1. HbA: 96-98%, Composed of two alpha(α) and two beta(β) globin chains.
2. HbA2: 1.5-3.5%, Composed of two alpha(α) and two delta(δ) globin chains.
3. HbF: < 1.0%, Composed of two alpha(α) and two gamma(γ) globin chains.

Hemoglobin can combine with other substances, some normally and some abnormally and can also occur as:

1. Oxyhemoglobin: Oxygen combined with hemoglobin.
2. Carboxyhemoglobin: Carbon monoxide (CO) combined with hemoglobin.
3. Carbaminohemoglobin: Carbon dioxide (CO₂) combined with hemoglobin.
4. Methemoglobin: Iron oxidized from its ferrous state to ferric state.
5. Sulfhemoglobin: Sulfur combined with the hemoglobin.
6. Cyanmethemoglobin: Methemoglobin bonded to cyanide ions.

Methods for Hemoglobin Estimation:

The different methods used for the estimation of hemoglobin can be divided as follows:

1. Visual methods:
   • Sahli’s method
   • Dare method
   • Haden method
   • Wintrobe method
   • Tallqvist method
2. Spectrophotometric methods:
   • Oxyhemoglobin method
   • Cyanmethemoglobin method
3. Gasometric method
4. Automated hemoglobinometry
5. Other methods:
   • Alkaline-hematin method
   • Specific gravity method
   • Lovibond Comparator method

Sahli’s method for hemoglobin estimation

Sahli’s method, also called as acid hematin method is the visual comparator method for the estimation of hemoglobin. As visual comparison may lead to unacceptable imprecision and accuracy, this method is not recommended nowadays and the use of spectrophotometric methods like Cyanmethemoglobin method is preferred to it.

Principle:

When the blood is added to dilute hydrochloric acid (HCl), hemoglobin present in the RBCs is converted into brown-colored acid hematin. The acid hematin solution is further diluted until it’s color matches exactly with the permanent standard brown glass compared by direct vision.

Requirements:

Specimen:

Capillary or venous blood. Venous blood should be anticoagulated with 1.5-1.8 mg EDTA per mL of blood and mixed immediately.

Instruments:

1. Sahli’s hemoglobinometer
   It is a set of devices that includes a comparator, hemoglobin tube, hemoglobin pipette, and stirrer.
   
   • Comparator: It is a rectangular plastic box with a slot in the middle which accommodates a hemoglobin tube. Brown standard glasses are provided on either side of the slot for color matching. White opaque glass is present at the back to provide uniform illumination.
   • Hemoglobin tube: Sahli’s graduated hemoglobin tube is graduated in one side in gram percentage (g%) from 2 to 24, and on the other side in percentage (%) from 20 to 140. The tube is also called Sahli-Adams tube.
   • Sahli’s pipette or hemoglobin pipette: It contains only one mark at 20μl or 0.02ml. Unlike WBC and RBC diluting pipettes, it contains no bulb.
   • Stirrer: It is a thin glass rod used for stirring the mixture inside the hemoglobin tube.

Reagents:

1. N/10 Hydrochloric acid (HCl): Mixing 36 grams HCl in distilled water to 1 liter gives 1 N HCl. Diluting it 10 times will give N/10 HCl.
2. Distilled water

Procedure:

1. Ensure that the hemoglobinometer tubes and pipette are clean and dry.
2. Fill the hemoglobinometer tube with N/10 HCl up to its lowest mark i.e. 2 g% or 10% mark with the help of a dropper.
3. Take blood up to mark in the Sahli’s pipette (20 μl). Wipe the extra blood outside the pipette and deliver it to N/10 HCl in the hemoglobin tube.
4. Mix and leave it for 10 minutes in order for a complete conversion of hemoglobin to hematin.
5. Add distilled water drop by drop and stir till color matches with the standard glass of the comparator.
6. Take the reading at lower meniscus, which directly gives the hemoglobin concentration in 100 ml of blood.

Advantages and Disadvantages of Sahli’s method

Advantages:

1. Easy to perform and convenient.
2. Not very time consuming. Can be performed within maximum 15 minutes.
3. Reagents and apparatus are cheap and easily available. Reagents are less harmful.
4. Can be used in mass surveys. Doesn’t require electricity.

Disadvantages:

1. Acid hematin is a suspension, not a true solution. So, some turbidity may result.
2. This method can’t measure all hemoglobins. It estimates only oxyhemoglobin and reduced hemoglobins. But carboxyhemoglobin, methemoglobin and sulfhemoglobin (which all constitute about 2-12% of total hemoglobin) are not converted to acid hematin.
3. HbF is not converted to acid hematin. Therefore, Shali’s method is not suitable for measuring hemoglobin in infants upto 3 months.
4. WBC counts >100,000/cumm will produce turbid solution of acid hematin, which will increase the hemoglobin report by 5-10%.
5. Chances of visual error are high. Color of acid hematin also fades gradually.
6. The color of glass standard may fade over time.

