Hemoglobin estimation

HEMOGLOBIN ESTIMATION
  • Hemoglobin is a conjugated protein that comprises of 90% of dry weight of RBC.
  • Hemoglobin is tetramer containing two pairs of similar polypeptide chains called globin chains. To each of the four chains is attached heme which is complex of iron in ferrous form and protoporphyrin.
  • Types of hemoglobins

  • Abnormal hemoglobins – Hb S, Hb C, Hb D, Hb E
  • Functions of hemoglobin
    • Carries oxygen to tissues
    • Carries carbon dioxide from tissues to lungs
  • Normal hemoglobin concentration
    • Men – 13 – 18g/dl
    • Women – 12 – 16g/dl
    • New born – 16 – 22 g/dl
    • Children – 12 – 14g/dl
  • WHO cut off value below which anemia is present
    • Men – <13g/dl
    • Women – <12g/dl
    • Pregnant – <11g/dl
  • INDICATIONS FOR HEMOGLOBIN ESTIMATION
    • Detremine the presence and severity of anemia
    • Screening for polycythemia
    • To assess response to specific therapy in anemia
    • Estimation of Red cell indices
    • Selection of blood donors

 

  • METHODS OF DETERMINATION OF HEMOGLOBIN
  • Visual method
    • Sahli’smethod
    • Dares method
    • Haldane method
    • Tallquists method
    • WHO hemoglobin color scale method
    • Spencer method
  • Gasometric method
  • Spectrophotometric method
    • Cyanmethemoglobin method
    • Oxyhemoglobin method
    • Alkaline hematin method
  • Specific gravity method
  • Chemical method
  • Hemoglobin estimation in autoanalyzers
  • Portable hemoglobinometer (hemocue system)

 

METHOD OF COLLECTION OF BLOOD FOR HEMOGLOBIN ESTIMATION
  • Finger prick (in children and adults)
  • From veins (venous blood)
  • Blood collected in EDTA (1.0 to 1.2mg/ml) or double oxalate (2mg/1ml) in appropriate proportion (Double oxalate is mixture of 3 parts of ammonium oxalate and 2 parts of potassium oxalate)
  • Heal prick (in infants)

 

SAHLI’S ACID HEMATIN METHOD
Principle           
N/10 HCl converts hemoglobin into soluble unstable acid hematin. The colour intensity of the acid hematin after dilution is compared with standard brown glass in the comparator
Apparatus required
  • Sahli’s hemoglobinometer
    • It contains hemoglobin tube, comparator, hemoglobin pipette and stirrer
    • Hemoglobin pipette – pipette has one mark indicating 20cumm (no bulb)

Hemoglobinometre pipette

 

Hemoglobinometre tube

 

  • Hemoglobin tube – it is graduated on both sides. One side is graduated in gram percentage from 2 – 22 (g%). Other side is graduated as percentage from 20 – 140 (%)
  • Comparator – central slot accommodates hemoglobin tube and on either side non-fading brown tinted glass pieces for colour matching are present
  • Stirrer – thin glass rod for stirring the solution

Sahlis hemoglobinometre

 

 

Sahlis hemoglobinometre tube

Hemoglobinometre tube

  • N/10 HCl-
    • To make 500 ml of N/10 HCl mix
      • Concentrated HCl – 4.5ml
      • Distilled water – 500ml
  • Distilled water
Procedure
  • Add N/10 HCl to the hemoglobinometer tube up to its lowest mark i.e 2g% (If N/10 HCl is taken above the mark the color of undiluted solution is lighter than standard and if N/10 HCl is taken less then all the hemoglobin is not converted).
  • Take blood up to 20 cu mm mark on the pipette and transfer it to the hemoglobinometer tube containing N/10 HCl.
  • Leave the solution for 10 minutes (for the conversion of hemoglobin to acid hematin)
  • After 10 minutes add distilled water drop by drop and mix it by stirrer until the color matches with the color of comparator. While matching the color glass rod should be removed from the solution.
  • The lower meniscus of the solution should be taken as the result which expresses hemoglobin content as g%
If the hemoglobin is too low (less than 3g/dl) then 40µl blood is added to HCl upto 20 marks in the tube. Color is matched and result is halved
Advantages
  • Inexpensive
  • Easy to perform
  • Requires no technical skill
  • Can be used as bed side procedure
  • Reagents and apparatus are free
Disadvantages 
  • As Acid hematin is unstable, color fades away quickly
  • Visual error is possible while matching with brown color of comparator box
  • Technical errors like improper mixing of blood, errors in pipetting and capillary blood with tissue fluid can give false results
  • No international standard for brown coloured comparator box
  • If the match point is passed then the whole process has to be repeated
  • Not all types of hemoglobin are converted to acid hematin (eg.sulfhemoglobin, methemoglobin)

 

HALDANE METHOD
Blood is mixed with hypotonic solution like distilled water to produce hemolysis of RBCs . Carbon monoxide is added to this mixture. The colour produced is compared with the standard one.
However commercially available standards are unreliable and fade quickly.

