Reverse engineering a Haemoglobin detection kit

Lab Aug 05, 2019 5 min read

Note: This is a slightly more technical article aimed at people who work in a biological laboratory - but I've tried to make it understandable by people who don't too! Please let me know how I can improve in the comments below!

Working in a Haematology lab means I sometimes have to measure certain blood parameters myself, and here's a story about how I managed to make a kit that normally costs $300 for basically nothing. Hopefully things like this will speed up research and help us discover better life-saving treatments! I work in a lab that focuses on ways to stop bleeding, and in order to do that, we need to measure bleeding.

Recently I was faced with the task of measuring the haemoglobin content in a large number of samples. These were very dilute, so sticking them in the normal blood analyzer machine did not work so well. I needed to do this manually. I had available to me a plate reader (to take lots of measurements of absorbance quickly) and the usual reagents around a biology lab.

Aims

  • To measure haemoglobin in really diluted samples (not regular blood samples)
  • To do this for lots of samples quickly and cheaply
  • If I had lots of the reagent required, I wouldn't have to worry about running out or ordering more (that's more admin work than lab work and I'd much rather do the latter!)

What is haemoglobin?

Haemoglobins are proteins in our blood that carry oxygen around the body. In my experiment, I needed to use it to measure how much blood was lost in a large volume of water. I had haemoglobin concentration from the veins, and I knew how large the volume the blood had been lost in. If I could work out what the haemoglobin concentration was in the water, I could work out exactly how much blood was lost. Eventually, we could use this to measure if treatments are working to help stop excess bleeding or not!

History of measuring haemoglobin

Haemoglobin was one of the first blood parameters to be measured routinely. There are lots of ways to do it! In this case, I needed something fast and scalable (on a 96-well plate reader). Normally, the absorbance spectrum of haemoglobin really varies, because it changes depending on if it bound to oxygen and various other substances. The goal of any reagent to help with this is to stabilise this to only one form, and hence one peak of haemoglobin in the absorbance spectrum.

The classic is the cyanide-based Drabkin's solution that turns haemoglobin into a form that has a well-defined spectrum. However, this is not that stable, contains cyanide and not usually available in most labs. In recent times, there have been many more methods at measuring haemoglobin, from using Sodium laurel sulphate (AKA Sodium dodecyl sulphate, SDS, which is common in Western blotting as a detergent) to HARBOE. There's actually a pretty good review on the topic here, but I will focus on one method that I found to be quick, cheap and effective.

Kits?

Often in biology these days, there are kits available to do common things that contain all the necessary reagents to do the experiment. This has undoubtedly been a huge time saver, but often comes at a cost. Both in terms of money and in the time it takes to deliver the kit. There's nothing worse than having lots of samples to test and have a kit run out because the manufacturer only provided enough for 100 tests before they wanted you to pay for another kit!

What's worse is that the costs of these kits are extortionate sometimes for the number of tests that one can do with them!

The kit in question.

The QuantiChrom Hemoglobin Assay kit (catalogue no.: DIHB-250 from BioAssay Systems) was the recommended kit to do this with. The manufacturer has recently become eco-friendly, which basically means they don't print out the instructions for you and put them in the box. The kit actually comes with two small bottles. A 50ml bottle labelled "Reagent" and a 10ml bottle labelled "Calibrator". That's it. $309 please. Enough for 250 tests. Which sounds like a lot, except everything needs to be done in duplicate on a plate reader, so it's really only 125 tests. Minus the calibrator and the water (not included) samples, it was only about enough for all of my samples (~100 of them) - from one experiment!

The bottle labelled "reagent" ran out first, since I only needed 200uL of the calibrator once here, as the manufacturer has helpfully demonstrated how the kit is linear up to 200mg/dL!

Background research

I read carefully in the datasheet, and it says it is "based on an improved Triton/NaOH method". That sounds interesting. I dug up a few papers on the "normal" Triton/NaOH method. Triton is a detergent commonly used in Biology and NaOH is, well, sodium hydroxide. Presumably the Triton is to lyse the red blood cells and the NaOH is the make the pH such that the haemoglobins oxidise and form a single peak in the spectrophotometer (rather than the multiple peaks that the different forms of haemoglobin when bound to oxygen give).

I found a paper on a reagent called "AHD575" which is a Triton/NaOH mixture (it stands for "alkaline hematin + detergent") that should be measured at 575nm. I wondered if the "improvement" came from the instructions in the kit that said to measure at 400nm instead. It's possible to buy this for about €36, but we can do better by just using common lab reagents (already available almost everywhere) making this basically free.

From https://www.karger.com/Article/Pdf/419579

Testing the hypothesis

So, I ran one of my samples both using the original reagent in the kit as well as the one I made up in the lab using 0.1mol/l NaOH and 2.5% Triton (to make up 100ml, we just need 0.4g NaOH [remembering from Chemistry that the mass to add = conc (M) x Mr x vol (litres)] and add 2.5ml Triton if in a liquid form or 2.5g if in a solid form).

Both the kit and the homemade reagent gave the same results, and the dilutions seem to have been accurate (and linear!)

I also did a spectral scan to see if the reagent did anything different to the rest of the spectrum of the haemoglobin sample. See the results for yourself!

Awfully similar spectrograms...

Conclusions

To make your own reagent for a haemoglobin quantification kit, you just need to add 0.1mol/l NaOH and 2.5% TritonX-100. The calibrator you can actually make up pretty easily too, if you have access to a blood testing machine - just test some blood on there, then dilute it as the kit suggests for using in the assay, and voila, a reference point!

Souradip Mookerjee

I'm an MB/PhD student at the University of Cambridge. I'm interested in stem cells, genomics and deep learning applied to medicine. I also write lots of code and get involved with startups.