TH2 cells, therefore mast cells act as mediators in
❖ Cytoplasm: colorless and contains large numbers of the both innate and adaptive immunity
characteristic large blue-black granules ❖ Act as immunologic “gatekeepers” because of their location in
➢ If any granules have been dissolved during the staining mucosal surfaces and their role in barrier function
process, they often leave a reddish-purple rim surrounding
what appears to be a vacuole

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT

B. Mononuclear Cells 2. Monocyte/Macrophage Kinetics

A. Monocytes ❖ Promonocyte pool consists of approximately 6x108 cells/kg, and

they produce 7x106 monocytes/kg per hour
➢ Make up between 2% and 11% of circulating leukocytes, with ❖ Mitotic Division
an absolute number of up to 1.3 x 109/L ➢ Under normal circumstances, promonocytes undergo 2
division in 60 hours to produce a total of 4 monocytes
1. Monocyte Development ➢ Under conditions of increased demand for monocytes,
promonocytes undergo 4 divisions to yield a total of 16
monocytes in 60 hours
❖ Similar to neutrophil development because both cell types are
❖ There is no storage pool of mature monocytes in the bone marrow,
derived from the GMP
and unlike neutrophils, monocytes are released immediately into
❖ Macrophage Colony-Stimulating Factor (M-CSF)
the circulation upon maturation
➢ Major cytokine responsible for the growth and differentiation
➢ Therefore, when the bone marrow recovers from marrow
of monocytes
failure, monocytes are seen in the peripheral blood before
❖ Monoblasts
neutrophils and a relative monocytosis may occur
➢ In normal bone marrow are very rare and are difficult to
❖ Like neutrophils, monocytes in the peripheral blood can be found
distinguish from myeloblasts based on morphology
in a marginal pool and a circulating pool
➢ Unlike with neutrophils, marginal pool of monocytes is 3.5
1. Promonocytes
times the circulating pool
➢ Monocytes remain in the circulation approximately 3 days
❖ 12 to 18 micrometer in diameter, and their nucleus is slightly
❖ Once in the tissues, monocytes differentiate into macrophages,
indented or folded
osteoclasts or dendritic cells depending on the microenvironment
❖ Chromatin Pattern: delicate, and at least one nucleolus is
of the local tissues
apparent
➢ Macrophages can be as large as 40 to 50 micrometer in
❖ Cytoplasm: blue and contains scattered azure granules that are
diameter
fewer and smaller than those seen in promyelocytes
➢ Life Span: depends on whether they are responding to
❖ Electron microscopic and cytochemical studies have found that
inflammation or infection, or they are “resident”
monocyte azure granules are heterogenous with regard to their
macrophages
content of lysosomal enzymes, peroxidase, nonspecific esterase
and lysozyme
3. Monocyte/ Macrophage Functions

❖ Innate Immunity
➢ Monocytes/ macrophages recognize a wide range of bacterial
pathogens by means of pattern recognition receptors (toll-like
receptors) that stimulate inflammatory cytokine production
and phagocytosis
➢ Macrophages can synthesize nitric oxide (cytotoxic against
viruses, bacteria, fungi, protozoa, helminths and tumor cells)
➢ Monocytes and Macrophages also have Fc receptors and
complement receptors
❖ Adaptive Immunity
➢ Both macrophages and dendritic cells degrade antigen and
present antigen fragments on their surfaces (antigen-
2. Monocytes presenting cells)
➢ They interact with and activate both T lymphocytes and B
❖ Appear to be larger than neutrophils (diameter of 15 to 20 lymphocytes to initiate the adaptive immune response
micrometer) because they tend to stick to and spread out on glass ➢ Dendritic cells are the most efficient and potent of the
or plastic antigen-presenting cells
❖ Slightly immature cells whose ultimate goal is to enter the tissues ❖ Housekeeping Functions
and mature into macrophages, osteoclasts, or dendritic cells ➢ Include removal of debris and dead cells at sties of infection
❖ Nucleus: round, oval, or kidney shaped but more often is deeply or tissue damage, destruction of senescent red blood cells
indented (horseshoe shaped) or folded on itself and maintenance of a storage pool of iron for erythropoiesis
❖ Chromatin Pattern: looser than in the other leukocytes and has and synthesis of a wide variety of proteins, including
sometimes been described as lace-like or stringy coagulation factors, complement components, interleukins,
❖ Nucleoli: generally not seen with the light microscope however, growth factors and enzymes
electron microscopy reveals nucleoli in roughly half of circulating
monocytes B. Lymphocytes
❖ Cytoplasm: blue-gray with fine azure granules often referred to as
azure dust or a ground-glass appearance
❖ Lymphocytes
➢ Small cytoplasmic pseudopods or blebs may be seen
➢ Are divided into three major groups:
➢ Cytoplasmic and nuclear vacuoles may also be present
▪ T cells and B cells major players in adaptive immunity
▪ Natural Killer (NK) cells make up small percentage of
lymphocytes and are part of innate immunity
❖ 3 Characteristics of Adaptive Immunity
➢ It relies on an enormous number of distinct lymphocytes
➢ Each having surface receptors for a different specific
molecular structure on a foreign antigen
➢ After an encounter with a particular antigen, memory cells are
produced that will react faster and more vigorously to that
same antigen on re-exposure and self-antigens are “ignored”
under normal circumstances (tolerance)

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT
❖ 2 Major Categories of Lymphocytes
➢ Participate in humoral immunity by producing antibodies and 2. T Lymphocytes
those that participate in cellular immunity by attacking foreign
organisms or cells directly ❖ Develop initially in the thymus – a lymphoepithelial organ located
❖ B lymphocytes in the upper mediastinum
➢ Antibody-producing lymphocytes, develop in the bone marrow ❖ Lymphoid progenitor cells migrate from the bone marrow to the
❖ Cellular Immunity (2 Types of Lymphocytes) thymic cortex, where, under the regulation of cytokines produced
➢ T cells: develop in the thymus by thymic epithelial cells, they progress through stages known as
➢ NK cells: develop in both the bone marrow and thymus pro-T, pre-T and immature T cells
❖ Lymphocytes are different from the other leukocytes in ➢ During these phases they undergo antigen receptor gene
several ways: rearrangement to produce T cell receptors that are unique to
➢ Lymphocytes are not end cells. They are resting cells, and each T cell
when stimulated, they undergo mitosis to produce both ❖ Apoptosis
memory and effector cells ➢ T cells whose receptors react with self-antigens are allowed
➢ Unlike other leukocytes, lymphocytes recirculate from the to undergo apoptosis
blood to the tissues and back to the blood ❖ Immature T Cells
➢ B and T lymphocytes are capable of rearranging antigen ➢ Proceed to the thymic medulla, whether further apoptosis of
receptor gene segments to produce a wide variety of self-reactive T cells occurs
antibodies and surface receptors ➢ The remaining immature T cells (antigen-naïve T cells) then
➢ Although early lymphocyte progenitors such as the common leave the thymus and migrate to secondary lymphatic organs,
lymphoid progenitor originate in the bone marrow, T and NK where they take up residence in specific zones such as the
lymphocytes develop and mature outside the bone marrow paracortical areas
❖ T cells comprise 51% to 88% of circulating lymphocytes
1. Lymphocyte Development ❖ T cells in secondary lymphoid organs or in the circulating blood
eventually come in contact with antigens
❖ Antigen-Independent Lymphocyte ➢ This results in cell activation and the production of either
➢ Development occurs in the bone marrow and thymus memory cells or effector T cells, or both
(sometimes referred to as central or primary lymphatic ❖ The transformation of resting lymphocytes into activated forms is
organs) the source of medium and large lymphocytes that have
❖ Antigen-Dependent Lymphocyte increased amounts of cytoplasm and usually make up only about
➢ Development occurs in spleen, lymph nodes, tonsils and 10% of circulating lymphocytes
mucosa-associated lymphoid tissue (peyer’s patches in ❖ Reactive Lymphocytes
intestinal wall, sometimes referred to as peripheral or ➢ The morphology of effector T cells varies with the subtype of
secondary lymphatic organs) T cell involved

1. B Lymphocytes

❖ Develop initially in the bone marrow and go through three stages

known as pro-B, pre-B, and immature B cells
➢ During these stages that immunoglobulin gene
rearrangement occurs so that each B cell produces a unique
immunoglobulin antigen receptor
❖ Immature B cells
➢ Have not yet been exposed to antigen (antigen-naïve B
cells) leave the bone marrow to migrate to secondary
lymphatic organs, where they take up residence in specific
zones such as lymph node follicles
➢ Also known as hematogones, have a homogenous nuclear Three cells representing lymphocyte activation
chromatin pattern and extremely scanty cytoplasm
➢ Normally found in newborn peripheral blood and bone marrow 3. NK Cells
and in regenerative bone marrows
➢ Leukemic cells from patients with acute lymphoblastic ❖ Heterogenous group of cells with respect to their surface antigens
leukemia can sometimes resemble hematogones, but the ❖ The majority are CD56, D16, CD3-, CD7+ large granular
leukemic cells can be distinguished from hematogones by lymphocytes
flow cytometry immunophenotyping ❖ The mature NK cell is relatively large compared with other resting
❖ Secondary lymphoid organs/blood lymphocytes because of an increased amount of cytoplasm
➢ B cells may come in contact with antigen, result in cell division ❖ Cytoplasm: contains azurophilic granules that are peroxidase
and the production of memory cells a well as effector cells negative
❖ Known as plasma cells and plasmacytoid lymphocytes ❖ Approximately 4% to 29% of circulating lymphocytes are NK cells
❖ Almost 3% to 21% of circulating lymphocytes are B cells

Large Granular Lymphocyte (Cytotoxic T lymphocyte or

a Natural killer lymphocyte)
Immature B Lymphocytes

Surell, R. – TRANSCRIBER
[HEMA311] 1.03 Lineage-Specific Hematopoiesis I Prof. Antonio C. Pascua, RMT, MSMT

2. Lymphocyte Functions A. Megakaryocyte Differentiation and Progenitors

❖ B lymphocytes ❖ Megakaryocyte progenitors arise from the common myeloid

➢ Essential for antibody production progenitor under the influence of the transcription gene product,
➢ Role in antigen presentation to T cells and may be necessary GATA-1, regulated by cofactor FOG1
for optimal CD4 activation ❖ Megakaryocyte differentiation is suppressed by another
➢ Produce cytokines that regulate a variety of T cell and antigen transcription gene product, MYB so GATA-1 and MYB act in
presenting cell functions opposition to balance megakaryocytes with erythropoiesis, where
❖ T lymphocytes the same progenitor cell is differentiated into the platelet cell line or
➢ CD4+ T cells the red blood cell line
▪ Subdivided into TH1, TH2, TH17 and Treg (CD4+ CD25+ ❖ 3 Megakaryocyte Lineage-Committed Progenitor Stages
regulatory T) cells ➢ Least mature burst-forming unit (BFU-Meg)
▪ TH1 cells mediate immune responses against ➢ Intermediate colony forming unit (CFU-Meg)
intracellular pathogens ➢ More mature progenitor Light Density CFU (LD-CFU-Meg)
▪ TH2 mediate host defense against extracellular ➢ All three progenitor stages resemble lymphocytes and cannot
parasites, including helminths be distinguished by Wright-stained light microscopy
• Important in the induction of asthma and other ➢ BFU-Meg and CFU-Meg are diploid and undergo normal
allergic diseases mitosis to maintain a viable pool of megakaryocyte
▪ TH17 involved in the immune responses against progenitors
extracellular bacteria and fungi ➢ LD-CFU-Meg undergoes endomitosis (nucleus can multiply
▪ Treg play a role in maintaining self-tolerance by regulating but cytoplasm cannot), a partially characterized form of
immune response mitosis unique to megakaryocytes in which DNA replication
➢ CD8 T cells
+ and cytoplasmic maturation are normal but cells lose their
▪ Capable of killing target cells by secreting granules capacity to divide
containing granzyme and perforin or by activating
apoptotic pathways in the target cell
▪ Sometimes referred to as cytotoxic T lymphocytes
❖ NK lymphocytes
➢ Function as part of innate immunity and are capable of killing
certain tumor cells and virus-infected cells without prior
sensitization
➢ Modulate the functions of other cells, including macrophages
and T cells

B. Thrombocytopoiesis (Platelet Shedding)

❖ During thrombocytopoiesis, a single megakaryocyte may shed

2000 to 4000 platelets
❖ An average size healthy human there are 108 megakaryocyte
Lymphocytes producing 1011 platelets per day
❖ The total platelet population turns over in 8 to 9 days (platelet
III. MEGAKARYOPOIESIS lifespan)
❖ In instances of high platelet consumption, such as immune
❖ Platelets thrombocytopenic purpura, platelet production may rise by as
➢ Nonnucleated blood cells that circulate at a concentration of much as tenfold
150 to 400x109/ L, with average platelet counts slightly
higher in women than in men and slightly lower in
members of both sexes who are older than 65 years
➢ Trigger primary hemostasis on exposure to subendothelial
collagen or endothelial cell inflammatory proteins at the time
of blood vessel injury
➢ Arise from unique bone marrow cells called Megakaryocytes
▪ Largest cells in the bone marrow and possess multiple
chromosome copies (polyploid)
▪ On a wright-stained bone marrow aspirate smear, each
megakaryocyte is 30 to 50 micrometer in diameter with
a multilobulated nucleus and abundant granular
cytoplasm
▪ Account for less than 0.5% of all bone marrow cells, and
on a normal Wright-stain bone marrow aspirate smear
two to four megakaryocytes per 10x low-power field may
be identified
❖ Maturation series of a hematological cell that is committed for
platelet production
❖ Production of megakaryocytes
❖ Responds to a growth factor called thrombopoietin
❖ As the cell matures:
➢ Cell size increases
➢ N:C ratio decreases
➢ Number of nucleus increases
Surell, R. – TRANSCRIBER
 IV. SUMMARIZATION

Hematopoiesis
Committed Progenitor Cell Growth Factors/ Interleukins Mature Cell
CFU-MEG Thrombopoietin, GM-CSF Thrombocytes
CFU-GM CFU-M GM-CSF, M-CSF, IL-3 Monocytes
CFU-GM CFU-G GM-CSF, G-CSF, IL-3 Neutrophils
BFU-E CFU-E Erythropoietin, GM-CSF, IL-3 Erythrocytes
CFU-Eo GM-CSF, IL-3, IL-5 Eosinophils
CFU-Ba IL-3, IL-4 Basophils

Table 4.0 CD Markers

CD2, CD3 Lymphoid, pan T cells
CD4 Helper/inducer T cells
CD8 Suppressor/cytotoxic T cells
CD13 Pan myeloid
CD11c, CD14 Monocytes
CD19, CD20 Lymphoid, pan B cells
CD33 Pan myeloid cells
CD34 Stem cell marker (lymphoid and myeloid precursor)
CD16, CD56 NK cells

Table 4.1 General Cell Maturation Characteristics for Leukocytes

Immature Cells Mature Cells
Cell is large Cell becomes smaller
Nucleoli present Nucleoli absent
Chromatin fine and delicate Chromatin coarse and clumped
Nucleus round Nucleus round, lobulated or segmented
Cytoplasm dark blue (rich in RNA) Cytoplasm light blue (less RNA)
High N:C ratio Low N:C ratio

A. GRANULOCYTES

Myeloblast Promyelocyte Myelocyte Metamyelocyte Band Neutrophil

Size (micrometer) 14-20 15 – 21 12 – 18 10 – 18 10 – 15
N:C ratio 7:1 – 4:1 3:1 2:1 1.5:1 1:2
Nucleus Round/oval Round/oval Round Kidney bean C or S
Chromatin Fine reddish-purple Slightly coarsening Coarse Coarse, clump Coarse, clump, lack segmentation
Nucleoli 2–5 1–3 Early: Visible Less than half the Greater than half the width
width
Cytoplasm Dark blue Dark blue Light blue-light pink Pink filled with pale Pink filled with pale blue – pink
blue-pink specific specific granules
Granules No Nonspecific Specific/secondary Nonspecific/ primary Nonspecific/ primary
(myeloperoxidase) (hydrolytic enzymes)
Location 1% bone marrow 2 – 5% bone marrow 13% bone marrow 16% bone marrow 12% bone marrow, 0 – 5% p.WBC

B. MORPHOLOGY OF MATURE GRANULES

Neutrophil Eosinophil Basophil

Size (micrometer) 10 – 15 12 – 16 10 – 15
N:C ratio 1:3 - -
Nucleus Coarse Bilobed -
Chromatin Clumped, 3 – 5 lobes - -
Cytoplasm Pink, pale blue to pink specific/secondary Bright red-orange, secondary granules Purple-black, secondary
granules contain enzymes and proteins granules contain heparin and
histamine
Granules Nonspecific/ primary but do not stain - Numerous
Location 12% bone marrow, 50-80% peripheral WBC 1% bone marrow, 5% peripheral WBC 0.1% bone marrow and
peripheral blood
C. MONOCYTES AND MACROPHAGES

Monoblast Promonocyte Monocyte Macrophage

Size (micrometer) 12 – 18 12 – 20 12 – 20 15 – 80
N:C ratio 4:1 3:1 - -
Nucleus Round/oval eccentric Irregularly shaped, indented Horseshoe, kidney bean Indented, elongated, egg-shaped
Chromatin Fine Fine Fine, lacy Fine
Nucleoli 1–2 0–1 - -
Cytoplasm Dark blue Blue to gray Blue to gray, Blue to gray, many vacuoles
pseudopods/vacuoles
Granules No cytoplasmic granules Fine azurophilic Fine azurophilic Coarse azurophilic
(ground glass appearance) (contain ingested material)

LYMPHOCYTES

Lymphoblast Prolymphocyte Lymphocyte

Diameter (micrometer) 10 – 18 9 – 18 7 – 18
N:C ratio 4:1 3:1 -
Nucleus Round/ oval eccentric Round or indented Round, oval, slightly indented
Chromatin Fine Coarse Condensed
Nucleoli 1 or more 0–1 -
Cytoplasm Dark blue Basophilic Scant to moderate blue
Granules No cytoplasmic granules No cytoplasmic granules Few azurophilic granules

E. ERYTHROCYTES

Pronormoblast Basophilic Polychromatophilic Orthochromatic Reticulocyte Erythrocyte

Normoblast normoblast normoblast
Size 20 16 12 10 10 6–8
N:C ratio 8:1 6:1 4:1 0.5:1 - -
Nucleus Dark areas DNA Centrally located Eccentric Eccentric No -
Chromatin Fine, uniform Coarse Clumping Condensed - -
Nucleoli 1–3 0–1 No No - -
Cytoplasm Deep blue Less blue, Begin to produce Pale blue to salmon - -
intensely hgb, Gray-blue
basophilic
Granules No - - - - -
 OLFU Erythrocyte Metabolism, Membrane Structure & Function
CLINICAL HEMATOLOGY 1 LEC 4 2021 – 2022
1st Semester
RMT 2023 Instructor: Prof. Antonio C. Pascua, Jr., RMT, MSMT TRANS 4 HEMA311
Date: October 7, 2021 LEC

Outline II. ENERGY PRODUCTION – ANAEROBIC GLYCOLYSIS

At the end of the session, the student must be able to learn:
I. Introduction ❖ Lacking mitochondria, the RBC relies on anaerobic glycolysis for
II. Energy Production – Anaerobic Glycolysis its energy
A. Three Phases of Glycolysis ❖ Exchange of O2 and CO2 is a passive function from high partial
III. Glycolysis Diversion Pathways (Shunts) pressure to low partial pressure; however, the cells metabolic
A. Hexose Monophosphate Pathway processes require energy:
B. Methemoglobin Reductase Pathway Table 1.0 Erythrocyte Metabolic Processes Requiring Energy
C. Rapoport-Leubering Pathway Metabolic Processes
IV. Red Blood Cell Membrane Intracellular cationic gradient maintenance
A. Red Blood Cell Membrane Deformability Maintenance of membrane phospholipid distribution
B. Red Blood Cell Membrane Lipids Maintenance of skeletal protein deformability
C. Red Blood Cell Membrane Proteins Maintenance of functional hemoglobin with ferrous iron
1. Transmembrane Proteins Protecting cell proteins from oxidative denaturation
2. Cytoskeletal Proteins Glycolysis initiation and maintenance
D. Osmotic Balance and Permeability Glutathione synthesis
Nucleotide salvage reactions
Note: as energy production slows, the RBC grows senescent and is
I. INTRODUCTION removed from the circulation
❖ Anerobic glycolysis or EMP
❖ RBC ➢ Requires glucose to generate ATP, a high-energy phosphate
➢ Approximately 5 million erythrocytes per microliter of source
circulating blood ➢ RBCs lack internal energy stores and rely on plasma glucose
➢ Primary cell in the blood to enter the cell to generate ATP
➢ Lacks a nucleus, has an average volume of 90fL ➢ Glucose enters the RBC through facilitated diffusion via the
➢ Biconcave shape transmembrane protein Glut-1
▪ Supports deformation ➢ Glucose is then catabolized to pyruvate (pyruvic acid) in the
▪ Enabling the circulating cell to pass smoothly through EMP, generating four molecules of ATP per molecule of
capillaries, where it readily exchanges oxygen (O2) an d glucose, for a net gain of two molecules of ATP
carbon dioxide (CO2) while contacting the vessel wall
➢ Cytoplasm A. Three Phases of Glycolysis
▪ Contains abundant hemoglobin (complex of globin,
protoporphyrin and iron) which transports O2 from the ❖ First Phase
lungs to the tissue ➢ Employs glucose phosphorylation, isomerization and
➢ Transports CO2 and bicarbonate (HCO3-) from the tissues disphosphorylation to yield fructose 1,6-biphosphate (F1, 6-
back to the lungs BP)
❖ Hemoglobin ➢ Intermediate Stages
➢ Has four globin chains, and each chain contains a heme ▪ Employs the enzymes hexokinase, glucose-6-phosphate
molecule with an iron in the ferrous state isomerase, and 6-phosphofructokinase
▪ Allows each hemoglobin molecule to carry four O2 ➢ Initial Hexokinase and 6-phosphofructokinase steps
molecules consume a total of 2 ATP molecules and limit the rate of
❖ Production glycolysis
➢ RBCs are produced through normoblastic proliferation and ➢ Fructose-biphosphate aldolase then cleaves F1,6-BP to
mature in the bone marrow produce glyceraldehyde-3-phosphate (G3P)
➢ The nucleus, present in maturing normoblasts, is extruded
as part of the RBC maturation process, typically as the RBC Table 2.0 Glucose Catabolism: First Phase
moves from the bone marrow to circulation Substrates Enzyme Products
➢ Cytoplasmic ribosomes and mitochondria also disappear Glucose, ATP Hexokinase G6P, ADP
24 to 48 hours after bone marrow release, eliminating the cells
G6P Glucose-6-phosphate isomerase F6P
ability to produce proteins or support oxidative metabolism
F6P, ATP 6-Phosphofructokinase F1, 6-BP,
❖ Without mitochondria for aerobic respiration via oxidative
ADP
phosphorylation, adenosine triphosphate (ATP) is produced
within the cytoplasm through anerobic glycolysis (Embden- F1, 6-BP Fructose-bisphosphate adolase DHAP, G3P
Meyerhof pathway, EMP) for the lifetime of the cell
❖ ATP drives mechanisms that slow down the oxidation of proteins ❖ Second Phase
and iron by environmental peroxides and superoxide anions, ➢ Converts G3P to 3-phosphoglycerate (3-PG)
maintaining hemoglobin’s function and membrane integrity ➢ In the first step, G3P is oxidized to 1,3-bisphosphoglycerate
❖ Oxidation takes a toll, limiting the RBC circulating life span to 120 (1,3-BPG) through the action of glyceraldehyde-3-phosphate
days, whereupon it is disassembled into its reusable components: dehydrogenase (G3PD) with the reduction of NAD to NADH
globin chains and iron from hemoglobin, and phospholipids and ➢ 1,3-BPG is dephosphorylated by phosphoglycerate kinase,
proteins from the cell membrane which generates two ATP molecules and 3-PG
❖ The porphyrin ring of hemoglobin is not reusable and is excreted
as bilirubin Table 2.1 Glucose Catabolism: Second Phase
Substrates Enzyme Products
G3P, NAD Glyceraldehyde-3- 1,3-BPG, NADH
phosphate dehydrogenase
1,3-BPG, ADP Phosphoglycerate kinase 3PG, ATP
1,3-BPG Bisphosphoglycerate mutase 2,3-BPG
2,3-BPG Bisphosphoglycerate 3-PG
phosphatase

Surell, R. – TRANSCRIBER
[HEMA311] 1.04 Erythrocyte Metabolism, Membrane Structure and Function I Prof. Antonio C. Pascua, RMT, MSMT
❖ Third Phase
➢ Converts 3-GP to pyruvate and generates ATP
➢ The 3-PG is isomerized by phosphoglycerate mutase to 2-
phosphoglycerate (2-PG)
➢ Enolase (phosphopyruvate hydratase) then converts 2-PG
to phosphoenolpyruvate (PEP)
➢ Pyruvate kinase (PK) splits off the phosphates, forming two
ATP molecules and pyruvate. PK activity is allosterically
modulated by increased concentrations of F1, 6-BP, which
enhances the affinity of PK for PEP
➢ When F1,6-BP is plentiful, increased activity of PK favors
pyruvate production
➢ Pyruvate may diffuse from the erythrocyte or may become a
substrate for lactate dehydrogenase (LD or LDH) with
regeneration of the oxidized form of nicotinamide adenine
H2O2 H2O
dinucleotide (NAD+)
➢ The ratio of NAD+ to the reduced form (NADH) modulates the
activity of LD
Glutathione peroxidase Hexose
Table 2.2 Glucose Catabolism: Third Phase
Monophosphate
Substrates Enzyme Products
3-PG Phosphoglycerate mutase 2-PG Pathway
GSH GSSG
2-PG Enolase (phosphopyruvate PEP
hydratase
PEP, ADP Pyruvate kinase Pyruvate, ATP Glutathione reductase
Pyruvate, Lactate dehydrogenase Lactate, NAD
NADH
NADPH
NAD
Embden- Glucose ATP
Meyerhof Hexokinase (-1 ATP)
Pathway ADP
Glucose 6- phosphate 6-phosphogluconate
Glucose-6-phosphate Glucose-6-phosphate dehydrogenase NADP
isomerase Fructose 6-phosphate
ATP 6-phosphogluconate NADPH
6-phosphofructokinase (-1 ATP) dehydrogenase
ADP CO2
Fructose 1,6-biphosphate
Fructose-biphosphate
aldolase Ribulose 5-phosphate
Dihydroxyacetone Glyceraldehyde
phosphate 3-phosphate
Triosephosphate isomerase
Methemoglobin
Glyceraldehyde NAD Methemoglobin
reductase +H
3-phosphate
NADH Methemoglobin
dehydrogenase Hemoglobin
reductase
Methemoglobin
1,3-bisphosphoglycerate Reductase
Bisphosphoglycerate
Pathway
mutase
Rapoport- ADP
Glucose
Leubering Phosphoglycerate (+2 ATP)
Pathway kinase ATP

2,3-bisphosphoglycerate
(2,3-BPG) 3-phosphoglycerate

Phosphoglycerate mutase

2-phosphoglycerate
Enolase
Phosphoenolpyruvate
ADP
Pyruvate kinase (+2 ATP)
Pyruvate ATP

Lactate dehydrogenase NADH

NAD
Lactate

Surell, R. – TRANSCRIBER
[HEMA311] 1.04 Erythrocyte Metabolism, Membrane Structure and Function I Prof. Antonio C. Pascua, RMT, MSMT

III. GLYCOLYSIS DIVERSION PATHWAYS (SHUNTS) ❖ Using H+ from NADH formed when G3P is converted to 1,3-BPG,
cytochrome b5 reductase acts as an intermediate electron
❖ Three alternative pathways, called diversions or shunts, branch carrier, returning the oxidized ferric iron to its ferrous, oxygen-
from the glycolytic pathway carrying state
❖ 3 Diversions: ➢ This enzyme accounts for more than 65% of the
➢ Hexose monophosphate pathway (HMP) methemoglobin-reducing capacity within the RBC
➢ Methemoglobin reductase pathway
➢ Ropoport – Luebering pathway C. Rapoport-Luebering Pathway

