HEMATOLOGY 1

MT 303
COURSE DESCRIPTION:
The course deals with the study of the disease affecting red blood cells, white
blood cells and platelets. It also includes the study of laboratory diagnostic
method, clinical manifestation and pathology.

COURSE OBJECTIVES:
At the end of the course, the learners are expected to:

COGNITIVE
1.) Analyze the given clinical manifestation, laboratory test and pathology to
be able to identify the type of abnormality or disease.
2.) Evaluate the laboratory tests results during the assessment of specimen
collection, processing and handling.
AFFECTIVE
1.) Realize the importance of understanding the pathophysiology of each
disease concerning the blood cells in our body.
2.) Manifest the values of honesty, integrity, patient’s confidentiality, critical
thinking, empathy and value of life.
3.) Manifest the values of being a soon to be ,RMTs upholding the code of
ethics of the profession.

PSYCHOMOTOR
1.) Apply the concepts in determining the laboratory results of the patient in
relation to ethical practices medical technology
2.)Perform the manual laboratory techniques applicable to be able to identify
the underlying cause of the disease in relation with its clinical manifestation
and history of the patient.
HEMATOLOGY
Hematology is the branch of medicine concerned with the study of the cause,
prognosis, treatment, and prevention of diseases related to blood.

HISTORY OF HEMATOLOGY

1.) ARISTOTLE - The human is made up pf 4 elements; Fire, Air, Water and Earth
2.) William Harvey- First completed the description of the circulatory system
3.) Gabriel Andral (1879) – First described the complete classification of
leukocytes
4.) Paul Ehrlich (1891)- found out that certain mixtures of acidic and basic dyes
produced better staining
5.) Athanasius Kircher- in 1646, he used microscope to study the blood of
plague victims; In his Scrutinium Pestis of 1658 ,he noted the presence of “little
worms” or “animalcules” in the blood.
6.) Karl Ludwig- (1816-1895) He established that hemoglobin carries oxygen
7.) Jan Swammerdam – (1658) He differentiated RBC’s from mother cells
8.) Eduard Friedrich Pfluger – (1816-1895) He demonstrated that Carbon
dioxide was taken from the tissue and released in the lungs
9.) Malphigi- Pioneered the study of coagulation
10.) Max Perutz- Discovered the structure and function of hemoglobin
11.) Antoine van Leeuwenhoek- (1674) First described the human erythrocyte
12.) Giulio Bizzozero – Discovered the function of platelets
13.) Wharton Jones- (1846) First described polymorphonuclear cells from other
cells
14.) James Homer Wright- Modification of Romanowsky stain and
megakaryocyte origin
THE CIRCULATORY SYSTEM
ARTERY - are the blood vessels that bring
oxygen-rich blood from your heart to all of
your body's cells.
VEINS- blood vessels located throughout your
body that collect oxygen-poor blood and
return it to your heart.
CAPILLARY- where oxygen and nutrients are
exchanged for carbon dioxide and waste
VENULE-small blood vessel in the
microcirculation that allows deoxygenated
blood to return from capillary beds to larger
blood vessels called veins
ARTERIOLE- small branch of an artery leading
into capillaries.
CELLULAR RESPIRATION - converts ingested
nutrients in the form of GLUCOSE(C6 H12 O6)
and oxygen to energy in the form of
adenosine triphosphate (ATP). Carbon
dioxide (CO2) is produced as a byproduct of
this reaction.
THE HEART
- The heart is a fist-sized organ that
pumps blood throughout your body. It's
the primary organ of your circulatory
system.

HEART’S LOCATION
-Our heart is located between your lungs in
the middle of your chest, behind and slightly
to the left of your sternum. The apex of the
heart is located in the 5th intercostal space of
the ribcage.
WOUND/TISSUE INJURY - an injury to living tissue caused by a cut, blow, or other
impact, typically one in which the skin is cut or broken.

NOTE: WOUND,CUTS, TISSUE INJURIES

WILL ALWAYS RESULT TO INFLAMMATION.

INFLAMMATION - a localized physical condition in

which part of the body becomes reddened, swollen,
often painful, especially as a reaction to
injury or infection.

Inflammation is our body’s response to

Infections.
TISSUE REPAIR
-the restoration of tissue architecture and function following an injury.
SERUM vs. PLASMA

Coagulation - the action or process of a liquid, especially blood, changing to a solid

or semi-solid state

WHY DOES BLOOD COAGULATE?

Platelets play a vital role in the early response to vascular injury; at a wound site
when in contact with air, platelets become activated, agglomerate, and form platelet
clots adhering to injured blood vessel wall components.

WHAT DOES AN ANTICOAGULANT DO?

-prevents the blood from clotting (e.g. Ethylenediaminetetraacetic acid)
EDTA- prevents coagulation by chelating calcium.
 PREFIXES SUFFIXES

PREFIX MEANING SUFFIX MEANING

a-/an- lack,without,absent,decreased blast primitive

Ante Before ectomy to cut,excision

Cyto cell itis inflammation
Dys abnormal (o)logy study of
Ferr Iron (o)pathy disease
Hyper above,beyond,extreme penia decrease,lack of
Iso Equal,alike,same plasia cell production or repair
Macro Large or long poietin stimulates production
Mega Large or Giant trophy nourishment
Mono One cyte cell
Myel(o) From bone marrow emia blood
Phleb Vein lysis destruction or dissolving
Poikilo Varied or irregular oma tumor,swelling

Aniso Unequal osis state, condition, increase

Brady Slow poiesis cell production,formation,development
Dia through stasis same,standing still
Erythro red
Hemo Pertaining to blood
Hypo Beneath, deficient
Leuko White
Mal bad or abnormal
Meta After,next or change
Morph Shape
Pan overall, all , all inclusive
Phago eat,ingest
Schis Split
Scler Hard
Splen spleen
Thromb(o) clot
THE RED BLOOD CELL (ERYHTROCYTES)
-anucleate, biconcave, discoid cells filled with a reddish protein called HEMOGLOBIN.
-The biconcavity of the rbc provides a large surface-to-volume ratio and facilitates
gas exchange
-SALMON PINK in color
-Thin plasma membrane
-Lipid bilayer covering the cell’s cytoplasm
-Do not have cell organelles
-7 to 8 um in diameter , 2.6 um thick in the rim, and 0.75 um in the center
-Central pallor occupies 1/3 of their center reflecting their biconcavity
- Their main function is to TRANSPORT OXYGEN throughout the body
- main cell to be examined incases of ANEMIA or POLYCYTHEMIA

NOTE: RBCs has a lifespan of 120 days.

