Various method of hb estimation Sahlis hematin method Cyanmethemoglobin method etc

1. ⚫ BY MAHESH KUMAR
⚫ ROLL NO. :: 06
⚫ BSC MLT 1ST YEAR
[DR. RPGMC TANDA KANGRA]

2. ◼method used to measure the concentration of hemoglobin in blood. It's essential for diagnosing
conditions like anemia and monitoring certain medical treatments.
VARIOUS METHODS:
1.Calorimetric Methods:
 In these methods, color comparison is made between the standard and the test sample by
calorimetric methods.
*Visual Method:
 Sahli's Acid-Hematin Method
*Photoelectric Methods:
 Cyanmethemoglobin
 Oxyhemoglobin method: HB liq. NH3 Oxyhemoglobin color. Obs. Photocalorimeter
540nm
*Alkaline Hematin method

3. 2.Gasometric Method:
 oxygen carrying capacity of blood is measured in a van slyke apparatus.
 1gm HB carries 1.34 ml of oxygen.
 This method measures only physiologically active hemoglobin ,which can carry oxygen.
 Result 2% less than other methods.
3.Chemical Method:
 Iron content of HB is estimated first.
 100 grams of HB contain 374 mg of iron.
4. Specific Gravity Method:
 This method is useful in mass screening like selection of blood donors
 copper sulphate technique is used to determine specific gravity of blood and rough estimate of
HB is obtained

4. SAHLI'S ACID HEMATIN METHOD:
 Principle:
 Blood is added to N/10 HCL which converts Hb into Acid-Hematin
 The brown color of Acid-Hematin is matched against the brown color of the standard of the
comparator.
 Apparatus:
 Sahli's Hemoglobinometer: Comparator, Sahli's HB Tube
 Dropper
 Glass Rod as Stirrer
 Reagent:
 N/10 HCL and Distilled Water
 Specimen:
 EDTA-Anticoagulated venous blood or from a finger prick.

6. Method:
• Add 0.N HCl with a dropper into the square HB meter tube up to the mark of 2
gm%
• Fill HB pipette with 0.02 ml of blood and wipe off the excess blood at the nozzle
of the pipette with moistened cotton.
• Blow off the blood in the pipette into the HB meter tube.
• Rinse the pipette by drawing in and discharging the blood acid solution at least
twice.
• Allow acid to act on RBCs for 10 minutes to lyse the red cells and convert HB
into acid hematin
• Match the color of the solution with that of the comparator in natural light.
• If the color is darker, add distilled water with the dropper and stir the solution
with the glass rod. Continue the process until the color of the solution matches
the comparator color.
• Take out the stirrer from the solution.
• Take reading from the upper meniscus in gm%

7. Sources of Error:
•If blood in EDTA tube is not mixed properly before filling the pipette, low/ high
may be obtained depending on whether the plasma or sedimented RBCs are taken
the pipette.
Advantages:
•Easy to perform and quick.
Disadvantages:
•Carboxyhemoglobin, methemoglobin, Sulfhemoglobin, and fetal hemoglobin are
converted to acid hematin by 0.1 N HCl.
•Color of brown glass standard fades with time.
•Personal visual error in matching colour is 10%
•Normal Range:
•Males: 13-18 gm%
•Females: 11-16 gm%
•Newborns: 14-19 gm%

8.  Clinical significance of HB:
 Low Hb values:
 Deficiency or hemolytic anemia
 High Hb values:
 Polycythemia vera
 Secondary erythrocytosis
 Hypoxia
 Cigratte smoking
 Precautions:
 Rinse the Hb pipette using tap water in a beaker.
This prevents blocking of the pipette.

9. CYANMETHAEMOGLOBIN METHOD :
 PRINCIPLE: When blood is mixed with Drabikin's reagent containing Potassium
Cyanide and potassium ferricyanide.
 Hemoglobin reacts with K.ferricyanide to form methemoglobin and is then
converted to stable cyanmethemoglobin (HICN) by potassium cyanide.
 The color of the solution is compared with known cyanmethemoglobin standard at
540 nm, and its intensity is directly proportional to the concentration of
hemoglobin in blood.
 Apparatus:
 Hb pipette
 Spectrophotometer

10.  Reagents:
 Drabkin's solution at ph( 7.0-7.4)
 Contains;
 POTASSIUM CYANIDE =100mg
 POTASSIUM
FERICYANIDE=400mg
 POTASSIUM DIHYDROGEN
PHOSPHATE =280mg
 STEROX SE =1ml
 D/W TO =2ltr's

11.  Sample:
 EDTA anticoagulated venous blood or blood from a finger prick or capillary blood.
 METHOD
 Take 5 ml of Drabkin's solution in a test tube.
 Add 0.02 ml of blood to it using an HB pipette.
 Wait for 10 minutes.
 Switch on the calorimeter with a green filter / 540 nm, and wait for 5 minutes.
 Adjust OD to 0 using Drabikin's solution as a blank.
 Pour the test tube solution into the cuvette and note down the OD of the test
sample.
 OD of the standard cyanmethemoglobin solution is taken against the blank.
 Calculate hemoglobin concentration using the standard curve.

12. •Preparation of standard curve
•1. Make serial dilutions of the reference solution eg 1in2, 1in4, 1in8 and so on with Drabkin’s solution.
•As the Hb present in the solution is known, the Hb concentration of each dilution will also be known.
• 2. Take the OD of each dilution in the colorimeter against a blank of Drabkin’s solution.
•3. Plot the OD against the Hb concentration on a linear graph paper. The absorbance is plotted on the
vertical axis and the hemoglobin concentration on the horizontal axis.The points should lie in a straight line
that passes through the origin.
• 4. From this graph a table of readings and the corresponding Hb value can be prepared. This is more
convenient than the graph specially when a large number of readings have to be taken. After OD of the
sample is taken, the corresponding Hb value can be directly read from the table

14.  Advantages:
 All forms of hemoglobin except sulfhemoglobin are converted to HICN, so accurate HB values are
obtained.
 Visual error is eliminated as no color matching is required.
 Disadvantages:
 Potassium cyanide (KCN) is a poisonous substance, and therefore Drabkin solution must never
be pipetted by mouth.
 Abnormal plasma proteins may cause turbidity when blood is diluted with Drabikin solution,
leading to erroneous results.
 Precautions:
 Never pipette Drabikin solution directly.
 Lipemic blood with high total leukocyte count (TLC) can cause turbidity, resulting in erroneous
results.


15.  MODERN HB ESTIMATION METHOD:
 Automated Hematology Analyzers:
These are sophisticated instruments
that use flow cytometry, impedance,
and/or spectrophotometry techniques to
measure hemoglobin levels along with
other blood cell parameters. They offer
high throughput and accuracy, requiring
minimal manual intervention.