Hey there,
I'm using commercially available Drabkin's reagent to measure the hemoglobin content of blood photometrically. The reagent I bought contains sodium bicarbonate, potassium ferricyanide and potassium cyanide. In my understanding, all different forms of hemoglobin (except sulfhemoglobin) are converted to methhemoglobin by potassium ferricyanide in a first step which reacts then with potassium cyanide to cyanomethemoglobin which shows a significant absorbance at 540 nm.
But sometimes I see a second peak around 575 nm (indicated by the red arrow in the graphic), sometimes very weak but sometimes also very strong and I wonder what it could be. Could this be some remainings due to uncomplete reaction or could it maybe be due to poor quality of the sample?
Thank you for your advice!
Non-hemoglobin absorbance is usually due to turbidity caused by abnormal plasma proteins, Hyperlipidemia, fatty droplets and leukocytosis. These factors increase light scattering which is detected as increased absorbance.
Oxy-Hemoglobin typically has peaks at 541 and 576 nm. Because these peaks are still present in the spectra you showed, your sample likely still has some amount of unreacted hemoglobin which will impact your results. Typically this lack of complete reaction is caused by 2 factors:
Shamsuz Zaman Dear Shamsuz,
thank you for your answer! Following the manufacturers instructions I also added the detergent Brij, which should reduce the turbidity of the sample. Therefore I think (or hope) that this is not due to turbidity...
Best
Caspar
Donald Belcher
Dear Donald,
thank you for your advice. Could the peak also derive from HbCO that shows a similar absorption spectrum to HbO2? Or would I expect only negligible amounts in the sample (non-smoker, sample stored for 5 days in the fridge)?
Best
Caspar
Caspar Hoyer
You bring up an excellent point. You may be able to differentiate by comparing where the peak location. Carboxyhemoglobin tends to have strong absorbance around 570 nm compared to oxyhemoglobin which has a strong absorbance at 576 nm. If you are dealing with CO bound samples you will likely need to allow your solutions to react for 1 to 2 hours to allow time for the CN to displace the CO. Here's an old study that studies this difference: http://clinchem.aaccjnls.org/content/clinchem/13/1/2.full.pdf
Donald Belcher
Dear Donald,
thank you very much, this helps me a lot! I think based on my absorption maximum it is oxyhemoglobin caused by an incomplete reaction, since I used the reagent in a modified application.
Best
Caspar
You are using Drabkin's reagent having alkaline pH (8.6 or so). At this pH Hb is converted to Oxy-Hb, which has peaks at 540 and 576. Ferricyanide converts it to Met-Hb. This reaction is slower at alkaline pH and it is advisable to take readings after 15 to 20 minutes. Conversion of Met-Hb to HiCN is instanstaneous. ICSH modification of Drabkins's reagent contains 140 mg KH2PO4 in place of NaHCO3. It has final pH of 7.0 to 7.4. At this pH the first reaction is faster and nearly full conversion of Hb to HiCN takes about 3 minutes. So if you are taking the readings earlier, some Oxy-Hb is likely to be there. My suggestion is that you take the readings at 5 minutes interval for 30 minutes and put the graphs on Researchgate for the benefit of all of us. Below is attached a figure showing absorbance graphs of different hemoglobin derivatives for your perusal. You have detected an interesting phenominon. I am curious to know the result of my suggestion. Every observation in science has its own charm. Thanks for enlightening us.
Ramesh Z. Patel Dear Ramesh,
thank you for your detailed answer. I didn't use the readily prepared Drabkin's reagent but I tried to mix the solid ingredients directly with blood for a time and cost effective approach, but obviously they did not solve properly and the reaction stopped at th Oxy-Hb. Maybe I will try it again and wait longer than the manual suggested 5 minutes, but sadly I can not do it right now because our lab is currently under construction :(
Best
Caspar
The alkaline Drabkin's reagenthas an advantage of not having turbidity due to protein precipitation. What I was preparing in my lab was from a very old text book of laboratory medicine. It contained Pot. ferricyanide - 200 mg, Pot Cyanide - 50 mg and Sod. bicarbonate 1 g per leter of deionised water. For every day, it was not possible to check pH with pH meter, hence I added a drop od 1 g/dl Phenophthalein in Alcolho to 5 ml drabkins and get satisfied to get reasonably good pink color !
Whenever you are able to do the experiment, please do put on Researchgate.
Please have a look at a table of conversion time with Drabkin's reagent
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