- Because of the lack of nucleus, erythrocytes cannot divide and thus need to
be continually replaced by new cells synthesized in the bone marrow.
-The development of erythrocytes from stem cells occurs in about 7 days via
the process called erythropoiesis.

RBC VOLUME IN THE BODY

WOMEN: 3.9-5.5 million/microliter

MEN: 4.1-6.0 million/microliter
AT BIRTH: 6.7 million/microliter

-Erythrocytes are flexible ,meaning, they tend to bend and adapt to small
diameters and irregular shapes of our blood vessels
RBC COMPOSITION
-62.5% WATER
-35% HEMOGLOBIN

The remaining 2.5% is :

-Glucose
-Cephalin, Cholesterol, Lecithin (LIPIDS)
-Glutathione (PROTEIN) –acts as reducing agent and prevents damage of
hemoglobin
-Carbonic Anhydrase and Catalase (ENZYMES)
-Na+ , K+ , Ca2+ PO43- (IONS)
The plasma membrane of erythrocyte is:
40% lipid
10% carbohydrate
50% water

-The PHOSPHOLIPID BILAYER membrane supports the structure of the RBCs

How is the shape of Erythrocyte maintained?

SPECTRIN
-principal membrane protein found in erythrocytes
-contractile protein
-maintains shape and flexibility of erythrocytes
-Responsible for manifesting surface antigens
FUNCTIONS OF RBC

-Respiratory
-Acid-Base Balance -Your blood needs the right balance of acidic and basic
(alkaline) compounds to function properly
-Maintain Viscosity
-Pigment –various pigments are derived from hemoglobin after disintegration
of RBC

NOTE: Normal blood PH is 7.35-7.45

FRAGILITY AND BREAKDOWN

HEMOLYSIS – breakdown of RBC and liberation of hemoglobin

Fragility – the susceptibility of an RBC to break easily (hemolyze)

Types of FRAGILITY

1.) OSMOTIC FRAGILITY – exposure to hypotonic saline (cell burst)

2.) MEACHANICAL FRAGILITY - trauma
Physiologic of increase count:
Age
Gender
High altitude
Exercise
Temperature
Meal

Physiologic decrease in count :

High barometric pressure
Pregnancy – Placental estrogen production = increased renal sodium reabsorption
Sleep
Pathological Variations

Increase: Polycythemia
Decrease : Anemia

Size variations :

Microcytes – smaller rbc

Macrocytes – bigger rbc
Anisocytes – constantly different sizes of rbc

Shape Variations:

Crenated RBC- due to hypertonic solution

Spherocytosis-Globular form in hypotonic solutions
Elliptocytosis
Sickle cell- crescent shape
Poikilocytosis-unusual shapes of RBC
FATE OF RBCs (Erythrocytes)

Lifespan – 120 days

Site of destruction – Reticuloendothelial system

RETICULOENDOTHELIAL ORGANS
Liver
Spleen (Graveyard of Erythrocytes)
Lungs
ANEMIA- a condition that
develops when your blood
produces a lower-than-normal
amount of healthy red blood cells.
Different types of anemia include:

• Anemia due to vitamin B12 deficiency

• Anemia due to folate (folic acid) deficiency
• Anemia due to iron deficiency
• Anemia of chronic disease
• Hemolytic anemia
• Idiopathic aplastic anemia
• Megaloblastic anemia
• Pernicious anemia
• Sickle cell anemia
• Thalassemia
POLYCYTHEMIA
- an abnormally high number of red blood cells in the blood, as a primary disease or
secondary condition (usually associated with lung or heart disease or living at high
altitude).
-High altitudes can cause low oxygen saturation levels or desaturation of an individual's
blood
- Also present in patients with chronic diseases

NOTE: HIGH ALTITIUDE = LOW OXYGEN SATURATION because of LOW ATMOSPHERIC

PRESSURE

OXYGEN SATURATION (SPO2)

-refer to the extent hemoglobin is bound or saturated to oxygen.

-measures oxygen bound hemoglobin vs. oxygen “not bound” to hemoglobin

Normal SPO2 level : 95%

NOTE: Lower than 95% requires immediate external oxygen supplementation to prevent
organ/systemic failure.
THE WHITE BLOOD CELL (LEUKOCYTES)
White blood cells, also known as leukocytes, are responsible for protecting
your body from infection.
As part of your immune system, white blood cells circulate in your blood and
respond to injury or illness.

What WBC are present in a peripheral blood smear?

GRANULOCYTES-type of white blood cell that has small granules inside them.
These granules contain proteins

AGRANULOCYTES-white blood cells that have no distinct granules in their

cytoplasm.
GRANULOCYTES
1.) NEUTROPHIL (SEGMENTERS)
-are the most numerous white blood cells, account for
50–70% of the WBC types. Neutrophils are about twice
as large as erythrocytes. The neutrophil cytoplasm
contains very fine granules (of two varieties) that are
difficult to see. Neutrophils get their name because their
granules take up both basic (blue) and acidic (red)
dyes. Together, the two types of granules give the
cytoplasm a lilac color..
Others, especially the smaller granules, contain
antimicrobial proteins, called defensins. Neutrophil
nuclei consist of three to six lobes.
Neutrophils are our body’s bacteria killers, and their
numbers increase explosively during acute bacterial
infections such as meningitis and appendicitis.

Neutrophils are chemically attracted to sites of

inflammation and are active phagocytes.
2.) EOSINOPHIL- account for 2–4% of all
leukocytes and are approximately the size
of neutrophils. Their nucleus usually
resembles an old-fashioned telephone
receiver— it has two lobes connected by a
broad band of nuclear material. Large,
coarse granules that stain brick red with
acid (eosin) dyes pack the cytoplasm.
These granules are lysosome-like and filled
with a unique variety of digestive enzymes.
However, unlike typical lysosomes, they lack
enzymes that specifically digest bacteria.
The most important role of eosinophils is to
lead the counterattack against parasitic
worms, such as flatworms (tapeworms and
flukes) and roundworms (pinworms and
hookworms) that are too large to be
phagocytized.
3.) BASOPHILare the rarest white blood cells,
accounting for only 0.5–1% of the leukocyte types.
Their cytoplasm contains large, coarse, histamine-
containing granules that have an affinity for the
basic dyes and stain purplish-black. Histamine is an
inflammatory chemical that acts as a vasodilator
(makes blood vessels dilate) and attracts other
white blood cells to the inflamed site; drugs called
antihistamines counter this effect.
• AGRANULOCYTES

