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On p. 563 of AABB Tech Manual 18th edition, it only mentions that titer methods other than "saline AHG 60 minute incubation" in tube may result in higher titers and "should be validated with clinical findings" (see Malcolm's post, above).  So it does not seem to say one cannot use gel or other methods, just that you need to document validation.
I have always been a bit uncomfortable with identifying an antibody with gel (for a prenatal), then doing the titers in tube.  But then again, I guess it is the comparison of the series of tube titers that they are looking at.
Scott

I have seen one in a hospital setting (she was pregnant).
Her antibody was very weak (and no Jo(a-) blood was available in the UK anyway, apart from a couple of units of Gy(a-) that we ahd frozen down) and, if she had required transfusion, we would have been quite happy to have given "least incompatible).
As far as I know, anti-Joa has never been implicated in a clinically significant transfusion reaction (and certainly not HDFN), although it was reported that one patient, when trialed with radio-labelled red cells, destroyed Jo(a+) red cells more rapidly than Jo(a-) red cells.

There is a procedure in the technical manual I believe.  Basically it is equal volumes of cells, plasma and peg.  Incubate for 15 minutes, centrifuge and remove supernatant (save the cells 'cuz you can reuse them if you need to repeat the absorption), test the supernatant using 4 drops (2d peg/2d plasma).  I use it for all my warm autos - it works excellently.  I do this testing in tubes - I can never absorb out all the autoab if I test in gel.
 
In another post somewhere on this site someone discussed the fact that if the absorption showed no allosensitization they just used the immediate spin xm and did not have to worry about any interference from the autoab (might have been jpcroke).
 
Cost:  I can't go there.  Warm is a bit expensive.  I only use it to destroy Kell system ags and use it well past its outdate (it still works and so the the controls I run).

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Remeber NOT to use EDTA anticoagulated samples though!

Not sure if you are still confused about IgM and one's ability to detect it.....
 
Paraphrasing Malcolm; complement is not required to be present for an IgM antibody to bind to it's cooresponding antigen.  With or without complement, the IgM antibody will bind to the antigen which most likely will result in visable agglutination.
 
If detecting hemolysis is your end goal, then yes you would need a source of complement (serum or antibody free "fresh serum") to accomplish that goal.

Thanks Malcolm, usually I learned much from this forum especially from you Malcolm.
Regarding to first point I aware and for autoanti P I know, only confusion about the second point.
Thanks for your clarification

Thanks Malcolm, usually I learned much from this forum especially from you Malcolm.
Regarding to first point I aware and for autoanti P I know, only confusion about the second point.
Thanks for your clarification

Well, there are two reasons.
 
The first, and main reason, is that we don't want to detect cold antibodies in the first place, because, unless they react at 30oC or higher, they are most unlikely to be clinically significant.  Therefore, detecting them, and then having to investigate them for specificity is a complete waste of time and reagents - and staff time is the single most expensive thing in the laboratory.
 
Secondly, it is highly unusual that you would only detect an IgM antibody by the presence of activated complement (not impossible, but highly unusual) and, therefore, the fact that EDTA chelates the Ca++, Mn++ and Mg++ ions required for the complement cascade to initiate, is irrelevant.  The IgM molecules will still be present in the plasma, and are normally easily detectable.
 
As I said above though, obviously, if we are trying to detect the biphasic anti-P that causes PCH, we would need complement present, and we would then use serum - BUT, very often in these cases, the patient's own complement is already exhausted, and so we would use the indirect two-stage DL test, where fresh complement from another source is added.

We use EDTA, unless, of course, we are doing a Donath-Landsteiner test.

Thank you for your kind comment David.
 
Anti-M is the bane of my life!
 
