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Guided By:
• Respected S.K SHARMA Sir
• Respected JOSEPH Sir




                             Presented By:
                             BISHWAS NEUPANE
                             B.sc MLT part I
                             PGIMER (NIPS)
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.
HEMOGLOBIN STRUCTURE

HEME (CONSTITUTES
 ABOUT 4% OF THE
    WT OF THE
    MOLECULE)


                    HEMOGLOBIN

     GLOBIN (
  CONSTITUTES
ABOUT 96% OF WT
  OF MOLECULE)
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.
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%
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.
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.
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
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.
Visual comparision method

   Talliquist scale method.
   Dare’s method.
   WHO haemoglobin colour scale.
   Spencer method.
   Acid haematin or Sahli’s method.
   Alkaline haematin method.
   Haldane’s carboxyhaemoglobin method
   BMS haemoglobinometer(compatator)
• 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.
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.
• 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.
• 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.
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.
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.
Reagents:
• N/10 Hydrochloric acid (HCl)

• Distilled water for dilution.

• Blood anticoagulated with
  EDTA
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.
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.
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.
•    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.
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.
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
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
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.
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.
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.
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.
 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
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.
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
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.
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.
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.
Cyanmethaemoglobin
       method
This is the preferred and the
most accurate method for
determining the
haemoglobin concentration
in laboratory.
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.
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.
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)
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.
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.
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
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.
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.
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.
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.
 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.
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)
 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.
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.
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
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.
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.
 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
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.
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.
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.
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.
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.
Faulty or dirty equipments

• Broken or chipped pipettes.

• Dirty pipettes.

• Dirty cuvettes.

• Dirty filters.

• A defective spectrophotometer,
  hemoglobinometer or colorimeter.
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.
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.
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.
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.
Haemoglobin estimation bishwas  neupane b.sc mlt part i

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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.
  • 3. HEMOGLOBIN STRUCTURE HEME (CONSTITUTES ABOUT 4% OF THE WT OF THE MOLECULE) HEMOGLOBIN GLOBIN ( CONSTITUTES ABOUT 96% OF WT OF MOLECULE)
  • 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.
  • 10. Visual comparision method  Talliquist scale method.  Dare’s method.  WHO haemoglobin colour scale.  Spencer method.  Acid haematin or Sahli’s method.  Alkaline haematin method.  Haldane’s carboxyhaemoglobin method  BMS haemoglobinometer(compatator)
  • 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.
  • 17. Reagents: • N/10 Hydrochloric acid (HCl) • Distilled water for dilution. • Blood anticoagulated with EDTA
  • 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.