1.) LYMPHOCYTE
-accounting for 25% or more of the WBC population, are the
second most numerous leukocytes in the blood. When stained, a
typical lymphocyte has a large, dark-purple nucleus that
occupies most of the cell volume. The nucleus is usually spherical
but may be slightly indented, and it is surrounded by a thin rim of
pale-blue cytoplasm.
Large numbers of lymphocytes exist in the body, but relatively
few (mostly the small lymphocytes) are found in the bloodstream.
In fact, lymphocytes are so called because most are closely
associated with lymphoid tissues (lymph nodes, spleen, etc.),
where they play a essential role in immunity.
T lymphocytes (T cells) function in the immune response by acting
directly against virus-infected cells and tumor cells.
B lymphocytes (B cells) give rise to plasma cells, which produce
antibodies (immunoglobulins) that are released to the blood.
4.) MONOCYTE
-account for 3–8% of WBCs. With an average
diameter of 18 μm, they are the largest
leukocytes. They have abundant pale-blue
cytoplasm and a darkly staining purple
nucleus, which is distinctively kidney shaped.
When circulating monocytes leave the
bloodstream and enter the tissues, they
differentiate into highly mobile macrophages
with prodigious appetites. Macrophages are
actively phagocytic, and they are crucial in
the body’s defense against viruses, certain
intracellular bacterial, parasites, and chronic
infections such as tuberculosis.
Continuation to RBC
-Since before 1900s, physicians and medical
laboratory professionals counted RBCs in measured
volumes to detect anemia and polycythemia.
-Historically, microscopists counted RBCs by carefully
pipetting a tiny aliquot of blood diluted in a Normal
Saline Solution (NSS) with a concentration of 0.85%.
-NSS matches the osmolality of blood; consequently,
the RBCs retain their morphology, neither swelling nor
shrinking.
- 1:200 dilution was typical for RBC count using a
thoma pipette
- The diluted blood is then transferred to a
Hemacytometer for reading
HEMACYTOMETER
- also known as “cell counting
chamber”
-a tool used for manual counting
of both WBC and RBC
- expensive
HEMOGLOBIN
- the protein contained in red blood cells that is
responsible for delivery of oxygen to the tissues

STRUCTURE OF HEMOGLOBIN
Heme- an iron-containing compound of the porphyrin class
which forms the nonprotein part of hemoglobin and some other
biological molecules.
Globin- protein/carrier
-4 polypeptide chains
-Each polypeptide chains has 1 heme group on it
-Each heme group is composed of an iron Fe2+
(FERROUS) and protoporphyrin
Among four globin molecules, two chains are alpha chains and two are non-alpha chains.
Based on the type of non-alpha chain present, three different types of hemoglobin are
found in normal adults

1.) HbA: >95%Composed of two alpha(α) and two beta(β) globin chains.
2.) HbA2: 0-3.5% Composed of two alpha(α) and two delta(δ) globin chains.
3.) HbF: 0-2.0% Composed of two alpha(α) and two gamma(γ) globin chains.

Hemoglobin can combine with other substances, some normally and some abnormally and
can also occur as:

Oxyhemoglobin: Oxygen combined with hemoglobin.

Carboxyhemoglobin: Carbon monoxide (CO) combined with hemoglobin. 210
Carbaminohemoglobin: Carbon dioxide (CO2) combined with hemoglobin.
Methemoglobin: Iron oxidized from its ferrous state to ferric state.
Sulfhemoglobin: Sulfur combined with the hemoglobin.
Cyanmethemoglobin: Methemoglobin bonded to cyanide ions.
Normal Values:
At birth: 25 g/dL (250 g/L)
At puberty: 14-16 g/dL (140-160 g/L)
Adult male: 13.5-18.0 g/dL (135-180 g/L)
Adult female: 12.0-15.0 g/dL (120-150 g/L)

How do we measure hemoglobin?

-Automated Hematology Analyzer
-Drabkin’s reagent
-Sahli’s hemoglobinometer set
DRABKIN’S REAGENT
- used for the quantitative, colorimetric determination of hemoglobin
concentration in whole blood at 540 nm.

COMPOSITION OF DRABKIN’S REAGENT

1.) Potassium cyanide
2.) Potassium Ferricyanide

PROCEDURE:
-a drop of whole blood is placed in the Drabkin’s reagent and mixed.
-The intensity of the solution is measured by a spectrophotometer at 540 nm
wavelength.
-The color intensity is measured by means of comparison to a known standard
and is mathematically converted to hemoglobin concentration.
SAHLI’S HEMOGLOBINOMETER METHOD

A common method for measuring the hemoglobin content of blood makes use of an
instrument known as a hemoglobinometer, which compares the color of light passing
through a hemolyzed blood sample with a standard color. The results of the test are
expressed as grams of hemoglobin per 100 ml of blood.

• Comparator: It is a rectangular plastic box with a slot in the middle which accommodates a
hemoglobin tube. Brown standard glasses are provided on either side of the slot for color
matching. White opaque glass is present at the back to provide uniform illumination.
• Hemoglobin tube: Sahli’s graduated hemoglobin tube is graduated in one side in gram
percentage (g%) from 2 to 24, and on the other side in percentage (%) from 20 to 140. The
tube is also called Sahli-Adams tube.
• Sahli’s pipette or hemoglobin pipette: It contains only one mark at 20μl or 0.02ml. Unlike
WBC and RBC diluting pipettes, it contains no bulb.
• Stirrer: It is a thin glass rod used for stirring the mixture inside the hemoglobin tube.
Reagents:
1.N/10 Hydrochloric acid (HCl): Mixing 36 grams HCl in distilled water to 1 liter
gives 1 N HCl. Diluting it 10 times will give N/10 HCl.
2.Distilled water

Procedure:
1.Ensure that the hemoglobinometer tubes and pipette are clean and dry.
2.Fill the hemoglobinometer tube with N/10 HCl up to its lowest mark 2 g% or
10% mark with the help of a dropper.
3.Take blood up to mark in the Sahli’s pipette (20 μl). Wipe the extra blood
outside the pipette and deliver it to N/10 HCl in the hemoglobin tube.
4.Mix and leave it for 10 minutes in order for a complete conversion of
hemoglobin to hematin.
5.Add distilled water drop by drop and stir till color matches with the standard
glass of the comparator.
6.Take the reading at lower meniscus, which directly gives the hemoglobin
concentration in 100 ml of blood.
HEMATOCRIT
-Also called as “Packed Cell Volume” (PCV)
- the ratio of the volume of packed RBCs to the volume of
whole blood
- the fractional volume of blood the erythrocytes occupy
-reliable index of red cell population

Normal Values:
Male: 40-54 % or 0.40-0.54 (L/L)
Female: 35-49% or 0.35-0.49 (L/L)
Infant: 30-43% or 0.30-0.43 (L/L)
Newborn: 53-65% or 0.53-0.65 (L/L)

Note:
Males have higher levels of androgens, like testosterone,
compared to females. Testosterone stimulates the release of
erythropoietin from the kidneys to stimulate the
erythropoietic process, resulting in an increased number of
red blood cells, a higher hematocrit, and a higher
hemoglobin content in the blood.
HEMATOCRIT DETERMINATION
It can be estimated by :

1.) Wintrobe’s method (MACRO)

2.) Microhematocrit (MICRO)

Microhematocrit (Micro-method)
-Hematocrit(HCT) is usually determined by spinning method using a blood- filled
capillary tube in a centrifuge machine

Procedure (reagents/instruments)
-Capillary tubes 75mm
-Microhematocrit centrifuge
-Microhematocrit reader
-Hematocrit tube sealant (Sealing clay)
NOTE:
BLUE Capillary tube does not have any
anticoagulant and does not have to be
mixed. RED Capillary tube (red band) has
heparin which prevents blood from clotting.

