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Saline incubation...why is this SOP still allowed?


labgirl153

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Dr. Pepper asked:

Think about it, there is no enhancement medium "in vivo" so what's wrong with using this technique?

Labgirl, although I like wine as well (almost as much as I like peppers), this quote should be attributed to its proper author, Likewine99. But I also like the line just before it in the post: "We have lots of tools on our antibody ID toolbelt and saline enhancement is just one of them." That was the major point of my post, that our jobs are to provide blood in a timely manner to our patients with the greatest degree of safety that we can, and some may involve using a technique that may be in some cases a little less sensitive than another. That's still better than issuing a 3+ incompatible (in terms of the autoantibody but not knowing if an allo is in there either) and hoping for the best. It's a good tradeoff.

As for your HDN example, you might think twice about calling an anti-K at 1:16 "wimpy". Many places consider a titer of 8 for anti-K to be significant. I agree that you never can tell how an antibody will react in vivo, but the common practice of not taking invasive action with prenatal antibodies until they reach a saline titer of 8 (anti-K) or 16 (the rest of them) has established, and validated, by decades of clinical observation. So your anti-c, detectable only with gel, should not be a problem.

At the worst, what generally happens if you transfuse a patient who has an antibody, too low-titered to be detectable by saline (or LISS, or PEG, or gel, or solid phase)? They have a secondary response and a delayed (usually serologic) reaction that you discover by chance a week later, not because they have had frank symptoms of a hemolytic reaction. Peter Issitt (I know, one of the sainted priesthood but still they were, and are, 10 times the blood bankers we can ever hope to be) once said that, in general, if you have to go to extraordinary lengths to detect an antibody, it is not going to cause much harm to your patient.

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An anti-K PN titer of 1:16 in a saline titration is anything but "wimpy" and should elicit a serious response from the mother's physician in regards to monitoring the well being of the fetus. As far as the usefulness of the saline technique, it is a valuable tool in the hands individuals who are confident in their abilities. I've worked in a reference laboratory for 24 years and have total confidence in the technique. If the need arises, I will not hesitate to drop ANY of the enhancement methods available to me and move to saline tube testing. I can't think of a single common clinically significant antibody that I haven't identified using saline techniques at one time or another. I'd like to say that for all clinically significant ones, but the opportunity just hasn't occured ;). Dr. Pepper has some wise observations with which I'm in total agreement.

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I absolutely agree with what Malcolm, Dr. Pepper, likewine, Mabel, Seven B and others have said. I would not lose much sleep if I was told I could only use saline IAT. Sometimes I wonder if all the new and improved enhancement methods cause more trouble than benefit. How many panels and tech hours have been utilized chasing down positive gel antibody screens without identifying a clinically significant antibody? One of my favorite saline IAT tricks was to start with a dry cell button and use 3 or 4 drops of plasma with a 45-60 minute incubation. I have detected many ant-Jk(a) antibodies this way. On the other hand, we all know there is no single method that is going to detect every clinically significant antibody. As Dr. Pepper said, a low-titered antibody not detected by a given method is unlikley to cause a serious problem. True in the vast majority of cases, but there are exceptions. Case in point is a patient who had several immediate intravascular hemolytic reactions with no demonstrable antibody using gel, PeG, LISS, saline, albumin, or ficin. This patient never had a positive DAT. Red cell phenotyping of the patient and all the units he received pointed toward anti-C. It was not until we sent a sample to George Garraty's lab for Polybrene testing was anti-C identified serologically! As said by someone in a previous post, we have a lot of tools we can use. We have to find the method or methods that are the best fit for our lab. Many factors enter into this decision. What I select for my lab may not be best for your lab.

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Thank you BMarotto for weighing in - and thanks too for the rest of you as well (yes, even to you Malcolm, you really are a super blood banker and we all idolize you). Now I'll 'fess up. I had an ulterior motive and used you all as guinea pigs. Knew I'd get "beaten up" for playing devil's advocate but then I enjoy that role :devilish:

Truth?