A. Hexose Monophosphate Pathway ❖ A third metabolic shunt generates 2,3-bisphosphoglycerate (2,3-

BPG; also called 2,3-diphosphoglycerate or 2,3-DPG)
❖ Oxidative glycolysis occurs through a diversion of glucose ❖ 1,3-BPG is diverted by bisphosphoglycerate mutase to form 2,3-
catabolism into the HMP, also known as the pentose phosphate BPG
shunt ➢ 2,3-BPG regulates oxygen delivery to tissues by competing
❖ HMP with oxygen for the oxygen-binding site of hemoglobin
➢ Detoxifies peroxide (H2O2) which arises from O2 reduction in ➢ When 2,3-BPG binds heme, oxygen is released, which
the cell’s aqueous environment enhances delivery of oxygen to the tissues
❖ H2O2 oxidizes heme iron to the non-functional ferric state and ➢ 2,3-BPG forms 3-PG by the action of bisphosphoglycerate
oxidizes and denatures proteins and lipids phosphatase
❖ HMP Shunt extends the functional life span of the RBC by ➢ This diversion of 1,3-BPG to form 2,3-BPG sacrifices the
maintaining membrane proteins and lipids, enzymes, and production of two ATP molecules
hemoglobin iron in the functional, reduced, ferrous state ➢ There is further loss of two ATP molecules at the level of PK,
❖ HMP diverts glucose-6-phosphate dehydrogenase (G6PD) because fewer molecules of PEP are formed. Because two
❖ In the process, oxidized nicotinamide adenine dinucleotide ATP molecules were used to generate 1,3-BPG and
phosphate (NADP) is converted to its reduced form (NADPH) production of 2,3-BPG eliminates the production of four
❖ NADPH is then available to reduce oxidized glutathione (GSSG) to molecules, the cell is put into ATP deficit by this diversion
reduced glutathione (GSH) in the presence of glutathione ➢ There is a delicate balance between ATP generation to
reductase support the energy requirements of cell metabolism and the
❖ Glutathione need to maintain the appropriate oxygenation and
➢ Is a cysteine-containing tripeptide, and the designation GSH deoxygenation status of hemoglobin
highlights the sulfur in the cysteine moiety ➢ Acidic pH and low concentrations of 3-PG and 2-PG inhibit
➢ Reduced glutathione becomes oxidized as it reduces the activity of bisphosphoglycerate mutase, thus inhibiting
peroxide to water and oxygen via glutathione peroxidase the shunt and retaining 1,3-BPG in the EMP
❖ Steady-State Glycolysis ➢ These conditions and decreased ATP
➢ 5% to 10% of G6P is diverted to the HMP activate bisphosphoglycerate phosphatase, which returns
➢ After oxidative challenge, HMP activity may increase up to 2,3-BPG to the glycolysis mainstrea
thirtyfold
➢ The HMP catabolizes G6P to ribulose 5-phosphate and IV. RED BLOOD CELL MEMBRANE
carbon dioxide by oxidizing G6P at carbon 1
❖ G6PD
A. Red Blood Cell Membrane Deformability
➢ Provides the only means of generating NADPH for glutathione
reduction, and in its absence erythrocytes are particularly
vulnerable to oxidative damage ❖ Red Blood Cells
➢ Normal G6PD Activity: the HMP detoxifies oxidative ➢ are biconcave and average 90fL in volume
compounds and safeguards hemoglobin, sulfhydryl- ➢ Their average surface area is 140 μm2, a 40% excess of
containing enzymes, and membrane thiols, allowing RBCs to surface area compared with a 90-fL sphere
safely carry O2 ➢ Red Blood Cell Deformability
➢ G6PD Deficiency: the most common inherited RBC enzyme ▪ This excess surface-to-volume ratio enables RBCs to
deficiency worldwide, the ability to detoxify is hampered, stretch undamaged up to 2.5 times their resting diameter
resulting in hereditary non-spherocytic anemia as they pass through narrow capillaries and through
splenic pores 2 μm in diameter
Table 3.0 Glucose Catabolism: Hexose Monophosphate Pathway ➢ The RBC plasma membrane, which is 5 μm thick, is 100
times more elastic than a comparable latex membrane, yet
Substrates Enzyme Product
it has tensile (lateral) strength greater than that of steel
G6P, NADP Glucose-6-phosphate 6-PG, NADPH ➢ The deformable RBC membrane provides the broad surface
dehydrogenase and 6- area and close tissue contact necessary to support the
phosphogluconolactonase delivery of O2 from lungs to body tissue and CO2 from body
6-PG, NADP 6-phosphogluconate R5P, NADPH, CO2 tissue to lungs
dehydrogenase ❖ Red Blood Cell Deformability depends not only on
RBC geometry but also on relative cytoplasmic
(hemoglobin) viscosity
B. Methemoglobin Reductase Pathway ❖ Normal Mean Cell Hemoglobin Concentration (MCHC)
➢ ranges from 32% to 36%, and as MCHC rises, internal
❖ Heme iron is constantly exposed to oxygen and peroxide viscosity rises
❖ Peroxide oxidizes heme iron from the ferrous (+2) to the ferric ➢ MCHCs above 36% compromise deformability and shorten
(+3) state the RBC life span because viscous cells become damaged
❖ Methemoglobin as they stretch to pass through narrow capillaries or splenic
➢ The affected hemoglobin molecule pores
❖ Although the HMP prevents hemoglobin oxidation by reducing ➢ As RBCs age, they lose membrane surface area, while
peroxide, it is not able to reduce methemoglobin once it forms retaining hemoglobin. As the MCHC rises, the RBC, unable to
❖ NADPH is able to do so, but only slowly pass through the splenic pores, is destroyed by splenic
❖ Methemoglobin Reductase (Cytochrome B5 reductase) macrophages
➢ The reduction of methemoglobin by NADPH is rendered more
efficient with its presence

Surell, R. – TRANSCRIBER
[HEMA311] 1.04 Erythrocyte Metabolism, Membrane Structure and Function I Prof. Antonio C. Pascua, RMT, MSMT

B. Red Blood Cell Membrane Lipids ❖ Glycolipids (sugar-bearing lipids)

➢ Make up 5% of the external half of the RBC membrane
❖ Besides geometry and viscosity, membrane elasticity (pliancy) ➢ Associate in clumps or rafts and support carbohydrate side
also contributes to deformability chains that extend into the aqueous plasma to anchor the
❖ The RBC membrane consists of approximately 8% glycocalyx
carbohydrates, 52% proteins, and 40% lipids ➢ May bear copies of carbohydrate-based blood group
❖ The lipid portion, equal parts of cholesterol and phospholipids, antigens, such as antigens of the ABH and the Lewis blood
forms a bilayer universal to all animal cells group systems
❖ Phospholipids form an impenetrable fluid barrier as ❖ Glycocalyx
their hydrophilic polar head groups are arrayed upon the ➢ Is a layer of carbohydrates whose net negative charge
membrane’s surfaces, oriented toward both the aqueous plasma prevents microbial attack and mechanical damage caused by
and the cytoplasm, respectively adhesion to neighboring RBCs or to the endothelium
➢ Reseal rapidly when the membrane is torn
➢ Asymmetrically distributed C. Red Blood Cell Membrane Proteins
❖ Their hydrophobic nonpolar acyl tails arrange themselves to
form a central layer dynamically sequestered (hidden) from the ❖ Proteins make up 52% of the membrane structure by mass
aqueous plasma and cytoplasm ❖ A proteomic study revealed there are at least 300 RBC membrane
❖ The membrane maintains extreme differences in osmotic proteins, including 105 transmembrane proteins
pressure, cation concentrations, and gas concentrations between ❖ Some proteins have a few hundred copies per cell, and others have
external plasma and the cytoplasm through the dynamic interaction more than a million copies per cell
of the lipids and proteins
❖ Cholesterol A. Transmembrane Proteins
➢ Esterified and largely hydrophobic, resides parallel to the acyl
tails of the phospholipids
➢ It is equally distributed between the outer and inner layers, ❖ Serve many functions including:
and evenly dispersed within each layer, approximately one ➢ Transport sites, Adhesion sites, Signaling receptors
cholesterol molecule per phospholipid molecule ❖ Any disruption in transport protein function changes the osmotic
➢ Cholesterol’s β-hydroxyl group tension of the cytoplasm, which leads to a rise in viscosity and loss
▪ Only hydrophilic portion of the molecule, anchors within of deformability
the polar head groups, while the rest of the molecule ❖ Any change affecting adhesion proteins permits RBCs to adhere to
becomes intercalated among and parallel to the acyl one another and to the vessel walls, promoting fragmentation
tails (vesiculation), reducing membrane flexibility and shortening the
▪ Cholesterol confers tensile strength to the lipid bilayer RBC life span
▪ As the cholesterol concentration rises, the membrane ❖ Signaling receptors bind plasma ligands and trigger activation of
gains strength but loses elasticity intracellular signaling proteins, which then initiate various energy-
❖ The ratio of cholesterol to phospholipids remains relatively dependent cellular activities, a process called transduction
constant and balances the need for deformability or elasticity and ❖ Glycosylation
strength ➢ The transmembrane proteins also support surface
❖ Membrane enzymes maintain the cholesterol concentration by carbohydrate, which join with glycolipids to make up the
regularly exchanging membrane and plasma cholesterol protective glycocalyx
❖ Deficiencies in these enzymes are associated with membrane ❖ Most transmembrane proteins assemble into one of two major
abnormalities such as acanthocytosis, as the membrane loses macromolecular complexes named by their respective cytoskeletal
tensile strength anchorages:
❖ Phospholipids ➢ Ankyrin complex
➢ Are asymmetrically distributed ➢ Actin junctional complex (protein 4.1 complex)
➢ Phosphatidylcholine and sphingomyelin: predominate in ❖ The anchoring of these transmembrane complexes to cytoskeletal
the outer layer proteins (adjacent to the inner or cytoplasmic side of the
➢ Phosphatidylserine (PS) and phosphatidylethanolamine: membrane) prevents loss of the lipid bilayer
form most of the inner layer ❖ Transmembrane proteins also provide vertical membrane
➢ Distribution of these four phospholipids is energy dependent, structure
relying on a number of membrane-associated enzymes, *For Table 4.0 Kindly refer to the last page
termed flippases, floppases, and scramblases for their
functions 1. Blood group Antigens
➢ When phospholipid distribution is disrupted, as in sickle cell
anemia and thalassemia or in aging RBCs, PS, the only ❖ Located in membrane macromolecular complexes that serve as
negatively charged phospholipid, redistributes to the outer transporters, structural components, enzymes, receptors and
layer adhesion molecules
➢ Splenic Macrophages possess receptors that bind to the PS ❖ The carbohydrate-defined blood group antigens are supported
displayed on senescent and damaged RBCs nd remove these in the RBC membrane by transmembrane proteins
from circulation ❖ Half of the known transmembrane proteins (approx. 25) are
➢ C-reactive protein and inflammatory conditions increase involved in the macromolecular complexes that define blood
the PS distribution in the outer layer of the RBC membrane antigen groups
leading to increased RBC death (eryptosis) ❖ Band 3 (anion transport) and Glut-1 (glucose transport)
❖ Membrane phospholipids and cholesterol may also redistribute ➢ Support the majority of ABH system carbohydrate
laterally so that the RBC membrane may respond to stresses and determinants
deform within 100milliseconds of being challenged by the presence ❖ Several transmembrane proteins provide peptide epitopes
of a narrow passage, such as capillary ❖ Glycophorin A carries the peptide-defined M and N determinants
❖ As the proportion of cholesterol increases, the RBC becomes ❖ Glycophorin B carries the Ss determinants, which together
more rigid and is unable to deform as readily comprise the MNSs system, whereas the minor glycophorins C and
❖ In liver disease, membrane cholesterol concentration becomes D carry the Gerbich System Antigens
increased because of an increased plasma bile salt concentration. ❖ The Rh system employs two transmembrane lipoproteins and one
As a result, the cell membrane surface area to volume ratio multipass glycoprotein, each of which crosses the membrane 12
increases giving the RBC a target cell appearance times

Surell, R. – TRANSCRIBER
[HEMA311] 1.04 Erythrocyte Metabolism, Membrane Structure and Function I Prof. Antonio C. Pascua, RMT, MSMT
*Refer to Table 4.1 Names and Properties of Cytoskeletal RBC
❖ The expression of the D and CcEe antigens requires the proteins
separately inherited glycoprotein RhAG, which localizes near the
Rh lipoproteins in the ankyrin complex
❖ Loss of the RhAg glycoprotein prevents expression of both the D 1. Spectrin Stabilization
and CcEe antigens (Rh-null) and is associated with RBC
morphologic abnormalities
❖ The secondary structure of both a-spectrin and B-spectrin
features triple-helical repeats of 106 amino acids each
2. The GPI Anchor and Paroxysmal Nocturnal Hemoglobinuria
➢ 20: repeats make up a-spectrin
➢ 16: make up B-spectrin
❖ A few copies of the phospholipid phosphatidylinositol (PI) reside ❖ Single Helix at the amino terminus of a-spectrin consistently binds
in the outer, plasma-side layer of the membrane a pair of helices at the carboxyl terminus of the B-spectrin chain,
❖ PI serves as a base on which a glycan core of sugar molecules it forming a stable triple helix that holds together the ends of the
synthesized, forming the glycosylphosphatidylinositol (GPI) anchor heterodimers
❖ More than 30 proteins bind to the GPI anchor and appear to float ❖ Joining these ends are actin and protein 4.1 in the actin junctional
on the surface of the membrane complex
➢ 2 of these proteins: ❖ Actin forms short filaments of 14 to 16 monomers whose length
▪ Decay-accelerating factor (DAF, or CD55) is regulated by tropomyosin
▪ Membrane inhibitor of reactive lysis (MIRL, or CD59) ❖ Adducin and Tropomodulin cap the ends of actin
▪ It protects the RBC membrane from lysis by ❖ Dematin appears to stabilize the acitn junctional complex and
complement helps maintain the RBC shape
❖ Phosphatidylinositol Glycan Anchor Biosynthesis Class A
(PIGA) 2. Membrane Deformation
➢ Gene codes for a glycosyltransferase required to add N-
acetylglucosamine to the PI base early in the biosynthesis of
❖ Spectrin dimer bonds that appear along the length of the
the GPI anchor on the membrane
molecules disassociate and reassociate (open and close) during
❖ Paroxysmal Nocturnal Hemoglobinuria
RBC deformation
➢ Acquired mutation in the PIGA gene affects the cells’ ability to
❖ 20 a-spectrin and 16 B-spectrin repeated helices unfold and
synthesize the GPI anchor
refold
➢ Without the GPI anchor, the cell membrane becomes
➢ These flexible interactions plus the spectrin protein 4.1
deficient in CD55 and CD59, and the cells are susceptible to
junctions in the actin junctional complexes between the
complement-mediated hemolysis
tetramers are key regulators of membrane elasticity and
mechanical stability
3. Nomenclature
❖ Abnormalities in any of these proteins result in deformation-
indued membrane fragmentation
❖ Numerical naming, for instance, band 3, protein 4.1, and protein ❖ Hereditary elliptocytosis arises from one of several autosomal
4.2, derives from historical (preproteomics) protein identification dominant mutations affecting the spectrin dimer-to-dimer lateral
techniques that distinguished membrane proteins using sodium bonds or the spectrin protein 4.1 junction
dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS- ❖ Horizontal Interaction defects inhibit the membrane’s ability to
PAGE) rebound from deformation
❖ Bands migrate through the gel, with their velocity a property of their ❖ Mild to Severe Hemolytic Anemia
molecular weight and net charge, and are identified using ➢ Ultimately, the RBCs progressively elongate to form visible
Coomassie blue dye elliptocytes
❖ Glycophorins with abundant carbohydrate side chains, are ❖ Hereditary Spherocytosis
stained using periodic acid-Schiff (PAS) dye ➢ Conversely, autosomal dominant mutations that affect the
❖ Band 3 and Protein 4.2 integrity of band 3, ankyrin, protein 4.2, or a or B-spectrin
➢ Major components of the ankyrin complex and link their ➢ In these cases, there are too few vertical anchorages to
associated proteins and the lipid bilayer membrane to the maintain membrane stability
spectrin cytoskeleton through ankyrin ❖ Blebs or Vesicles
➢ Band 3, Protein 4.2, Adducin and Actin are major ➢ Lipid membrane peels off in small fragments and immediately
components of the actin junctional (4.1) complex and link the reseals keeping the cytoplasmic volume intact
lipid membrane to the spectrin cytoskeleton through protein ➢ This results in a reduced surface area-to-volume ratio and the
4.1 formation of spherocytes
▪ 4.1 anchorage also includes the protein dematin, which
links the transmembrane protein Glut-1 D. Osmotic Balance and Permeability

B. Cytoskeletal Proteins ❖ RBC membrane is impermeable to cations Na1, K1 and Ca2-

❖ It is permeable to water and the anions bicarbonate (HCO3-) and
chloride (Cl-), which freely exchange between plasma and RBC
❖ a-spectrin and B-spectrin
cytoplasm
➢ principal cytoskeleton proteins
❖ Aquaporin 1
➢ Assemble to form an antiparallel heterodimer held together
➢ A transmembrane protein that forms pores or channels whose
with a series of lateral bonds
surface charges create inward water flow in response to
➢ Antiparallel means that the carboxyl (COOH) end of one
internal osmotic changes
strand associates with the amino (NH3) end of the other, and
➢ Decrease in aquaporin 1 expression has been linked with
the two heterodimers self-associate head-to-head to form a
hereditary spherocytosis
tetramer
❖ ATP-dependent cation pups Na+, -ATPase and K+- ATPase
❖ Ends of Spectrin Tetrameter
regulate the concentrations of Na+ and K+, maintaining intracellular-
➢ Linked in the actin junctional complex, forming a hexagonal
to-extracellular ratios of 1:2 and 25:1, respectively
cytoskeletal lattice adjacent to the inner (cytoplasmic) lipid
❖ Ca2+ -ATPase expels calcium from the cell, maintaining low
bilayer
intracellular levels of 30 to 60nM compared with 1.8mM in the
➢ Provides lateral or horizontal membrane stability
plasma
➢ Because the cytoskeletal proteins do not penetrate the
bilayer, they are also called peripheral proteins

Surell, R. – TRANSCRIBER
[HEMA311] 1.04 Erythrocyte Metabolism, Membrane Structure and Function I Prof. Antonio C. Pascua, RMT, MSMT

❖ Calmodulin, cytoplasmic Ca2 -binding protein, controls the

function of Ca2 -ATPase. These pumps, in addition to aquaporin,
maintain osmotic balance in the RBC
❖ The cation pumps consume a significant portion of RBC ATP
production
❖ ATP loss or pump damage permits Ca2 and Na influx, with water
following osmotically
❖ Colloid Osmotic Hemolysis
➢ The cell swells, becomes spheroid, and eventually ruptures
❖ Defects in the ion channels result in the red cell volume disorders,
such as overhydrated stomatocytosis (hereditary hydrocytosis)
and dehydrated stomatocytosis (hereditary xerocytosis)
❖ Sickle Cell Disease
➢ Also provides an example of increased cation permeability
➢ When hemoglobin S polymerizes on deoxygenation, the cell
deforms into a sickle shape and the membrane becomes
more permeable to Ca2+
➢ This causes a downstream effect of increased Na+ and K+,
resulting in hemolysis

Surell, R. – TRANSCRIBER
Table 4.0 Names and Properties of Selected Transmembrane RBC Proteins
Transmembrane Gene Band MW (kD) Copies/Cell (x103) % of TP Function
Protein
Aquaporin 1 AQP1 28 120-160 Water transporter, Colton antigen
Band 3 (anion SLC4A1 3 90-102 1200 27% Anion transporter, location of ABH
exchanger, AE1) antigens
Ca2+ - ATPase ATP2B1 110 Ca2+ transporter
Duffy FY, DARC, 35-43 6-13 G protein-coupled receptor,
ACKR1 chemokine receptor, Duffy antigens,
receptor for malarial parasites
Glut-1 SLC2A1 4.5 45-75 200-700 5% Glucose transporter, location of ABH
blood group antigens
Glycophorin A GYPA PAS-1 36 1000 85% of GP Sialic acid transporter, location of MN
blood group antigens
Glycophorin B GYPB PAS-4 20 250 10% of GP Sialic acid transporter, location of Ss
blood group antigens
Glycophorin C GYPC PAS-2 14-32 135 4% of GP Sialic acid transporter, location of
Gerbich system antigens
ICAM-4 ICAM4 Integrin adhesion
K+ -Cl- cotransporter Transports K+, Cl-
Kell KEL 93 3-18 Zn2+ -binding endopeptidase, Kell
antigens
Kidd SLC14A1 43 14 Urea transporter, Kidd (Jk) antigens
Na+, K+, -ATPase ATP1A2 140 0.5 Na+, K- transporter
Na+, K+ -2Cl- Na+, K-, Cl- transporter
cotransporter
Rh RHCE, RHD 30-45 200 D an CcEe antigens; stabilizes band
3 and Rh macrocomplexes
RhAG RHAG 45-100 100-200 D and CcEe antigen component; CO2
cation, nd ammonium transporter

Table 4.1 Names and Properties of Selected Cytoskeletal RBC Proteins

Skeletal Protein Gene Band MW (kD) Copies/Cell (x103) % of TP Function
a-spectrin SPTA 1 1 240-280 242 16% Filamentous antiparallel heterodimer, primary
B-spectrin SPTB 2 220-246 242 14% cytoskeletal proteins

Adducin ADD1 2.9 80-103 60 4% Caps actin filament, binds Ca2+/ calmodulin
ADD2
Ankyrin ANK1 2.1 206-210 124 4.5% Anchors band 3, protein 4.2 and other proteins
in the ankrin complex to spectrin
Dematin EPB49 4.9 43-52 140 1% Actin bundling protein
B-actin ACTB 5 42-43 500 5.5% Binds B-spectrin
G3PD GAPD 6 35-37 500 3.5% Carbohydrate metabolism, phosphorylates G3P
Protein 4.1 EPB41 4.1 66-80 200 5% Anchors the actin junctional complex to spectrin
tetrameters, RCB cytoskeleton shape
Protein 4.2 EPB42 4.2 72-77 250 5% Part of ankyrin complex, ATP binding protein
(protein kinase)
Tropomodulin TM0D1 5 41-43 30 - Caps actin filament
Tropomyosin TPM3 7 27-38 70 1% Stabilizes and regulates actin polymerization
 HEMATOLOGY 1  Hb is slighlty soluble in water = 3mL O2/L of blood
 0.08 mmol of O2/ L of blood
MIDTERM LECTURE WEEK 7
 16 g/dL Hb = 2.41 mmol of O2
 8 g/dL Hb = 1.22 mmol of O2
Hemoglobin
Hemoglobin Functions
Topic Outline
 Oxygen Transport
 Hemoglobin
 Carbon Dioxide Transport
- Components
 Nitric Oxide Transport
- Role of Iron
- Hemoglobin Variants
Components:
- Oxyhemoglobin Dissociation Curve
1. Globin
- Hemoglobin Derivatives
2. Protoporphyrin IX
3. Iron
 For red cells to be functional and to be able to
**combination of protoporphyrin IX and Iron you will
transport and carry oxygen, inside it should contain
form the molecule called heme
hemoglobin
 Through enzymatic reaction you will form this
 Each hemoglobin molecule is a mixture of heme and
heme, and through the bonds and connections you
globin and it should always come in pairs
will form your hemoglobin
 1 heme can carry 1 mole of Oxygen
 Additional extra component necessary to make your
 1 Hemoglobin = 4 heme = 4 moles of O2
hemoglobin able to transport oxygen to your tissue
 Heme is the one that makes the red cells red
is 2,3DPG which facilitates the affinity of the oxygen
 It’s a globular protein which weighs around 68,000
to the hemoglobin
Daltons and it almost occupy 1/3 of the red cells
weight
 As to the numbers, each red cell contains millions of
Globin
hemoglobin (roughly 640M of Hgb)
• Varied sequence of amino acids
 For every red cell that you have, it will contain a lot
• Difference in globin chains designation relates both
of hemoglobin that the carries a lot of moles of
to the sequence and number of amino acids
oxygen
 Hemoglobin it’s not just about the delivery of
 Globin makes your hemoglobin a protein
oxygen, it has also an ability to transport waste
 Globin determines the type of hemoglobin you have
products, particularly carbon dioxide (CO2) because
 So for you to know if it is gower I, gower II, or is it
CO2 adds up to the acidity of our body
Portland hgb what we should check is the globin
 If we have red cell, it has hgb inside which can carry
component of your hemoglobin, by doing so we can
oxygen. And once this hgb releases out the oxygen
the identify which is which
going into your tissues later on hgb will then carry
 Globin is a protein and is made up of amino acids
carbon dioxide so it can be excreted out of the body
 Production of protein, it will all start from DNA it
 If your red cells already delivered oxygen into the
will be later on transcribed to become mRNA and
tissues, in turn your hgb may carry carbon dioxide
then later on it will be translated to become amino
that will transported back to the lungs so you can
acid and through peptide bond later on you form
then eliminate it or excreted out.
proteins
 In 1862, Felix Seyler is the one that identified
 Remember, only immature cells are capable of
respiratory protein called hemoglobin. He was the
producing hemoglobin, because only immature red
one who discovered the colored characteristics
cell do contain that nucleus that will then contain
spectrum of hemoglobin and proved that this was
your DNA
the true coloring matter of the blood.
 Mature RBC are not capable of synthesizing
 What makes the blood red particularly according to
hemoglobin, only the immature forms particularly
Felix Seyler is because of hemoglobin
those cells that contains nucleus
 Reticulocytes are cells without the nucleus but still
Adults at rest needs around 250mL of O2/ min.
capable of synthesizing hemoglobin

-CACL
 So, within this globins, there are two important
chromosomes necessary for the generation or
production of these globins. We have chromosome
no. 16 and chromosome no. 11
 Ch. 16 and Ch. 11 = these are the two important
chromosomes so we can generate the following
hemoglobin
 Chromosome no. 16 is the one who is responsible
for the synthesis or generating alpha globins, zeta
globins
 Chromosome no. 11 is the one who synthesized or
generate the beta, delta, epsilon, gamma
 The specific sequence of these amino acids is
known and is important in the identification of
abnormal hemoglobins involving substitution of
specific amino acids
 Let’s say I’m on the beta globin, on beta globin I
have 146 amino acids, remember they are  Upper figure is representing your beta globins
organized, they are arranged specifically produced by chromosome no. 11
 So for every amino acid here, they have their own  The 2 gamma globin genes here are important
specific location. So the moment you change a during the fetal growth because this will give you
certain amino acid on a certain position, or a HgF
certain place, it will then create changes on the  Lower figure, the alpha globins are important for
structure of hemoglobin making them susceptible adults
to lysis  Remember, after birth that’s the only time we can
 Example: HbS found on beta globins or 6th position then have an increased production of the beta
which is the glutamic acid. If you replace the globins. That’s why after birth the HgA1 will
glutamic acid with valine increase
 Because of that replacement it makes your
hemoglobin and your entire red cell susceptible to Globin Chain Structure
lysis  Primary Structure
 These varied sequence of amino acid should always - Specified sequence of amino acid residues
be organized and you should not change any of this  It’s the specific sequence of amino acid
amino acid or else it will then make your red cell  Together they form a certain type of globin
susceptible to lysis. chain
 For every hgb molecule you have 4 units of heme  This tells the identity of your globin, then it
and 4 units of globin tells you the identity of your hemoglobin
 Among adults, the most abundant Hgb is HgA1
which has 2a and 2b globin. SO if you will do the  Secondary Structure
math - Dividing the chain into 8 separates helical
segments (A to H)
 Makes your hgb structure basically flexible
 Because our hemoglobin should be always
flexible so that it can accept oxygen and at the
same time it can release out oxygen
 So when the individual heme groups unload
oxygen in the tissues, the beta chains are pulled
apart
 If there’s an oxygen will enter the hemoglobin
the beta chains will move closer to one another

-CACL
  If your red cells unload oxygen the beta chains Heme Production
will move apart, so that 2,3DPG can now enter • Requires the formation of protoporphyrin IX and
your molecule the availability of iron.
 2,3DPG it will prevent the affinity of oxygen • The synthesis of heme begins in the mitochondria
with hemoglobin with the formation of D-ALA from glycine and
 Lets say your tissue is hypoxic/ hypoxia then it’s succinyl coenzyme A.
a must that your hemoglobin will the let go of  Remember heme synthesis occurs in most body
oxygen or your oxygen should not bind more cells except for mature RBC
with your hemoglobin and it should be  Of all the body tissues, it is then your bone marrow
delivered into tissues particularly the red bone marrow and the liver
 So when beta chain will move apart, that’s the which are then considered as the most
time your 2,3DPG can now enter your hgb predominant heme producers particularly referring
molecule, so that oxygen will not easily bind to porphyrin
with hemoglobin so that it will be delivered to  Heme is a combination of protoporphyrin IX and
tissues iron
 What if tissues is already oxygenated, that’s the  What we are actually producing is porphyrin and
time the beta chains will move closer to one what we source our from our diet and from the
another, so that 2,3DPG cannot enter which damaged or lysed red cells is iron
result for the more oxygen to bind on to your  The synthesis of heme begins at your mithochodria
hemoglobin. in the formation of D-ALA
 Within mithochodria, it will then allow the mixture
of glycine and the succinyl conenzyme A. And
because of the different enzymatic activity later on
you will form D-ALA and then later on form your
Protophophyrin IX
 The rate of heme formation depends on the rate of
the synthesis of D-ALA
 Meaning, the more D-ALA you form the more you
can synthesized heme.

