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Posts posted by Clarest
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I am wondering if your lab uses the buffered gel cards to test with enzyme treated panel cells in parallel with non-treated panel cells to differentiate multiple antibody specificities, i.e., observing increased or reduced/eliminated reactivity of antibodies. I am still confused whether the buffered gel card can detect IgG antibodies as there is no anti-IgG.
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I am drafting a new SOP for functional calibration of serologic centrifuges and have the following questions.
- Prior to determining the optimal time for centrifugation, we need to find the optima dilution of the antisera. We will use the the centrifuge to be calibrated to determine the optimal dilution with its current set up of the rpm and centrifugation time, e.g., the dilution that gives 1+ reaction at 3400 rpm for 15 seconds. Then, with this optimal dilution, we will check tubes after centrifugation at different time intervals (e.g., 15, 20, 30 and 45 seconds). My question is that since we already used the 15 seconds as the centrifugation time to get the 1+ reaction in the first step, will the optimal centrifugation time be 15 seconds, which is kind of predetermined in the first step?
- In many reagent inserts, they already indicate the suggested centrifugation (e.g., approximately 15-30 seconds at 3400 9rpm (900-1000 rcf)). If the time and rpm of the centrifuges we use are already calibrated with a standard timer and tachometer regularly, what's the reason for doing the functional calibration?
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I was trying to look into the difference between the MTS Diluent 2 vs. 2 Plus and found that 2 Plus contains EDTA to prevent complement activity and hemolysis in test procedure. However, if a sample collected in EDTA tube is used, the calcium has already been chelated in the sample tube. Is it still necessary to use the diluent 2 plus that is formulated specially with EDTA?
In addition, let's talk about the use of EDTA sample tube vs. observing hemolysis as a positive reaction. If an EDTA sample is used, theoretically, we should not be able to observe hemolysis as no calcium is available for the complement activity. Please feel free to share your thoughts.
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As per "standards", ABO confirmation testing is only required for red blood cell transfusion. So, I have seen some institutions just require ABO confirmation specimen for red blood cell not plasma or platelet transfusion, or for red blood cell and plasma but not platelet transfusion. Or they give multiple group A instead of AB plasma units for patients with unknown blood group during massive hemorrhage events and they claim there has been almost no transfusion reaction observed for giving group mismatched plasma units. I understand when there is a massive hemorrhage, a lot of transfused mismatched product could be bled out or diluted, or even our body's nature is busy coping with bleed instead of having strong immune response. I am still wondering if there is really so much difference of transfusing equal volume of mismatched RBCs vs. plasma/platelets.
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Thank you! Your responses have confirmed some of my thoughts and assumptions. Thanks again!
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Hi all,
Below is what it says in the Instructions for Use (IFU) of MTS Anti-IgG Card.
"The use of enzyme-treated red blood cells with the MTS™ Anti-IgG Card may detect clinically insignificant antibodies. The MTS™ Buffered Gel Card is recommended when using enzyme treated cells."
I am confused
If a buffered gel card is used, does that mean it is NOT an indirect antiglobulin test as I don't see where the AHG reagent is added. On the other hand, in the IFU of MTS Buffered Gel Card, it has the following statement.
"This MTS™ Buffered Gel Card can be used in ABO Serum Grouping as well as direct agglutination i.e., cold and warm antibody detection."
I would assume the warm antibody mentioned here is not necessary to be an IgG antibody?!
Then, how would we use the enzyme panel to identify the clinically significant IgG antibody with MTS gel method?
Thank you for your input!
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Thank you all for responding to this topic. Fortunately, the patient survived probably due to bleeding at that moment. Both poly- and mono-specific DATs were done and they (i.e. poly DAT, IgG, complement and saline control) were all weakly positive. An eluate was tested against A1, A2 and B cells and reacted with A1 and B cells, not A2 cells. The reaction strength with A1 cells was stronger than B cells. The possible explanation for eluate reacting with B cells is that the anti-A,B in this O patient's plasma coated on the A donor cells got eluted and reacted with group B reagent cells.
