GEN CAMATO

BLOOD VOLUME

HEMATOLOGY 1 | LECTURE

BLOOD VOLUME
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Total amount of blood in the circulation
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Normal blood volume

Male: approx. 5.8 L

Female: approx. 4.5 L

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Definition of terms:

Normovolemia
Normal volume of blood in the body

Hypovolemia

Decrease in the volume of the blood

Hypervolimia

Excessive volume of blood in the body

Oligemia

Total reduction of blood volume

A condition in which the total volume of

the blood is reduced

NORMAL MATURATION OF CELLS

3 Principles:
1) Cytoplasmic Differentiation
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Several Factors:

Loss of Basophilia

Associated with color blue

Nucleus
Acidic part of the cell
Methylene Blue

Cytoplasm
Basophilic part of the
cell
Eosin

Cytoplasmic Granules

Increase in granules, the

more mature

Elaboration of hemoglobin

Increase in hemoglobin, the

more mature

FACTORS RELATED TO DECREASE () BLOOD VOLUME

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Loss of whole blood

Hemorrhage
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Loss of RBC

Spherocytosis

An auto-hemolytic anemia
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Loss of plasma

Burned patients, Dehydration

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Loss of body water

Decrease consumption of water

2)

Nuclear Maturation
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Subdivisions:
a) Structure & Cytochemistry

Presence of round/oval
nucleus

Young cell-compact

Large nucleus to cytoplasm

ratio

The larger the

nucleus, the more
immature

The larger the

cytoplasm, the more
mature

Nuclear chromatin

Rich in RNA

The more nuclear

chromatin, the
younger the cell

Decrease in the number of

Normal neutrophil
nucleoli

2-5 lobes

Found on the most

Hypersegmented neutrophil
immature stage

More than 5 lobes

The more lobulated,

Pernicious Anemia
the more mature

b) Changes in the shape of nucleus

Lobulation of nucleus

The more lobulated,

the more mature

FACTORS RELATED TO INCREASE () BLOOD VOLUME

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During blood transfusion
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Intravenous fluid
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Due to increase fluid intake
FORMULA:
Plasma Volume + Packed Cell Volume = Total Blood Volume
Blood = 45% Formed Elements (Cells) + 55% Liquid Elements (Plasma)

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Plasma volume should comprise 55% of the total blood

volume
Packed cell volume should be round 45% of the total blood
volume

Total reduction of blood volume

Packed Cell Volume

Measured by counting with use of radioactive

materials

Radioisotopes for the determination

of Packed Cell Volume (PCV)

Examples:
Cr-51, P-32, Radio Iron
Inject Radioisotopes: attach to
RBC
Plasma Volume

Measured by intravenous injection of foreign

substances of known amount and concentration
that will allow the measurement of the body fluid

Could be radio active material (radioisotope iodine

131 and with the use of dyes)
Examples of Dyes:

Evans Blue

Congo Red

Labeled Human Serum Albumin

3)

Reduction of cell size

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The smaller the cell, the more mature
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The larger the cell, the more immature

GEN CAMATO

BLOOD VOLUME

HEMATOLOGY 1 | LECTURE

HEME SYNTHESIS
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FACTORS AFFECTING THE HEMOGLOBIN AFFINITY (ATTACHMENT)

WITH OXYGEN

First Step

Condensation of succinyl co-enzyme A + glycine in

the presence of pyroxidal phosphate and delta
aminolevulinic acid synthesis will react to co-enzyme A
and glycine to produce delta aminolevulinic acid
dehydrogenase

I.

Body Temperature
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An increase in the body temperature causes the
hemoglobin to release oxygen more readily
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* In Body temperature = in Oxygen released

Shift to the right

In the release of Oxygen &

release of uptake in Carbon
dioxide

II.

Blood pH/Bohr Effect

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The relationship of the blood pH and oxygen affinity
is what we call the Bohr effect which tells us an
increase in pH favors the uptake of Oxygen
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* pH = Alkaline
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* Blood pH = uptake of Oxygen

Shift to the left

in the uptake of Oxygen &

release of Carbon dioxide

III.

2,3 Diphosphoglycerate (DPG)

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An increase amount of DPG level will cause a
diminish oxygen affinity

Shift to the right

DPG = Oxygen (release)

affinity

IV.

Carbon Dioxide
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An increase of Carbon dioxide will diminish the
affinity of Oxygen

Shift to the right

V.

Presence of HbF (Fetal Hemoglobin)

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An increase of HbF in the fetus will cause increase
oxygenation of the fetal blood

Shift to the left

*For adults: Shift to the right (Not Normal)

VI.

