Jump to content

Recommended Posts

Hi all,

Do you check microscopic reaction for all DAT?

Do you use Ortho/Immucor/Other reagent?

Do you receive specimen fro API/CAP for proficiency testing?

Is there a requirement to check microscopic reaction / mixfield reaction?

If yes, is this CAP/AABB/CLIA/other? \

Thanks

Share this post


Link to post
Share on other sites


3 minutes ago, Eagle Eye said:

Hi all,

Do you check microscopic reaction for all DAT?

Do you use Ortho/Immucor/Other reagent?

Do you receive specimen fro API/CAP for proficiency testing?

Is there a requirement to check microscopic reaction / mixfield reaction?

If yes, is this CAP/AABB/CLIA/other? \

Thanks

Microscopic check?  No.  As far as I know, no regulatory agency requires it for DATs.  A mixed field reaction is a presumptive positive.  We use Ortho reagents, proficiency used to be from CAP, we have since switched to API.  

Share this post


Link to post
Share on other sites

I will quote (one of my favourite quotes) from Issitt Peter D and Anstee David J.  Applied Blood Group Serology.  4th edition, 1998, Montgomery Scientific Publications, pages 63-64 (only because I cannot get to my copies of earlier editions at the moment - as while looking for them, a large number of my text books almost knocked me sideways, as they fell off the top of my wardrobe, and my wife nearly "brained" me too for making our bedroom look a mess!).

"Reading Methods for In Vitro Tests.

Now that most routine tests are carried through to an antiglobulin reading the question of how to read them does not often arise.  They are usually read macroscopically in order that the cells and serum are left in the tube for progression of the test.  A few cells may, of course, be removed and examined microscopically at any stage if this type of reading is required.  However, one of us (PDI) has believed for years that routine use of the microscope in the blood bank creates far more problems than it solves.  Almost any cell suspension, including those in which washed cells have never been exposed to antibody, if examined carefully enough under the microscope will be found to contain a few small clumps of red cells.  Thus, while reading aids such as mirrors or hand lenses are acceptable, routine use of the microscope is not condoned.  This reasoning also applies to the reading of antiglobulin tests.  Again if agglutination cannot be seen with the naked eye, a hand lens, a convex mirror, or the type of microscope in which the contents of the tube are viewed while still inside the tube by placing the tube itself on the microscope slide, IT IS NOT THERE.  Were it not for special tests, such as those in which mixed-field reactions may have occurred or when a small percentage of fetal cells might be present in a maternal sample, the microscope should be banned from the blood bank.

Enzyme tests for agglutination or following conversion to antiglobulin reading, should NEVER be read microscopically."

Several things should be noted about this quote.

Firstly, there is NO distinction here between the indirect and direct antiglobulin test.  Peter Issitt (see PDI, although I happen to know that Dave Anstee agrees with him) talks about reading antiglobulin tests.

Secondly, this quote was originally written well before 1998.  Since that time, there have been huge steps made in improving the sensitivity of tests within blood transfusion and blood group serology (often at the expense of specificity, and certainly at the expense of making diagnosis from the results of these tests straightforward - see the introduction of the term "delayed serological transfusion reaction", when there is laboratory evidence of a transfusion reaction, but no clinical evidence of a "delayed haemolytic transfusion reaction" (Petz LD and Garratty GImmune Hemolytic Anemias, 2nd edition, Churchill-Livingstone, 2004).

Thirdly, both Peter Issitt (1986) and Dave Anstee (1997) have been awarded the AABB Emily Cooley Memorial Award and Lectureship (amongst numerous other international awards - Dave was the President of the British Blood Transfusion Society, the Kenneth Goldsmith Award in 1985 and the James Blundell Award in 2003; these two are far from idiots!

Lastly though, looking down a microscope is no panacea to detecting a transfusion reaction.  I would draw your attention to Sachs UJH, Röder L, Santoso S, Bein G. Does a negative direct antiglobulin test exclude warm autoimmune haemolytic anaemia? A prospective study of 504 cases.  British Journal of Haematology 2006; 132: 651-661.  This paper talks about the production of de novo antibodies and anamnestic antibody production post-transfusion, as well as WAIHA.  It comprehensively debunks the fallacy that a mixed-field reaction can, in all cases, be detected by the use of a microscope.

