Enzymes in UK antibody screening

[blut]

Hi all,

I'm hoping that UK colleagues involved in NEQAS can help with the following issue:

Background: I've corresponded with a colleague in Switzerland to explain why transfusion services in Canada and the USA rely on gel-IAT to detect unexpected antibodies and do NOT do an extra enyme-IAT gel antibody screen, as her hospital does, to detect possible antibodies in the Kidd system that may be too weak to be detected by gel-IAT without enzyme-treated red cells (summary of reasons below).

My colleague offers this evidence for why her hospital's transfusion service lab includes enzyme testing:

"The UK NEQAS report published in Transfusion Medecine 2002; 12:11-23 states that sample 988RO Anti-Jk(a)

Detection rate by technology

* LISS tube, NO (%) 143 (89)

* Diamed (Geltest) 34 (19,3)"

From the cited statistics, if 0.89n=143, LISS n=160.7 and if 0.193n=34, Gel n=~176 (where n=no. of labs performing each method).

My questions:

1. Has Diamed gel consistently performed that poorly for Jka compared to LISS methods?

2. If yes, it it possible that human and systemic error involved in the Diamed test played a role in this particular survey, now ~10 yrs old/ (e.g., wrong LISS diluent, wrong pipetting technique)

3. My sense is that labs in Europe use enzyme-treated rbc in gel (and possible other) testing more than in NA, where enzymes are not used for routine antibody screening but reserved for more complex antibody identification. Is this true for the UK, and if so, what are the rationales?

4. Did NEQAS or influential professionals suggest that the cited study provides a valid rationale for including enzyme-IATs when antibody screening by gel?

Thanks for any comments you can offer.

Cheers, Pat

http://www.ualberta.ca

TraQ: http://www.traqprogram.ca

----------------------

SUMMARY (omitting enzymes in routine antibody screening):

Kidd antibodies are relatively rare and ones too weak to be detected by LISS-IAT, Gel-IAT, PEG-IAT, etc., are rarer still. Today most NA labs rationalize testing so as to stay within constrained budgets.

For example, papers on test rationalization by John Judd and colleagues on

(i) why doing a routine autocontrol (~DAT) when antibody screening is not cost effective:

http://www.ncbi.nlm.nih.gov/pubmed/3705135

(ii) why reading antibody screen tests at 37C (applicable only to standard saline and LISS tube tests) is not effective:

http://www.ncbi.nlm.nih.gov/pubmed/10204593

Similar arguments can be made for not adding an enz-IAT just in case gel-IAT or LISS-IAT alone do not detect weak Kidd antibodies.

The clinical significance of weak antibodies missed by IAT testing by any method using anti-IgG (e.g., LISS, PEG, gel) is open to question.

Nancy Heddle, for example, showed that most delayed hemolytic transfusion reactions were actually "delayed serological transfusion reactions". Antibodies detected 5-7 days post-transfusion hardly ever caused clinical symptoms in the patient (only one in her large study):

http://www.ncbi.nlm.nih.gov/pubmed/8547111

The risk of missing a weak anti-Jka that needs enzymes to be detected is small. Even when missed, how much red cell destruction the antibody would cause is debatable. Enzymes detect many clinically insigniifciant antibodies. Eliminating enzyme tests has a low risk and significant benefits.

--------------------------------------------

[David Saikin]

I am replying from the USA - I have not used enzymes for routine antibody screenings since 1977. During the intervening years (decades) - I have worked in 40-700 bed institutions. In the smaller venues, BB was at its most basic. Even in the tertiary care settings, enzymes were/are only used when deemed necessary. The reasons for this I could not tell you. I only use enzyme (specifically ficin) in selected instances. I do know that when we used enzymes routinely we worked up way too many non-specificities. There is some bad-mouthing of gel's ability to detect abs to Jka (and E). I have not found that to be the case.

[pluto]

Have been using Diamed for years now never had an incorrect NEQAS exercise yet, do not use enzyme treated cells either any more

on exercise 98RO we were on Biovue at the time , Biovue only gave 1+ results in IAT in the panel at the time and 4 Jka+b+ cells were negative as well . Had enzyme panel at time and still 2 Jka+b+ cells were negative

Can't find NEQAS report so unsure what they recommended after this exercise

[blut]

Thanks David & Pluto for replying.

Still would like to learn how many gel users in the UK use enzymes routinely...

[Malcolm Needs ☆]

I am now at a Reference Laboratory, where all of our panels include an enzyme phase, but when I was working in the hospital environment, the screeing was only ever done by IAT (non-treated red cells), but panels were performed using both IAT (non-treated red cells) and direct agglutination with enzyme-treated red cells.

[Fluffy agglutinates]

Hi Blut

It's definitely a NEQAs reply you need - they should have quite accurate figures of just who is still doing enzyme screening in the UK.

When I'm teaching I always ask the audience if anyone still does an enzyme screen routinely - I haven't had a yes in 6 years! So that may be some indication at least.

As for picking up otherwise undetected Kidd antibodies, I personally would only be truly confident if it was an 'ENZ with AHG phase' screen in use. I'm pretty sure no one is doing this...

Robina

[blut]

Thanks, Robina.

