Haemoglobin estimation bishwas neupane b.sc mlt part i
1. Guided By:
• Respected S.K SHARMA Sir
• Respected JOSEPH Sir
Presented By:
BISHWAS NEUPANE
B.sc MLT part I
PGIMER (NIPS)
2. INTRODUCTION
Haemoglobin (Hb),is a chromoprotein.
Molecular weight 64,458 Dalton.
Each haemoglobin molecule carries four molecule of oxygen
and each gram of haemoglobin can carry 1.34 ml of oxygen.
Each RBC contains between 27 to 32 pico grams of Hb.
About 6.25 grams of haemoglobin is synthesized each day to
replace the haemoglobin lost due normal destruction of
erythrocytes.
Synthesis begins from Proerythroblast to Reticulocytes.
4. Haemoglobin derivatives
a) Methaemoglobin –
Here the iron is in the ferric state and is incapable of
reversibly combining with oxygen.It is dark brown in colour.
Normal concentration : 1-2%
b) Sulphaemoglobin –
It is formed when sulphur combines with the heme of
haemoglobin. It is green in colour, cannot carry oxygen and is the
only haemoglobin not measured by cyanmethhaemoglobin
method.It is formed by the action of certain drugs and chemicals
such as sulphonamides and presence of sulphur in air.Once it is
formed,it is irreversible and remains in the carrier RBC.
5. c) Carboxyhemoglobin -
It is formed by the exposure of normal
haemoglobin to carbon dioxide or carbonmonoxide. In
high concentrations it imparts a cherry red colour to
blood and skin.The affinity of haemoglobin for carbon
monoxide is more than for oxygen (200 times)
therefore it readily combine with CO even when there
it is present in low concentration.It is reversible.
Normal Ranges:
General people: 0.16%
Smokers and mine worker: 1-10%
6. Functions of haemoglobin
It imparts red color to the blood.
It buffers blood pH.
And the main function is to
deliver Oxygen to the tissue and
carbon dioxide from tissue to the
lungs.
7. Purpose of estimating haemoglobin
To detect the oxygen carrying capacity of
blood.
The result assists in detecting diseases, which
causes a deficiency or excess of haemoglobin.
Studying changes in haemoglobin
concentration before or after operations and
blood transfusions.
To detect anaemia and its severity and to
monitor an anemic patients response to
treatment.
To check haemoglobin level of blood prior to
donating blood.
To calculate red cell indices.
8. Normal Values
The normal value depends on the age and sex of the
individuals:
Men: – 13-18 gm/dl
Women :– 11-16 gm/dl
Full term / cord blood :– 13-19 gm/dl
Children 1 year :- 11-13 gm/dl
Children 10 - 12 years :- 12-15gm/dl
9. Methods of Estimation of
Haemoglobin
Colour based: -
Based on the colour of haemoglobin or a derivative of haemoglobin.
Physical method: –
Based on specific gravity.
Chemical method: –
Based on iron content of Haemoglobin.
Gasometric method :–
Based on oxygen combining capacity of Haemoglobin.
Spectrophotometric method:-
Based on measurements using spectrophotometric devices.
11. • Talliquist scale method:
Talliquist method involves direct visual matching of red
colour of a drop of whole blood on a filter paper with colour
standards on paper.The technique is totally unsatisfactory
with high degree of error ± 20% to 5o%.
• Dare’s method:
Here undiluted blood is spread in thin films between glass
discs for direct matching.This method is inaccurate.
12. WHO Haemoglobin Colour Scale:
This technique of estimating
haemoglobin is based on
comparing the colour of a
drop of blood absorbed on a
particular type of
chromatography
paper,against a printed scale
of colour corresponding to
different levels of
haemoglobin ranging from 4-
14 gram/dl.
13. • The new WHO haemoglobin colour scale uses
modern materials and techniques to provide a
simple,inexpensive and reliable method of
estimating Hb in community where photometric
measurement is not possible.
