Haemoglobin Estimation

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

Presented By:BISHWAS NEUPANEB.sc MLT part IPGIMER (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.

HEME (CONSTITUTES ABOUT

4%

OF THE WT OF THE MOLECULE)

GLOBIN

( CONSTITUTES

ABOUT 96%

OF WT OF

MOLECULE)

HEMOGLOBIN

HEMOGLOBIN STRUCTURE

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 haemoglobinIt 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.

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)

Visual comparision method

• 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

N/10

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. 16Advantage 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 1000Where,A540 = absorbance of solution at 540nm.16114 = monomeric molecular weight of haemoglobin.11.O = millimolar coefficient extinction.d = layer thickness in cm1000 = 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.