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Posts posted by exlimey
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1 hour ago, Baby Banker said:
As for the panel/screen, I take the antigen profile and circle all the required antigens. Then I select a cell that is homozygous for each one. I sometimes have to use cells from other panels or screens. I know the rule is that you can substitute two heterozygous cells for one homozygous cell, but I never do that if I can help it.
A sound approach.
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19 hours ago, Baby Banker said:
If you know she has anti-E, you can probably put together a custom screen of E negative cells. That screen would only be positive if she developed another antibody. Be careful that you cover all the antigens that the FDA requires. That list used to be in the Technical Manual. I think it is D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Fya, Fyb, Jka, Jkb.
15 hours ago, pbaker said:We do as Baby Banker does, create a selected cell panel to rule out everything else. The game we play is how few cells can we run and have a valid rule out panel
We have had several patients that we do every 3 days until delivery. One of our patients had Anti-c and Anti-E.
I like the approach taken/suggested by Baby Banker and pbaker, but it does need a moderately high skill set to make up the selected panel. Perhaps that's not possible in the "average" blood bank?
A follow-up question: Are you performing titrations (potency) of the antibodies that are identified? If so, how often ?
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1 hour ago, TreeMoss said:
I suspect because anti-A, anti-B, and anti-A,B are usually cold-reacting. That would be similar to washing with cold saline if you were working with a cold autoantibody in preparation to performing a cold auto-absorption, I think.
I think I understand what you're saying - we don't want to miss isogglutinins, but why is this practice only applied to cord samples ?
If you want to perform a cold autoadsorption, preparing the cells (washing) with WARM saline would be better - this would theoretically eluate off some of the already bound immunoglobulins and free-up antigens for binding of additional (auto)antibodies. You would have to "cool-off" the warm-washed cells before actual use in the adsorption.
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16 hours ago, Malcolm Needs said:
Obviously I don'y know for certain, as I don't work at the same venue as TreeMoss, but I suspect that it is to try to ensure that ABO antibodies are not washed off the red cells. Maternal ABO antibodies that can cause HDFN have to be IgG, but, of course, ABO IgG react quite happily in the cold, so I don't really think that washing in cold saline has any actual benefit, as they also react quite happily in the warm.
15 hours ago, gene20354 said:I agree with Malcolm's statement about trying to preserve ABO antibodies on the cells. This made me think for a second. At my facility when we prepare eluates we use cold saline for the first wash and then cold working wash solution for the remaining washes. I looked at the Immucor Elu-Kit package insert. It has the following statement in the limitations section.
The degree of dissociation of antibody that occurs during the washing procedures. This may be minimized by washing in 1° to 10°C Working Wash Solution. In most cases, satisfactory eluates can be made after washing the cells with Wash Solution at room temperature.
The question was somewhat rhetorical: I know why it's done and agree that the approach is quasi-logical (as Malcolm points out). There appears to be more emphasis on detecting isoagglutinins on cord cells than in older patients, perhaps with good reason.
I was attempting to make folks think about the logic of some of the tests/processes that have become "normal". One could argue that we should want to detect isoagglutinins on any patient's cells, regardless of age, and therefore we should use "cold" wash solutions universally - what's good for the goose is good for the gander.
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On 7/10/2017 at 9:29 AM, rravkin@aol.com said:
jwnola I believe, though practice of procedures from different blood banks, that washing the cells eight times is considered excessive such that the antigen, if present, might be partially wash away or altered rendering weak reactivity. The maximum number of washes when testing Cord blood cells is four times, as I have practiced. Do you know of any studies or articles stating that eight wash cycles is practical and does not alter antigens?
In my opinion: Antigens are unlikely to "wash away" or be "altered" by washing with normal saline. [One exception: Lewis antigens may be liberated during washing.] Antibodies, on the other hand, are more likely to be eluted from red cells by excessive washing with acidic saline. I doubt any publications exist that prove excessive washing has the effects you describe, but I would love to be proved wrong.
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2 hours ago, TreeMoss said:
The AABB Technical Manual says to wash cells once and make a 3-5% suspension. Then to wash one drop of cells (for each the DAT and DAT Control) a total of 3 more times prior to adding the AHG and spinning. Our AHG requires two drops. I have also read that the cord cells should be washed in cold saline to prevent eluting of the antibody while washing the cells. We did this years ago but somehow got away from that practice.
What is the reasoning behind using "cold saline" for cords and not other samples?
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16 hours ago, kris6026 said:
For those of you treating cells with DTT for patients taking daratumumab/darzalex: Have you come across a positive autocontrol, with treated and untreated cells?
