can wash change the strength of the reaction

[jwnola]

I performed a DAT with IgG on a cord blood sample.  I washed the cells about 4 times before making 3-5% cell suspension.  I washed that one drop of cells 4 times before adding IgG for DAT.  The DAT result was negative. 

I tried again.  This time, I put two drops of unwashed cells into a tube and washed the two drops of cells 4 times before I added IgG. This time the result was M+. 

What caused the differences?  Do you think the washing itself can change the result? 

 

Thanks.

 

[exlimey]

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). 

[yan xia]

i guess the difference is because the second time's cells is more than the first time( two drops vs. one)

[exlimey]

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.

[yan xia]

 

58 minutes ago, exlimey said:

Please explain your logic.

just my personal opinion, when the antigens and antibodies reaction, there is a formula, in this case, the antigens( the binding antibodies on the cells) is fewer,( we can see this from the reaction strength w+), so add more antigens will give stronger reaction. of course it is in a range.

[galvania]

First of all, what do you mean by M+?  Do you mean it was microscopically (only) positive?  If so, then really really weak. 

In the first test you washed 8 times.  In the second, four times.  My guess is that your final cell suspension in test 1 was weak to the point of almost non-existent, whereas in test 2 you would have had a decent quantity of cells.  So the difference between very very weak and negative is possibly just down to that.  Plus, tube technique is much more difficult to standardise anyway than gel.

Plus it won't be clinically significant - why waste all that time and effort?

anna

 

[jwnola]

Another tech performed the test and I tried to use this as a part of competency. 

We use the same saline for cell washer and manual washing.  I am not sure about the pH of the saline.  It is not PBS. 

[rravkin@aol.com]

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?

[jwnola]

The sample is cord blood.  Cord blood sample has to be washed several times before making cell suspension.  After the cell suspension is made, can you use one drop of that blood to perform DAT without any further washing?  The washing before making the cell suspension is sufficient? 

[TreeMoss]

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.

[gene20354]

We perform tube testing for cord blood DATs.  Wash cord cells once by hand washing and then make a 3-5% suspension.  Place one drop of the suspension in a tube and wash 4 times in a cell washer.   We don't use the microscope to read the tubes. 

[exlimey]

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?

[exlimey]

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.

[Malcolm Needs ★]

22 minutes ago, exlimey said:

What is the reasoning behind using "cold saline" for cords and not other samples?

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.

[gene20354]

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.

[exlimey]

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.

[TreeMoss]

On ‎7‎/‎18‎/‎2017 at 0:01 PM, exlimey said:

What is the reasoning behind using "cold saline" for cords and not other samples?

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.

[exlimey]

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.

[rravkin@aol.com]

On ‎7‎/‎18‎/‎2017 at 2:11 PM, exlimey said:

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.

Exlimey, thank you for pointing this out. It is definitely the weakly reacting antibody that may be washed away with excessive washing.