 

DARE METHOD
In this method small glass chamber is filled with whole blood by capillary action. Then glass chamber is illuminated by battery bulb. Colour of the blood  is matched with standard after seeing through eye piece.

 

TALLQUIST METHOD
In this method drop of blood is placed on filter paper and the colour is matched with standard
Though it is quickest method, high degree of errors are possible

 

WHO HEMOGLOBIN COLOR SCALE METHOD
This technique of estimating hemoglobin is based on comparing the color of a drop of blood absorbed on a particular type of chromatography paper against a printed scale of color corresponding to different levels of hemoglobin ranging from 4 to 14gm/dl

 

SPENCER METHOD
In this method the color of diluted oxyhemoglobin is matched visually. This method is less accurate than sahlis method. It is more difficult for the human eye to accurately grade and match small differences in red color than brown color of acid hematin.

 

SPECTROPHOTOMETRIC METHODS
CYANMETHEMOGLOBIN METHOD
This is the most accurate method.
  • Principle –  when the blood is mixed with a solution  containing potassium ferricyanide and potassium cyanide, all types of hemoglobins except sulfhemoglobin is converted to cyanmethemoglobin. The intensity of color is proportional to hemoglobin concentration and it is compared with a known cyanmethemoglobin standard at 540nm (green filter) in a photoelectric colorimeter.
  • Reagents used
    • Drabkins reagent
      • Distilled water – 1000ml
      • Potassium ferricyanide – 200mg
      • Potassium dihydrogen phosphate – 140mg
      • Potassium cyanide – 50mg
      • Non-ionic detergent – 1ml
    • pH of Drabkins solution is 7.0 to 7.4
    • Drabkins reagent is clear and pale yellow.
    • It should be stored in brown borosilicate bottles as it is unstable if exposed to light.
    • If temperature is high should be refrigerated at 2 to 80 (but never freeze) and should be brought to room temperature before using it.
    • When measured in a spectrophotometer against water as a blank at the wavelength of 540nm, the absorbance must be zero
    • Drabkins solution should not be used if
      • pH is outside the range
      • fluid is turbid
      • its absorbance in spectrophotometer is not zero at 540nm
  • Cyanmethemoglobin standard solution
    • commercially available
    • Standard is directly pipetted in a cuvette and optical density measured at 540nm (green filter). Reading obtained corresponds to 15g/dl of hemoglobin for dilution of 1:250
    • Necessary to store this hemoglobin standard at 2 – 80C
    • Bring this standard solution to room temperature before recording its optical density on photometer.
Procedure
  • 20µl of blood is added to 5ml of Drabkins reagent and mixed well. Leave it for 5 minutes. Now measure the absorbance of this solution and standard in spectrophotometer at 540nm after adjusting the OD at 0 by using Drabkins solution as blank
  • Calculation – Hbg g/dl –  15 X OD test / OD standard
Advantages
  • Visual error is absent as there is no color matching
  • Cyanmethemoglobin is stable so it does not fade away quickly
  • All forms of hemoglobin except Sulfhemoglobin are converted to cyanmethemoglobin
  • Reliable standard reference is available from WHO for direct comparision
Disadvantages
  • Potassium cyanide is poisonous and should not be pipetted by mouth
  • After dilution solution should be kept for some time for complete conversion
  • Rate of conversion of blood containing carboxy hemoglobin is long (30 minutes)
  • Blood with abnormal plasma proteins and high leukocyte count may cause turbidity on dilution of blood

 

OXYHEMOGLOBIN METHOD
  • 0.007N Ammonium hydroxide is added to blood which causes hemolysis of red cells and converts hemoglobin to oxyhemoglobin. The color of the solution is measured in the spectrophotometer at wave length of 540nm.
  • Advantage – simple , quick and reliability is not affected by increased bilirubin level
  • Disadvantages –
    • Oxyhemoglobin solution fades quickly.
    • Method does not give satisfactory results in the presence of methemoglobin, sulfhemoglobin and carboxyhemoglobin.
    • Stable standard solution cannot be prepared

 

ALKALINE HEMATIN METHOD
  • Hemoglobin is converted to alkaline hematin by adding alkali such as sodium hydroxide. Then the color is measured in spectrophotometer at 540nm.
  • Standard solution used in this method is Gibsons and Harrisons solution. This is mixture of Chromium potassium sulphate, Cobaltous sulphate and Potassium dichromate in aqueous solution. This solution is equal in color to 1 in 100 dilution of containing 16.0 gm of Hbg/dl
  • Advantage – It gives true estimate of total hemoglobin including sulfhemoglobin and methemoglobin. It is not affected by plasma proteins and lipids
  • Disadvantage – less accurate than cyanmethemoglobin. Some forms of hemoglobin like HbF are resistant to alkali denaturation.