 If hemoglobin is denatures, it will then loose its

ability to carry oxygen  Succinyl CoA + glycine = D-ALA
 because of the oxidative stress hemoglobin maybe  You will form D-ALA with the help of ALA synthetase
denatured and Vitamin B6 (vitamin B12 in red cell is for dna
 Its important that we can prevent oxidative synthesis so that later on you can generate globin)
mechanism or oxidative stress so that red cells can  The synthesis of heme is a complex process that will
continuously carry and transport oxygen involve different enzyme that are needed so you
-CACL
 can then start producing each of the following  So iron uptake and release from the body should be
substances here always carefully controlled
 Once D-ALA is formed it will undergo other  And iron uptake is precisely controlled so we can
enzymatic activities. then maintain iron balance because we cannot
 ALA-dehydrase = Porphobilinogen afford to have less iron and at the same time we
 Uroporphyrinogen decarboxylase = cannot afford to have extra iron and excessive
uroporphyrinogen amount of iron in our body
 Coprophyrinogen oxidase = coprophyrinogen  One of the most important source of iron will be
 Protophyrinogen oxidase = protophyrinogen always our diet (green leafy vegetables particularly
 Later on protoporphyrin IX + Iron (with the help of malunggay)
heme synthetase you will form heme  You also source iron from all those damaged or
 Remember, iron should be always on its ferrous lysed RBCs that you had
state, to maintain the ferrous state we will use an  The iron that will come out form red cell
enzyme called ferrochelatase destruction will then be recycled so even if you
 That will make sure once protoporphyrin IX and iron lysed your red cell, those iron that comes out will
binds with the help of heme synthetase you can then be used again or recycled
form a heme that’s capable of carrying and  When it comes to absorption especially from the
transporting oxygen. food we eat, iron may be stored as ferritin in the
 If iron is on its oxidized state or the ferric form, enterocytes or exported into the circulation by an
oxygen cannot bind iron transport protein called ferroportin
 Ferroportin is very important as the last step in the
Protoporphyrin IX instestinal iron absorption so for you to be able
• Nitorgenous substance synthesized partly in absorb that iron you need ferroportin and later on it
mitochondria and partly in the cytoplasm of a will allow macrophages to recycle iron by moving it
nucleated red cell out from the macrophages in your liver, spleen and
 Again this is what you produce from your cells, to even your bone marrow
form that heme from you liver and to your bone  For the iron to be recycled ferroportin will help to
marrow what will you produce is porphyrin move it out from liver, spleen or BM
particularly protoporphyrin IX  So once its in the circulation again you can then
reuse it or recycle it
The Role of Iron in Heme Synthesis

 An important hormone is necessary when it comes

to the activity of the ferroportin which is called
hepsidin
 Hepsidin is a liver produce hormone which is said
 Iron is the most abundant transition metal in the to be the master regulatory hormone of systemic
body iron metabolism
 And majority of the iron remember are present  Hepsidin controls your plasma iron concentration
inside the red cells by inhibiting the activity of ferroportin
 And majority of your iron is within your rbcs
-CACL
 If you have hepsidin you will inhibit iron to got to Hemoglobin Structure
circulation • Primary: specified sequence of amino acid residues
 Deficiency in hepsidin, the ferroportin will then just • Secondary: dividing the chain into 8 separate helical
continuously export out iron into circulation. If segments (A to H)
that’s the case there will be iron overload • Tertiary: arrangement of the helices into a pretzel-
 Increased hepsidin, you will lose ferroportin, if like configuration
that’s the case you will have the decrease release of • Quarternary: describes the complete hemoglobin
iron into circulation molecule
 If iron becomes decreased in the circulation, or
decreased in the production of heme you then end
up with anemia
 Hepsidin will help you regulate the levels of iron in
our circulation and the levels of iron in our plasma.
 The liver and the reticuloendothelial macrophages
function as your major iron stores
 Around 1-2mg of iron is absorb and at the same
time lost everyday
 The total amount of iron in the body can be
regulated by absorption only whereas iron loss
occurs only possibly when you bleed
Normal Hemoglobin variants
• Embryonic (3 months)
IRON – Gower I
• In the ferrous form is required to convert – Gower II
protoporphyrin to become heme. – Portland
• Locations: could be present in red cells, liver, bone • HbF (most abundant in fetal life)
marrow stored as ferritin, macrophages • Hb A1(most abundant in adults)
• Sources: • Hb A2
• Transport: Transport iron with the help of protein
called transferrin, ferroportin,
• Immature red cell with ferritin: Immature cell Normal Hemoglobin Forms
contain iron deposit inside we call it as sideroblast A. Oxyhemoglobin: hgb with ferrous iron and oxygen
• Mature red cell: mature cell containing iron inside it which is seen in arterial circulation
is called siderocyte  It contains oxygen
• Stain with: if you want to see granules or inclusions
inside the cells are really iron deposist then you B. Deoxyhemoglobin: hemoglobin with ferrous iron
stain your sample using Prussian blue or Perl’s stain but no oxygen which is seen in venouscirculation
 no oxygen
 Has 2,3DPG which prevent the affinity of
oxygen to hemoglobin

• The connection of heme and globin through

chemical bonds forms the basis of hemoglobin
molecule
 65% of hemoglobin synthesis occurs among
nucleated red cells particularly on your
polychromatophilic normoblast and ortochromic
normoblast
 35% will be on your reticulocyte

-CACL
Regulation of Hemoglobin Production 4 factors tha affect the affinity of Oxygen
• Heme Regulation  pH (Bohr effect)
– HEME: inhibits transcription of the ALA  LEFT: if pH (alkaline) is 
synthase gene, ALA dehydrase and PBG  RIGHT: if pH (acidic) is 
deaminase  CO2 (Haldane effect facilitates CO2 binding in your
• Globin Regulation tissues)
– controlled at the transcription level - (adds up to the acidity of our body)
– KLF1, GATA1, Ikaros, TAL1, p45-NF-E2, and LDB1 - CO2 diffuses to red cells so that hgb can act as a
– Locus Control Region buffer
**Heme  LEFT: 
 RIGHT: 
Oxyhemoglobin Dissociation Curve **If hgb carries oxygen, it cannot carry CO it cannot
• Hemoglobin has the ability to bind large quantities carry at the same time
of O2, however, hemoglobin must be willing to ** do if you pH is , it means to say that your CO2
release O2 when needed. levels are low, so if you have less CO2 in blood it means
• Shift to the left: oxygen binds more with to say that what my red cells are carying is O2 so that’s
hemoglobin. High affinity of O2 w. hgb why it if shift to the left (oxygen binds more w/ hgb)
• Shift to the right: oxygen binds less with **if my pH is , it means to say that the CO2 is high
hemoglobin. There’s low affinity of O2 w/ hgb (adds to acidity if high), so the more acid I have the
 Both of them are equally important because more my pH is low. If CO2 is too much in my body, it is
when we say shift to the left we are conserving then the responsibility of hgb to balance out pH by
oxygen or bind more with hgb. It implicates that carrying CO2. So if red cells carries now CO2 it means to
your tissues are already oxygenated. say that, oxygen will not easily bind to your RBCs and
 If shifts to the right it means O2 has low affinity because of that lower affinity, it makes your curve shifts
to hgb and it should be important whenever to the right
your tissues are hypoxic/hapoxia then it’s a
must that oxygen is delivered on to your  2,3DPG
tissues. So is you need to deliver O2 to your  LEFT: 
tissues, your Hemoglobin should let go of ** the less 2,3DPG you have the more your O2 will bind
oxygen. to hgb
 So if there is hypoxia, what you need is shift to
the right  RIGHT: 
 If the tissues are already oxygenated, what I **the more 2,3DPG you have, the more your oxygen
need is shift to the left. will not bind to you hgb

 Temperature
 LEFT: 
**if the temperature is low, the more your oxygen
binds with hgb
 RIGHT: 
**the more the temp. is high the more it will allow O2
to be release to your tissues.
**during fever, namumula because you’re constantly
transporting oxygen to your tissues.
 Normal oxygen dissociation curve of an adult blood
 The oxygen tension at 50% oxygen saturation is *** In HgF – causes shift to the left because it has a high
approximately 27 torr affinity with oxygen because during pregnancy, the
 As the curve shifts to the right, the oxygen affinity more your HgF binds with O2 it then increases placental
of the hemoglobin decreases and the other way oxygen
around
 If the curve shifts to the left, the oxygen affinity
with hemoglobin increases
-CACL
 Hemoglobin Derivatives  As to studies, sulfhemoglobin may be form
because of cyanosis, constipation, or
Carboxyhemoglobin Clostridium perfringens infection, or oxidative
• Carbon monoxide will bind with oxygen stress that produce “Heinz bodies”
• Cannot bind and carry oxygen
• Gasoline motors, gas heaters, defective stoves, References
smoking • Clinical Hematology: Theory and Procedures, 5th
 Hgb binds more with carbon monoxide as Edition, Turgeon, M.L.
compared with oxygen, because it is lighter as • Rodak’s Hematology: Clinical Principles and
compared with O2 Applications, 6th Edition
 200times more hgb will bind to carbon
monoxide
 Usually people with carbon monoxide
poisoning, usually will have a cherry red
discoloration of the blood
 If you are smoking, continuous inhalation of
gasoline motor, pollution
 This is reversible, can still go back o normal hgb
form

Methemoglobin
• Derivative of hemoglobin in which the ferrous ions
is oxidized to ferric state
• Chocolate brown discoloration of blood, cyanosis
and functional anemia
 Reversible
 Your iron is oxidized to its ferric state
 Out methemoglobin pathway makes sure our
iron stay at its ferrous state
 If a lot of methemoglobin in blood it can cause
chocolate brown discoloration
 hbM maybe be responsible for
methemoglobinemia at birth

Sulfhemoglobin
• Mixture of oxidized, partially denatured forms of
hemoglobin that form during oxidative hemolysis
• Associated with sulfonamides administration,
severe constipation, Clostridium perfringens,
enterogenous cyanosis
• Cannot be reduced back to hemoglobin
 During the oxidation of hemoglobin, sulfur is
incorporated into the heme rings of hgb
resulting to a green hemechrome
 In blood, the color is mauve lavender
 With sulfhemoglobin it cannot transport O2 but
it may combined with carbon monoxide =
carboxysulfhemoglobin
 Irreversible, meaning you cannot reduce it back.
So if you want to get rid of this, you need to
destroy your RBCs

-CACL
  Red cells should be normal in structure, normal in
HEMATOLOGY 1 morphology together with hgb so they can survive
MIDTERM LECTURE WEEK 8 in the circulation and bale to transport your oxygen.
 There are times there are problem in morphology
RED BLOOD CELL ABNORMALITIES or physical appearance of your RBC.
 So when we say morphologic abnormalities you are
dealing with the physical features of RBC  NORMAL RED BLOOD CELL:
 Diameter: roughly 6-8 um
RED BLOOD CELLS  Pallor area at the center of RBC - 1/3
 Non-nucleated, biconcave disc-like cell (biconcave) – to increase the surface area of
 6-8 microns in diameter RBC.
 N/N  Extra surface area or compartment. So when
 Central area of pallor hypotonic fluid enters your RBC, at first it can
prevent lysis because you have extra space.
 Red cells is important for the delivery of oxygen  If more and more hypotonic fluid enters RBC,
because they have protein called hemoglobin which definitely there’s a breaking point that RBC will
can bind and carry and transport oxygen. be destroyed.
 What makes our red cell functional is because of  The rest is red color because of hemoglobin.
hgb
 This size, this color, shape of RBC sometimes maybe
 Red cells also need other components for them to then be abnormal. Instead of 6-8um diameter,
be able to survive in our peripheral blood. If you sometimes it is smaller or larger and the pallor area
want your red cell to survive, aside from having hgb sometimes may be then larger or smaller than 1/3
 Your red cell should have:
1. Intact red cell membrane  If you want your RBC to have this normal
**Components of membrane: Proteins, lipids appearance, in size, shape, appearance. You need
and carbohydrates to provide RBC essentials minerals and vitamins
 If you want your red cell to be intact and survive needed for their survival, especially during their
in circulation then you will need to have those maturation period.
three
2. Red cells need energy, and the energy obtained  Vitamin B12, vitamin B6, folic acid, iron these will
would be derived from your carbohydrate, for make your RBC intact and be able to transport
the glucose that will then enter your RBC, it will oxygen.
then be broken down through glycolysis and
emden-meyer pathway you can then come up Nutritional Requirements:
with ATP, giving your cells energy  CHON and amino acids
3. Red cell needs protection against oxidative  Vit B12, folic acid, Vit. B6
stress, against those chemicals or oxidants that  Fe++
can denatured and harm your RBCs. The  Riboflavin, panthotenic acid, nicotinic acid
pathway that protects them from oxidative
stress mechanism is the hexose Variations of RBCs
monophosphate shunt or pentose phosphate  Size: Anisocyte
pathway. - Normocyte
 If you want your red cell to be fully functional in - Microcyte
transporting our oxygen aside from hemoglobin, - Macrocyte
other chemicals and components should also be  Hb content: anisochromia
there to make sure that your red cell can survive  Shape: Poikilocyte
within your peripheral circulation.

 If red cells are easily lysed, do not survive in our

peripheral blood circulation expect that delivery of
oxygen will be also affected.

-CACL
 ANISOCYTE (SIZE) ** The red cell size is normal and has enough
 Normal diameter: 6-8um hemoglobin, but there are conditions that looks are
- We measure your cells volume to know if the normal but the no. is low resulting to anemia.
size is normal
- In CBC parameter, what we measure is the MCV  MICROCYTIC
- The volume of the RBC is directly reflects the  Red cells are smaller than the usual size
size of your RBC  RBC is normal in size because they have enough
- expected MCV of a normal RBC is 80-100fL hemoglobin
- anything in between is called Normocytic  In this case, your red cell is smaller than the usual
- < 80fl = microcytic size because your hemoglobin concentration is low
- >100fl = macrocytic or is not enough or insufficient.
**You associate for microcytic RBC to those conditions
**ANISOCYTE: term use to denote variation in RBC size or conditions that results with less hgb production

 Normocytes:  Conditions: TICS

 sometimes normal in size, meaning the MCV is A. Thalassemia
normal but then there are disorders experience by - Problem is globin
your patient - In normal hgb there are 4 globin
 “looks can be deceiving” - In thalassemia, the disease is present if
 can be normal looking RBC but deep inside there’s there’s a deficiency with your globin chains
on going pathologic disease (less than 4)
 Because as you produce, as you undergo - So if you don’t have enough globin, you will
erythropoiesis, you still have essentials mineral and not have enough hgb
vitamins and enough hemoglobin that’s why the B. Iron Deficiency Anemia (IDA)
size of your RBC is normal or it looks normal. - Lack iron,
 “Quality over Quantity” = meaning your red cells - Iron is necessary in heme
have enough hemoglobin but the no. of circulating - Less iron = less heme = less or decrease hgb
or producing RBC is very low.
 Normal size RBC but the no. of circulating red cell C. Anemia of chronic disease & Sideroblasctic
is low anemia
- You have iron but you cannot use iron for
**We can associate normocytic RBC in conditions AHA hgb synthesis, so you still ended up with
anemia because you don’t create enough
 Conditions: AHA (The no, of circulating Red cells is hgb
low) ** all of this condition will result to a smaller size of
A. Aplastic Anemia RBC
 There is aplasia, there is absence of hematopoietic ** MCV values will be less than 80fL
cells
 If you still produce some, still the looks or  MACROCYTIC
appearance of RBC is still normal  Your red cell is larger than the expected one
 In aplastic anemia your marrow cannot produce  In erythropoiesis, the development of red cell they
those cells so it ended up with the transportation of become smaller as they mature
oxygen resulting to anemia  In here, you will encounter such abnormality if your
B. Hemolytic Anemia red cell did not mature properly.
 Lysing or destroying your RBC could be external  If you fail to give them vitamin B12 or folic acid.
factors or because of antibodies  The mature size of RBC is same size of your
 Still the red cell is normal in appearance immature RBC
C. Bleeding (Acute of Chronic)  You fail to have enough vitamins necessary for
 Loosing blood or red cells, you end up anemia, less growth, in RBC their immature forms are large and
delivered oxygen they become smaller when they mature. So since
 Still RBC is normal appearance. your red cell did not mature properly, expect the

-CACL
 size they will assume will be the size of your  A little concentration of hypotonic will easily result
immature forms, meaning the RBC is larger in size. to lysis of RBC
 It does not mean it has excess hemoglobin, they  Hyperchromic RBC, it does not mean you have
are large because there is improper/incomplete excess hemoglobin, it’s more of a description that
maturation among RBC your RBC do not have pallor area anymore.
 Conditions:  Low surface area to volume ratio
A. Liver problems  Because hgb will not exceed 32-36g/dL
- Since your liver provides you a lot of
minerals and a lot of vitamins needed for  POLYCHROMASIA
erythropoiesis  Many color
B. Megaloblastic state  Polychromatic RBC in other terms it actually refers
- Cells in bone marrow did not mature to reticulocytes
properly ***If your RBC is polychromatic it is reticulocytes
- Improper maturation of hematopoietic cells and it is normally present in blood
*** if too much polychromatic cells in blood,
ANISOCHROMIA (COLOR) meaning there’s is an overproduction/
 Anisochromia: color of RBC uncontrolled of cells from your bone marrow
 That color reflect the hgb content of your RBC
 In your CBC parameter, to check the concentration VARIATION IN RED CELL COLOR
of your hemoglobin what you have is MCH/ MCHC 1. Polychromasia
 MCH- is just a mere measurement of your hgb - variation in hemoglobin content showing a
weight slight blue tinge (wright stain), gray-blue and
 MCHC – measure the actual concentration of hgb larger than normal; residual RNA
2. Hypochromasia
 NORMOCHROMIC: normal in color - larger than normal central area of pallor - IDA,
 Normal hemoglobin inside thalassemia, anemia of chronic disease,
 Same condition with Normocytic RBC = AHA sideroblastic anemia, myelodysplastic anemia
 If you classify anemia: you pair the size and color of
rbc (Normo/Normo) Hypochromasia Grading
 1/3 pallor area

 HYPOCHROMIC
**denote the color of RBC (not denoting the color of
pallor area)

 > 1/3 pallor area: Hypochromic (less color RBC or

pale color looking, leaving less concentration of hgb
in RBC)
 Microcytic – you don’t have enough hgb
 So the disease associated with Hypochromic RBC Polychromasia Grading
still same with microcytic = TICS
- Thalassemia
- Iron deficiency Anemia
- Anemai of Chronic Disease
- Sideroblastic Anemia

 HYPERCHROMIC
 < 1/3 or totally none pallor area: Hyperchromic
(Sterocytes- no palor area, which means it is
susceptible to lysis)
 It became susceptible to lysis because you don’t
have extra surface area
-CACL
Hemoglobin content Poikilocytes secondary to Membrane Abnormalities
 Hyperchromic
 -central pallor: 2. Acanthocytes
 condition

VARIATION IN RED CELL SHAPE

 Poikilocytes secondary to Developmental
Macrocytosis
 Poikilocytes secondary to Membrane Abormalities
 Poikilocytes secondary to Trauma
 Poikilocytes secondary to Abnormal Hb Content

 There are instances that you red cell vary in shape  Irregular spikes at the membrane of your RBC
because of  3-12 rbc irregular spikes
1. Abnormal development or maturation  Because of such appearance RBC sometime referred
2. Abnormality or deficiency or excess to as spur and thorn cells
concentration of the components of your cell  The problem in acanthocytes, is you have an
membrane (carbohydrates, lipids, protein) abnormal ratio of the different lipids in your cells
3. If they are exposed to injuries or trauma that membrane
results to the alterations in the shape of RBC  Specifically, abnormal ratio of lecithin and
4. Certain abnormal forms or types of hgb sphingomyelin on the cell membrane of your RBC
that results to the formation of those spikes
 No pallor area anymore making them susceptible to
1. Oval Macrocytes (OVALOCYTES) lysis
 Markedly increased MCV  Associate to conditions like cirrhosis, alcoholism,
 Megaloblastic erythropoiesis hemolytic anemia, malabsroption condition,
 No central area of pallor hepatitis, any problem in the liver which results to
 Megaloblastic anemia acanthocytes.
 Abetalipo proteinemia- LDL (bad cholesterol) –
 abnormal development of cells transports cholesterol from liver going to your
 Red cell is oval in shape tissues
 Elongated, the diameter is broad **its not bad but it is bad if it has excess
 Report as oval macrocyte concentration cholesterol
 Macrocyte- red cells did not mature properly  Insufficient delivery of cholesterol into tissues
 Ovalocyte forms also when RBC did not mature especially needed for cell production/ maturation,
properly expect that there will be imbalances in your cell
 There is nuclear maturation defect in immature membrane
cells as you undergo hematopoiesis  In acantocytes, it results to abnormal ratio of the
 As to its components, because of this nuclear lipids particularly the lecithin and sphingomyelin
maturation defect, there will less concentration
of cholesterol in cell membrane resulting to
elongation of red cell membrane
 Associate this ovalocyte in cases of
Megaloblastic, liver disorders, alcoholism, and
deficiency in vitamin b12 or in folic acid.
*** Some literature, ovalocyte is a bit thinner
you call it pencil cell or oat cell
 Still MCV value is > 100fL

-CACL
3. Echinocytes 5. Spherocyte

 You have spikes but shorter than acanthocytes and

evenly distributed all over your cell.
 Entire cell have those short projections or spikes
 You form this echinocytes also called as crenated
cells, or sea urchin cells
 We associate this in cases of exposure of RBC to
hypertonic solution, decrease or less energy or less
ATP concentration in RBC probably because of the
changes in the osmotic environment of RBC
 Sometimes you form this echinocytes if your air dry
your blood smear in front of electric fan or in front
of the aircon
 If you see a lot of crenated RBC or echinocytes and
the patient happened to have a kidney problem/
renal disease this can be called as Burr cells
(associated with kidney disease)

4. Codocytes
 Red cell do not have pallor area which makes your
rbc susceptible to lysis
 It can see in cases of herridetary spherocytosis,
Autoimmune hemolytic anemia, or in blood banking
you form spherocytes if the blood bag was already
stored for too long in laboratory
 With blood bag they contain chemicals that will
then support the survival of RBC, if that blood bag
was stored for too long sometimes those chemicals
may be not enough and it will then change the
environment of the entire blood that will affect the
 RBC: you have peripheral rim of hgb, and followed integrity of cell membrane resulting to the presence
by the clear space, and central hemoglobinized area of spherocytes.
 Not equal distribution of RBC  Increased temp. about 45°C
 In this case, there is an excess cholesterol and  Deficiency of cytoskeletal protein (spectrin)
excess phospholipids in red cell membrane, it  If there will be certain antibodies that binds on your
resulted to imbalances in the distribution of RBC (because it has certain antigens)
hemoglobin in your RBC
 In books, you may also called it as target cells/
Mexican hat cell (top view codocytes)
 Encounter such abnormalities in such conditions
like liver disease, thalassemia(most common),
hemoglobinopathy, iron deficiency anemia
-CACL
6. Stomatocyte Poikilocytes secondary to Trauma

8. Schistocyte

 You also call it as mouth cell, or lips

 You form this abnormality because of osmotic
changes in your rbc, so there will be some cationic
imbalances
 Potassium should be inside and Sodium should be  Greek term Schistos means split
outside or RBC  **within the vessel your red cell will circulate, we
 Because of the osmotic changes you have within makes sure that that blood vessel are intact and
your RBC environment there will be cationic smooth and should also repel from your RBCs. Our
imbalances so your sodium will easily enter your blood vessel is negatively charged, and the RBC will
cell. It has increased permeability of sodium into not bind to blood vessel because they are both
your red cell membrane. negatively charged.
 You see this such conditions with liver problems,  Normally, if you don’t have problem with your
alcoholism blood vessels, it is smooth and intact, red cells can
 The most prominent one associated with easily circulate.
stomatocytosis are for those people classified to be  If your blood vessel has clot, injuries, or prosthetic
Rh null heart valves, then as red cells pass on through
 Example you are blood type A (+), in your red cell these clot, injuries, valves, or unwanted material
membrane you have A antigens and then you have on your vessel, expect that the rbc will be
their Rh antigens that’s why you are (+). fragmented or damaged.
 In Rh null (Rh negative), it simply means you don’t  Schistocytes are formed whenever passed on
express Rh antigens on your RBCs. Among people through those unwanted material or components
who Rh null expect presence of stomatocytes. within your blood vessel.
 You can encounter schistocytes in cases of:
7. Elliptocyte  DIC (disseminated intravascular coagulopathy)-
you keep on forming clots even if you don’t
need them
 MAHA (microangiopathic hemolytic anemia)
** angio refers to blood vessels
** the cause of hemolytic anemia here or the
lysis or destruction of RBC because of those
unwanted material in your blood vessels or
altered structure of blood vessels.

 Elongated, rod shape or cigar shape

 Elongated but the diameter is narrow
 You form this because a problem with peripheral
protein band 4.1, so there is a deficiency in a
protein band 4.1 resulting to the elongation of
your red cell membrane.
 Usually you see this in hereditary ellitocytosis,
Thalassemia, or iron deficiency anemia
-CACL
9. Dacryocyte
OTHER POIKILOCYTES

11. Blister Cell

 Or sometimes you call it dacrocyte

 You can also called it as tear drop cell because of tis
appearance
 When red cell squeeze and pass through your  Helmet cells appearance
spleen they will then be fragmented and if the red  Actually this is a precursors of schistocyte
cell is lucky enough to leave the spleen after  Before it become schistocytes (red cell are split),
squeezing and after fragmentation, they will look before it totally split, it is looks like this blister cells.
like this.
 Expect that you form this tear drop cell if your 12. Degmacyte (bite cell)
spleen is enlarge, thalassemia or
 Myeoloid dysplasia – capable of producing cells,
but the ones you are producing are abnormal cells
which will then be remove by spleen and if the y
lucky enough to pass through they will look like this.

