LAL (Limulus Amebocyte Lysate) Test problem

[merveilleuse]

Hello everyone,

I have just found out this forum while searching for LAL test. My name is Merve SARIAHMET, I live in Turkey and I am a biologist. I work in the microbiology department of a pharmaceutical company.

We are building a new area for sterile production. It will be ready in a few months and our demo production is about to start. Now, we are testing WFI points of this area with LAL test. We are using gel-clot method (later we will use turbidimetric method). Our LAL sensitivity is 0.125 EU / ml, and our stock solution of endotoxin is 5000 EU/ml ( according to its certificate.) Our dilutions are below : (We take 0.1 ml of stock solution.)

1.tube : 100 EU/ml (0.1 ml of endotoksin stock solution + 0.9 ml LRW),
2.tube : 10 EU/ml (0.1 ml of 10 EU/ml solution + 0.9 ml LRW),
3.tube : 1 EU/ml (0.1 ml of 10 EU/ml solution + 0.9 ml LRW),
4.tube : 0.5 EU/ml (0.5 ml of 1 EU/ml solution + 0.5 ml LRW),
5.tube : 0.25 EU/ml (0.5 ml of 0.5 EU/ml solution + 0.5 ml LRW),
6.tube : 0.125 EU/ml (0.5 ml of 0.25 EU/ml solution + 0.5 ml LRW),
7.tube : 0.06 EU/ml ((0.5 ml of 0.125 EU/ml solution+ 0.5 ml LRW),
8.tube : 0.03 EU/ml ((0.5 ml of 0.06 EU/ml solution + 0.5 ml LRW)
standart solutions. We are performing this test in the following way :

To test our standart solutions, we add 0.1 ml lysate to 0.1 ml standart solution.
To test our LRW, we add 0.1 ml lysate to 0.1 ml LRW.
To test samples, we add 0.1 ml lysate to 0.1 ml sample.
To test spike solutions of sample, we add 0.1 ml lysate to 0.05 ml sample and 0.05 ml of 0.25 EU /ml standart solution .


And our test results are : We have 4 WFI points and all of them always pass the test. no clotting.
LRW is always proved to be endotoxin free as expected.
And the standart solutions, our 0.5 EU/ml, 0.25 EU/ml solutions always clot and 0.06 EU/ml and 0.03 EU/ml solutions never clot as expected. But our 0.125 EU/ml dilution and accordingly our spike solutions never clot which it is not expected. We think our dilutions are ok, but we couldn't be able to find the reason.

I need an opinion immediately, if anyone could help i will be really appreciated.

Thank you, Merve.

 

[somashekar]

Hello everyone,

I have just found out this forum while searching for LAL test. My name is Merve SARIAHMET, I live in Turkey and I am a biologist. I work in the microbiology department of a pharmaceutical company.

We are building a new area for sterile production. It will be ready in a few months and our demo production is about to start. Now, we are testing WFI points of this area with LAL test. We are using gel-clot method (later we will use turbidimetric method). Our LAL sensitivity is 0.125 EU / ml, and our stock solution of endotoxin is 5000 EU/ml ( according to its certificate.) Our dilutions are below : (We take 0.1 ml of stock solution.)

1.tube : 100 EU/ml (0.1 ml of endotoksin stock solution + 0.9 ml LRW),
2.tube : 10 EU/ml (0.1 ml of 10 EU/ml solution + 0.9 ml LRW),
3.tube : 1 EU/ml (0.1 ml of 10 EU/ml solution + 0.9 ml LRW),
4.tube : 0.5 EU/ml (0.5 ml of 1 EU/ml solution + 0.5 ml LRW),
5.tube : 0.25 EU/ml (0.5 ml of 0.5 EU/ml solution + 0.5 ml LRW),
6.tube : 0.125 EU/ml (0.5 ml of 0.25 EU/ml solution + 0.5 ml LRW),
7.tube : 0.06 EU/ml ((0.5 ml of 0.125 EU/ml solution+ 0.5 ml LRW),
8.tube : 0.03 EU/ml ((0.5 ml of 0.06 EU/ml solution + 0.5 ml LRW)
standart solutions. We are performing this test in the following way :

To test our standart solutions, we add 0.1 ml lysate to 0.1 ml standart solution.
To test our LRW, we add 0.1 ml lysate to 0.1 ml LRW.
To test samples, we add 0.1 ml lysate to 0.1 ml sample.
To test spike solutions of sample, we add 0.1 ml lysate to 0.05 ml sample and 0.05 ml of 0.25 EU /ml standart solution .


And our test results are : We have 4 WFI points and all of them always pass the test. no clotting.
LRW is always proved to be endotoxin free as expected.
And the standart solutions, our 0.5 EU/ml, 0.25 EU/ml solutions always clot and 0.06 EU/ml and 0.03 EU/ml solutions never clot as expected. But our 0.125 EU/ml dilution and accordingly our spike solutions never clot which it is not expected. We think our dilutions are ok, but we couldn't be able to find the reason.

I need an opinion immediately, if anyone could help i will be really appreciated.

Thank you, Merve.

Perhaps you are still in the very old method. There are quick test kits of gelclot tests for fixed EU/ml and perhaps 0.125 EU/ml is included. If you have confidence over your dilution and as you say you have 2 positive controls and 2 negitive controls answering correctly, why do you use the 0.125 control ?
Can you tell me who has provided you this protocol and would you discuss this with ACC or ENDOSAFE people ? Did you look at your MVD calculations as well ...

 

[Ajit Basrur]

Welcome to the Cove, Merve

First of all, there is no requirement to use up to 100 EU/mL as far as the standard series is concerned. WFI limit is 0.25 EU/mL and so standard series up to 0.5 EU/mL is absolutely okay.

There was no mention about clotting at 0.125 EU/mL as part of the standard series. As you are getting the clot with 0.3 and 0.6, there should be a spike with with either 0.3 and/or 0.6 EU/mL.

BTW, did you perform a method verification to see that there is no inhibition/exhibition. Usually WFI is neutral.

 

[merveilleuse]

Thanks for your replies Somashekar and Ajit,

Someshekar, yes we know there are quick test kits for gel-clot, but we are using this method temporarily. We will use turbidimetric method for LAL test, but our device is out of order right now.

We don't calculate MVD for this test because we are told that we don't have to calculate it while testing for WFI. We will calculate it when we will start to test products.

Ajit, we don't use 100, 10, 1 EU/ml for standart, I just underspecified We just use these solutions to get 0.5 Eu/ml. We use 0.5, 0.25, 0.125, 0.06 and 0.03 Eu/ml solutions to verify our LAL sensitivity.

Well I think we were just misguided about clotting at 0.125 Eu/ml. Because we are told that we must get clot at this one just because our LAL sensitivity is 0.125 EU/ml. But as I understood, there is no requirement for this. So can I tell I verify my LAL sensitivity? And one more question, do I have to do this verification all the time I run the LAL test or is it enough to verify it just because I use new LAL and CSE vial?

Thanks, Merve.