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LAL Update - Associates of Cape Cod, Inc.

LAL UpdateLALU pdateNovember 2006 Volume 23, OF CAPE COD, From the EditorDear LAL User:In this issue of the LAL Update we pres-ent articles written by two of ourTechnical Service Fields addresses testing of blood samples(whole blood, serum, or plasma).His articleexpands on a topic that was included in the articleon interferences with the LAL test in the October2005 issue of the LAL Update ( Volume 22, no. 3).Kathleen Brosnahan writes about calculationsperformed by the software used in photometric LALtests and discusses the importance of knowingwhat is being calculated, how it is being calculatedand the appropriateness of the you wish to discuss the issues raised in these arti-cles, or any other LAL / endotoxin related issues, orto talk about our Northstar BioProducts line ofproducts for glycobiology and glycoprotein charac-terization, please call our technical Servicesdepartment at 800-848-3248.

LAL Update www.acciusa.com LAL Technical Report 2 The first procedure to try is heat inactivation, which is both effective and relatively easy to perform. This protocol for heat inactivation is …

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Transcription of LAL Update - Associates of Cape Cod, Inc.

1 LAL UpdateLALU pdateNovember 2006 Volume 23, OF CAPE COD, From the EditorDear LAL User:In this issue of the LAL Update we pres-ent articles written by two of ourTechnical Service Fields addresses testing of blood samples(whole blood, serum, or plasma).His articleexpands on a topic that was included in the articleon interferences with the LAL test in the October2005 issue of the LAL Update ( Volume 22, no. 3).Kathleen Brosnahan writes about calculationsperformed by the software used in photometric LALtests and discusses the importance of knowingwhat is being calculated, how it is being calculatedand the appropriateness of the you wish to discuss the issues raised in these arti-cles, or any other LAL / endotoxin related issues, orto talk about our Northstar BioProducts line ofproducts for glycobiology and glycoprotein charac-terization, please call our technical Servicesdepartment at 800-848-3248, or one of our mainnumbers, 888-395-2221 / best wishes,Michael E.

2 Dawson, : Mark Fields Introduction Blood samples (whether whole, serum, plasma, orfractions) can be among the most difficult samples toanalyze by the LAL test. This article describes some ofthese difficulties and offers tips on how to prepare andtest such Blood samples contain a number of substances that caninterfere with the LAL example, certain proteinsin blood have the ability to neutralize endotoxin,2,3which can be troublesome when attempting to quantifythe endotoxin concentration of a sample. Furthermore,blood contains serine proteases that are also known tointerfere with the LAL assay. Some of these proteasesdegrade the proteins of the LAL enzyme cascade,resulting in inhibition; others activate the cascade,resulting in false it is usually necessary to treat the sample insome way, as will be discussed, it is worth testing thesample before employing any inactivation can vary widely in the degree of interferenceand, depending on the dilution at which the sample isbeing tested, treatment may not be necessary.

3 As withmany samples in which there is interference with the LALtest, sometimes the best solution for testing blood orserum is to dilute the samples until the interference isovercome. However, it is quite likely that it will benecessary to treat the sample prior to testing toneutralize interference. There are several treatments thatdenature or inactivate the , 5 Testing Blood Samplesfor EndotoxinPN001978 Rev000 LAL technical Report2 The first procedure to try is heat inactivation, which isboth effective and relatively easy to perform. Thisprotocol for heat inactivation is described in detail in aprevious LAL Update ,6and we have used it successfully inour laboratory for testing plasma and sera.

4 In some cases,however, the heat inactivation protocol can affect theapparent concentration of endotoxin because certainproteins bind endotoxin to a greater degree whenheated. One approach is to test a sample that has beenheat inactivated and compare it with an untreatedsample to see if this protocol is inactivation protocols involve chemical treatments,such as the addition of acid or chloroform,5but these arerecommended only when heat inactivation is noteffective. A treatment that we have found to besuccessful in reducing interference in a wide variety ofwhole-blood samples includes the use of nitric acid,detergent, and sodium treatmentis used, it is important that positive product controls beincluded for every sample tested.

