My company tests leukapheresed plasma in BacT/ALERT bottles. Recently, we have see heavy turbidity in the aerobic bottle only after 3-5 days of incubation; the instrument never calls the bottles positive. We don't see anything when we gram stain the turbid media and get no growth on subculture. Has anyone else experienced this phenomenom?

Thanks!

I would refer to the Package Insert for the bottle that you are using. If they are like the bottles that we use for Platelet testing, they state something to the effect of, "On rare occasions, organisms may be encountered that grow in the BacT/ALERT culturebottle growth media but do not produce enough carbon dioxide to be determined positive." Also, "BacT/ALERT [positive] specimens may contain organisms that are not seen with routine smear methods and may require both specialized smears and subculturing media for detection and recovery." However, these incidents should be rare, and for both of them to occur simultaneously on multiple occasions is probably excessively improbable. I would get in touch with bioMerieux and see if they can help.

Thanks, Heather,

I'm a long-time user of BTA, so I realize that some organisms won't turn the sensor yellow. I'm also a microbiologist, so I understand that some organisms might be hard to grow. But I'm facing a very baffling situation right now. We are seeing gross turbidity with most of our plasma samples 3-5 days after inoculating into the aerobic BTA bottle. With the amount of turbidity present, organisms should be seen on a smear and should grow on subculture, but we're having no luck in either area. I've talked to bmx and they haven't had any similar complaints, though I am ready to talk to them again. Since I know the blood bank group often tests plasma specimens, I was hoping your group might have encountered similar situations in the past.

Thanks!

I'm not familiar with a leukapheresed plasma product. How is the product collected and prepared and what anticoagulant is used? What does the final product contain in addition to plasma?

The plasma product is collected using the auto-PBMS procedure on a leuk machine. We are interested in getting lots of WBCs, especially monocytes. The anticoagulant in the bags is ACD-A. We are seeing this issue with plasmas drawn from 3 different sites, so it not site-dependent. As far as we know, the maker of the leuk bags has not made any changes, but we may have to dig into that issue more.

Our final product contains dendritic cells (we have matured these from the monocytes collected during the leuk) diluted in the plasma. USP sterility and mycoplasma testing on the final product is always no growth, which is another reason why we think we don't have a plasma contamination issue.

The package insert for the BacT/ALERT discusses its use with leukoreduced platelet products and that is the product sampled by blood centers. Since you report inoculating a white cell rich product, my guess is what you're seeing has something to do with that white cell content and the breakdown or release of "things" from those white cells that's causing the turbidity. Is the patient/donor stimulated with anything prior to the collection which might also be present in the final product and/or is anything else used in the collection process, e.g., a hetastarch?

LeeHL,

Have you made Wright Giemsa smears?

LeeHL,

Have you made Wright Giemsa smears?

Also, have you considered protien electrophoresis to rule out the possiblity of the presence of a protein conjugate?

We have not done Wright-Giemsa stains, but all our testing has proven that we are not dealing with any type of microbe.

We did play with adding Urea and EDTA to separate bottles, with the thought that either would break up clear the media if the turbidity was due to protein, but that didn't show anything. We had the same result when we added Tween to see if the turbidity was due to lipids.

We did extensive experimenting today to narrow down what part of our process is leading to the turbidity. We definitely know that some substance in our process is reacting to something in the BTA media (we do not get turbidity if we test plasma in plain TSB). And we are working with biomerieux R&D, but they have no resolutions yet.

I'll let you know more next week!

The patients are not stimulated prior to donation and nothing else is used during the collection process.

We have not done Wright-Giemsa stains, but all our testing has proven that we are not dealing with any type of microbe.

We did play with adding Urea and EDTA to separate bottles, with the thought that either would break up clear the media if the turbidity was due to protein, but that didn't show anything. We had the same result when we added Tween to see if the turbidity was due to lipids.

We did extensive experimenting today to narrow down what part of our process is leading to the turbidity. We definitely know that some substance in our process is reacting to something in the BTA media (we do not get turbidity if we test plasma in plain TSB). And we are working with biomerieux R&D, but they have no resolutions yet.

I'll let you know more next week!

LeeHL,

The Wright-Giemsa stain would stain any cellular constituents differentially, so I would think that you might want to try this to see the type of cells that are present, if any. Also, if the cloudy precipitate were the product of conjugated coagulation factors the EDTA would be of no consiquence in breaking them up and I'm not sure that the Urea would be either. The Urea, and correct me if I am wrong, will effect the nitrogen component of a protein such that the protein would break up and macroscopically resolvate producing a clear solvent. However, in order for the Urea to act in this manner on the protein the nitrogen component would have to be accessible. When the prossibility is a conjugated protein I'm not sure that the nitrogen components would be readily accessible, therefore protein electrophorises may be a better method of analysing a possible protein constituent. I hope this helps and again please let me know what you find out as I look forward to next week.:)

Edited November 18, 201014 yr by rravkin@aol.com