Microhematocrit centrifuge is designed for

spinning only capillary tubes.

Sealing Clay is used as a stopper.

PROCEDURE (SAMPLE)
-Capillary and venous blood in Lavender top or
directly collected blood.

PROCEDURE (STEP-BY-STEP)
1.) Mix the anticoagulated venous or capillary blood
gently 5 times.
2.) Fill 2/3 the capillary tube with blood (prefer to use
2 tubes for each sample. AVOID BUBBLES as it may
cause interference to the result.
3.) Seal one end of the tube with sealing clay + PERLA
and place these tubes in the microhematocrit
centrifuge.
4.) Centrifuge for 5 minutes at 10,000 rpm.
5.) Place the tube in the microhematocrit reader.
6.)The bottom margin of red cells layer is against the 0
mark of the scale, while the top margin should be at
100 mark of the scale.
7.) Adjust the sliding line so that it cuts between the
red cell and the buffy coat
8.) The reading should be in %.
PRECAUTIONS:
-Incomplete sealing of the capillary tube will give falsely low results.
-Shortened spin time or slowed centrifugation speed may yield falsely
elevated results.
-Microhematocrit centrifuge should not be forced to stop.

Source of Errors:
-Incorrect anti-coagulant concentration
-Incorrect mixing
-Storage for 6-8 hours
-Incorrect centrifugation
-Hemolysis
-Clots in the blood sample
WINTROBE METHOD (MACRO)

-A Wintrobe tube is a narrow glass tube

measuring 110-mm-long, with graduation from 0
to 100 mm in both ascending and descending
order. This method has been succeeded by the
“micro-hematocrit” method which uses a small
capillary tube instead of a Wintrobe hematocrit
tube.
-Also used for Erythrocyte Sedimentation Rate
RED BLOOD CELL INDICES

-”INDEX” ; a measure of something.

-Initial classification for anemia
-To determine the average volume and hemoglobin content and
concentration of the red blood cells in the sample
-Quality control check
-MCV,MCH, MCHC
MCV (Mean Corpuscular Volume)
-measures the average volume of the red blood cell
-Expressed in femtoliters (fL)
Formula:

REFERENCE VALUES:
Normal: 80 to 100 fL
Microcytic: <80fL
Macrocytic: >100fL

Sample problem:
RBC : 6.9 x 1012
HCT: 0.45 L/L
MCV interpretation: 65 fL; Microcytic
MCH (Mean Corpuscular Hemoglobin)
-Average weight of hemoglobin in a red blood cell
-Expressed in picograms (pg)

Formula:

Reference value:
Normal: 26 – 32 pg

Sample Problem:
Hgb: 15.6 g/dL
RBC: 4.5x1012
MCH interpretation: 34 pg
MCHC (Mean Corpuscular Hemoglobin Concentration)
-Average concentration of hemoglobin per red blood cell
-expressed as g/dL (formerly %)

Formula:

Reference value:
32-36 g/dL

Sample problem:
Hgb: 15.5
Hct: 45%
MCHC interpretation: 34 g/dL
HEMATOPOIESIS
-continuous, regulated process of renewal, proliferation, differentiation, and
maturation of all blood cell lines.
-these processes result in the formation, development and specialization of all
functional blood cells that are release from the bone marrow into the
circulation.
-Mature blood cells have limited life span and a cell population capable of
self-renewal that sustains the system
-A hematopoietic stem cell (HSC) is capable of renewal (replenishment) and
directed differentiation into all required cell lineages
-Hematopoietic stem cell serves as a functional model to study stem cell
biology, proliferation and maturation and their contribution to disease and
tissue repair.
STAGES OF HEMATOPOIESIS

1.) MESOBLASTIC PHASE

-Begins during embryonic development in blood islands of the yolk sac

-Starts: 9-20 days of GESTATION (conception to birth)
- Characterized by the development of primitive erythroblasts that produce
hemoglobin ( Portland, Gower-1, Gower-2)

EMBRYONIC HEMOGLOBIN (HBE)

Embryonic hemoglobin is a tetramer produced in the blood islands in the embryonic yolk
sac during the mesoblastic phase.
PORTLAND 1
PORTLAND 2
GOWER 1
GOWER 2
H-GOWER 1
-primary embryonic hemoglobin
-2 zeta and 2 epsilon chains
-relatively “unstable” ; breakdown easily

H-GOWER 2
-relatively low amount in embryonic and fetal life
-2 alpha and 2 epsilon chains
-although unstable but not as much as the GOWER 1

H-PORTLAND 1
-low levels during embryonic life
-2 zeta 2 gamma chains

H-PORTLAND 2
-low levels in embryonic life
-2 zeta and 2 beta chains
-very “unstable” that it rapidly breaks down during stress
2.) HEPATIC PHASE

-Begins at 4th to 5th week of gestation

-Clusters of developing erythroblasts, granulocytes and monocytes
-Definitive hematopoiesis
-Appearance of lymphoid cells
-Liver is the major site of hematopoiesis and retaining activity until 1-2 weeks after birth
-The spleen, kidney, thymus, and lymph nodes contribute to the hematopoiesis process

NOTE: Primitive hematopoiesis generates

mainly nucleated primitive erythrocytes.
Definitive hematopoiesis generates all
hematopoietic lineages, including nucleated
erythrocytes.
3.) MEDULLARY PHASE (MYELOID)

-At 5th month of development, hematopoiesis begins in the bone marrow

-Myeloid : Erythroid ratio (M:E ratio) reaches adult levels of 3:1 at 21 weeks of gestation
-Measurable levels of HBA1 , HBF, and HBA2
-Measurable levels of Erythropoietin (EPO) , Granulocyte colony stimulating factor
(G-CSF), and Granulocyte-macrophage colony stimulating factor (GM-CSF) is seen

NOTE:
Myeloid : Erythroid ratio is a comparison of relative proportions of WBC(Myeloid) and RBC (Erythroid).
-Constant normal value is 3:1

-Increased M:E ratio (6:1) may be seen in infection, chronic myelogenous leukemia, and erythroid
hypoplasia.