I don't mind using the saline 37C 30-60 min. technique at all. On the contrary, I think it's a fine technique. BMarotto summed it up best. I put this pseudo concern to you all because over the years, have seen the same techs on one hand go overboard with technique after technique hour after hour to generate sheets of panels - and little if anything is achieved. Certainly from a clinical standpoint, it didn't benefit the patient, particularly if the etiology was autoimmune in nature. On the other hand, those same techs will take a case and throw all techniques to the wind - by just using the old-fashioned 37C 30 min. saline technique. Sometimes this makes sense, but the 180 degree contrast is striking at times and confusing to newer techs.

Mea culpa if I got anyone's BP jumpy or if anyone wanted to find me on the map and commit a criminal act. I promise to be "good" :floating: from here on out. At least I finally got some honest to goodness opinions.

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For the past several years, as required by CAP, our facility does a semiannual method comparison. We run 3 positive and 3 negative antibody screens with the following techniques: ProVue, manual gel, Peg, LISS, and enhancement free (better known as saline). Gel is our primary method so all of the positive reactions were initially discovered in gel. We have seen excellent correlation in antibody detection with all methods although the strength of reactivity is admittedly weaker in LISS and saline. On one occasion, there was an antibody that was not detected in the saline technique, but was detected with the other 4 methods.

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What! Should we really be catering to the stalwart pagan last-century anarchists, who waste time practicing useless cold-reacting IgM antibody immunohematology psuedo-testing, wallowing in thier cobwebbed beds of antiquated techniques straight out of the dark ages! Perish the thought!

Scott

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John Judd's biggest complaint about what he called the "pre-fried" technique was that it was used (and abused) indiscriminately. People would use it just to make reactivity "go away" when they had no idea what they might be dealing with and if that didn't work, they would raise the saline temperature to 40C (just a bit of a fever eh?) to make it go away. Sort of similar to Labgirl's logic about the saline technique being applied without a lot of thought.

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I think we were talking about the 'classic' tube IAT, with cells suspnded in saline and incubated for 30 minutes at 37C. We then seemed to have an argument about something we all agreed on!

Thank you very much.

I have met some warm auto patient, the "classic" tube IAT seems can't avoid the autoantibody's interference, is there an auto control tube , to see if the auto is stronger than test tube?

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Auto-antibody is a very headache question worry me, if saline incubation can resolve it , I will be very happy.

I am very interesting in how to do it, and is it useful when the auto is 37 degree reactive and very strong. Thanks for your opinion.:P

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Hi Yanxia - The problem with many techniques (LISS, PEG, enzyme, gel) is that while they do a good job of enhancing the reactions of significant antibodies that you do want to detect, they also will enhance reactions of antibodies that you do not want to detect, like IgG autoantibodies. Basically it is tube technique without any enhancement media. Tests are incubated longer (30-60 min) at 37o and some workers use 3-4 drops of serum/plasma to increase sensitivity. We use 4 drops of serum with one drop of saline-suspended red cells, a 60 minute incubation at 37o, followed by washing and AHG. If the autoantibody is strong it's probably going to still react anyway, but sometimes a weaker autoantibody will not react with the technique.

Edited by Dr. Pepper
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do you suspend reagent red cells (panel cells) in saline or use it from the vial?

We use the cells straight from the vial - do not add additional saline - just don't add any other enhancement media (LISS, PEG, ALB, etc). We incubate for 60 mins at 37C, wash and add IgG coombs reagent, spin and read. Strong auto immunes will still react as noted previously - those get sent to the reference lab for them to play with!

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We use the cells straight from the vial - do not add additional saline - just don't add any other enhancement media (LISS, PEG, ALB, etc). We incubate for 60 mins at 37C, wash and add IgG coombs reagent, spin and read. Strong auto immunes will still react as noted previously - those get sent to the reference lab for them to play with!

