QC of Anti-C3b, C3d

[Brenda K Hutson]

Am updating some procedures and want some input. In looking at a Control for Anti-C3b, -3d, it states: A positive control of anti-complement activity is a test with red cells sensitized with complement (low ionic strength or low ionic strength plus trypsin treatment method). Hmmm..seems like at past facilities, we just add Complement Control Cells to Anti-C3b, -3d as a control.

Ok, so I am probably humiliating myself by asking this, but what do you all use to QC this reagent?

Thanks,

Brenda Hutson, CLS(ASCP)SBB

[swede]

I'll be humiliated right along with you....we use complement control cells!

[JOANBALONE]

Is this a trick question? We use complement control cells too.

JB

[David Saikin]

Commercial Complement control cells too. Making C activated cells is too spurious and they are notoriously unstable.

[Brenda K Hutson]

Is this a trick question? We use complement control cells too.

JB

No, not a trick question. That is an exact quote from the Manufacturer's Insert.

Brenda Hutson, CLS(ASCP)SBB

[Shari]

We, too, use Complement Control Cells

[Eagle Eye]

We use complement control cells.

[phouck]

We use complement control cells, too.

[SMW]

.....: A positive control of anti-complement activity is a test with red cells sensitized with complement (low ionic strength or low ionic strength plus trypsin treatment method). ....

Aren't the reagent complement control cells "red cells sensitized with complement"? Perhaps the manufacturer of the anti-complement reagent you're using does not produce/distribute reagent complement control cells so is being somewhat vague to not appear to be referring you to use another company's product?

[Mary**]

Immucor does not recommend the use of their complement control cells with another manufacturer's anti-C3B,C3d.

[pmata]

Thank you, Mary... this explains why they often do not work with the Ortho reagent.

[David Saikin]

They often do not work (in tubes) with the Immucor reagent too. 4+ in buffered gel with anti-C3b,-C3d, even after 5' incubation.

[Brenda K Hutson]

Right, I know this has been an ongoing problem. When I was a Supervisor at a Reference Lab, I put on 2 Seminars a year, inviting various Blood Bank speakers from all over. There were 6-1 hr. sessions and the last 1 hour, I called "Ask the Experts." It was a questoin and answer forum with myself, and the head of the other 2 local Reference Labs. At one of these meetings, I recall someone bringing up this very issue, and you could hear agreement throughout the room (and we are talkng over 200 people). We did mention the fact that they should be incubated at RT for 5', but as you said, even that sometimes does not work.

But here is why we have 2 Manufacturer's: in looking into this more (since these orders have been in place long before I came), it appears that Ortho does not have a Complement Control Cell (and we do use their Anti-C3b, 3d) and the Anti-C3b, 3d by Immucor, would cost us MUCH more than what we are purchasing from Ortho. So that is a dilemna. I am thinking it is odd that Ortho would sell the antisera but not the control cells. I do use Gel, but only for Antibody Screen, Antibody ID; we do not purchase any of the other cards. Someone in this Thread mentioned using the cards for C' testing. I think I need to investigate that before I put out a lot of $$ for Immucor Anti-C3b, 3d.

Brenda

[AMcCord]

Complement Control Cells are 'designed' to react 1-2+ according to the insert, so they aren't going to be really strong reactors. I find that your shaking technique can make a huge difference in what your reaction strengths are with these cells. When I have a tech who consistently gets a 1+ at best (or less), I watch their technique and often they are pretty enthusiastic shakers. I have them ease up and they usually get better results. I also use Comp Control Cells to educate students about shaking out tubes properly.

If the control cells are really fresh, I can almost always get a 2+, sometimes a 3+ (unless the cells were shipped on a HOT summer day - takes the stuffing right out of them!). I tend to open the new lot when it comes in, rather than waiting for the previous lot to outdate before switching lots. I think that helps quite a bit, too. I do incubate patient tubes for 5 minutes with anti-C3 prior to reading, but I don't incubate for 5 minutes with the control cells.

[Malcolm Needs ☆]

Complement Control Cells are 'designed' to react 1-2+ according to the insert, so they aren't going to be really strong reactors. I find that your shaking technique can make a huge difference in what your reaction strengths are with these cells. When I have a tech who consistently gets a 1+ at best (or less), I watch their technique and often they are pretty enthusiastic shakers. I have them ease up and they usually get better results. I also use Comp Control Cells to educate students about shaking out tubes properly.

If the control cells are really fresh, I can almost always get a 2+, sometimes a 3+ (unless the cells were shipped on a HOT summer day - takes the stuffing right out of them!). I tend to open the new lot when it comes in, rather than waiting for the previous lot to outdate before switching lots. I think that helps quite a bit, too. I do incubate patient tubes for 5 minutes with anti-C3 prior to reading, but I don't incubate for 5 minutes with the control cells.

