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In this issue:Choosing an LAL test methodCSE ActivityMaster Card/VisaCalendar

pfiJL LUPD TEVolume 13, No September 1995

Fig. The LAL Clotting Reaction original model

A Wealth Optionshoosing an L L Test Method

by Michael E. Dawson, Ph.D.

Coagulin-clot

Active clotting enzyme

Endotoxin

Coagulogen

Inactive clotting enzyme

Selecting the appropriate method can be difficult,especially for someone new to the LAL test. This articledescribes the choices, starting from the fundamentals of theLAL/endotoxin reaction, and includes a table summarizingthe differences between the methods.There are three principal LAL test methods: gel-clot,turbidimetric and chromogenic. The essentials of all threeare included in the original model for LAL clotting proposedby Levin 1979 Fig. 1 . Endotoxin activates the clottingenzyme which then cleaves a soluble substrate. Theresulting insoluble clotting protein forms a gel-clot.

Since this model was first proposed, it has beendemonstrated that activation ofclotting enzymeis notdirect.There are intermediate steps prior to the activation of theclotting enzyme. A more complex model, complete with theadditional steps and an alternate pathway Fig. 2 , is nowaccepted. The intermediate steps amplify the response toendotoxin and help to give the LAL test its extraordinarysensitivity. Activated clotting enzyme cleaves a peptidefrom the inner portion ofthe coagulogen molecule. The tworemaining peptides are linked by disulfide bridges to form

Another summer has ended on Cape Cod and with itanother successful crab bleeding season. Al though oursummer was exceptionally hot and dry, the crabs stayedcooland moist in our specially designedfacility until returning tothe ocean after their blood donation.

In addition to our normally hectic summer pace,Associates continued to expand its operation. We wereexceptionally pleased when Dr. Jack Levin, co-discovererofLAL, accepted a posi tion on our Board of Directors. Dr.Levin s ties to Woods Hole extendback to the 1960 s with hisintroduction toLimulus by the lateDr. FrederikBang. Duringhis WoodsHole summer research sojourns, Dr. Levin becameacquainted with Associates founder, the late Dr. Stanley atson Together they co-edited several proceedings of theWoods Hole conferences on Limulus and LAL. Dr. Levinbrings toAssociatesofCape Codhis exceptional expertise andunderstanding ofL L In addition, his knowledgeofmedicine,especially endotoxemia, sepsis and infectious disease, willhelp us develop clinical applicationsfor LAL.

I am also pleased to announce that Dr. Jack Sloyer hasbecome our Director of Product Development. Dr. Sloyercomes to uswitha solidbackgroundin clinical diagnostics andindustrial microbiology and will be working on some newbacterial detection products. Jack is also no stranger to LAL,having published several papers in the field, including oneofthe early studies on turbidimetric LAL kinetics.

This LAL UPDATE contains a summary of LAL testmethods by Dr. Michael Dawson. syou may have noticed,more ofthe articles in the LAL UPDATE are beingwritten bymy very capable colleagues, Dr. Michael Dawson (VicePresident, Production) and Dr. Marilyn Gould (VicePresident, RegulatoryAffairs and Responsible Head). I amsure they would appreciate your comments, and, as always,entertain any questions you may have.

Dear LAL User,

Thomas Novitsky, Ph.D.Editor

Sincerely,

o 1995, Associates of CapeCod, Inc., Woods Hole Massachusetts All rights reserved

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insoluble coagulin. Coagulin molecules associate to form agel. The gel is fragile, particularly when it first forms. Incontrast, when a horseshoe crab is injured innature, the clotis apparently stabilized by the membranes of degranulatedamebocytes and is quite resilient.Endotoxin

\Factor C Factor Ca p-glucan \ Factor B Factor B Factor G Factor Ga a \

Clotting enzyme Clotting enzymea

\

The gel-clot test yields a binary result, either positive ornegative. A tube is scored as positive + if the clotwithstands 1800 inversion without breaking. All otherconditions are scored as negative - , even if the clot almostremains intact but then collapses. This is not a subjectivetest despite some assertions to the contrary.The test can be set up as a limits tes t to pass or fail aproduct at a particular endotoxin concentration. In this case,a negative result means that the sample contains less than thelabeled sensitivity multiplied by the dilution factor of thesample. A positive result is reported as greater than or equalto the sensitivity times the dilution factor. Quantitation oran assay is achieved by testing a series of twofold dilutionsof sample. The results of a typical gel-clot assay arepresented in Table 1. The endpoint is the greatest dilutionto give a positive test. The endotoxin concentration of theoriginal sample is calculated by multiplying the reagentsensitivity by the dilution factor at the endpoint. Byconvention, results are reported as a specific value. In Table1,the result is 2.0 EU/mI, not 2>2.0 EU/mI but

