Type of hemoglobin estimation

1. ANIL KHADKA
B.SC.MLT STUDENT

2. INTRODUCTION
 Haemoglobin is the major constituent of the red blood cell cytoplasm accounting for
approximately 90% of the dry weight of the mature cells.
It comprised of heme and globin
Haemoglobin is a protein( heme protein)
It is two type of protein are found in haemoglobin
- globular proteins( functional proteins)
- linear proteins( structure proteins)
Haemoglobin is considered of globular proteins
Mature RBCs do not be synthesis haemoglobin, while immature RBCs synthesis haemoglobin.
Haemoglobin synthesis is Polychromatic stage of erythropoiesis.
Mitochondria is very important for heme synthesis

3. STRUCTURE OF
HAEMOGLOBIN
• Hemoglobin is a conjugative
protein due to attachment of
prosthetic group.
• Hemoglobin contain 2 parts
heme and globin.
• Heme part is prosthetic group
and globin is protein parts.
• Iron containing pigment called
heme attached with the protein
globin.
• Ferrous form iron found in
hemoglobin.

4. Function of
haemoglobin:
haemoglobin carries oxygen lung to
tissue.
It carries carbon dioxide back from the
tissue to the lungs.
Oxygenated haemoglobin give red colour
to the blood.
It acts as a chemical buffer helps in
the regulation of pH or acid base
balance.

5. Haemoglobin
derivatives:
1. oxyhaemoglobin: haemoglobin with Fe++ + O2
seen in arterial blood circulation.
2. Deoxyhaemoglobin: haemoglobin with
Fe++ but no O2 seen in venous blood
circulation.
3. Carboxyhaemoglobin: haemoglobin with Fe++
and carbon monoxide(CO) haemoglobin has
200 times more affinity for CO than O2,
so CO is carried instead of O2 can result in
death but is reversible if give pure O2.
4. Sulf-haemoglobin: haemoglobin with sulfer
can not transport O2 caused by drugs and
chemicals.
5. Methaemoglobin: haemoglobin with Fe+++
can not transport O2 increased level caused
cyanosis and anaemia.

6. BLOOD COLLECTION OF ESTIMATION OF HAEMOGLOBIN
• Capillary blood collection
• Venous blood collection
• Arterial blood collection
INDICATION FOR HAEMOGLOBIN ESTIMATION
• Determine presence and severity of anaemia.
• Screening for polycythaemia.
• Response to specific therapy in anaemia
• Estimation of red cell indices.
• Selection of blood donors

7. METHODS OF HEMOGLOBIN ESTIMATION
1. Physical method( based on specific gravity)
2. Chemical method( based on iron content of haemoglobin)
3. Gasometric method( based on oxygen combining capacity of haemoglobin)
4. HemoCue method
5. Colorimetric method( based on colour)
Colorimetric method divided in to two method
1. Visual method:
* direct matching method
* carboxy haemoglobin method
* acid hematin method
2. Photo colorimetric method
* alkaline hematin method
* oxy haemoglobin method
* cyanmeth haemoglobin method

8. PHYSICAL METHOD
 It is based on specific gravity.
 Specific gravity of given sample of blood depend upon the
concentration and the weight of the material present in haemoglobin.
 Specific gravity of blood is 1.060, so change in the specific gravity of blood
is mainly due to change in concentration of haemoglobin.
 A solution od copper sulphate(cuso4) having specific gravity of 1.093 is
prepared. Thee solution is equivalent to 12.5 gm of haemoglobin. When a
drop of blood is dropped into cuso4 solution, if it falls to the bottom its
specific gravity is more that of the solution if it remains on the surface the
specific gravity is less.
Advantage
- This method is quickly and easy
- It is used in blood bank for donor screening.
Disadvantage
- It does not give exact vale
- Not accurate
- Not used routinely

9. CHEMICAL METHOD
- It is based on the iron containing of haemoglobin.
- In this method the amount of iron present in a blood sample is determined chemically and the
amount of haemoglobin is calculated indirectly.
- 1 gm of haemoglobin contain 3.47mg of iron. (0.347gm iron/ 100 gm of haemoglobin)
- The haemoglobin is then calculated by using the formula.
Hb concentration/100ml of blood =
𝑏𝑙𝑜𝑜𝑑 𝑖𝑟𝑜𝑛 𝑐𝑜𝑛𝑡𝑒𝑛𝑡 𝑖𝑛 𝑚𝑔
𝑑𝑙
3.47
Advantage
- Very accurate method
- Used as a reference method i.e. a method used to check the accuracy of other method
Disadvantage
- Complex
- Time consuming

10. GASOMETERIC METHOD
- It is based on oxygen combining capacity of haemoglobin.
- It is done by using van slyke apparatus.
- One molecules of haemoglobin binds 4 molecules of oxygen. Thus oxygen
combining capacity.
- It is indirectly measure the amount of haemoglobin
- This method involves the oxygen binding capacity of haemoglobin and direct
calculation of haemoglobin in the blood.
- 1 gm of haemoglobin contain 1.34 ml of oxygen, hence the amount of oxygen
present in the particular volume of blood is determined by
Concentration of Hb in gm%=𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑂2 𝑟𝑒𝑙𝑒𝑎𝑠𝑒𝑑 100𝑚𝑙 𝑜𝑓 𝑏𝑙𝑜𝑜𝑑
1.34

11. Continue
Disadvantage
- It is time consuming method and required expensive
instrument.
- Methaemoglobin and carboxy haemoglobin cannot be
determined.

