LAL Update. Letter From the President. Dear LAL User:

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1 LAL Update ASSOCIATES OF CAPE COD, INCORPORATED OCTOBER 00 VOLUME 0, NO. Lette Fom the Pesident Dea LAL Use: This Update will claify some of the statistics used with tubidimetic and chomogenic LAL tests. Most LAL uses ae concened with epeatability, i.e. checking eplicate deteminations fo outlies and, epoducibility, checking whethe two laboatoies (o technicians) ae obtaining significantly diffeent esults. Unfotunately, thee is little LAL-specific guidance povided by eithe the FDA o the USP in this egad othe than limit values which indicate a valid test, i.e. a coelation coefficient of = 0.98 and a spike ecovey of 0 to 00%. The FDA in thei Guidance fo Industy --- Validation of Analytical Pocedues: Definition and Teminology uses the tems Pecision, Repeatability, Intemediate Pecision, and Repoducibility. The latte thee tems ae all subsets of Pecision which is defined as " the vaiance, standad deviation o coefficient of vaiation of a seies of measuements." Thus, the LAL use is mainly left to his own devices as to how to analyze esults to detemine whethe they ae in contol. At least one LAL manufactue ecommends in a chomogenic poduct inset unde "Pefomance Chaacteistics" that: "Replicate samples should be un to establish good technique and low coefficient of vaiation." This manufactue futhe ecommends: "The of [these] absobance values should be less than 0%. With expeience, values of -% should be attainable." In a tubidimetic poduct inset, this same manufactue indicates that the " () equals the "sample" standad deviation of the eactions times divided by the mean " Although no examples ae povided, it is obvious that this manufactue ecommends aw values, i.e. optical density values (fo chomogenic endpoint) and time of onset values fo kinetic tubidimetic to calculate It should be noted that these ecommendations ae not geneal equiements fo all LAL eagents, but ae specific to the LAL poduct(s) coveed by thei espective poduct insets and would be suppoted by data geneated by this manufactue. Associates of Cape Cod, Inc. (ACC) on the othe hand does not include its expectations fo in its poduct inset but does include this calculation in the softwae which accompanies the LAL-000 and Pyos Kinetix machines as a convenience fo ou customes. ACC's s howeve ae calculated fom EU/ml values athe than time of onset. Although s should be a elatively simple and convenient check fo pecision, due to a lack of claification on the pat of LAL manufactues and the FDA, thee is definitely some confusion among LAL uses. In addition, some LAL uses may be using s incoectly, leading to a false sense of secuity elated to the epoducibility (o lack theeof) of thei assays. Hopefully this UPDATE will claify the use of s. Fo this UPDATE I have elied heavily on the expeience of M. Keith Richadson, ACC's Instumentation Manage. Keith has been with ACC fo yeas, actually beginning as a pat-time hoseshoe cab "bleede" and woking his way though ou Technical Sevices Depatment. Although Keith is cuently esponsible fo machine manufactue, maintenance, and calibation, he is still ACC's "kinetic assay expet" and continues to help with technical inquiies elated to the LAL-000, Pyos Kinetix machine, and plate eades, and paticipates in wokshops and site visits. Duing his tenue at ACC he has accumulated moe expeience with kinetic data than anyone else in the company and possibly moe than any othe LAL use. This UPDATE concludes with an aticle on Simple Statistics fo the LAL Use - Standad Deviation, Repeatability, Repoducibility and a claification of the Coefficient of Vaiation. Sinceely, Thomas J. Novitsky, Ph.D.

