GEN CAMATO

BLOOD VOLUME
HEMATOLOGY 1 | LECTURE

BLOOD VOLUME NORMAL MATURATION OF CELLS

} Total amount of blood in the circulation 3 Principles:
} Normal blood volume 1) Cytoplasmic Differentiation
Male: approx. 5.8 L } Several Factors:
Female: approx. 4.5 L Loss of Basophilia
} Definition of terms: Associated with color blue
Normovolemia Nucleus
Normal volume of blood in the body - Acidic part of the cell
Hypovolemia - Methylene Blue
Decrease in the volume of the blood Cytoplasm
Hypervolimia - Basophilic part of the
Excessive volume of blood in the body cell
- Eosin
Oligemia
Total reduction of blood volume
Cytoplasmic Granules
A condition in which the total volume of
Increase in granules, the
more mature
the blood is reduced
Elaboration of hemoglobin
FACTORS RELATED TO DECREASE () BLOOD VOLUME Increase in hemoglobin, the
} Loss of whole blood more mature
Hemorrhage
} Loss of RBC 2) Nuclear Maturation
} Subdivisions:
Spherocytosis
a) Structure & Cytochemistry
An auto-hemolytic anemia
} Loss of plasma
Presence of round/oval
nucleus
Burned patients, Dehydration
} Loss of body water
Young cell-compact
Decrease consumption of water
Large nucleus to cytoplasm
ratio
FACTORS RELATED TO INCREASE () BLOOD VOLUME The larger the
} During blood transfusion nucleus, the more
} Intravenous fluid immature
} Due to increase fluid intake The larger the
cytoplasm, the more
FORMULA: mature
Nuclear chromatin
Plasma Volume + Packed Cell Volume = Total Blood Volume
Rich in RNA
The more nuclear
Blood = 45% Formed Elements (Cells) + 55% Liquid Elements (Plasma)
chromatin, the
younger the cell
} Plasma volume should comprise 55% of the total blood Decrease in the number of
volume Normal neutrophil nucleoli
2-5 lobes
} Packed cell volume should be round 45% of the total blood Found on the most
volume Hypersegmented neutrophil
More than 5 lobes
immature stage
Total reduction of blood volume Pernicious Anemia The more lobulated,
} Packed Cell Volume the more mature
Measured by counting with use of radioactive b) Changes in the shape of nucleus
materials Lobulation of nucleus
Radioisotopes for the determination The more lobulated,
of Packed Cell Volume (PCV) the more mature
Examples:
- Cr-51, P-32, Radio Iron 3) Reduction of cell size
- Inject Radioisotopes: attach to } The smaller the cell, the more mature
RBC } The larger the cell, the more immature
} 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

 GEN CAMATO

BLOOD VOLUME
HEMATOLOGY 1 | LECTURE

HEME SYNTHESIS FACTORS AFFECTING THE HEMOGLOBIN AFFINITY (ATTACHMENT)

WITH OXYGEN
} First Step
Condensation of succinyl co-enzyme A + glycine in I. Body Temperature
the presence of pyroxidal phosphate and delta } An increase in the body temperature causes the
aminolevulinic acid synthesis will react to co-enzyme A hemoglobin to release oxygen more readily
and glycine to produce delta aminolevulinic acid } * In Body temperature = in Oxygen released
dehydrogenase Shift to the right
In the release of Oxygen &
Synthesis of nucleus release of uptake in Carbon
Spleen graveyard of RBC dioxide

RBC (120 days) II. Blood pH/Bohr Effect

} The relationship of the blood pH and oxygen affinity
Hemoglobin is what we call the Bohr effect which tells us an