I'm sorry; I know I'm a pain, but I am a little worried about the word "shaking" (although I do know what you mean). There are so many people nowadays who are very "anti-tube" that I would prefer the term "gentle resuspension by a slight rolling motion of the tubes".

There; that's got that off my over-pedantic chest!!!!!!!!!!!!!!

:redface::redface:

[AMcCord]

I do agree with you Malcolm, "shaking" is the problem. I was taught to tip, observe for sheeting, tremble with your dainty fingertips, repeat the tip.... The first thing I do with a new tech or a student in Blood Bank for their clinicals is to correct their bad "shaking' habits. Most of them arrive with a lot of wrist action. It's also amazing how many of my long term Blood Bankers pick up that bad habit over time.

[Malcolm Needs ☆]

I do agree with you Malcolm, "shaking" is the problem. I was taught to tip, observe for sheeting, tremble with your dainty fingertips, repeat the tip.... The first thing I do with a new tech or a student in Blood Bank for their clinicals is to correct their bad "shaking' habits. Most of them arrive with a lot of wrist action. It's also amazing how many of my long term Blood Bankers pick up that bad habit over time.

Oh phew! I thought that you could take offence (although it was not meant in any way like that)!

Thanks.

Now, about my dainty fingertips. Imagine four salamis, touched off with a log, and you've got my fingers and thumbs.....

:disbelief:disbelief:disbelief

[Brenda K Hutson]

Complement Control Cells are 'designed' to react 1-2+ according to the insert, so they aren't going to be really strong reactors. I find that your shaking technique can make a huge difference in what your reaction strengths are with these cells. When I have a tech who consistently gets a 1+ at best (or less), I watch their technique and often they are pretty enthusiastic shakers. I have them ease up and they usually get better results. I also use Comp Control Cells to educate students about shaking out tubes properly.

If the control cells are really fresh, I can almost always get a 2+, sometimes a 3+ (unless the cells were shipped on a HOT summer day - takes the stuffing right out of them!). I tend to open the new lot when it comes in, rather than waiting for the previous lot to outdate before switching lots. I think that helps quite a bit, too. I do incubate patient tubes for 5 minutes with anti-C3 prior to reading, but I don't incubate for 5 minutes with the control cells.

Well, if you are able to get > 2+ reactions with your complement control cells, without a 5' incubation, you are doing better than most Hospitals. If I am correct, the Manufacturer's Insert also refers to incubating for 5'. Even then, it can be troublsome (and I think I have the hang of resuspending cells at this stage of the game).

Brenda Hutson, MT(ASCP)SBB

[David Saikin]

Oh man, I was always taught to resuspend tubes like I had the "DTs". It was an axiom where I trained (decades ago) that if you could shake it off, it isn't there. The only tubes we resuspended gently were cord DATs.

[eric1980]

My lab makes complement-coated cells for controls...

[jeloweryii]

As David Saikin stated, "Making C activated cells is too spurious and they are notoriously unstable. " For those of you who are making your own C3 control cells successfully, I am curious as to which methods are being used.

[AMcCord]

Well, if you are able to get > 2+ reactions with your complement control cells, without a 5' incubation, you are doing better than most Hospitals. If I am correct, the Manufacturer's Insert also refers to incubating for 5'. Even then, it can be troublsome (and I think I have the hang of resuspending cells at this stage of the game).

Brenda Hutson, MT(ASCP)SBB

I don't incubate for 5' for QC of the reagent. If I can't get a good reaction with QC straigt up, then I would consider my reagent to have a problem (deterioration, contamination, whatever), though that hasn't happened so far...knock on wood. All patient tests (and my negative reagent control) get the 5' incubation.

[SMW]

I don't understand the rationale of not following the manufacturer's instructions when performing the quality control testing . If the manufactuer says to add 2 drops of a reagent to the test system, do you only add 1 drop to your control test? If the manufacturer states to incubate your Hepatitis B surface antigen assay for 3 hours, do you only incubate your controls for 30 minutes and if they work then the patient assays at 3 hours must be OK? It's one thing to select a control that will detect/express a weak antigen (single dose expression vs. double dose) or be capable of detecting a low level of antibody when following the manufacturer's instructions. It's a whole other animal to decide to test your controls in a different manner than your test samples. Since you are not challenging the actual testing process, how can you interpret the control results as valid?

[eric1980]

The recipe for the C'-coated cells we use in my lab is as follows:

-Make 10% sucrose solution.

-Extort a tube of blood from a O RH(D) POS colleague.

-Mix 5ml of sucrose solution with 5 drops of packed donor RBC and 20 drops of donor serum.

-Incubate 15mins@37C.

-Wash with saline and then lastly resuspend with a preservative to ~3% HCT. (We use DiaMed CellStab)

-Controls are anti-IgG and anti-C'.

The C'-coated cells should not react with anti-IgG but should react well with the anti-C'. The shelf-life should end when there is haemolysis or after a week, whichever comes first.