Currently, patient on DARA: initial screen-panagglutinin, auto-positive.
Testing with treated cells: screen-negative, auto-positive.
QC-valid (testing Kpos and Epos cell) antigen typing for K-neg, E-pos
I have been unable to find any research with this information. We are currently in the process of validating DTT treatment.
Any advice would be welcomed.
Thank you
Based on your initial screen, it appears that the patient's cells are probably already coated with antibody (DAT-positive). Unless you are DTT-treating the patient's cells - not typically part of the testing protocol - they will remain DAT-positive and therefore reactive in the second series of tests using the DTT-treated screening cells.
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11 hours ago, yan xia said:
i guess the difference is because the second time's cells is more than the first time( two drops vs. one)
Please explain your logic.
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Questions:
- Is the saline used for tube washing the same as that used in the automated cellwasher ?
- What is the pH of your saline(s) ?
- Why was testing repeated ? What was "wrong" with the original results ?
If the pH of the saline used is low (acidic), it can cause elution of bound antibodies. A total of eight washes in mildly acidic saline in the first case may have resulted in a negative DAT. A weakly positive result in the second case may be because the cells were not exposed to the same degree of acidity (fewer washes, less time exposure).
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5 hours ago, kate murphy said:
Oddly enough, Wisconsin seems to have lots of Yta neg donors. We had a patient a few years ago, and Community Blood Center of Appleton Wisc was able to supply us many liquid units. AABB members can use the National Blood Exchange to facilitate this. So if you do decide to have blood on hand for 4-6 weeks, give them a call.
5 hours ago, Malcolm Needs said:Yes, we also have over 30 Yt(a-) donors that we can call upon when we may need them.
If you have a decent anti-Yta available, and a goodly number of donors, when you screen for them, Yt(a-) donors are surprisingly common at just under 1%. For example, if you have 1000 donors, you will, potentially, detect about nine Yt(a-) individuals. Doing this by genotyping is swifter and, probably, more cost effective.
Don't forget....in this case......Rh-negative units are required. Multiply by 0.15 !!!
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19 hours ago, Mabel Adams said:
Since this antibody doesn't cause HDFN due to the antigen not being well-developed in the fetus, why should a miscarriage have sensitized her? Are they ever naturally occurring? If so, does that correlate to significance?
As I understand it, IgG2 and IgG4 do not cross the placenta easily, whereas IgG1 and IgG3 are "actively" transported from Mother to baby. Obviously the baby benefits from this passive immunity. Since most examples of anti-Yta are predominantly IgG4, that's why they don't cause HFDN, especially since, as you mentioned, the Yta antigens are poorly expressed in utero.
Never say never, but I doubt that anti-Yta occurs naturally. Cartwright system antigens are poor immunogens, so from my point of view, the antibody's existence implies repeated stimulation. That being said, your patient may be a "super-responder". I don't if anyone really understands why immune responses vary or how some result in different IgG subclasses.
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1 hour ago, Mabel Adams said:
If she requires transfusion, should we make an effort to match her for K, C, E, Jkb etc.? Can we assume that she is a "responder"? If she makes another antibody it will be very hard to figure out what she has quickly (and locally) in the presence of that anti-Yta. She's young. Matching K, C & E would be easy, the Jkb a bit less so, especially among Rh neg units. Is the anti-Yta likely to remain detectable for decades?
I think matching Rh and K is a good idea. My foggier-by-the-day memory leads me to believe that most of the examples of anti-Yta that I've seen were single specificity. I do remember a couple with anti-D and at least one with anti-c.
As to longevity......anti-Yta tends to fade away over time in the absence of additional stimulation. In your patient, additional pregnancies may be a source of re-stimulation, so her antibody might be more persistent.
As usual, these are generalized statements and opinions. There are always antibodies that don't read the literature.
- Malcolm Needs and jojo808
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7 hours ago, Mabel Adams said:
We have recently identified (with reference lab confirmation) an anti-Yta in an A neg pregnant woman. She has had one prior pregnancy--miscarriage-- and no transfusions. We are 3.5 hours from our blood supplier (over a mountain pass) in good weather and she is due about Christmas. We are convinced that the risk of HDFN is nil so I am devising a plan for managing the patient if she should bleed during delivery. We can have the MMA done for significance but it is expensive and maybe not worth it when the patient isn't all that likely to bleed excessively. I am about ready to decide that we should just wing it because I can't get in compatible units "just in case" because they probably would have to be deglycerolized and thus would have a 24 hr expiration. If she massively hemorrhages, I won't have any choice but to give her Yta untested units. If she doesn't have a life-threatening bleed there might be time to get in Yta negative blood in a day or 2 to fill her back up. Autologous donation would give us only maybe 1 liquid unit and they probably would want to transfuse it even if she didn't need it. Not sure if there is a family member but they can only donate every 56 days and I would want a liquid unit for the month around her due date. Maybe a sibling could donate a double red cell but that's about the best it will get (assuming there is a Yta neg sibling). We would try to send out an antibody workup in the last 2 weeks before her due date just to make sure there aren't any new antibodies that we will want to honor. Of course she will have RhIG on board by then I'm sure. I will happily take all suggestions and input.