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recommended for blood products, many other biologicalmaterials, and for electrolyte solutions containing traceelements. Less sensitive reagent is commonly used forwater, saline, and for many drug products. Ultimately, thesamplemust betested to assure that it doesnot interferewiththe test at a dilution at which the endotoxin limit can bedetected. Itmay benecessary to try a more sensitive reagentif interference cannot be overcome. One approach is toalways use a sensitive reagent. The disadvantage of thisstrategy is that greater sensitivity means more dilutions ofsamples and standards, which is unnecessary work andconsumes supplies. The selected sensitivity ultimatelydepends upon the type of sample to be tested and uponpersonal preference. Associates of Cape Cod s technicalrepresentatives are always available and willing to discussthe issues involvedand advise on the choice of sensitivity.Turbidimetric Methods

As the concentration of insoluble coagulin increasesduring the LAL reaction, the turbidity of the reactionmixture increases. The rate at which turbidity increases isrelated to the endotoxin concentration in the sample and isthe basis of the methods. There are two variations of theturbidimetric method.Endpoint Turbidimetric MethodAfter the sample and LAL reagent have incubated for afixed period of time, the absorbance is read in aspectrophotometer at a wavelength less than 450 nm (405nmor belowis preferable). Higher endotoxinconcentrationsin samples or standards give greater absorbance. Standardcurves are constructed by plotting the absorbance againstthe known endotoxin concentrations of standards. Becausestandard curves allow interpolation between the standardconcentrations, the method has greater resolution than thegel-clot method. This is also true for the kineticturbidimetric and chromogenic methods. The endpointturbidimetric method requires that timing of incubation andreading be controlled carefully because development ofturbidity can not be stopped. Consequently, each sampletube or well can only be read once. The range of endotoxinconcentrations that can be detected is limited. Only thoseconcentrations that are sufficient to cause development ofmeasurable turbidity within the incubation time, but are notso highthat the reaction has reached completion (saturation)may be quantified in a given assay. Concentrations belowthe minimum are indistinguishable from negative controls.At concentrations above the maximum, turbidity approaches saturation and it is not possible to differentiatebetween them. The range of concentrations that can bedetected is generally limited to approximately a factor oft n(e.g. 0.01 - 1.0 EU/ml or 0.5 - 5 EU/ml) for a givenincubation period. Longer incubations give a more sensitive

LALUPDATE ol 13,Number3, page3

test up to a limit of 0.001 EU/ml.Kinetic Turbidimetric MethodIn contrast with the endpoint turbidimetric method, inwhich a singlereading is taken for each sample, inthe kineticturbidimetric methodreadings are taken throughout the test.This requires an optical reader which incubates the reactionmixture at 37C and stores readings at regular intervals(typically every 10 seconds). The time taken to reach aspecific optical density threshold (the onset time) isrecorded. At higher endotoxin concentrations, the reactionis rapid and the onset time is relatively short. Standardcurves are constructed by regressing the log of the onset timeonthe log of the concentration of standard endotoxin. As theoptical density is read throughout the assay, not after a fixedincubation time, the detection range of this method is verywide, from 0.001 EU/ml up to 100 EU/ml. Standard curvesmay cover part or all of the range. Kinetic methods requiresophisticated software to analyze the results. Pyros forWindows for the LAL-5000 (Associates of Cape Cod,Inc.) is very easy to use and provides data summaries andreports. hromogenicMethods

Chromogenic methods utilize a synthetic substratewhich is added to the lysate. Pyrochrome Y (Associates ofCape Cod, Inc.) has the substrate co-lyophilized with theLAL reagent, which enables itto beused for all chromogenicmethods. The substrate has an amino acid sequence that ishomologous to one of the points of cleavage of the naturalsubstrate, coagulogen. A terminal chromophore paranitroaniline,pN is linkedto this peptide. The intact substrateis colorless, whereas free pN is yellow with a peakabsorbance at 405 nm. When chromogenic LAL reagentreacts with endotoxin, the cascade is initiated and clottingenzyme is activated as in the other methods. The clottingenzyme acts on the synthetic substrate, liberating thechromophore which turns the reaction mixture yellow. Thereaction proceeds more rapidly at higher endotoxinconcentrations, so the rate of development of the yellowcolor is greater. There are three variations of thechromogenic test, two endpoint methods and a kineticmethod.Endpoint Chromogenic MethodsIn the endpoint methods, the reaction is stopped after aspecific incubation period by the addition of acid. Theabsorbance of the yellow color is measured in a microplatereader or spectrophotometer. The higher the endotoxinconcentration in the sample, the greater the amount of pNliberated at the end of the incubation and consequently, themore intense the yellow color. The ability to stop thereaction is a great advantage over the endpoint turbidimetric