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On 7/12/2022 at 8:28 AM, Arno said:
This phenomenon is also described with the monoclonal antibody therapy anti-CD47 (Hu5F9-G4) where red cells are so heavily loaded with IgG that it creates steric hindrance. Basically, antibodies bound to the red cells hinder the binding sites of the anti-human globulin leading from very weak to negative DAT. Anti-CD47 can be eluted off the red cells and it gives very strong reaction in IAT. Of note: it is not the same mechanism involved with the anti-CD38 (another monoclonal antibody therapy often called DARA) where here the DAT can be negative too because of down-regulation of CD38 expression onto the red cell membrane.
Hi Arno,
Thank you for the explanation! Actually I had a sample from a patient on DARA yesterday showed 1-2+ positivity with all screening and panel cells, but DAT was negative.
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On 7/19/2022 at 9:07 AM, Malcolm Needs said:
I am uncomfortable with the use of the term "child-bearing age" because, if the bit in the brackets isn't properly interpreted, a four-year-old D Negative female (for example) might be given D Positive blood because she is NOT of child-bearing age, but is, of course, of child-bearing potential.
Yes, I agree with you Malcolm. We use child-bearing potential in our policy.
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I was thinking this phenomenon could be a part of transfusion reaction caused by the ABO incompatible red blood cell transfusion. However, in order for us to report a valid DAT result, the saline control should be negative. We could try to use the warm saline to wash the cells, then the DAT including the saline control might become negative. I doubt the negative result would represent the true situation of the transfusion reaction. How do you think?
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Hi Malcolm,
Thank you for your response. I kind of understand the theory for every antigen site being occupied by the maternal antibody, so nothing available for binding the antibody in the antiserum. However, for DAT, we add the anti-IgG to bind the antibody. Is the antibody still available to react with anti-IgG? I was thinking if the false negative result is caused by insufficient wash by normal wash cycles?
In addition, I just usually consider the interference of positive DAT to indirect testing but have never thought that could interfere with direct agglutination testing, such as ABO, RhD, K phenotyping.
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If patient's red cells are heavily coated with antibody (e.g., cord blood cells coated by maternal antibody(ies) with high titer), is it possible to cause a false negative DAT result?
Thank you!
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On 8/5/2021 at 3:32 AM, Ensis01 said:
It is used to provide a negative control for the forward grouping, but is therefore only needed for AB pos patients. It is usually/often easier to require the control every time so it is not forgotten when the occasional AB pos does occur.
The control's use in weak D testing is useful when the patient has a positive DAT.
Thank you Ensis01 for your response.
We do not do weak D testing if the patient has a positive DAT. We only do weak D testing on the cord blood samples when possible. However, I would like to know how you would interpret the weak D testing when the patient has a positive DAT. Based on the inserts of the RhD typing reagents we used, there are the following statements.
"An Indirect Antiglobulin Test result with cells that demonstrate a positive Direct Antiglobulin Test cannot be reliably interpreted with respect to weak D."
"A positive Indirect Antiglobulin Test for weak D must be validated by a macroscopically negative direct antiglobulin test or a negative indirect antiglobulin test using an appropriate control."
Red blood cells coated with alloantibodies or autoantibodies of the same or similar specificity as the reagent (i.e., cells that are DAT positive) may give weak reactions. This is due to decreased availability of antigen sites because of antigen blocking or steric hindrance. In extreme cases, false-negative results may occur.
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I have more questions on this topic.
1). Is the monoclonal control only required for AB pos cord but not for full blood grouping test (including forward and reverse grouping)?
2). Is the monoclonal control required for weak D test? For what I understand, the monoclonal control is mainly used to detect spontaneous agglutination that could cause mistyping. If your RhD typing at immediate-spin is negative (or even there is negative reaction with ant-A and/or anti-B reagent), does that prove there is no spontaneous agglutination? If yes, what's the purpose of using monoclonal control in the weak D test?