Presence of Hemoglobin Variance

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The number of normal hemoglobin balance will show
either shift to the left or right
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Different types of Hemoglobin

HbM & HbO Right

HbA2 & HbA Left

VII.

Decrease affinity of oxygen and increase carbon dioxide

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Haldane Effect

Affinity = Right

Synthesis of nucleus

Spleen graveyard of RBC

RBC (120 days)

Hemoglobin

heme globin (reabsorbed protein)

Iron (Fe) Porphyrin

HEMOGLOBIN
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Red coloring pigment of the blood
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Main component of RBC

35% of the total RBC

Sometimes called respiratory pigment because of its capacity to

transport O2 and CO2

A gram of hemoglobin can carry approximately 1.34mL of

Oxygen and 3.47mg of Iron

The adult RBC mass of 600grams hemoglobin can carry

approximately 800mL of Oxygen

Women need 400-800g of iron supplement

Conjugated CHON (Protein) that is made up of 1 globin molecule of

heme groups and the globin is divided into 4 polypeptide chains

Each chain is attached to 1 heme group

Heme

Heme

1 Globin Molecule
Heme

Heme

Polypeptide chain

Made up of amino acid

In normal adult hemoglobin, it is made up of

2 alpha and 2 beta chain

Alpha chain

Made up of 141 amino acid

First Amino Acid Valine

Last Amino Acid Arginine

Beta chain

Made up of 146 amino acid

First Amino Acid Valine

Last Amino Acid Histidine

Body Temperature shift to the LEFT

HbF adult shift to the RIGHT
Haldane Effect shift to the RIGHT
Oxygen uptake shift to the LEFT
2, 3 DPG shift to the LEFT

The rest of the globin chain contains 146 amino acids

2

GEN CAMATO

BLOOD VOLUME

HEMATOLOGY 1 | LECTURE

CLASSIFICATION OF HEMOGLOBIN
A.

Normal Hemoglobin
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Capable of transporting blood gases particularly
Oxygen & Carbon dioxide

According to Function

Oxyhemoglobin
Arterial blood
Bright red
Oxygen

Reduced Hemoglobin
Venous blood
Dark red
Carbon dioxide
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Hemoglobin in Adult (post-neonatal)

HbA

Constitutes majority of adult Hg

96-98% Hg

2 & 2 chains

HbA2

Normal adult Hg

1.5 3% total types of Hg

2 & 2 (delta) chains

HbF

Major hg in fetus (90-95% hg)

0.5 1% hg

2 & 2 (gamma) chains

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B.

Abnormal/Non-functional Hemoglobin
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Formed due primarily to a defect in the
polypeptide chain of hemoglobin molecule
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Category:
Carboxyl Hemoglobin (HbCO)

Reacts with Carbon monoxide in

which the affinity of CO 218x
greater than Oxygen at 37C

Do not transport oxygen that may

lead to hypoxia & finally death of
the patient

Produces cherry red blood color

Concentration to non-smokers:
0 2.3%

Smokers: 2.1 4.2%

Test for HbCO

Katayamas Test
a. 10 mL Distilled H2O
b. + 5 drops of blood
from patients/ control
blood
c. + 5 drops of
Ammonium sulfide
d. + Acidify with acetic
acid

3.

Met Hemoglobin

A.k.a.

Hemiglobin

Oxidized Hemoglobin

Ferri Hemoglobin

Reversible reaction

Normal individual
approximately 1% of
methylene blue

Characteristic color:
Chocolate brown

Hemoglobin C

Found also among blacks, rare in whites

Appears also in homozygous & heterozygous

state

If present, it demonstrate target cell

Target cell

Condensation of Hg at the
center/periphery

A.k.a.

Bulls eye cell

Codocyte

Leptocyte

Mexican hat cell

Happy Hormones:
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Serotonin
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Adrenaline
{
Epinephrine

if NORMAL BLOOD : Dirty greenish, Brown

if HbCO (+): Rose red color
Other Test for HbCO:
r
Sanderman Dethionite Test
r
Palmers Test

Yersinia enterocolitica

Hemoglobin D

Most common for HbD Purijab & HbD Los Angeles

Both alpha & beta chain abnormally have been

reported

Shows no sickling of RBC

Blood pH becomes basic

Humble yourselves, therefore, under Gods mighty hand, that he may lift you
up in due time. Cast all your anxiety on him because he cares for you.

