Hemoglobin estimation by Sahli's method ( Sahli’s haemoglobinometer )

1. Hemoglobin estimation by Sahli's method(Sahli’shaemoglobinometer) DR. MOHAMMED CHYAD AL-NOAEMI

2. Hemoglobin Estimation • Haemoglobin concentration provides information about the status of anaemia in the population. Normal adult Hb: • In male, it is 14-18 g/100ml of blood (dl). • In female it is 12-16 g/100ml of blood.

3. Hemoglobin is estimated By either • Sahli’s Method • Colorimetric Method. • Autoanalyzer. • Hemoglobin and Hematocrit (HCT) Test Meter Kit • Sahli’s method were compared to Haemiglobincyanide (HiCN) method of same subjects. Results: Sahli’s method is less accurate and has lower values than Haemiglobincyanide method

4. Sahli’s Method • Hermann Sahli (May 23, 1856 – April 28, 1933) was a Swiss internist. Sahli,s apparatus (1930)

5. Materials used • 1. Sahli’s haemoglobinometer. • 2. Two Pasteur pipettes (one for HCl and one for distilled water). • 3. Glass rod to stir ( stirrer ) • 4. 0.1 N - Hydrochloric acid • 5. Distilled Water • 6. Comparison tube. • 7. Pipette ( Hemoglobin pipette with rubber tubing and mouthpiece )

6. Procedure • 1. Add 0.1N.hydrochloric acid (1: 10 diluted) to the haemometer tube (comparison tube) up to lowest graduation ( 0.02 gramme ). • 2. Sterilize the fingertip with Isopropyl alcohol, surgical spirit and allow it to dry • 3. Using sterile lancet prick the finger tip • 4. Wipe away first few drops of blood • 5. Suck blood into the hemoglobin pipette ( capillary pipette ) up to 20 cu.mm( avoid air bubbles coming into a tube ) • 6. Wipe the outside tip of pipette, clean with tissue

7. Cont. Procedure • 7. Immediately transfer the blood to the comparison tube • 8. Suck blood back into the pipette several times and blow out again into the tube(to mix blood with HCl ) • 9. Place the haemometer tube in the stand and allow for 5-10 minutes (during this period HCl lysis red cell, and released hemoglobin on reacting with HCl forms a dark brown colored Acid Hematin). • 10. Now using a Pasteur pipette to add a few drops of distilled water (or 0.1N HCl) and stir the contents with a glass rod .

8. Cont. Procedure 11. Continue to add water (or acid) drop by drop and stir the contents each time until the solution is just darker than the standard . 12. Carefully add one or two drops of water till the color exactly matches with that of the Standard and note the reading 13.while taking the reading hold the haemoglobinometer against good daylight at arms length 14. The comparison tube represent 100 percent haemoglobin with reference to a standard which is 14. 8 g Hb / 100 ml of blood

9. Summary-Sahli’s Method • It measures acid hematin. A sample of 0.02 mg of whole blood taken with a pipette, is mixed with a small quantity of 0.1mol/litre HCl. After few minute, distal water or 0.1 mol/lit HCl is added drop by drop and mixed, until the color of the solution matches the color of the two identical standards placed to the left and right of the dilution tube. Hemoglobin concentration is then read from the graduated scale on the dilution tube.

10. Drabkin's is more accurate than sahli’sMethod • Colorimetric Method: Using potassium ferricyanide will produce Cyanmethemoglobin. • Comparative study of hemoglobin estimated by Drabkin's and Sahli's methods. • P Balasubramaniam, A Malathi. • Year : 1992 | Volume : 38 | Issue : 1 | Page : 8-9 • http://www.jpgmonline.com/article.asp?issn=0022-3859;year=1992;volume=38;issue=1;spage=8;epage=9;aulast=Balasubramaniam

11. Hemoglobin • The name hemoglobin is the combination of heme and globin, reflecting the fact that each subunit of hemoglobin is a globular protein with an embedded heme (or haem) group. Each heme group contains one iron atom, that can bind one oxygen molecule. The most common type of hemoglobin in mammals contains four such subunits.

12. Hemoglobin (Hb): • Each RBC contains 200-300 millions molecules of Hb, which represents about one third of the RBC mass. Each Hb molecule consists of globin [globular protein; consists of 2 alpha (141aa and 2 beta (146aa) subunits] and four heme molecules. Each heme molecule is attached to one subunit. Each heme contains an iron atom to which an oxygen molecule could be combined. Hence, one Hb molecule can carry maximally four oxygen molecules. • [If Valine replace Glutamine → Sickle cell anemia]. • When blood is pumped to the lungs, Hb becomes saturated with oxygen, forming oxyhemoglobin (bright red in color). When this blood is pumped to the body tissues, some its oxygen dissociates from Hb, and this reduced Hb is dark red in color.

13. Hemoglobingreen = heme groupsred & blue = protein subunits

14. Types of Hemoglobins • HemoglobinA (Hb A), (α2alphaβ2beta), about 97%. • Hemoglobin A2 (HbA2), (α2δ2 delta), . About 2%. • Hemoglobin F (HbF), (α2γ2Gamma), or fetal Hb. It is less than 1% in adults but it is the dominants Hb in fetal life. Glycosylated hemoglobin (HbA1c): normally it is about 5-6%. In which Hb A is combined with glucose. It is increase in diabetes mellitus. • [Alpha-α , Betaβ-, Delta-δ, Gamma- γ-globin.]

15. Glycosylated Hemoglobin • Long-term control of blood sugar concentration can be measured by the concentration of Hb A1c. Measuring it directly would require many samples because blood sugar levels vary widely through the day. Hb A1c is the product of the irreversible reaction of hemoglobin A with glucose. A higher glucose concentration results in more Hb A1c. Because the reaction is slow, the Hb A1c proportion represents glucose level in blood averaged over the half-life of red blood cells, is typically 50-55 days. An Hb A1c proportion of 6.0% or less show good long-term glucose control, while values above 7.0% are elevated. This test is especially useful for diabetics.[23]

16. Hemoglobin and, Hematocrit (HCT) Test Meter Kit HIGH PRECISION PROFESSIONAL AND HOME USE PORTABLE METER

17. Glucometer