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Drabkin’s Solution for Hemoglobin, Preparation of Drabkin’s Solution

November 16, 2023HematologyLab Tests

Table of Contents

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  • Drabkin’s Solution for Hemoglobin Estimation
        • What sample is needed for hemoglobin estimation?
      • What precautions will you take to keep the Drabkin’s solution?
      • What is the principle of Drabkin’s solution?
      • How will you manually make Drabkin’s solution?
      • How to estimate hemoglobin by Drabkin’s solution (Procedure)?
      • Normal hemoglobin:
      • Physiological variation of Hb:
      • False causes of raised Hb:
      • Questions and answers:

Drabkin’s Solution for Hemoglobin Estimation

  • There are commercially available kits, which make it easy to make the solution.
  • When blood runs in the hematology analyzer, these instruments also estimate hemoglobin.

What sample is needed for hemoglobin estimation?

  • Whole blood is needed for hemoglobin estimation.

What precautions will you take to keep the Drabkin’s solution?

  1. Keep the solution in a dark-colored bottle and in the dark to protect it from the light.
  2. If the solution is cloudy after adding the blood, centrifuge before the reading; this may be due to nonhemolyzed RBCs or globulins.
  3. Spectrophotometer cells should be fingerprint-free; otherwise, the reading will be high.
  4. Drabkin’s solution is a pale yellow clear fluid; it should not be used when it is cloudy.
Drabkin's solution kept in colored bottle

Drabkin’s solution is kept in a colored bottle.

What is the principle of Drabkin’s solution?

  1. Whole blood is diluted 1 into 200 dilutions with Drabkin’s solution, containing potassium ferricyanide and potassium cyanide.
  2. RBCs are hemolyzed by Drabkin’s solution.
  3. Now hemoglobin is oxidized, and its derivatives, except sulfhemoglobin, form methemoglobin in the presence of alkaline K-ferricyanide. 
  4. The methemoglobin reacts with K-cyanide to form a very stable compound, cyanmethemoglobin, and this complex has maximum absorption at 540 nm.
Drabkin's solution principle

Drabkin’s solution principle

How will you manually make Drabkin’s solution?

  1. Hemoglobin (Drabkin’s)  solution can be prepared in the laboratory.
  2. Drabkin’s solution reagents needed are:
    1. Potassium ferricyanide = 200 mg
    2. Potassium cyanide = 50 mg
    3. Potassium dihydrogen phosphate = 140 mg
    4. Non-ionic detergent = 1 ml
      1. https://patents.google.com/patent/US4341527A/en
    5. Distal water = Make up to 1000 ml (1 L)

How to estimate hemoglobin by Drabkin’s solution (Procedure)?

  1. Take 20 microliter of blood + Drabkin 4 mL = 1: 200 dilution.
  2. OR take 20 microliter of blood + Drabkin 5 mL = 1: 250 dilution.
  3. Now mix well.
  4. Read within 6 hours of mixing the blood with Drabkin’s solution.
  5. Read on a spectrophotometer at filter 540.
  6. Read against the blank of Drabkin’s solution (Drabkin solution can be used as blank).
  7. Also, read the standard solution (12 G/dL) with the same dilution as the test sample.
  8. Read by the spectrophotometer; the reading is called optical density (OD).
Hemoglobin procedure by Drabkin's solution

Hemoglobin procedure by Drabkin’s solution

Hemoglobin calculation formula

Hemoglobin calculation formula (OD = optical density)

Normal hemoglobin:

  1. Adult male = 14 to 18 g/dL
  2. Adult female = 12 to 16 g/dL
  3. 10 years old child = 12 to 14.5 g/dL
  4. 3 months old infants = 9 to 14 g/dL
  5. Newborn = 17 to 23 g/dL

Physiological variation of Hb:

  1. Strenuous physical exercise.
  2. There is a diurnal variation, with the highest level in the morning and low in the evening.
  3. High altitude increases the Hb concentration.

False causes of raised Hb:

  1. Hemoconcentration due to dehydration and burns.
  2. Immediately after hemorrhage.
  3. If taken during the I/V infusion, if it contains iron.

Questions and answers:

Question 1: What is the end result of KCN reaction for hemoglobin?.
Show answer
Methemoglobin is the colored stable compound to measure.
Question 2: What is the physiological variation in the hemoglobin?
Show answer
These are strenuous exercise, diurnal variation, and high altitude.

Possible References Used
Go Back to Hematology

Comments

Victor Ngowi Reply
June 9, 2020

You shows the principle of drabkin solution but not show how we are apply this solution

Dr. Riaz Reply
June 9, 2020

Dear
As I have understood from your question, that you want the procedure by Drabkin’s solution. It is already given there. Or please explain your question.

Japhet Damas Reply
June 18, 2020

I got good news

Dr. Riaz Reply
June 18, 2020

ok Thanks.

Dr Aadil Reply
September 2, 2020

Can we take 2ml reagent and multiple the reading with 20

Dr. Riaz Reply
September 3, 2020

You have to keep the same dilution, that is very important.

Dhansukh Rana Reply
September 23, 2020

Sir how to measure bilirubin ??

