exlimey

Question to those wiser than I. Is the primary reason for these required validation comparisons to ensure we in the lab know how to follow the package inserts and so use the reagents correctly? I ask because I can't imagine any lab will be able to test the reagents to the level the manufacturer can with respect to numbers and variants (see package insert).

We have educated our multiple myeloma specialists to send a type and screen before administering the first dose of a daratumumab (Darzalex).  Our standard operating procedure is to have a panel of three cord blood cells (we have a large OB service) that is a laboratory developed test of sorts.  Cord cells do not express CD38 at interfering levels.
As it turns out we have made more of an issue of this than it warrants.  Patients who have negative antibody screens essentially never develop new antibodies to red cells after being started on daratumumab probably because it potential inhibits B cells function.  Minimal B cell function apparently yields little ability to make antibodies to red cell antigens, which are relatively weak alloantigens, especially when there is no adjuvant or inflammation in the recipient.  That said, a manufacturer is making a soluble CD38  analog that will inhibit the anti-CD38 activity and make testing easier from what I've read.  DTT treatment is also reasonable.  But the good news is that patients on this drug do not make new antibodies. There are literature references to this, and we have probably tested about 500 patients with no new alloantibodies. Mostly non-transfused patients, obviously.

I was thinking of antigens such as M, N, S, s, Fya, Fyb, Jka and Jkb.  However, they would only tell you if the patient is likely to be a chimera.

As far as what to give the patient, you are right in saying that these results would not help too much, if at all.

From what you have described, group A, D Positive cross-match compatible units should be fine.

A3 cells are characterized by mixed field reactions with anti-A and anti-A,B. I don't know what they do with anti-A1 lectin and it may vary depending upon which manufacturer's product you use. As Yanxia pointed out....crude Dolichos extract is NOT specific. Manufacturers formulate (dilute) it to differentiate between A1 and A2 expressions of antigen.

Dolichos biflorus (Horse gram) is amongst the best known lectin in the serologist’s tool kit, but beware!.

The lectin also agglutinates A, B, AB and O red cells that are Cad+ or Tn+.
In addition, as Yanxia so correctly says, it will react with some red cells that are A2 (or, indeed, other subgroups of A) unless the reagent is suitably diluted.

By he description of the agglutinates, I would also favour a possible chimera, as my own experience of A3 is that the agglutinates are usually quite small.  However, as you yourself say, it could be the result of a stem cell transplant of some kind.  I did my project for Fellowship of the Institute of Biomedical Science on blood groups of bone marrow transplant recipients when I was at Westminster Hospital (way back in the last century - well in the 1970's anyway) and found that group A recipients of group O bone marrow transplants, if they were Secretors, sometimes retained a sort of chimera that reacted with both anti-A and anti-A,B as a result of adsorbing soluble A substance onto the group O red cells (with no other apparent mixed-field reactions with other specificities), but did not appear to produce an anti-A post-transplant, or, if they did, it seemed to be adsorbed onto the (apparent) group O red cells coated in soluble group A substance, and had a weakly positive DAT.  Having said all that though, the female patients were usually sterile, and required either donated ova, or had their own eggs frozen prior to the transplant treatment.

That's a VERY specialized rack. I suspect the originals must have come from Ortho many moons ago. If Ortho doesn't have any more, I doubt any of the typical suppliers would carry them. Perhaps one of the new suppliers of gel cards might have something (e.g., Grifols) ?
One can always get something custom-made, but $$$$$.
Good luck.

Do you think somebody should tell them that ABO HDFN sometimes gives a Negative DAT result in the fist two or three days of life, and that they might actually be better off looking for clinical signs, rather than performing diagnoses on the result of pathology results???????

I think that it depends upon the state of the baby.  For example, if the mother is D Negative, and has been in receipt of anti-D immunoglobulin, and the baby has a positive DAT, but is showing no other signs of HDFN, then we wouldn't perform any further testing.

If the baby has a lowish Hb, and the mother is group O and the baby group A or B, we may take a look at the mother's IgG ABO status, and then perform an eluate on the baby.

Where we might really "go to town" is if the baby is showing overt signs of HDFN, we might well go the "whole hog" and perform an elution, just in case there is a maternal alloantibody directed against a low prevalence antigen also expressed on the red cells of the father.  If this is suspected, it would be easy enough to adsorb out any IgG anti-A or anti-B on the baby's red cells, without adsorbing out the potential antigen against a paternal low prevalence antigen.  The specificity of such an antibody would be interesting, but not necessarily vital, as, should the baby require a transfusion, suitable blood should be easy to obtain.

