Transcript Hemoglobin

Photometry
• Photometry means “the measurement of light”
• If a substance can be converted to a soluble, colored
material, its concentration may be determined by the
amount of color present in the solution.
• Photometer & Spectrophotometer are instruments used
for this type of measutment, in which a photocell or
photomultiplier tube is used to detect the amount of light
that passes through a colored solution from a light
source.
Characteristics of Light
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Light is a form of electromagnetic energy that travels in waves.
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Objects that appear colored absorb light at particular.
The wavelength of light is the distance between two beaks of
the light wave, it is inversely proportional with its energy.
Approximately
wavelength
< 0.1
0.1-10
Ultraviolet
Visible
Infrared
<380
380-750
>750
radiowaves
>25 x 107
Wavelength
Types of
radiation
Gamma
X-rays
Energy
Table-1(wavelengths of various types of Radiation)
Table –2 (the visible Spectrum)
Approximately
wavelength
400-435
Color of absorbed
light
Violet
Color of reflected
light
Green–Yellow
435-500
Blue
Yellow
500-570
Green
Red
570-600
Yellow
Blue
600-630
Orange
Green blue
630-700
Red
Green
Beer’s law
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When the light of an appropriate
wavelength strikes a cuvet that contains
a colored sample, some of the light is
absorbed and the rest is transmitted
through the sample to the detector.
% percent transmittance which
represents the proportion of light reaches
the detector.
% T = (It \ Io) x 100 %
Where:
Io: is the intensity of light striking the
sample.
It: is the intensity of transmitted
light.
Io
It
Beer’s law
If the concentration of a solution is increased, the It will decrease
and then % T is decreased.
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The relationship between the concentration and %T is not linear,
but if the logarithm of the %T is plotted against the concentration,
a straight line is obtained.
-The term absorbance is used to represent – log % T
A = - log % T
= 1/ log % T
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Then we can determine the concentration of x substance
by measuring both sample and standard absorbance, which
can be made by spectrophotometers.
UV – Visible photometry
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Typical coloremetric instruments contain five
components:
Stable source of radiation energy.
A transparent container for holding the sample.
A device that isolates a restricted region of the
spectrum for measurement.
A radiation detector which converts radiant
energy to electrical signals.
A signal processor and read out which displays the
transudated signals, a meter scale, a digital meter or a
recorder chart.
UV – Visible photometry
Radiation sources
- In UV region:
The most commonly used is deuterium lamp or hydrogen
lamp. In which a continues spectrum is produced by the
excitation of deuterium (D2) or hydrogen at law pressure,
and then produced light with (160-375) nm.
- In visible region:
Tungeston filament lamp is the most commonly used and
produces light at (350-2500) nm.
Note:
Colorimeters
Photometers
Spectrophotometer
- Used filters as
wavelength selector
- Used monochromators as
Wavelength selector
Sample containers:
• Cuvetes that hold the samples must be made of material that
passes radiation in the spectral region of intrest.
• Quartz or fused silica may be used in the spectral region (350-3000
nm), mean it may be used in the UV, visible and a part of infrared.
• Silicated glass used in (350- 2000 nm) region.
• Plastic is used in the visible region
Radiation detectors and read out.
Phototubes
Photomultiplier tube
Photoconductivity detector
silicon diode electrode
Hemopoiesis
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Is the process of blood cell formation which takes
place during the embryonic life in the yolk sac;
mesenchyme and blood vessels; liver; spleen, thymus
and lymph nodes; bone marrow.
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While in late fetus & adult takes place in bone
marrow and lymphtic tissues in normal situation
(medullary hemopoiesis).
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In pathological conditions hemopoiesis is
(extramedullary) in the liver, spleen and lymph nodes.
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Hemoglobin (Hb) is the standard abbreviation for
hemoglobin, the oxygen-carrying pigment and
predominant protein in the red blood cells.
Hemoglobin is the protein that carries oxygen from
the lungs to the tissues and carries carbon dioxide
from the tissues back to the lungs.