Dr. Riaz Reply
September 23, 2020

Dear nowadays nobody make reagents themselves. Ready-made kits are available in the market. Definitely you need a lab facility.

Nasr-Eldeen khalil Reply
October 14, 2020

what is the function of potassium dihydrogen phosphate in drabkin’s solution ?

Dr. Riaz Reply
October 15, 2020

This has the buffering action.

Adam Reply
October 21, 2020

Can drabkins reaction work with freeze dried hemoglobin instead?

Dr. Riaz Reply
October 22, 2020

I think if you thaw the frozen Hb, then try with control. That may work.

Leyal Reply
March 30, 2021

What’s the quality control of drabkin solution?

Dr. Riaz Reply
March 30, 2021

You can run known control of hemoglobin. These known controls are available from various companies.

hiam najem Reply
June 18, 2021

what is the function of the non-ionic detergent in the drabkins solution ?

Dr. Riaz Reply
June 18, 2021

Please see the following link for your question.
https://patents.google.com/patent/US4341527A/en

Han Reply
August 6, 2021

May i ask sir, i read that a source of error when using the Drabkin’s reagent was a possible increase of globulins which can then be fixed using the dihydrogen potassium phosphate – is this true? and if so, how come what’s the principle behind it? Thank you !!

Dr. Riaz Reply
August 6, 2021

I have given diagrammatically the principle of Drabkin’s solution.
https://labpedia.net/haemoglobin-part-3-hemoglobin-solution-drabkins-solution-preparation/

Maryam Ch Reply
October 11, 2021

Can we prepare the standard solution of cyanmethemoglobin process in lab…if it is not available commercially?

Dr. Riaz Reply
October 11, 2021

You can prepare Drabkin’s solution. I have described the procedure.

amir Reply
December 4, 2021

sir, what is OD in formula?

Dr. Riaz Reply
December 4, 2021

OD is the reading by the spectrophotometer, called optical density. Read the article again.
https://labpedia.net/drabkins-solution-for-hemoglobin-estimation/

Bindu Reply
January 28, 2022

Sir is Hb levels of 17.4 normal according to Drabkins method sir?

Dr. Riaz Reply
January 28, 2022

This is a higher limit. Please keep on checking Hemoglobin to rule out the possibility of polycythemia. It would be better to consult a hematologist.

Sagar Reply
February 17, 2022

Sir
Can hemoglobin react with potassium thiocyanate? Please

Dr. Riaz Reply
February 17, 2022

You can use sodium cyanide instead of potassium cyanide.

Chetan Khadka Reply
August 21, 2022

The procedure for commercially available cyanmethemoglobin standard soln is differ from the above mentioned one…
because if we use commercially available std. soln we should use directly if the soln. is in RT.

Dr. Riaz Reply
August 21, 2022

You can use a ready-made solution.

Manon Sorais Reply
January 23, 2023

Hi, thank you for this useful publication. If I want to measure hemoglobin from plasma that was mixed in Drabkin reagent, would it be suitable to produce a standard by mixing a known quantity of lyophilized hemoglobin in Drabkin reagent? This way, I could build a standard curve with increasing hemoglobin concentrations, am I right?

Dr. Riaz Reply
January 24, 2023

https://labpedia.net/laboratory-part-1-serum-plasma-preparation-specimen-storage-and-precautions/
Please see this topic and it will answer your question.

Dr. Riaz Reply
January 24, 2023

https://labpedia.net/laboratory-part-1-serum-plasma-preparation-specimen-storage-and-precautions/
Please see this topic, and it will answer your question.

Muhsina Reply
July 14, 2023

How to discard Drabkin’s reagent after use

Dr. Riaz Reply
July 14, 2023

Section 13. Disposal Considerations
In the fume hood, add the Cyanide solution to a solution of 1% Sodium Hydroxide (about 50 mL/g of Cyanide). Household bleach (about 70 mL/g of Cyanide) is
slowly added to the basic Cyanide solution with stirring. When the addition of the bleach is complete, the solution can be tested for the presence of Cyanide
using the Prussian Blue test: to 1 mL of the solution to be tested add 2 drops of freshly prepared 5% aqueous Ferrous Sulfate solution. Boil this mixture for at
least 60 seconds, cool to room temperature, and then add 2 drops of 1% Ferric Chloride solution. The resulting mixture is made acid to litmus with 6 Molar
Hydrochloric Acid (prepared with equal amounts of concentrated Hydrochloric Acid and water ). If Cyanide is present, a deep blue precipitate will form.
(Concentrations of greater than 1 ppm Cyanide can be detected.) If the test is positive, more bleach is added to the Cyanide solution, and the test is repeated.
Continue until no Prussian Blue precipitate is formed. Wash the solution down the drain with excess water. Always dispose of in accordance with local, state
and federal regulations.
Section 14. Transport Information

https://us.vwr.com/assetsvc/asset/en_US/id/8452666/contents
Please read this article.

CR Reply
November 16, 2023

What can be used instead of potassium dihydrogen phosphate?

Dr. Riaz Reply
November 16, 2023

Please read this article, and it may help you.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC477205/pdf/jclinpath00098-0110b.pdf

CR Reply
November 17, 2023

I want to try using actual whole blood with the above method.
Do I need to process the blood in any way before using it?

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