12. HAEMOCUE
METHOD:
- It consists of a pre-calibrated, portable
battery operates spectrometer
- No dilution required because blood run
by capillary action directly into a cuvette
containing sodium nitrite and sodium
azide which convert haemoglobin to
azidemethemoglobin
- The absorbance is measured at
wavelength of 565nm

13. Continues----------
Advantage:
• Quickly result are available
• Can be used for mass
screening purpose.
• No precision equipment is
required.
• Use only for single drop of
blood.

14. VISUAL
COLORIMETRIC
METHOD
1. Direct
marking method
It is also called tallquist method this involves the direct visual matching
of red colour of a drop of whole fresh blood on a filter paper
with different colour standard.
The amount of haemoglobin present in the sample corresponds to that of
band with which the colour is matched.
WHO haemoglobin colour scale:
Based on comparing the colour of a drop of blood absorbed o a particular
type of chromatography paper. Against a printed scale of colour are
observed to different levels of haemoglobin ranging from 4-14 gm/dl.
figure of WHO haemoglobin colour scale next slide

15. Continues------

16. 2. CARBOXY
HAEMOGLOBIN:
In this method haemoglobin is converted to
carboxy haemoglobin by carbon monoxide in
dilute blood. Bright red colour produced is
matched by adequate dilution with colour
standard and report is given in gm%

17. 3. ACID HEMATIN METHOD
It is also known as shali’s method.
Principle
When blood is added to 0.1N HCl, haemoglobin is converted to brown
coloured acid hematin. The resulting colour after dilution is compared
with standard brown glass reference blocks of a sahli’s
haemoglobinometer.
The concentration of haemoglobin is read directly from the calibration
tube. The intensity of brown colour is proportion to the concentration
of haemoglobin present in the sample.
Hb + 0.1N HCl acid haematin( brown colour)

18. REQUIREMENT
Sahli’s haemoglobinometer contains
Graduate glass tube
Shali’s Pipette
Comparator block
Glass stirrer
0.1N HCl
Distilled water for dilution
EDTA anticoagulated blood

19. Fig: SHALI'S HAEMOGLOBINOMETER

20. PROCEDURE
Take 0.1N HCl up to lowest mark of graduated glass tube.
Draw the blood specimen into Sahli’s pipette till the mark of 20µl .
Wipe outside of the pipette by using tissue paper.
Transfer the blood in to the acid solution inside the bottom of the tube.
Mix the acid and blood by shaking the tube well.
Allow to stand for 10 minute after dilute the solution with in distilled water
drop by drop till the colour of diluted blood matches the colour of the standard
block.
Reading should be done against natural light.
Take the reading in g/dl or gm%.

21. NORMAL RANGE
Male: 13-17 gm%
Female: 12-16gm%
At Birth: 14- 22gm%
At one month: 11.5- 16.5gm%
At one year: 11.1 – 14.1 gm%
At 6-12 year : 11.5- 16.5 gm%

22. CLINICAL SIGNIFICANCE
1. INCREASED CONDITION
Polycythemia
Hypoxia
Dehydration
2. DECREASED CONDITION
Anemia
Hemodilution
Pregnancy

23. PRECAUTION
 Immediately after use, rinse the haemoglobin pipette by using the tap water to
prevent the blocking of the pipette.
 the brown colour is not stable, so the reading should be taken with a
short time.
ADVANTAGE
ü very easy method
üRequires little amount of blood
üReagent are cheep

24. DISADVANTAGE
Visual error is vey high.
Technical error:
 improper mixing of blood.
 Error in pipetting.
Tissue fluid contaminating while capillary blood is used .
The brown colour is not stable, so the reading should be taken with in a short
time
Carboxy haemoglobin, meth haemoglobin, sulf haemoglobin are not converted
to acid hematin.

25. SOURCE OF ERROR
Clotted blood
Visual error
Faulty eye problem
Improper mixed blood
Quality of comparator
Pipetting error
Time delay in reading

26. PHOTOELECTRIC METHOD
1. Alkaline hematin method
• Haemoglobin is converted to alkaline hematin by the addition of strong
alkali(N/10) NaOH the brown colour produced is measured
calorimetrically or comparator standards.
Advantage:
This method is accurate and can measured all type of haemoglobin except
fetal haemoglobin.

