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Jermin

Rh+K Phenotype Validation

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Hi All,

I am about to venture into performing my first ever validation of a test. I have been tasked to validate Rh+K phenotype testing on the BioRad IH-1000 (two of them). We have been, until now, performing the test manually, but as work is getting busier, performing it on the analyser might prove easier (or at least motivate people to perform phenotype). I have been given advise by my senior on how to go about it:

  • Select 10 Donor Red Cell units which have Rh+K phenotype performed
  • Perform the phenotype manually
  • Perform the phenotype on the analyser
  • compare the result
  • pat myself on the back, provided I don't mess it up

The issue I have is donor red cells doesn't indicate if they are K+, and I wanted some K+ as well as K-. I could always keep testing a lot of donor units until I come across a K+ unit, but I don't want to was a lot of cards (but that might be my last option). If I choose the NBS or BioRad Antibody Panel Cells, then the issue is the strength of the test cells, as they are, I think, 0.8%, and it does not fully represent the way we perform our phenotype manually, as it uses around 5%. I can always try and make the strength of the solution stronger, that is another option. 

So this is where I am at. If anyone has a suggestion, or better a complete plan, then I am happy to hear it. Also if there is any point I missed or need clarification, please feel free to ask, I'm fixed to this specific thread all night long.

 

Cheers,

Jermin

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Hi Jermin,

First of all, good luck with your first validation; I'm sure it will go well.

All units of blood from the NHSBT have been typed for D, C, c, E, e and K, but, whereas the full Rh type is recorded on the label (whether positive or negative for the D, C, c, E and e antigens), only if the unit is K Negative is this recorded on the label.  If you take a look at all of the units in your stock, some will have other antigen types on the unit label, but the only ones that are printed on there are those that are negative.  Some units will also have a sort of "luggage label" also attached, and on this, the antigens for which the units is positive, as well as those that are negative are often printed (although very few show the full type).

I'm not certain about this, but I think there is some kind of rule that says, except for the five Rh antigens mentioned above, only those antigens that have been tested on this particular donation, and found to be negative, are allowed to be printed on the actual label on the unit (as opposed to the "luggage label").  I think the "powers that be" do not believe that trained and educated scientists can read!

Any how, this rather long post boils down to me saying that, in the VAST MAJORITY of cases, if the unit does not state, on the actual unit label, that it is K-, it is almost certainly K+.

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Food for thought: Are the reagents licensed/approved for use on the instrument ?

If "Yes", then minimal validation is required (and is perhaps better called "verification"), but 10 samples is probably too small (even if you select/cherry-pick the phenotypes). You would have a very hard time covering all of the most common Rh haplotypes/phenotypes with such a small sample size.

If "No", then you're definitely in the validation realm and 10 samples is way too small to achieve appropriate statistical confidence levels. If you have a pet statistician, it might be worth talking to them.

BTW, if the phenotypes of the selected test samples are already known, there is no need to re-test them manually as part of this process. As  Malcolm suggests......there are some not-so-secret ways to collect that information.;)

A final brain-dripping: You will need a plan to deal with discrepancies. No two techniques are the same, nor are the formulations of the reagents (monoclonal cell lines and other secret ingredients).

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Exlimey - Yes, of course the reagents are licensed to use on the instrument.  This is BioRad reagents in the UK.  And there are already LOTS of labs in the UK working with these reagents on this instrument.  And the cards used on the Instruments are the SAME cards that are used manually; and the Diluent is the same in the two techniques too, so no differences there to worry about

Jermin - you could never use reagents as positive controls on the instrument.  First of all, if you leave it in the reagent bottle, then the instrument will be looking for something in the sample rack and give you an error message.  If you transfer it to a tube and put it in the sample rack, the Instrument will dilute it down - then you will end up with a 1% suspension of a 1% suspension - and you won't be able to see anything!  Don't you have some QC tubes that are K+?  I presume you have been correctly doing QC for your manual technique?

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3 hours ago, galvania said:

Exlimey - Yes, of course the reagents are licensed to use on the instrument.  This is BioRad reagents in the UK.  And there are already LOTS of labs in the UK working with these reagents on this instrument.  And the cards used on the Instruments are the SAME cards that are used manually; and the Diluent is the same in the two techniques too, so no differences there to worry about.

No need to get so snippy. Unlike you, I am not intimately familiar with the state of things in the UK - that's why I asked.

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On 12/16/2017 at 8:03 AM, galvania said:

sorry Exlimey, didn't mean to be snippy.

anna

No worries, perhaps I was just a little sensitive.

May I presume that your detailed knowledge of the reagents and platform (and the Swiss flag) is a result of an association with the company (formerly know as DiaMed) ??

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Thanks for the replies.

On 15/12/2017 at 7:26 AM, Malcolm Needs said:

Any how, this rather long post boils down to me saying that, in the VAST MAJORITY of cases, if the unit does not state, on the actual unit label, that it is K-, it is almost certainly K+.

Thanks for the insight Malcolm, I always assumed that there was a lot of K- units on the donor units that did not have K antigen status indicated. I will use the donor units in that case, and see where I get.

On 15/12/2017 at 12:10 PM, exlimey said:

Food for thought: Are the reagents licensed/approved for use on the instrument ?