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On 2/25/2021 at 8:35 AM, galvania said:
true. Should have read that more carefully.
Well the answer is still - yes and no. If you are using monoclonal reagents, and assuming the anti-D has the same formula as the other reagents (barring the actual antibody obviously) then most results will have at least one negative well that can serve as a control in any case. You could just then put up a neg control on the AB+ results. But the majority of cards/cassettes do have a control on them anyway. If there's only anti-A-B-D, that's only supposed to check a group that is known i.e. has already had at least one full group with a control. But there too - only a problem if AB+. And if you are using an Rh pheno card, the control well on the grouping card is valid provided that the formula for all reagents is the same
Even with a AB+, if the forward matches to the reverse grouping, I am wondering if the control is still needed.
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Thank you Malcolm and David for sharing your experience with me.
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21 hours ago, exlimey said:
NicolePCanada - I agree with both Ward_X and Malcolm's comments. There are definitely situations (patient groups, diagnoses) where a less sensitive methodology like LISS-IAT can be useful to work around "junk" that may be detected in Gel, Solid Phase or PEG test systems. But...they should be employed only by operators who understand the consequences of such actions, AND have the support of their medical staff.
I totally agree with exlimey for keeping LISS-IAT as a backup and less sensitive method to the more sensitive ones like solid phase and others.
Since the LISS-IAT is our backup method, we usually use it after the first solid phase panel combined with a tube DAT as no autocontrol is set up with the solid phase panel. Now, we are having a debate on whether we need to follow the statement on the Gamma N-HANCE package insert "An autologous control ... is recommended for compatibility and antibody identification tests." I was thinking that if the DAT has already been done on the same specimen, we do not need to set up an autocontrol when using the LISS IAT. As a common Blood Bank practice, when an autocontrol is positive, a DAT is the next step to differentiate if the positivity is an in vitro or in vivo phenomenon. However, there could be a scenario that the specimen has a positive autocontrol but negative DAT (DAT-negative AIHA?). If as what I thought in above, that means we might miss this type AIHA. In fact, our current practice (no autocontrol set at all) before switching to Gamma N-HANCE from ImmuAdd, we have missed this
Any input on this debate will be much appreciated!
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14 hours ago, Malcolm Needs said:
Clarest, does the N-HANCE package insert mention anywhere just WHY an optical aid is required? For example, does it explain why it is such an inferior reagent to all the others, including normal ionic strength saline and low ionic strength solution, that an optical aid is required?
Hi Malcolm Needs,
I would like to see an explanation for “why” on the package insert. Unfortunately, no! It only mentions an optical aid may be used. I am afraid of over reading by the staff. Particularly, we are planning to use this enhancement medium in our IAT corssmatch. Currently, we use saline IAT crossamtch.
By the way, how would you define a microscopically positive reaction. How many cells clumping together would be a positivity. I find some staff even call occasionally seen “kissing cells” a positive reaction.
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On 3/29/2020 at 8:15 AM, Kathyang said:
Yes. We just had to validate the Gamma N-Hance since we had been using ImmuAdd.
Hi Kathyang,
When you use either ImmuAdd or Gamma N-Hance, do you do the microscopic check for the negative reaction at AHG phase?
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Hello! Our lab currently uses Immucor's ImmuAdd for our LISS enhancement. Now, Immucor has discontinued ImmuAdd and has a new product called N-HANCE which is still a LISS enhancement. My question is, do I need to perform a validation or comparison study on the new N-HANCE product when we receive it?
Hi, our lab has the same situation as yours. I am wondering if you do the microscopic check for the negative reaction at AHG phase when using LISS enhancement.
On the ImmAdd package insert, it doesn't mention the use of an optical aid, but the N-HANCE one does.
Thank you in advance for your response.
