Preparing red cell suspension for grouping

[Kandahlawi]

Hi, just wanna ask your practice in preparing red cells suspension for routine ABO blood grouping (pretransfusion testing) by tube method. In our SOP (reference: AABB Technical Method), we must wash the red cells 3 times before preparing the suspension. But in practice, we actually skip the wash step, and straight away proceed to suspense the cell in saline. We feel the wash step is a bit tedious. What do you all think of this practice? 

[Malcolm Needs ☆]

The wash step used to be vital in the days when we used human-derived polyclonal ABO grouping reagents, as there were occasions recorded in the literature whereby the ABO soluble substance in some individual's serum/plasma was in sufficient amounts to adsorb the anti-A or anti-B, and thus cause either falsely weak, or even false negative reactions on some occasions, leading to ABO mismatched transfusions.

The monoclonal ABO grouping reagents used now are very different, being much more avid and much more specific to the Type 2 ABO structures expressed on the red cells, and so the washing step is no longer required.

[exlimey]

As Malcolm points out, washing is not really important for ABO grouping in the modern era of tube testing. The reagents are formulated to tolerate a degree of neutralization. I assume this is true also for the reagents used in gel cards. Washing is important when using the cells in any test using antiglobulin reagents (as LIMPER55 notes).

Perhaps more of a concern is that your in-house procedures should not contradict the reagent manufacturers' instructions. One could even argue that an SOP is not really required if you follow the package insert - that's your SOP!

Whichever approach you take, you should definitely be following your SOP. I can see and hear the regulatory folks cringing in their seats as they read your post.

[David Saikin]

I only wash if I get a discrepancy I cannot resolve easily

[Malcolm Needs ☆]

Excellent point David.  Actually, we do wash the red cells in PBS pre-warmed to 37^oC several times over, if there is a cold-auto-antibody present that is interfering with the results.

[AMcCord]

The package insert for the reagents we use says wash a minimum of 1 time, so that's what we do with patients - 1 wash. If there is a discrepancy that isn't easily resolved we would wash the specimen 3 times - haven't had to do that in a loooong time.

[MAGNUM]

I wash once, mainly due to habit.

[jayinsat]

I say change your SOP to match your practice, as long as it has been validated, passes daily QC and doesn't contradict manufacturers requirements.  I don't know of any transfusion services that wash the cell suspensions routinely anymore.

[exlimey]

2 hours ago, MAGNUM said:

I wash once, mainly due to habit.

I'd like to be a fly-on-the-wall when you have that conversation with an Inspector......just kidding. I suspect many folks are doing just as you describe and for the same reason.

[exlimey]

1 hour ago, jayinsat said:

I say change your SOP to match your practice, as long as it has been validated, passes daily QC and doesn't contradict manufacturers requirements.  I don't know of any transfusion services that wash the cell suspensions routinely anymore.

What do you mean by "validated" ? Have you actually validated your ABO grouping process?

[carolyn swickard]

EXCEPT - for cord bloods - they ALWAYS have to be washed at least 3 times!  I was just doing one and noticed that we forgot to mention them as the exception to the "rules" being discussed here. 

For adults - I do not wash - just make a 3% suspension in PBS from 1 drop of packed cells.  I always do tell my students that if they use too many drops of packed cells, they should wash 1 time at least.

For cord bloods, I make a 3% suspension and then drip that out to the test tubes (1 drop each) and then let the cell washer wash the those tubes 4 times.  Very easy.  (But it always requires telling the students that they must not wash the 3% suspension directly in the cell washer!!!)

 

[Dansket]

9 minutes ago, cswickard said:

EXCEPT - for cord bloods - they ALWAYS have to be washed at least 3 times!  I was just doing one and noticed that we forgot to mention them as the exception to the "rules" being discussed here. 

For adults - I do not wash - just make a 3% suspension in PBS from 1 drop of packed cells.  I always do tell my students that if they use too many drops of packed cells, they should wash 1 time at least.

For cord bloods, I make a 3% suspension and then drip that out to the test tubes (1 drop each) and then let the cell washer wash the those tubes 4 times.  Very easy.  (But it always requires telling the students that they must not wash the 3% suspension directly in the cell washer!!!)

 

I stopped "washing cord blood samples 3 times" over 20 years ago.  Routine washing of cord blood red cells is a "solution" looking for a problem.  If you have documented evidence in your facility that unwashed cord red cells produce a high incidence of false-positive test results, then wash, wash, wash.  Otherwise, I would wash cord red cells only when a discrepancy is detected (positive Rh control test).

[AMcCord]

18 hours ago, Dansket said:

I stopped "washing cord blood samples 3 times" over 20 years ago.  Routine washing of cord blood red cells is a "solution" looking for a problem.  If you have documented evidence in your facility that unwashed cord red cells produce a high incidence of false-positive test results, then wash, wash, wash.  Otherwise, I would wash cord red cells only when a discrepancy is detected (positive Rh control test).

The cord blood collection techniques used at my facility leave something to be desired. I swear they wring the sample out of the cord...after it has sat around for awhile. We still wash cord cells for manual testing because we've had some samples that have given us problems. Easier for us to wash than for me to clean up reporting errors made by generalists who are seeing those kinds of problems once in a blue moon. We do not, however, wash samples that go on the Echo, though we do get a sample or two a month that doesn't give valid results. We make sure we wash those samples well prior to tube testing.

[mollyredone]

AMcCord, 

Who manufactures your reagents?  We use Immucor and they state you "may" wash the cells, but it isn't required.  We do wash cord bloods 3 times.  The staff in family birth usually get as much blood on the outside of the tube as in the tube!  Gross!

[jayinsat]

On ‎10‎/‎20‎/‎2016 at 11:16 AM, exlimey said:

What do you mean by "validated" ? Have you actually validated your ABO grouping process?

Yes I have for both our automation and back up methods.  Any methodology, or change in methodology, used in blood bank must be validated before it is implemented.  That means, you must run tests using the new method in parallel with the old method and prove that it yields acceptable accurate results, sensitivity and specificity.  After that initial validation, you only need to perform QC on a daily basis.

Edited October 25, 2016 by jayinsat

[ANORRIS]

Donor blood????

[carolyn swickard]

Since we also do a DAT on each of our cord bloods that we test (O Pos and RH Neg moms only) - it just works best to wash all the tubes and do the testing all at once.  You have to wash the DAT at least 3X.

[mollyredone]

19 hours ago, cswickard said:

Since we also do a DAT on each of our cord bloods that we test (O Pos and RH Neg moms only) - it just works best to wash all the tubes and do the testing all at once.  You have to wash the DAT at least 3X.

Carolyn,

That's what we do as well.