With your reply and Malcom's I'm confident that enzymes are not used for routine antibody screening in the UK. I'll let my Swiss colleague know.

Cheers, Pat

UA: http://www.ualberta.ca/~pletendr/

TraQ: http://www.traqprogram.ca/

[blut]

Thanks, Malcom. See reply to Robina.

About doing an enzyme *panel* with all investigations submitted to your reference lab - I'm curious, as to my knowledge this is not standard practice in Canada (or the USA), where typically enzymes are used when initial panel results are not clear cut.

Why was this implemented, e.g., based on experience, it turned out to save time in the long run? Or you wanted to be sure you had covered all bases when reporting antibodies found? Or?

Thanks.

Cheers, Pat

UA: http://www.ualberta.ca/~pletendr/

TraQ: http://www.traqprogram.ca/

TM Blog: http://traq.blogspot.com/

Twitter: http://twitter.com/bogeywheels

[Malcolm Needs ☆]

Hi Pat,

Actually, the "enzyme panel" was not introduced per se, but has always been "encouraged" in UK Pre-transfusion Testing Guidelines, because it does save time in the long run, as you will detect some Rh antibodies that are not detected by IAT, and you have more chance of detecting weak Kidd antibodies.

What has happened, since, I think 1996 (but I am unsure of the accuracy of that date, is that the same Guidelines no longer require that enzyme-treated red cells are used in either the screen or in the serological cross-match.

That having been said, we still get a remarkable number of samples referred to us that contain, for example, an anti-E that, try as we may, we can only detect using enzyme-treated red cells, so quite how the Hospitals detected this in a screen using only the IAT does escape me!!!!!!!!!!!!!

Incidentally, when we are using the enzyme-treated panel (and, of course, this is always done in parallel with an IAT panel with untreated red cells), we do use an IAT cassette, so we are actually performing an enzyme-IAT panel. There are two reasons for this.

Firstly, as Fluffy says, you are much more likely to detect weak Kidd antibodies by this technique.

Secondly, and quite empirically, we reckon that the reactions are far "crisper" than using enzyme-direct agglutination, and you get less "anti-tat"!!!!!!!!!

[blut]

Thanks, Malcolm.

I figured enzyme-IATs.

For fun, a trip down memory lane....I'm so ancient that when I first started in the mid-60s as a child prodigy at Canadian Red Cross BTS (now Canadian Blood Services) in Winnipeg, Canada, where the BTS was (and still is) a combined blood donor centre and transfusion service for the entire city, we used capillary tubes to determine ABO and Rh groups, but also for antibody screening (IATs, enzymes, saline)

For enzymes, I believe the order was serum, enzyme, rbc suspension, then invert and stick the capillary tube into plasticine on a special box with a back light, let sit, and read with a magnifying glass. Quite often index fingers would get punctured (enough to draw blood) as some cap tubes were jagged. All this a "good grief" to today's workers.

Prehistoric paper by Brits evaluting the capillary method:

http://tinyurl.com/3sj2lar

Cheers, Pat

UA: http://www.ualberta.ca/~pletendr/

TraQ: http://www.traqprogram.ca/

[Malcolm Needs ☆]

Yes, it was Bruce Chown (a Brit, if I remember correctly), and you are quite correct: I think today's "intake" would wonder what on Earth we are talking about!!!!!!! Capillary methods????????????????!!!!!!!!!!!!!!!!!!!!!!!!!!!!!!

Chown B, Lewis M. The slanted capillary method of Rhesus blood-grouping. J Clin Pathol 1951; 4: 464-469.

Chown B. A rapid, simple and economical method for Rh agglutination. Amer J Clin Path 1944; 14 (11): 114-115.

Here is photo I took of the capillary IAT method (VERY effective with weak antibodies).

It was Joyce Poole who introduced me to the use of this method for the IAT.

Edited June 7, 2011 by Malcolm Needs
Remembering my age.

[blut]

Thanks once again, Malcolm. I haven't see one of those for more than 30 yrs. I had not realized capillary tubes were adopted to some extend in the UK. In Canada, only workers in Winnipeg used them, as the "Rh Institute" with Chown & Lewis had great influence.

As it turns out, unlike many of the physicians who developed Canada's fledgling blood system in the 1940s and 50s, Bruce Chown was born a Canadian. Many transfusion specialists were British, since that's where our immigrants mostly came from in the early post WWII years.

[Malcolm Needs ☆]

Whoops, sorry; I could have sworn that Bruce Chown was a Brit - back to school for me!

Sadly, I don't think anywhere in the UK now uses the technique (although Joyce may still use it occasionally; I'm not sure). We have been overtaken by column agglutination technology, national Standard Operating Practices and stifling accreditation that rules out any deviation from the straight and narrow, whether this deviation will be for the benefit of the patient or otherwise.

The photograph was taken when I was putting together an article on the IAT about three years ago. The rest of the Laboratory staff were a bit agog, and wondered what the old fool was up to this time!!!!!!!!!!!!!!!!!!!

[adiescast]

This isn't relevant to the original conversation, but one hospital I worked at used capillary methods for antigen typing to preserve expensive reagents. I wonder if others are doing that?

Edited June 8, 2011 by adiescast
Word confusion