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method but all of the endpoint methods share the limitationof a detection range of approximately 1 log for a givenincubation time. The maximum sensitivity is 0.005 EU/mI.Ina variation, the diazo endpoint method, liberatedpis coupled to a diazo compound after stopping the reaction.This result is an intense magenta color with a greaterabsorbance at 540 nm comparedwithp at 405 nm. Thisassay may be used to test samples which absorb at 405 nm,avoiding interference due to color.Standard curves are constructed and unknownsquantified as described for the endpoint turbidimetricmethod.Kinetic ChromogenicMethodThe kinetic chromogenic method is very similar to thekinetic turbidimetric method described above, except thatthe optical density is due to the yellow color ofp ratherthan turbidity. The test is typically performed using anincubating microplate reader. The range of endotoxinconcentrations that can be quantified is 0.005 - 50 EU/mI.Correlations etween Methods

Results obtained for the same sample generallycorrelatewell. When comparing results, it is important to rememberthe error associated with each method. The plus or minustwofold error of the gel-clot method has been noted, but it isoften forgotten that there are errors associatedwith the othermethods as well. If the criterion for spike recovery is +/50 , as insome cases for kinetic methods, this is the errorofthe method. Thus, results of 0.1 EU/ml and 0.5 EU/mI maynot be significantly different; the actual concentrationmightbe 0.3 Eo/mt. If results appear to be substantially differentby different methods, it is important to insure thatinterference is controlled for all methods.While results should be similar, the degree of inhibitionor enhancement caused by a given product frequently variesbetween methods. The dilution required to overcomeinterference may be quite different for different methods able 2).

T LE 2. Dilutions required to overcome interferencefor two parenteral products using three L L methods

The results in Table 2 suggest that the endpointchromogenic test is the least sensitive to interference, butgiven the test errors, the differences are not significant.Generally, the gel-clot method shows the least interference.The various methods have different sensitivities.Consequently, the maximum valid dilution MVD) at whichthe endotoxin limit of a product can be detected is differentfor each method. Higher test sensitivity allows for a greaterdilution to overcome interference.T LE 3. Maximum valid dilutions MVD) of two drugproducts for three L L methods

Sensitivity A) Penicillin ClindamycinEU/ml)Gel-clot 0.03 640 2,784EndpointChromogenic 0.005 4,000 17,400KineticTurbidimetric 0.001 20,000 87,000

Most samples can be tested by any of the methods.However, for some samples, one method is preferable to theothers. Determination of the preferable method is often aquestion of trial and error, but it is not necessary to test allsample types by all methods. If a product cannot bevalidated and tested by a particular method, another shouldbe tried. The gel-clot is often the best method to start withwhen uncertain aboutwhich one to use. It is straightforwardto perform, the results are easy to interpret, and itincorporates the principle ofall methods. Also, if a producthas been validated initially by gel-clot and later by anothermethod, the gel-elotmethod is always available as a back-upmethod in the event of equipment failure. Ifyou needhelp inthe selection of a suitable test method, our technical servicerepresentatives at Associates of Cape Cod are alwaysavailable to help

Penicillin200,000 Units/ml)

Gel-clot 1:16Endpoint Chromogenic 1:10Kinetic Turbidimetric 1:200

LAL UPDATEVol. 13,Number3, page 4

Clindamycin50 mg/ml)1:321:201:100

References1.Levin,J. 1979.Thereactionbetweenbacterialendotoxinandamebocyte lysate, p. 131-146. In E. Cohen ed.), BiomedicalApplications of the Horseshoe Crab Limulidae), Progress inClinical and BiologicalResearch,Vo 29 Alan R. Liss, Inc.,NewYork.2. Iwanaga, S., T. Morita, T. Miyata and T. Nakamura.1985.Hemolymph coagulationsystem in Limulus Microbio -ogy:29-32.

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omparison L L Test MethodsGel-clot Endpoint Turbidimetric KineticTurbidimetric Endpoint Chr omogen ic Kinetic C hromogeni c

ost Lowest equipment cost Relatively inexpensive, Moderate t o expensive Relatively inexpensive, Most e xpensivewidely available instrumentation widely available instrumentationinstrumentation instrumentation

Sensitivity Sensitive - up to 0.03 EU/ml. Most sensitive (detection limit Most sens it ive(detec tion l imit More sensi tive (detec tion More sensi tive (detec tionSensitivity standardized by of 0.001 EU/ml) of 0.001 EU/ml inthe LAL- limit of 0.005 EU/ml) limit of 0.005 EU/ml)the manufacturer 5000)

