hmust1 Posted March 3, 2008 Share Posted March 3, 2008 Hello all,I'm reviewing our Antibody Titration procedure and read in the Technical manual that using enhancement media (LISS or albumin) is not recommended. Currently, we treat a titer the same as an antibody screen and read IS, 37 degree LISS, and AHG reactions. It seems simpler to just incubate "plain" for 60 minutes as recommended and only read once. What is everyone else doing?Thanks!Heather Link to comment Share on other sites More sharing options...
GilTphoto Posted March 3, 2008 Share Posted March 3, 2008 One reading at IAT, 60 minutes, no LISS. The technical manual also states to read the endpoint as the last tube showing a 1+ macroscopic reaction. This is not always the case.There are several reasons for performing titers.1. Pregnant mother with significant antibody. Read macro only2. To tell whether an anti-D is passively acquired due to RhIG or immune. <1:4 is RhIG. read microscopically - last positive. Only done if reaction >2+ or reactive in a phase other than IAT (IS or 37C)3. To tell whether a high incidence weakly reactive antibody is a HTLA antibody (>64) read microscopically - last positive.4. Titers used to be performed on cold agglutinins for mycoplasma pneumonia before better tests were available. Titers >1000 usually indicate CAD (Cold Agglutinin Disease or CHD). DAT is normally positive with C3 and it can cause a hemolytic anemia. A blood warmer is only recommended for cold autos when they are this strong and don't prewarm away.Gil Link to comment Share on other sites More sharing options...
Mary Posted March 4, 2008 Share Posted March 4, 2008 Sending them out to the Blood Center Reference Lab. Link to comment Share on other sites More sharing options...
Mabel Adams Posted March 6, 2008 Share Posted March 6, 2008 We got re-educated quite a few years ago and quit doing LISS titers. Now it's saline, 60 min, IgG, endpoint is last tube showing 1+ or greater.Titers < 4 don't speicifically determine RhIG, but it has been used as a reasonable assumption. Link to comment Share on other sites More sharing options...
Cathy Posted March 6, 2008 Share Posted March 6, 2008 We do the same as Mabel. After getting too high on a survey, we reviewed the Technical Manual and changed our procedure accordingly. Link to comment Share on other sites More sharing options...
David Saikin Posted March 7, 2008 Share Posted March 7, 2008 We use 6-8% albumin as diluent (but otherwise no enhancements) with anti-IgG after 60 minutes. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 6, 2009 Share Posted July 6, 2009 One reading at IAT, 60 minutes, no LISS. The technical manual also states to read the endpoint as the last tube showing a 1+ macroscopic reaction. This is not always the case.There are several reasons for performing titers.1. Pregnant mother with significant antibody. Read macro only2. To tell whether an anti-D is passively acquired due to RhIG or immune. <1:4 is RhIG. read microscopically - last positive. Only done if reaction >2+ or reactive in a phase other than IAT (IS or 37C)3. To tell whether a high incidence weakly reactive antibody is a HTLA antibody (>64) read microscopically - last positive.4. Titers used to be performed on cold agglutinins for mycoplasma pneumonia before better tests were available. Titers >1000 usually indicate CAD (Cold Agglutinin Disease or CHD). DAT is normally positive with C3 and it can cause a hemolytic anemia. A blood warmer is only recommended for cold autos when they are this strong and don't prewarm away.GilAnother reason for performing titrations that is becoming more common is for major side ABO incompatible renal transplants (i.e. A2 donor, O recipient). Link to comment Share on other sites More sharing options...
BBK710 Posted July 6, 2009 Share Posted July 6, 2009 Is anyone using the "standardized uniform procedure"? That incubation is only 30 minutes, no potentiator and w+ read macroscopically as the endpoint. Link to comment Share on other sites More sharing options...
L106 Posted July 6, 2009 Share Posted July 6, 2009 Yes, just this year. Link to comment Share on other sites More sharing options...
YorkshireExile Posted July 7, 2009 Share Posted July 7, 2009 So is it wrong to use the Diamed cells and reagents for an antibody titration? We do the doubling dilutions in saline, pick the appropriate Diamed Screening cell reagent, add them to an AHG gel card and then incubate for 15 minutes, then spin and read. Just like the procedure for the routine antibody screen. End point is a 1+ reaction. Should we be resuspending our cells in saline and using a longer incubation time with tubes?? Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 7, 2009 Share Posted July 7, 2009 So is it wrong to use the Diamed cells and reagents for an antibody titration? We do the doubling dilutions in saline, pick the appropriate Diamed Screening cell reagent, add them to an AHG gel card and then incubate for 15 minutes, then spin and read. Just like the procedure for the routine antibody screen. End point is a 1+ reaction. Should we be resuspending our cells in saline and using a longer incubation time with tubes??No, you are perfectly okay doing your titrations in DiaMed columns BoroCliff.We have been doing this for at least two years (probably longer) in the NHSBT, following a national study and change control. The correlation between our tube and DiaMed titration end points was almost perfect. Obviously, there was the odd antibody that was a couple of dilutions more or less in either method (sometimes the tubes gave, for example, a titre of 64 and the DiaMed 256, sometimes the tubes gave, for example, a titre of 256 and the DiaMed 64 - 2 dilutions difference, so the actual titre was probably 128; this was using master dilutions), but we already know that certain antibodies react differently by certain techniques (tile antiglobulin, tube antiglobulin, capillary tube antiglobulin, liquid-phase microplates, solid-phase microplates, column agglutination technology - you name it).We actually use Diluent2 as our suspension medium, but there is no reason why you should not use the cells in the diluent in which they are supplied (we looked at that too). We just feel, subjectively, that we get crisper results with Diluent2.:) Link to comment Share on other sites More sharing options...
