galvania

Did you try with a new EDTA tube. It MIGHT be an anti-EDTA - but it might be that there was a problem with that specific tube

Well, I would also say Congratulations, except I know that you don't WANT to retire. Health Service's loss. But do enjoy your retirement. There are plenty of interesting places to visit, posts to write.......You know. Try not to drive your wife crazy!

Two hours later does not seem extreme to me - depending on the state of the patient. But was 98.7 not high already? Sorry - can't remember normal values in °F

There is a very good reason not to use pooled cells. Many antibodies only react with cells with a double dose of the relevant antigen. That does not mean that these antibodies are not clinically significant. If you mix cells together, even if, for example cell I is Jka+b- and the other Jka-b+, you are diluting out the concentration of each antigen and you will miss the antibody if it is there. Same goes for weak antibodies in general. Pooled cells are OK if you are only looking for the type of antibody that jumps out of the tube at you - as for example for donors, and even then, it's not ideal. You would need a good system for detecting DPs! I don't know anybody who tests ante-natals only using pooled cells. If they are they should be closed down!

What is that?????

This is maybe a silly question, but I presume you have checked that the antibody she has made herself (as opposed to the proph) really is an anti-D and not an anti-G....

Concerning the eluate - I was thinking more of a pre-and post- to see if there was a difference between them. Just as an example, if all the cells were reacting at ++ pre-transfusion and now suddenly all the Jka+ cells are reacting at ++++, that might give a clue....

Have you done an eluate?

Surely you could just give the organ donor a number? And you would surely want to make sure of his group in case you get some funny results after the recipient is transplanted.....

If 84 different labs saw the same thing then I can't see how it's down to you (and presumably the other 84 labs) to all independently look for the root cause. If I were you, I would talk with the manufacturer, send them some of the sample and ask for their help. Whatever they find will be valid for ALL of the labs, surely anna

If anyone says that it's reasonable for me to have a baby (or even want one) at my age (61) then I'm quite happy to accompany them to the local psychiatrist for happy pills

I would check again now and see if there's any change. Also, if you can, test the platelet donor for antibodies against low frequency antigens, and maybe re-test his anti-A titre. Maybe the red cell donor was an A2 and the patient is an A1 so the anti-A would hit the patient cells more than the donor cells. And how did you do the eluate? Maybe not the best method for detecting anti-A???

I don't have any specific information for you about these instruments. What I would just like to point out to you is that TAT is a parameter that is almost impossible to calculate as it will depend on what profile of tests you select (rather than the individual tests), how many samples you load at any one time, and what else the instrument is doing when you loaded those samples. TAT for a single panel carried out on its own, when the machine was doing nothing else will not be the same as TAT for a single panel loaded when the machine is already carrying a whole series of other tests

Could also be a mutation in the H-gene, meaning that there was less H to compete for.

there was an article on this in the last issue of the biomedical Scientist. When I read it, I have to confess, a - I didn't really understand the point and b - moved one step nearer to total despair about the useless requirements that might have sense in chemistry but in IH just get in the way of doing useful work I'm ranting again

Basically Lingkwyz, an auto control looks for antibodies that are present in the patient's plasma that are going to stick on to the patient's red in vitro in the same conditions that you have used to perform your antibody identification. The DAT looks for antibodies that are present in the patient's plasma that have stuck on to red cells that are present in the patient's circulation in vivo. Although you often DO get the same results for both, it is not necessarily the case and the results do not have the same significance

When you do a tube technique, the cells and the plasma are all sitting in the mix together. If you add AHG to this the plasma is omnipresent, and has probably, after incubation, ended up sitting on top of the red cells, which have sedimented down because heavier - so the plasma is the first thing the AHG comes into contact with. In gel, the AHG is in the gel. You pipette the red cells first, then the plasma, so when you centrifuge, the AHG first comes into contact with the red cells. So it can react with sensitised cells before coming into contact with the plasma

Hi Well let me deal with the 'ergo' part first. Depending ion the circumstances, enzyme-IATs are ALSO done on gel. If you have the slightest doubt about Kidd antibodies, this is one of the best methods for finding them. It is just not very common (but not unknown) for this to be used as a routine method for ALL antibody screens So why is gel more sensitive than tube. First of all this ONLY applies to atypical antibodies. the reason lies in the method. Firstly, the tube technique tends to be (in most people's hands) very imprecise, with most people using drops (1 or 2 or 4.....) of plasma and drops (1 or 2...) of cells (3 or 4 or 5%.....) with or without LISS; if with LISS then either with LISS addition or by suspension of the cells in LISS; incubating for x minutes where x can be from 5 to 60 mins. Then the tubes are washed 3 or 4 times with a spin cycle that is xxx(?) rpm for yy minutes - you can fill in your own values, removing some/most/all of the liquid between washes.- Then to your possibly quite diluted remaining red cells, from which you may have washed off weakly attached antibodies anyway, you add 1 or 2 drops of AHG and spin with a spin cycle that is xxx(?) rpm for yy minutes then read by eye/with a magnifying glass/with a microscope after re-suspending the cell button from so gently that you can't see anything to so hard that all your weak agglutinates have been shaken away................I will admit the variation in any one site is much less than this, but globally there is just no standardisation. Gel is standardised, can be done on an instrument, and there is no washing. But there are still good reasons for gel users to revert to tube techniques from time to time. Does that answer your question? (And yes, I've done literally millions of tube IATs in my time; and no, I would not go back to using them as a routine method EVER!)

