Hemoglobin estimation BMLT


Hemoglobinometry


measurement of the hemoglobin content of the blood, usually with a hemoglobinometer after the hemoglobin has been converted to cyanmethemoglobin.

Haemoglobin

Haemoglobin is the iron-containing protein attached to red blood cells that transports oxygen from the lungs to the rest of the body. 
Haemoglobin binds with oxygen in the lungs, exchanges it for carbon dioxide at cellular level, and then transports the carbon dioxide back to the lungs to be exhaled.

Indications for hemoglobin estimation

  • To determine presence and severity of anemia
  • To assess response to specific therapy in anemia
  • Screening for polycythemia
  • Estimation of red cell indices
  • Selection of blood donors

METHODS OF HAEMOGLOBIN ESTIMATION

  • Colorimetric method
  • Gasometric method
  • Specific gravity/ physical method
  • Chemical method

METHODS OF HAEMOGLOBIN ESTIMATION

  • A. COLORIMETRIC METHOD
  1. Visual colorimetric method. -Sahlis method or acid hematin method.-Alkaline hematin method.
  2. Photocolorimetric (photoelectric) method -Cyanmethemoglobin (HiCN) method-Automated analyser - Heme -cue blood haemoglobin system  - Oxyhemoglobin method-Haldane method
  • Physical method – Copper sulfate specific gravity – Used in blood donors.
  • C.Chemical method – Iron content of hemoglobin

  • D. Gasometric method – Oxygen combining capacity of hemoglobin measures the Hb.

COLORIMETRIC METHOD 


Visual colorimetric method:


Sahlis method or acid hematin method
Most Popular colorimetric method.

Principle: When blood is added to N/10HCl, hemoglobin is converted to acid hematin. The resulting brown color of acid hematin is matched against the standard brown colour of the comparator. The intensity of the brown colour depends on the amount of acid hematin produced, which in turn depends on the amount of hemoglobin in the blood sample.

Sahli's Hemoglobinometer

Sahli's Hemoglobinometer aims to estimate the haemoglobin content in blood by Sahli's method. 



Materials of the experiment

Sahli's haemoglobinometer consists of:
  • Haemoglobin pipette which has one mark of 0.02ml (20cumm)
  • A comparator which has a flat surface and a standard brown color.
  • A square/ round tube with markings of hemoglobin %(one side), Hb in gm(on other side).
  • Glass rod for mixing the blood in the square/round tube.
  • A dropper to take and transfer N/10HCl and distilled water into the square tube. 


REAGENTS

  1. N/10 Hydrochloric acid (HCL)
  2. Distilled water for dilution
  3. Blood anticoagulated with EDTA

Procedure

  • Place N/10 HCL in tube up to the lowest mark.
  • Draw up to 20mm mark in the pipette and transfer it to the acid in the tube.
  • Rinse the pipette well by drawing up to 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.
  • Add distill Water drop by drop , mixing well by a glass rod and compare the colour of this tube with the standard tube.
  • After 5 minutes, read the haemoglobin content from the scale on the graduated mixing tube. Read the lower meniscus.
  •  - Haemoglobin is recorded as gram/dl (or gm%) 

Advantages

  • Simple, easy and inexpensive method
  • No colorimeter is required
  • Small quantity of blood is needed
  • No sophisticated equipment is required.
  • Can be repeated often.

Disadvantages

  • Technical error
  • Visual error
  • Quality of color comparators can affect the reading
  • Insufficient time
  • Time delay

Alkaline Hematin method

Principle : Hb is converted to alkali hematin by the additon of a strong alkali (N/10 NaOH). The alkali hematin formed is brown in color and is read against comparator standards or in a colorimeter. This method is not in use.

Photoelectric method

CYANMETHEMOGLOBIN METHOD:
-Most accurate, convenient, readily available and preferred method for estimation of hemoglobin concentration. It is the standard method used in most of the centres.

