Using complement coombs control cells with polyspecific antisera.

[mlapierre]

Looking to see what the current thinking is on using control cells for direct coombs testing. We were recently sited on our CAP inspection for only running an IgG coombs control on our polyspecific direct coombs testing. We only use complement coombs control when we specifically test with anti-complment antisera. We use Ortho bioclone polyspecific antisera for the preliminary DAT and follow-up with monospecific testing if the test is positive. For negative reactions, the polyspecific AHG product insert only refers to using Ortho Coombs Control-Group O cells sensitized with IgG.

[David Saikin]

You only have to run the IgG sensitized cells when using poly ahg. The inspector should read the commentary associated with the ahg standards. The complement control cells only need to be run when using the specific anti-Complement reagent. You should also be aware of the commentary for that standard and could have rebutted the citation on the spot. The inspectors (esp those from CAP) are not always on top of the inspection game. It behooves you to have a handle on the inspection process . . . it never bothers me to butt heads with the inspector if they do not understand a standard or are just plain "iggerant!"

[tricore]

I think we need to ask for a clarification from CAP

40200 is about control of anti-IgG. 40201 is about control of anti-C3

"Complement-coated red blood cells must be used to confirm all negative antiglobulin test

results when the antiglobulin reagent used for testing has anti-C3 reactivity." If you are not testing for C3 why use polyspecific reagent?

TRM.40200 DAT Controls Phase II

When performing an antiglobulin test with anti-IgG or polyspecific antiglobulin reagents,

IgG-coated red blood cells are used as a control in all negative antiglobulin tests.

NOTE: IgG-coated red blood cells must be used to confirm all negative antiglobulin test results

when the antiglobulin reagent used for testing has anti-IgG reactivity. Tests found negative by tube

methodology must be verified by obtaining a positive test result after adding IgG-coated (control)

red blood cells. If a licensed blood typing system is used that does not require verification of negative

test results using IgG-coated red blood cells, an appropriate quality control procedure must be

followed, as recommended by the manufacturer.

Evidence of Compliance:

✓ Patient records/worksheet documenting confirmation of negative antiglobulin tests

TRM.40210 DAT Phase II

When performing an antiglobulin test with anti-C3 antiglobulin reagents, C3-coated red blood

cells are used as a control in all negative antiglobulin tests.

NOTE: Complement-coated red blood cells must be used to confirm all negative antiglobulin test

results when the antiglobulin reagent used for testing has anti-C3 reactivity. Tests found negative

by tube methodology must be verified by obtaining a positive test result after adding C3-coated

(control) red blood cells. If a licensed blood typing system is used that does not require verification

of negative test results using C3-coated red blood cells, an appropriate quality control procedure

must be followed, as recommended by the manufacturer. If a polyspecific antiglobulin reagent is

used, refer to checklist item TRM.40200.

[tricore]

PS - how can you use both IgG coated and C-3 coated cells in the same tube?

[Malcolm Needs ☆]

The thing is that most people now use EDTA samples for testing these days, and so the need for anti-C3 in the AHG is obsolete, as EDTA is antagonistic to the complement pathway, as it chelates Ca++, Mg++ and Mn++, all of which are required as cofactors in this pathway, and so Michele is absolutely correct to question the need for the use of polyspecific AHG in the first place.

The only time you really require a polyspecific AHG containing anti-C3 is when you are performing a DAT, but then you should go on to test with monospecific AHG reagents, if the DAT is positive.......

......and your P.S. is very apposite Michele!

[mollyredone]

We QC our poly with complement check cells every morning, but only check patient poly DATs with IgG check cells. But we do know that our poly is working properly. I agree about disagreeing with inspectors. They are usually just techs like us and can be misguided or ill-informed.

[Dansket]

The thing is that most people now use EDTA samples for testing these days, and so the need for anti-C3 in the AHG is obsolete ...

I thought the presence of EDTA in a freshly drawn blood sample was to prevent in-vitro complement binding.

[Malcolm Needs ☆]

I thought the presence of EDTA in a freshly drawn blood sample was to prevent in-vitro complement binding.