This a classic case of Sensitivity vs Specificity. Clinical assays (including those in the transfusion medicine arena) are often designed to be as sensitive as possible, especially those intended to be frontline screens. Nobody wants to miss anything. However, the downside of this approach is that sometimes specificity suffers - more positives are obtained than desired.
Solid Phase, Gel and PEG assays are super-sensitive, but may detect "nuisance" antibodies like weaker autoantibodies (warm and cold), or things like anti-P1, anti-Leb, or "HTLA-like" antibodies.
Less sensitive assays that employ no enhancement (saline) or thing like LISS are sometimes used strategically to avoid the nuisances. Many institutions have policies that allow the deliberate down-regulation of sensitivity to ameliorate such problems. For example, performing crossmatches in LISS rather than Gel. Certainly, there's a risk that something might be missed using a less sensitive assay, but I think it's important to realize that in years past, the use of those less sensitive assays didn't leave a trail of bodies.
In patients with long-term autoantibodies, it's not unusual for the autologous cells (DAT/autocontrol) to react weaker than Screening Cells or panel cells. The appears to be some kind of weakening of native antigens to which the autoantibody is directed. Sometimes, that may even mean the DAT is negative, but still yields a reactive eluate .

This a classic case of Sensitivity vs Specificity. Clinical assays (including those in the transfusion medicine arena) are often designed to be as sensitive as possible, especially those intended to be frontline screens. Nobody wants to miss anything. However, the downside of this approach is that sometimes specificity suffers - more positives are obtained than desired.
Solid Phase, Gel and PEG assays are super-sensitive, but may detect "nuisance" antibodies like weaker autoantibodies (warm and cold), or things like anti-P1, anti-Leb, or "HTLA-like" antibodies.
Less sensitive assays that employ no enhancement (saline) or thing like LISS are sometimes used strategically to avoid the nuisances. Many institutions have policies that allow the deliberate down-regulation of sensitivity to ameliorate such problems. For example, performing crossmatches in LISS rather than Gel. Certainly, there's a risk that something might be missed using a less sensitive assay, but I think it's important to realize that in years past, the use of those less sensitive assays didn't leave a trail of bodies.
In patients with long-term autoantibodies, it's not unusual for the autologous cells (DAT/autocontrol) to react weaker than Screening Cells or panel cells. The appears to be some kind of weakening of native antigens to which the autoantibody is directed. Sometimes, that may even mean the DAT is negative, but still yields a reactive eluate .

Hi Exlimey, this makes sense. Thank you for taking the time to explain it thoroughly. I appreciate it! 

This a classic case of Sensitivity vs Specificity. Clinical assays (including those in the transfusion medicine arena) are often designed to be as sensitive as possible, especially those intended to be frontline screens. Nobody wants to miss anything. However, the downside of this approach is that sometimes specificity suffers - more positives are obtained than desired.
Solid Phase, Gel and PEG assays are super-sensitive, but may detect "nuisance" antibodies like weaker autoantibodies (warm and cold), or things like anti-P1, anti-Leb, or "HTLA-like" antibodies.
Less sensitive assays that employ no enhancement (saline) or thing like LISS are sometimes used strategically to avoid the nuisances. Many institutions have policies that allow the deliberate down-regulation of sensitivity to ameliorate such problems. For example, performing crossmatches in LISS rather than Gel. Certainly, there's a risk that something might be missed using a less sensitive assay, but I think it's important to realize that in years past, the use of those less sensitive assays didn't leave a trail of bodies.
In patients with long-term autoantibodies, it's not unusual for the autologous cells (DAT/autocontrol) to react weaker than Screening Cells or panel cells. The appears to be some kind of weakening of native antigens to which the autoantibody is directed. Sometimes, that may even mean the DAT is negative, but still yields a reactive eluate .

This a classic case of Sensitivity vs Specificity. Clinical assays (including those in the transfusion medicine arena) are often designed to be as sensitive as possible, especially those intended to be frontline screens. Nobody wants to miss anything. However, the downside of this approach is that sometimes specificity suffers - more positives are obtained than desired.
Solid Phase, Gel and PEG assays are super-sensitive, but may detect "nuisance" antibodies like weaker autoantibodies (warm and cold), or things like anti-P1, anti-Leb, or "HTLA-like" antibodies.
Less sensitive assays that employ no enhancement (saline) or thing like LISS are sometimes used strategically to avoid the nuisances. Many institutions have policies that allow the deliberate down-regulation of sensitivity to ameliorate such problems. For example, performing crossmatches in LISS rather than Gel. Certainly, there's a risk that something might be missed using a less sensitive assay, but I think it's important to realize that in years past, the use of those less sensitive assays didn't leave a trail of bodies.
In patients with long-term autoantibodies, it's not unusual for the autologous cells (DAT/autocontrol) to react weaker than Screening Cells or panel cells. The appears to be some kind of weakening of native antigens to which the autoantibody is directed. Sometimes, that may even mean the DAT is negative, but still yields a reactive eluate .