2 T E C H N I C A L R E P O R T Simple Statistics fo the LAL Use - Standad Deviation, Repeatability, Repoducibility and a Claification of the Coefficient of Vaiation. By Keith Richadson & Thomas J. Novitsky Using the standad deviation and coefficient of vaiation The standad deviation (actually the standad deviation of the mean as used in this context) is calculated accoding to the fomula(s) in Figue. Thee ae at least two ways to calculate standad deviation depending on whethe the entie population (n) is measued o only a epesentative sample (n-) is used. It can be agued that the values (e.g. times of onset, EU/ml) geneated fo each eplicate in an LAL assay epesent the entie population (the population in this case being all the eplicates fom a single concentation). On the othe hand, the volume of endotoxin standad solution used in the actual LAL test usually epesents only a small pecentage of the oiginal dilution. The test also employs only a single vial fom lage lots of CSE, LAL, LRW, etc., theefoe it may be moe appopiate to use equation. Note that fo small numbes of eplicates, equation is significantly moe consevative, i.e. esults in highe values fo standad deviation and subsequently coefficient of vaiation (). than equation. Fo these easons, Associates of Cape Cod, Inc. (ACC) pefes to use equation. The standad deviation has a unit value that is the same as that attibuted to the mean, e.g. if the mean of the times of onset is used to detemine the standad deviation, seconds would be the unit; likewise, if endotoxin units wee used, EU/ml would be the unit. The standad deviation is also sometimes efeed to as the standad eo. It is a diect measue of pecision, i.e. the lowe the standad deviation, the highe the pecision. The standad deviation is also a measue of vaiation, i.e. the lowe the standad deviation, the lowe the vaiation (vaiability). The is the standad deviation expessed as a pecentage of the mean. It is useful when compaing means that diffe substantially. Although it is expessed as a pecentage and is thus unitless, units ae implied. Theefoe s calculated fom times of onset cannot be compaed to s calculated fom optical densities o EU/ml. While it is easy to use the standad deviation when compaing one set of LAL measuements to anothe, if the two measuements diffe substantially in thei means, a diect compaison of the standad deviation is not possible. This is because values obtained at the extemes of a measuement spectum could be expected to have moe vaiation. Fo example, measuement of a numbe of eplicates of EU/ml (vey shot time of onset) esulted in a standad deviation of 0. and a of.9% (calculated fom endotoxin values as EU/ml) while eplicates at 0.0 EU/ml (modeate time of onset) had a standad deviation and of 0.0 and % espectively. Thus, in this example, if only standad deviations ae compaed, the vaiability of the standad fo EU/ml looks wose than that at 0.0 EU/ml when in fact the indicates moe vaiability fo the lowe concentation. Theefoe if a technician is inteested in assessing thei LAL assay skills, they should use standad deviations as a measuement of pecision only when compaing a single concentation fom one assay with the same concentation in a sepaate assay. To compae pecision between concentations, the analyst would need to use s. It should be pointed out at this point (so as not to discouage ou eades) that excellent (i.e. low) values can be obtained acoss the spectum of LAL concentations. Thus, although highe values tend to be obtained at the ends of the test ange, a technician with good laboatoy skills can expect low values at all points on the standad cuve. Thee is howeve no absolute that is indicative of a "good" o a "bad" test. As a ule of thumb, a "< 0%" value is consideed "good". Howeve, as I will illustate in the next example, not all s ae ceated equal. As mentioned above, although s ae unitless, units ae implied. We have also leaned that we cannot compae s that wee calculated using diffeent units. But what units should be used fo this calculation (o does it even matte)? The answe to this question is not as staightfowad as one might think since the choice of units affects the size of the value. In Vol. 9 of the Endosafe Times, s wee used to compae standads pefomed by an analyst vs. those obtained by a obot. To pefom the compaison, time of onset values wee used. The esulting s fo all assays wee less than 0% although the analyst was geneally bette than the obot (8 out of paied s wee lowe fo the analyst although this obsevation was not highlighted by the Endosafe Times autho). Inteestingly, one of the conclusions dawn fom this study was that since all s wee less than 0%, the obot method could be intechanged with the analyst method. But is this tue? What if the statistician had decided