• Validation studies in blood transfusion centres have
shown the scale be more reliable and easier to use
than copper sulphate method in selecting Blood
donors.
14. • Spencer method:
In this method,the colour of diluted
oxyhaemoglobin is matched visually.This
method is less accurate than Sahli’s
method because it is more difficult for the
human eye to accurately grade and match
small differences in red colour than the
brown colour of acid haematin.
15. Sahli’S Method or acid heMatin
method
Principle
• Haemoglobin is converted to acid haematin
by N/10 HCl, the resulting brown colour is
compared with standard brown glass
reference blocks.
• The intensity of the brown colour depends
on the amount of acid haematin produced,
which in turn depends on the amount of
haemoglobin in the blood sample.
16. Instruments
Sahli’s Haemoglobinometer –
• The main parts of which are a
graduated glass tube, colour
comparators, glass stirrer and Sahli’s
pipette to measure 20 µl of blood.
• The tubes commonly used are square
with graduations in percent on one
side and grams per 100 ml on the
other.
• The colour comparators are made of
brown coloured glass and some
better instruments have glass prisms.
18. Procedure
Place N/10 HCl in the tube up to the lowest mark.
Draw blood up to 20 mm mark in the pipette and transfer it to the acid in
the tube.
Rinse the pipette well by drawing up the acid and re expressing it. Mix the
acid and blood by shaking the tube well.
Allow it to stand for at least 10 minutes - to allow brown colour to
develop due to the formation of acid haematin.
95% of Hb is converted at the end of 10 minutes, 98% of the colour
develop at the end of 20 minutes, and the maximum colour is reached
after about 1 hour.
Now dilute the solution with distilled water drop by drop with continuous
mixing, using the glass rod provided.
19. Procedure
(contd....)
• Match the color with that
of the glass plates in the
comparator.
• While comparing,take care
not to leave the glass rod
inside the glass tube.
• Reading is taken when the
color of the solution in the
tube exactly matches the
comparator. Matching
should be done at eye level
against natural light.
• The level of the fluid at its
lower meniscus is noted
and the reading on the
scale corresponding to this
level is read as gram/dl.
20. Sources of Errors
• Technical errors
Improper mixing of blood,
Errors in pipetting,
Tissue fluid contaminating capillary blood.
• Visual errors –
Taking the reading is very subjective, as it is a
comparison of colours. It can vary from
person to person. So the results may not be
accurate.
• Quality of the color comparators can affect
the reading – If the glass blocks are old or
faded it can cause wrong results.
21. • Insufficient time allowed for the conversion of Hb to acid haematin.
A minimum of 10 minutes is required for the reaction to be almost
complete, otherwise biological false negative result is obtained.
• Carboxy haemoglobin, methaemoglobin and sulphaemoglobin are
not converted to acid haematin.
• Non-haemoglobin substances such as protein, liquid and cell
stroma interfere with the colour of blood diluted with acid and
hence give false results.
• Time delay - The brown colour of acid haematin is not stable, so
undue delay in reading the test result is not allowed.
22. Alkaline Hematin method
In this method the Hb is converted to alkali haematin by
the addition of N/10 NaOH.
The alkali haematin gives a brown colour that can be
read against comparator standards or in a colorimeter
Apparatus:
Photo electric meter with green filter.
N/10 NAOH
0.05 ml pipette
Standard(Gibson’s and Harrison’s):This is a mixture of
chromium potassium sulphate,cobaltous sulphate and
potassium dichromate in aqueous solution.The solution
is equal in colour to 1 in 100 dilution of blood
containing 16.0 Hb per dl.
23. Technique:
Add 0.05 ml of blood to 4.95 ml of N/10 NAOH.
Mix well and boil for 4 minutes, along with 5 ml standard solution.
Cool quickly in cold water,and match the test against standard using
colorimeter using green filter.If the test give too high value add 5.0 ml of
water and read again.
