Drabkin’s solution is used to estimate the amount of hemoglobin that is present in the blood of the individual. Drabkin’s solution helps in the detection of many processes such as hemolysis in RBCs, etc.

Drabkin’s Solution For Hemoglobin Estimation

There are kits available commercially, which makes it easy to prepare the solution. When blood passes through the analyzer, these instruments estimate its level of hemoglobin.

Sample For The Estimation Of Hemoglobin

A sample of blood is required for the estimation of hemoglobin.

Manual Method For The Preparation Of Drabkin’s Solution

For the preparation of Drabkin’s solution, follow the steps below.

  1. Put in one vial of Drabkin’s reagent to 1000 ml of water.
  2. Add 0.5 ml of 30% of Brij L23 solution, catalog number B4184, with the mixture of drabkin’s reagent and water.
  3. Mix well and if insoluble particles are remaining, filter those as well.
shows the different constituents and their quantity required in the preparation of Drabkin's solution.
Figure: shows the different constituents and their quantity required in the preparation of Drabkin’s solution.

Precautions For Drabkin’s Solution

  1. Avoid contact with eyes and skin.
  2. Make sure that you don’t breathe in the vapors.
  3. Good ventilation is needed to prevent vapors from forming.
  4. Don’t eat, drink, or smoke while using this product.
  5. In case, the solution becomes cloudy after the addition of blood, centrifuge before taking the readings. This occurs because of the non-hemolyzed RBCs or globulins.
  6. Clean the spectrophotometer cells so that they would be free of fingerprints, or else the reading will be high
Figure shows the backside of Drabkins reagent packaging that includes all the warnings
Figure : shows the backside of Drabkin’s reagent packaging that includes all the warnings.

Principle Of Drabkin’s Solution

Drabkin’s procedure is based on the oxidation of hemoglobin and its derivatives. However, sulfhemoglobin is not oxidized in the Drabkin’s solution. Sulfhemoglobin is a pigment that occurs in the blood. It forms methemoglobin in the presence of potassium ferricyanide.

The reaction of methemoglobin and potassium cyanide forms a stable product, cyanmethemoglobin. This complex compound has a maximum absorption of 540nm. 

Methemoglobin + potassium cyanide = cyanmethemoglobin.

Figure showing the basic principle of Drabkin s solution for the estimation of Hb image
Figure: showing the basic principle of Drabkin’s solution for the estimation of Hb.

Procedure to estimate Hemoglobin (by Drabkin’s solution)

  1. Take 0.02ml of blood + 4 ml Drabkin’s solution= 1:200 dilution.
  2. Or, you can also take 0.02 ml of blood + 5 ml of Drabkin’s solution= 1:250 dilution.
  3. Mix the solution.
  4. Within 6 hours of mixing the blood with the Drabkin’s solution, take readings.
  5. At filter 540, record the readings on the spectrophotometer.
  6. Read against blank of Drabkin’s solution.
  7. Read the standard (12 G/dL) solution with the identical dilution as that of the test sample.
  8. Take readings from spectrophotometer, which are called the optical density (OD).
Figure shows the results of hemoglobin oxyhemoglobin carboxyhemoglobin and methemoglobin calculated by Drabkins solution

Hb (Hemoglobin) calculation formula (optical density – OD)

Normal Hemoglobin Level

Hemoglobin levels are usually the same in both male and female infants which gradually rises with the increase in age. The normal level of Hb of the respective gender and age is given in the table below.

GenderAgeNormal Hb level:
Male & female1-5 years10.9 – 15.0 g/dl
Male & female5-11 years11.9 -15.0 g/dl
MaleAdult14 – 18 g/dl
FemaleAdult12 – 16 g/dl

Physiological Variation Of Hb

Factors causing an increase in the level of Hb

  1. Respiratory and heart diseases
  2. Testosterone and other hormones
  3. Cigarette smoking
  4. Kidney diseases
  5. High altitude
  6. Polycythemia

Factors causing a decrease in the level of Hb

  1. Genetic disorders in the production of hemoglobin
  2. Gastric Bypass Surgery
  3. Iron deficiency
  4. Blood loss
  5. Increased levels of heavy metals and toxins in the body
  6. Aging
  7. Some medications
  8. Pregnancy
  9. Bone marrow disorder
  10. Chronic kidney diseases
  11. Other vitamin and mineral deficiencies
  12. Disorders and diseases that disrupt the nutrient absorption
  13. Abnormal disruption of RBCs
  14. Hypothyroidism
  15. Chronic inflammatory diseases
  16. Obesity

False Causes Of raised Hemoglobin

  1. After hemorrhage
  2. Dehydration 
  3. Tested during I/V infusion, if the I/V contains iron
  4. Burns

Frequently Asked Questions

Q1. What methods are used to estimate the level of hemoglobin?

Ans. There are many methods available by which the be level of hemoglobin can be estimated like the Sahli’s/acid hematin method and cyanmethemoglobin method.

Q2. Is Drabkin’s solution sensitive to light?

Ans. Yes, Drabkin’s solution is sensitive to light. It is advised to be stored at room temperature in a place that is away from light. If it is stored in the recommended conditions then it will remain stable for 6 months.

Q3. What is Drabkin’s reagent composed of?

Ans. Drabkin’s reagent is composed of potassium ferricyanide, potassium cyanide, potassium hydrogen phosphate, and distal water.

Q4. Why is Drabkin’s method more reliable than hemoglobin estimation?

Ans. Drabkin’s method is more reliable as its readings are comparatively precise. This is because a spectrophotometer is used, so error due to subjective visual matching is eliminated.

Q5. Is sulfhemoglobin irreversible?

Ans. Yes, sulfhemoglobin is irreversible. It means that sulfhemoglobin lasts up to the lifetime of an erythrocyte.

Q6. Is sulfhemoglobin normal hemoglobin?

Ans. No, sulfhemoglobin is a rare, abnormal form of hemoglobin. It doesn’t carry oxygen. The formation of sulfhemoglobin may result from particular medications such as metoclopramide, sulfonamide, dapsone, nitrates, etc.

Q7.  Can drinking too much water lower the level of hemoglobin?

Ans. Yes. The level of hemoglobin can be dropped by as much as a gram to a gram and a half, with the intake of a sufficient quantity of water.

Q8. Which foods lower the level of hemoglobin?

Ans. Some foods lower the level of hemoglobin in your body, which are listed below
1. Eggs
2. Nuts
3. Meat
4. Whole foods
5. Cheese

Q9. What is the pH of Drabkin’s reagent?

Ans. Drabkin’s reagent is slightly alkaline with a pH of 8.6.

Q10. What is cyanmethemoglobin?

Cyanmethemoglobin is a comparatively nontoxic compound of cyanide with methemoglobin. It is formed when methylene blue is given in cases of cyanide poisoning.

References

  1. https://www.healthline.com/health/normal-hemoglobin-levels
  2. https://www.sciencedirect.com/topics/biochemistry-genetics-and-molecular-biology/hemoglobin-blood-level
  3. https://labs.selfdecode.com/blog/symptoms-low-high-hemoglobin-diseases-increase-decrease/
  4. https://www.sigmaaldrich.com/US/en/product/sigma/d5941
  5. https://www.ncbi.nlm.nih.gov/books/NBK259/
  6. https://medicostimes.com/satyanarayana-biochemistry-pdf/

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