INTRODUCTION TO HEMOGLOBIN
Hemoglobin (Hb or Hgb) is a red color pigment present in red blood cells (RBCs) comprises Fe2+ and Globin protein. It is Hemoglobin in RBCs that carries the oxygen from the lungs to the tissues and CO2 from body tissues to the lungs for excretion.
Hemoglobin (Hb or Hgb) is responsible for the appearance of Red color RBCs and blood. Hemoglobin is a chromoprotein consisting of Globin molecule attached to 4 red colored Heme molecules. Hemoglobin synthesis requires the coordinated production of Heme and Globin. Heme is a prosthetic group that medicates reversible binding of oxygen by hemoglobin. Globin is the protein that surrounds and protects the Heme molecule.
The Estimation of hemoglobin in the blood is commonly prescribed in various physiological and pathological conditions and as both diagnostic and prognostic test especially in case of suspected Anemia which can be caused by various factors.
Nowadays in many laboratories, the Hemoglobin estimation is done by using Automatic Hematology Analyzers but still in many other labs the following method is Commonly used to determine the Hemoglobin concentration in patient’s blood.
- Sahli’s Method a.k.a. Acid Hematin Method
- Cyanmethemoglobin Method (CMG) a.k.a Drabkin’s Method
ESTIMATION OF HEMOGLOBIN BY SAHLI’s METHOD
PRINCIPLE OF SAHLI’s METHOD / ACID HEMATIN METHOD
The principle of Sahli’s Method or Acid hematin method is quite easy that when the blood is added to N/10 Hydrochloric acid (HCl), the hemoglobin present in RBCs is converted to acid hematin which is a dark brown colored compound. The color of the formed acid hematin complex corresponds to the Hemoglobin concentration in the blood and is matched with the standard which is a reference brown glass given in the Sahli’s apparatus by diluting with N/10 hydrochloric acid or distilled water until the color of acid hematin complex match with the color of the standard.
REAGENTS REQUIRED FOR SAHLI’s METHOD / ACID HEMATIN METHOD
- N/10 hydrochloric acid (It is prepared by diluting concentrated hydrochloric acid 0.98 ml in distilled water and volume is made up 100 ml).
- Distilled water
APPARATUS & EQUIPMENTS REQUIRED FOR SAHLI’s METHOD / ACID HEMATIN METHOD
- Sahli’s Apparatus
- Hemoglobin pipette (0.02 ml or 20 µl capacity)
- Sahli’s graduated Hemoglobin tube
- Thin glass rod Stirrer for Hemoglobin Tube
- Sahli’s Comparator box with brown glass standard
- Spirit swab
- Blood Lancet
- Dry cotton swab
- Pasteur pipette
PROCEDURE OF SAHLI’s METHOD / ACID HEMATIN METHOD
⇒ N/10 Hydrochloric acid is taken in Hemoglobin tube (has two graduations – one side gm/dl, and other side shows the Hb %age), up to the mark 20 – the lowest marking (yellow marking).
⇒ Venous or Capillary blood is drawn up to 20µl mark of hemoglobin pipette exactly.
⇒ For capillary blood draw, boldly prick the tip of the middle or ring finger with the help of Blood lancet or pricking needle. Wipe out the first drop of blood and suck the blood from the second drop in Hb pipette up to the mark of 20 µl. Fill the Hb pipette by capillary action.
⇒ Wipe out the surface of the pipette with the help of tissue paper/ cotton so that excess blood may not be added to the Hb tube.
⇒ Dispense the blood into N/10 hydrochloric acid taken in the hemoglobin tube, rinse the pipette with the same solution and mix properly with the help of stirrer.
⇒ Place the tube at room temperature for 10 minutes for complete conversion of hemoglobin into acid hematin.
⇒ After the reaction completes, place the Hb tube in the column in Sahli’s Comparator box and start diluting the dark brown coloured compound (Acid Hematin) formed in the Hb tube using the N/10 HCl or distilled water by adding drop by drop of it into the solution and mix with the help of stirrer after each addition.