Calculation:
If the OD of test =21
OD of standard =28
As the standard is equivalent to 16 gm per 100 ml,the haemoglobin of test
will be
21 X 16 g per 100 ml =12 g per 100 ml.
28
24. Again, 16 gm per 100 ml = 100%
12 gram per 100 ml =12 X 100 =82 percent.
16
Advantage
1) Unlike Sahli’s method, carboxyhaemoglobin, sulphaemoglobin are
converted to alkali haematin.
2) Foetal haemoglobin is resistant to denaturation by alkali and this
method is used to determine the level of foetal haemoglobin in
blood.
Disadvantage:
The solution of Hb in alkali has to be heated to ensure complete
denaturation.
Note:
Matching should be done within 30 minutes after boiling
25. Acid Alkali method
• In Alkaline haematin method the solution of
Hb has to be heated to ensure complete
denaturation.This procedure can be omitted if
the blood is collected first into acid and after
standing for 20-30 min,sufficient alkali is
added to neutralize acid and convert acid
haematin to alkaline haematin.
26. Procedure
• Add 0.05 ml of blood to 4.95 ml of 0.1 N HCL and
immediately mix well.
• After standing for 20-30 mins,add 0.95 ml of 1 N
NAOH and the tube is inverted several times.
• After standing for not less than 2 mins, the test
sample can be matched in photoelectric
colorimeter using yellow-green filter using
Gibson And Harrison’s standard.
27. Haldane carboxyhaemoglobin
method:
The haemoglobin is converted to carboxy
haemoglobin by the action of coal gas on
diluted blood.
Apparatus and requirements:
Haldane’s graduated tube and standard.
Approximately 0.4% ammonia in D/W.
0.02 ml pipette.
28. Technique:
Fill the graduated tube upto the 20 mark with ammoniated
D/W.
Add 0.02 ml of the patients blood and mix well.
Pass coal gas through this solution for 2-3 minutes.By
means of rubber tubing attaching pasteur pipette to the
gas supply.Dip the end of the pipette into caprylic alcohol
and gently bubble the gas through the blood solution.The
caprylic acid prevents frothing.
Add 0.4% ammonia drop by drop,mixing between each
addition,until the solution,viewed in diffuse day
light,matches the standard.
29. Read the amount of solution in the calibrated tube.The calibrations
gives the Hb concentration as a percentage.
Calculate the amount of haemoglobin in gm per 100ml of blood.
Eg: if the colour standard is defined as corresponding to14.6
g/100ml and reading is 95,then Hb content is calculated as:
100 % = 14.6 g/100 ml
95 % =14.6 X95 = 13.87 g of Hb per 100 ml blood.
100
30. Portable Haemoglobinometers
The HemoCue system is a well established method for
haemoglobinometry.It consists of a precalibrated,
portable,battery operated spectrometer;no dilution
required because blood is run by capillary action directly
into a cuvette containing sodium nitrite and sodium
azide,which convert haemoglobin to azidemethaemoglobin.
The absorbance is measured at wavelength of 565 nm.
Measurement is not affected by high level of
bilirubin,lipids,or WBC.
Cuvettes must be stored in a container with a drying agent
and kept within the temperature range of 15 – 300 C.
31. Spectrophotometric method
The esimation is based on Beer’s and Lambert’s law i.e. the optical
density (OD) of a coloured solution is directly proportional to the
concentration of the coloured material in the solution and the
pathlength.i.e diameter of cuvette.Here pathlength is constant i.e 1
cm.
1) Cyanmethemoglobin method
2) Oxyhemoglobin method
32. Oxyhaemoglobin method
Principle:
Blood is diluted in weak alkali(0.04% ammoniun hydroxide,sp gravity:
0.88)which lyses the red blood cells and oxyhaemoglobin is released into
solution.This conversion is complete and immediate and the resulting colour
is stable.
Standard:
The Hb value of normal anticoagulated blood is first determined by using
HiCN method.