heme globin (reabsorbed protein)
increase in pH favors the uptake of Oxygen
} * pH = Alkaline
Iron (Fe) Porphyrin } * Blood pH = uptake of Oxygen
Shift to the left
in the uptake of Oxygen &
HEMOGLOBIN release of Carbon dioxide
} Red coloring pigment of the blood
III. 2,3 Diphosphoglycerate (DPG)
} Main component of RBC } An increase amount of DPG level will cause a
diminish oxygen affinity
} 35% of the total RBC Shift to the right
DPG = Oxygen (release)
} Sometimes called respiratory pigment because of its capacity to
affinity
transport O2 and CO2
IV. Carbon Dioxide
} A gram of hemoglobin can carry approximately 1.34mL of
} An increase of Carbon dioxide will diminish the
Oxygen and 3.47mg of Iron
affinity of Oxygen
} The adult RBC mass of 600grams hemoglobin can carry Shift to the right
approximately 800mL of Oxygen
V. Presence of HbF (Fetal Hemoglobin)
} Women need 400-800g of iron supplement } An increase of HbF in the fetus will cause increase
oxygenation of the fetal blood
} Conjugated CHON (Protein) that is made up of 1 globin molecule of Shift to the left
heme groups and the globin is divided into 4 polypeptide chains *For adults: Shift to the right (Not Normal)

} Each chain is attached to 1 heme group VI. Presence of Hemoglobin Variance

} The number of normal hemoglobin balance will show
either shift to the left or right
Heme Heme
} Different types of Hemoglobin
HbM & HbO Right
1 Globin Molecule HbA2 & HbA Left
Heme Heme VII. Decrease affinity of oxygen and increase carbon dioxide
} Haldane Effect
Affinity = Right
} Polypeptide chain
Made up of amino acid
Body Temperature shift to the LEFT
} In normal adult hemoglobin, it is made up of HbF adult shift to the RIGHT
2 alpha and 2 beta chain Haldane Effect shift to the RIGHT
Oxygen uptake shift to the LEFT
Alpha chain 2, 3 DPG shift to the LEFT
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

} The rest of the globin chain contains 146 amino acids

 GEN CAMATO

BLOOD VOLUME
HEMATOLOGY 1 | LECTURE

CLASSIFICATION OF HEMOGLOBIN 2. Sulfhemoglobin

Blood with combined sulfide
A. Normal Hemoglobin Benign condition
} Capable of transporting blood gases particularly With significant effect of
Oxygen & Carbon dioxide cyanosis
According to Function Cause & effect of drug
Oxyhemoglobin Mauve lavender color
- Arterial blood
- Bright red 3. Met Hemoglobin
- Oxygen A.k.a.
Reduced Hemoglobin Hemiglobin
- Venous blood
Oxidized Hemoglobin
- Dark red
- Carbon dioxide
Ferri Hemoglobin
} Hemoglobin in Adult (post-neonatal) Reversible reaction
HbA Normal individual
Constitutes majority of adult Hg approximately 1% of
methylene blue
96-98% Hg
Characteristic color:
2 & 2 chains
Chocolate brown
HbA2
Normal adult Hg VARIANCE OF HEMOGLOBIN
1.5 3% total types of Hg } Hemoglobin S
2 & 2 (delta) chains Causes sickling of RBC
HbF Confined to blacks
Major hg in fetus (90-95% hg) Homozygous state causes sickle cell anemia
0.5 1% hg Heterozygous state causes/shows sickle cell
2 & 2 (gamma) chains fomite
Heterozygous carrier parents
} Embryonic Hemoglobin (Pregnant) If both parents HbS(+) = Sickle Cell Anemia
Gower 1 HbS + HbA = Sickle cell Trait
2 (epsilon) & 2 (zeta) chains HgS + HgS = Sickle cell Anemia
Gower 2 HgS + HgA = Sincle cell shape (carrier)
2 & 2 chains
Hemoglobin Portland } Hemoglobin C
2 (zeta) & 2 gamma chains Found also among blacks, rare in whites
Appears also in homozygous & heterozygous
B. Abnormal/Non-functional Hemoglobin state
} Formed due primarily to a defect in the If present, it demonstrate target cell
polypeptide chain of hemoglobin molecule Target cell
} Category: Condensation of Hg at the
center/periphery
1. Carboxyl Hemoglobin (HbCO) A.k.a.
Reacts with Carbon monoxide in Bulls eye cell
which the affinity of CO 218x
Codocyte
greater than Oxygen at 37C
Leptocyte
Do not transport oxygen that may
lead to hypoxia & finally death of
Mexican hat cell
the patient Yersinia enterocolitica