5 In addition to proteins, normal blood contains low levelsof (1,3)- -D-glucan (BG), a component of fungal cellwalls that activates the LAL enzyme cascade. Fungalinfections can increase BG levels, and even low levels ofBG can introduce a testing artifact when BG-sensitive LALreagents are used to measure ,when testing blood or serum samples for endotoxin, anendotoxin-specific test is strongly recommended. ACC'sGlucashield product is a reconstitution buffer that canbe used in conjunction with all of our multitest LALreagents (Pyrotell 5mL and 2mL, Pyrotell -T,Pyrochrome , and Chromo-LAL). When reconstitutedwith Glucashield ,the LAL enzyme cascade is renderedinsensitive to BG, producing a more accuratemeasurement of endotoxin in the chemistry is highly variable; it is influenced by thephysiological state of the patient or donor, which is itselfinfluenced by many factors, including age, diet, geneticmake-up, health, fatigue, and lifestyle.

6 Consequently,although a treatment may be effective for many or mostsamples, it is quite likely that there will be some samplesfor which the treatment will not overcome was noted in a recent LAL Update on interference,6treatments other than dilution should be validated byadding a known amount of endotoxin to a knownvolume of sample, performing the treatment, anddemonstrating recovery of the added endotoxin withinpre-specified limits (usually 50%-200%). RecommendationsOf the LAL methods (gel clot, chromogenic, andturbidimetric), gel clot is widely considered to be themost robust and least prone to interference, making it alogical choice for such difficult samples as blood andserum.

7 The chromogenic method is also a good choicefor testing these samples, provided that potential opticalinterference is taken into thechromogenic method is read at 405 nm, certain plasmaor serum samples that absorb at this wavelength mayinterfere with the technique. Although sufficient dilutionwill eliminate this problem, another possible solution is touse ACC's Pyrochrome kit with Diazo reagents. Thecolor produced in this variant of the test is magenta,which absorbs in the 540-550 nm range, thus avoidingthe interference at 405 , however, that this isan endpoint test and so has a narrower range ofdetection than kinetic using one of the photometric methods ( , thechromogenic and turdidimetric techniques) to quantifyendotoxin in whole-blood samples, it is generallynecessary to centrifuge the sample to avoid the opticalinterference caused by blood cells.

8 The photometricmethods are also likely to provide valid results fromseveral of the dilutions tested (that is, the positiveproduct control "spike" is recovered within the specifiedlimits, and the sample result falls within the standardcurve). However, it is not unusual to find that althoughspike recovery is consistent from dilution to dilution, themeasured endotoxin concentration (after correction fordilution) changes with dilution of the unspiked the concentration increases with dilution. Thus,although spike recovery indicates that there is nointerference, the endotoxin measured in the sampleclearly indicates that interference is still present.

9 Theadded endotoxin spike is behaving differently from theendotoxin in the sample. The interference can sometimesbe removed by diluting the sample until the endotoxinconcentrations are consistent in both the spike and theunspiked sample. At other times dilution is not effectiveand another treatment must be sought to overcome theinterference. In the absence of an effective alternativetreatment, the only appropriate course is to report theresult as greater than or equal to the highest endotoxinconcentration Rev000 LAL technical Report3 SummaryIn conclusion, blood (or blood-derived) samples canpresent a challenge to the LAL test.

10 Some methoddevelopment work is often necessary to find anappropriate test technique. A number of possiblestrategies are available for overcoming interference, butthe simplest approach that can be validated is always thebest. Thus the choices, in order, should be dilution, heatinactivation, and chemical treatments. The TechnicalService staff at Associates of Cape Cod are always readyto advise you on a testing strategy, and the Contract TestService has a wide range of experience testing differenttypes of blood-derived samples from a range of Ketchum, P. A. and T. J. Novitsky. 1999. Assay of endotoxin byLimulus Amebocyte Lysate.


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