The blood is then diluted 1:201 by pipetting 0.20 ml of well mixed blood
into 4 ml of ammonia and serial dilution is made in ammonia and
absorbance is read in spectrometer at 540 nm and plotted in graph.
haemoglobin values are obtained from tables prepared from calibration
graph.
A neutral grey screen of 0.475 density(Ilford and chance) can also be used
as a 14.6 g/dl(100%) standard.
33. Procedure
• Add 0.02ml of blood into a tube containing 4
ml of 0.04 ammonia(SG 0.88) with a tightly
fitting rubber bung.
• Mix by inverting several times,the solution of
HbO2 is ready for matching with yellow-green
filter in colorimeter.
• If the absorbance of Hb solution exceeds
0.7,the blood should be further diluted with an
equal volume of water.
34. Advantages:
No time is required for colour development.
This is simple and accurate method(error of 2-3%).
Economical and easy to perform.
It is fast and accurate method than visual comparative method.
Disadvantages:
Standard solution not easily available and unstable.
Methhaemoglobin and carboxyhaemoglobin are not accurately
detected.
35. Cyanmethaemoglobin
method
This is the preferred and the
most accurate method for
determining the
haemoglobin concentration
in laboratory.
36. Principle:
Blood is diluted in a solution of potassium ferri
cyanide and potassium cyanide.
The ferri cyanide oxidizes haemoglobin to
methaemoglobin.
Potassium Cyanide provides cyanide ions (CN–) to
form Cyanmethaemoglobin.
The absorbance of the solution is then measured in
a spectrophotometer at a wavelength of 540 nm or
in a colorimeter using a yellow-green filter.
37. Reagent
Haemoglobincyanide standard
Detergent modified Drabkin’s
solution(Van kampen and Zijlstra)
Potassium Ferri cyanide – 200 mg
Potassium Cyanide – 50 mg
Potassium Dihydrogen Phosphate –
140mg
and a non ionic detergent – 1 ml
Distilled water up to – 1000 ml
Note - In the place of Non-ionic detergent,
Sterox SE - 0.5 ml, Triton X-100 – 1 ml, or
Saponin - 1 ml may be used.
38. Properties of the reagent
The reagent should be clear, pale yellow, pH 7-7.4.
When it is measured against water as blank in
photometer at 540 nm the absorbance must read
zero.
Storage:-
It is stored at room temperature in a brown
borosilicate glass bottle away from direct
sunlight. It should periodically be checked for
turbidity, pH change, absorbance variation and if
these are noted the solution should be
discarded.(if freeze this can result decolorization
with reduction of ferricyanide)
39. Advantages of modified solution
This is less likely to cause plasma protein
precipitation which could interfere with test
results. The detergent enhances complete lysis of
red cells.
Shortens the reaction time and ensures
complete conversion of Hb to HiCN.The time
needed for complete conversion of Hb to HiCN is
shortened from 10 minutes to 3 minutes.
40. Procedure
Take 4.98(approximately 5) ml of Drabkin’s solution in a
large sized test tube.
Add 20 micro litres of well mixed anticoagulated
venous blood.
Rinse the pipette and mix well.
Allow it to stand at room temperature for 3 - 10
minutes.
Absorbance is measured against reagent blank at 540
nm either in a spectrophotometer or in colorimeter.
41. Calculation
The result is calculated by using the formulae
Hb gm/dl = OD of sample × conn of std in mg/dl × dil factor
OD of std 1000
= OD of sample × 60 × 250
OD of std 1000
Concn of Hb = OD of sample × 15
OD of std
42. Advantages
All forms of Hb except SHb are readily converted
to HiCN.
Direct comparison with HiCN standard possible.
Stability of the diluted sample.
Easy to perform the test.
Reagents are readily available.
The standard is stable.
43. Disadvantages
Increased absorbance not due to haemoglobin may be
caused by turbidity due to abnormal plasma proteins,
hyperlipaemia, high WBC count or fat droplets.