~ x i m u m t s t r n g NA I log e.g. 0.1 -I or 0.01 -0.1 0.001 - 100 EU/ml I log e.g. 0.1 l or0.01 -0.1 0.005 - 50 EU/mlEU/ml EU/mlResolution plus or minus one twofold 1-25 1 25 or50 * 1-25 1 25 or50 *Sus cep ti bi li ty to Resilient - o ften less a ffected May show more interference than gel-clot, b ut greater sensitivity g ives more scope for dilution t oovercome itinterference by interference than other

methodsTiming Mus t be on hand to read test Cr iti cal - reaction must be Automated inst rumentation C ri ti cal- reaction must be Automated instrumentation

after one hour timed carefully handles timing timed carefully but can be handles timingstopped for reading - easierthan end-point turbidimetric

Reaction vessel Soda l ime g lass culture tubes Borosilicate g lass c ulture Borosilicate g lass culture Microplate**, sometimes Microplate** - allows f ortubes or microplates** tubes ( in LAL-5000) or glass culture tubes quick, easy dilutions

microplate**

Other Comments USP compendial method. LAL-5000: Diazo option allows testingofResults are easy to interpret. - very good temperature samples that absorb at 405Can process many samples at control nma time. - individually controlled

timing for each well- samples can beadded to atest in progress

*The resolutionofkineticmethodsdepends on the spike recovery range used. The 1987 FDA Guidelineon Validation of the Limulus Amebocyte Lysate Test .... specifies that spikesbe recovered within +/-25 . This was increased to 1- 50 in the 1991 FDA Interim Guidance for Human and Veterinary Drug Products and Biologicals: KINETIC LALTECHNIQUES . This change did not apply to medical devices.**Microplatescannotbe practicallydepyrogenatedbythe user. Occasional contaminatedwells ( hot wells ) are to be expected they are used. appropriate source of relatively cleanplates is necessary. Pyroplates are available from Associates of Cape Cod, Inc. and are provided with a certificate of analysis.

L L UPD TE Vol. 13, Number 3, page 5

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Syringes Inhibit CSE ActivityIfyou are having problems with the potency and

stability of reconstituted Control Standard Endo-toxin CSE), the syringes used during reconstitutionof the CSE may be the cause. While severalcustomers had reported isolated problems withsyringes, ACC was unable to reproduce them until afew months ago when our technicians experiencedproblems. Potencies were reduced by more thantwofold and tended to decrease over time. Ourproblem was traced to syringes located in the middleofan otherwise good box. Subsequentwork showedthat potencies were more reproducible when CSEwas reconstituted using glass pipets thanwhen usingthese syringes.Notice of this problem has been included withCertificates ofAnalysis to make customers aware ofthe issue. If you experience similar problems, werecommend that pipets be substituted for the syringeand needle to determine whether or not you shouldcontinue to use syringes. To reconstitute CSEwith apipet, either lift the CSE stopperjust enough to breakthe vacuum or insert a needle through the stopper tobreak the vacuum. Then remove the stopper. AddLRW with a pipet and seal the vial with ParafilmM Follow the instructions in the CSE insert forthe remainder of the reconstitution protocol.If you experience different activities between

vials ofCSE reconstituted with a syringe and thosereconstituted with a pipet, please contact ourtechnical services department and inform them ofthedetails. We would like to knowthemanufacturer andlot number ofany suspect syringes to help us and themanufacturer s) determine what substance s) maybe causing the problem. We would also like to knowof any adverse experiences with RSE lot F EC-5).Note that RSE lot G EC-6) is not stoppered undervacuum and is usually reconstituted with a pipet.If you have any questions regarding syringes or

CSE potency, please contact our Technical ServicesDepartment at 800-848-3248.

LAL UPDATE Vol. 13, um er 3, page6

CalendarDefense and Civil Institute of

Environmental MedicineSepsis and Septic ComplicationsEndotoxin and Sepsis:A Multi-center Clinical Trial of SepTestby Paul A. Ketchum, L.S. Stotts, TJ Novitsky,

1. Parsonnet and AMCC Sepsis Investigators.Toronto, CanadaOctober 12-13, 1995

Come visit us at the PDA 1995 Annual Meeting

Hynes Convention Center, Boston, MABooth 310

November 13-15, 1995Poster: Water Determinations in Lyophilized

Product Using Near Infrared Spectroscopy NIRS)by Marilyn 1. Gould, Ph.D.

andKevin 1. Barry

The American AutomobileManufacturers AssociationThe Industrial MetalworkingEnvironment:Assessment ControlPoster: Rapid Determination 60 seconds) ofBacterial Contamination in Industrial Fluidsby Dr. Jack SloyerHyatt Regency Dearborn, MINovember 13-16, 1995

The 4th Conference of theInternational Endotoxin SocietyOctober 22-25, 1996Nagoya, Japan