-Decreased M:E ratio (<2:1) may be seen in decreased granulocytic activity (WBC) or increased
erythroid cells (RBC).
THE BONE MARROW

Bone marrow is a spongy substance found in the center of

the bones. It manufactures bone marrow stem cells and
other substances, which in turn produce blood cells.

Resorption of cartilage and endosteal bone creates a

central space within the bone. (OSTEOCLASTS breaks
down bone tissues transferring calcium to the blood)

Projections of calcified bone called the trabeculae

radiate outside the bone cortex into the central space,
forming a three-dimensional matrix resembling a
honeycomb appearance.

The trabeculae provide a structural support for the

developing blood cells.
ADULT HEMATOPOIETIC TISSUES

In adults, hematopoietic tissue is located in the bone marrow, lymph nodes,

spleen, liver and thymus. The bone marrow contains developing erythroid,
myeloid, megakaryocytic, and lymphoid cells.

Lymphoid development occurs in both primary and secondary lymphoid

tissue.

Primary lymphoid tissue consists of the bone marrow and thymus and is where
T and B lymphocytes are matured.

Secondary lymphoid tissue, where lymphoid cells respond to foreign antigens,

consists of the spleen, lymph nodes, and mucosa-associated lymphoid tissue.
THE BONE MARROW
-Normal bone marrow has 2 major components:
A.) RED MARROW – developing blood cells and their progenitors (active)
B.) YELLOW MARROW- adipocytes (fat) with undifferentiated mesenchymal cells and
macrophages (inactive)

Between 5 to 7 years of age, adipocytes become more abundant and begin to

occupy the spaces in the long bones previously dominated by the active red marrows.
This process is called retrogression.
Yellow marrow is capable of reverting back to active red marrow in cases of increased
demand on the bone marrow, such as in excessive blood hemolysis.

Bone marrow contains hematopoietic cells, stromal cells and blood vessels (arteries,
veins, vascular sinuses).
Stromal cells originate from mesenchymal cells that migrate into the central cavity of
the bone.

BONE MARROW MICROENVIRONMENT

MESENCHYMAL CELLS - Mesenchymal stromal cells (MSCs) are the spindle shaped
plastic-adherent cells isolated from bone marrow, adipose, and other tissue sources,
with multipotent differentiation capacity in vitro. They are also considered as non-
hematopoietic stem cells.

STROMAL CELLS - include endothelial cells, adipocytes (Fat) ,macrophages and

lymphocytes, osteoblasts, osteoclasts, and reticular adventitial cells (fibroblasts).
STROMAL STEM CELLS GIVES RISE TO:

A.) Endothelial cells – broad flat cells that form a single continuous layer along the inner surface
of the arteries, veins, and vascular sinuses. It regulate the flow of particles entering and leaving
hematopoietic spaces in the vascular sinuses
B.) Adipocytes - large cells with a single fat vacuole; they play a role in regulating the volume of
the marrow in which active hematopoiesis occurs. They also secrete cytokines or growth factors
that positively stimulate HSC number and bone homeostasis.
C.) Macrophages – function in phagocytosis and both macrophages and lymphocytes secrete
various cytokines that regulate hematopoiesis. They are in the marrow space.
D.) Osteoblasts – are bone forming cells
E.) Reticular adventitial cells (ARCs) – form an in complete layer of cells on the abluminal
surface of the vascular sinuses
THE LIVER

-major site of blood cell production during 2nd trimester of fetal development
-Hepatocytes (liver cells) have many functions including protein synthesis and
degradation, coagulation factor synthesis, carbohydrate and lipid
metabolism, drug and toxin clearance, iron recycling and storage, and
hemoglobin degradation in which bilirubin is conjugated and transported to
the small intestine for eventual excretion.
-The lumen of the sinusoids contains Kuppfer cells (macrophage in the liver)
that maintain contact with the endothelial lining.
-Kuppfer cells removes debris from the blood that circulates through the liver,
and they also secrete mediators that regulates protein synthesis in the
hepatocytes.
THE SPLEEN
-graveyard of RBCs
-the largest lymphoid organ in the body
-lies beneath the diaphragm, behind the fundus of
the stomach in the upper left quadrant of the
abdomen
-rich blood supply; 350mL/min

3 TYPES OF SPLENIC TISSUE

1.) WHITE PULP – consists of scattered follicles with germinal centers (lymphocytes,
macrophages and dendritic cells)
2.) RED PULP – consists of vascular sinuses separated by cords of reticular cell
meshwork (cords of Billroth) containing loosely connected specialized macrophages.
This created a sponge-like matrix that functions as a filter for blood passing through the
region.
3.) MARGINAL ZONE – surrounds the white pulp and forms a reticular meshwork
containing blood vessels, macrophages, memory B-cells and CD4 T-cells
THE LYMPH NODES

-organs of the lymphatic system located along the

lymphatic capillaries that are parallel to but not part
of the circulatory system.
-These bean-shaped structure (1-5mm in diameter)
are typically present in groups or chains at various
intervals along the lymphatic vessels.
-Lymph nodes can be divided into an outer region
called the cortex and an inner region called the
medulla. An outer capsule forms trabeculae that
radiate through the cortex and provide support for
the macrophages and lymphocytes located in the
nodes.
-The medullary cord lie toward the interior of the
lymph node. These cords consists primarily of plasma
cells and B cells.
THYMUS

-Located in the chest, between the lungs and behind

the breastbone or sternum
-first, the thymus originates from the endodermal and
mesenchymal tissues
-second, the thymus is populated initially by primitive
lymphoid cells from the yolk sac and the liver
-This increased population of lymphoid cells physically
pushes the epithelial cells of the thymus apart;
however, their long processes remain attached to one
another by desmosomes (CELL STRUCTURE SPECIALIZED
FOR CELL-TO-CELL ADHESION)
-In adults, T-cell progenitors migrate to the thymus from
the bone marrow for further maturation.
-In other words, the thymus is responsible for making T-
cells
NOTE:

-Both T-cell and B-cell are produced in the bone marrow

-But the path in which they are distributed is different
-The T-cell is produced mainly in the bone marrow but later on, transported in
the thymus for further maturation whereas, the B-cell, also was produced in the
bone marrow but :