Potassium cyanide in the solutions is poisonous, though
it is present only in a very low concentration hence the
reagents should be handled carefully.
Explosion can occur if undiluted reagents are poured in
the sink. Hydrogen cyanide is released by acidification
and the gas if it accumulates can result in explosion.
Reagents and samples should be disposed along with
the running water in the sink.
44. Direct Spectrometry
• The haemoglobin of a diluted sample can be determined by
spectrometry without the need for a standard,provided that the
spectrometer has been correctly calibrated.The blood is diluted 1:250
by cyanide-ferricyanide reagent and the absorbance is measured at
540 nm.
• Calculation:
• Hb = A540 HicN x 16114 x Dilution factor
11.0 x d x 1000
Where,
A540 = absorbance of solution at 540nm.
16114 = monomeric molecular weight of haemoglobin.
11.O = millimolar coefficient extinction.
d = layer thickness in cm
1000 = conversion of mg to g.
45. Specific gravity method (Physical
method)
Haemoglobin being the largest single constituent, affects
the specific gravity of blood more than other substances.
Serum proteins are the next heaviest constituents of blood.
It is assumed that (which is not always true) the level of
serum proteins and other smaller constituents remain the
same, so any change in the specific gravity of blood is
mainly due to change in concentration of haemoglobin.
46. Hence this method uses the principle
that when a drop of whole blood is
dropped into a solution of copper
sulphate,which has a given specific
gravity,the blood will maintain its own
density for approximately 15 seconds.
The density of the drop is directly
proportional to the amount of
haemoglobin in that drop.
If the blood is denser than the specific
gravity of the solution,the drop sinks to
the bottom,if not it will float.
47. Preparation of Stock solution
170 grams of CuSO4 is dissolved in
1006.0 ml of distilled water to make
a stock solution of specific gravity
1.100 at 250 C.
The specific gravity of copper
sulphate solutions corresponds the
13 gram/dl and 12gram/dl for men
and women are 1.055 and 1.053
required to donate blood.
(assuming a serum total protein of
7.0 gram/dl)
48. For SG 1.053,measure 52.25 ml of stock soln.
and make up to 100 ml in a volumetric flask.
For SG 1.055,measure 54.3 ml stock solution
and made up to 100 ml with distilled water.
100 ml of this solution is kept in bottles and
this will be enough for 100 tests, after which it
is discarded and a fresh solution prepared.
49. This procedure does not give the exact
Hb value, as it is not accurate. So it is
not used as a routine test.
Use in blood donor screening
It is used in blood banks as a
screening procedure to ensure that
the donor is not anaemic.
Since there is no need to know the
individuals exact Hb value of the
donor, the blood bank sets a cut off
value for men and women and copper
sulphate solutions with corresponding
specific gravity are prepared.
50. Chemical methods
(estimation of the iron
content)
The principle is based on the fact that each
molecule of haemoglobin contains 4 atoms of iron
or 0.347 grams of iron per 100 grams of
haemoglobin.
The iron present is detached from the
haemoglobin and measured.
The haemoglobin is calculated by using the
formula.
Hb(gm/dl) = Blood iron content in mg/dl blood
3.47
51. This is a complex method which is difficult
and time consuming but very accurate and is
therefore used as a “reference method “
especially by those who are preparing the
Cyanmethaemoglobin standard.
It is almost never done in routine clinical
Laboratory.
52. Gasometric methods
(Measurement of Oxygen combining
capacity)
• It is done by using van
Slyke apparatus.
• This is a reference
method as it is very
accurate, however it is
not used for routine
laboratory work.
53. The principle is based on the fact that one
molecule of O2 binds to each iron atom.
So one molecule of haemoglobin binds 4
molecules of oxygen. Thus oxygen combining
capacity thus indirectly measures the amount
of Hb.
It is estimated that 1 gram of haemoglobin binds
about 1.34 ml of oxygen.
From this the haemoglobin concentration is
calculated by using the following formula.