27. 2.
OXYHAEMOGLOBIN
METHOD:
- Haemoglobin is converted to oxy haemoglobin
by dilute solute of sodium carbonate or
ammonium hydroxide.
- This solution or mixture is read
by photoelectric
colorimeter using green filter.
Disadvantage:
There are not stable standards.
It can not measure carboxy haemoglobin
methaemoglobin and sulf-haemoglobin

28. 3. CYANMETH HAEMOGLOBIN METHOD:
 recommended method by ICSH( The International Committee of standardization
of haematology).
Most accurate and preferred method.
Commonly used method for haemoglobin estimation.
principle:
When a blood mixed with Drankin’s reagent containing potassium cyanide(KCN)
and potassium ferric cyanide( K3{Fe(CN)6}, haemoglobin react with potassium
ferric cyanide to form methaemoglobin which is not stable .
Methaemoglobin is converted to stable cyanmethemoglobin(HiCN) by react with
potassium cyanide.
Haemoglobin(Fe++) + K3{Fe(CN)6} methaemoglobin(Fe+++)
Methaemoglobin +KCN cyanmethemoglobin(HiCN)
Cyanmethemoglobin also known as hemiglobincyanide

29. Continues-----------------------
• the intensity of the colour is proportion to haemoglobin concentration and it is
compared with a known cyanmethemoglobin and it is compared with a known
cyanmethemoglobin standard at 540 nm( green filleter)
Specimen:
Capillary blood / thoroughly mixed EDTA anticoagulated venous blood.
Note: EDTA( Ethylene diamine tetra acetic acid)
Requirement:
1. Drabkin’s reagent
2. Haemoglobin standard
3. haemoglobin pipette
4. Test tube
5. Photometer/ spectrophotometer

30. Composition of drabkin’s fluid
- Potassium ferricyanide = 200mg
- Potassium cyanide = 50mg
- Potassium dihydrogen phosphate = 140 mg
- Non ionic detergent = 1 ml
- Distilled water up to = 1000ml
function:
- potassium ferricyanide and cyanide to help the haemoglobin converted (cyanmethemoglobin).
- The detergent lyses the RBCs quickly and it also dissolve the cell.
- Potassium dihydrogen phosphate acts as a buffer to control the pH.
Note: in the place of non-ionic detergent sterox SE-0.5ml , triton X- 100-1 ml or saponin 1ml
may be used
haemoglobin standard are commercially available. This standard is directly pipette in a cuvette
and optical density is measured at 540nm .
Haemoglobin standard or cyanmethemoglobin standard concentration = 60mg/dl

31. Properties of reagent
• The reagent should be clear, pale yellow and pH -7-7.4
• when it is measured against water as a blank in photometer at 540 nm the
absorbance must red 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.
Advantage of modification drabkin’s fluid:
This is less likely to cause plasma protein precipitation which could interfere
with test results.
The detergent enhance complete lysis of red cells.
Shortens the reaction time and ensure complete conversion of haemoglobin to
HiCN is shortened from 10 minutes to 3 minutes.

32. Procedure:
Pipette in the tubes labelled as blank and test.
Mixed the contents in the tube labelled as test thoroughly and wait form 5
minutes.
Read absorbance of test by setting blank at 100% T at 540nm.
Read absorbance of standard by pipetting it directly in cuvette.
Reagent name Blank Test
Drabkin’s fluid 5ml 5ml
EDTA blood ---- 20µ

33. calculation:
Haemoglobin(gm/dl)=
𝑂.𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡
𝑂.𝐷 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
×
𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑 𝑚𝑔/𝑑𝑙
1000
× 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
=
𝑂.𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡
𝑂.𝐷 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
×
60
1000
×250
=
𝑂.𝐷 𝑜𝑓 𝑡𝑒𝑠𝑡
𝑂.𝐷 𝑜𝑓 𝑠𝑡𝑎𝑛𝑑𝑎𝑟𝑑
× 15

34. Preparation of standard curved:
When a graph of concentration of the haemoglobin standard solution on the
test X- axis and optical density of standard on the Y- axis is plotted a straight
line passing through the original is obtained which is called the standard curve
of haemoglobin.
A unknown haemoglobin concentration may be calculated from the measured
optical density and can be read from standard calibration curve directly.
Dilution the known haemoglobin standard and read the optical density.
Plot the graph of optical density of haemoglobin standard on the vertical axis
and the haemoglobin standard with gm/dl on the horizontal axis.
A straight line passing through the origin is obtained this graph can be used in
determining the haemoglobin concentration of blood sample.

35. Continues----
-------------

36. precaution:
- The reagent is poisonous so handle it carefully
- Mix anticoagulation blood by swirling properly before pipettes.
- Do not discard drabkin’s reagent in the sink poisonous cyanide gas is related if
the sink has an acidic solution.
- If capillary blood is used keep the drabkin’s reagent ready in the test tube.
Advantage:
- Most accurate method.
- The colour produced is stable for a considerable period.
- All form of haemoglobin except sulf haemoglobin are readily converted to
cyanmethemoglobin.
- Standard is stable.

37. Disadvantage:
Drabnkin’s reagent is highly poisonous.
Abnormal plasma concentration or high leukocyte
count may result in turbidity so it will cause false
high value.

38. Reference
Note books B.SC.MLT
Dacie and Lewis practical
haematology book.
All photos are copied by google.