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konkous ☆ Greece, 2012-06-19 16:59 (4321 d 22:10 ago) Posting: # 8790 Views: 8,906 |
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Dear forum members The recent EMA guideline on bioanalytical method validation clearly states that full validation should be performed using matrix which has the same anticoagulant as the study samples. The general approach employed is that blood is collected into vacutainers which contain EDTA as an anticoagulant agent. However the plasma collection bags contain citrate-phosphate buffer. Is it possible (does the guideline allow it?) for a lab to conduct full validation with spiked plasma samples, with citrate-phosphate buffer as anticoagulant, and then run the main study samples, with EDTA as anticoagulant and not face major invalidation issues during an audit? Would a series of QC samples, covering the whole quantification range, prepared by spiking EDTA-plasma be enough to prove that there is no interference? The same guideline later on states that only a partial validation is required when there is a change in the anticoagulant. In the case that the guideline is binding regarding the above matter could someone propose a solution, as we have not been able to find large volumes of blank plasma (to be used for pre-study validation) which has been treated with EDTA. Thank you in advance Constantinos |
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Dr_Dan ★★ Germany, 2012-06-19 17:36 (4321 d 21:33 ago) @ konkous Posting: # 8793 Views: 7,954 |
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Dear Constantinos the Guideline on bioanalytical method validation EMEA/CHMP/EWP/192217/2009 states in paragraph 4.2. Partial validation: "In situations where minor changes are made to an analytical method that has already been validated, a full validation may not be necessary, depending on the nature of the applied changes. Changes for which a partial validation may be needed include transfer of the bioanalytical method to another laboratory, change in equipment, calibration concentration range, limited sample volume, another matrix or species, change in anticoagulant, sample processing procedure, storage conditions etc. All modifications should be reported and the scope of revalidation or partial validation justified. Partial validation can range from as little as the determination of the within-run precision and accuracy, to an almost full validation." I hope this helps Kind regards Dan — Kind regards and have a nice day Dr_Dan |
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Helmut ★★★   Vienna, Austria, 2012-06-19 17:41 (4321 d 21:28 ago) @ konkous Posting: # 8794 Views: 7,861 |
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Γεια σου Κωνσταντίνο ❝ […] The general approach employed is that blood is collected into vacutainers which contain EDTA as an anticoagulant agent. Why “general”? Vacutainers are available with various anticoagulants. ❝ Is it possible (does the guideline allow it?) for a lab to conduct full validation with spiked plasma samples, with citrate-phosphate buffer as anticoagulant, and then run the main study samples, with EDTA as anticoagulant and not face major invalidation issues during an audit? No, IMHO. ❝ Would a series of QC samples, covering the whole quantification range, prepared by spiking EDTA-plasma be enough to prove that there is no interference? I don’t think so. ❝ The same guideline later on states that only a partial validation is required when there is a change in the anticoagulant. In such a case (change of anticoagulant) you may only skip those parts where the anticoagulant plays no role, e.g., stock solution stability, autosampler stability,… ❝ In the case that the guideline is binding regarding the above matter could someone propose a solution, as we have not been able to find large volumes of blank plasma (to be used for pre-study validation) which has been treated with EDTA. You can get certified plasma with any type of anticoagulant from commercial sources. We routinely use samples for PAA Laboratories (in Greece: KNT Biologiki, Korgialeniou Str. 1-3, 115 26 Athens, T 210-699-34-39, kntbiol ![[image]](https://static.bebac.at/img/at.png) otenet.gr).— Dif-tor heh smusma 🖖🏼 Довге життя Україна! ![[image]](https://static.bebac.at/pics/Blue_and_yellow_ribbon_UA.png) Helmut Schütz ![[image]](https://static.bebac.at/img/CC by.png) The quality of responses received is directly proportional to the quality of the question asked. 🚮 Science Quotes |
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konkous ☆ Greece, 2012-06-19 18:19 (4321 d 20:50 ago) @ Helmut Posting: # 8797 Views: 7,691 |
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Thank you very much for all the help and guidance! |
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Ohlbe ★★★ France, 2012-06-19 19:44 (4321 d 19:26 ago) @ Helmut Posting: # 8802 Views: 8,021 |