Poikilocytes secondary to abnormal hemoglobin

content

10. Drepanocyte

 Abnormal hgb content

 Menisocyte or Sickle cells  Beaten piece of donut appearance
 Holly leaf like appearance of red cell  Spleen beaten the rbc because of the Heinz bodies
 And your form this sickle cell because of the  Heniz bodies are example of denatured hgb, your
polymerization of abnormal hemoglobins red cells are exposed to oxidative stress forming
 Specifically the abnormal hemoglobin associated your Heinz bodies.
with sickle cell is hemoglobin S, whenever you  Your spleen can remove those heinz bodies but it
have polymerization of that deoxygenated HbS, will leave a permanent mark or a permanent scar or
you will form sickle cells damaged among your RBC
 Associated with Sickle cells disease

-CACL
 Poikilocytes
 Proper way on how we should grade for your
poikilocytes
 So you need to give approximation on how much
poikilocytes are present within your patients
specimen
 You can grade it semi-quantitatively

 Slight: less than 5%

 Moderate: 5-15%
 Marked: more than 15%

 Particular variation:
 Occasional: <1%
 Few: 1-5%
 Frequent: 5-10%
 Many: >10%

-CACL
  Megalobalstic states (did not develop propeprly
HEMATOLOGY 1 because of defieciencies to ceratin minerals like
MIDTERM LECTURE WEEK 9 vitamins b12, folic acid)
 thalassemia, hemolytic anemia
Continuation of RBC abnormalities…  Most common poikilocytes seen in cases of
thalassemia= Target cell/ Codocytes
INCLUSIONS  You can also have your howell-jolly bodies to one of
 if you will have your RBC definitely what you should those common anomaly you can in cases of
have inside as you observed it under the thalassemia
microscope will be just your hemoglobin
 Any materials, substances or unwanted structures Basophilic Stipplings
inside RBC are what we call as inclusion bodies
 Generally, when you talk about inclusion bodies
they are formed primarily because of incomplete or
improper development or maturation of your cells
 So during erythropoiesis there might be some
deficiencies, or excessive concentration of a
particular component that’s why they are then
retained (left over components) during your
maturing cells

Howell-Jolly bodies  Dark blue granules inside your RBC

 Blue berry bagel appearance (bread with
blueberries on top)
 You called it basophilic because of the color of the
granules
 it is formed because of the precipitation of the
ribosomes and RNA (only immature cells have it)
 the precipitation of those ribosome and RNA then
resulted to the formation basophilic stipplings

TWO CLASSIFICATIONS:
 Round fragments of the nucleus which is a product  FINE:
of kayorexis or nuclear break down - you form this because of polychromasia/
 Normally our mature red does not have nucleus, increased polychromatophilia (talking about
but the immature forms they do contain the reticulocytes)
nucleus. So if there will be remnants of that nuclear - Within reticulocytes they have remnants of
breakdown or karyorexis then what you will form RNA, because of the precipitation of those RNA
the left over portions will then be called as howell- and a lot of polychromatic RBC, then possibly
jolly bodies you can see basophilic stipplings
 There are the nuclear remnants of DNA, fragments  COARSE:
of the nucleus resulting from karyorexis - impaired (problem with) hgb synthesis
 To make sure that round fragment is really DNA, - If a person is intoxicated with lead (lead
use special stain = Feulgen stain (for DNA remnants) poisoning), it then leads to the problem in the
 In general, if you want you identify those diseases formation of heme particularly affecting the
associated with howell-jolly bodies, this will be formation of D-ALA
associated for those diseases characterized from - In hgb synthesis, the rate of the hemoglobin
improper or incomplete development of your cells formation, specifically heme formation depends
and the most common example is the Megaloblastic of D-ALA
anemia - If your are intoxicated with lead it will affect
heme synthesis and the formation of D-ALA
thus resulting to an impaired hgb synthesis
-CACL
 resulting to the precipitation of ribosomes and  It could be associated with defects and problems in
RNA your hexose monophosphate shunt
 Or G6PD deficiency, in cases of thalassemiam
 Encounter in such intoxicated lead, thalassemia, hemoglobinopathy or unstable hgb
heavy metal poisoning, certain enzyme deficiency  Its golf ball/ pitted gold ball appearance
related to the synthesis of hgb  If you want to demonstrate heinz bodies use
Supravital stain (Methylene blue and brilliant cresyl
Cabot Ring blue)
 If there are Heinz bodies within you cell and your
spleen started to remove that Heinz bodies, in
poikilocyte you will form bite cell (degmacyte)

Hemoglobin H inclusion

 Looks like a figure of 8

Heinz Bodies

 This represent precipitated hgb H

 This is a problem with your hemoglobin particularly
your globin (kinukulang)
 This associated with thalassemia
 Easily recovered using supravital stains

Hb CC crystals

 Another example of unstable or abnormal hgb

 Certain changes in the structure of hemoglobin
making them unstable
 Hgb they are made up of amino acids, and the
globins are made up of specific number of amino
 You form this when your red cells are exposed to acids
oxidative stress resulting tot the denaturation of  Example beta globins has 146 amino acid, these AA
hemoglobin are arranged accordingly, meaning if I have 1 beta
-CACL
 globin in room no.1 and another beta globin in Ringed sideroblast
room no.2 , whatever AA I have in room 1 will be
the same set of AA I have in room 2
 whatever arrangement of AA I have in room no. 1
will be the same arrangement in room 2
 Meaning when it comes to globins, you will have
the same structure, same number of AA, same type
of AA and same arrangement
 Unstable hgb – those are disease called as
hemoglobinopathy , where there is a change in
structure of hgb
 Example: On beta globin you have 146, the 6th AA if
glycine, then because of certain genetic changes or
abnormalities instead of glycine of 6th AA, it  Sideroblast = substance accumulating in your cell is
becomes valine >> this creates changes in the iron
structure of hgb, then it makes your entire red cell  Immature cells containing iron deposits inside
susceptible to lysis (imbalance distribution of hgb)  It could be due to a problem, a defective heme
 Hexagonal with bland end, stains darkly and synthesis
described as a “bar of gold”  If you want to form heme your iron will then
combined with protoporphyrin IX
Hb SC crystals  Your prophorphyrin has problem, or D-ALA,
intoxicated with lead tendency is iron that you have
may then accumulate within your cell
 Use Prussian blue or pearl’s stain to easily
demonstrate iron
 You can encounter this in cases of sideroblastic
anemia, myelodisplastic syndrome

Pappenheimer bodies

 Another example of hemoglobinopathy/ unstable

hgb
 dark colored crystals of condesned hgb which
distorts or changes the structure of RBC
 at times it has crystaline projection
 “Washington monument” shape like crystals

 Basophilic inclusions (dark bluish to purplish) which

aggregates near the periphery of your cell
 Formed because of the accumulation of
mitochondria a little iron, little ribosomes
 You can see this in megaloblastic states,
erythropoiesis cycle is abnormal, sideroblastic
anemia, thalassemia

-CACL
 Others

Agglutination :

 Infection to Plasmodium spp. may result to ceratin  In blood smear examination, if there will any
inclusion bodies inside your RBC presence of agglutinated RBCs it should be reported
 Ringed form (malarial infection) because it may then be associated with certain
 Plasmodium falciparum is a lot deadlier compared antibodies binding or affecting or attaching on your
with other species because in P. falciparum doses RBCs
not chose RBC either young or old RBC will be  In cases of agglutinins, a typical pneumonia or
targeted hemolytic anemia
 P. vivax it targets your young RBC
 P. malariae targets old RBC Rouleaux:

 Rouleux formation- if there will be increase plasma

proteins parsticlulary plasma globulins
 Red cells forms a stack
 Seen in cases of disease like multiple myeloma
(many proteins)
Babesia
RBC morphology grading

 tick bite
 because of its appearance it is mistaken as
plasmodium
 to differentiate babesia and plasmodium, in babesia
there it maltese cross formation
 MOT: tick borne (babesia) mosquito borne
(plasmodium)
-CACL
 HEMATOLOGY 1  Then because of the release of those enzyme you
have in your lysosome, you can now then kill the
MIDTERM LECTURE WEEK 9
ingested foreign material through oxidative
mechanisms.
WHITE BLOOD CELL ABNORMALITIES
 Later it will undergo exocytosis to remove all those
 When it comes to identifying WBC on your smear
fragments afte killing or destroying the unwanted
under the microscope you always need to check
material
two things = (1) Nucleus and (2) cytoplasm
particularly the granules
Circulating Kinetics and Morphology
 Bilobed nucleus with red orange granules –
 BAND CELLS /STAB CELLS
eosinophil
- cell undergoing granulopoiesis, derived from a
 A white cell covered with darkly stained
metamyelocyte, and leading to a mature
purplish granules to the point you can no longer
granulocyte
clearly observed the nucleus of the cell –
basophil
 Left shift: ↑’d release of precursors from the bone
 When it comes their activity/function- we have
marrow
white cell to remove any of those unwanted
 the immediate precursor of neutrophils is band
materials in our system or blood (soldiers of our
cell
body)
 you can easily identified that there is shift to
 The first reach the tissues or destroyed foreign
the left if in your smear/ cell counting you have
material is the will always be the neutrophils, first
lots of band cell (normally present in blood)
to attack the foreign or unwanted materials
 If you have increased band cell in peripheral
 Also reason why Neutrophils are the most
blood, it may then be associated with an
abundant WBC
increase release of cells from your bone
 Hypersensitivity – basophils
marrow.
 Parasitic infection – eosinophil
 If you have shift to the left, it does not
 Antibody production – lymphocytes
necessarily mean that you’ll have neutrophillia
 After neutrophil the next thing to attack foreign
(neutrophil count is high
body is the monocytes
 Right shift :characterised by the presence of
 Monocytes is same with macrophages different
hypersegmented polymorphonucleocytes
is the location (monocytes= blood,
 when your neutrophils did not mature/ develop
macrophage= tissue)
completely
 Neutrophil has around 3-5 lobes
WHITE CELLS
 Shift to the right indicates you have
- major function of neutrophils is to respond rapidly
hypersegmentation of nucleus
to microbial invasion to kill the invaders
 Meaning you have more than 5 lobulations
(phagocytosis)
inside your cell
Steps:
 seen in cases of megalobalstic state (its not
1. adherence
only red cell are affected, other cell like WBC
2. migration
and megakaryocyte they have nucleus so they
3. recognition and phagocytosis (or ingestion)
need B12, folic acid if lack of these nutrients it
4. degranulation
will also affects other cells in your marrow
5. oxidative metabolism and bacterial killing
containing nucleus)
 Then chemotaxis and diapsedesis will occur
 In the circulatory system :
 Chemotaxis – white cells move toward the site
 Marginating pool
of infection because of chemical attractants
 Circulating pool
 Diapedesis – white cell leave your blood vessels
 Neutrophils are believed to survive 2-5 days
going to the site of infection or damaged
after entering the tissues
without causing any injuries/damage
 Marginating pool (50%)
 Then you will have adherence, enhanced by your
 you have neutrophils remain in your circulatory
opsonins and engulf by WBC, fusion of phagosome
system looking for an area of inflammation.
and lysosome forming your phagolysosome
-CACL
  When inflammation is found, your neutrophils  Caused by high stress conditions like burns, drug
leave your circulation by diapedesis and enter reactions, MDS, malignancy, myeloproliferative
those affected tissues disorder
 Simply they go to tissues
 Nucleus also failed to divide in to segments
 Circulating pool (50%)  Same characteristics with pelger-huet
 neutrophils leave blood stream by random  This is more of associate with diseases like
migration and they will not returned back to myelodisplactic syndrome (MDS)
your blood.  At some point the function of neutrophil may
 Even there is no inflammation they leave the be affected
bloodstream.  Genetically, acquired anomaly because of
underlying disease
NUCLEAR ABNORMALITIES
 It could be nuclear abnormalities and cytoplasmic Hypersegmentation
abnormalities  Megaloblastic anemia
 The point of distinction would be the number of  Hypersegmentation is sometimes referred to as a
lobulations (normal 3-5 lobes) myeloid "right shift".
 Also called polycytes or macropolycytes
Pelger Huet
 A.k.a.
 Nuclei are round oval or bilobed which are
spectacle-like, pince-nez like, dumbbell or peanut
with intense nuclear clumping of chromatin
 Pelger-Huet anomaly is also described as a blood
laminopathy associated with the lamin B receptor.

 associate with shift to the right

 Characterized by an abnormal synthesis of DNA
 So in your cells maturation there has been an
incomplete or improper development resulting to
the hypersegmentation of the nucleus
 When you have hepersgementation you always see
macrocyte or ovalocyte (these are also seen
whenever your cells did not mature properly
commonly observed in megaloblastic states)
 < 3 lobes – pelger huet or hyposegmentation
 Nucleus fail to divide into segments beyond two
lobed stage
 Associate with dumbbell shaped, peanut shaped,
pience-nez appearnce
 Associated with prolong use of certain drugs
particularly the chemo therapeutic drug
 Your neutrophil still functions normal
 As to certain genetic association it has been
described as a problem or mutation in Lamin gene
coding for nuclear lamina

Pseudo-Pelger-Huet anomaly
 Acquired anomaly

-CACL
 CYTOPLASMIC ABNORMALITIES
 Its an evidence of primary granules fusion
Alder-Reilly Granules  In neutrophil development your start to see
 Large purple-black coarse cytoplasmic granules granules
 Accumulation of degraded mucopolysaccharides  Promyelocyte (primary granules)
 Primary grnauled fused with one another and
tested with peroxidase (+) you form this
 You only see this in myeloid and monocytic lineage
form of leukemia
 If cell contain as mass of auer rods called as faggot
cell

Chediak_Higashi granules
 Large purple-black coarse cytoplasmic granules  Giant red, blue to grayish round inclusions
 They are mucopolysaccahride  These bodies are formed by aggregation and fusion
 Generally we have those mucopolysaccharide in of the primary and secondary specific granules.
cells the thing is we can then metabolized it  Neutropenia and thrombocytopenia are the
normally if we are equipped with the enzymes complications
needed to metabolized them, then they will not  The primary defect is in special granules present in
accumulate skin pigment cells and certain white blood cells
 The problem here is that you have certain enzyme  Chédiak–Higashi syndrome is caused by mutations
deficiency resulting to failure to metabolized and in the LYST gene.
this leads to the accumulation of those
mucopoplysaccharide in your cell
 Alde riley- sometimes mistaken as toxic granules
(seen in cases if infections or toxic states like
burned)

 Alder-Reilly Granulation

 Figure shows fused primary and secondary granules (left) and

image of a child affected by chediak higashi syndrome.

 Fusion of primary and secondary granules

 And result to the less chemotactic activity or
neutrophils they become less chemotactic because
of membrane receptor defects in chediak higashi
anomaly
 Patient with this is always susceptible to
staphylococcus aurues infection
 Manifest albinism
Auer Rods  Does not only affect RBC, also platelets. Because
 Pink or red rod shaped structures platelets also do contain granules
 Fused primary granules:  Genetically, mutations in LYST gene – makes
 Found in myeloid and monocytic series only instruction for the traffic regulation of proteins in
your cell
 LYST gene provide instruction for making protein
called as your lysosomal trafficking regulator.

-CACL
Dohle Bodies  These are reaction of neutrophils in cases of those
 Single or multiple blue inclusions toxic states / infection
 Aggregates of free ribosomes of rER  Sometimes when too much toxic granules it could
 Conditions: just be artifactual granulation, it could be because
 Confused with May-hegglin of certain technical error or medtech errors
encounter in the lab (the smear is not stained
properly)

LE cells
 Neutrophil with large purple homogenous round
inclusion

 Aggregate of ribosome of ER
 You form this, in cases of infection and toxic toxic
states (burned patient)
 Because of its appearance it sometimes confused
as may-hegglin animally  stand for lupus erythematous cell
 Neutrophil with an engulf purple homogenous
May-Hegglin anomaly round body probably from an immune complex/
 Pale-blue inclusion activity
 Derived from:  If you want easily recover your LE cells, you can go
 With the presence of giant platelets with buffy coat as specimen (white cells are
concentrated)
 Staining pattern looks smooth and evenly stained

TART CELL:
 Monocyte with ingested lymphocyte

 Basically derived from your RNA of cells

 For some unknown reasons, may-hegglin anomaly
is accompanied by thrombocytopenia (giant
platelets)  Monocyte with an ingested lymphocyte
 Genetic wise has been assocated woth mutations  Staining pattern in unevenly stained and it is rough
of MYH9 genes looking
 Sometimes also called as pseudo-LE cell
Toxic granules
 Large purple to black granules
 toxic granulation is clinically significant because it
appears to reflect a poorer prognosis.
 Conditions:

 Usually seen in cases of infection and toxic states

-CACL
 LYMPHOCYTE ABNORMALITIES Basket cell/ Smudge Cell
 Degenerated nucleus
Reactive lymphocyte  Pressure in making smear
 Atypical lymphocyte  CLL
 Stimulated
 Variant lymphocyte

 seen one – called as basket cell

 formed because of excessive pressure applied on
your smear
 To avoid you slide is added with bovine albumin
 So many degenerated lymphocyte you now call it as
 Lymphocytes are stimulated and lymphocytes are  associates with chronic lymphocytic leukemia
reacting into something
 In laboratory reporting you can add on your result Hairy cell
that lymphocytes are atypical (unusual appearance  Lymphocyte with hair-like cytoplasmic projections
of lymphocytes) surrounding the nucleus
 Associated with downey cell  Hairy cell leukemia:

Downey cell
 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  B-cells with hair like projection
 Commonlyobserved in cases of haary cell leukemia
 To confirm this anomaly, perform specailstaining
TRAP (Tartrate Resistant acid phosphatase)
 TRAP (+)

Sezary cell
 Round lymph cell with nucleus that is grooved or
convoluted
 Type 1:  Sezary syndrome
- downey cells with blocks of chromatin bodies  Mycosis fungoides
inside
 Type 2: call as IM cells (infectious mononucleosis)
 With your EBV infection
 If you are infected with EBV ccuasing IM your
target is the B-cell
 the type 2 downey cell are T-cells which reacts
to b-cells infected with EBV
 Type 3:
- atypical lymphocyte with (bilog bilog/ butas
butas) vacuoles inside  Having convolutions or grooves in nucleus will then
be associated with sezary cell if you have cerebry
formed nucleus

-CACL
PROBLEM WITH PLASMA

Flame cell
 Plasma cell with red to pink cytoplasm
 Associated with increased Ig
 Multiple myeloma

 The one that provides antibodies

 Your plasma cell has that red colored cytoplasm
 This caused by an excessive production of
antibodies/ immunoglobulins
 Associated with Multiple myeloma

Grape cell
 A.k.a:
 Plasma cell with vacuoles
 Large protein globules
 Multiple myeloma

 Berry cell, morula cell

 Plasma cell with accumulation of antibodies
 Russel bodies – are the individual globules of
immunoglobulins/ antibodies
 Associated with multiple myeloma
 Morula cell with accumulation of antibodies

References
 Rodak’s Hematology: Clinical Principles and Applications,
6th Edition
 Clinical Hematology: Theory and Procedures, 5th Edition,
Turgeon, M.L.
 Steininger, Cheryl et al. Clinical Hematology: Principles,
Procedures and Correlations.

-CACL
 HEMATOLOGY 1 Materials Needed
MIDTERM LECTURE WEEK 10 Prof. Antonio Pascua Jr.
 Hemacytometer
 Thoma Pipet
Hematologic Procedures  Suction device
Topic Outline  Thick coverslip
 Routine Complete Blood Count
 Cell counts
 Cell Counter
- RBC Count  Diluting fluids
- Platelet Count
- WBC Count
- Differential Count
 Hemoglobin
 Hematocrit
 Red Cell Indices
 Special Hematologic Tests HEMACYTOMETER
 Automation

I. Complete Blood Count

 Cell counts
 RBC Count
 Platelet Count
 WBC Count
 Differential Count
 Hemoglobin
 Hematocrit
 Red Cell Indices

A. MANUAL CELL COUNT

 The improve neubauer counting chamber, has 2 counting
 Red Blood Cell Count area (top and bottom chamber)
 Platelet Count  Primary square contains 9 squares which is your
 White Blood Cell Count secondary squares
 Whether it is WBC, RBC, or manual platelet counting they  As to the numbers, every secondary squares is equivalent
will have the same principles, concept, and the to 1mm X 1mm
procedure  So your primary square is equivalent to 9mm2 (3mm X
 The difference are would then be with diluting fluid that 3mm)
you are about to use  As for the 4 CORNERS are said to be the WBC Secondary
 most routine counting cell procedures in hematology Squares
laboratory today is with automation but still we should ** Inside WBC Secondary squares has 16 smaller squares
still know about the manual methods which refers to Tertiary Squares
 When you perform manual cell counting you will then be ** In every WBC Secondary Squares it contains 16
using a hemacytometeror we call it as counting chamber Tertiary Squares inside.
 Hemacytometer is said to be the heart of the manual cell  At the CENTER is your RBC Secondary Squares
counting because this allows us to count cells in an **RBC squares contains 25 Tertiary Squares inside
organized manner. **Every Tertiary Squares in your RBC Secondary Squares
 With all those lines and squares it will helps us and guide will further contain 16 smaller squares inside which is
us to count the cells in an organized manner your Quaternary squares
 As you perform manual cell counting, your specimen will  The vertical and horizontal lines will serve as your guide
also be later on diluted, so we are using specialized or mark that will tell you have reached the end of the
pipette called as Thoma pipette (RBC and WBC pipette). squares or dot
Automated pipettes can also be used.  In your Improve Neubauer Counting Chamber you will
have 2 PRIMARY SQUARES
** Each primary square has 9 SECONDARY SQUARES
 Aside from the Improve Neubauer Counting Chamber,
there are other counting chamber that you can use such
as Fuchs Rosenthal Counting Chamber, Speirs-Levy
counting chamber, Bass-Jones counting chamber
 But the most commonly used in manual cell counting is
your Improve Neubauer counting chamber

-CACL
 **this indicates a more than 10 difference of the
two counting chamber meaning your count is
INVALID
**If the counting is VALID, you need to get the
AVERAGE of the 2 counting chambers

 DILUTION FACTOR: depends on the dilution of your

specimen. If you use thoma pipette and you have
diluted your specimen
- Usually if it is a RBC PIPETTE, Dilution = 1:200
- In RBC pipette you aspirate the blood routinely
up to 0.5 mark and the diluting fluid is up to 101
mark. So it will be 0.5:101.
- As for the rule, we will deduct (minus) 1
(because you need to discard first the 3-5
drops).
 If you will be counting cells manually, this would be - It will now be 0.5:100, this equivalent to 1:200
the expected dilution that you will be then using - It’s not always 200, because it depends on the
amount of the blood that you will use
CALCULATIONS  Eg. Instead of aspirating the blood at 0.5 mark, you
 Whether it’s a WBC, RBC, or Platelets counting you aspirated blood up to 1 mark (you aspirated blood
will just make use of the same formula as to the entire stem of your pipette_
 CONSTANT number is the DEPTH FACTOR (0.1) ** Because it has 10 lines and every line is the equal
**DEPTH FACTOR is the distance between your to 0.1
coverslip and your counting chamber **When you aspirated it all, the total volume of
 as with the derivation, the depth factor can be blood that you have used is one = 1:101
placed in numerator (rule: is if you put the number  Instead of 0.5, you aspirated a blood only up to the
from the denominator to numerator you just get 0.4/ 0.6/ 0.7 it will cause for it to cause them to
the reciprocal) that’s why it became 10 change the dilution factor
 This will be the formula we are using (square)
- In WBC PIPETTE, is up to 11 mark. You also
aspirate blood up to 0.5 mark the ration will be
0.5:11
- **as for the rule you also deduct 1 because you
need to discard 3-5 drops
- **so the dilution will be 0.5:10, simplified form
will be 1:20 dilution

 CELLS COUNTED: it depends on the number of the  AREA FACTOR: it refers to the area of the squares
cells you have counted that you have counted and it will all depend on the
**In neubauer counting chamber you have 2 area of the squares in your neubauer
counting areas  The SECONDARY SQUARE is equivalent to 1mm X
** Both should be counted so you can check the 1mm in size so the AREA is = 1mm2
distribution of your cells  E.g. you counted 3 WBC secondary squares what is
**the difference between the 2 chamber (top and your area factor?
bottom chamber) should be only 10% **answer is 3mm2
**Example:  In RBC, you only count 5 tertiary squares (4 corners,
Top Chamber = 100 cells 1 central square)
Bottom chamber = 200  RBC Area factor
** The area of the Secondary Square is 1mm
** In RBC secondary you have 25 tertiary squares
** 1 divided by 25 = 0.04 mm2
-CACL
 ** So for every tertiary square is equal to 0.04mm2  The thing with N-RBC is that they are not lysed WBC
 Routinely, In RBC you need to count 5 squares. diluting fluids
**so you multiple 0.04 mm2 to 5  In this case, even you make use of hypotonic
** so the AREA FACTOR IF YOU COUNTED 5 RBC solution, still N-RBC will not be lysed
 The presence of N-RBC will require as to correct the
TERTIARY SQUARES would be 0.04 X 5 = 0.2mm2
WBC count
 If there will be 5 or more N-RBC per 100 WBC are
seen or observed in smears, you need to correct
your WBC count
 There are times the WBC count is high not because
your patient has infection but you WBC count is
high because if N-RBC

 Adults: if > 5 N-RBC for every 100 WBC

 Newborns: > 10 N-RBC for every 100 WBC
 This indicates there is a NEED to CORRECT WBC
 AREA FACTOR STILL DEPENDS ON THE AREA OF
COUNT
SQUARES THAT YOU HAVE COUNTED
 What if you counted 10 RBC tertiary squares it will
be 0.04 X 10 = 0.4
 After computing using the formula is it expressed in
CUBIC MILLIMETER (cu.mm.) and convert it to SI
unit
 EXAMPLE:
 RBC SI unit = x1012/L
Initial WBC count: 15 x 109/L
** Simply multiply your answer to 0.000001
After blood smear examination you see 20 N-RBC
 WBC and Platelets SI unit = x109/L ×10 1500
** Simply multiply you answer to 0.001 15 20+100
= 120
= 𝟏𝟐. 𝟓 × 𝟏𝟎𝟗 /𝑳

 In RBC counting, you make use of ISOTONIC FLUID B. INDIRECT PLATELET COUNT
so you can preserve your RBC  Aside from using hemacytometer in counting
 In WBC counting, you make use of HYPOTONIC platelets, you also count platelets by examining
SOLUTION because you need to lyse your RBC (such your blood smear
as acetic acid, hydrochloric acid)  Just simple count or observed 10 OIO fields
**or use TRUK’S DILUTING FLUID which contains  After counting and identifying platelets just simply
water, acetic acid and genshin violet so you can get your average multiply it by 20,000
enhance the morphologic feature of your WBC  EXAMPLE:
 In PLATELET counting, you make us of ISOTONIC 90 platelets
SOLUTION
**difference from RBC, in platelets you add certain 90 divided by 10 = 9
stains on isotonic fluid so you can easily count and 9 x 20,000 = 180, 000 per cu.mm.
identify your platelets 180,000 x 0.001 = 180 x 109/L
 If you have counted your platelets in your blood
N-RBC smear whatever value you have there will just be
 Falsely counted as WBC considered as an ESTIMATE
 Not lysed by WBC diluting fluids  Because in your blood smear the distribution of
 CORRECT THE WBC COUNT: your cells will vary

 For WBC count, take note of N-RBC (Nucleated RBC)

 N-RBC are falsely counted as WBC, it make WBC
count falsely elevated

-CACL
 Platelet estimate of Report platelet estimate as  So from 100, you can count 200 especially of WBC
0 – 49,000/uL Markedly Decreased  count is high
50,000 – 99,000/uL Moderately Decreased   If WBC count is high, it suggest infection. If too high
you can increase the number of WBC to be
100,000 – 149,000/uL Slightly Decreased 
differentiated
150,000 – 199,000 /uL Low Normal
 If WBC count is low, you lower it <100
200,000 – 400,000 /uL Normal (N)
 Through WBC differential counting, as you perform
401,000 – 599,000/uL Slightly Increased 
it manually, this is the time how we can then
600,000 – 800,000/uL Moderately Increased  further determine or identify the presence of N-RBC
Above 800,000/uL Markedly Increased   Since you are examining your blood smear, you can
always check the morphologic appearance of your
 You only use this table is you counted you platelet cells
in your blood smears (INDIRECT PLATELET  Blood smears gives you a picture of the true
COUNTING) condition of your blood
 If you use Neubauer chamber (DIRECT PLATELET
COUNTING)

Disposable Blood Cell Count Dilution Systems

 Capillary pipette and diluent reservoir systems
 WBC count and platelet count
 1.98mL of 1% ammonium oxalate
 Blood: 20uL  WBC abnormalities are also reported

 Capillary pipette that is calibrated to accept about Reporting:

20uL  Reactive Lymphocytes:
 If you are using this disposable blood cell dilution,  separate %, as a % of total lymphocytes
you dilution is automatically set as 1:100 dilution  Semi-quantitatively: occasional to many
 Diluting fluid is always set at 1.98mL
 In this system, the WBC and Platelet count can be  Toxic granulation:
done in the same diluted specimen  Present
 You follow it using hemacytometer  Semi-quantitatively: slight to marked / 1+ to 3+

C. Differential Count  Specimen

 One hundred WBCs are counted and classified  Peripheral blood
through the use of push-down button counters  Bone marrow
 Results are reported as percentages  Body fluid sediments
 Principle: A stained smear is examined to determine
the percentage of each type of leukocyte present
and assess the erythrocyte and platelet
morphology.