3 to use calculated EU/ml values instead of times of onset? Since EU/ml ae calculated fom time of onsets, what diffeence could it make? Table. uses the data fom the "Compaison: Reaction Times of ic vs. Analyst Cuves" as published in Vol. 9, No. of the Endosafe Times conveted to EU/ml. s wee then calculated fom the standad deviations of the means and compaed to the s calculated fom the times of onset. The esults ae damatic. Although thee is definitely a coelation between the two sets of s, i.e. high s calculated fom time of onset data coespond to high s calculated fom EU/ml values, the EU/ml s ae substantially geate than those fom times of onset (ange of s fom time of onset = 0. to.% while those calculated fom EU/ml ange fom 0.98 to.%). Thus if one used EU/ml values to calculate, the "geneal ule" of <0% would have failed some of the assays. It is inteesting to note that only out of assays fo the "analyst" esulted in s >0% (one was close at 0.%) while out of assays fo the "obot" would have failed. Should the statistician theefoe choose the data set that gives the lowest values when calculating the? Thee is no solid answe to this although if one is compaing an assay o standad whose intention is to measue endotoxin concentation, then it seems easonable to use endotoxin concentations fo the statistical analysis. This is the position taken by ACC. The calculation fo included in Pyos (and Pyosoft ) employs the standad deviation of the means in EU/ml units. Table. illustates an actual kinetic tubidimetic assay that compaes s calculated fom the time of onset vs. those calculated fom endotoxin values. Note that although thee is a coelation between s calculated fom the diffeent data sets, the actual values fo those calculated fom time of onset ae always of a lowe magnitude. Since the EU/ml values ae obtained fom a standad cuve that employs the time of onset, ACC believes using these tansfomed values povides a moe igoous test of pecision. In ode to be useful, values need to be compaed with those detemined afte pefoming the test on a lage numbe of samples. At ACC, values (calculated fom EU/ml) unde 0% ae outinely obtained fo all standad concentations, howeve, values between 0 and 0% ae common fo the low concentations, e.g. EU/ml. Uses of ou eagent and softwae should expect simila esults afte sufficient pactice. The eal value of s, howeve, is as a continual check fo pecision and with the new Pyosoft softwae uses will be able to tend thei own s. Repeatability and Repoducibility Calculations The FDA in thei "Guidance fo Industy-Validation of Analytical Pocedues defines Repeatability as measuement that " expesses the pecision unde the same opeating conditions ove a shot inteval of time. Repeatability is also temed inta-assay pecision." In othe wods, epeatability is the pecision within a laboatoy. Repoducibility on the othe hand is defined as a measuement that " expesses the pecision between laboatoies (collaboative studies, usually applied to standadization of methodology)." The FDA povides an additional definition of "Intemediate pecision" defined as a measuement that " expesses within-laboatoies vaiations: diffeent days, diffeent analysts, diffeent equipment, etc." s of couse with the cautions noted above could be compaed to get an idea of the pecision fo all these goups. Refeences ) Biomety, Sokal, Robet R., and F. James Rohlf, second ed. W.H. Feeman and Company, New Yok. 98. ) Guidance fo Industy Validation of Analytical Pocedues: Definition and Teminology. Final Guidance. U.S. Depatment of Health and Human Sevices, Food and Dug Administation, Cente fo Veteinay Medicine, July 999. ) Endosafe Times Volume 9, Numbe. Mach 00. Figue. whee: Fomulas fo the Calculation of Standad Deviation )* Standad Deviation = )* Standad Deviation = n x - ( x) n n x - ( x) n(n-) n = sample size, e.g. numbe of eplicates x = mean (of time of onset, EU/ml, etc.) * Fomula used to calculate Standad Deviations and subsequent s in Refeence.

4 T E C H N I C A L R E P O R T Table. "Compaison: Reaction Times of ic vs. Analyst Cuves" Using s Fom EU/ml.* Day. Using Regession Y - intecept Day. Using Regession Y - intecept Day. Using Regession Y - intecept Day. Using Regession Y - intecept * Raw data fom Ref.

5 T E C H N I C A L R E P O R T Table. Compaison of s Calculated Fom Onset Time and Endotoxin Concentation File: Test.lv Well Desciption Standad Conc. Units Raw Onset Adjusted Onset CV Onset Calc. Endotoxin CV Endotoxin Neg. Ctl. Neg. Ctl. EU/ml EU/ml EU/ml EU/ml EU/ml EU/ml EU/ml EU/ml : Y Intecept: Coelation Coefficient: New CFR Pat Compliant Softwae Call us o talk to you local sales epesentative about Pyosoft- and see how ou newly designed softwae implements the equiements of CFR pat and its featue set. Pyosoft TM - CFR Pat Compliant Runs on Access and SQL Database Fomats Multiple Access Levels fo Impoved Secuity Detailed System, Use, and Assay Audit Tails Built in Tending And Much, Much Moe...

6 CALENDAR OF EVENTS DECEMBER 00 Decembe 9- PDA Annual Meeting New Oleans Maiott Hotel New Oleans, LA Booth # 00 MARCH 00 Mach 7-9 PDA Sping Confeence Paadise Point Hotel San Diego, CA Booth # 0 Fo custome sevice: call (800) LAL TEST o (08) 0. Fo technical sevice: call (800) 88 8 o (08) 0. Please visit ou website! 00 Associates of Cape Cod, Inc. All ights eseved. Pinted on ecycled pape. 70 Main Steet Falmouth, MA 00 VOLUME 0, NO.

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