Iodine - Human Plasma Blood Purification

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Iodine-Mediated Inactivation of Lipid- and Nonlipid-Enveloped Viruses inHuman Antithrombin I11 ConcentrateBy F. Highsmith, H. Xue, X. Chen, L. Benade, J. Owens, E. Shanbrom, and W. Drohan

Human plasma-derived protein concentrates ntended forclinical use mustbe treated for viral inactivation o ensurepatient safety. This study explored the use of liquid iodinefor inactivation of several lipid- and nonlipid-enveloped vi-ruses in an antithrombin 111 (AT-Ill) concentrate. odine atlevels of0.01% to 0.02% caused between43% and94% lossof AT-Ill activity, as ell as degradation ofAT-Ill as shownbysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot analysis. However, additionof up to 0.1% human albumin protected the AT-Ill against

HE PAST DECADE has seen great advances in donorscreening, blood testing, and viral inactivation proce-

dures; however, the potential for disease transmission re-mains an area of concern in the use of human plasma-derivedproducts. Although usually considered safer in this respect,even products produced in tissue culture or transgenic ani-mals carry the possible risk of infection from crossover ofnonhuman viruses. Current viral inactivation techniques forbiologic products, such as solvenddetergent treatment andpasteurization, have improved the safety of many products.However, the potential transmission of nonlipid-envelopedviruses that are not inactivated by solvenddetergent, thermo-resistant viruses such as parvoviruses, prions, and other newinfectious agents are still potential obstacles in the path toproduction of safer products. Recent reports on the transmis-sion of human parvovirus B19,'" hepatitis and hepatitisB or C6,7 n plasma-derived protein concentrates treated forviral inactivation have led to increased efforts by manufac-turers to develop improved viral inactivation procedures. Onthe other hand, no licensed antithrombin I11 (AT-111) producthas been associated with transmission of hepatitis B, humanimmunodeficiency virus (HIV), or non-Ahon-B viral hepati-tis.8 This has been confirmed by viral inactivation studiesand patient follow-up stu di e~ .~ .' ~

There are currently only a limited number of techniquesavailable for viral inactivation or removal in biologic prod-ucts. The major difficulty is inactivating viruses while notdamaging the product. In fact, manufacturers realize a loss inyield of their product due to using many of these techniques.Iodine has a long history of use as a disinfecting agentagainst both bacteria and viruses.LL3L2o date, we have evalu-ated crosslinked polyvinylpyrrolidone-iodine,'3 crosslinked~tarch-iodine,'~nd liquid iodine for inactivating lipid- andnonlipid-enveloped viruses in normal human plasma andplasma-derived protein concentrates. This study explores theuse of liquid iodine for the inactivation of several lipid-and nonlipid-enveloped model viruses in a human plasma-derived AT-I11 concentrate.

AT-I11 is an anticoagulant used clinically for the preven-tion and treatment of thrombosis in congenitally deficientpatients. AT-I11 is a major inhibitor of serine proteinases andplays a vital role in the regulation of hemostasis. AT-I11 waschosen as a model for other plasma-derived proteins becauseof its availability as a high purity concentrate, its stabilityat room temperature, and its therapeutic importance.

Five different viruses were evaluated: HIV plus four oth-

T

Blood, Vol 86, No 2 (July 15). 1995: pp 791-796

both inactivation and fragmentation. t albumin levelssuf-ficient to retain greater than 75% of AT-Ill activity, greaterthan 6 logs of sindbis, encephalomyocarditis, and vesicularstomatitis viruses, greater than 4 logs of pseudorabies, andgreater than3 logs of human immunodeficiency virus wereinactivated.Except with sindbis virus, his represented com-plete inactivation of all the viruses spiked into the AT-Illconcentrate.0 1995 by The American Societyof Hematology.

ers that are often used as models for human pathogenicviruses. HIV is the retrovirus that causes AIDS. Encephalo-myocarditis virus (EMCV) has been well characterized phys-ically and biochemically and has been shown to be similarto hepatitis A virus.15 Sindbis virus (SINV) is related tohepatitis C virus.'6 Pseudorabies virus (PRV) is from thesame family as cytomegalovirus and herpes virus and is usedas a model for these.I7 Vesicular stomatitis virus (VSV),although not a model for any particular human pathogen, isoften used as a model for lipid-enveloped viruses in gen-eral."

MATERIALS AND METHODSAT-I11 conce ntrate. The AT-I11 used was a pasteurized, freeze-dried concentrate purified from human plasma by heparin-Sepharosechromatography , according to the method of Wickerhauser and Wil-liams." The concentrate was made for the American Red Cross byBaxter Healthcare Corp (Glendale, CA). After reconstitution, theproduct is in phosp hate-buffered saline (PBS) at pH 7.3 and has anAT-III potency of 58 IU/mL, a protein concentration of 11.9 mg/

mL and a specific activity of 4.9 IU/mg. The product is essentially100% AT-III by sodium dodecyl su lfate-polyacrylamide gel electro-phoresis (SDS-PAGE; Fig l ) .AT-111 assay . AT-111 concentration w as de termined as h eparincofactor activity using chromogenic substrate S-2238 (Kabi Phar-macia, Chromogenix Division, Franklin, OH) according to themethod of Abildgaard et a as modifiedby Wickerhauser andWilliams.'' Aliquots from a pool of fresh-frozen human plasma,standardized against the First International Reference Preparationfor AT-111: were used as a reference. Potency was expresse d ininternational units (IU).