ANTIGEN DEPENDENT: SPLEEN, LYMPH NODES, TONSILS, PEYER’S PATCHES; OCCURS

FOLLOWING B-CELL ACTIVATION BY ANTIGEN BINDING AND CO-STIMULATION

ANTIGEN INDEPENDENT: BONE MARROW ; MATURATION OF LYMPHOID PROGENITORS

TO MATURED NAÏVE B-CELL
STEM CELL THEORY
-1961
-Till and McCulloch
-irradiated spleens and bone
marrows of mice, creating a state
of aplasia
-given intravenous injection of
marrow cells
-HSC were seen 7 to 8 days
-CFU-S (Colony forming unit
spleen)
-The CFU-S represents what we
now refer to as COMMITTED
MYELOID PROGENITORS OR
COLONY FORMING UNIT-
GRANULOCYTE, ERYTHROCYTE,
MONOCYTE AND
MEGAKARYOCYTE (CFU-GEMM)
-These cells can give rise to
multiple lineages of blood cells
CULTURED COLONY FORMING UNITS (COIN TERM)

• CFU-GEMM – Colony forming unit Granulocyte, Erythrocyte, megakaryocyte, monocyte

• CFU-E – Colony forming unit Erythrocyte
• CFU-Meg – Colony forming unit Megakaryocyte
• CFU-M – Colony forming unit Monocyte
• CFU-GM – Colony forming unit Granulocyte, Monocyte
• CFU-BASO – Myeloid to Basophil
• CFU-G – Myeloid to neutrophil
• CFU-EO - Myeloid to eosinophil
• CFU pre-T – T lymphocyte
• CFU pre B – B- lymphocyte
Cytokines - small proteins that are crucial in
controlling the growth and activity of other
immune system cells and blood cells. When
released, they signal the immune system to do its
job. Cytokines affect the growth of all blood cells
and other cells that help the body's immune and
inflammation responses
CYTOKINE SOURCE TARGET CELL BIOLOGIC ACTIVITY CURRENT/POTENTIALTHERAPEUTIC
APPLICATION

ERYTHROPOIETIN KIDNEY (Peritubular BFU-E and CFU-E Stimulates proliferation of Anemia secondary to
erythroid progenitors and CKD
(EPO) epithelial cell) prevents apoptosis of CFU-E
GRANULOCYTE COLONY Endothelial cells, placenta, Neutrophil precursors, Stimulates granulocytes Congenital neutropenia,
STIMULATING FACTOR (G-CSF) monocytes, macrophages fibroblasts, leukemic colonies Idiopathic neutropenia
myeloblasts
GRANULOCYTE, T-CELLS, MACROPHAGES, Bone marrow Promotes antigen Chemotherapy induced
MACROPHAGE COLONY ENDOTHELIAL CELLS, treatment, Leukemia
STIMULATING FACTOR (GM- FIBROBLASTS, MAST CELLS progenitor cells, NKT- presentation, T- cell treatment
CSF) cells homeostasis
IL-2 (Interleukin 2) CD4 T cells, NK cells, T-cells, NK cells, B Cell growth/activation Metastatic melanoma,
B- cells cells, Monocytes of CD4 and CD8 T cells Renal cell carcinoma

IL-3 (Interleukin 3) Activated T cells, NK Hematopoietic stem Proliferation of Stem cell

cells cell hematopoietic mobilization, Bone
progenitors marrow failure
IL-6 (Interleukin 6) T-cells, macrophage, T-cells, Macrophages Co-stimulation with other Stimulation of platelet
fibroblasts cytokines, Megakaryocyte production
maturation, T and B cell
growth
IL-10 (Interleukin 10) CD4 and CD8 T-cells T-cells macrophages Inhibits cytokine Target lymphokines in
production prevention of B-cell
lymphoma
IL-12 (Interleukin 12) Macrophages T-cells T-cell differentiation Allergy treatment

IL15 (Interleukin 15) Activated CD4 T cells CD4 and CD8 T cells CD4 and CD8 proliferation Melanoma, Rheumatoid
arthritis
CYTOKINES SOURCE TARGET CELL BIOLOGICAL CURRENT/POTENTIALTHE
RAPEUTIC APPLICATION
ACTIVITY

IFN –a Dendritic cells, NK Macrophages, NK Antiviral, Enhances Adjuvant treatment

cells, T cells, B cells, cells MHC expression for stage II/III
Macrophages, (Major melanoma
Fibroblasts, Histocompatibility
Endothelial cells, complex)
Osteoblasts
Erythropoiesis
-It is the process of formation or production or RBC or Erythrocytes.

Terminology
-RBCs are formally called erythrocytes. Nucleated RBC precursors, normally
restricted to the bone marrow are called erythroblasts.
-They also may be called normoblasts, which refers to the developing RBC
precursors with normal appearance

3 Nomenclatures of RBC development

1.) Normoblastic – commonly used in the USA
2.) Rubriblast – parallels the nomenclature used for granulocyte development
3.) Erythroblast – commonly used in Europe
Morphologic determination of blood cells depends on a well-stained
peripheral blood smear. The stage of maturation of any blood cell is
determined by careful examination of the nucleus and cytoplasm. The most
important features in the identification of RBCs are the nuclear chromatin
pattern (texture, density and homogeneity), nuclear diameter, nucleus :
cytoplasm ratio (N:C ratio) presence or absence of nucleolo and cytoplasmic
color.
THE MATURATION
SEQUENCE (ERYTHROCYTE)
1.) Pronormoblast /
Proerythroblast / Rubriblast
-14 – 20 um in diameter
-Deeply basophilic
cytoplasm
-Non-granular
-N:C ratio is 8:1
-Fine chromatin
-Usually 1-2 nucleoli
2.) Basophilic Normoblast /
Basophilic Erythroblast /
Prorubricyte
-12-17um in diameter
-intensely basophilic cytoplasm
-N:C ratio 6:1
-Chromatin is slightly coarse
-Nucleoli is usually not visible
3.) Polychromatophilic Normoblast /
Polychromatophilic Erythroblast /
Rubricyte
-10-15um
-blue-gray to pink-gray cytoplasm
-N:C ratio is 4:1
-Last stage of mitosis
4.) Orthochromatophilic
Normoblast / Orthochromic
Erythroblast / Metarubricyte
-7-12um in diameter
-pink cytoplasm
-N:C ratio is 2:1
-Small pyknotic nucleus
-Last nucleated stage
5.) Polychromatic Erythrocyte / Diffusely basophilic Erythrocyte / Reticulocyte
-7-10 um in diameter
-pink to slightly pinkish gray cytoplasm
-contains fine basophilic reticulum RNA which is only demonstrated in a
supravital stain
6.) MATURE RBC / ERYTHROCYTE
-6-8 um in diameter (Rodak’s)
-salmon pink in color
-no nucleus , round, biconcave
BLOOD- participates in the physiologic and pathologic activities in the body