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Our PreAdmit patient samples are valid for 30 days after collection if patients have not been pregnant or transfused in the last 3 months. We usually perform the group and screen test on the day of collection and if applicable, antibody workup ASAP. If required, we usually do the crossmatch one the day before the scheduled surgery date. Previously, we had separated plasma from cells for all samples. After we did validation to show that the reactions strength of ABO antibodies in the unseparated samples on their 30th days is comparable to that in the separated plasma, we stopped separating plasma if the antibody screen is negative and only immediate-spin crossmatch is required.
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Below is the source of my 1st question.
" If the patient has been transfused within the past three months, but not within the last three weeks, an elution may be omitted if all of the following apply:
a). The DAT was positive on the last specimen tested (i.e. collected more than three weeks ago) and was investigated, and
b). The positive DAT (i.e. strength of the reaction) on the current specimen is not stronger than the DAT performed on the last specimen, and
c). The antibody screen (i.e. strength of the reaction) on the current specimen is the same as on the last specimen.
Note: Clinical circumstances, evaluation of transfusion, test result history and/or specimen history may override the above criteria and elution may be desirable for selected patients."
Could any one explain why "three weeks" is specified here but a longer or shorter duration?
Thank you.
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Hi Malcolm,
Look at what I found in the link below
http://www.transfusion.ca/Resources/CSTM-Blog/January-2017/I-will-remember-you-Malcolm-Needs
Clarest
- bldbnkr and Malcolm Needs
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8 hours ago, Malcolm Needs said:
1. Without a doubt! The thing is that not all antibody specificities will become detectable at exactly the same time (they are a pain, as they do not read the text books or, if they do, they do not take any notice of the bits they do not like)! For example. if you detect an anti-D in the first sample, but nothing else, it does not mean that there is not some other "nasty" bubbling under the level of ability to detect, The next time, there may be, for example, an anti-D and an anti-Jka. It is always better to detect the anti-Jka in vitro, rather than in vivo!
2. In contrast, no, we would not perform an elution every time on such a case, because, in the situation you describe (which, incidentally, working in a Reference Laboratory, we saw only a very regular basis) the patient's immune system is rarely, if ever, working at what may describe as "full capacity". We match them for Rh and K to reduce the chances of "common" antibody production, but that is about all. We would test an eluate about once a month, UNLESS there was clinical evidence of a transfusion reaction (which, given that they are haemolysing their own red cells, may be difficult to detect), or if the time between the need for transfusion becomes noticeably closer, in which case we will do elutions more frequently, but, it should be noted, as there will almost certainly be a panaggltutinin present, the eluate itself may have to undergo alloadsorption, which could weaken the reaction of any underlying atypical alloantibodies.
Hi Malcolm,
1. I agree with you if we suspect there is a transfusion reaction which causes the positive DAT with IgG, it makes sense to do an elution on the new sample in case some other antibody(ies) are being detected this time but not from the previous sample.
2. Even we test an eluate once every month for the patient with an autoantibody, it is exactly as what you said the eluate shows pan- agglutination. Unfortunately, we do not perform alloadsorption on site. So, the eluate really does not mean that much for detecting underlying alloantibody(ies) developed due to transfusion reaction, especially when the auto is strong and the eluate always shows 3 to 4+ reaction strengths. Do you think it's necessary to send to our Reference Laboratory once every month for an alloadsorption or just send out when there is a sign of hemolysis process going on?
Clarest
Dealing With Cold Agglutinins
in Transfusion Services
Posted
As the prewarming technique was also mentioned here, I am wondering if my confusion with the Notes below in the Method 3-6 Prewarming Procedure of AABB Technical Manual (20th ed.) could also be discussed here.
"2. Cold-reactive antibodies may not be detectable when room temperature saline instead of 37 C saline is used in the wash step.3 The use of room temperature saline may avoid the elution of clinically significant antibody(ies) from reagent red cells that can occur with the use of 37 C saline. Some strong cold-reactive autoantibodies, however, may still react and therefore require the use of 37 C saline to avoid their detection."
Why are the cold-reactive antibodies not be detected when washing with RT saline, but are detected with a warm saline wash? I was thinking the opposite way
And the last sentence in the above notes seems to confirm my thoughts 