So, perhaps the question should be, if the DAT is negative to the naked eye, should we perform an elution in each case?  I REALLYREALLY hope that nobody (seriously) answers YES to that suggestion (believe me, it was made with my tongue very firmly in my cheek)!

Sorry about the rant!

Share this post


Link to post
Share on other sites

Quote:  Again if agglutination cannot be seen with the naked eye, a hand lens, a convex mirror, or the type of microscope in which the contents of the tube are viewed while still inside the tube by placing the tube itself on the microscope slide, IT IS NOT THERE.  Were it not for special tests, such as those in which mixed-field reactions may have occurred or when a small percentage of fetal cells might be present in a maternal sample, the microscope should be banned from the blood bank.

Is this section clearly referring to rolling a tube on a microscope under a 10X lens?? - As opposed to pouring it out on a slide and using a higher power???

If so, does this quote condone the use of a microscope under certain restrictions and/or for certain tests (notably the Fetal Screen - which it must be used for) - or is it truly interpretable as "a scope should not be used at all"?

Just checking ------

And for the start of the thread -

Yes - we check our DATs under the scope - tube rolling only; we use Immucor reagents and use CAP and API for various Proficiencies (API for the DATs).  I don't know of a Joint Standard that requires microscopic reading, but the inspectors always check that a full DAT investigation includes both IgG and Complement reagents (Polyspecific or the individual monospecific reagents (and you have to have complement dependent check cells too)). 

Share this post


Link to post
Share on other sites

There are microscopes that have a contraption that can be attached to the stage, where there are two sort of parallel metal bars that are just far enough apart from one another that a tube can lie between them, but the sides of the tube just lie on the metal bars, rather than on the stage itself.  The bars are also slanted, so that the contents of the tubes slide towards the tube opening (but not slanted sufficiently to allow the contents to spill out).  The trick is to get  the contents of the tube in focus.

The contents of the tube has to be examined under low power, otherwise all tests will appear positive.  In over twelve years of using such a microscope, I found it useful just once, when I was dealing with an anti-Lub, as it showed the distinctive Lutheran type of agglutination.  Other than that, there was no occasion when looking down such a microscope helped me in any way.  However, Peter was NOT suggesting that a microscope should not ever be used to read a serological test, but suggested that such use should be exceptional, rather than the rule.

Share this post


Link to post
Share on other sites

For tube testing

Immucor states: Negative tests may be examined using an optical aid. 

Ortho states: Examine negative tests with an optical aid. 

I believe a concave mirror or a microscope would qualify as an optical aid. Our policy does not define optical aid though.  If the tech feels they need to look under the scope we do not discourage for DATs. 

Share this post


Link to post
Share on other sites
2 hours ago, Patty said:

For tube testing

Immucor states: Negative tests may be examined using an optical aid. 

Ortho states: Examine negative tests with an optical aid. 

I believe a concave mirror or a microscope would qualify as an optical aid. Our policy does not define optical aid though.  If the tech feels they need to look under the scope we do not discourage for DATs. 

I would say that an electron microscope or a telescope would qualify as an optical aid also, but I am pretty sure that is not the intent of Immucor using that term either.

The problem with using, say, a high-dry (40x) microscope objective on a specimen from a tube on a slide is that you will have to define how you are going to deal with a false positive.  Because as it has been pointed out above, most specimens examined by tilt-tube, be they ABO typings or something involving AHG, will be seen to have at least a few small agglutinated clumps of RBCs if you look at it this closely.  To create a procedure for using a scope, one would have to arbitrarily define what can and cannot be ignored under these conditions.

Scott

Share this post


Link to post
Share on other sites

What has been your experience with microscopically (only) positive direct antiglobulin tests?  Does anyone have any long-term data supporting the clinical significance of a positive DAT test that is detectable only by a microscope?  Do you report these as positive in the say way as you would report a macroscopic result of 1+ to 4+?

Share this post


Link to post
Share on other sites

I have never looked at a DAT test down a microscope.  As far as I know, and I probably WOULD know, I have never "missed" either a clinically significant haemolytic transfusion reaction, or a clinically significant case of haemolytic disease of the foetus and newborn.  If I had, I would have been in DEEP trouble.