Functions of blood
-Respiration
-Nutrition
-Excretion
-Buffer
-Transports human and other endocrine substances needed for normal body
functions
-Maintenance of a constant body temperature and slight alkalinity of tissues
-Protection
COMPONENTS OF BLOOD

1.) Plasma (55%) – 90% water and 8% solutes (protein, gas, electrolytes, organic nutrients, hormones
and metabolic wastes)
2.) Buffy coat (<1%) – platelets and leukocytes
3.) Formed elements (45%) – Red blood cell, white blood cell and platelets

BLOOD VOLUME
1.) Normovolemia – 5-7L
2.) Hypovolemia
-<5L ; decreased blood volume
-Small amount of nutrients to be given to organs and tissues
-Tachycardia

Causes:
A.) Loss of whole blood
B.) Loss of body fluid
C.) Loss of plasma
3.) HYPERVOLEMIA
->7L ; increased amount of blood / blood volume
-Strain on heart because of the weight
-Cases of cardiac arrest (Too much fluid will increase your blood pressure and
force your heart to work harder.)

Causes:
A.) Pregnancy (Pregnancy retains sodium, thus, retains water)
B.) Intravenous fluid ingestion
C.) Massive blood transfusion
ORDER OF DRAW
-Blood collection tubes must be drawn in a specific order to avoid cross-
contamination of additives between tubes.

1.) YELLOW TOP ; Blood culture bottle/Microbiology (SPS)

2.) LIGHT BLUE TOP (Sodium Citrate)
3.) RED/PLAIN TOP (No additive)
4.) GOLD TOP (No additive but with GEL SEPARATOR)
5.) DARK/LIGHT GREEN TOP (Sodium Heparin)
6.) LAVENDER TOP (EDTA)
7.) GRAY TOP (Sodium Fluoride)

NOTE: Tube must be thoroughly mixed with its

corresponding anticoagulant/additive.
 Tube Color Anticoagulant/Additive Mechanism of Action Specimen Type Use/s

RED (Glass) None N/A Serum Chemistry,

Serology
RED (Plastic) Clot Activator Provides surface for the Serum Chemistry,
platelets to enhance Serology
clotting
Lavender (Glass) K3 EDTA – Sequestrene Chelates calcium ions Whole blood Hematology
(LIQUID)

Lavender (Plastic) K2 EDTA- Versene Chelates calcium ions Whole blood Hematology
(Spray dried form)
Pink K2 EDTA- Versene Chelates calcium ions Whole blood Blood bank and
(Spray dried form) molecular
diagnostics
White EDTA and gel Chelates calcium ions Plasma Molecular
diagnostics
Light blue Sodium Citrate Chelates or binds calcium Plasma Coagulation studies
ions ; PT and APTT
Black Sodium Citrate Chelates or binds calcium Plasma ESR
ions
Light green Lithium heparin Inhibits thrombin Plasma Chemistry,
Ammonia, HLA
Tube Color Anticoagulant/Additive Mechanism of Action Specimen Type Use/s

Dark green Sodium Heparin Inhibits thrombin Plasma Routine Chemistry,

Blood gas analysis

Royal Blue K2 EDTA , Sodium Inhibits calcium ions Plasma Toxicology,

Heparin
and inhibits thrombin Heavy metals
Gray Sodium Fluoride / Inhibits glycolysis Plasma Glucose
Potassium oxalate
testing
Yellow Sodium Inhibits complement, phagocytes
and certain antibiotics
Serum (Sterile) Blood culture
Polyanetholsulfonate

Yellow Acid Citrate Dextrose WBC preservative Plasma HLA phenotyping,

Paternity testing

Tan (glass) Sodium heparin Inhibits thrombin Plasma Lead testing

Tan (plastic) K2 EDTA Chelates or bind Plasma Lead testing

calcium
Orange Thrombin Clot activator Serum Chemistry
SPECIMEN COLLECTION
• Fasting – NPO (Non-per-orem) “Nothing by mouth” ; 8-14 hours
• Random – Collected anytime
• Basal state – Early morning
• Post-prandial – collected after meals

CLASSIFICATION OF SAMPLE REQUIREMENTS

• Macromethod- installs blood sample 1mL and above
• Micromethod – installs blood sample from 0.1 to 0.9 mL
• Ultramicromethod – blood sample from 0.01 to 0.09 mL
• Nanoliter method – blood sample from 0.001 to 0.009 mL
GOING BACK TO RBC

RBC MEMBRANE
-allow ion and nutrient passage
-allow cell to deform when required
-Sperate intra- and extracellular
Fluid environment of the plasma
Structure: Trilaminar (3 layers)

3 layers of the cell membrane

1.) Outermost layer : Glycolipids , glycoprotein
2.) Central layer: Cholesterol, Phospholipids
3.) Inner layer: Cytoskeleton

Also present in the membrane is :

1.) Integral protein

2.) Peripheral protein
INTEGRAL PROTEINS
-Glycophorins
-Band 3 protein ( chloride shift )

PERIPHERAL PROTEINS
-Trophomyosin
-Spectrin
-Actin
-Protein 4.1
-Protein 4.2
-Ankyrin
INTEGRAL PROTEINS (ZETA POTENTIAL)
-Glycophorins
-Band 3 protein ( chloride shift)
CYTOSKELETON
-Formed by structural protein
-Hexagonal lattice with 6 spectrin molecules (Basic Unit)
-Tail end: tetramers linked to actin and protein 4.1
-Head end: spectrin linked to ankyrin

PLANE OF DESIGN
1.) VERTICAL INTERACTION – Stabilize the lipid bilayer membrane
2.) HORIZONTAL INTERACTION – Maintain the biconcavity of RBC
DEFECTS IN RBC CELL MEMBRANE

1.) HEREDITARY SPHEROCYTOSIS

-Hereditary hemolytic disorder (most common)
-Mutation in membrane protein
-Ankyrin deficiency
-Alpha or Beta spectrin deficiency
-Band 3 abnormalities
-Protein 4.2 abnormalities
2.) HEREDITARY ELLIPTOCYTOSIS
-Oval shape RBC
-Rare
-Alpha or Beta spectrin mutants leading to defective spectrin dimer formation
-Alpha or Beta spectrin mutants leading to defective spectrin-ankyrin
associations
-Protein 4.1 deficiency or abnormality

3.) South-East Asian Ovalocytosis

-Band 3 deletion
RBC ENZYME SYSTEM
1.) Na+, K+ ATPase (Sodium-Potassium Pump)
-Controls active transport of SODIUM out of the cell and POTASSIUM into the
cells, therefore maintaining high levels of INTRACELLULAR POTASSIUM
-Increased SODIUM without POTASSIUM loss causes cells to swell and lyse
-helps to maintain osmotic equilibrium and membrane potential in cells.
-ACTIVE TRANSPORT
-Uses ATP
2.) Ca+,Mg+ ATPase (Calcium pump)
- Acts calcium out of the cell to the plasma against a high concentration
gradient
-Calcium helps regulate and stabilize membrane phospholipid structure
-Increased intracellular calcium prevents cell deformity
-Magnesium converts inactive form of Vitamin D (Ergocalciferol and
Cholecalciferol) into its active form (calcitriol) so that it can help absorb
calcium
-ACTIVE TRANSPORT
-Uses ATP
What is ACTIVE TRANSPORT?
-In cellular biology, active transport is the movement of molecules or ions
across a cell membrane from a region of lower concentration to a region of
higher concentration against the concentration gradient.
-It requires energy, therefore, ATP.