- 1 Peter 5:6,7

Sulfhemoglobin

Blood with combined sulfide

Benign condition

With significant effect of

cyanosis

Cause & effect of drug

Mauve lavender color

VARIANCE OF HEMOGLOBIN
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Hemoglobin S

Causes sickling of RBC

Confined to blacks

Homozygous state causes sickle cell anemia

Heterozygous state causes/shows sickle cell

fomite
Heterozygous carrier parents
If both parents HbS(+) = Sickle Cell Anemia
HbS + HbA = Sickle cell Trait
HgS + HgS = Sickle cell Anemia
HgS + HgA = Sincle cell shape (carrier)

Embryonic Hemoglobin (Pregnant)

Gower 1

2 (epsilon) & 2 (zeta) chains

Gower 2

2 & 2 chains

Hemoglobin Portland

2 (zeta) & 2 gamma chains

1.

2.

GEN CAMATO

BLOOD VOLUME

HEMATOLOGY 1 | LECTURE

HEMOGLOBINOMETRY
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Screening test associated with anemia
FACTORS ASSOCIATED WITH INCREASED OF HEMOGLOBIN
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Hyperchromia

Increase intensity in the color of hemoglobin

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Polycythemia

Increase in RBC
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High altitude

Depletion of oxygen

Hypoxia will trigger the release of erythropoietin

in kidney Bone marrow RBC
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Dehydration

Loss of fluid within the body

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Heart disease

Patient with heart problem will have

hemoglobin pigmentation because of the
compensation of erythropoietin
FACTORS IN DECREASE HEMOGLOBIN CONCENTRATION
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Pregnancy

Competition for hemoglobin with the fetus

Ferric sulfide/iron supplements needed ^

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Anemia
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Women

Females naturally have low hemoglobin

because of menstruation

Males naturally have high hemoglobin because

of testosterone (which is associated with
maturation/production of RBCs)

Normal low hemoglobin counts

It is quite common for women to experience low

hemoglobin counts during pregnancy.

Some people might also experience this as a

natural way of life it is simply how their body
works.

Low counts in these cases shouldnt be alarming.

Conditions and diseases that cause fewer than normal
red blood cells

Some conditions can cause lower numbers of red

blood cells which can lead to a low hemoglobin
count

Anemia, cancer, cirrhosis (scarring/ tumor in the liver |

Kirros means yellow), kidney disease and lead
poisoning are just a few reasons.

Conditions and diseases that destroy red blood cells

Sometimes your body cant produce red blood cells

fast enough.

Conditions like Sickle cell anemia,

vasculitis(Inflammation of blood vessels) and an enlarged
spleen can all destroy red blood cells quickly
which leads to low hemoglobin.

Blood loss

Losing large amounts of blood can easily lead to

low hemoglobin.

This can also be a warning sign of internal blood

loss.

Vitamin deficiency

Vitamins such as B12 or foliate help your body

create red blood cells. If you arent getting enough,
your hemoglobin levels can drop

Blood disorders

Some conditions, such as some cancers, can lead

to low hemoglobin. These blood disorders mean
that your bone marrow isnt producing red blood
cells fast enough.

Aplastic anemia

Bone marrow disorder

Incapable of releasing RBC

Vitamin C (Ascorbic acid)

This is a vitamin that helps your body absorb iron,

which is essential to hemoglobin production.

Eat foods that contain Vitamin C and take a

supplement that helps your body get even more of
it.

Other foods

The staples of a healthy diet can keep your

hemoglobin high.

Nuts of all kinds, fish and lean meats, poultry,

eggs, organ meats and even fortified cereals can
help.

Herbs

Iron deficiency

Iron is required to create hemoglobin, so if your

body needs more iron, your hemoglobin counts
are probably low.

In fact, this is the most common cause of

anemia

HOW TO INCREASE HEMOGLOBIN COUNTS

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Fruits and vegetables

Look for fruits of all kinds and vegetables that are

bursting with leafy green color.

Cabbage, peas, beans, lettuce, broccoli and

tomatoes are good places to start.

You can opt for all kinds of fruits, such as

grapefruits, oranges, bananas, raspberries, kiwi
and mango

CAUSES OF LOW HEMOGLOBIN COUNTS

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There are many herbs that up your hemoglobin

counts,
Including fenugreek seeds, rosemary, basil,
thyme, sage, nettle leaf and yellow dock root,
among others.

GEN CAMATO

BLOOD VOLUME

HEMATOLOGY 1 | LECTURE

METHODS OF HEMOGLOBIN DETERMINATION

I.
Colorimetry
II.
Specific gravity method
III.
Gasometric
IV.
Chemical method

THINGS TO AVOID
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Iron blockers

Anything that blocks iron absorption is bad.

Avoid things like coffee, foods with high levels of

fiber, tea, and some antacids and medications
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I.

Foods with oxalic acid

This can prevent absorption, and should be

avoided.

