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Hemoglobindetermination

This document describes several methods for determining hemoglobin levels and counting red blood cells and reticulocytes. It discusses colorimetric, specific gravity, gasometric, and chemical methods for measuring hemoglobin. For reticulocyte counting it covers wet and dry methods. The red blood cell count section explains hemocytometry using a hemocytometer, Thoma pipettes, diluting fluids, and the counting procedure and calculations.

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161 views24 pages

Hemoglobindetermination

This document describes several methods for determining hemoglobin levels and counting red blood cells and reticulocytes. It discusses colorimetric, specific gravity, gasometric, and chemical methods for measuring hemoglobin. For reticulocyte counting it covers wet and dry methods. The red blood cell count section explains hemocytometry using a hemocytometer, Thoma pipettes, diluting fluids, and the counting procedure and calculations.

Uploaded by

Liazq
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© © All Rights Reserved
We take content rights seriously. If you suspect this is your content, claim it here.
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HEMOGLOBIN

DETERMINATION

BOND KING
NITISH
RAHUL DEV

I. COLORIMETRIC METHOD
A. Direct visual colorimetric Method
Tall quist method
Dares Hemoglobinometer
Acid Hematin method
Alkaline Hematin method

B. Photoelectric colorimetric method


1. Oxyhemoglobin method
Measures normal hemoglobin
Used 0.007 N NH4OH or 0.1% Na2Co3
Read with the wavelength at 540 nm

2. Cyanmethemoglobin (HiCN)
Also known as hemiglobin cyanide or
ferrihemoglobin cyanide
All forms of hemoglobin are measured except
sulfohemoglobin
Uses Drabkins solution
Potassium ferricyanide
Potassium cyanide
Dihydrogen potassium phospate
Distilled water
PH = 7.0-7.4
( blood capacity )
Used sahli pipet= (0.02ml or 20 micro liter)

PROCEDURE
Place 5ml of Drabkins reagent into a

testube
Get 0.02 ml of whole blood using sahli pipet
Place the 0.02 ml of blood in to drabkin's
reagent through rinsing it.
Mix and let it stand for 10 minutes
Read in a spectrophotometer at 540 nm.

II. Specific gravity method/Gravitational


method CUSO4 method
Specific gravity of copper sulfate = 1.053

with an hemoglobin equivalent of 12.5 gm%


Mass blood
Procedure
Collect blood sample
Drop a blood into the solution
Observe the activity of the blood
Within 12 seconds, describe how the drop
of blood behaves.

Interpretation : Maintain = equal to 1.053 = 12.5 gm%


Sink = > 1.053 = >12.5 gm%
Float = < 1.053 = <12.5 gm%

III. Gasometric Method


Indirect method
Based on the assumption that 1gm Hb can
carry approximately 1.34 ml O2.
IV. Chemical Method
Indirect method
Based on the assumption that 1gm Hb
contains approximately 3.47 mg iron.

RETICULOCYTE COUNTING

I. Wet method
New methylene blue method
Cook, meyer and tureen
seiverds method
Procedure
Get blood sample
Secure equal proportion of blood and stain.
Mix it and letit stand for 10 minutes
Make a smear.
Dry the smear
Examine under microscope using OIO
Count reticulocytes in relation to 1,000 RBC.

II. Dry method


Schilings rapid method =(BCB method).
Sabins method = (janus green /neutral
red)
Seiverds method =(BCB method)
Osogood- wilhelm method = (new
methylene blue method)
BCB = Brilliant crystal Blue

PROCEDURE
Spread stain thinly on a glass slide and air
dry.
Place a small drop of blood upon the layer of
the dried stain.
Place a cover slip on the drop of blood.
Allow to stand for 10 minutes
Examine under the microscope under OIO
Count reticulocytes in relation to 1,000 RBCs.

COMPUTATION

% Reticulocytes count = no.of retics.counted X 100


1000 RBCs
Example :12/1000X 100 = 1.2% normal in adult

RBC COUNT
A. Hemocytometry method (microscopic

method)

(used hemoglobinometer)

. Diluting fluids
. Thoma pipets
. Counting chambers / Hemocytometer

1.Diluting fluids
Hayerms
Gowers
Toissons
Bethels
Formol-citrate/Dacies solutn
NSS
3.8 % sodium citrate
- easy to prepare
- must have preservative method
- must be safe
- no corrosive, non-caustic
- should be isotonic

2.Thoma pipet
Bead - identification of type of pipet
- used for mixing
- seperating color
Upper calibration of RBC pipet = 101
Capacity of bulb is 100 times capacity of stem
Constant volume of RBC pipet = 100[ 101-1]
RBC thoma- red bead
WBC thoma white bead

Thoma pipet
Bulb/ mixing
chamber

Short stem
Bead
Long
stem

3. Counting chamber
Raised platform

Drawing ruled area

Counting
chamber

H-shaped
moat

Counting chamber
Improved neubaber- commonly used
Cover slip= depth of the counting chamber

(0.1mm)
1 ruled area = 1mm2
1 large square width and length 1mm
Center of large square have 25 small squares
and each 25 small square has 16 small square
which is used in RBC count.
Total 400 small square are found in center of
large square.

WBC

WBC

R
R
R

WBC

WBC

Procedure
Collect blood
Suck blood to 0.5 mark of the pipet.
Suck diluting fluid to 101 mark.
Shake pipet for 2 minutes.
Discard first few drops.
Charge the counting chamber at an angle from
30- 35 degree.
Count the RBC under HPO using 5 RBC squares of
central large square
Compute.

Computation
RBC count = RBC counted X DCF X VCF
DCF = Volume of blood / amt of blood

sucked
VCF = volume desired / area x depth of the
counting chamber x nos of squares used.
DCF= diluting correction factor
VCF = volume correction factor
For RBC pipet DCF = 200 and VCF is 50
VCF = 1/ 0.04x 0.1x 5 =50

Errors
Technical error
Pipetting
Shaking the pipet
Charging the counting chamber
Application of cover slip
Counting of the cells
Computation
Reporting of results.

Never leave that till tomorrow


which you can do today.

Thank you.

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