As Peter Issitt said, such "kissing red cells" (I paraphrase) happen in ALL tests, if you look hard enough and, if these are genuine cases of agglutination, they are, at worst, cases of serological transfusion reactions (see George Garratty).  Even in the paper by Sachs et al I quoted above. the worse scenario was that a de novo antibody was being formed, WHICH DID NOT RESULT IN A CLINICALLY SIGNIFICANT HAEMOLYTIC TRANSFUSION REACTION.

Share this post


Link to post
Share on other sites

Malcolm--

How many times over the years, here on this most excellent internet informational exchange site, have you responded to this particular issue?

Scott

Share this post


Link to post
Share on other sites

I'd say that you have to consider the capabilities of your staff. I do ask my techs to use the microscope for DATs. They are all generalists and their time in blood bank is limited. Some of them shake too hard, in spite of my best efforts to fix that problem. They use a mirror, but some don't use a mirror well. So, in order to not miss weak positive reactions they use the scope with a tube roller. We also have a definition for microscopic agglutination (right out of the Technical Manual) that says it is a clump of 4-5 cells (though I do tell them that they should be cautious with this - if tests look suspicious, check them out, don't blindly ignore what you see). When I train, I stress the difference between a clump of cells that are friendly/kissing and a clump of cells that 'love each other' (agglutination). They do very well - false positives are rare. I don't see a lot of unnecessary work being done.

 

 

Share this post


Link to post
Share on other sites

I have to say that I disagree with the following statement:

  •  
  • galvania
  • Members
  • 728
  • 832 posts
  • Joined: June 13, 2007

And never EVER , under ANY circumstances look at gel tests under a microscope or a magnifying glass - unless you want to call absolutely everything positive and waste everybody's time

I have personally seen this to be untrue. I worked with a former colleague (a 30 year Blood Bank veteran) who used to inspect questionable gel card results with the agglutination mirror that is normally used for looking at tube testing results. She once had a gel card reaction that was nothing more than a little speck at the bottom of the card, and because she used the mirror to inspect the gel card, she saw the small speck, and decided to set up a ficin panel, which led to her identify an Anti-Jka.

Share this post


Link to post
Share on other sites

Well you carry on doing just that then klsmith, seeing as you had ONE example where it came up with something that would have otherwise been missed, and you clearly think that that justifies it. Actually why not put up enzyme IATs routinely as well.   But please do not complain when you have to put up panels on 90% of your samples and get inconclusive results on all of them

Share this post


Link to post
Share on other sites

Yes unfortunately we look at negative reactions  under the microscope. I really want to stop doing this and thank you for the reference to support my plan. It is correct reading microscopically causes more trouble then it is worth.

Answers to the other questions"

> Yep we get a CAP survey

>And as far as I know reading under the microscope isn't a CAP or AABB requirement. 

Note: I am the most senior Blood Banker here now (30 years) and this process has been in place when I started ( I think). Anyway I will be seeing one of the retired Supervisors today and will ask why we started doing that

Share this post


Link to post
Share on other sites

Well you carry on doing just that then klsmith, seeing as you had ONE example where it came up with something that would have otherwise been missed, and you clearly think that that justifies it. Actually why not put up enzyme IATs routinely as well.   But please do not complain when you have to put up panels on 90% of your samples and get inconclusive results on all of them

I am not complaining, nor am I the tech who discovered the antibody by the means which you say are incorrect! I am just telling you what I have witnessed by a reputable tech. BTW, how do you know that this situation doesn't happen more frequently than you are aware of?? Sometimes you really do need to think outside of the box, perhaps not everything is as cut and dry as we would like for it to be....

Share this post


Link to post
Share on other sites

Create an account or sign in to comment

You need to be a member in order to leave a comment

Create an account

Sign up for a new account in our community. It's easy!

Register a new account

Sign in

Already have an account? Sign in here.