Principle: Blood is diluted in a stable standard solution of potassium ferricyanide and potassium cyanide. Hemoglobin is first oxidised to methemoglobin by the potassium ferricyanide. The cyanide ions of the potassium cyanide convert methemoglobin to stable cyanmethemoglobin. The cyanmethemoglobin has a broad absorption, maximum being at a wavelength of 540nm. Thus the color of this solution is compared against a standard HiCN solution of known Hb value in a spectrophotometer or photoelectric colorimeter at540nm.

Apparatus: Photoelectric colorimeter with a green filter or spectrophotometer at a wavelength of 540nm is used.

Reagents: Diluent known as Drabkin solution.

-Potassium Ferricyanide  - 400mg
-Potassium Cyanide – 100mg
-Potassium dihydrogen phosphate (anhydrous)-280mg 
-Non ionic detergent (sterox SE) – 1mL
-Distilled water – 2L

Technique: 

  • Take 5 mL of Drabkin solution in a test tube.
  • Take twenty microliter (0.02 ml) of blood in a hemoglobin pipette and add it to the above test tube. Rinse the Hb pipette at least twice by drawing in Drabkin solution.
  • Thoroughly mix the blood with Drabkin solution.
  • Allow it to stand for 10 mins at room temperature.
  • Switch on the photoelectric colorimeter at 540 nm or with an appropriate filter (green) colorimeter
  • Adjust optical density at 0 using Drabkin solution as blank.
  • Pour test solution into the cuvette and note down OD of the test sample.

  • OD of standard Cyanmethemoglobin solution is taken against the blank.

  • Hb=OD of test sample x concentration of standard x 250
       -------------------------------------------------------------------------
       OD of standard x 1000

Advantages

  • Hb value will be accurate.
  • -Visual error during matching as in Sahlis is eliminated as this is colorimetric.
  • -Cyanmethemoglobin is a stable compound. Colour doesnt change with time.
  • -Easy to perform
  • All forms of hemoglobin can be estimated.

Disadvantages

  • Turbidity interferes with the reading – due to plasma proteins, hyperlipidemia, high WBC or fat droplets.
  • Poisonous diluents. – Potassium cyanide is poisonous.
  • Explosive reagent- Undiluted reagents poured in sink can explode.

Automated analyser.

  • Along with Hb, cell counts, PCV and absolute values (MCV, MCH, MCHC ) are also calculated.
  • Most of the cell counters utilize a cyanide free biodegradable reagent.

Hemo-cue blood hemoglobin system.

  • WHO approved method for estimating Hb in blood donors.
Principle: Reaction in the cuvette is a modified azidemethemoglobin reaction. The microcuvette contains 3 reagents in dried form which convert Hb into azidmethemoglobin. The photometer uses a double wavelength measuring method. , 570 nm and 880nm and determines Hb.

Oxy hemoglobin method

  • Principle : 0.04% liquid ammonia is used to lyse the red cells. Oxyhemoglobin formed is then read in the colorimeter.
  • Advantages
  • Test can be read within few seconds
  • Colour is stable for a longer duration

Haldane method:

Principle: Red cells are lysed and carbon monoxide is added which converts hemoglobin to carboxy hemoglobin. The colour is compared with that of a given standard.

Specific gravity method

  • A rough estimate of hemoglobin is obtained from the specific gravity of blood as determined by using copper sulphate solution of known specific gravity (1.053)
  • Normal specific gravity of blood 1.048 to 1.066

chemical method

  • Iron content of hemoglobin is first estimated
  • Value of hemoglobin is then derived directly from the formula that 100gm of hemoglobin contains 374 mg of iron
  • Tedious time consuming
  • Useful in mass screening of blood donors

Gasometric method

  • Van- slyke method
  • Oxygen carrying capacity of blood is measured in a van slyke apparatus 
  • The amount of hemoglobin id then derived from the formula that 1gm of hemoglobin carries 1.34ml of oxygen 
  • Measures only physiologically active hemoglobin which carry oxygen
  • Time consuming and expensive.