Prevention is better than Treatment

Hemoglobin Normalization (Hb+)

Hemoglobin Normalization - The Highlights

Points to consider:

What can this patent do for you, how can it be used to make your life easier?

• This patent practically eliminates all needs for a separate Hb determination and all calculations associated with it. Thus it helps to present a test which has less labor, cost and time, in short: a simpler and more effective test.

• It automatically corrects all mistakes related to wrong sample volume, i.e. partially covered dried blood spots (wrong disk cuts), a misperforming channel in a multichannel pipette  etc. This is extremely important for large screening centers, which perform thousands of liquid transfers per day - and mistakes are bound to happen. With this test, you need worry no more - any mistake done is automatically taken care for during the calculation step.

• Because the Hb determination is performed in the same sample that the activity is measured tests using this patent are far more accurate than the competition. No other method can guarantee correct results, since any mistake made (either in the Hb test or the separate G-6-PD test) will give wrong results. This is extremely important in borderline values.

• It can be incorporated in the program of automated analyzers for all examinations that need to be expressed in relation to the Hb content of the sample (G-6-PD, Pyruvate kinase etc.)

• Results are expressed directly in U/g Hb the only units that are universally accepted and allow for direct comparison of results from different laboratories.

• Recent work supports our view that quantitative G-6-PD testing will face a tremendous boost in years to come. Not only it is now shown that G-6-PD probably has an embryoprotective role (hence many miscarriages, abortions and prenatal deaths are due to its deficiency) but it has also shown that screening with semiquantitative tests misses most females (who are classified as normal instead of partially deficient). Both papers were published in 2000 and are included in this site (click on the links).

Is it normal for a normal whole blood sample to give more mOD/min than the Sigma G6PDH Normal level control? This is what I usually get. Does that mean that the majority of the samples have very high activity? These are some questions most often asked by people using our kits for the first time.

It is absolutely NORMAL for a normal sample to give MORE mOD/min than the Sigma normal control. This is a proof that the person performing the test is doing it correctly. He should definitely get this sort of results. I get these results all the time. And it is because of these results I understood that we have to normalize for Hb content. However, this does not mean that these samples have more U/g Hb than the controls. On the contrary, the majority of these samples have a lower activity than the controls.

This is because of two reasons (please contact us if you need more info).

Firstly, if the Sigma control is rated at 13 U/g Hb (at 37oC) there are quite a few individuals who may have more activity than that. I, for example, have a G-6-PD activity of 15.5 - 16.5 U/g Hb and I consistently give (as a sample) far more mOD/min than any Sigma Normal control. This is reason Number 1 and it is the easy part of the answer.

The second reason (which is FAR more important for marketing these kits) is that a sample may have just 10 U /g Hb and the Sigma Control may be rated at 13 U/ g Hb. However, all whole blood samples have MORE Hemoglobin / sample as compared to Sigma control. According to my experience (more than 10.000 samples), Sigma controls give an OD at 405 about 0.710 while the whole blood samples give an OD at 405 between 0.900 and 1.300 (which means there are a lot more Red Blood Cells in the whole blood sample). Hence, although the overall activity of the whole blood sample is higher than the Sigma control, when you make your calculations to express it in relation to the Hb content it may fall below the Control values.

Example:

Sigma Normal Control (rated at 16 U / g Hb): 4 mOD/min at 340 nm, OD 405 nm = 0.710

Whole blood sample (A): 6 mOD/min at 340 nm, OD 405 nm = 1.300 (in this sample, there is 50% more activity than the control but there is also 83% more Hemoglobin. So, on a "per Hemoglobin" basis, this sample has less activity / Hb unit). So the value should be BELOW the value of the control. Actually it should be 13,12 U/g Hb if the control was rated at 16 U/g Hb.

Whole blood sample (B): 6 mOD/min at 340 nm, OD 405 nm = 0.900 (in this sample there is also 50% more activity than the control and there is only 27% more Hemoglobin, so there is more activity per Hemoglobin unit than the control). So the value should be ABOVE the value of the control. Actually it should be 18.9 U/g Hb if the control was rated at 16 U / g Hb.

Whole blood sample (C): 2.5 mOD/min at 340 nm, OD 405 nm = 1.300 (in this sample there is 37% LESS activity than the control but there is also 83% more Hemoglobin, so there is even less activity per Hemoglobin unit than the control). So the value should be FAR BELOW the value of the control. Actually it should be 5.92 U/g Hb if the control was rated at 16 U / g Hb. Definitely a borderline value / possible a partially deficient female.

This is the main advantage of the Normalization procedure. If the normalization is not employed in this test you will conclude that both samples (A and B) have more activity than the control which is an ERROR. This error would be fatal in the case of blood sample C above. If we do not normalize for Hb then we assume this sample to have just 37% less activity than the control and we would give the result = 10.08 U / g Hb and classify it as normal instead of 5,92 U/g Hb (which is a borderline value, possibly partially deficient).

As a conclusion, performing quantitative analysis of G-6-PD enzyme activity only makes sense if the information obtained makes sense. No matter if you use an expensive kit or a cheap kit, the point is always the same : accuracy. To the best of our knowledge, this can only be done with the use of the Normalization procedure. Any other method will include a certain degree of inaccuracy which, in the case of borderline or partially deficient samples may be dangerous. We therefore recommend the use of R&D Diagnostics MMR series of kits for the determination of the enzymatic activity of G-6PD along with the patented "Hb Normalization" method.

Since we were the ones to develop this method, we know it better than anyone else. We would be really happy to discuss it with all users and help them to get the most out of it - which is accurate results that make sense.

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Page last edited on 05/07/2005