Until relatively recently, if an anti-M was referred to my laboratory, we would test it first by Bio-Rad (Used to be Dia-Med) gel IAT and enzyme technique (enzyme technique to see if anything else is there - even I know that the M antigen is destroyed by papain!!!!!!!!!) to make sure that there was an anti-M present.  If there was an anti-M present, we would then test it by pre-warmed, warm-washed LISS tube IAT at 37oC.  If we saw no reactions by this technique, we would advise that cross-match compatible blood would be suitable, and safe, for transfusion.  If we did see reactions by this technique, we would advise that M- typed blood should be cross-matched, and those compatible transfused.
 
The reason for the difference between the gel technique and the tube technique is two-fold.
 
Firstly, in the gel technique, the reactants (plasma and red cells) are introduced to each other at room temperature, and then the cassettes are put into a 37oC incubator.  "Cold-reactive" antibodies can sensitise red cells in "peco-seconds" (I may have exaggerated a bit there!!!!!!!!!!!), but the antibody-antigen complex takes a bit of time to dissociate (longer than the incubation time), and so a "false IAT positive reaction" is seen.  On the other hand, if the reactants are introduced to each other, already at 37oC, "cold-reacting" anti-M will (eventually) sensitise red cells, but not in the time allowed for incubation, and so the reaction would be negative.
 
The second reason is that the column containing the AHG is slightly acidic, and if there is one antibody/antigen reaction that likes acidic reactions, it is that between anti-M and the M antigen.
 
I was quite happy with this situation, BUT, and my own line manager is well aware of this, so I don't mind saying it publically, NHSBT has decided, on the grounds that most of our hospitals use gel techniques, we now recommend giving M- typed blood to anyone who has an anti-M, which I think is a total waste of units of blood that have been typed for all sorts of other antigens and have been found to have (presumed) homozygous expression for other, more useful, antigens.
 
I am well aware of the fact that anti-M can cause haemolytic transfusion reactions (IF serologically reactive at 37oC) and can cause clinically significant haemolytic disease of the foetus and newborn (IF serologically reactive at 37oC), and, in the case of HDFN, particularly amongst ethnicities in the Far East, such as the Japanese, irrespective of titre, but these cases are very, very rare.

Hi NAN47,
Any antibody that reacts at STRICTLY 37oC should be titrated during pregnancy (except, of course, for anti-D and anti-c, which, in the UK, are measured by quantification against NIBSC standards).
The titration should take place at first identification and then again at 28 weeks gestation. If the titre is less than 32, then the antibody is unlikely to cause clinically significant alloimmune haemolytic anaemia and disease of the foetus and newborn. Only rarely is an antibody titre high enough to warrant further titration after 28 weeks gestation.
The obvious exception to this is anti-K (and other Kell-related antibodies). These should be titrated when first identified, and then every four weeks to 28 weeks gestation, and then 2 weekly thereafter, until delivery. Indeed, women with anti-K who are pregnant, and who have a partner who is K+k+ or K+k- should be referred to a Specialist Foetal Medicine Unit, so that the pregnancy can be monitored by experts in the field.
It is always recommended that the previous sample is titrated in parallel with the present sample (or quantified, in the case of anti-D or anti-c), as this will show up genuine rises in the titre, as opposed to rises due to operator differences and errors and changes due to the different expression strengths of an antigen from one red cell sample to another. Where possible, the red cells used should have heterozygous expression of the antigen, although, in certain cases, this can be difficult to do (e.g., if the antibody is anti-U, it is not possible to tell whether the red cells used for the titration are U+/U+ or U+/U-).
In rare cases, titration of the previous sample, in parallel with the present sample, may show up a second specificity. For example, if the pregnant woman is known to have an anti-E, with a titre of, for example, 4 against r"r red cells, and then all of a sudden the titre goes up to 128, but ALSO shows as 128 with the previous sample run in parallel, it may be that the r"r red cells used on this occasion also express an antigen against a low prevalence antigen, against which the woman has also produced an antibody (for example, an anti-Wra).
The end point of a titration is usually given as the reciprocal of the last dilution in which agglutination can be seen with the naked eye.
We are actively re-writing the BCSH Guidelines (technical guidelines) as I post, and the complementary RCOG Guidelines (clinical guidelines) are also in their final draft.