So if there is an ACTIVE TRANSPORT, there is also a PASSIVE TRANSPORT.

-Passive transport is the movement of ions without the use of ATP.
Example:
-Diffusion
-Osmosis

So how do RBCs get ATP?

ENERGY METABOLISM
Energy is required for the following:
-preserve the membrane shape
-Enzymatic reactions
-Movement of Calcium, Sodium and Potassium in and out of the cell
-Reduce oxidized proteins in Hemoglobin
-Hemoglobin must be maintained in its reduced state for proper function
-Ferrous to ferric state results in the formation of methemoglobin

2 sites prone to oxidation

1.) Iron atom in the heme ring (Acquired or hereditary)
2.) Sulfhidryl group of the globin (Causes “Heinz bodies” or precipitated HGB)
GLYCOLYSIS
-is the process in which glucose is broken down to produce energy. It
produces two molecules of pyruvate, ATP, NADH and water.
-The process takes place in the cytoplasm of a cell and does not require
oxygen.

Importance of glycolysis in the red blood cells:

-Energy production: It is the only pathway that supplies ATP (energy) to RBCs
-Reduction of methemoglobin: Glycolysis provides NADH for reduction of
methemoglobin by NADH-cytochrome b5 reductase
-2,3-disphosphoglycerate (2,3 DPG) accumulates in mammalian erythrocytes,
where it facilitates the supply of oxygen to the tissues by binding to
hemoglobin. In glycolysis, it binds to hemoglobin causing Oxygen to increase
its affinity to hemoglobin.
EMBDEN-MEYERHOF PATHWAY (ANAEROBIC GLYCOLYSIS)

So how does it work?

-RBC doesn’t have the ability to undergo the citric acid cycle or
the so-called KREB’S CYCLE because RBCs doesn’t have a nuclei,
ribosomes and mitochondria. A basic CELL has the ability to do
such, therefore, for the RBCs to generate ATP, they must undergo
anaerobic glycolysis.

KREB’S CYCLE – uses OXYGEN

EMP – doesn’t use OXYGEN

GLUCOSE – enters the RBC through a protein transmembrane

called GLUT-1
 1ST PHASE

SUBSTRATES ENZYME PRODUCT

GLUCOSE, ATP HEXOKINASE G6P,ADP

G6P G-6-P ISOMERASE F6P

F6P, ATP 6-PHOSPHOFRUCTOKINASE F1,6-BP,ADP

F1,6-BP FRUCTOSE-BIPHOSPHATE ALDOLASE DHAP,G3P

1ST PHASE: 2 ATP WAS CONSUMED

2ND PHASE: 2 ATP IS SUBSTRATES ENZYME PRODUCT
GENERATED
G3P GLYCERALDEHYDE-3- 1,3-BPG
PHOSPHATE
DEHYDROGENASE

1,3-BPG,ADP PHOSPHOGLYCERATE KINASE 3PG,ATP(2)

1,3-BPG BIPHOSPHOGLYCERATE 2,3-BPG

MUTASE

2,3-BPG BIPHOSPHOGLYCERATE 3-PG

PHOSPHATASE
3RD PHASE: 2 ATP IS GENERATED

SUBSTRATES ENZYME PRODUCT

3-PG PHOSPHOGLYCERATE MUTASE 2-PG

2-PG ENOLASE PEP

PEP,ADP PYRUVATE KINASE PYRUVATE, ATP (2)

EMP needs 2 ATP to Generate 4 ATP

GROSS ATP GENERATED: 2 ATP
PYRUVATE TO LACTATE
-Pyruvate may diffuse from the erythrocyte or may become a substrate for
lactate dehydrogenase with regeneration of the oxidized form of NAD.
- Lactic acid fermentation converts the 3-carbon pyruvate to the 3-carbon
lactate and regenerates NAD+ in the process, allowing glycolysis to continue
to make ATP in low-oxygen conditions.
GLYCOLYSIS DIVERSION PATHWAYS

Three alternate pathways, called the diversion or shunts, branch from the
glycolytic pathway. The three diversions are the:

1.) Hexose Monophosphate Pathway

2.) Methemoglobin reductase pathway
3.) Rapoport-Luebering pathway
HEXOSE MONOPHOSPHATE PATHWAY

-also known as the pentose phosphate shunt

-Peroxide or (H2O2) arises from oxygen reduction . It oxidizes and destroys
heme iron.
-HMP detoxifies peroxide to prolong the functional life span of the RBC.
-HMP diverts Glucose-6-phosphate (G-6-P) to ribulose-5-phosphate by the
action of Glucose-6-phosphate dehydrogenase (G6PD).
-In the process, oxidized NADP is converted to reduced form NADPH.
-G6PD provides the only means of generating NADPH for glutathione
reduction. (GSSG to GSH).
-Without the reduced form of glutathione, RBCs become vulnerable to
oxidation.
METHEMOGLOBIN REDUCTASE PATHWAY

-Heme iron is constantly exposed to oxygen and peroxide

-Peroxide oxidizes heme iron from FERROUS to FERRIC STATE
-Although HMP prevents hemoglobin damage through reducing peroxide, it is
not able to reduce METHEMOGLOBIN once it forms
-NADPH is able to do so
-NADPH with the presence of methemoglobin reductase (cytochrome b5-
reductase)
-Using HYDROGEN (H+) from NADH formed when G3P is converted to 1,3-BPG,
cytochrome b5 reductase acts as an intermediate electron carrier, returning
the ferric state to ferrous state.
RAPOPORT-LUEBERING PATHWAY

-generates 2,3-bisphosphoglycerate or 2,3 diphosphoglycerate (2,3-DPG)

-1,3-BPG is diverted by bisphosphoglycerate mutase to form 2,3-DPG
-2,3-DPG regulates oxygen delivery to tissues by competing with oxygen for
the oxygen-binding site of hemoglobin.