The DiaMed reagents are, but not the NBS reagents. Thanks for that question, as it slipped my mind.

On 15/12/2017 at 12:10 PM, exlimey said:

If "Yes", then minimal validation is required (and is perhaps better called "verification"), but 10 samples is probably too small (even if you select/cherry-pick the phenotypes). You would have a very hard time covering all of the most common Rh haplotypes/phenotypes with such a small sample size.

If "No", then you're definitely in the validation realm and 10 samples is way too small to achieve appropriate statistical confidence levels. If you have a pet statistician, it might be worth talking to them.

Yeah, I think 10 might be too small as well, but as I don't want to use excess cards, and I want to perform the test manually and two analysers, I can't see myself getting more than 10, but I will go back to the list of possible genotypes and review the number. In the end, there is no point in being frugal if it means that the whole validation process was not undertaken properly.

On 15/12/2017 at 12:10 PM, exlimey said:

validation is required (and is perhaps better called "verification")

I had it in my mind that validation is what the laboratory conducts, and verification is done by the manufacturer. 

On 15/12/2017 at 12:10 PM, exlimey said:

BTW, if the phenotypes of the selected test samples are already known, there is no need to re-test them manually as part of this process. As  Malcolm suggests......there are some not-so-secret ways to collect that information.;)

 

Yeah, all Rh phenotypes are clearly indicated, but as for K antigen status, it is not always clear on the donor units. I will see if I can get that information from the the National Blood Service

On 15/12/2017 at 12:10 PM, exlimey said:

A final brain-dripping: You will need a plan to deal with discrepancies. No two techniques are the same, nor are the formulations of the reagents (monoclonal cell lines and other secret ingredients).

I wish I knew how I could do that. :(  I will need to probably check with the manufacturer's reagent sheet and see what sort of limitations and discrepancies there might be, otherwise it might be beyond me.

On 15/12/2017 at 1:29 PM, galvania said:

Jermin - you could never use reagents as positive controls on the instrument.  First of all, if you leave it in the reagent bottle, then the instrument will be looking for something in the sample rack and give you an error message.  If you transfer it to a tube and put it in the sample rack, the Instrument will dilute it down - then you will end up with a 1% suspension of a 1% suspension - and you won't be able to see anything!  Don't you have some QC tubes that are K+?  I presume you have been correctly doing QC for your manual technique?

 

I was planning on getting the reagent cells, centrifuge them to separate from the preservative it is in, then resuspend it in PBS. But I might go ahead with using donor units, so this might not be an issue anymore 

 

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Hi Jermin

I think there are a couple of points here that seem to me to be a bit confused.

1.  'The DiaMed reagents are, but not the NBS reagents. Thanks for that question, as it slipped my mind. '  You won't be using NBS reagents to do your Rh phenotype on the IH1000.  All you will be using are the cards, which are what you are using now, and diluent 2, which you are using now.

2.  I was planning on getting the reagent cells, centrifuge them to separate from the preservative it is in, then resuspend it in PBS.  No, Jermin you really cannot do this.  It is not the preservative that is the issue, but the concentration.  The samples you put on the analyser need to be packed cells - and you need enough packed cells for the instrument's needle to be able to aspirate the correct amount.  If you spin down reagents, even if they are at 3-5%, you will not have enough cells in the pellet; even less so if you them dilute them with PBS - which you should never use with the cards anyway as it can cause trailing.

3.  I wish I knew how I could do that. :(  I will need to probably check with the manufacturer's reagent sheet and see what sort of limitations and discrepancies there might be, otherwise it might be beyond me.   You are currently using the same cards and the same Diluent to carry out your manual testing as you will be using on the Instrument.  Any discrepancies you see will come about because of manipulation error only.

 

Can I suggest that you contact the local Biorad rep who installed the instruments and ask them to help you with this?

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1 hour ago, galvania said:

Can I suggest that you contact the local Biorad rep who installed the instruments and ask them to help you with this?

An excellent idea ! No need to re-invent the wheel.

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5 hours ago, galvania said:

Can I suggest that you contact the local Biorad rep who installed the instruments and ask them to help you with this?

Sounds like a very good idea indeed.

5 hours ago, galvania said:

No, Jermin you really cannot do this.  It is not the preservative that is the issue, but the concentration.  The samples you put on the analyser need to be packed cells - and you need enough packed cells for the instrument's needle to be able to aspirate the correct amount.  If you spin down reagents, even if they are at 3-5%, you will not have enough cells in the pellet; even less so if you them dilute them with PBS - which you should never use with the cards anyway as it can cause trailing.

 

 Point taken. I should have also clarified that I was actually going to use the BioRad diluent (we just so used to calling everything PBS). Also I was not going to dilute it too much, so the concentration would be similar to a pRBC (how would I achieve such consistency? God knows). In the end it was just all a rough draft, which I can can assure you I will not be carrying out, and I am glad you have given me such pointers. 

I will contact the BioRad and see what they suggest. If they can't help much, I will use the donor units with known Rh phenotype.

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3 hours ago, Micper81 said:

If anyone is interested, we have x 3 used Bio-Rad IH-1000 for sale in an excellent condition.

These posts are not allowed here.

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