References:

1. Pal, G.K., 2006. Textbook Of Practical Physiology-2Nd Edn. Orient Blackswan.
2. Ghai, C.L., 2012. A textbook of practical physiology. JP Medical Ltd.
3. A. V. Naigaonkar. A Manual Of Medical Laboratory Technology.
4. Cheesebrough, M., 1998. District laboratory practice in tropical countries, part II. Cambridgeshire Tropical Health Technology, Cambridge, UK, 231.
5. Laboratory Procedures in Clinical Hematology By United States. Department of the Army
6. Mohan, H. and Mohan, S., 2011. Practical Pathology for Dental Students. JP Medical Ltd.

The post Sahli’s Method For The Estimation Of Hemoglobin appeared first on LaboratoryTests.org.

1. HbA: 96-98%, Composed of two alpha(α) and two beta(β) globin chains.
2. HbA2: 1.5-3.5%, Composed of two alpha(α) and two delta(δ) globin chains.
3. HbF: < 1.0%, Composed of two alpha(α) and two gamma(γ) globin chains.

Hemoglobin can combine with other substances, some normally and some abnormally and can also occur as:

1. Oxyhemoglobin: Oxygen combined with hemoglobin.
2. Carboxyhemoglobin: Carbon monoxide (CO) combined with hemoglobin.
3. Carbaminohemoglobin: Carbon dioxide (CO₂) combined with hemoglobin.
4. Methemoglobin: Iron oxidized from its ferrous state to ferric state.
5. Sulfhemoglobin: Sulfur combined with the hemoglobin.
6. Cyanmethemoglobin: Methemoglobin bonded to cyanide ions.

Methods for Hemoglobin Estimation:

The different methods used for the estimation of hemoglobin can be divided as follows:

1. Visual methods:
   • Sahli’s method
   • Dare method
   • Haden method
   • Wintrobe method
   • Tallqvist method
2. Spectrophotometric methods:
   • Oxyhemoglobin method
   • Cyanmethemoglobin method
3. Gasometric method
4. Automated hemoglobinometry
5. Other methods:
   • Alkaline-hematin method
   • Specific gravity method
   • Lovibond Comparator method

Cyanmethemoglobin method for hemoglobin estimation

The cyanmethemoglobin method or hemoglobincyanide method is the most accurate method of choice for the measurement of hemoglobin. It is a type of colorimetric/spectrophotometric method which has the following three major advantages over other methods:

1. Measures all forms of hemoglobin except sulfhemoglobin, which is normally not present in the blood.
2. Can be easily standardized, and
3. Cyanmethemoglobin reagent is very stable.

Principle:

Blood is diluted 1:201 in a solution containing potassium ferricyanide (C₆N₆FeK₃) and potassium cyanide (KCN). Potassium ferricyanide oxidizes hemoglobin in the sample to methemoglobin. The methemoglobin further reacts with potassium cyanide to form a stable-colored cyanmethemoglobin (hemiglobincyanide- HiCN) complex. The intensity of the colored complex is measured at 540 nm which is directly proportional to the amount of hemoglobin present in the specimen.

Requirements:

Specimen:

Capillary or venous blood. Venous blood should be anticoagulated with 1.5-1.8 mg EDTA per mL of blood and mixed immediately.

Reagents:

1. Drabkin’s reagent, pH 7.0–7.4
   The original cyanmethemoglobin technique was proposed by Stadie in 1920. This method used separated alkaline ferricyanide and cyanide reagents. A single reagent was introduced by Drabkin and Austin in 1935.

   This fluid contains:

   • Potassium ferricyanide = 200 mg
   • Potassium cyanide = 50 mg
   • Potassium dihydrogen phosphate = 140 mg
   • Non-ionic detergent = 1 ml
   • Distilled or deionized water = to 1 liter
2. Hemoglobin standard

Procedure:

1. Label three clean, dry test tubes as Blank (B), Standard (S), and Test (T).
2. Pipette as follows:

	Blank	Standard	Test
Drabkin’s Reagent	5 ml	5 ml	5 ml
Hemoglobin standard	–	20 µl	–
Sample	–	–	20 µl

3. Mix well and allow to stand at room temperature (25⁰C) for 5 minutes.
4. Measure the absorbance of the standard and test sample at 540 nm (green filter) against blank in a colorimeter. The color will be stable for up to several hours.

Calculation:

Calculate the concentration of hemoglobin in the specimen using the following formula:

Alternatively, a calibration graph can be prepared using multiple standards of varying concentration and the result can be obtained quickly by checking hemoglobin concentration which corresponds to obtained absorbance. This is markedly acceptable when a large number of specimens are daily processed on the same instrument.

References:

1. Pal, G.K., 2006. Textbook Of Practical Physiology-2Nd Edn. Orient Blackswan.
2. DR .Sara Fadhil Bunea, Medical laboratory techniqes, Human physiology practical.
3. Haemoglobin Reagent Product Insert – Tulip Diagnostics

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