I echo Malcolm's sentiments - you appear to in control of the situation. One wrinkle, perhaps: Are you able to "sell it" to the physicians ?
It has been many moons, but I have performed dozens of MMAs on examples of anti-Yta. Almost all were considered insignificant and even those that were over the threshold were barely above. Add the fact that they are almost universally IgG4 - no harm to the baby and minimal risk of a transfusion reaction. The examples that did just make it into the "significant" interpretation usually had a touch of IgG1 and/or IgG3. I have no hard data to support it, but I'm quite sure that many of the patients (even those with "significant" results) received Yt(a+) blood without consequence.
If the case arises and in my opinion, giving Rh-negative blood is way more important than fussing about anti-Yta.
Given your geography, it might wise, albeit extremely cautious, to suggest the patient move to the "big city" (Portland) for Christmas, but that would only hold true if the physicians want easier access to Yt(a-) blood.
- Malcolm Needs and Yanxia
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14 hours ago, Quality_Lady said:
Exlimey - What I was referring to is the confirmation / validation that the use of the materials as described in the post works in the manner they are being used, provides equivalent results, and there is parallel testing with accepted methods (e.g., buffering with NaOH) showing that the use of either solution gives comparable results in the hands of the lab..
Catchnenow51 - Did not intend to hit your hot button, just wanted to give you food for thought before an inspector asked the same questions. I came from medical devices so I have heightened expectations of regulatory impact. Plus there is no data to support the expiration date and stability of the PBS prepared in this fashion.
Bottom line: If a facility "manufactures" a reagent, it is obliged to prove that it functions as designed and can be made repeatedly/reliably. However, the rules and regulations that apply to licensed and registered reagents do not necessarily apply to "home-brew" materials.
Providing the material will only be used in-house and not distributed, there is no need for "parallel testing" of any other formulation, no matter how crude the manufacturing process appears to be. Such testing is only required for licensed or registered reagents and devices (under the umbrella of Design Control).
I'm assuming (correct me if I'm wrong) that the PBS used in preparation of the DTT is made immediately before use, and therefore the stability of it is irrelevant. Stability of the finished reagent may be important if the "manufacturers" wish to assign an expiration date. This may not be necessary, since in the case of DTT, controls are included EVERY time it is used - the operator has a current and immediate indication of whether the reagent worked or not.
Validation is essential to provide confidence when it is impossible to directly prove that a process has worked. Think of vaccine manufacture: One cannot test every vial/dose to see if it meets specifications - all of the product would be compromised. When integral testing proves that a process works each time, validation requirements are minimized.
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19 hours ago, Quality_Lady said:
Use of either pHix or Buffering solution as described is outside of IFU and would require extensive in-house validation to support their use. Not only would you have to provide "in-use" testing, you would also have to determine stability data.
What do you mean by "extensive in-house validation" ? What is "in-use testing" ?
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On 6/20/2017 at 1:54 PM, amym1586 said:
We went and bought the BIO-RAD DTT
I started looking for a package insert and now I see it says For research only.
Can we not use this for patient testing?
In my opinion: Yes, you can. That statement on chemicals is meant to tell you that you can't use it in a medical or nutritional fashion.
You will not find a package insert for any raw chemical - the supplier has absolutely no idea what the buyers are going to do with the materials. If you buy plain old sodium chloride (NaCl), it doesn't have a package insert, exactly for the reasons stated above.
Anything anyone is doing with DTT and/or other exotic chemicals in the Blood Bank realm is completely out of the typical regulated environment. These chemicals assist in a complex investigation, they are not making a diagnosis. Sometimes it is necessary to go beyond the use of licensed, registered, validated reagents to best serve a patient.
That being said, the DARA issue has brought DTT use into some routine labs. Complex serological investigations should be left to experts - the high level Reference Laboratories, who understand the pros, cons and limitations of the specialized reagents they use.
End of rant.☺
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9 hours ago, MOBB said:
I heard last week that there is another method some of the US ref labs are either using or want to start using for the Dara patients and Kell isn't affected, but I can't for the life of me remember what they said and I haven't had any luck with google.