 What we differentiate here is would be your WBC

because it has many different type (Neutrophils,
basophils, eosinophil, lymphocytes, and monocytes)
 Manullay in differential counting, we need to count
about 100 WBC and we need to classify them, and
specifically identify what type of white cell you have
 As for the reporting of result, it will expressed in
PERCENT (%)
 If want to have a better accuracy of your manual
WBC differential counting, you can then increase
the number of WBC that you will then count
-CACL
D. Absolute count
 More accurate than the relative count F. Hematocrit
 Actual number of specific WBC in a liter of blood  “Packed Cell Volume”
 Absolute count = percentage x WBC Count  Volume of packed RBCs that
 Routinely, we count 100 WBC but this does not occupies a given volume of
represent the entire white cell in your blood whole blood
**100 WBC count is called as RELATIVE COUNT  Reported: percentage(%) or L/L

 Example
In 100 WBC, 50% N, 30% L, 20% M
Your WBC count is 10 x109/L
Rule of Three
50% X 10 = 5 x109/L  you only make us of this if Red cells are normocytic
30% X 10 = 3 x109/L and normochromic
20% X 10 = 2 x109/L  Theoretical representation about the relationship of
those parameters
 If you will add your Absolute count it should be  Applicable if red cells are normocytic-
equal to you WBC count normochromic
 The value of the hematocrit should be three times
EOSINOPHIL COUNT the value of the hemoglobin plus or minus 3
 50-350 x106/L
 To assess your adrenocortical function of you
adrenal galnds

Diluting Fluids Compositions

 Phloxine/eosin/neutral red: stain
 Propylene glycol
 Na2CO3
 Heparin G. Red Cell Indices
 Helps determine the size and color of your RBC
Diluting Fluids  This measure of indices will help you classify
 Pilot’s different type of anemia
 Manner’s  Serves as quality control check to assess the
 Randolphs’s parameters of CBC
 Hinkleman  Used to define the size and hemoglobin content of
 Tannen’s the red blood cell
 Aids in diagnosing and differentiating anemia
E. Hemoglobin  MCV
 Higher in the morning and lower in the evening  MCH
 Screen for anemia and may detect RBC breakdown/  MCHC
hemolytic anemia
 Higher in high altitudes increase in strenuous 1. Mean Cell Volume
 Muscular activity  Average volume of red blood cells expressed in
 Standard method is CYANMETHOGLOBIN femtoliters (fL)
 Important to lyse your RBC  Volume reflects the size of your RBC
 NV: 80-100 fL
 Between 80-100fL = NORMOCYTIC
 < 80fL = MICROCYTIC
 >100fL = MACROCYTIC

-CACL
2. Mean Cell Hemoglobin (MCH)  Anisocyte with size below or above the normal
 Average weight of hemoglobin in a red blood cell range= abn. MCV and RDW
expressed in picograms (pg)
 NV: 26-32 pg
Representative Critical Values
 Hemoglobin: less than 5.0 g/dL
 Hematocrit: less than 15%
 Platelet count: less than 30,000 per microliter
 WBC count: less than 2,500 per microliter and
greater than 30,000 per microliter
 Immediately notify attending physician right
3. Mean Cell Hemoglobin Concentration (MCHC) away because it may mean the lives of patient
 Average concentration of hemoglobin in each
individual red blood cell expressed in grams per II. Reticulocyte Count
deciliter (g/dL)  An indicator of the rate of erythrocyte production
 Concentration of the hemoglobin itself  Use of supravital stains
 We use this more in classifying anemia  The count is expressed as a percentage of total
 NV: 32-36 g/dL erythrocytes
 > 36 g/dL does not mean you have
excessive amount of hgb but it actually  Not part of CBC
means your red cell are spherocytic or it  Used to assess the erythropoietic activity of our
loses its pallor area bone marrow
 MCHC will not fall below 22%, it will lower if  Whether your bone marrow is active in production
you abnormal hemoglobins like Hgb S or of RBC
Hgb C or specimen is lypemic  In cases of hemolysis red cells are lysed so if you
want to know whether your body is responding or
not compensating on loss of RBC we then measure
your reticulocytes
 Reticulocyte is normally seen in our peripheral
blood but what separates them from RBC is that the
reticulocytes still contains remnants or RNA
 In reticulocyte counting we prepare a smear and we
mix our blood and supravital stain, we prepare the
smear and count about 1000 RBC
 Out of that 1000 RBC we just need to count how
many of them contains remnants of RNA inside to
cell

H. Red Cell Distribution Width

 It reflects the degree of red cell variation in size
 Reflects the degree of anisocytosis
Example:
 RDW = S.D./mean MCV x 100
- There are 15 reticulocytes counted in 8 months old
 NV: 9.0-14.5
infant.
 Higher than the NV indicates you have a lot
- Retics count (%) = 15 reticulocytes / 1,000 = 0.015 x
of RBC which varies in size
100 = 1.5%
- Interpretation: NORMAL
 Microcytic/macrocytic – normal RDW + dec or inc
MCV
 Anisocyte with size w/in ref range= inc RDW + N
MCV

-CACL
Reference range
 Adult: 0.5-1.5%
 Newborn: 2.0-6.0% (bone marrow is 100% active)  The reticulocyte count here refers to your
Relative count
 Because that reticulocyte is just a
representation of 1000 cells, it does not
represent the entire numbers of cells or
reticulocytes in your blood

II.B – Absolute Reticulocyte Count (ARC)

 the actual number of reticulocytes in 1 liter (L) or 1
II.A – Miller Disc microliter (mL) of blood
 Designed to reduce the labor-intensive process of  reflects the actual number of reticulocyte in 1
counting reticulocytes liter of blood
 Disc is inserted into the eyepiece of the microscope
 Minimum of 112 cells should be counted in the
small square

 Compose of two square, inserted to the

eyepiece of your microscope  UNIT is x10^9 /L
 Smaller square where you supposed to count  Any values between 20 and 115 are within your
your RBC reference interval for most populations
 Larger square where reticulocyte are counted
 About a minimum 112 RBC should be counted II.C – Corrected Reticulocyte Count (CRC)
in your small square because it is equivalent to  In specimens with a low hematocrit, the percentage
1008 RBC (according to College of American of reticulocytes may be falsely elevated because the
Pathologist) whole blood contains fewer red blood cells.
 9 is constant it’s the area of small square  A correction factor is used, with the average normal
hematocrit considered to be 45%.
 When the hct is mababa, tendency is the
percentage of reticulocyte maybe falsely
elevated
 If your hct is low it means that there is less red
cell
 If our red cell is decreased it may appear like
that you have a lot of reticulocytes
 So you need to correct your reticulocyte count
-CACL
  Maturation time – checks patients hematocrit
 In RPI, if the value is greater than 3 it means to say
that there is an adequate bone marrow response
meaning to say it reacts properly
 If your RPI is < 2 it means to say that there is
 If you use decimal multiply it by 100 at the end
inadequate response from your bone marrow
 If you use percent, it does not need to be multiplied  In cases of anemia, if the RPI is > 3 meaning your
by 100 bone marrow is responding

II.D – Reticulocyte Production Index (RPI) II.E – Automated Reticulocyte Count

 Measures erythropoietic activity when stress  Analyzers evaluate reticulocytes using optical
reticulocytes are present scatter or fluorescence after the red blood cells are
 What are “shift reticulocytes”? treated with fluorescent dyes or nucleic acid stains
 Normally as you undergo erythropoiesis your to stain residual RNA in the reticulocytes.
reticulocyte will stay in your bone marrow for about
 Retics percentage and absolute retics count can
1-2 days then they will to your blood and after 1 day
they mature as RBC
be computed immediately
 When we say shift reticulocyte or stress reticulocyte
you are referring to those reticulocyte who shifted III. Erythrocyte Sedimentation Rate (ESR)
from your bone marrow going into your blood  Non-specific measurement used to detect and
 Pinabilis yung paglabas ng reticulocyte from the monitor an inflammatory response to tissue injury
bone marrow going into your blood  Distance in millimeters at which the RBCs fall in 1
 So instead of staying in your bone marrow for two
hour
days, mga isang araw lang lumabas na sya from the
 Sometimes it is called as “sed rate”
bone marrow
 The problem is once that reticulocytes goes into  Measure the rate of settling of RBC after an
your blood, instead of 1 day interval before it hour
become RBC, but in this case it will take you about 2-  It reflects the distance travelled by your red cell
3 days more before that reticulocyte lose their in mm/hour
reticulum and mature as RBC  One of the most important factor affecting your
 If you will count reticulocytes expect them to be ESR will always be the plasma components of
elevated because they stay more in the blood your patients’ blood
 That usually happens if there is anemia particularly  Plasma components it is where acute phase
hemolysis
reactant (substances that become elevated
 If there is hemolysis your bone marrow tends to
whenever you have an inflammation )
compensate and react to create more cells
 The higher those proteins the more you pushed
down your RBC making your ESR elevated
suggesting that there is an inflammation

-CACL
Stages of ESR  There are certain factors that may affect
 Initial rouleaux formation (10 minutes) hematocrit value here example if there is a low
 Rapid settling of RBCs (40 minutes) total protein tendency your hematocrit will be
 Final sedimentation of RBCs (10minutes) falsely decreased
 If your WBC count is high, it will also falsely
IV. Osmotic Fragility Test (OFT) increased your hematocrit
 A measure of the ability of red cells to take up fluid  Even the cold agglutinins it will decreased your
without lysing hematocrit
 Employed to help diagnose different types of  Although it is available but there are factors
anemia, in which the physical properties of red cells that may limit our test
are altered.
 Increased OFT (decrease resistance): found in  Hemoglobin
hemolytic anemias and hereditary spherocytosis - measured by modified hemoglobinometers or
and whenever spherocytes are found by oximeters integrated with a blood gas
 Decreased OFT (increase resistance): occurs analyzer
following splenectomy and in liver disease, sickle  through geometrics analysis we can measure
cella nemia IDA and thalassemia how much hemoglobin is present
 Used to detect spherocytosis so you can check
the ability of your red cells to resists lysis  Cell Counts
 In cases of spherocytes you don’t have that - Traditional cell counting methods
pallor area, a little hypotonic fluid entering your - employs a buffy coat analysis method
cell it will results to the lysis of your RBC
 If OFT is elevated  indicates cannot technically V. Automation
resists your lysis you find this one in cases of
 Automation in hematology laboratory, particularly
spherocytosis or in cases of hemolytic anemia,
in CBC
particularly your Autoimmune Hemolytic
 With our current hematologic analyzer it uses
Anemia
basically nowadays a combination of different
 To confirm if AIHA you go with antiglobulin test
principles
 If OFT is  it means you have an increased
 Like principles of light scattering, electrical
resistant, your pallor area is high, your surface
impedance, fluorescence, electrical conductivity
area to volume ratio is high
and etc.
 Hypochromic – 3rd to lysed
 Normochromic – 2nd to lysed
 Two General Principles:
 Hyperchromic – 1st to be lysed
 Electronic resistance ( impedance)
 Measures cells volume
Point of Care Tests
 Optical/Light Scatter
 Offers a rapid and accurate results of testing
 After with the cell size and other
certain hematologic parameter
characteristics of your cell
 Although its not applicable to all hema
 For conductivity, it goes with the internal structure
parameter
of your cell
 Point of care testing meaning you can perform
 In cytochemistry, you use enzymes as you employ it
the test while bedside of your patient
with light scattering
 Commonly is the monitoring of sugar, especially
 With fluorescence it combined with light scattering
among diabetics (glucometer)
so you can analyze specialize component of your
 In hematology also has POCT
cell like RNA
 Hematocrit
- Centrifuge-based device
- Conductivity Method
 Plasma conducts electrical current whereas
your WBC later on acts as insulators

-CACL
 With automated analyzers for CBC, one of the  As you count your WBC here, you count it alongside
classic difference or types would be: with your hemoglobin because in hemoglobin and
1. 3 part differential machine WBC counting you both need to lyse your RBCs
- It means you are differentiating your WBCs that’s why they are counted together by the
machine
into 3 types (Monocytes, Lymphocytes,
 The height of that pulse is directly proportional to
granulocytes) the volume of the cells and that volume is a
2. 5 part differential machine reflection of the size of your cell
- Differentiating your WBC into 5 types  Also you have here is the HYDRODYNAMIC
(monocytes, lymphocyte, neutrophil, FOCUSING – it ensures your cells will pass on
basophil, eosinophil) through a single file going into your cells. SO you can
- Specifcy your WBC better measure the volume and the size of your cell
- For 6th measure id for Nucleated RBC (N-
RBC) since they are not lysed by hypotonic
solution
- Electrical impedance most commonly used
methodology in CBC, whereas optical
scattering uses both laser and non-laser
light in measuring different parameter in
your CBC

1. Electronic Impedance
 Utilizes non-conductive properties of blood cells
 as blood cell passes through orifice of aperture it
displaces its own volume
 RBCs and Platelets counted together, separated by
pulse heights
 Hydrodynamic focusing forces cells to pass single  Platelets: 2-20fL
file through sensing zone  MPV(Mean platelet volume): 6.8– 10.2fL
 Widely known as COULTER PRINCIPLE  The average volume of platelet
 It utilizes non-conductive properties of blood cells in  Red Cells: 36fL
short you consider your blood cells as non-  Lymphocytes: 35-90fL
conductors because of thar there will be an increase  Mononuclear cells: 90-160fL
resistance between electrode which later on results  Granulocytes: 160-450fL
into electrical pulse
 E.g. you have electrical current and our cell pass  The thing about electrical impedance is that, it’s
through that (red cells are considered as
more of your 3 part differential machine. If you
nonconductor) there will be resistance or impedance
 And because of that impedance it will then create
want this to be more specific or to be a 5 part
those different pulses differential machine, later on the electronic
 In electrical impedance, this pulses will reflect the impedance and should be combined with another
size of your cell, the volume of your cell principle , if purely coulter or purely electrical
 What you measure here is the volume that directly resistance only you just go with your lymphocytes
reflects the size of your cell monocytes and granulocyte
 In short, thisis based on the detection and
measurement of changes in your electrical
resistance produced by your cells as they pass on
through your electrical current
 RBC and Platelets are counted together which then
later on separated by pulses, to know if its RBC or
platelet >> they base it on the volume
 If the volume of the cell is about 2-20fL automatic
your machine will count it as platelets
 Higher than 36fL, automatic our machine will count
it as red cells

-CACL
  from time to time machines has self-
cleaning mechanism
 Cell carryover
 Coincident passage loss
 It’s the passage of more than one cell, what
happens is that the volume will increased and
the pulse height also increases
 Falsely elevated volume it makes your cell count
falsely decreased
 Orientation of the cell in the center of the aperture
 Deformability of the RBC
 Which then can be altered by hemoglobin
 Recirculation of cells back into the sensing zone

2. Optical Scatter
 Aside from measuring the size of your cell you can
also measure other characteristic of the cell
particularly the internal components of your cell
 Pass through your sensors and interrupt the light
source that the beam of light, light later on is
scattered
 Cells counted as passed through focused beam of
light
 Display the pulses generated, for every pulse you  A hydrodynamically focused sample stream is
have there is an equivalent volume of your cell directed through a quartz flow cell past a focused
 They identify the cell based on its reflected volume light source
 Cells counted as passed through focused beam of
Factors Affecting Volume Measurement light (LASER)
 Aperture diameter – RBC and platelets are smaller  Sum of diffraction, refraction and reflection
as compared to your WBC aperture that can then Diffraction-bending of light around the corner
increased your platelet counting, it will then affect Refraction- bending of the light due to the change
the measurement of your cell of speed
 Kaya meron ka talagang kailangan Reflection light rays turned back due to obstruction
hydrodynamic focusing  Multi angle polarized scatter separation (M.A.P.S.S)
 Red cell/platelet  Forward-angle light scatter ( 0° )
 White cell  Forward low-angle light scatter (2° to 3°).
 Protein build-up: decreases diameter (that  Forward high-angle 5° to 15°.
results to falsely elevated volume)  Orthogonal light scatter (90°)
 Kapag mataas ang volume nung cell or
mataas ang measurement ng volume it then  It measuring your cells here you’ll be using low
makes your cell count lower kasi angled light and high angled light
nagdidikitdikit na yung cell natin  With your low angled lightbasically you are
 Mataas nga ang volume nya but the count measuring the volume of the size of your cell
itself is lower kasi nagdidikit dikit sya but if you are with your side scatter light or 90°
because of that protein built up light what we measure is the internal
 If theres a protein built up it causes falsely complexity of your cell so it well then help
elevated volume of your cell making your you measures structures such as the
count falsely lower nucleus and the cytoplasmic granules
 to avoid that the machines will then require especially of your WBC
regular clean up. Burn circuits so you can  Automatic for optical scatter we use 5 part
then slow down protein built up differential machine

-CACL
3. Radiofrequency
 Used in conjunction with electrical impedance
 Cell interior density is proportional to pulse height
or change in the RF signal
 You will have in here a conductivity that will
help you analyze the nuclear density and
cytoplasmic granulation of your cells

 Usually combined with electrical impedance

 With radiofrequency it can the help you analyze the
internal complexity of the cell which can help you
later on convert it into a five part differential
machine

-CACL
-CACL
-CACL
 VI. CBC Parameters

CBC Quality Control Advantages of the automated analyzers

 rapid, objective, statistically significant
 Commercial Controls:  Not subject to the distributional bias of the manual
- 3 levels (low, normal, high) count
- Values stored in instrument computer  more efficient and cost effective
- Levey-Jennings graph generated and stored for  The precision of the automated differential makes
each parameter the absolute leukocyte counts reliable and
- **tells you whether your instruments is in good reproducible.
condition or not
- **low control= results is low Disadvantages of the automated analyzers
 Mode to Mode QC:  Produce cell counts which are falsely increased or
- Most automated hematology instruments have decreased.
a primary and secondary mode of sample  Some analyzers check only the volume and number
aspiration. Controls must be run on BOTH and of particles.
correlate.  Platelet clumps may be misclassified as leukocytes
- Primary=Automated or Closed (you don’t need or erythrocytes, and nucleated red blood cells can
to open the tube) be misclassified as leukocytes or, specifically,
- Secondary=Manual or Open (remove the cover lymphocytes.
of the tube let the machine aspirate)
 Delta Checks References
- When the Laboratory Information System (LIS)  Rodak’s Hematology: Clinical Principles and Applications, 6th
Edition
and the instrument are interfaced (connected)  Clinical Hematology: Theory and Procedures, 5th Edition,
delta checks are conducted by the LIS on select Turgeon, M.L.
parameters.  Steininger, Cheryl et al. Clinical Hematology: Principles,
Procedures and Correlations.

-CACL
 HEMATOLOGY 1  ABSOLUTE = true anemia. Low red cells mass and
MIDTERM LECTURE WEEK 13 Prof. Antonio Pascua Jr., RMT normal blood volume
 Meaning your problem really deals with RBCs
RBC DISORDERS  Low RBC delivery to the circulation = your problem is
production. There could be a problem in your bone
Topic Outline marrow and you failed to synthesize enough amount of
 Introduction to Anemia RBCs. In your circulation, there is low level of RBCs; RBC
 Microcytic-Hypochromic Anemia count is low
 Normocytic-Normochromic Anemia  Loss of RBC from the circulation – Bone marrow was
 Macrocytic Anemia able to synthesized red cells it’s just that on blood
 Thalassemia circulation there are things or factors that destroyed the
 Hemoglobinopathies RBCs
 Example you are infected with parasite Plasmodium
falciparum = that lysed/damaged your RBCs; as a result
ANEMIA
less red cells will circulate in your body.

 Reduction of red cell mass  Or you have a problem with the synthesis of hgb and
 Decreased concentration of hemoglobin other nutrients like iron, folic acid, vitamin B12

 RELATIVE: normal rbc mass, volume is low SIGNS AND SYMPTOMS

 transient
 normal retics  Easy fatigability
 Dyspnea on exertion
 ABSOLUTE: low RBC mass, normal volume  Bounding pulse
 low rbc delivery to circulation  Palpitations
 loss of rbc from circulation  Systolic murmur
 Faintness and vertigo
 Generally, there’s a problem with the ability of RBCs to  Pallor
deliver oxygen
 Low BP
 Remember the main function of our RBC is to deliver
oxygen; and red cell are able to do so because it has
 Headache
hemoglobin inside  Spoon nails
 When we say anemia, its whenever there’s a reduction
in red cell mass which refers to hemoglobin content  Palpitations – less oxygen will be delivered to your
 If you want to screen or identify persons with anemia or tissues and send signal to your heart to work harder or
with at risk for anemia the first parameter you need to faster
check is your hemoglobin  Spooning nails – clumped shaped that resembles a
 If hgb is low, that gives you an idea, that your patient has spoon. The curvature is very obvious.
anemia
 If <12g/dl hgb = suspect it as anemia Evaluation of Anemia
 The first parameter we should check is hgb if suspecting
 RELATIVE – something temporary or transient. RBC mass for anemia
is normal (normal hgb content) but blood volume is low
 Example if you are dehydrated, remember majority of
 Red cell count
our plasma or blood is made up of water; the water will
allow free circulation of red cells to deliver oxygen onto  Red cell indices
our tissue; so, even if our red cells have normal mass or  Hemoglobin and hematocrit
normal hgb content but the amount of blood/ water  RDW
circulating in your body is low you may end up with less  PBS
delivery of oxygen to your tissues.  Reticulocyte count
 Whenever we talk about anemia, our main concern is  OFT
the less delivery of oxygen to tissues.
 Bone marrow examination
 When you supply back water in your body, then expect
that slowly your anemia will be corrected.

- Cris Anne C. Legaspi Page | 1

 Red cells indices- (MCV, MCH, MCHC) can classify types I. MICROCYTIC HYPOCHROMIC
of anemia/ differentiate anemia
A. Iron Deficiency Anemia
 RDW- tells the degree of anisocytosis; how much red cell
B. Chronic Disease
vary in size and shape
 PBS (Peripheral Blood Smear) – used for the qualitative C. Sideroblastic Anemia
description of the appearance of the blood cells; confirm D. Thalassemia
the size, shape, color, maturity of cells; Blood smear
gives us a picture of the true condition of blood. A. Iron Deficiency Anemia (IDA)
 OFT – evaluates the relationship of red cells surface area  Most common cause of anemia
to its volume; Testing how capable our red cells are in
preventing lysis/ in protecting themselves against lysis;  Iron is an important component of heme; if you want to
(identify like hereditary spherocytosis) form heme, you need iron together with protoporphyrin
 Reticulocyte count – used to assess the erythropoietic IX (PIX)
activity of the bone marrow; index of RBC production; if  Enough iron and PIX can easily synthesized heme and
you have anemia, your retics count is normal or higher bind to globin and form hgb
that means your bone marrow is responding/ capable of  If you lack iron you can’t synthesized enough heme; if
producing RBCs you don’t have enough heme you will not be able to
 Bone Marrow Examination – if your problem is with the synthesized enough hgb; without enough hgb it will
number of circulating RBCs; If the number of red cells are result to anemia = less distribution of oxygen onto your
high or low you can always check your BM; Because RBCs tissues
are synthesized in your BM; check the maturation of red
cells, number of red cells; red cell production.
 Due to:
 If the no. of red cells are high or low, you can then always
check your bone marrow (because RBC are made from  dietary inadequacy
bone marrow)  malabsorption
 increase iron loss
Classification of Anemia  increased iron requirement
 Physical Characteristics
 Dietary inadequacy – one of the major source of iron is
 *Degree of Hemoglobinization: diameter of central from eating green leafy vegetables
pallor  Malabsorption of nutrients in your system particularly
 *Size of red cells problem with absorption of iron
 Hookworms may cause IDA because they compete with
 PATHOGENESIS your body’s iron
- Disorders of red cell formation  Increase iron loss when you bleed = (in women
- Excessive loss of red cell menstruation)
 Increased iron requirement, need to synthesized more
- Abnormal distribution of red cells
red cells; if your dehydrated or your in higher altitude
area = you will need more iron to synthesized hgb
 We will classify anemia based on physical characteristics
 If you fail to meet the iron requirements of the body =
of the red cells
you may end up with IDA
 We will check the size, the color, and hemoglobin
content of our RBC
 LAB FINDINGS:
 Microcytic – hypochromic = they are small and pale  CBC: decreased () hemoglobin
because there’s less production of hgb (TICS)  Retics: low () to normal
 OFT: decreased
 Normocytic – normochromic = normal in size, normal  Bone Marrow: Erythroid hyperplasia
hgb content, problem is the number of the circulating  Others: Decreased () serum iron, %saturation,
RBCs (AHA), you suffer from anemia even your red cell serum ferritin
are normal in appearance/ morphology, you suffer from  Increased () TIBC, FEP
anemia because of the number of circulating RBCs (fail to
produce them or you are destroying them)
 If your iron is low – it’s NOT A CONFIRMATORY that you
really have IDA; if serum iron level is low it does not
confirm that you really have IDA

- Cris Anne C. Legaspi Page | 2

 To confirm IDA you should check the serum ferritin  Immature cells contains iron deposits inside
(storage form of iron); since you lack iron, your ferritin  In IDA you don’t really have iron, in ACD you have iron
will should be also decreased but it was not able to enter your cell;
 In here you have iron and it was able to enter your cell
 Normal retics count = your problem is not with the but iron don’t have that partner or do not meet PIX
production of red cells; your problem is with the hgb inside; so your iron will just accumulated on your cell
content  In short what’s missing here is your PIX
 Since red cells are microcytic and hypochromic = you will  If you want to confirm the granules is iron use a stain
have more pallor area  your OFT becomes low = you Prussian blue or Pearls stain
cannot be easily lysed because you have big pallor area
 It could be inherited, which is sex linked, very rare
 Increase – TIBC (total iron binding capacity – used so  PRIMARY – idiopathic (unknown cause)
that later on you can store iron as ferritin); in IDA you  SECONDARY – can be associated with diseases like
can’t use the TIBC because you don’t have ferritin leukemia or disorders of hgb synthesis (lack or B12, folic
 FEP (free erythrocyte protoporphyrin) – if you want to acid or exposed to lead poisoning)
create heme you will need iron and PIX; if you don’t have
iron, the PIX will just accumulate; that’s why if you  RDW () increased
measure FEP it will be elevated  Hypercellular BM
 Normal retics
B. Anemia Chronic Disease  High () serum iron, serum ferritin and LDH
 Due to chronic infections, inflammatory process  (+) stain Prussian blue or pearls stain
and malignant neoplasms  Pappenheimer bodies on Giemsa
 Blockage in delivery of iron to the developing red
cell
 Low () serum iron and TIBC COMPARISON OF 3 TYPES
 High () serum ferritin
Serum TIBC Serum FEP RDW
 Immature forms of RBC are the ones who are capable iron Ferritin
creating hgb
 You have iron, so in chronic disease you have a lot of IDA Low High Low High High
iron, the problem is that the chronic disease that you
will have is which could be an infection or an Chronic Low Low High High Normal
inflammation or cancer, this will block the delivery on Disease
the entry of iron into your cell.
 That’s why this immature cell don’t have iron inside, you Sideroblas High Normal High Low High
won’t be able to synthesized hgb tic
 The values are low serum iron because those iron that
you did not use will be stored as ferritin; that’s why your
ferritin levels are high II. NORMOCYTIC NORMOCHROMIC
 Low TIBC because this iron was then stored as ferritin A. Aplastic Anemia
B. Hemolytic Anemia
C. Renal Disease
C. Sideroblastic Anemia D. Acute Blood Loss

 Whenever red cells are normal in size (6-8um) and

 Abnormalities in heme metabolism
normal pallor area (1/3) and the rest is red filled up with
 Adequate iron stores but unable to incorporate it
hgb
into hgb.  Px may still suffer from anemia not because of the hgb
 Presence of nucleated RBC with iron granules content and the morphologic structure of red cell
 PRIMARY – idiopathic (unknown cause)  But because of the decreased number of circulating
 SECONDARY – associated with leukemia or RBCs
disorders of hgb synthesis (lack of B12, folic acid or
exposed to lead poisoning)

- Cris Anne C. Legaspi Page | 3

A. Aplastic Anemia Pure Red Cell Aplasia
 Hypoplasia of erythrocyte precursors only
 it all deals with your bone marrow; your BM is aplastic or  Severe anemia with reticulocytopenia
there is aplasia  Associated with:
 Aplastic marrow – there is very few or total absence of  hemolytic anemia, parvovirus infxn, drugs,
cells within bone marrow thymoma
 If you have aplastic anemia it then be associated with
 *Diamond-Blackfan Syndrome:
PANCYTOPENIA = all of blood cell count is low (Low
RBC, Low WBC, low platelets); because all of this cells
are derived from the bone marrow  what’s down are only your RBCs
 If your bone marrow is totally down or there is aplasia =  within your BM there is an absence of erythroid
expect that all cell counts will be decreased precursors with normal myeloid and platelet elements
 Retics counts is low; our BM is hypocellular there is  Infected with parvovirus
totally none hematopoietic stem cells within your BM  If not treated right away it could lead to aplastic anemia
 Congenital form = Diamond Blackfan Syndrome
 Associated with:
 drug exposure B. Hemolytic Anemia
 viral infections  Shortened red cell survival
 exposure to toxins  Intracorpuscular or extracorpuscular defects
 tuberculosis  lysing the red cells and destroying red cells so that’s
why there is shortened red cell survival because of
 chemicals
factors that resulted to the lysis or destruction of
RBCs
 Drugs – like chloramphenicol (antibiotic, side effect is to
 And this hemolysis is could be due to INTRINSIC
damaged your BM) and sulfonamides
factors and EXTRINSIC factors
 Viral infections – like parvo virus B19 (one of the most
common viral organism that results to aplastic anemia);
cytomegalovirus, Epstein-Barr virus  Classification:
 Exposure to certain toxin or chemicals – benzene,  Intrinsic: RBC themselves have abnormality
herbicides, pesticides, due to radiation through  Extrinsic: external factors that destroy RBC
chemotherapy (performed to kill cancer cells, that BM
can be affected)  Intravascular:
 Aplastic anemia could be true with people 50 y/o and  red cells are lysed while they are inside the
above (common) blood vessels
 Extravascular:
 Congenital form of AA: Fanconi’s anemia  Red cells are lysed outside your blood vessels
 Common to 50 y/o or above
 50% person diagnose with this has 6 months mortality;  INTRINSIC FACTORS: RBC really lysed themselves;
because no red cells mean less oxygen delivery, no white red cells themselves have abnormality and lead
cells means infection, no platelets means you bleed them to be susceptible to lysis
 Example: red cell are spherocytic – you don’t have
 Since your problem here is bone marrow – common enough pallor area, meaning your red cell is
treatment is bone marrow transplantation susceptible to lysis
 So when the problem lies within the red cell itself it
 Severe if 3 of the 4 criteria are present: is called as intrinsic hemolytic anemia
 neutrophil count: <500/uL
 platelet count: <20,000/uL  EXTRINSIC FACTORS: red cells are normal and
 reticulocyte count: <10,000/uL healthy but there are external factors / forces that
 markedly hypocellular marrow destroys your RBC
 Example: Plasmodium falciparum infection =
damaged and affect your RBC
 **you consider AA as critical or severe if the 3
values are met = meaning your BM is totally down