This a classic case of Sensitivity vs Specificity. Clinical assays (including those in the transfusion medicine arena) are often designed to be as sensitive as possible, especially those intended to be frontline screens. Nobody wants to miss anything. However, the downside of this approach is that sometimes specificity suffers - more positives are obtained than desired.
Solid Phase, Gel and PEG assays are super-sensitive, but may detect "nuisance" antibodies like weaker autoantibodies (warm and cold), or things like anti-P1, anti-Leb, or "HTLA-like" antibodies.
Less sensitive assays that employ no enhancement (saline) or thing like LISS are sometimes used strategically to avoid the nuisances. Many institutions have policies that allow the deliberate down-regulation of sensitivity to ameliorate such problems. For example, performing crossmatches in LISS rather than Gel. Certainly, there's a risk that something might be missed using a less sensitive assay, but I think it's important to realize that in years past, the use of those less sensitive assays didn't leave a trail of bodies.
In patients with long-term autoantibodies, it's not unusual for the autologous cells (DAT/autocontrol) to react weaker than Screening Cells or panel cells. The appears to be some kind of weakening of native antigens to which the autoantibody is directed. Sometimes, that may even mean the DAT is negative, but still yields a reactive eluate .

I guess the solid phase is more sensitive to detect the auto in this patient. I will use the same method as detecting the antobodies to do crossmatch. Of course I will do saline and AHG crossmatch as well. Just my humble opinion.

Hemoglobin and hematocrit re-equilibrate over minutes to an hour.  Usually minutes.  Not five hours. That's not compatible with what we know.  Transfusing a patient in this setting is more likely to cause inflammation, thrombosis, congestive heart failure, etc., than help, although it is understandable that the surgeon is trying to "do something" for a patient who is not responding to treatment.  The hemoglobin may or may not be precise, but it tells you that the circulating red cell mass isn't likely the problem.  If the patient is in shock and not bleeding, the problem is almost certainly not fixable by transfusion of red cells is my thought.  But desperation leads people to try stuff that is unlikely to help and may, in some cases, harm.  Likely cause(s) of the shock in such patients is cardiac dysfunction, sepsis or something else not easily fixable.  Not due to anemia/lack of red cell mass obviously.

I guess if there was carry-over it is the same as  using the same tube during the whole washing process.

Genius !

Thank you for you responses. I was always taught to use a clean pipette, but the IFU doesn’t state specifically. I wasn’t trying to get the older tech in trouble just saw something that was different. Thank you again.

Using a new pipette each time is how I keep track of how many times I've washed!..........4 washes with Working Wash? - Lay out 4 pipettes!........AND close my drawer so I won't - out of habit - reach in and get one! LOL!
(I'm and "older tech" too!  )
 
but as to your question........I don't know the true answer......... we always use a new one.  But, if it does not state specifically in the IFU and their eluates / LW's give valid results.........I'd say it doesn't matter..........???

I believe the old saying is, "If the computer ain't happy, ain't nobody happy!!"

Giving O units (or A2) keeps our computer happier on patients with documented anti-A1.  Of course, that is the prime objective these days, right?  We bow to our computers!  

I went back to your original post and it clearly said "Trigger" for some posters.  My lab only uses the microscope for FMH screening tests and rarely (like once in a blue moon) looking for mixed field during post-transfusion reaction investigations.  I would consider it to be looking for Zebras, as Malcom said, to want to see rouleaux or a cold-reactive antibody by letting tubes "sit before looking under the microscope" (slightly adulterated version, my apologies).  I didn't see any mention of "only for ABO discrepancies" or even "reverse type".
I've seen cold reactive antibodies look like rouleaux and rouleaux that gave the same appearance as cold reactive antibodies. Some are helped to be clearer with saline replacement, some not.  That's probably where I stopped looking at these things under a microscope.  We predominantly test using automated gel method with tube usually being the "come to the rescue" method to not see just those things you were talking about.  Do I saline replace or pre-warm the test components for those things?  If I need to, but I try not to wake the sleeping beasts so I don't see them in the first place.

I think we're confusing ourselves. I was referring to the original post which suggested a modification of procedure in order to enhance rouleaux/colds.
Serological assays are typically read "without undue delay", i.e., immediately. If some techs have a practice to "let tubes sit before looking at them", that could be construed as deviating from the procedure, unless they have some bizarre and very specific local instruction to do so.

I think we're confusing ourselves. I was referring to the original post which suggested a modification of procedure in order to enhance rouleaux/colds.
Serological assays are typically read "without undue delay", i.e., immediately. If some techs have a practice to "let tubes sit before looking at them", that could be construed as deviating from the procedure, unless they have some bizarre and very specific local instruction to do so.