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Dear Constantinos, I fully agree with Helmut, and particularly in the case of CPD. The problem with CPD plasma is the large volume of buffer added to blood. It basically results in a 30 % dilution of plasma compared to EDTA-coated vacutainers. This can create differences in matrix effects and in recovery, depending on your sample preparation method. I've seen a presentation by a French inspector at the EBF meeting in Barcelona in 2009. He reported two-fold differences in internal standard response between CPD and EDTA plasma, with also differences in retention times and peak width. You can expect this issue to be raised in case of an inspection. Regards Ohlbe — Regards Ohlbe |
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Helmut ★★★ Vienna, Austria, 2012-06-19 21:48 (4321 d 17:22 ago) @ Ohlbe Posting: # 8806 Views: 7,785 |
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Dear Ohlbe! ❝ […] The problem with CPD plasma is the large volume of buffer added to blood. It basically results in a 30 % dilution of plasma compared to EDTA-coated vacutainers. This can create differences in matrix effects and in recovery, depending on your sample preparation method. Agree – but only if you compare different methods. If a method for citrate-plasma is fully validated according to current requirement I don’t see any reason why we should not be happy with it. ❝ […] a presentation by a French inspector at the EBF meeting in Barcelona in 2009. He reported two-fold differences in internal standard response between CPD and EDTA plasma, with also differences in retention times and peak width. Do I recall it correctly that the method was validated in CPD plasma and partial (cross-?) validation (one batch acc/prec) was done with EDTA plasma of unknown origin? Or – more precisely – it wasn’t even clear whether the anticoagulant was EDTA at all? Of course it’s bullshit to ‘evaluate’ unknown samples with one anticoagulant (EDTA) based on calibrators/QCs with another (CPD). All the points you mentioned above might cut in. But this does not speak against CPD as such (I got this impression from your post’s subject line). If a method is validated with any given anticoagulant I would be happy to defend it against an inspector. What arguments would he/she have? “CPD is uncommon; I have seen only methods for this drug with EDTA until now.” — Dif-tor heh smusma 🖖🏼 Довге життя Україна! Helmut Schütz The quality of responses received is directly proportional to the quality of the question asked. 🚮 Science Quotes |
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Ohlbe ★★★ France, 2012-06-19 23:45 (4321 d 15:24 ago) @ Helmut Posting: # 8808 Views: 7,696 |
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Dear Helmut, ❝ Do I recall it correctly that the method was validated in CPD plasma and partial (cross-?) validation (one batch acc/prec) was done with EDTA plasma of unknown origin? Or – more precisely – it wasn’t even clear whether the anticoagulant was EDTA at all? Correct. That's what he said, if I remember correctly. ❝ Of course it’s bullshit to ‘evaluate’ unknown samples with one anticoagulant (EDTA) based on calibrators/QCs with another (CPD). All the points you mentioned above might cut in. But this does not speak against CPD as such (I got this impression from your post’s subject line). If you compare heparin, EDTA and CPD plasma, CPD is certainly the one showing the more difference from the other two. If a lab disregards the EMA guideline, or is working for other markets, and wants to prepare CCs and QCs in a different anticoagulant because they can obtain it more easily, CPD is the worst choice you can get if the subject samples were collected on another anticoagulant. ❝ If a method is validated with any given anticoagulant I would be happy to defend it against an inspector. What arguments would he/she have? “CPD is uncommon; I have seen only methods for this drug with EDTA until now.” Agreed. If the subject samples were collected on CPD, then the CCs and QCs should be prepared on CPD plasma - with the same proportion of buffer as in the vacutainers during blood collection... Regards Ohlbe — Regards Ohlbe |
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konkous ☆ Greece, 2012-06-22 19:10 (4318 d 19:59 ago) @ Helmut Posting: # 8829 Views: 7,549 |
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The whole debate on the subject has been more than enlightening. Could anyone propose a commercial plasma supplier or blood collection bag supplier which uses EDTA as anticoagulant, preferrably in Europe? Thank you Constantinos |
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Helmut ★★★ Vienna, Austria, 2012-06-23 15:00 (4318 d 00:10 ago) @ konkous Posting: # 8834 Views: 7,625 |
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Γεια σου Κωνσταντίνο ❝ Could anyone propose a commercial plasma supplier or blood collection bag supplier which uses EDTA as anticoagulant, preferrably in Europe? Try Sera Lab or Europa Bioproducts. Pretty expensive stuff. In method development we therefore routinely used pooled blank (i.e, pre-dose) samples from previous studies. — Dif-tor heh smusma 🖖🏼 Довге життя Україна! Helmut Schütz The quality of responses received is directly proportional to the quality of the question asked. 🚮 Science Quotes |
Ing. Helmut Schütz