Has anyone heard anything similar?
You may be referring to trypsin-treatment of the red cells (screening cells). Apparently CD38 is destroyed/inactivated (along with Lutheran system determinants) but Kell system antigens remain intact. Other blood group antigens are also affected by trypsin, so I think the modified approach involves testing the patients' samples against both DTT-treated and trypsin-treated cells.
To further complicate matters.....manufacturing a reliable, consistent trypsin reagent is VERY difficult. The enzyme activity of source material varies immensely and, as with other enzymes, stability is a problem.
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51 minutes ago, natalynn said:
Dissolve 1 gram DTT into 32 ml PBS. Aliquot into 2 ml samples and store at -20ºC or below.
Expiration is 1 year from preparation.
http://www.sigmaaldrich.com/catalog/product/sial/d0632?lang=en®ion=US
^ you can buy it pre packaged in 1g amounts. (the price has gone up! ha supply and demand I guess)
You performed stability testing on your home-made material ?
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Feisty rainbows? Some grayling? I'm very jealous - haven't made it to Alaska yet.......
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16 hours ago, Malcolm Needs said:
I don't know of ANY "serological" laboratory that has the ability (or the necessity) to test the whole gamut of Weak D types (and I don't think anyone could - as many of them are unique to the proband), but, I would have thought, as long as you know you can detect a Weak D Type 2 (which is the "weakest" of the normal "Weak D" types, you should be covered) - you should be okay.
I concur. A good writer should be able to finagle that logic into a validation plan. Getting hold of said cells may still be problematic, but at least it might be simpler than an expedition to catch the Loch Ness Monster.
- MaryPDX and Malcolm Needs
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1 hour ago, cswickard said:
Yeah - I knew that, but you are going to need a whole year (or even two) to get enough real specimens for validation without some help from a manufacturer or a reference lab - maybe?
First, a disclaimer: I am not a Regulatory expert.
Perhaps you're over-stretching? I don't think you're required to validate the new system in the sense that the manufacturer has to do for licensing - that's a LOT of work and an attempt to cover every variable imaginable.
In your case, perhaps just proving the system works in your facility is enough. That would probably only need to include a couple of examples of weak-D, rather than the whole gamut.
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43 minutes ago, Malcolm Needs said:
I'm sure you are right about exaggeration. This is something that genuinely worries me about Health and Safety (amongst other things). If they keep making all sorts of claims where things are DANGEROUS, the old thing about "crying wolf" will occur when something that is REALLY DANGEROUS gets ignored, because people get apathetic about H&S because of exaggeration.
Well said. The world is so "dangerous" these days, it's a wonder we're still around.
- Malcolm Needs, R1R2 and gagpinks
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1 hour ago, MAGNUM said:
I have had mine for about 6 or 7 years, and have always used deionized water.
Contrary to the manufacturer's instructions ? That's really living on the edge. ☺
- David Saikin and John C. Staley
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3 hours ago, Malcolm Needs said:
As I understand it, when liquid nitrogen boils, the gaseous nitrogen vapours, which are, of course, invisible, do not mix immediately with the rest of the air. This nitrogen gas is "heavy", and sinks to the floor, and anyone not aware of the situation can become asphyxiated, as the oxygen level at "breathing height" (for want of a better way of putting it) plunge. There was a near fatal case of this at the NHSBT National Frozen Blood Bank a few years ago.
I must confess.....I was poking the bear. I have worked with LN2 for 25+ years and personally believe the risk of asphyxiation is highly over exaggerated. Any room with adequate ventilation (and that's a very loose term) is safe. Most interactions with an LN2 environment are short term, except perhaps for the worker(s) at the NHSBT National Frozen Blood Bank where special ventilation systems should have been designed into their operation.
I was expecting answers involving cryogenic burns, too.
- Malcolm Needs and Ensis01
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We have had several patients that we do every 3 days until delivery. One of our patients had Anti-c and Anti-E.
Blood Bank staff
in Transfusion Services
Posted
There is a very good reason why "generalists" avoid Blood Bank and transfusion medicine - it's complicated and you need a lot of specific training to do it well. Even today, with a significant level of automation, a warm body is often needed to interpret results and give recommendations. And then add the fact that there is a seemingly endless list of "exceptions", "equivocal", "indeterminate", and other levels of results that confound even a trained (SBB) person, let alone an "every other weekend, third shift" employee.
Cross-training is a must for very small, low volume facilities. No question. However, once work gets to a certain level of complexity and volume, institutions should seriously consider having dedicated staff.
I don't know how "generalists" manage to maintain their legally-required competency levels.