 Tx: bone marrow transplantation

- Cris Anne C. Legaspi Page | 4

Intrinsic Hemolytic Anemia  Lab findings:
 Hereditary defects  Increased: OFT, B1, LDH, urobilinogen
 Abnormalities of red cell membrane:  Decreased: haptoglobin
spherocytosis and elliptocytosis
 Inherited RBC enzyme defect:  Clinical features of HS
 Disorders of Hgb production:  Spleen will be enlarge because those products are being
Hemoglobinopathies removed by the spleen to the point your liver will be
 Acquired defects: Proxysmal Nocturnal affected that’s why there will be jaundice
 OFT is HIGH = Hereditary Spherocytosis, Presence of
Hemoglobinuria
AIHA (Autoimmune Hemolytic Anemia)
 **to differentiate you can perform COOMBS TEST (+) =
 Hereditary Defects
AIHA (you are destroying your own red cell)
 Such as abnormality of red cell membrane: just like
spherocytosis and elliptocytosis (protein band 4.1)
which makes them susceptible to lysis Hereditary Elliptocytosis
 Inherited RBC enzyme defect: there are certain enzyme  Inherited disorder
deficiency within your red cell which makes them  Defective membrane protein skeleton structure,
susceptible to lysis elongated elliptical cells
 Hgb production problem: hemoglobinopathies  Variant: hereditary pyropoikilocytosis
 Acquired defects: Paroxysmal Nocturnal Hemoglobonuria
 Problem with your protein band 4.1 which make red
cells susceptible to lysis
Hereditary Spherocytosis
 Autosomal dominant trait in whites G6DP Deficiency
 Causes: Deficiency in spectrin  X-linked disorder
 Inability to neutralize oxidation stress:
 Loss of red cell membrane resulting in decreased  Hemoglobin molecule is unstable, susceptible to
surface area hemolysis
 Cells are less deformable:  OFT  Rapid intravasular destruction
 Clinical Features:
 jaundice, splenomegaly, skeletal abnormalities,  Very important enzyme so that later on you can prevent
chronic leg ulcers, gall stones, spherocytes and oxidative stress among your red cells
stomatocytes in PBS  Involved in pentose phosphate pathway that will later on
provide you glutathione to make sure that you can then
prevent oxidative stress on your red cell
 Caused: Deficiency in spectrin or there is thermal injury
 If you have G6PD deficiency you have problem with
(red cells ae exposed to high temperature) that’s why
oxidative stress; where hgb will be denatured and then
they have lost their pallor area, decreased surface area
it loses its ability to carry and deliver oxygen
to volume ratio making them susceptible to lysis
 Most common enzyme deficiency affecting
 Testing: OFT to diagnose hereditary spherocytosis
hematopoietic cells
 Since cells are less deformable: it makes OFT value
 Aging cells/ old cells are more susceptible to the
elevated
destruction or oxidative stress
 Its inability to reduce oxidation stress because reducing
 Since we are talking about hemolytic anemia, red cells
NADPH or glutathione is not formed
are lysed all of its content will come out, like enzymes,
 Those denatured hgb, will then be manifested in your red
LDH, potassium and etc.; if you will measure them in the
cytoplasm and you call it as Heinz bodies
blood expect them to be elevated
 Heinz bodies are further evaluated with the use of
 If you want to assess further bone marrow response in supravital stain
cases of hemolytic anemia, the best procedure to check
is counting reticulocyte whether our bone marrow is  Clinical Patterns
responding well or not in case of anemia.  neonatal jaundice
 congenital hemolytic anemia
 drug-induced hemolysis
 -favism
- Cris Anne C. Legaspi Page | 5
  Confirmatory test: PK Assay
 Associated with drug induced hemolysis – if you are
taking primaquine (use for malaria)  Main problem id the Emden- Meyer pathway
 Favism is a mediteranian enzyme variant; it is called as  Since this is an essential enzyme for the production of
such whenever you consume “fava” beans results to ATP/ energy
oxidative stress  This is an autosomal recessive disease and its effects is
more profound among older RBCs
 One good thing with G6PD = you are protected with  As red cells age, their metabolism slows down
plasmodium infection; you are resistant to p. falciparum  With the failure to generate sufficient ATP = it will then
further allow sodium and calcium enters your result =
Classes of G6DP Deficiency: the more it will result to damaged or lysis
 Class I: severe deficiency
 Class II: severe deficiency  Whenever there’s Hemolytic anemia, the liver will have
jaundice, spleen is bigger because those by products will
 Class III: mild deficiency
be eliminated by the spleen
 If red cells are lysed especially when extravascularly, you
 If you are measuring enzyme in lab, you measure their will form more and more bilirubin that why you form
activity and not their absolute value jaundice

 CLASS I and II = < 10% activity of G6PD

 Class I = hemolysis is chronic Paroxysmal Nocturnal Hemoglobinuria
 Class II = hemolysis is intermittent (pabalik balik)  Increased susceptibility to complement mediated
 Class III = 10-60% activity; hemolysis will only occur if red cell lysis
there are stressors/ or factors causing oxidative stress  -EM: protuberances on the red cell surface
on your RBCs  Chemical abnormalities
 -deficient acetylcholinesterase activity &
 Lab Diagnosis abnormally constituted glycoproteins
 Inclusion body: Hein bodies
 Increased retics, bilirubin, serum LDH  Rare acquired disorder characterized by the proliferation
 Hemoglobinuria of abnormal clones of hematopoietic cells within the
 Decreased () haptoglobin bone marrow which may be manifested by hemosiderin
 Negative Coomb’s test in your urine after a night’s sleep
 Confirmatory: G6PD Assay  You may have a presence of hemosiderin granules or hgb
granules in your urine after a night sleep
 Decreased is haptoglobin because intravascular  PNH = your red cells are lysed because of the increased
whenever red cells are lysed and hgb will come out and susceptibility to complement
bind with haptoglobin and will then be phagocytized by **complement is a protein that once activated, will then
WBCs result to
 Confirmatory test: G6PD Assay – you are after with **normally, and active complement will not lyse your
fluorescent testing, if there will be fluorescence on your RBC, the problem with your PNH is that complement will
red cells and that means you have G6PD; a little flourest easily lysed your RBC because within your red cell
or non means deficient G6PD activity membrane there is a deficiency in DAF (Decay
Accelerating Factor) = lysis
 Ascorbate cyanide test – nonspecific – where your use  DAF regulates complement system
sodium cyanide and sodium ascorbate (serves as
stressors)  Lab Diagnosis
 Solution RED = meaning red cells are not lysed  Sugar of Sucrose Lysis Test – SCREENING
 Solution turns BROWN = red cells are lysed because of - excessive hemolysis when exposed to low
this two stressors ionic strength solution
 Ham’s acid hemolysis test – CONFIRMATORY
Pyruvate Kinase Deficiency - excessive hemolysis when exposed to
 Problem in E-M pathway complement containing serum at low pH
 Failure to generate sufficient ATP results in
defective control of ions
 Jaundice and splenomegaly
- Cris Anne C. Legaspi Page | 6
 Sugar of Sucrose Lysis Test – Px RBC + ABO compatible IMMUNE HEMOLYTIC ANEMIA
serum + sucrose
 AIHA associated with cold antibody
**serum is the source of complement
- due to IgM cold reactive antibodies.
** if you observed lysis = PNH
**because if compatible why they are lysed
 AIHA - cold antibody referring to IgM (cold reacting Ab),
 Confirm: Ham’s acid serum/Hemolysis test – this reacts best in temperature below 32°C and may
(1) px rbc + normal serum + .2 N HCL (weak acid) occur in association with infection, malignancy, or any
autoimmune disorder
(2) Px rbc + own serum + 0.2N HCL
**(own serum so you can rule out autoimmune  Laboratory findings:
disease) - Direct Antiglobulin test is positive (+)
- Cold agglutinins titer is increased ()
(3) Px rbc + inactivated serum + 0.2N HCl
**inactive means no complement (heat serum at  To confirm = perform COOMBS TEST or DIRECT
56°C) ANTIGLOBULIN TEST (+)
 The antibody that are associated is anti I and anti i (cold
 (-) PNH = no hemolysis in all this 3 agglutinins)
 (+) PNH = there will be hemolysis on tube 1 and 2
and no hemolysis in tube 3  Anti I = the one associated with infection with
mycoplasma pneumoniae (primary causing atypical
Extrinsic Hemolytic Anemia pneumonia)
 Non-Immune  If you are infected you may develop anti I that may later
 mechanical on affects your RBCs
 microangiopathic
 Anti i = associated with infections with infectious
 chemical & toxic agents mononucleosis caused by Epstein-barr virus
 infections  If you are infected you may develop anti i that may later
 hypersplenism on affects or damaged your RBCs
 systemic disease
 Immue  Peripheral smears:
 Auto - polychromatophilia
- primary - spherocytosis
- secondary - agglutination of red cells
- drug induced
- infections
 Allo Warm Autoimmune Hemolytic Anemia
- transfusion reactions
 RBCs react with IgG and/or complement
 Idiopathic cases or secondary
 Red cells are lysed because of external factors
 Red cells are healthy, there are external factors that  Laboratory Findings:
cause lysis on your RBC  HIGH(): OFT, bilirubin, retics
 External factors could be Immune or Non-immune  DAT positive
 IMMUNE – it has something to do with your antibodies;
RBC has antigen on its surface so if there is antibody  Warm antibody = IgG
against that antigen there could lysed your RBC  RBCs will react with IgG and complement that will lead
 Could be Autoimmune or Aloimmune to hemolysis
AUTO= you produce antibody against your own  Secondary which is caused by lymphoma or caused by
ALO = the antibody destroying your antigen was from leukemia
another individual; due to incorrect transfusion of blood.  Confirm autoimmune HA use DAT (direct antiglobulin
test) = differentiate autoimmune HA and hereditary
 NON IMMUNE – nothing to do with your antigen spherocytosis
antibody activity

- Cris Anne C. Legaspi Page | 7

 Paroxysmal Cold Hemoglobinuria
- this is a rare state in w/c hemolysis occurs when  External pressure or trauma that then affected your
blood is warmed after previous exposure to blood vessels resulting to the damaged of your RBCs
chilling.  This transient HA – may be due to marathon runner,
triathlon damaged, boxers,
 Antibody associated is IgG (it’s still with warm AHA)  Wearing blender syndrome = hemolytic anemia cause by
 This cause by the presence of an autohemolysin prosthetic heart valves
(antibody) in the plasma that becomes attached to the
red cells while in a cold environment  MAHA (micro angiopathic HA) causes formation of
 Red cells exposed to COLD environment, this antibody schistocytes ; if there are unwanted clots or unwanted
IgG WILL BIND ON YOUR RED CELLS products on the walls of your vessel, red cells will pass
through in that area resulting to lysis
 When red cells are WARMED, this Ab CAUSED LYSIS in
the presence of complement  DIC (disseminate intravascular coagulopathy)
 In a cold temp. that is when your IgG attached in your  Within in your BV you are forming unwanted clot, you
RBC, in warm environment it will lyse your RBC because are active your coagulation mechanism unwantedly that
this Ab reacts best in warm temp. why you form clot in your BV wall
 Anti-P called as Donath-Landstainer antibody is an IgG
which fixes the complement to RBC in the cold and lysed  HUS = lysis of red cells in your kidneys
when placed in warm
 TTP = you breakdown your von willebrand factor = leads
increase destruction on your blood vessels resulting to
 Laboratory findings: clot formation and passing through RBC will be lysed
- elevated () Reticulocyte count
- Increased () concentration of Indirect Hemorrhagic Anemia
Bilirubin  body adjusts, increased heart rate
- Hemoglubinuria  expanding circulatory volume
- Positive (+) Donath-Landsteiner of Rosenbach  **hematologic response to acute blood loss
or Ehrlich or Sanford test  -increase plt, circulating granulocytes
- Positive (+) for methemalbumin  -increased () EPO levels in 6 hours
 -reticulocytosis in 24 hours
Transfusion reaction
 Blood incompatibility C. Renal Disease
 Lab. Findings:  Decreased release and production of erythropoietin
 DAT positive (EPO)
 High hemoglobin  Decreased in RBC count

 Because px has antibodies against antigen from the

blood donor
 (+) DAT, elevation hgb, presence of Ab, IgM which could
III. MACROCYTIC ANEMIA
be due to ABO incompatibility or Rh incompatibility
 Megaloblastic Anemia
NON-IMMUNE - Vit B12 deficiency: Pernicious,
 Transient forceful contact of the body with hard  Non=pernicious
surfaces - Folate deficiency
 Mechanical trauma: chemicals, drugs, snake venom,  Non-Megaloblastic Anemia
prosthetic heart valves - Anemia of Liver Disease
 Thermal Burns
 Infections: damages red cell membrane
 DIC: fibrin is deposited in small vessels
 Hemolytic Uremic Syndrome: renal damage
 Thrombotic Thrombocytopenic Purpura: deficiency
of enzyme ADAMST13

- Cris Anne C. Legaspi Page | 8

A. MEGALOBLASTCI ANEMIA Differentials of Erythrocytosis
 Deficiency in:
- Vitamin B12 (Cobalamin)  Primary Polycythemia: PV
- Folic acid  Secondary: high altitude, CPD, cyanotic congenital
 Defective DNA production heart dse, low cardiac output, hypoventillation
 *Earliest signs: hypersegmented PMNs, oval syndrome, high affinity Hgb variants, neoplasms-
macrocytes renal artery stenosis, renal transplant, renal cyst
 Relative erythrocytosis: dehydration, stress
B. VITAMIN B12 Deficiency
 Intrinsic factor: needed to absorb Vit. B12 in Refractory Anemia
terminal ileum  Abnormal cell production, unresponsive to
 Pernicious anemia: most common cause treatment
 gastrectomy, atophic gastritis  Hallmark is ineffective erythropoiesis, with
 Veganism markedly hypercellular marrow and abundant
 D. latum infection abnormal erythroid precursors
 Features: jaundice, sore tongue, numbness and  Precursors are megaloblastic
other CNS disease
Porphyrias
C. Folate Deficiency  Usually a genetically acquired inborn error of
 Due to: metabolism
 dietary (most common)  Deficiencies of enzymes involved in porphyrin-
 intestinal malabsorption heme biosynthetic pathway
 increased demand  Dx: Spectroscopy and biochemical analysis of blood,
 excess loss urine and stool
 defective synthesis  Urine phorphobilinogen is markedly elevated

 Lab Diagnosis: Genetic Porphyrias

 macroovalocytes  Manifestations may be neurologic (excruciating
 WBCs and plts are low pain and other neurologic symptoms), cutaneous
 hypersegmented PMNs  *Acute intermittent porphyria (AIP)

 Low retics Lead Poisoning

 BM: marked erythroid hyperplasia  Adults-occupational: Children-PICA
 Blood Chem: increased B1, serum iron and LDH  *Enzymes depressed
 INCLUSION: Howell-Jolly bodies (DNA remnant),  -delta amino levulinic dehydratase
pappenheimer, cabot ring  -heme synthetase
 Features: abdominal pain, weakness
D. Liver Disease
 Non- megaloblastic anemia  Lab Diagnosis
 Decreased cholesterol synthesis  Anemia (micro/normo)
 Spurr cells, acanthocytes  Increased reticulocytes
 Decreased OFT
 INCLUSION: Basophilic stippling
ERYTHROCYTOSIS  Blood lead usually >40 ug/mL
 Excess production of red cells  BM: erythroid hyperplasia; ringed sideroblasts
 Primary Erythrocytosis: uncontrolled growth of cells
for no apparent purpose
 -polycythemia vera

- Cris Anne C. Legaspi Page | 9

Syndromes of Iron Overload 1. Alpha Thallasemia
 Hemochromatosis  Each individual has 4 genes for hemoglobin
-  GIT iron absorption and systemic iron  Severity of disease depends on the number of
overload. Iron deposits in liver genes/globin chains defective or missing
 Hemosiderosis  If 2 = alpha trait (minor)
- Secondary iron accumulation. Iron deposits in  If 3 = H hemoglobin (intermedia)
parenchymal cells and Kupffer cells in the  If 4 = Barts hemoglobin (major)
portal tract
 A problem with your alpha globins
THALASSEMIA SYNDROME  Hgb H can be observed using supravital stain
 Characterized by decreased rate of production of TYPES OF ALPHA THALLASEMIA
globin chains
 Gene for synthesis is located in Chr. 11 and Chr. 16

 *Alpha thalassemia – decreased production of

alpha chains
 *Beta thalassemia – decreased production of beta
chains

 Demographics: Southeast Asia and Mediterranean

region

 Total deficiency or absence of globin chains

 1 hgb = 4 globins
 If < 4 globin it is associated with thalassemia  The more genes are missing the more your anemia will
 Problems with amino acids of your globin, that is under be severe
the thalassemia  Alpha thalassemia trait = two alpha genes are missing
 Any problem with your globin is then will be associated (homozygote or heterzygote)
with thalassemia  Hydrops fetalis = all genes are missing
 Less globin = less hgb produced = less oxygen distributed
to your tissues
2. Beta Thallasemia
 Chromosome 11 (beta, delta, epsilon, gamma) and
Chromosome 16 (alpha and zeta)  Production of B chain will occur only at 3-6months
 Problems with this chromosomes will then result to a after birth
problem with your globin chains  Homozygous beta thalassemia (major, cooley’s
 Among us adults, the most abundant hgb is HgbA1 (alpha anemia, medittarenean anemia) severe lifelong
and beta) anemia
 The most common form of thalassemia could be Alpha  Heterozygous : one normal & one abnormal beta
Thallasemia or Beta Thallasemia chain (minor)

 Beta globins are affected

 Clinical Presentation:  Homozygote = both of beta globins are abnormal
 MINOR: mild anemia (confused with IDA) **alpha may bind to other forms of globins
 INTERMEDIA: moderate anemia ** alpha + gamma = Hgb F (among us adults we cannot
 MAJOR: severe anemia (Hydrops Fetalis) afford to have Hgb F, because we cannot survive)
**Hgb F is selfish = oxygen binds more with Hgb = O2
 The severity of anemia is depends on the number of delivery is low
globins present **much severe
 If 1 globin is only affected = mild form of thallasemia  Heterozygote = one beta is normal and the other is
 If more globin is missing = the more severe abnormal
 Totally no globin = could lead to death (Hydrops Fetalis)

- Cris Anne C. Legaspi Page | 10

 c. Hemoglobin E-Thalassemia
- co-inherited of Hemoglobin E and β thalassemia
that results to a marked reduction of β chain
production.

Laboratory Findings of Thalassemia

1. CBC
 ↓ Hb and Hct (no globin produced)
 ↑ RBC count
 ↓ RBC indices (MCV and MCHC)
 ↑ RDW (many poikilocyte)

2. Peripheral Smear
 Cooley’s trait = heterozygote - microcytic hypochromic
 Cooley’s anemia = homozygote (blood transfusion
- exhibits anisocytosis and poikilocytosis (target
dependent; every 2-3 months)
cells and elliptocytes)
- presence of NRBC
3. Hereditary Persistence of Hb F (HPHF) - polychromasia and basophilic stippling
 thalassemia with increased levels of fetal
hemoglobin 3. Increased Reticulocyte count
 partial or total suppression of beta and delta  Elevated because your cells are lysed
chains and HB F increased to compensate  And as a response of your bone marrow

 more Hgb F = less delivery of oxygen into our tissues 4. Bone marrow examination
 partial or toatal suppression of beta globin synthesis - shows hypercellular with extreme erythroid
 Test for Hgb F hyperplasia
- Alkali denaturation test (Hgb F is alkali resistant)
(A) Singer
5. Decreased OFT
(B) Betke
 Because RBC is pale, few hgb and has more pallor =
- Acid elution test (Hgb F may resist acid elution; acid
the more you can resist lysis
like citric acid phosphate buffer)
**If many Hgb F = solution turns to pink-red,
meaning there is lysis 6. Supravital stain
- shows Hb H inclusions
4. Hemoglobin Lepore
7. Electrophoresis
 a rare class of thalassemia caused by crossing over
- differentiates hemoglobin variants
of beta and delta genes
 its about migration pattern
 there are time certain abnormal hgb migrates the
5. Hemoglobinopathy + Thalassemia same pattern with normal hgb
a. Hemoglobin S- Thalassemia
- is a double heterozygous abnormality 8. Mass Spectrophotometry
- the abnormal genes for Hb S and thalassemia - assess the difference in mass of the globin
are co- inherited chains
Types: - detects single amino acid substitutions in the
1. Hb S-α-thalassemia globin chains
2. Hb SS-α-thalassemia
3. Hb S-β-thalassemia 9. DNA analysis
- identify globin gene mutations
b. Hemoglobin C-Thalassemia a. PCR
- β thalassemia with inherited Hb C b. Signal Amplification System

10. Increased Indirect Bilirubin

- Cris Anne C. Legaspi Page | 11
HEMOGLOBINOPATHIES + Thallasemia  Clinical considerations:
 *Homozygous S (SS) – lifelong, severe,
 These are a group of inherited disorders causing hemolytic anemia
structurally abnormal globin chain synthesis due to - hemolytic crisis, vaso-occlusive episodes,
amino acid substitutions (qualitative defect); prone to infection (pneumococcus),
changes in RBC deformability and electrophoretic gallstone is common, bone pain and
mobility can occur. tenderness
 Homozygous/ disease conditions (both globin - heterozygotes, one Hgb S gene, one Hgb A
chains affected) are more serious than
heterozygous/trait conditions (only one globin chain  *Sickle cell trait (Hgb AS)
affected). - usually with no apparent disease
- protected from Plasmodium falciparum
 When we say hemoglobinopathies, the problem is the infection
amino acids you have in your globins - normal PBS
 Globins are proteins which is made up of amino acids - positive sickle cell solubility test
 There is substitution of amino acid within your globins
 Example: In bet globins you have 146 AA (arranged  Lab Diagnosis
accordingly)  PBS (peripheral blood smear)
 E.g. 1st = A, 2nd = B, 3rd = C (AA set)
- marked poikilocytosis(target cells),
**If you have another red cell and beta globins there
inclusion bodies, sickle cells (6-18%)
should be a same set of AA
**you change the AA on 3rd position from C to E - increased WBCs, platelets, retics
** If letter E replaces C, expect its structure will be
different, changing its structure will then result to  Bone Marrow:
increase susceptibility to lysis - marked erythroid hyperplasia
- high iron storage
Sickle Cell Disease
 Hgb S – most common abnormal hemoglobin  Blood Chemistry:
- normal glutamic acid at 6th position in the B - increased B1, serum iron
chain is replaced by valine
 Results in: altered solubility, altered ability to  Hgb C disease:
withstand oxidation, instability, increased  resembles Hgb S but instead of valine, LYSINE is
propensity for methemoglobin production, seen on the 6th position of B chain
increased or decreased oxygen affinity  mild hemolytic anemia
 Hgb A is lacking, Hgb S is present  Hgb C crystals and clam shaped cells
 Sickling is increased
- low oxygen tension  On beta globin you replaced your glutamic acid with
- low pH lysine forming Hgb C
- increased body temp  Homozygote = Hgb CC
** you don’t have hgb A
***confers protection against falciparum malaria
**little Hgb F  Hgb A2
 Heterozygote = Hgb AC
 associated with abnormal Hgb S
** can produce hgb A and C
 on your bet globin, what’s normal is that in 6 th position
you have Glutamic Acid
 In Hgb S, instead of GA you replaced it with Valine  Laboratory: Normochromic/ normocytic anemia
 Homozygote = HgbSS (both beta globin are affected, with target cells; characterized by intracellular
both are replaced with valine) rodlike C crystals
 Heterozygote = HgbAS (one normal and one abnormal)  Hgb C migrates with hemoglobins A2, E, and on
 If you have Hgb S you have resistance in P. falciparum alkaline hemoglobin electrophoresis; can
because for then to survive they need healthy RBC differentiate hemoglobins using acid
 TEST: Na metabisulfite Test = RBC is sickling shape (+) electrophoresis.
 Solubility Test = if there is increased turbidity (+); use
reagent is sodium dithionate
- Cris Anne C. Legaspi Page | 12
Hgb SC Disease

 Hgb SC disease is a double heterozygous condition

where an abnormal sickle gene from one parent
and an abnormal C gene from the other parent
inherited.
 Laboratory: Moderate to severe
normocytic/normochromic anemia with target cells;
characterized by SC crystals; may see rare sickle
cells or C crystals; positive haemoglobin solubility
screening test.

 One beta is replaced by valine, and one beta is replaced

with lysine
 Severity : S > SC > C

Other Hemoglobinopathies
 Hemoglobin E
- Caused when lysine replaces glutamic acid at
position 26 on the beta chain.
- Homozygous condition results in mild anemia
with microcytes and target cells; heterozygotes
are asymptomatic.
- Hgb E migrates with hemoglobin A2, C, and O
an alkaline hemoglobin electrophoresis.

 Hemoglobin D (Punjab)
- Caused when glycine replaces glutamic acid at
position 121 on the beta chain.
- Hgb D migrates with Hgb S and Hgb G on
alkaline hemoglobin electrophoresis.

References:
 Rodak’s Hematology: Clinical Principles and
Applications, 6th Edition
Clinical Hematology: Theory and Procedures, 5th
Edition, Turgeon, M.L.
 Steininger, Cheryl et al. Clinical Hematology:
Principles, Procedures and Correlations.

- Cris Anne C. Legaspi Page | 13

 HEMA311LAB: Hematology 1 LABORATORY

RBC Disorders
1st Semester | SY 2022-2023 Transcribed by: Stephanie D. Robles
Lecturer: Prof. Antonio Pascua Jr.
Topic Outline
 Introduction to Anemia  PATHOGENESIS
 Microcytic-Hypochromic Anemia - Disorders of red cell formation
 Normocytic-Normochromic Anemia - Excessive loss of red cell
 Macrocytic Anemia - Abnormal distribution of red cells
 Thalassemia
 Hemoglobinopathies Microcytic Hypochromic
 Iron Deficiency Anemia
Anemia  Chronic Disease
 Reduction of red cell mass  Sideroblastic Anemia
 Decreased concentration of hemoglobin  Thalassemia
 Problem with the ability of the red cells to deliver oxygen Iron Deficiency Anemia (IDA)
 <12g/dL: hemoglobin is down, suspect to anemia  Most common cause of anemia
 Due to:
 RELATIVE: normal rbc mass, volume is low - dietary inadequacy
- transient - malabsorption (problem in absorbing nutrients in the
- normal retics system)
- something temporary - increase iron loss
- increased iron requirement
 ABSOLUTE: low RBC mass, normal volume - hookworms: parasitic organisms that may cause IDA
- low rbc delivery to circulation (problem with
production) Lab Findings
- loss of rbc from circulation  CBC: decreased hemoglobin
- true anemia  Retics: low to normal
 OFT: decreased
Signs and Symptoms  Bone Marrow: Erythroid hyperplasia
- Easy fatigability  Others:
- Dyspnea on exertion - Decreased - serum iron, %saturation, serum ferritin
- Bounding pulse o * serum iron low: not a confirmatory that
- Palpitations you have IDA instead, go with serum ferritin
- Systolic murmur - Increased - TIBC, FEP
- Faintness and vertigo o Total iron binding capacity (TIBC) – used to
- Pallor store iron as ferritin
- Low BP o Free erythrocyte protoporphyrin (FEP)
- Headache
- Spoon nails (clump shape like a spoon) Anemia of Chronic Disease
 Due to chronic infections, inflammatory process and
Evaluation of Anemia malignant neoplasms
 Red cell count  Blockage in delivery of iron to the developing red cell
 Red cell indices  Low serum iron and TIBC; High serum ferritin
 Hemoglobin and hematocrit
 RDW (degree of anisocytosis or how red cells vary in size Sideroblastic Anemia
and shape)  Abnormalities in heme metabolism
 PBS (peripheral blood smear – used for the qualitative  Adequate iron stores but unable to incorporate it into hgb.
description of the appearance of blood cells)  Presence of nucleated RBC with iron granules
 Reticulocyte count  PRIMARY: idiopathic or cause is unknown
 OFT (evaluates the relationship of red cells surface area to  SECONDARY: associated with diseases like leukemia or
its volume) disorders of hemoglobin synthesis
 Bone marrow examination
 RDW increased
Classification of Anemia  Hypercellular BM
 Physical Characteristics  Normal retics
o Microcytic & hyopochromic: small and pale bec.  High serum iron, serum ferritin and LDH
less production of hemoglobin (TICS)
 (+) stain: Prussian Blue Pearl stain
o Normocytic & normochromic: normal in size &
 Pappenheimer bodies on Giemsa
normal hemoglobin (AHA)
 Problem is the number of circulating
RBCs

 *Degree of Hemoglobinization: diameter of central pallor

 *Size of red cells
 HEMA311LAB: Hematology 1 LABORATORY

RBC Disorders
1st Semester | SY 2022-2023 Transcribed by: Stephanie D. Robles
Lecturer: Prof. Antonio Pascua Jr.
Comparison of 3 Types  Intravascular: red cell are lysed INSIDE the blood vessels
Serum TIBC Serum Fep RDW  Extravascular: red cell are lysed OUTSIDE the blood
Iron Ferritin vessels
IDA Low High Low High High
Chronic Low Low High High Normal Intrinsic Hemolytic Anemia
Disease  Hereditary defects
Sideroblastic High Normal High Low High o Abnormalities of red cell membrane:
o Inherited RBC enzyme defect:
Normocytic Normochromic o Disorders of Hgb production:
 Aplastic Anemia  Acquired defects:
 Hemolytic Anemia o paroxysmal nocturnal dyspnea (PNH)
 Renal Disease
 Acute Blood Loss Hereditary Spherocytosis
 Autosomal dominant trait in whites
Aplastic Anemia  Causes:
 Deals with bone marrow o Deficiency in spectrin
 Very few or total absence of cells within the bone marrow o Thermal injury
 Pancytopenia: all of the blood cell count is low o Testing: OFT
 Associated with: o Loss of red cell membrane resulting in decreased
- drug exposure surface area
o chloramphenicol & sulfonamides o Cells are less deformable:
- viral infections  Clinical Features:
o parvo virus B19 o jaundice, splenomegaly, skeletal abnormalities,
- exposure to toxins chronic leg ulcers, gall stones, spherocytes and
o benzene, herbecides, due to exposure to stomatocytes in PBS
radiation (chemotherapy)  Lab findings:
- tuberculosis o Increased: OFT, B1, LDH, urobilinogen
- chemicals o OFT high: hereditary spherocytosis or
 true among people 50y/o above (case to case basis) autoimmune hemolytic anemia (AIHA)
o may have 6 months mortality rate  Perform coombs test: (+) AIHA
 fanconi’s anemia: congenital forms of aplastic anemia o Decreased: haptoglobin

