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25 views18 pages

Publication 11 16425 1196

Uploaded by

roomasa99
Copyright
© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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Introduction To Medical

Technology

Hemoglobin Concentration
Determination
Hemoglobin (Hb)

 Hemoglobin (Hb) is the standard abbreviation for


hemoglobin, the oxygen-carrying pigment and
predominant protein in the red blood cells.
 Hemoglobin carries oxygen from places of high
oxygen pressure (lungs) to places of low oxygen
pressure (tissues), where it readily releases the
oxygen. Hemoglobin also returns CO2 from the
tissues to the lungs.
 A hemoglobin molecule consists of 1 molecule of
globin and 4 molecules of heme (each containing
1 molecule of iron in the ferrous state).
Structure of hemoglobin

 “Globin" consists of four polypeptide


chains: two alpha chains, each with
141 amino acids and two beta chains,
each with 146 amino acids.
 The α and β globin chains are very
similar in structure and each one of
them is linked with a heme molecule.
Each heme group can combine with 1
molecule of oxygen or CO2.
 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.
Normal Ranges
In the very common laboratory tests 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.
The Normal Ranges For Hemoglobin Depend On:
 The age.
 Altitude
 The sex of the person.
 Normal values in an adult are 12 to 18 grams per
deciliter (100 milliliters) of blood.
The normal ranges
Newborns 17-22gmdl
One (1) week of age 17-22gm\dl
One (1) week of age 15-20gmdl
One (1) month of age 11-15gm\dl
Children 11-13gm\dl
Adult men 14-18gm\dl
Adult women 12-16gm\dl
Men after middle age 12.4-14.9gm\dl
Women after middle age 11.7-13.8gm\dl
Above-normal hemoglobin levels is called polycythemia
which is may be:

I. 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.
II. Primary polycythemia which is due malignant
variation in blood cells production in bone
marrow
Below-normal hemoglobin levels may lead to anemia
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 Cyan-methemoglobin Method for Hb determination is


the reference method.
Principle:
 Whole blood is diluted in a solution of potassium
Ferricyanide and potassium cyanide.
 The Hb is oxidized to met-hemoglobin by the
potassium Ferricyanide.
 The potassium cyanide then converts the met-
Hb (Fe ++) K 3 Fe (CN)6 Methemoglobin
hemoglobin to cyanmet-hemoglobin. (Fe +++ )

KCN

Cyan-Methemoglobin
 The absorbance of the cyanmet-hemoglobin at
540 nm is directly proportional to the Hb
concentration.
 Sulf-hemoglobin is not converted to cyanmet-
hemoglobin; therefore, it can not be measured
by this method.
Procedure of standard curve

A. Standard Curve Preparation


1. Create a standard curve, using a commercially
available cyan-methemoglobin standard which, has
constant concentration 25g/dl, the following dilutions
should be made to get the line between the
concentration & the absorbance of the standard using
also drabkin reagent as shown:
Procedure

Hb Absorban
Volume of V of Drabkin
concentration ce
St / ml reagent / ml
g/dl reading
0 0 5 0
5 1 4 0.125
7.5 2.5 2.5 0.188
10 2 3 0.250
15 3 2 0.375
20 4 1 0.500
Procedure

2. Allow the tubes to stand for 10 minutes.


3. Transfer the dilutions to cuvettes. Starting with
the blank, zeroing the spectrophotometer with
the BLANK solution, then measure the
absorbance on a spectrophotometer at 540 nm.
4. Plot absorbance on the y-axis and the Hb
concentration on the x-axis. The Hb
concentrations of the patients’ samples and
controls can be read from this standard curve.
Standard Curve
B. Patient Sample Preparation

1. Pipette 5 ml of Cyan-methemoglobin reagent


into a tube. Add 20 µl of the sample into the
tube.
2. Allow the tube to stand for 10 minutes.
3. Read Absorbance (A) in the
spectrophotometer at 540 nm, zeroing the
spectrophotometer with the BLANK solution.
Standard Curve calculation

∆Y
∆X

Concentration of Standard
Calculation
Discussion
 mechanical sources of error:
 Pipetting error.
 Use of dirty or scratched cuvettes.
 Use of deteriorated reagents.
 Before the test sample is read, the solution should
be clear:
 A high WBC count: centrifuge specimen and
use the supernatant for reading.
 Hemoglobin S (HbS) and Hemoglobin C (HbC),
dilute the mixture 1:1 with distilled water and
then read in the colorimeter; multiply the
reading by 2.
 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
 Drabkin’s reagent is sensitive to light. It should be
stored in a brown bottle or in dark place.
 Carboxy-hemoglobin takes up to 1 hr to convert to
cyan-methemoglobin and therefore, theoretically
could cause erroneous results in the samples from
heavy smokers. However the degree of error is
probably not clinically significant.
 Because Drabkin’s reagent contains cyanide, it
must be used cautiously; a minimum of four L of
reagent is lethal.
 Acid free sinks should be used for disposal of
reagent and samples, because acidification of
cyanide releases hydrogen cyanide gas. Copious

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