Hb in gm/dl = O2 binding capacity in ml/dl blood
1.34
54. Though it is difficult and time consuming
method, it is a reference method because of
its accuracy.
Disadvantages:
This method measures only functional
Haemoglobin and not Sulphaemoglobin or
carboxyhaemoglobin, which does not bind
with with oxygen.
55. Condition that decreases
haemoglobin concentration
• Physiological:
1)pregnancy (due to hemodilution).
2)women have lower values than men because the
total RBC count is less.(testosterone stimulates
erythropoiesis in men but Estrogen inhibits
erythropoiesis in female).
• Pathological:
1)Different types of anaemia.
2)Excess ADH secretion as seen in pituitary tumour.
56. Condition that increases haemoglobin
concentration
• Physiological:
1)high altitute(due to hypoxia)
2)newborns and infants.
• Pathological:
1)condition that produce haemoconcentration(due to
loss of body fluid)for eg,Severe diarrhoea,Vomiting.
2)condition that produce hypoxia for eg:Congenital
heart disease,emphysema.
3)polycythaemia vera.
57. Sources of error in
Haemoglobinometry
Errors in sampling
Inadequate flow of blood from the finger
prick.
Excessive squeezing of the finger after
pricking.
Prolonged use of tourniquet when
collecting venous blood,which leads to
concentration of blood cells.
Insufficient mixing of venous blood,which
has sediment after collection.
Small clots in venous blood due to
inadequate mixing with EDTA after
collection.
58. Contd…
Adding too little blood to Drabkin’s diluting fluid
(pipetting or dilution error).
Air bubbles trapped in pipettes.
Reagents left on bench exposed to prolonged light or
allowed to freeze.
Reference preparation out of date or deterioration
,especially if it has been left standing on the bench for
sometime after opening the vial.
59. Faulty or dirty equipments
• Broken or chipped pipettes.
• Dirty pipettes.
• Dirty cuvettes.
• Dirty filters.
• A defective spectrophotometer,
hemoglobinometer or colorimeter.
60. Faulty technique
• Using a dilution factor different from the one for which the spectrophotometer,
haemoglobinometer or colorimeter was calibrated.
• Inadequate mixing of reagents.
• Air bubbles in the cuvette.
• Using a standard filter from another spectrometer or haemoglobinometer for adjustment.
• Using wrong filter for the colorimeter.
• Improper instrument calibration.
• Main voltage variations.
• Non linearity.
• Cuvettes incorrectly positioned.
• Cuvettes dirty or scratched.
61. ways to minimise technical errors
Technical errors can be reduced by
good training, understanding the
clinical significance of the test and
the necessity for a dependable
method,adherence to oral and
written instructions and familiarity
with the equipment and with sources
of errors.
Automated instruments are widely
used now-a-days and this eliminates
most of these errors.
62. Quality Control
The important aspect of quality control is to identify
those steps in which the likelihood of error is high
and to consider ways to minimize that likelihood.
Some of the measures followed are:
Duplicating samples.
Hemolysate of known value are run with batches of
tests.
Haemoglobin values are compared with other
values. For example PCV = 3 x Hb. This is true unless
there is marked microcytosis or macrocytosis.
If haemoglobin values are abnormal either too low
or high, check peripheral smear to look for other
associated abnormalities.
63. Summary
Haemoglobin (Hb) is a chromoprotein and the main
content of the red cell, which carries oxygen to the
tissues and carbon dioxide away from tissue.
It can be estimated on the basis of colour, specific
gravity, oxygen binding capacity,iron content method.
Visual comparative methods is not satisfactory
because it has high degree of error.
Cyanmethaemoglobin method is the internationally
recommended method for determining haemoglobin
concentration of blood in laboratory.
Estimation of haemoglobin is helpful for diagnosis
and prognosis of anaemia.
Haemoglobin helps to study changes in Hb
concentration before and after operations and blood
transfusions.