Produces cherry red blood color

} Hemoglobin D
Concentration to non-smokers:
Most common for HbD Purijab & HbD Los Angeles
0 2.3%
Smokers: 2.1 4.2%
Both alpha & beta chain abnormally have been
reported
Test for HbCO
Shows no sickling of RBC
Katayamas Test
a. 10 mL Distilled H2O
b. + 5 drops of blood Happy Hormones:
from patients/ control { Serotonin
blood { Adrenaline
{ Epinephrine
c. + 5 drops of
Ammonium sulfide [ Blood pH becomes basic
d. + Acidify with acetic
acid
if NORMAL BLOOD : Dirty greenish, Brown
if HbCO (+): Rose red color 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.
Other Test for HbCO: - 1 Peter 5:6,7
r Sanderman Dethionite Test
r Palmers Test

 GEN CAMATO

BLOOD VOLUME
HEMATOLOGY 1 | LECTURE

HEMOGLOBINOMETRY } Iron deficiency

} Screening test associated with anemia Iron is required to create hemoglobin, so if your
body needs more iron, your hemoglobin counts
FACTORS ASSOCIATED WITH INCREASED OF HEMOGLOBIN are probably low.
} Hyperchromia In fact, this is the most common cause of
Increase intensity in the color of hemoglobin anemia
} Polycythemia
Increase in RBC } Vitamin deficiency
} High altitude Vitamins such as B12 or foliate help your body
Depletion of oxygen create red blood cells. If you arent getting enough,
Hypoxia will trigger the release of erythropoietin your hemoglobin levels can drop
in kidney Bone marrow RBC
} Dehydration } Blood disorders
Loss of fluid within the body Some conditions, such as some cancers, can lead
} Heart disease to low hemoglobin. These blood disorders mean
Patient with heart problem will have that your bone marrow isnt producing red blood
hemoglobin pigmentation because of the cells fast enough.
compensation of erythropoietin Aplastic anemia
Bone marrow disorder
FACTORS IN DECREASE HEMOGLOBIN CONCENTRATION Incapable of releasing RBC
} Pregnancy
Competition for hemoglobin with the fetus
Ferric sulfide/iron supplements needed ^ HOW TO INCREASE HEMOGLOBIN COUNTS
} Anemia } Fruits and vegetables
} Women Look for fruits of all kinds and vegetables that are
Females naturally have low hemoglobin bursting with leafy green color.
because of menstruation Cabbage, peas, beans, lettuce, broccoli and
Males naturally have high hemoglobin because tomatoes are good places to start.
of testosterone (which is associated with You can opt for all kinds of fruits, such as
maturation/production of RBCs) grapefruits, oranges, bananas, raspberries, kiwi
and mango
CAUSES OF LOW HEMOGLOBIN COUNTS
} Vitamin C (Ascorbic acid)
} Normal low hemoglobin counts This is a vitamin that helps your body absorb iron,
It is quite common for women to experience low which is essential to hemoglobin production.
hemoglobin counts during pregnancy. Eat foods that contain Vitamin C and take a
Some people might also experience this as a supplement that helps your body get even more of
natural way of life it is simply how their body it.
works.
} Other foods
Low counts in these cases shouldnt be alarming.
The staples of a healthy diet can keep your
} Conditions and diseases that cause fewer than normal hemoglobin high.
red blood cells Nuts of all kinds, fish and lean meats, poultry,
Some conditions can cause lower numbers of red eggs, organ meats and even fortified cereals can
blood cells which can lead to a low hemoglobin help.
count
} Herbs
Anemia, cancer, cirrhosis (scarring/ tumor in the liver |
Kirros means yellow), kidney disease and lead
There are many herbs that up your hemoglobin
poisoning are just a few reasons. counts,
Including fenugreek seeds, rosemary, basil,
} Conditions and diseases that destroy red blood cells thyme, sage, nettle leaf and yellow dock root,
Sometimes your body cant produce red blood cells among others.
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.