In order to function most efficiently, hemoglobin
needs to bind to oxygen tightly in the oxygen-rich
atmosphere of the lungs and be able to release oxygen
rapidly in the relatively oxygen-poor environment of
the tissues.
It does this in a most elegant and intricately
coordinated way.
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Hemoglobin forms an unstable, reversible bond with
oxygen. In the oxygenated state it is called
oxyhemoglobin and is bright red.
In the reduced state it is called deoxyhemoglobin and
is purple-blue.
A hemoglobin molecule consists of four polypeptide
chains: two alpha chains, each with 141 amino acids
and two beta chains, each with 146 amino acids.
The protein portion of each of these chains is called
"globin".
The α and β globin chains are very similar in structure
and each one of them is liked with a heme molecule.
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A heme group is a flat ring molecule containing
carbon, nitrogen and hydrogen atoms, with a single
Fe2+ ion at the center.
Without the iron, the ring is called a porphyrin.
Changes in the amino acid sequence of these chains
results in abnormal hemoglobin's.
For example, hemoglobin S is found in sickle-cell
disease, a severe type of anemia in which the red cells
become sickle-shaped when oxygen is in short
supply.
Normal Human Hemoglobin's
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Adult hemoglobin's
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Hemoglobin A
hemoglobin A2
hemoglobin F
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{2, 2}
{2, 2}
{2, 2}
>95%
<3.5%
1-2 %
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In the very common laboratory test for hemoglobin (Hb), it is measured as
total hemoglobin and the result is expressed as the amount of hemoglobin
in grams (gm) per deciliter (dl) of whole blood, a deciliter being 100
milliliters.
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Adult men
Adult women
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14-18 gm/dl
12-16 gm/dl
polycythemia
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Is Above-normal hemoglobin levels
Secondary polycythemia which is may be due to:
• Dehydration (sever burns, diarrhea, vomitting,
…etc.).
• Severe lung or heart disease.
• Living at high altitudes.
• Heavy smoking.
• Primary polycythemia which is due malignant
variation in blood cells production in bone marrow.
anemia
• Below-normal hemoglobin levels that can be the result of
• Iron deficiency or deficiencies in essential vitamins of other
elements, such as B12, folate, B6.
• Inherited hemoglobin defects, such as sickle cell anemia or
Thalassemia.
• Other inherited defects affecting the red blood cells.
• Excessive bleeding.
• Excessive destruction of red blood cells.
• Kidney disease.
• Bone marrow failure or aplastic anemia.
• Cancers that affect the bone marrow.
Measurement of hemoglobin
The Cyanmethemoglobin Method for Hb determination
is the reference method.
 Specimen
• Whole blood, using EDTA as the anticoagulant.
Capillary may also be used.
 Principle
Hb (Fe++) K3Fe (CN)6
methemoglobin (Fe+++ )
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KCN
Cyanmethemoglobin
Procedure
Create a standard curve, using a commercially available
cyanmethemoglobin standard which, has constant
concentration 25g/dl,
Hb concentration
g/dl
Volume of
St / ml
V of Drabkin
reagent / ml
0
5
10
15
20
0
1
2
3
4
5
4
3
2
1
Procedure
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Put 5 ml of pre-prepared working reagent in a test
tube . Then add 20 µL blood to the test tube .
Mix well and leave it at room temp. for 3 minutes.
Measure the absorbance for cyanmethemoglobin at
wave length 540 nm against reagent blank.
Calculation
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Test Hb concentration = Abs. of test /Abs. of standard
* conc. of standard
OR
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Test Hb concentration = Abs. of test * factor obtained
by the standard curve
Discussion
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Before the test sample is read, the solution should be
clear.
A high WBC count: centrifuge specimen and use the
supernatant for reading.
Lipemia can also interfere, and a false result can be
corrected by adding 0.02 ml of the patient’s plasma
to 5 ml of the cyanmethemoglobin reagent, this
solution being used as the reagent blank.
Carboxyhemoglobin takes up to 1 hr to convert to
cyanmethemoglobin and therefore, theoretically
could cause erroneous results in the samples from
heavy smokers. However the degree of error is
probably not clinically significant.