Give it about 10-20 years playing with the things and listening to people like Malcolm - you'll get there too!

-+Okay - so going back to AHG and enzymes in answer to your last question: Enzymes will destroy a large number of antigens, so you would not be able to pick up most of the 'normal' antibodies to antigens in the Duffy and MNS blood group systems. So you can never JUST do an enzyme technique. On the other hand some antibodies will be enhanced using enzyme techniques; that used to be much more important in the 'old days' when the AHG reagents were less sensitive than they are today. Now AHG reagents will (should!) pick up very low levels of clinically significant antibodies; for example they are (should be!) standardised to be able to detect anti-D at 0.05IU/ml, which is extremely low indeed. As Malcolm has already said in a previous post, most enzyme-only antibodies are not clinically significant. So, what this means is that you can do an IAT on its own; or you can do an IAT technique and an enzyme technique; or you can do an IAT and an enzyme-IAT. Regulations change from country to country as to whether an enzyme technique for the antibody screen is a requirement or not. What you cannot do is JUST an enzyme technique or JUST an enzyme-IAT technique or even a combination of these two. When you add AHG to the enzyme tube test, you are effectively carrying out an enzyme-IAT. As long as this is not being done instead of a simple IAT, then that's fine but is definitely not common practice everywhere. Also, bear in mind that for antibody screens, tube is not as sensitive as gel..... Then for your indeterminate group. You have a positive reverse group in a patient with an anti-PP1Pk. Well, as all cells will be positive for this antigen, and the antibody is presumably active at room temperature, then her plasma is reacting (and will always react as long as her antibody is visible) with the reverse grouping cells. On a more urgent note, if she needs transfusion then you are in big trouble. I would strongly advise you to test her siblings, and close relations (and if she's from a village or area where there's a lot of marrying within cousins, then people from her village too) to see if any of them are compatible. Malcolm - do you know if there's any frozen blood anywhere?

Whatever the reason, do not even think of ever transfusing with group AB blood! anna

I would like to come back to your question about neutral cards not containing AHG. (And apologies for the delay, have been inundated with my day to day work recently). So - IgG antibodies can not usually agglutinate red cells directly. They stick on to the red cells and can cause their destruction, but for us to be able to see that the IgG antibodies are present, we need agglutination. So in order to make IgG antibodies agglutinate, we have to add something that will help them. This 'something else' is either AHG or enzymes. The two react in different ways. The AHG makes a bridge between the IgG molecules attached to the red cells; and the enzymes remove the outer negatively-charged layer of the red cell membrane so that the distance between them is reduced and IgG molecules can agglutinate directly. So, for the AHG test, you need a source of AHG. In the old days (and still in many places today), where the test was done in tubes, this was added to the test after incubation and washing as a liquid. In gel, the AHG is already in the cards. So the 'helper' is present in the gel. For enzymes, the 'helper' is already in the cells, which have been pre-treated with enzymes. Therefore you do not need to have a second helper in the gel. If you put AHG in the gel as well as using enzyme treated cells you are using two different helpers at the same time, when for most cases one is enough. You can choose to use both in an enzyme Coombs technique; it can be helpful for identifying Kidd antibodies; but it is not a good idea to use this as a routine technique unless you want to increase considerably the number of identification panels you have to do, most of which will lead to nothing. Hope that helps anna

I am going to have a rant here!! I think using this type of reagent for routine blood banking (as opposed to quantification techniques) is WAY over the top. Metrologicals are all very relevant in chemistry where you are looking for a number but totally meaningless in most areas of immunohaematology. With the exception mentioned by Malcolm, IH is NOT a quantitative technique. So just what are you trying to prove with this expensive and precious material ? You want to compare that your instrument is getting the same results as your manual technique? So use a series of weak antibodies. The actual strength does not really matter, does it? What you want to know is - for the same cells and the same technique, did my instrument detect the same antibodies, and was the reaction strength the same? Rant over!

1. Test in gel with enzyme treated red cells on Coombs, with 15mins incubation at 37°C. If that is negative I would suspect a false pos in your solid phase due to parabens. 2. Check to see whether this patient has ever been transfused or pregnant before. If not, then confirms that she can't have an anti-Jkb

First of all, I would read the package insert very carefully. I do not know this particular reagent, but I do know that sometimes panel cells, depending on what they are suspended in and their concentration, just are not suitable for use as controls with some reagents. Secondly, a Ee cell had been chosen to test the antiserum. Some of the posts above were worried that this cell might have been used to rule out an anti-e. Well, I hope nobody is using Ee cells to rule out the presence of anti-e with ee cells being in such abundance. Thirdly, you have probably got a second lot of panel cells. You should test the anti-e with the Ee cell on the other panel. If that is reacting strongly, then it is possible that the original Ee cell was in fact a previously undetected RH:CE variant and the manufacturer should be notified. That should not stop you using the cell, however, as you would look at the panel results as a whole, and not just this cell in isolation, and the other antigens (outside the Rh system) would not be affected. On the other hand, if none of your Ee cells are reacting and you are following the instructions in the package insert to the letter, then you probably do have a problem with you anti-e. You should stop using it and contact the manufacturer. They may well need examples of the cells that you are finding negative to see if they can reproduce your reactions on their retained samples.