Spinach is the biggest culprit among foods with

oxalic acid

Dont combine leafy green with fruits because it causes

very high oxalic acid

Foods with gluten

Wheat products, such as breads or pasta can

cause trouble for those who have gluten allergies.

But they can also lead to problems with iron, and

they can lead to low hemoglobin.

Reduce your intake of foods that contain gluten

Dares method

The blood is drawn through capillary

action between 2 glass plates

c)

Acid hematin

Hemoglobin in a known quantity of

blood in the presence of acid such as
0.1 N HCl is converted to acid
hematin. Which is brown colored
solution diluted with distilled water
drop by drop until it matches the
standard solution

d)

Sahli hellige method

Sahli hayden method
Sahli adam s method
Osgood-haskin method
New comer

Alkaline hematin

Same principle except we use an

alkaline solution produces alkaline
hematin
D.1 Wu method
D.2 Clegg and king method
Equivalent hemoglobin

Symptoms in children

The symptoms in children can be more severe, as

low hemoglobin can lead to long-term
consequences. Look for paleness, a rapid heart
beat, a lowered ability to concentrate, poor
neurological development and disturbed
behavior patterns
High hemoglobin in children are NORMAL

b)

C.1
C.2
C.3
C.4
C.5

Rare symptoms

Other symptoms that might occur but are relatively

rare include excessive sweating, vomiting and
persistent heartburn(because of too much acidity or very
low/decrease of hemoglobin).

You might experience swelling in your arms or

legs, and could even notice bloody stool when you
go to the bathroom

2 types
1) Direct visual colorimetry
a) A tallguist method

Measures hemoglobin using an

absorbent pad.

The color is match using a standard

White pasta/white bread inhibit proper absorption

SYMPTOMS OF LOW HEMOGLOBIN COUNTS

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General symptoms

In general, the signs of low hemoglobin include

dizziness, headache, fatigue and a general
feeling or tiredness. It might be tough to
concentrate. You can experience an irregular
heartbeat or notice the skin, gums or nail beds
are very pale in color.

In addition, you might experience shortness of

breath, palpitations and chest pain with more
severe cases of low hemoglobin.
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COLORIMETRY

14-18

gms

/dL

Male

12-16

gms

/dL

Female

14-26

gms

/dL

Children

GEN CAMATO

BLOOD VOLUME

2)

HEMATOLOGY 1 | LECTURE

Photoelectric colorimetry
a) Oxyhemoglobin method

Measures only normal hemoglobin

Reagent:

0.007N NH4OH or 0.1%

NaCo3 solution

Wavelength: 540nm

III.

GASOMETRIC METHOD
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b)

Cyanment hemoglobin

Also known as ferriHbCN

Considered as the most accurate

method of hemoglobin determination
since It measures all Hb except the
Sulfhemoglobin

Reagents:

Drabkins reagent
KCN2 (Potassium cyanide)
K3Fe(CN)6 (potassium ferricyanide)
NaHCO3 (Sodium bicarbonate)
Distilled water
Principle: blood is diluted in a solution K3Fe(CN)6, KCN,
K3Fe(CN)6 oxidizes hemoglobin to hemoglobin. The
gemiglobin will further oxidize the KCN to form
cyanmethemoglobin and that cyanmethemoglobin has a
borad absorption max 540.

II.

IV.

SPECIFIC GRAVITY = DENSITY OF SUBSTANCE

DENSITY OF WATER

CuSO4 (Copper sulfate) METHOD

Commonly used in blood banks

Easily acquire fast

May start 1.055 in counting

Approximation of donors

Heavy CUSO4, the blood will sink & vice versa

Sink if:

>12.5 g/dL (can donate blood)

Suspended blood if:

<12.5 g/dL (rejected)

Another indirect method based on the principle that gram

of hemoglobin can carry 3.47 mg of Fe (Iron)
Ex.

Wongs

kennedys method

ABNORMAL HEMOGLOBIN
1) Several test for abnormal hb pigments:
a. Naked eye test
b. Dilution test
c. Alkali spot plate test
d. Katayamas test (Important, nakakalason sa dugo)
e. Hand spectroscopic test
f.
Spectrophotometric method

Ratio of the weight of a volume of the blood to the

ratio of the weight to the same volume of water at
room temperature.

CHEMICAL METHOD
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SPECIFIC GRAVITY METHOD

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Base on ability of hemoglobin to carry 1.34mL of

oxygen/gram of hemoglobin.
Also called indirect method
Disadvantage:

Abnormal hemoglobin may not be measure

Ex.

vanSlyke,

Haldane

smith

Be strong and of a good courage, fear not, nor be afraid of them:

for the LORD thy God, he it is that doth go with thee; he will not
fail thee, nor forsake thee.

- Deuteronomy 31:6
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