 Severe if 3 of the 4 criteria are present: Hereditary Elliptocytosis

- neutrophil count: <500/uL  Inherited disorder
- platelet count: <20000/uL  Deficiency with protein band 4.1 which makes the red cell
- reticulocyte count: <10000/uL susceptible to lysis
- markedly hypocellular marrow  Defective membrane protein skeleton structure, elongated
 Tx: bone marrow transplantation elliptical cells
 Variant: hereditary pyropoikilocytosis
Pure Red Cell Aplasia
 Hypoplasia of erythrocyte precursors only G6PD Deficiency
 Absence of erythroid precursors with normal myeloid and  Most common enzyme deficiency affecting hematopoietic
platelet elements cells
 RBC precursors are only down  Aging cells are more susceptible to oxidative stress
 Severe anemia with reticulocytopenia  X-linked disorder
 Associated with:  Inability to neutralize oxidation stress:
- hemolytic anemia, parvovirus infxn, drugs, thymoma o Reducing NADPH or glutathione is not formed
 *Diamond-Blackfan Syndrome: congenital form of pure  Hemoglobin molecule is unstable, susceptible to hemolysis
red cell aplasia  Rapid intravascular destruction
 One good thing: protected against plasmodium infections
Hemolytic Anemia (resistant) particularly with falciparum
 Shortened red cell survival
 Lysing the red cells  Clinical Patterns
 Intracorpuscular or extracorpuscular defects o neonatal jaundice
 Best procedure: counting reticulocytes o congenital hemolytic anemia
 Classification: o drug-induced hemolysis
- Intrinsic: problem lyse with red cells themselves o particularly primaquine (malaria)
o EX: spherocytes o favism
- Extrinsic: there are external factors that destroy the o Mediterranean enzyme variant
RBCs o Whenever consume “Fava” beans
o EX: Plasmodium falciparum
 HEMA311LAB: Hematology 1 LABORATORY

RBC Disorders
1st Semester | SY 2022-2023 Transcribed by: Stephanie D. Robles
Lecturer: Prof. Antonio Pascua Jr.
Classes of G6PD Deficiency - (+): observed lysis
If you will be measuring enzymes, measure their activity and not  Ham’s acid hemolysis test – CONFIRMATORY
their absolute values - excessive hemolysis when exposed to complement
 Class I: severe deficiency containing serum at low pH
o chronic 1) px red cell + normal serum ABO compatible + 0.2
<10% activity of G6PD
 Class II: severe deficiency N hydrochloric acid
o Intermittent 2) px red cell + own serum + 0.2 N hydrochloric
 Class III: mild deficiency – 10-60% acid: to rule out autoimmune disease
o Hemolysis will only occur will only happen if 3) px rbc + inactivated serum (no compliment) + 0.2
there is stressors N hydrochloric acid
- Normal: no hemolysis on the 3 test (PNH)
Lab Findings - Has PNH: hemolysis in tube 1 &2 but no on tube 3
 Inclusion body:
 Increased reticulocytes, bilirubin, serum LDH Extrinsic Hemolytic Anemia
 Hemoglobinuria Non-Immune Immune
 Decreased haptoglobin - mechanical *Auto ( produce antibody on
 Negative Coomb’s test - microangiopathic own)
 Confirmatory: G6PD Assay - chemical & toxic - primary
o Fluorescence testing agents - secondary
 Ascorbate cyanide test: - infections - drug induced
o Non specific - hypersplenism - infections
o Sodium cyanide and sodium ascorbate - systemic disease
(stressors) *Allo (destroying was from
o Normal: Solution will remain red (red cells were another individual)
not lysed) - incorrect transfusion
o Brown: lysed of blood
- Transfusion reactions
Pyruvate Kinase Deficiency
IMMUNE
 Problem in E-M pathway
 Failure to generate sufficient ATP results in defective  Has something to do with antibodies
control of ions  AIHA associated with cold antibody
 Autosomal recessive disease and its effects is more - due to Ig M cold reactive antibodies.
profound among older RBCs - Reacts best at <32°C and may occur in association
 It will then further allow sodium as well as calcium to with infection, malignancy or any autoimmune
enter the cell, which results to damage/lysis disorders
 Jaundice and splenomegaly - Perform coomb’s test or direct antiglobulin test
o (+): has AIHA and elevated cold agglutinins
 Confirmatory test: PK Assay
 Laboratory findings:
Paroxysmal Nocturnal Hemoglobinuria - Direct Antiglobulin test is positive
- Cold agglutinins titer is increased
 Increased susceptibility to complement mediated red cell
o Anti-I: associated with infections with
lysis
mycoplasma pneumonia
 Rare acquired disorder characterized by the proliferation
o Anti-i: associated with infections with
of abnormal clones of hematopoietic cells within the bone
infectious mononucleosis
marrow which may be manifested by hemosiderin in urine
after a night sleep
 Peripheral smears:
 Red cells are lysed because of the increased susceptibility
of complement - polychromatophilia
 There is a deficiency in an important component called - spherocytosis
DAF (decay accelerating factor) - agglutination of red cells
o Results to complement lysis on red cells
- EM: protuberances on the red cell surface Warm Autoimmune Hemolytic Anemia
 Chemical abnormalities  RBCs react with IgG and/or complement
- deficient acetylcholinesterase activity & abnormally  Idiopathic cases or secondary
constituted glycoproteins  Secondary: lymphoma or leukemia
 Laboratory Findings:
Lab Diagnosis - HIGH: OFT, bilirubin, retics
 Sugar of Sucrose Lysis Test – SCREENING - DAT positive
- excessive hemolysis when exposed to low ionic  Paroxysmal Cold Hemoglobinuria
strength solution  this is a rare state in w/c hemolysis occurs when blood is
- px red cell + ABO compatible serum (source of warmed after previous exposure to chilling.
compliment) + sucrose
 HEMA311LAB: Hematology 1 LABORATORY

RBC Disorders
1st Semester | SY 2022-2023 Transcribed by: Stephanie D. Robles
Lecturer: Prof. Antonio Pascua Jr.
 Caused by the presence of the auto hemolysin in the Macrocytic Anemia
plasma that becomes attached to the red cells while in a  Megaloblastic Anemia
cold environment - Vit B12 deficiency: Pernicious, Non=pernicious
 Laboratory findings: - Folate deficiency
- elevated Reticulocyte count  Non-Megaloblastic Anemia
- Increased concentration of Indirect - Anemia of Liver Disease
Bilirubin
- Hemoglubinuria Megaloblastic Anemia
- Positive Donath-Landsteiner of Rosenbach or
 Does not only affect the RBC, it affects also the other cells
Ehrlich or Sanford test
 Deficiency in:
- Anti-P: Donath-Landsteiner antibody
- Vitamin B12 (Cobalamin)
o IgG antibody which fixes the
- Folic acid
complement rbcs in the cold
 Defective DNA production
environment and it will be lysed when
 *Earliest signs: hypersegmented PMNs, oval macrocytes
placed in warm temp. 37°C
- Positive for methemalbumin
 Vitamin B12 Deficiency
Transfusion Reactions - Intrinsic factor: needed to absorb Vit. B12 in terminal
 Blood incompatibility ileum
 Lab. Findings: o Needs to be released by parietal cells
- DAT positive - Pernicious anemia: most common cause
- High hemoglobin o Condition characterized by a deficiency in
intrinsic factor
- gastrectomy, atophic gastritis
NON-IMMUNE - Veganism
- D. latum infection
 Nothing to do with antibodies
- Features: jaundice, sore tongue, numbness and other
 Transient forceful contact of the body with hard surfaces
CNS disease
 Mechanical trauma: chemicals, drugs, snake venom,
- Schilling’s test: expected to be abnormal in cases of
prosthetic heart valves (Waring blender syndrome) vit. B12 deficiency
 Thermal Burns
 Folate Deficiency
 Infections: damages red cell membrane
- Due to:
 DIC: fibrin is deposited in small vessels
- dietary (most common)
 Hemolytic Uremic Syndrome: renal damage
- intestinal malabsorption
 Thrombotic Thrombocytopenic Purpura: deficiency of - increased demand
enzyme ADAMST13 - excess loss
 Transient non immune hemolytic anemia: may be due to - defective synthesis
marathon runners, triathletes
o Excessive use of muscles there will be tear on Lab Diagnosis
the muscles or tissues that will affect the blood  macroovalocytes
vessels resulting to the damage of RBCs
 WBCs and plts are low
 Microangiopathic hemolytic anemia (MAHA): formation
 hypersegmented PMNs
of schistocytes
 Low retics
 BM: marked erythroid hyperplasia
Hemorrhagic Anemia
 Blood Chem: increased B1, serum iron and LDH
 Bleeding: acute & chronic  Pancytopenia: all cell counts are low
o Acute bleeding: sudden loss of blood from
 INCLUSION:
injuries or trauma
o Howell jolly bodies: most common inclusion
o Chronic bleeding: long term or gradual (ex.
bodies seen in megaloblastic state
Gastrointestinal bleeding)
 body adjusts, increased heart rate Liver Disease
 expanding circulatory volume
 Non- megaloblastic anemia
 **hematologic response to acute blood loss  Decreased choleterol synthesis
 increase plt, circulating granulocytes
 Spurr cells, acanthocytes
 increased EPO levels in 6 hours
 reticulocytosis in 24 hours ERYTHROCYTOSIS
 Excess production of red cells
Renal Disease  Primary Erythrocytosis: uncontrolled growth of cells for
 Decreased release and production of erythropoietin no apparent purpose
 Decreased in RBC count - polycythemia vera
 HEMA311LAB: Hematology 1 LABORATORY

RBC Disorders
1st Semester | SY 2022-2023 Transcribed by: Stephanie D. Robles
Lecturer: Prof. Antonio Pascua Jr.
o uncontrolled production of cells from the Thalassemia Syndromes
bone marrow resulting to the elevation of  thalassemia: deficiency or total absence of globin chains
all cell counts  Characterized by decreased rate of production of globin
chains
Differentials of Erythrocytosis  Gene for synthesis is located in ____
 Primary Polycythemia: PV  *Alpha thalassemia – decreased production of alpha
 Secondary: high altitude, CPD, cyanotic congenital heart chains
disease, low cardiac output, hypoventilation syndrome,  *Beta thalassemia – decreased production of beta chains
high affinity Hgb variants, neoplasms-renal artery stenosis,  Demographics:
renal transplant, renal cyst - Southeast Asia and Mediterranean region
 Relative erythrocytosis: dehydration, stress  Clinical Presentation:
- MINOR: mild anemia (confused with IDA)
Refractory Anemia - INTERMEDIA: moderate anemia
 Abnormal cell production, unresponsive to treatement - MAJOR: severe anemia (Hydrops Fetalis)
 Hallmark is ineffective erythropoiesis, with markedly
hypercellular marrow and abundant abnormal erythroid Alpha Thalassemia
precursors  Each individual has 4 genes for hemoglobin
 Precursors are megaloblastic  Severity of disease depends on the number of
 If it not managed, it could lead to leukemia genes/globin chains defective or missing
 Produces more of immature cells  If 2 = alpha trait (minor)
 If 3 = H hemoglobin (intermedia)
Porphyrias  If 4 = Barts hemoglobin (major)
 Usually a genetically acquired inborn error of metabolism
 Deficiencies of enzymes involved in porphyrin-heme Types of Thalassemia
biosynthetic pathway Alpha (α) Thalassemia
 Dx: Spectroscopy and biochemical analysis of blood, urine Description Hemoglobin present
and stool a. Silent Carrier - deletion of one α birth: 1%-2% Hb
 Urine phorphobilinogen is markedly elevated (- α/ α α) globin gene, leaving Bart’s
 Genetic Porphyrias 3 functional α adult: normal Hb A,
- Manifestations may be neurologic (excruciating pain globin genes Hb Bart
and other neurologic symptoms), cutaneous b. α Thalassemia Trait - deletion of two α birth: 2%-10% Hb
- *Acute intermittent porphyria (AIP) homozygous (- α/- α) globin gene Bart’s
- Genetic: fail to inherit certain genes essential for heterozygous(--/ α α) adult: normal Hb A
formation of enzymes needed for heme synthesis c. Hemoglobin H - caused by the birth: 10%-40% Hb
- Acquired: associate with LEAD POISONING Disease presence of only Bart’s
(--/- α) one gene producing replaced by Hb H 30-
Lead Poisoning α chains. 50%
 Adults-occupational: Children-PICA remainder: Hb F,
 *Enzymes depressed HbA₂,
- delta amino levulinic dehydratase Hb Bart’s and Hb A
- heme synthetase adult: 70% Hb A
 features: abdominal pain, weakness d. Hydrops Fetalis -results in the birth: 80%-90% Hb
 basophilic stippling: common inclusion body observed if a (--/--) absence of all α Bart’s
person is intoxicated with lead chains synthesis 5%-20% Hb Portland
Lab Diagnosis - incompatible with
 Anemia (micro/normo) life
 Increased reticulocytes Beta Thalassemia
 Decreased OFT  Production of B chain will occur only at 3- 6months after
 INCLUSION: birth
- Blood lead usually >40 ug/mL  Homozygous beta thalassemia (major, cooley’s anemia,
- BM: erythroid hyperplasia; ringed sideroblasts medittarenean anemia) severe lifelong anemia
o Both of the beta globins are abnormal
Syndromes of Iron Overload  Heterozygous: one normal & one abnormal beta chain
 Hemochromatosis (minor)
- incomplete GIT iron absorption and systemic iron Other Names Description
overload. Iron deposits in liver A. Minor Heterozygous -results when one of
 Hemosiderosis Cooley’s trait the 2 genes that
- secondary iron accumulation. Iron deposits in Rietti-Greppi-Micheli produce beta globin
parenchymal cells and Kupffer cells in the portal tract disease is defective
hepcidin: hormone that regulate iron in the body
-usually presents a
 HEMA311LAB: Hematology 1 LABORATORY

RBC Disorders
1st Semester | SY 2022-2023 Transcribed by: Stephanie D. Robles
Lecturer: Prof. Antonio Pascua Jr.
mild, asymptomatic - shows Hb H inclusions
anemia 7. Electrophoresis
B.Intermediate Thalassemia -more severe but do - differentiates hemoglobin variants
Intermedia not require regualr 8. Mass Spectrophotometry
Transfusion - assess the difference in mass of the globin chains
- detects single amino acid substitutions in the globin
-occassional chains
transfusions 9. DNA analysis
C. Major Homozygous -decrease or - identify globin gene mutations
Cooley’s complete lack of a. PCR
Mediterranean beta chains b. Signal Amplification System
Target Cell Anemia 10. Increased Indirect Bilirubin
-most severe form
-TRANSFUSION HEMOGLOBINOPATHIES
dependent anemia  These are a group of inherited disorders causing
structurally abnormal globin chain synthesis due to amino
3. Hereditary Persistence of Hb F (HPHF) acid substitutions (qualitative defect); changes in RBC
- thalassemia with increased levels of fetal hemoglobin deformability and electrophoretic mobility can occur.
- partial or total suppression of beta and delta chains and  Homozygous/ disease conditions (both globin chains
HB F increased to compensate affected) are more serious than heterozygous/trait
- alkali denaturation test: to test Hb F conditions (only one globin chain affected).
o singer test  Problem is more on the amino acid have on the globins
o betke test  There is a substitution of amino acids within the globins
- acid elution testing: also to test Hb F
o pink to red: excess Hb F (meaning there is lysis) Sickle Cell Disease
4. Hemoglobin Lepore  Hgb S – most common abnormal hemoglobin
- a rare class of thalassemia caused by crossing over of beta - normal glutamic acid at 6th position in the B chain is
and delta genes replaced by valine
5. Hemoglobinopathy + Thalassemia  Results in: altered solubility, altered ability to withstand
a. Hemoglobin S- Thalassemia oxidation, instability, increased propensity for
- is a double heterozygous abnormality methemoglobin production, increased or decreased
- the abnormal genes for Hb S and thalassemia are oxygen affinity
co-inherited  Hgb A is lacking, Hgb S is present
 Types:  Sickling is increased
- Hb S-α-thalassemia  low oxygen tension
- Hb SS-α-thalassemia  low pH
- Hb S-β-thalassemia  increased body temp
b. Hemoglobin C-Thalassemia  ***confers protection against falciparum malaria
- β thalassemia with inherited Hb C  Test for Hgb S: sodium metabisulfite test ((+) RBCs
c. Hemoglobin E-Thalassemia becomes sickling) or solubility test((+) increase in
- co-inherited of Hemoglobin E and β thalassemia turbidity/ reaget: sodium dithionate)
that results to a marked reduction of β chain
production.  Clinical considerations:
- *Homozygous S (SS) – lifelong, severe, hemolytic
Laboratory Findings of Thalassemia anemia
1. CBC - hemolytic crisis, vaso-occlusive episodes, prone
- ↓Hb and Hct to infection (pneumococcus), gallstone is
- ↑RBC count common, bone pain and tenderness
- ↓RBC indices (MCV and MCHC) - heterozygotes, one Hgb S gene, one Hgb A
- ↑RDW - *Sickle cell trait (Hgb AS)
2. Peripheral Smear - usually with no apparent disease
- microcytic hypochromic - protected from Plasmodium falciparum infection
- exhibits anisocytosis and - normal PBS
- Poikilocytosis (target cells and elliptocytes) - positive sickle cell solubility test
- presence of NRBC
- polychromasia and basophilic stippling Lab Diagnosis
3. Increased Reticulocyte count - PBS-
4. Bone marrow examination - marked poikilocytosis, inclusion bodies, sickle cells (6-
- shows hypercellular with extreme erythroid 18%)
hyperplasia - -increased WBCs, platelets, retics
5. Decreased OFT - Bone Marrow:
6. Supravital stain - marked erythroid hyperplasia
 HEMA311LAB: Hematology 1 LABORATORY

RBC Disorders
1st Semester | SY 2022-2023 Transcribed by: Stephanie D. Robles
Lecturer: Prof. Antonio Pascua Jr.
- high iron storage
- Blood Chemistry:
- increased B1, serum iron

Hgb C disease:
- resembles Hgb S but instead of valine, lysine is seen on the
6th position of B chain
- mild hemolytic anemia
- Hgb C crystals and clam shaped cells
- Laboratory: Normochromic/ normocytic anemia with
target cells; characterized by intracellular rodlike C crystals
- Hgb C migrates with hemoglobins A2, E, and O on alkaline
hemoglobin electrophoresis; can differentiate
hemoglobins using acid electrophoresis.

Hgb SC Disease
 Hgb SC disease is a double heterozygous condition where
an abnormal sickle gene from one parent and an abnormal
C gene from the other parent inherited.
 Laboratory: Moderate to severe
normocytic/normochromic anemia with target cells;
characterized by SC crystals; may see rare sickle cells or C
crystals; positive haemoglobin solubility screening test.

Other Hemoglobinopathies:
 Hemoglobin E
- Caused when lysine replaces glutamic acid at position
26 on the beta chain.
- Homozygous condition results in mild anemia with
microcytes and target cells; heterozygotes are
asymptomatic.
- Hgb E migrates with hemoglobin A2, C, and O an
alkaline hemoglobin electrophoresis.
 Hemoglobin D (Punjab)
- Caused when glycine replaces glutamic acid at
position 121 on the beta chain.
- Hgb D migrates with Hgb S and Hgb G on alkaline
hemoglobin electrophoresis.
 HEMATOLOGY 1 | LECTURE FINALS 2

WBC DISORDERS
Leukocyte Disorders Abnormalities in macrophages/monocytes
Quantitative disorders: - Lipids Storage Diseases
- Affects function of monocytes (blood),
• Numbers: high or low macrophages (tissues)
• Increased WBC: - After neutrophils, next to attack is the
• proliferative: reactive leukocytosis (white cells are monocytes
reacting), neoplasms (Cancer; WBCs are - Due to certain enzyme deficiency, there
uncontrolled) might be accumulation of certain
• Decreased WBC: leukopenias components within the cell (naiipon ang mga
components)
Qualitative disorders Disease Accumulation Enzyme deficient Characteristic
Gaucher’s Wrinkled/
• Affecting phagocytic activities of WBCs disease: glucocerebroside
glucocerebrosid
Crumpled
ase
Cytoplasm
Leukopenia Niemann-
A. Decreased production: sphingomyelinas Foamy
Pick sphingomyelin
o Bone marrow is not synthesizing enough e cytoplasm
disease
WBCs Tay-Sach’s Hexosaminidase
o Aplastic Anemia: disease A
glycolipids and Vacuolated
▪ irradiation, drugs, viral infection,
Sandhoff’s ganglioside Hexosaminidase cytoplasm
congenital
disease A&B
B. Ineffective production:
Sea Blue
o Can produce cells, but abnormal and Blue-green
Histiocyte lipids
immature cells cytoplasm
s
▪ Ineffective BM, other cells productions
are also “ineffective”
o Megaloblastic anemia DISEASE DEFECT
▪ deficiency in vitamins needed for the Leukocyte Adhesion Def. B chain of CD11/CD18
maturation of cells (Vit B12, Folic Acid) 1 integrins
▪ If one is ineffective, more or less, other Leukocyte Adhesion Def. Sialylated oligosaccharide
cells production will also be ineffective 2 selectin
o Myelodysplastic syndromes PMN Specific Granulocyte
Defective chemotaxis
C. Increased destruction:
o Substances or chemicals are destroying CGD Decrease oxidative burst
WBCs -X linked -membrane component
o Isoimmune neonatal -Acquired -cytoplasmic component
o Autoimmune
o Complement-activation-induced hemolysis MPO Deficiency Absent MPO-H2O2 system
D. Splenic sequestration Chediak Higashi Multiple defects
o Enlarged spleen = more white cells are
sequestered = ↓WBC count Acquired
E. Increased margination • Decreased WBC count; Destroyed WBC
o One reason for ↑ WBCs precursors in BM • Thermal injury
compared to RBCs
• DM
o ↑ Margination = ↑ WBC will leave the blood =
• Malignancy
↓ WBCs in circulation
• Sepsis
Abnormal in Function • Malnutrition
A. Job’s syndrome:
o normal random activity; Leukemia
o abnormal chemotactic (WBCs move to the • Affects all formed elements
site of infection) activity • In BM, most abundant are WBC precursors
B. Lazy leukocyte syndrome: • Reversed hematopoiesis (orderly, controlled,
o both abnormal regulated process of producing cells):
o Tamad ang WBCs, no random and uncontrolled, unregulated (produces cells even if
chemotactic activity the body does not need them)
C. Chronic Granulomatous Disease (CGD): • Abnormal, uncontrolled proliferation and
o Paasa; WBCs can move, phagocytize but accumulation of one or more of the hematopoietic
what’s inside cannot fully phagocytize/ kill cells
o inability to phagocytized microorganisms • Symptoms: fever, weight loss, increased
▪ can ingest but cannot kill sweating
o impaired NADPH oxidase o Bone pain from large leukemic mass
o problem with respiratory burst mechanism • Classify anemia by checking its origin
o Patients with CGD are always at risk with
catalase-positive microorganisms (nocardia, PHSC
pseudomonas, listeria, aspergillus, candida,
E. coli, staphylococcus, serratia, B. cepacia
and H. pylori.) Myeloid Lymphoid
o Normal morphology of WBCs, Abnormal
function Incidence
o Diagnostic Test: Nitroblue tetrazolium • Dominant cause of cancer death in children
(NBT Dye test) under 15
▪ Blood (buffy coat) + bacterial
• ALL: most common in children
suspension
• AML: adults under 60
• CLL: adults over 60
 Classification Chronic Leukemias
• Predominance of mature cells in the peripheral
A. Duration (period before death)
blood
o Acute (days to 6 months)
• Lymphoproliferative
o Subacute (2 months – 6 months)
• Myeloproliferative
o Chronic (1 – 2 years)
B. Number of WBC in the peripheral blood
o Normal: 10 – 11 x 109 / L Laboratory Findings of Chronic Leukemia:
o Leukemic: (>15,000/uL of blood) • WBC count: high
o subleukemic, aleukemic (<15,000 uL/ • Platelet count: normal to increase
blood) • Mild anemia or no anemia
▪ subleukemic: low WBC count + RBCs have problem with oxygen delivery
abnormal and immature cells present in
peripheral blood
▪ aleukemic: low WBC count + no Treatment for Acute Leukemia:
abnormal and immature cells (just inside
the BM) • Bone Marrow Transplantation
C. Types of WBC involved • Radiation
o Acute: accumulation of immature cells • Chemotherapy
▪ Immature cells should not occur in • Supportive treatment (blood transfusion)
peripheral blood, it should be inside the
bone marrow; spleen will remove
abnormal cells and eventually will not be Acute Lymphoblastic Leukemia (ALL)
able to handle the increase load of
abnormal cells → increased spleen → • Primarily a disease of the children and young
immature WBCs will infiltrate other adults
organs → destruction → organ failure • 3 Types according to Fab: L1, L2, L3
▪ Reason why px with acute leukemia dies o To differentiate: check morphological
faster features of lymphoblast
o Chronic: abundant of mature cells • According to morphology of lymphoblasts
▪ Saturation point: mapapagod ang bone o Pre-B cell ALL: t(9;22)
marrow in producing cells even if it is not o B cell ALL: t(4:11)
needed ☹ o T cell ALL: t(7;11)
▪ From chronic, it will turn into acute Prognosis
• More than 90% of children with ALL can be cured
FAB Classification (French American British) • ALL in children between 2-10 years old with early
pre-B phenotype and hyperdiploidy in the range
• It divides acute leukemias into lymphoblastic of 51-60 chromosomes: Most Favorable
and myeloblastic (depending on origin) L1
• subdivided according to cellular morphology,
cytochemical staining results, cytogenetic o Childhood ALL
studies and T and B lymphocytes markers o Lymphoblast: Small and Homogenous
• Acute: ≥ 30% blasts in BM to diagnose leukemia

WHO Classification
• Stricter: ≥20% blast in BM to diagnose leukemia
• Standard in diagnosing leukemia (takes genetic
aspect of the disease)
• Based on cell morphology, cytochemical stains,
immunologic probes of cell markers,
cytogenetics, molecular aspects and clinical L2
manifestations. o Adult ALL
o Lymphoblast: Large and heterogenous (vary in
To identify types of leukemia, check the second letter size)
• -ML: myeloid
• -LL: Lymphocytic cells
• First letter:
o AML, ALL: Acute Myelogenous Leukemia
o CML, CLL: Chronic Lymphoblastic leukemia

Acute Leukemia
• Accumulation of blasts due to: L3 (Burkitt Type)
o clonal expansion of transformed stem cells o Rare
o failure of maturation
o Lymphoblast: Large and homogenous (vary little
o prolonged generation time
in size), presence of vacuoles
• FEATURES:
o t(8;14) with a rearrangement on MYC oncogene
o Organomegaly → Organ failure
o bone pain and tenderness
o organ infiltration
o symptoms related to depressions of normal
marrow function

Laboratory Findings of Acute Leukemia:

• WBC count: varying blast
• Platelets: usually decreased
• Anemia: present
(erythroid precursors cannot mature)
 Acute Myeloblastic Leukemia (AML)
M5b
• Immature forms of myeloid lineage
• FAB: 30% Blast o Acute Monoblastic
Leukemia with maturation
• Primarily in adults between 15-39 years old
o 20->80% monocytic cells
• Morphology:
o *monoblasts: <80%
o more abundant cytoplasm
o auer rods
o MPO granules
M6
o delicate nuclear chromatin
• Chromosomal Abnormalities o Erythroleukemia
o 90% of AML (DiGuglielmo’s Syndrome)
o -t(9;22)9q34;q11) Ph’ 10-15% of M1 Poor o (RBC precursors)
Prog o 30% blasts
o -t(8;21)(q22;q22)20-25% of M2 favorable o >50% erythroblastic
prog precursors
o -t(15;17)(q22;q21)70-80% M3 RAR-PML
fusion gene M7
o Abn. of Ch.16 20-25% M4 favorable prog o Acute Megakaryocytic
o Abn. of Ch.11 30-40% M5 poor prog Leukemia
o Absence or deletions of Ch.5 or 7 poor prog o Platelets
o Megakaryocyte
M0 (largest cell in BM)
o Accumulation of undifferentiated blasts/ o Many, Large cells =
immature cells easier to damage
M1 BM
o 30% blasts
o Acute Myeloblastic Leukemia o >30% Megakaryocytes
without maturation
o Myeloblast; too much
blasts = leukemia RECAP
o >30% blast in the BM o ALL: Acute Lymphoblastic Anemia
o <10% granulocytic cells o What predominates? IMMATURE CELLS
(mature cells are very low) of all cells under LYMPHOID LINEAGE
o AML: Acute Myelogenous Leukemia
M2 o What predominates? IMMATURE CELLS
o Acute Myeloblastic of all cells under MYELOID lineage
Leukemia with maturation (almost all cells except lymphocytes)
o >30% blast in BM
o >10% granulocytic cells
Chronic Myeloproliferative Disorders (CML)
M3 • Myeloproliferative: increased production of cells
o Acute Promyelocytic Leukemia (Promyelocytes) from myeloid stem cell
o Primary granules start to appear • Chronic: increased production of mature cells
o Presence of fusion of primary granules: Auer • Clonal neoplasia of multipotent stem cell
rods • With differentiation and maturation
o Cell filled with Auer rods: Faggot Cells • Bone marrow fibrosis: reactive process
o Associated with T(15,17) • Chronic Myelogenous Leukemia
o Associated with Disseminated intravascular o Stem cell disorder affecting the
coagulation (DIC) granulocytic, erythrocytic, and
o >30% blast megakaryocytic cell lines
o >10% Granulocytic cells o Associated with Philadelphia chromosome
o >50% promyelocytes ▪ One arm of chromosome 22 is
translocated to chromosome 9
▪ Determine prognosis of the patient
- (-) Philadelphia chromosome = poor
prognosis; do not respond well to
treatment
M4
Clinical Features
o Acute
• Chronic phase (3 years)
Myelomonocytic
o expansion of the granulocytic mass
Leukemia
o marked increase cellularity
(Myelocytes)
o basophilia (increased basophils)
o Granulocytic and
o predominantly myeloid hyperplasia
monocytic cells
o splenomegaly
o 30% blasts
o total absence of LAP
o 20-<80% monocytic cells (Nageli PML?)
o M4e • Accelerated phase (50%)
o Presence of numerous eosinophils o gradual failure of response to treatment
o increasing anemia and thrombocytopenia
M5a o additional cytogenetic abnormalities
o later on go to blastic crisis
o M5: MONOBLASTS • Blastic crisis
o Acute Monoblastic Leukemia o BM now produces immature cells
without maturation o AML (70%), ALL (30%)
o 20->80% monocytic cells o Usually ineffective to treatment
o *monoblasts: >80%
Essential Thrombocythemia Blasts
• Problem with increased number of platelets Blasts in in
Ringed
(increased chances of unwanted clot formation) MDS peripheral Bone
Sideroblasts
• Dominant proliferation of megakaryocytic cell line blood (%) Marrow
• no Philadelphia chromosome (%)
• platelet count >1000 x 109/L Refractory
• with spontaneous aggregation of abnormal Anemia/
platelets Refractory <1% <5% +/-
• associated with genetic abnormalities: JAK Cytopenia
(Janus-kinase) genes (RA/RC)
Refractory
Myelofibrosis with Myeloid Metaplasia Anemia with
• Fibrosis and granulocytic hyperplasia of the ringed <1% <5% >15%
bone marrow with granulocytic and sideroblasts
megakaryocytic proliferation in the liver and (RARS)
spleen Refractory
o Px may suffer bleeding in liver and spleen Anemia with
<5% 5-20% +/-
(Poikilocytes) excess blasts
• Giant platelets and occassional circulating (RAEB)
megakaryocytic fragments Refractory
Anemia with
Polycythemia Vera 20-
excess blasts in >5% +/-
transformation 30%
• Exact opposite of aplastic anemia (↓↓ cell count)
• Characterized by an absolute increase in red (RAEBIT)
blood cells, white blood cells and platelets Chronic
o Pancytosis: ↑↑ all cell counts in the blood myelomonocytic
<5% 5-20% -
o Panmyelosis: ↑↑ all cell counts in the BM leukemia
• common thrombosis, infarction gastric ulcer, high (CMML)
BP, stroke, heart attack
A. absolute: true polycythemia (vera = truth) Leukemoid Reaction
B. relative: Temporary • Sometimes mistaken as leukemia (CML)
A. Absolute o Differentiate using leukocyte alkaline
o Increase hematocrit = increase BM phosphatase (LAP) test
production ▪ ↑↑ LAP score: Leukemoid reaction
o Primary: Panmyelosis ▪ ↓↓ Lap score: CML
▪ ↑↑ BM production of RBC, WBC and • Excessive leukocytic response in the blood.
platelets • WBC ct. >50 x 109/L
▪ ↓↓ EPO (Erythropoietin, hormone for RBC • Exaggerated response to infection
production)
• Not a disease (only a description)
- Low level of EPO but increased
• Not related to leukemia
production of RBC (uncontrolled
production of cells) • Blood picture resembles leukemia:
o Secondary with appropriate EPO • (inc. WBC count w/ shift to the left)
production:
▪ response to hypoxia Chronic Lymphocytic Leukemia (CLM)
▪ patients with pulmonary or cardiac • Most indolent form of all leukemias (silent
diseases type)
▪ ↑↑ BM production of RBC, WBC and • 25% of all cases of leukemia in Western
platelets countries
▪ EPO:
• Median age: 60 years (Common among adults)
o Secondary with inappropriate production of
Chromosomal Abnormalities
EPO
▪ tumors of kidneys, liver, brain, adrenals • 50% abnormal karyotype
▪ ↑↑ RBC (but no hypoxia) • Most common:
▪ Normal WBC and platelets o deletion on chromosome 11, 13, 17
▪ EPO: o some studies: Trisomy 12
B. Relative • Require early treatment and have significantly
o Temporary, transient shorter survival
o ↑↑ Increase hematocrit, decrease plasma Clinical Features
volume • Often asymptomatic
▪ ↑↑ cells not due to ↑↑ production of cells • Non-specific symptoms
o Due to: • LAD (lymphadenopathy) with
▪ dehydration hepatosplenomegaly (50-60%)
▪ stress o (enlarged spleen and liver due to ↑↑
▪ spurious polycythemia accumulation of lymphocytes)
▪ anxiety
Laboratory Features
o Associated with Jak genes (Janus-kinase
genes) • Absolute lymphocytosis
o Therapeutic blood collection not intended for • Often hypogammaglobulinemia, inc
transfusion to avoid accumulation bacterial infection
• 5% monoclonal serum IgG
Myelodysplastic Syndromes • 10-15% autoantibodies to RBC, platelet
• Myelodysplastic: pre-leukemic state (producing • (CD5+ cells)
immature cells) → results to anemia (refractory • Smear: Smudge/ basket cells
anemia)
• Clonal stem cell disorders characterized by:
o maturation defects
o ineffective erythropiesis
o peripheral pancytopenia in spite of marrow
cellularity
Prognosis A. Myeloperoxidase (MPO): specimen should
• Median survival: 4-6 years always be fresh
• Not altered by therapy (usually discovered late) B. Sudan Black B: staining fats
• Depend primarily on stage ▪ (+) AML, (-) ALL
• Transformation to acute leukemia with blast C. Terminal Deoxyribonucleotidyl Transferase (TdT)
crisis is rare ▪ (+) ALL, (-) AML
D. PAS: Periodic Acid Schiff
• Super imposed large cell lymphoma
▪ Used for M6 (Erythroleukemia )
• Transformation to proplymphocytic leukemia
Esterases:
E. Napthol AS-D Chloroacetate Esterase
Prolymphocytic Leukemia
▪ Specific esterase:
• Affects B and T cells ▪ (+) Granulocytic cells (mature, immature)
• Rare, common among aged men ▪ (-) Monocytic cells (mature, immature)
• Massive hepatosplenomegaly with minimal LAD F. α-Naphthyl Acetate Esterase
• Poor prognosis G. α-Naphthyl Butyrate Esterase
• Absolute lymphocytosis ▪ Nonspecific esterase
▪ (+) Monocytic, (-) granulocytes
Hairy Cell Leukemia H. Acid Phosphatase
▪ Cannot be stored
• Hairy cell: B-cell with hairlike
▪ Treated with Caltrate → TRAP (+) Hairy
projections
cell leukemia
• “Leukemic Reticuloendotheliosis” I. Leukocyte Alkaline Phosphatase (LAP):
• (+) TRAP (Tartrate-resistant acid o PMN only contains this activity
phosphatase) o Can differential CML to Leukemoid reaction
• Fine hair-like cytoplasmic membrane projections o Count 100 neutrophils, check for red or
• Prominent spleenomegaly brown precipitate
• Tx: spleenectomy o Grading x number of cells counted
Mycosis Fungoides - Ex. 0 x 50 = 0, 1 x 35 = 35
• (+) PAS ▪ 0 - no red/brown ppt
• Incidence: elderly men ▪ 1+ - slightly diffused red ppt
• Cutaneous T cell lymphoma + circulating ▪ 2+ - moderately diffused
lymphoma cells ▪ 3+ - heavily diffused
o Begins with skin itching → ulcerative tumor ▪ 4+ - very heavily diffused
• Splenomegaly & LAD  NORMAL LAP SCORE: 30 – 185
▪ ↑ Lap score, ↑ WBC = leukemoid
• Types: pre-mycotic and mycotic
reaction
• Associated with Sensory cells
▪ ↓ Lap score = CML
▪ Increase in: Leukemoid reaction, PV, 3rd
Lymphoma trimester in pregnancy
• Different from leukemia ▪ Decrease in: CML, PNH
• Proliferation of malignant cells in solid lymphatic J. Toluidine Blue:
tissues (localized tissues then spread in BM and o binds with acid mucopolysaccharides in
blood) blood cells
• Malignancy of lymphoid tissues o used for recognition of mast cells (tissue)
A. Non-Hodgkin’s: and basophils (blood)
o Proliferation of Neoblastic lymphocytes
(cancer cells)
o Classification: Rappaport
o Associated with Burkitt lymphoma
B. Hodgkins:
o Proliferation of normal cells reacting to
cancer cells
o Diagnostic cell when examining
biopsy:
▪ REED-STERNBERG CELL
o Also associated with EBV virus
• Dx: definitive-lymph node biopsy
o Classification
▪ *Rye: appearance on histologic lymph
node biopsy
▪ *Ann Arbor: staging based on tissue
involvement

Special Stains
• Helps enhance particular structures of cells
OUR LADY OF FATIMA UNIVERSITY
Bachelor in Medical Laboratory Science
HEMATOLOGY 1
TOPIC: Leukocyte Disorders

Electrophoresis – checking the migration of

the charge particles

Alkaline environment – hemoglobin’s

that migrate on the same pattern
→ For example, the Hemoglobin A2 can also
co-migrate with hemoglobin C, E, O
meaning the Hemoglobin C is an abnormal
hemoglobin may migrate at the same
pattern as with the normal hemoglobin A2
 OUR LADY OF FATIMA UNIVERSITY
Bachelor in Medical Laboratory Science
HEMATOLOGY 1
TOPIC: Leukocyte Disorders

Leukocyte Disorders o When you count the WBC count and it is

A. Quantitative disorders: increase margination, then the WBC count
• proliferative: reactive leukocytosis, neoplasms will be low
o increase in the number of WBCs ▪ Because the white cells have moved
o reactive leukocytosis: if there’s an from the blood going into the tissues
infection it’s the job or the response of the
bone marrow to produce more white cells Abnormal in Function
▪ increase WBC because the white cell A. Job’s syndrome:
are reacting into something • normal random activity but they lack chemotaxis
o neoplasms: it is uncontrolled o chemotaxis: the white cells move to the site
• leukopenias: WBC is low of infection because they are attracted to the
• numbers: the count could either be high or low chemicals release or present on that
B. Qualitative disorders microorganism
• The abnormalities affecting the phagocytic ▪ without causing or damage within the
activities of the WBCs blood vessel
o whenever it has a chemo attractant, now the
Leukopenia WBC cant go to the site of infection
A. Decreased production: • abnormal chemotactic activity
• The bone marrow in not creating or synthesizing B. Lazy leukocyte syndrome: both abnormal
much of the WBCs • It don’t have random activity and chemotactic
• There’s a problem with the production of enough activity
white cells in the body • Both random and chemotactic activities are
• Associated with aplastic anemia when there is a affected
suppression of bone marrow if the patient is C. Chronic Granulomatous Disease (CGD): inability to
exposed to irradiation, drugs, viral infection, phagocytized microorganisms
congenital • the white cell can move and phagocytize but one
• The number of circulating WBCs are low inside it cannot fully kill or phagocytize that
B. Ineffective production: megaloblastic anemia, microorganism it can go to the cytoplasm but it
myelodysplastic snydromes cannot fully phagocytize them
• It can produce cells but it produce abnormal cells o because of impaired NADPH oxidase
• If one cell production in the bone marrow is o problem on the oxidative metabolism or
ineffective more or less other cell production are respiratory burst among the WBCs
also called as ineffective • for people with CGD they are always at risk in
• Megaloblastic anemia: the patient has deficiencies acquiree infection with the catalase (+) organisms
with certain vitamins necessary for the • to check or to diagnose CGD: nitroblue
maturation for the development of the cells tetrazolium test
o Example: B12 or folic acid o use a buffy coat and mix with the bacterial
C. Increased destruction: isoimmune neonatal, suspension
autoimmune, complement- activation-induced o blue precipitate
hemolysis
• There is a substances or chemicals that destroying Abnormalities in macrophages/monocytes
the WBCs it could either be immune or non- • Affecting the function of white cells called lipid
immune factors storage diseases/disorders
o Immune: connections with the antibodies or • After neutrophils the next cell to attack that
antigens microorganism which enter the body will be the
o Autoimmune: antibodies itself monocytes while in the tissue it is macrophages
• The white cell count maybe decreased if there is • Because of certain enzyme deficiency there will be an
something that destroys the white cells accumulation of certain components within the cell
D. Splenic sequestration o It is called storage because those components
• When the spleen is enlarge expect that the more accumulate, it cannot metabolize and digest them
white cells can be sequestered making the WBCs because it lacks an enzyme needed for that
count low mechanism
E. Increased margination
• Margination: the reason why we have a lot of A. Gaucher’s disease: accumulation of glucocerebrosidase
white cell precursors in the bone marrow • Wrinkled/Crumpled Cytoplasm
compared to the red cells B. Niemann-Pick disease: accumulation of sphingomyelin
o because of margination • Foamy cytoplasm
o whenever the white cell leave our C. Tay-Sach’s disease: accumulation of glycolipids and
bloodstream and go to the cite of ganglioside
infection/inflammation • Vacuolated cytoplasm
• If there an increased margination more white cells • Deficiency in the enzyme called hexosaminidase A
will leave the blood resulting to a decrease D. Sandhoff’s disease: accumulation of glycolipids and
number of white cell now in the circulation ganglioside
 OUR LADY OF FATIMA UNIVERSITY
Bachelor in Medical Laboratory Science
HEMATOLOGY 1
TOPIC: Leukocyte Disorders

• Vacuolated cytoplasm o Acute: accumulation of immature cells

• Hexosaminidase A and B ▪ Dies faster because of organ failure
E. Sea Blue Histiocytes: accumulation of lipids ▪ The immature starts to travel/leave the
• Blue-green cytoplasm bone marrow and go to the blood
▪ Once the immature cells start to
Disease Defect infiltrate other organs it may then lead
to destruction
Leukocyte Adhesion Def. 1 B chain of CD11/CD18
o Chronic: abundant is the mature cells
integrin
▪ Saturation point
Leukocyte Adhesion Def. 2 Sialylated oligosaccharide
▪ From chronic turn into acute
selectin → The bone marrow is tired, then the
PMN Specific Granulocyte Defective chemotaxis bone marrow maybe damage and
CGD Decrease oxidative burst once damage the one will come out
• X linked • Membrane component now from the bone marrow will
• Acquired • Cytoplasmic component then be immature cell
MPO Deficiency Absent MPO-H2O2 system ▪ There will be a transition
Chediak Higashi Multiple defects
French American British (FAB) Classification
Acquired • It divides acute leukemias into lymphoblastic and
There will be low WBC count it will cause leukopenia it myeloblastic
destroy the WBCs • subdivided according to cellular morphology,
o Thermal injury cytochemical staining results, cytogenetic studies
o DM and T and B lymphocytes markers
o Malignancy • for them to consider as acute, only > or ≥ 30% blasts
o Sepsis in the bone marrow it will be classified as acute
o Malnutrition leukemia

Leukemia WHO Classification

• Abnormal, uncontrolled proliferation and • Based on cell morphology, cytochemical stains,
accumulation of one or more of the hematopoietic cells immunologic probes of cell markers, cytogenetics,
• Symptoms: fever, weight loss, increased sweating molecular aspects and clinical manifestations
• Before we commonly think of white cells • ≥ 20% blasts in the bone marrow it will be classified as
• It is not just about the WBCs, its affecting all of formed acute leukemia
elements • Standard in diagnosing leukemia
• Bone pain when it is acute leukemia • More strict compared to FAB
• If you want to differentiate or classify types of
leukemia, check the origin of the cell Identify types of leukemia:
• ML – myelogenous meaning within the myeloid stem
Incidence cell lineage
o Dominant cause of cancer death in children under 15 • LL – lymphoid within the lymphocytic cells
o ALL: most common in children • A – first letter means it is acute or chronic
o AML: adults under 60 o AML – Acute myelogenous leukemia
o CLL: adults over 60 o ALL – Acute Lymphoblastic Anemia
o CLL – Chronic Lymphoblastic Anemia
Classification
A. Duration Acute Leukemia
• Acute: days up to 6 months Accumulation of blasts due to:
• Subacute: 2 months up to 6 months • clonal expansion of transformed stem cells
• Chronic: variable • failure of maturation
• Period • Features: bone pain and tenderness, organ infiltration,
B. Number of WBC in the peripheral blood symptoms related to depressions of normal marrow
• Leukemic: WBC count is high > 15 k/uL function
• subleukemic, aleukemic • Prolonged generation time for them to mature
o Both of WBC count is < 15 k/uL • Depression of the normal hematopoietic cycle
o Subleukemic: WBC count is low and it is • Organomegaly resulting to damage to total organ
accompanied by abnormal and immature failure
cells are present in the peripheral blood
o Aleukemic: there is no abnormal or Laboratory Findings
immature cells • WBC count: varying blast
▪ The abnormal and immature cells • Platelets: usually decreased
are in the bone marrow • Anemia: present
C. Types of WBC involved
• Acute or Chronic
 OUR LADY OF FATIMA UNIVERSITY
Bachelor in Medical Laboratory Science
HEMATOLOGY 1
TOPIC: Leukocyte Disorders

Chronic Leukemias L3 (Burkitt Type)

• Predominance of mature cells in the peripheral blood • Rare
• Lymphoproliferative • Lymphoblasts: large and homogenous
• Myeloproliferative o The cytoplasmic appearance, appears to be
vacuolated
Laboratory Findings • t (8;14) with a rearrangement on MYC oncogene
• WBC count: high • Burkitt disease
• Platelet count: normal to increase
• Mild anemia
o Problem on transferring or delivering of oxygen

Treatment
• Bone Marrow Transplantation
• Radiation
• Chemotherapy
• Supportive treatment: blood transfusion

Acute Lymphoblastic Leukemia

• Primarily a disease of the children and young adults
• According to morphology of lymphoblasts
Acute Myeloblastic Leukemia
• Pre-B cell ALL: t (9;22)
• Primarily in adults between 15-39 years old
• B cell ALL: t (4:11)
• Morphology:
• T cell ALL: t (7;11)
• more abundant cytoplasm, auer rods, MPO granules,
delicate nuclear chromatin
Prognosis
• More than 90% of children with ALL can be cured
Chromosomal Abnormalities
• ALL in children between 2-10 years old with early
• 90% of AML
pre-B phenotype and hyperdiploidy in the range of
• t (9;22)9q34; q11) Ph’ 10-15% of M1 Poor Prog
51-60 chromosomes: Most Favorable
• t (8;21) (q22; q22)20-25% of M2 favorable prog
L1 • t(15;17) (q22;q21)70-80% M3 RAR-PML fusion
• gene
• Childhood ALL
• Abn. of Ch.16 20-25% M4 favorable prog
• Lymphoblasts: small and homogenous • Abn. of Ch.11 30-40% M5 poor prog
• Common in the children • Absence or deletions of Ch.5 or 7 poor prog

M0
Accumulation of undifferentiated blasts

M1
• Acute Myeloblastic Leukemia
without maturation
• >30% blast in the BM
• <10% granulocytic cells
o Granulocyte: mature cells
L2
• Predominant cells: myeloblast
• Adult ALL • It is normal to have a blast in the bone marrow but they
• Lymphoblasts: large and heterogenous should not be too much
o If the blast is too much then it is the start to
associate it to leukemia

M2
• Acute Myeloblastic Leukemia with
maturation
• >30% blast in BM
• >10% granulocytic cells
 OUR LADY OF FATIMA UNIVERSITY
Bachelor in Medical Laboratory Science
HEMATOLOGY 1
TOPIC: Leukocyte Disorders

M3 M7
• Acute Promyelocytic Leukemia • Acute Megakaryocytic Leukemia
o Chromosome 15 & 17 o Megakaryocyte: the
o Promyelocyte – primary granules start to appear largest cell in the bone
• >30% blast marrow
• >10% granulocytic cells • 30% blasts
• >50% promyelocytes • >30% Megakaryocytes
• Presence of fusion of primary granules which is called
as auer rods Chronic Myeloproliferative Disorders
• It is associated with Disseminated Intravascular • Increase in the production of the cell particularly on the
Coagulopathy (DIC) myeloid stem cells
• Clonal neoplasia of multipotent stem cell
• With differentiation and maturation
• Bone marrow fibrosis: reactive process

Chronic Myelogenous Leukemia

• Stem cell disorder affecting the granulocytic,
erythrocytic, and megakaryocytic cell lines
• Philadelphia chromosome
o One arm of chromosome 22 is translocated to
chromosome 9
M4 o Determine the prognosis of the patient
• Acute Myelomonocytic ▪ (-) poor prognosis: it does not respond with
Leukemia to therapy
• 30% blasts ▪ zGood prognosis: responding
• 20-<80% monocytic cells
• Nageli type of AML Clinical Features
A. Chronic phase (3 years)
M4e • expansion of the granulocytic mass
Accompanied by eosinophilia • marked increase cellularity
• basophilia
M5a • predominantly myeloid hyperplasia
• Acute Monoblastic • splenomegaly
Leukemia without • total absence of LAP
maturation
• 20->80% monocytic cells B. Accelerated phase (50%)
• monoblasts: >80% • Start to have a poor prognosis
• Gradual failure of response to treatment
• Increasing anemia and thrombocytopenia
• Additional cytogenetic abnormalities
M5b C. Blastic crisis
• Acute Monoblastic Leukemia • AML (70%), ALL (30%)
with maturation • Usually ineffective to treatment
• 20->80% monocytic cells
• Coming out from the marrow are blasts (immature)
• monoblasts: <80%
Essential Thrombocythemia
• Dominant proliferation of megakaryocytic cell line
• no Philadelphia chromosome
• platelet count >1000 x 109/L
M6
• with spontaneous aggregation of abnormal platelets
• Erythroleukemia • has been associated with genetic abnormalities
• 30% blasts o janus kinase (JAK) genes
• >50% erythroblastic
precursors Myelofibrosis with Myeloid Metaplasia
• Di Guglielmo’s • Fibrosis and granulocytic hyperplasia of the bone
marrow with granulocytic and megakaryocytic
proliferation in the liver and spleen
o The patient may suffer from bleeding because of
that megakaryocytic proliferation in the liver and
in the spleen
 OUR LADY OF FATIMA UNIVERSITY
Bachelor in Medical Laboratory Science
HEMATOLOGY 1
TOPIC: Leukocyte Disorders

▪ The red cells that pass through the spleen, it MDS Blasts in Blasts Ringed
may form poikilocytes particularly peripheral in Bone Sideroblasts
dacrocyte blood (%) Marrow
• Giant platelets and occasional circulating (%)
megakaryocytic fragments Refractory < 1% < 5% +/-
Anemia/Refractory
Cytopenia (RA/RC)
Polycythemia Vera
Refractory Anemia < 1% < 5% > 15%
• Opposite of aplastic anemia with ringed
o In anemia all the cell count are low sideroblasts
• All the cell count are high (RARS)
• Pancytosis: all the cell count in the blood are elevated Refractory Anemia < 5% 5-20% +/-
• Panmyelosis: increase number of cells in the bone with excess blasts
marrow (RAEB)
• Characterized by an absolute increase in red blood Refractory Anemia > 5% 20-30% +/-
cells, white blood cells and platelets with excess blasts
in transformation
• common thrombosis, infarction gastric ulcer, high BP, (RAEBIT)
stroke, heart attack
Chronic < 5% 5-20%
• absolute: a true polycythemia, the problem there is myelomonocytic
uncontrolled production of cells in the bone marrow leukemia (CMML)
• relative: temporary
Leukemoid Reaction
Absolute • not a formed of leukemia
• Increase hematocrit = increase BM production • Excessive leukocytic response in the blood
• WBC ct. >50 X 109/L
A. primary: Panmyelosis • Exaggerated response to infection
• inc BM production of RBC, WBC and platelets • Not a disease (only a description)
• EPO: low • Not related to leukemia
• Blood picture resembles leukemia: CML
B. Secondary with appropriate EPO production: • ( inc. WBC count w/ shift to the left)
response to hypoxia • LAP: Leukocyte Alkaline Phosphatase
• patients with pulmonary or cardiac diseases
• inc BM production of RBC, WBC and platelets Chronic Lymphocytic Leukemia
• EPO: elevated or high • Most indolent form of all leukemias
• Common among adults
C. Secondary with inappropriate production of EPO • 25% of all cases of leukemia in Western countries
• tumors of kidneys, liver, brain, adrenals • Median age: 60 years
• Inc RBC, Normal WBC and platelets
• EPO: elevated or high Chromosomal Abnormalities
• 50% abnormal karyotype
Relative
• Most common: deletion in chromosome 11, 13, 17 or
• Increase hematocrit, decrease plasma volume Trisomy 12
• dehydration • Require early treatment and have significantly shorter
• stress survival
• spurious polycythemia
• anxiety Clinical Features
• Often asymptomatic
Myelodysplastic Syndromes • Non-specific symptoms
• Clonal stem cell disorders characterized by: • LAD with hepatosplenomegaly (50-60%)
• maturation defects o The spleen and liver will become large because of
• ineffective erythropoiesis the increase accumulation of lymphocyte
• peripheral pancytopenia in spite of marrow cellularity
• called pre-leukemic state Laboratory Features
• it produces cells but it produces immature cells
• Absolute lymphocytosis
• result to anemia
• Often hypogammaglobulinemia, inc bacterial infection
o refractory anemia: in the bone marrow it able to
• 5% monoclonal serum IgG
produce cell but it produce immature form
• 10-15% autoantibodies to RBC, platelet
• (CD5+ cells)
• Smear: can see a smudge cell
 OUR LADY OF FATIMA UNIVERSITY
Bachelor in Medical Laboratory Science
HEMATOLOGY 1
TOPIC: Leukocyte Disorders

Prognosis • Rye: appearance on histologic lymph node biopsy

• Median survival: 4-6 years • Ann Arbor: staging based on tissue involvement
• Median survival: 4-6 years
• Not altered by therapy Special Stains
• Depend primarily on stage • It helps to enhance a particular component or structure
• Transformation to acute leukemia with blast crisis is of the cell
rare o those special structure are not usually visible if you
• Super imposed large cell lymphoma are just using ordinary wright stain
• Transformation to prolymphocytic leukemia
Myeloperoxidase (MPO) and Sudan Black B
Prolymphocytic leukemia o (+) AML
• A problem affecting the B or T cell o (-) ALL
• It respond poorly to treatment given o Myeloperoxidase: the specimen must be fresh within
• Common among men 24 hrs
• Rare, common among aged men o Sudan Black B: staining fats
• Massive hepatosplenomegaly with minimal LAD
Terminal Deoxyribonucleotidyl Transferase (TdT)
• Poor prognosis
o (+) ALL
• Absolute lymphocytosis
o (-) AML
Hairy Cell Leukemia
PAS: M6
• “Leukemic
Reticuloendotheliosis”
Napthol AS-D Chloroacetate Esterase: specific esterase
• (+) TRAP: Tartrate Resistant (+) granulocytic cells: mature cells and immature forms
Acid Phosphatase (-) monocytic cells: mature and immature forms
• Fine hair-like cytoplasmic
membrane projections α-Naphthyl Acetate Esterase and α-Naphthyl Butyrate
• Prominent splenomegaly Esterase: non specific esterase
• Tx: splenectomy (+) monocytic cells
• B Cell: CD19 or CD20+ (-) granulocytic cells

Mycosis Fungoides Acid Phosphatase: TRAP → (+) hairy cell Leukemia

• (+) PAS
• Incidence: elderly men Leukocyte Alkaline Phosphatase (LAP): PMN only
• Cuataneous T cell lymphoma + circulating lymphoma contains this activity
cells characterized by the enlargement of the spleen o Differentiate CML from leukemoid reaction
and lymphadenopathy o Count 100 neutrophils and grade it as:
• Starts with itching that leads to ulcerative tumors ▪ 0 – no red/brown ppt
• Splenomegaly & LAD ▪ 1+ - slightly diffused red ppt
• Types: pre-mycotic and mycotic ▪ 2+ - moderately diffused
• Form Cezary cells ▪ 3+ - heavily diffused
▪ 4+ - very heavily diffused
Lymphoma NV: 30-185
• Malignancy of lymphoid tissues • Increase in: Leukemoid reaction, PV, 3rd
• localized on the tissue later on will spread into trimester in pregnancy
the bone marrow or in the blood and the more • Decrease in: CML, PNH
disease deadly
• conduct biopsy • J. Toluidine Blue: binds with acid mucopolysaccharides in
blood cells
A. Non-Hodgkin’s: o used for recognition of mast cells and basophils
• Classification: Rappaport
• It proliferates neoplastic lymphocyte: cancer cells
or abnormal lymphocyte
• Associated with Burkitt’s type
B. Hodgkins:
• REED-STERNBERG CELL
• Epstein-Barr Virus
• Dx: definitive-lymph node biopsy
• Proliferation of cells reacting to cancer cells
• Normal cells that react against cancer cells

Classification