 GEN CAMATO

BLOOD VOLUME
HEMATOLOGY 1 | LECTURE

THINGS TO AVOID METHODS OF HEMOGLOBIN DETERMINATION

} Iron blockers I. Colorimetry
Anything that blocks iron absorption is bad. II. Specific gravity method
Avoid things like coffee, foods with high levels of III. Gasometric
fiber, tea, and some antacids and medications IV. Chemical method

} Foods with oxalic acid I. COLORIMETRY

This can prevent absorption, and should be } 2 types
avoided. 1) Direct visual colorimetry
Spinach is the biggest culprit among foods with a) A tallguist method
oxalic acid Measures hemoglobin using an
Dont combine leafy green with fruits because it causes absorbent pad.
very high oxalic acid The color is match using a standard

} Foods with gluten b) Dares method

Wheat products, such as breads or pasta can The blood is drawn through capillary
cause trouble for those who have gluten allergies. action between 2 glass plates
But they can also lead to problems with iron, and
they can lead to low hemoglobin. c) Acid hematin
Reduce your intake of foods that contain gluten Hemoglobin in a known quantity of
White pasta/white bread inhibit proper absorption blood in the presence of acid such as
0.1 N HCl is converted to acid
hematin. Which is brown colored
SYMPTOMS OF LOW HEMOGLOBIN COUNTS solution diluted with distilled water
} General symptoms drop by drop until it matches the
In general, the signs of low hemoglobin include standard solution
dizziness, headache, fatigue and a general
feeling or tiredness. It might be tough to C.1 Sahli hellige method
concentrate. You can experience an irregular C.2 Sahli hayden method
heartbeat or notice the skin, gums or nail beds C.3 Sahli adam s method
are very pale in color. C.4 Osgood-haskin method
C.5 New comer
In addition, you might experience shortness of
breath, palpitations and chest pain with more
d) Alkaline hematin
severe cases of low hemoglobin.
Same principle except we use an
} Rare symptoms alkaline solution produces alkaline
hematin
Other symptoms that might occur but are relatively
rare include excessive sweating, vomiting and
D.1 Wu method
persistent heartburn(because of too much acidity or very
D.2 Clegg and king method
low/decrease of hemoglobin).
You might experience swelling in your arms or
legs, and could even notice bloody stool when you Equivalent hemoglobin
go to the bathroom gms
14-18 /dL Male
gms
} Symptoms in children 12-16 /dL Female
The symptoms in children can be more severe, as 14-26
gms
/dL Children
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

 GEN CAMATO

BLOOD VOLUME
HEMATOLOGY 1 | LECTURE

2) Photoelectric colorimetry
a) Oxyhemoglobin method III. GASOMETRIC METHOD
Measures only normal hemoglobin } Base on ability of hemoglobin to carry 1.34mL of
Reagent: oxygen/gram of hemoglobin.
0.007N NH4OH or 0.1% } Also called indirect method
NaCo3 solution } Disadvantage:
Wavelength: 540nm Abnormal hemoglobin may not be measure
Ex.
b) Cyanment hemoglobin vanSlyke,
Also known as ferriHbCN Haldane
Considered as the most accurate smith
method of hemoglobin determination
since It measures all Hb except the IV. CHEMICAL METHOD
Sulfhemoglobin } Another indirect method based on the principle that gram
Reagents: of hemoglobin can carry 3.47 mg of Fe (Iron)
Drabkins reagent } Ex.
KCN2 (Potassium cyanide) Wongs
K3Fe(CN)6 (potassium ferricyanide) kennedys method
NaHCO3 (Sodium bicarbonate)
Distilled water
Principle: blood is diluted in a solution K3Fe(CN)6, KCN, ABNORMAL HEMOGLOBIN
K3Fe(CN)6 oxidizes hemoglobin to hemoglobin. The 1) Several test for abnormal hb pigments:
gemiglobin will further oxidize the KCN to form a. Naked eye test
cyanmethemoglobin and that cyanmethemoglobin has a b. Dilution test
borad absorption max 540. c. Alkali spot plate test
d. Katayamas test (Important, nakakalason sa dugo)
II. SPECIFIC GRAVITY METHOD e. Hand spectroscopic test
} Ratio of the weight of a volume of the blood to the f. Spectrophotometric method
ratio of the weight to the same volume of water at
room temperature.

} 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)

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