Sign In Now

  • Similar Content

    • By SusieQ132
      Our facility is evaluating making a change to our process for Weak D testing for patients with a positive DAT.  For years, if we were required to do a Weak D, but the patient had a positive DAT, we used to cancel the Weak D as invalid.  Another hospital in our system mentioned that they tended to perform the Weak D, but then only cancel as invalid if the Weak D is positive.  We are thinking about changing to this process, as we now have to result many babies as "Rh Unknown" and give their mothers Rhogam.  
      Per our Anti-D's package insert:  "Red blood cells coated with alloantibodies or autoantibodies of the same or similar specificity as the reagent (i.e. cells that are DAT positive) may give weak reactions. This is due to decreased availability of antigen sites because of antigen blocking or steric hinderance. In extreme cases false-negative results may occur." 
      I'm worried about the "extreme cases" where a false negative could occur, but I cannot see this being common.  Also, would you think that if the cells were coated with that much antibody, that we would see any other odd reactivity in the ABO/Rh?  What do other facilities do?
      Thank you in advance,
      Susan
    • By ejani
      I am sorry if this has been discussed previous... I searched and didn't find anything.  Quick question...
      We are transfusion service that performs DAT's using poly-clonal IgG... if it is positive, we run the mono-clonal IgG, however, we do not run the C3d.    How many of you would and/or do run the complement control cells for DAT QC in addition to Check Cells?  
       
    • By Jermin
      Hi All,
      I have a question, but firstly good old story time for some context. I came across a patient who had positive antibody screen on all three screening cells used (BioRad). I was concerned this may be an auto and pan-reactive, and required units. Performed a monospecific DAT, showing a positive reaction to IgG only. By this time antibody panel finished cooking and showed the patient may have anti-Fya , but couldn't do phenotype. By this time I was nearing my shift so handed it over to my colleague and asked for some units to be crossmatched. However, he refused as DAT was positive and said he rather send the sample to reference laboratory for them to crossmatch. The next day I crossmatched units to verify if it could have been done in our laboratory (just because I am sad that way), and turn out the unit I crossmatched was compatible (which I wasn't surprised about)
      Question
      Why does positive DAT (or the cause of positive DAT) sometimes interfere with IAT techniques (such as antibody panel and crossmatch) and sometimes it does not? If both use AHG, then wouldn't positive DAT with IgG cause antibody panels shows pan-reactive with red cells? But obviously it doesn't, but I'm trying to figure out why, and I'm sure the answer is quite obvious. 
      My laboratory seems very hesitant whenever they see anything regarding autoantibodies or positive DAT, and thinks that sample cannot be crossmatched in-house and needs to be sent off without even trying to investigate. Hopefully, by me asking this question, I can explain it back to my colleagues (but obviously take all the credit).
      Cheers in advance,
      Jermin
       
    • By Jermin
      Hi All,
      Its been a while since I came back to this forum, but glad I feel like I have gained a lot more insight. I feel like I'm a bottomless cup. 
      So I come with a question, for which there is not going to be a definitive answer (but with BB, is there ever one?), but hopefully, I would gain a bit of understanding.
      Background: So, our laboratory has started sending samples to reference laboratory for genotyping of the foetus by FDNA, which is great, since we would figure out the Rh(D) status of the baby (on most occasions) before they are born! So our laboratory has set up a flow chart which basically mentions that you do not need to send cord sample of Rh(D) negative mother if the baby is shown to be Rh(D) positive (or D positive, I am quite wary when trying to talk about Rh group), and only send cord if baby of Rh(D) negative mother if the FDNA shows the baby is Rh(D) negative, just to confirm the accuracy of FDNA. It sounds kinda counterintuitive, but we will soon be just not processing any cord sample for the ones we performed FDNA on. That means no cord Blood Group or DAT on a lot of post-delivery patients.
      Question:  By missing out DAT, we would possibly be missing out on detecting ABO incompatible HDN. How significant do you think it is in the early stages? Is it OK to wait to see if the patient shows signs of jaundice and for them to send a DCT sample afterwards? 
      Bonus Question: What does your Hospital/Laboratory do in the event of positive DAT on cord sample, and why do you do it?
      I had a read through one of the articles stating about the significant of DAT, but they called the Rh blood group as Rhesus, so I'm not going to take them too seriously

      Cheers,
      Jermin
    • By kimg
      We are  having an issue reporting out grossly blood urines.  Techs tend to call everything "indeterminate" which I feel is unacceptable.  We have, in the past, been taught to put a drop of urine on a glass slide with a coverslip but then there is the problem of quantitating. How are other labs handling this?
  • Advertisement

×

Important Information

We have placed cookies on your device to help make this website better. You can adjust your cookie settings, otherwise we'll assume you're okay to continue.