⇒ This process is done until the endpoint comes matching the color of standard with the color of the test.
⇒ Once the color is matched with the standard brown glass, lift the stirrer up and note down the reading in Sahli’s Hb tube by taking the lower meniscus in consideration.
Note: Usually in colored solution, the upper meniscus is considered for taking the reading but in this case, it is a transparent color solution and lower meniscus can be recorded in order to give the exact reading.
⇒ Now add one more drop of distilled water and mix it properly with the help of stirrer. If color is still matching with the standard add another drop till it matches with the standard and note down the reading and, if it gets lighter after adding the first extra drop, it shows reading taken before dilution was correct. Note down that reading as the final result.
⇒ Reading of this method is expressed in Hemoglobin gm/dl (gram/100 ml) of blood.
PRECAUTIONS TO BE TAKEN WHILE PERFORMING ESTIMATION OF HEMOGLOBIN BY SAHLI’s METHOD / ACID HEMATIN METHOD:
⇒ Sahli’s apparatus especially the Hemoglobin pipette and Sahli’s Hemoglobin tube should be clean and dry before use.
⇒ Suck the blood exactly up to the mark of 20 µl (0.02 ml) and air bubbles should not be present in the pipette with blood.
⇒ Mix well the acid and blood and wait for at least 10 minutes after adding the blood in acid.
⇒ Add distilled water drop by drop and mix well after each dilution. Avoid over dilution of the content.
⇒ The matching of color should be done against the natural source of light or electrical tube light (white light) to avoid any visual errors.
⇒ Blood sample and N/10 HCl acid should be taken in an accurate and precise amount in the Hb tube.
⇒ The Hb pipette should be wiped off properly in order to avoid the excess addition of blood in the Acid.
ADVANTAGES OF SAHLI’s METHOD / ACID HEMATIN METHOD
⇒ It is the simple and easy method and may be done at any place because apparatus can be picked up anywhere.
DISADVANTAGES OF SAHLI’s METHOD / ACID HEMATIN METHOD
⇒ Visual intensity may be different for different individuals by this method, we are not able to measure the inactive hemoglobin.
⇒ This method estimates only oxy Hemoglobin. Carboxyhemoglobin and methemoglobin cannot be estimated.
⇒ The endpoint disappears soon so it is difficult to know the actual endpoint and also the Proper stable standard is not available
⇒ The resulting solution is not a clear solution but a suspension due to the action of hydrochloric acid on the proteins and lipids.
NORMAL VALUES OF HEMOGLOBIN
- Adult Male: 14-16 gm/dl
- Adult Female: 13-15 gm/dl
- Newborn: 16-18 gm/dl
CLINICAL SIGNIFICANCE OF HEMOGLOBIN ESTIMATION
Hemoglobin estimation gives a brief idea of the pathological conditions to the physician so that your physician can easily understand the cause of pathology and prescribe an effective treatment for it.
Raised Hemoglobin Content
- Polycythemia Vera
- Associated with Hypoxia
- Cyanotic Congenital Heart disease
- High Altitudes
- Heavy smoking
- Elevated erythropoietin levels
- Tumors of Kidney, Liver, CNS, Ovary etc.
- Renal Diseases (Hydronephrosis & Vascular impairment)
- Adrenal hypercorticism
- Therapeutic androgens
- Relative causes of high hemoglobin content
- Dehydration – Water deprivation, Vomiting, Diarrhea
- Plasma loss – Burns, Enteropathy
Reduced Hemoglobin Content
Low Hemoglobin value means anemia caused by the following conditions
- Iron deficiency anemia
- Parasitic infections severely in hookworm infection
- Sickle cell anemia
- Aplastic anemia
- Hemolytic anemia
- Loss of blood
FACTS ABOUT HEMOGLOBIN
- Each gram of hemoglobin carries 1.34 ml oxygen
- Each gram of Hb contains 3.33 mg of iron
- Total Hb content in a healthy body is approximately 600 gms.