 

GASOMETRIC METHOD
  • It is an indirect method of estimation of hemoglobin by using Van slyke apparatus.
  • This method is based on assumption that 1gm of hemoglobin carries 1.34ml of oxygen
  • It is not routinely used method as it is time consuming and complex process
  • It is used for research purpose and for preparation of standard solutions

 

SPECIFIC GRAVITY METHOD
  • In this method copper sulfate solution is used .
  • Specific gravity of copper sulfate solution is 1.053 which is equivalent to 12.5g% of hemoglobin.
  • Procedure – Drop of blood is dropped into the copper sulfate solution. The density of drop is directly proportional to the amount of hemoglobin in that drop. If the drop is denser than the specific gravity of the solution, it will sink to bottom. If the density is less then it will float
  • This technique is routinely used to screen the blood donors for anemia.

 

CHEMICAL METHOD
  • It is an indirect method which is based on the assumption that 0.347 mg of iron is compared with 100g of hemoglobin.

 

HEMOGLOBIN ESTIMATION IN AUTOANALYZERS (SYSMEX SERIES OF INSTRUMENT)
  • Principle: Electrical impedance principle
  • In this method blood is diluted using reagent diluent and then Sodium lauryl sulphate (SLS) hemoglobin reagent is added.
  • SLS reagent has surfactant which lyse RBC’s and release hemoglobin. It also reduces the turbidity developing from plasma lipids and cell membrane
  • Sodium Lauryl sulphate converts Ferrous (Fe+2) to ferric (Fe+3) forming methemoglobin. Methemoglobin combines with Sodium Lauryl sulphate forming SLS hemichrome molecule.
  • The absorbance of this molecule is measured at 555nm to determine hemoglobin
  • Advantage
    • Free hemoglobin is rapidly converted to detectable chromogen decreasing the measurement time
    • Reagent is cyanide free
  • Disadvantage – False elevation is noted when there is high WBC count (>30,000/µl)

 

PORTABLE HEMOGLOBINOMETER (HEMOCUE SYSTEM)
  • It consists of precalibrated portable battery operated spectrometer
  • No dilution is required
  • Blood is run by capillary action directly into cuvette containing sodium nitrate and sodium azide which convert hemoglobin to azide methemoglobin
  • The absorbance of this is measured at wavelength of 565nm.

 

PRINCIPLE SOURCES OF ERROR IN ESTIMATION OF HEMOGLOBIN
  • Errors of sampling including presence of micro-clots in EDTA/oxalated blood due to inadequate mixing
  • Presence of excess of tissue fluid diluting the capillary blood due to squeezing in finger prick method
  • Error while diluting the blood
  • Error in estimation of color intensity
  • Improperly lysed red cells, nucleated red cells, paraproteins, lipids may give erroneous high results.

 

 QUALITY CONTROL
  • Use of appropriate anticoagulant (citrate samples should not be used)
  • Sample should be checked for clots
  • Use of standards and controls in analyzers
  • Storage and stability of standard solutions should be maintained
  • Blood samples and controls must be brought to room temperature before testing samples.

 

CONDITIONS WITH VARIATIONS IN HAEMOGLOBIN LEVEL
  • Conditions where hemoglobin are raised
    • Chronic obstructive pulmonary disease
    • Congenital  cyanotic disease of heart
    • Smokers polycythemia
    • Renal cell carcinoma – due to ectopic secretion of erythropoietin
    • Pheochromocytoma
    • Polycythemia vera
    • People living in high altitude
  • Conditions where hemoglobin is falsely raised
    • Burns
    • Severe dehydration
    • Immediately after acute hemorrhage
    • Blood taken during the intravenous infusion of iron containing drugs
  • Conditions with false anemia
    • Pregnancy – due to increase in plasma volume leading to fall of 1 to 2 g/dl
    • Hypervolemia – due to disproportionate increase in plasma volume and RBC volume
  • Causes for decreased hemoglobin concentration
    • Anemia due to
      • Blood loss
      • Parasitic infection
      • Drugs and lead poisoning
      • Dietary deficiency (iron, copper, vitamins)
      • Malabsorption of nutrients
      • Chronic disease (diseases of liver, kidney and cancer)
  • Physiological variations
    • Strenuous physical exercise
    • Diurnal variation – highest in morning and lowest in evening
    • High altitude – increase with increase in altitude
References
  1. Tanushree Srivastava, Himanshu Negandhi, Sutapa B Neogi, Jyoti Sharma and Renu Saxena. Methods for Hemoglobin Estimation: A Review of “what works”. Journal of Hematology and Transfusion 2014;2(3):1028.
  2. Sabitri sanyal, Aparna Bhattacharya.Clinical pathology A practical manual 2017. Third edition.
  3. Praful B. Godkar, Darshan P. Godkar.Textbook of medical laboratory technology 2007. Second Edition
By
  • Dr. V. Shanthi (Professor of Pathology, Narayana Medical College, Nellore)
  • Dr. B. Syam Sundara Rao (Professor of Pathology, Narayana Medical College, Nellore)