THAT is EXACTLY what I meant. The point is that, if blood is drawn into EDTA, you will only detect IgG antibodies (well, okay, IgM and IgA too), but NOT "complement only" antibodies (by that, I mean things like an anti-Vel that can only be detected in serum, but not EDTA plasma), so anti-C3 in AHG is a waste of time if you are using EDTA blood. HOWEVER, of course, EDTA will not prevent in vivo C3 coating, and so you WILL detect complement on red cells in the DAT. I thought that was what I said in my earlier post?

[Dansket]

Malcom,

Thanks for your clarification.

My comment was meant to be in the context of this thread, i.e., Direct Antiglobulin testing.

I stoppped using/stocking polyspecific antiglobulin reagents many years ago, just because of the issues raised by CAP requirements. We routinely test with anti-IgG and anti-C3 reagents separately whenever a DAT is requested by a physician on an adult patient.

Dan.

[tricore]

The purpose of using coated cells (check cells for us oldsters) when you perform a DAT is to ensure that the cells to which the antiglobulin sera was applied were thoroughly washed and that no antibodies, plasma, serum remain in the tube that could have neutralized the antiglobulin giving you a false negative reaction. Therefore IgG coated cells serve to show that the washing procedure was complete for both anti-IgG and polyspecific.

This is different than QC in which you need to demonstate that the poly anti-globulin sera contains anti-IgG and anti-complement

[Malcolm Needs ☆]

Malcom,

Thanks for your clarification.

My comment was meant to be in the context of this thread, i.e., Direct Antiglobulin testing.

I stoppped using/stocking polyspecific antiglobulin reagents many years ago, just because of the issues raised by CAP requirements. We routinely test with anti-IgG and anti-C3 reagents separately whenever a DAT is requested by a physician on an adult patient.

Dan.

Sorry Dan, you are completely correct. I missed the bit about the DAT. Need to change my specs again!!!!!!!!!!!!!!!

- - - Updated - - -

The purpose of using coated cells (check cells for us oldsters) when you perform a DAT is to ensure that the cells to which the antiglobulin sera was applied were thoroughly washed and that no antibodies, plasma, serum remain in the tube that could have neutralized the antiglobulin giving you a false negative reaction. Therefore IgG coated cells serve to show that the washing procedure was complete for both anti-IgG and polyspecific.

This is different than QC in which you need to demonstate that the poly anti-globulin sera contains anti-IgG and anti-complement

Agreed Michele.

[Mabel Adams]

very succinctly stated, Michele.

[goodchild]

We use Ortho Polyspecific AHG and the manufacturer's instructions says to test it daily with both IgG-coated cells and C3-coated cells as well as a negative control.

 

If you're not running both you might want to check your reagent package insert.

[AMcCord]

You can call or email CAP at any time and ask them what they expect for compliance. The questions are referred to the person specializing in whatever area your question is in. They always answer emails promptly and have been very helpful on the phone. Several years ago I did ask them specifically about the use of C-3 coated cells with Poly - I was told that I must use them for QC purposes. Wouldn't hurt to ask again and see if there has been a change.

 

"Tricore  PS - how can you use both IgG coated and C-3 coated cells in the same tube?"

You can't. You have to set up 2 tubes - 1 for IgG coated cells and 1 for C-3 coated cells.

[tricore]

You can call or email CAP at any time and ask them what they expect for compliance. The questions are referred to the person specializing in whatever area your question is in. They always answer emails promptly and have been very helpful on the phone. Several years ago I did ask them specifically about the use of C-3 coated cells with Poly - I was told that I must use them for QC purposes. Wouldn't hurt to ask again and see if there has been a change.

 

"Tricore  PS - how can you use both IgG coated and C-3 coated cells in the same tube?"

You can't. You have to set up 2 tubes - 1 for IgG coated cells and 1 for C-3 coated cells.

This comment was tongue in cheek because when you perform a DAT on a patient using polyspecific you have only one tube to "control". It is amazing how the wording of a standard, FDA regulation, or inspection/assessment question can make us crazy, especially when we are inspected/assessed by people who do not have an understanding of what they are inspecting/assessing (see David's post above).

[BBCLS]

We QC our Poly  DAT daily . One tube for IgG coated and one tube for C-3 coated cells.  We use the same C-3 coated cells vial for the monospecific  anti-C3.