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Hemoglobin
is a conjugated protein present in Red blood cell. It carries oxygen from the
lung to the
tissue cell and carbondy oxeyde the gaseous waste from the cells to lung. Hemoglobin contents to components Hem(Iron+Protophyrin+Globulin+Protein / amino acid chain)
tissue cell and carbondy oxeyde the gaseous waste from the cells to lung. Hemoglobin contents to components Hem(Iron+Protophyrin+Globulin+Protein / amino acid chain)
Specimen :- EDTA anti coagulated
vein us blood is commonly use but other anticoagulants heparin , Double oxalate
alasoyiclds the same result capillary blood can be used directly.
Method :- Cyanmethaemoglobin.
Equipments:-
1)
Photometer
with 540 nm filter
2)
Blood
pipette (Sahlys) of 20 micro l capacity
3)
Test
tube
4)
Surgical
Gauze/Cotton
5)
Other
Glassware
Reagent :-
1)
Drabkin’s
Solution (PIOSION)
2)
Cyanmethemoglobin
standard
Procedure :-
1)
Two
clean and dry test tube are taken and marked as “B” (blank) & “T” (Test).
2)
5
ml of Drabkin’s solution is pipette into the two tube
3)
The
blood specimen is well mixed by gently swinging.
4)
An
aliquot 20 micro l of well mixed EDTA anti coagulated blood specimen is drawn
into the pipette and out site of the
pipette is cleaned by use of gauze to avoid volume error.
5)
The
pipette blood sample is added to the “T” marked tube.
6)
The
contents of tube is well mixed and waited for 10 minutes.
7)
The
colorimeter is turned on and allowed to warm up for 10 minutes. The wavelength
selected filter is used in this range.
8)
The
instrument is set to “Zero -0” with out cuvette using the first control knob.
9)
The
both solution “B – Blank”, “T- Test” are transferred to two matched cuvettes.
10)
The
instrument “Zero -0” is rechecked , the cuvette with Blank solution “B” is
inserted in to the colorimeter cuvette socket.
11)
The
reading is adjusted to “Zero-0)” absorbance with the second control knob.
12)
The
cuvette with test solution “T” is inserted in to the socked of the colorimeter
and the absorbance reading is recorded . absorbance can be rechecked using
again the Blank solution.
13)
The
reference of the calibration curve is taken to find out the concentration of
the unknown. This can be simplied find by applying the flowing formula ……..
Abs of Test
Hb%.con (g/dl) =
--------------------- X 15*
Abs of stander
In
the above formula the value of stander and Absorbance of stander remain
constant Hence, it can be calculated as……
Con of std
-------------- = factor
Abs of std
There
fore the formula can be written as
hb.con (g/dl)=Abs of the test X factor.
Calculation – OD
of Test
Hb (g %) =
-------------------- X con of
std X 0.251
OD of std
OD of Test
= ------------------- X 60
X0.251
OD of std
OD of Test
= ----------------------- X
15.06
OD
of std