YorkshireExile Posted July 8, 2009 Share Posted July 8, 2009 From a first-time poster - thank you Malcolm for your excellent reply. I was beginning to wonder what was going on with people talking about 60 minute incubations and only using tubes! Link to comment Share on other sites More sharing options...
David Saikin Posted July 8, 2009 Share Posted July 8, 2009 The problem with using gel for titers in the US is that our physicians are not used to the increase in titer seen in gel. It will take a sgnificant education process for them to realize that what was in tube a titer of 16 may be a titer of 32 or 64 in gel. I know some folks here are performing comparison studies, but unless we are all on the same page with these, there is the risk of interventions when they may not be necessary. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 8, 2009 Share Posted July 8, 2009 True, there may be David, but surely they would not intervene physically, unless they had performed other studies such as MCA Doppler/ultrasound?I know our doctors would only use the titre as a guide, rather than the be all and end all. Link to comment Share on other sites More sharing options...
David Saikin Posted July 8, 2009 Share Posted July 8, 2009 You are correct . . . but it would avoid the "unnecessary" extra stuff (that is certain to be expensive here in the US of A). Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 8, 2009 Share Posted July 8, 2009 Very true. I tend to forget that over this side of the puddle! Link to comment Share on other sites More sharing options...
clmergen Posted July 8, 2009 Share Posted July 8, 2009 We ran into the problem with a patient having a titer of 1:64 (can't remember which antibody)t at a different hospital. She ws referred to High Risk OB doctor who sends all of his work to us. We got a titer of 1:2. Difference of Gel vs Tube. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 8, 2009 Share Posted July 8, 2009 clmergen, I wouldn't mind betting it was a mixture of an IgG and IgM, and that you were detecting the IgM in the gel (same specificity, but a mixture of immunoglobulins). Link to comment Share on other sites More sharing options...
clmergen Posted July 8, 2009 Share Posted July 8, 2009 I thought gel was only supposed to detect IgG, much like solid phase. The gel testing was done at another hospital, we do tube titers at my facility. It did cause a lot of concern amongst the staff until I called and asked how the testing was done. Then it was like tring to compare apples and oranges. So I wonder if the patient would have been referred to the specialist if the typing had been done in tube to begin with. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 8, 2009 Share Posted July 8, 2009 No, not necessarily.We use cassettes that contain anti-IgG and anti-C3d (although cassettes containing anti-IgG only are available). But we often find that when an IgM antibody is present, particularly if it reacts better in the cold (well, cool) as most of them do, the sensitisation occurs before we put the cassettes in to incubate at 37oC, and you then get agglutination when you centrifuge. This is why did so much comparative work and change control.With experience, you can usually tell (although I'm sure we don't detect every time there is an IgM present). Link to comment Share on other sites More sharing options...
clmergen Posted July 8, 2009 Share Posted July 8, 2009 I forgot you can get those anti-IgG and anti-C3d cassettes. The only hospital we have in our system using Gel uses IgG only cards. I personally haven't worked with Gel since the 2000, and at that time I remember we did on occasion detect a cold reacting atibody. Of course, I can't remember if we had anti-IgG only or the dual anti-IgG/C3d. Link to comment Share on other sites More sharing options...
Malcolm Needs ☆ Posted July 8, 2009 Share Posted July 8, 2009 In a way though, the anti-C3d is irrelevant. If there is an IgM element to the antibody, as well as an IgG (as can often happen with an antibody produced de novo during a pregnancy) you may still get agglutination prior to 37oC incubation. Link to comment Share on other sites More sharing options...
GilTphoto Posted July 8, 2009 Share Posted July 8, 2009 Is anyone using the "standardized uniform procedure"? That incubation is only 30 minutes, no potentiator and w+ read macroscopically as the endpoint.Just saw that in the last CAP Titration Survey instructions.Of course I had already performed the titer using 60 minutes, titration using a 100 ul pipettor and 1 drop of cells.THEN I READ THE DIRECTIONS!!!Repeated using same 100 ul pipettor and a 50 ul pipettor for the cells, incubating 30 minutesThe first titer was one tube higherBTW we use w+ to denote microscopic readings only. The last macroscopic reading as 1+. Link to comment Share on other sites More sharing options...
Mabel Adams Posted July 9, 2009 Share Posted July 9, 2009 I wondered how they can call it the "uniform" procedure when it is not even in the Technical Manual. In fact there are 2 different methods for titration in the Tech Manual and they sort of contradict each other as to which is more appropriate for following pregnant women.To respond to the post further up, I am pretty sure I know of doctors that would order amniocentesis if the titer were 64--trained a long time ago and haven't kept up. I hope I am wrong. Link to comment Share on other sites More sharing options...
John C. Staley Posted July 9, 2009 Share Posted July 9, 2009 I'm curious, can anyone site a current/recent reference study indicating the titer significance when "enhancement" techniques are used for titration studies of antibodies during pregnancy?Point #1: The original and only antibody studied for HDN is Anti-D.Point #2: The technique utilized was saline and 60 minutes incubation.Point #3: This is the study most OB docs are made aware of during training and they were trained to those numbers. Any numbers provided to them using alternate techniques will be confusing to them and possibly dangerous to the fetus.A little historic food for thought. Link to comment Share on other sites More sharing options...
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