Same for us.

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Increasing the serum to cell ration when using the saline tube technique is, in itself an enhancement technique. This could mean the difference between resolving a warm auto problem at your own facility within an hour or two using two drops of serum/plasma or shipping it to a reference lab and delaying patient care because four drops of serum/plasma were used along with an extended incubation.

I liken this to when a facility uses LISS/PEG/Gel to test whether their warm autoadsorption worked only to find that it hasn't...a bit self defeating.

Issitt/Anstee made a statement in Applied Blood Group Serology, 4th Edition, that the saline/IAT test can be equivalent to LISS/PEG/Polybrene methods only if increased serum:cell ratios and extended incubations were used....otherwords, it becomes an enhancement technique; not what you want to use when trying to eliminate reactivity due to a warm autoantibody.

My recommendation: two drops serum/plasma to one drop cells, 30 minutes at 37C and on to AHG (obviously with a wash). If nervous...extend to 60 minutes but do not increase the serum:cell ratio.

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As a student I initially thought saline replacement meant you did not use patient serum in the testing. This was very confusing to me in how it was supposed to yield meaningful (or any kind) of results... LOL

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As a student I initially thought saline replacement meant you did not use patient serum in the testing. This was very confusing to me in how it was supposed to yield meaningful (or any kind) of results... LOL

What's being talked about here is not the same as saline replacement.

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Increasing the serum to cell ration when using the saline tube technique is, in itself an enhancement technique. This could mean the difference between resolving a warm auto problem at your own facility within an hour or two using two drops of serum/plasma or shipping it to a reference lab and delaying patient care because four drops of serum/plasma were used along with an extended incubation.

I liken this to when a facility uses LISS/PEG/Gel to test whether their warm autoadsorption worked only to find that it hasn't...a bit self defeating.

Issitt/Anstee made a statement in Applied Blood Group Serology, 4th Edition, that the saline/IAT test can be equivalent to LISS/PEG/Polybrene methods only if increased serum:cell ratios and extended incubations were used....otherwords, it becomes an enhancement technique; not what you want to use when trying to eliminate reactivity due to a warm autoantibody.

My recommendation: two drops serum/plasma to one drop cells, 30 minutes at 37C and on to AHG (obviously with a wash). If nervous...extend to 60 minutes but do not increase the serum:cell ratio.

Very good point StevenB. I think one factor here, though, is that your warm autantibodies usually have a specificity within the Rh system, and using the other techniques, particularly PEG, will really enhance Rh antibodies, so with the 3-4 drops and 60 min you may increase your sensitivity to pick up other IgGs but not enhance the warm auto enough to detect. You can always back off to 2 drops and 30 min.

Best is to do a PEG autoadsorption (with the usual caveats) and the auto is gone, gone, gone. The technique only needs a vial of PEG, is quick and easy, and you don't have to worry about any of the issues discussed above.

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  • 8 years later...
On 8/5/2012 at 8:43 AM, labgirl153 said:

Well, that's mighty nice of you to suggest busy work for me RRavkin. My question relates to literature in the past half century. Surely, in the 67 years since its introduction, someone has made a comparison of the saline incubation technique to other methods. If not, then that speaks favorably toward tradition but is not so flattering toward the upper echelon in blood banking. Believe I'll take this SOP question up with the AABB folks and possibly CBBS. Hopefully I'll get a response directly pertaining to "papers" and "research". If not, then the answer will finally be clear.

I know this thread is several years old but I just want to say I agree with Malcom. 
 

Also, i just want to add that I have been researching this method for the last few days, and found out that many labs use this method. 
 

The main idea here is that alloantibodies usually have a stronger reaction that WAAs. So if you don’t use saline without any enhancements, you will only get the alloantibodies reacting. This especially works well when the underlying alloantibodies are 2+ or greater and the WAAs are 1+ when done with enhancement. 
 

It’s a “quicker” way to identify alloantibodies without doing an autoadsorption.

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