Electrophoresis, C oomassie staining, and W estern blotting. Sam-ples were separated under reducing and nonreducing conditions bySDS-PAGE on 8% to 16% gradient gels.** The gels w ere stainedwith Coomassie Brilliant Blue R-250 (BioRad Laboratories, Rich-

From the Holland Laboratory, Plasma Derivatives D epartment,Submitted August 31, 1994; accepted F ebruary 20, 1995.Address reprint requests to Frank A. Highsmith, Holland Labora-tory, American Red Cross, 15601 Crabbs Branch Way, Rockville,MD 20855.The publication costs of this article we re defrayed in part by pagecharge payment. This article must therefore be hereby marked"advertisement" in accordance with 18 U.S.C . section 1734 solely toindicate this fac t.

American Red Cross, Rockville. MD.

0 1995 by The American Society of Hematology.0006-4971/95/8602-0014$3.00/0

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HIGHSMITH ET AL92

A.

4-c

1 2 3 4 5 6 1 8 9 1 0 1 2 3 4 5 6 7 0 9 1 0

- 97.4K-60.m- 4% l-L- 31K- 21.w- 14.4K =+

- 97.M- 66.2K- m- 31K- 21 m- 14.M

Fig l. SDS-PAGE under nonreduced (A) and reduced (B) ondkions: lane 1, AT-Ill control; lane 2, 0.08% albumin; lane 3, AT-Ill + 0.08%albumin; lane 4, 0.08% albumin + 0.188 mmol/L ascorbic acid; lane 5, AT-Ill + 0.788 mmol/L ascorbic acid; lane 6, AT-Ill + 0.02% iodine +0.788 mmo l/L ascorbic acid; lane 7,0.08% albumin + 0.02% iodine + 0.788 mmol/L ascorbic acid; lane 8, AT-Ill + 0.08% albumin + 0.02% iodine+ 0.788 mmol/L ascorbic acid; lane 9, AT-Ill + 0.05% albumin + 0.02% iodine + 0.788 mmol/L ascorbic acid; and lane 10, molecular weightstandards. Twenty micrograms of total protein was loaded in each lane. Lanes 1, 5, an d 6 contain 20 p g AT-Ill; lanes 2, 4, an d 7 contain 20pg albumin; lanes 3 an d 8 contain 14.6 p g AT-Ill plus 5.4 p g albumin; and lane 9 contains 16.6 pg AT-Ill plus 3.4 pg albumin.

mond, CA) or transferred to nitrocellulose filters (Hybond-ECL; then placed at 37C in 5% CO z overnight. Plaques were determinedAmersham Corp. A rlington Heights, IL) in the nonredu ced state for as described by Benade.I7Western blots. SDS-PA GE-prestained molecular weight markers EMCV tissue culture infecrious dose (TCID,) assay. Vero cells(BioRad Lab, H ercules, CA ) were used for molecular weight deter- were propagated in T-75 tissue cultu re flasks containing nonessentialmination. amino acid MEM with 10% fetal calf serum (FCS; Irvine Scientific,For Western blots, the nitrocellulose filters were blocked u sing Santa Ana, CA). Cells were seeded into 96-well tissue culture plates10% (wt/vol) skim milk powder inPBS (Life Technologies Inc.Grand Island, NY). The blocked filters were serially reacted witheither ( 1 ) 1:5,000 dilutions of sheep antihuman AT-Ill polyclonalantibody (Affinity Biologics, Hamilton, Ontario, Canada) and horse-radish peroxidase (HRP)-conjug ated rabbit antisheep polyc lonal an-tibody (Kirkegaard and Perry, Gaithersburg, MD) or with (2 ) 1 5 0 0dilutions of mouse antihuman serum albumin polyclonal antibody(Zymed Laboratories, Inc. San Francisco, CA) and H RP-conjugatedgoat antimouse polyclonal antibody (Kirkegaard and Perry). Theblots were developed with the Enhanced Chemilum inescence System(Amersham).

Cells andviruses. VSV (ATCC VR-1.58). SINV (ATCC VR-68) . PRV (ATCC VR-135). EMCV (ATCC VR-I29B), and Verocells (ATCC CC L 81) were obtained from the American Type Cul-ture Collection (ATCC) in Rockville, MD. Human T-cell lympho-tropic virus type lIlB (HTLV-IIIB), a well-established laboratorystrain of HIV, was obtained from UBI (Rockville, MD). MT2 cellswere obtained from the National Institutes of Health (NIH) AIDSResearch and Reference Reagent Program operated by Ogden Bio-Services (Rockville, MD). All cells and viruses w ere handled ac-cording to standard labo ratory practices as described by Benade."

VSV, PRV. and SINV plaque assay. Vero cells were propagatedin McCoy's 5A medium supplemented with 10% fetal bovine serum(FBS) and 2 mmol/L L-glutamine (GIBCOIBRL, Gaithersburg,MD ). Cells were seeded into six-well tissue culture plates and grow nto confluency. The monolayers were then infected with the testsample in duplicate for each dilution. A 250-pL inoculum was ap-plied, and infection was allowed to continue w ith intermittent rock-ing at 37C n 5% COz for 45 minutes. The inoculum was removed by

and grown to confluency. The monolayers were infected with hetest sample in quadrup licate for each sam ple dilution. A 50-pL inoc-ulum was app lied into the first well of the first four horizontal rowsof the 96-well plate, and this was repeated for the second fourhorizontal rows (two sam ples per plate). Samples were then seriallydiluted along the plate and incubated for 24 to 48 hours at 37C in5% CO z. Results were read using an inverted microscope accordingto the methods of Reed and Muench?'

HIV syncytial cell assay. The growth and assay of HIV n heMT2 cell line was performed according to the method of Harada etal,24 as modified by Benade.I7 Th e assay is based on the particularcytopathologic effects (appearance of syncytia) induced when thesecells are infected by HIV. Briefly, six to eight serial 2X dilutionsof the test sample were made across microtiter plates. MT2 cellswere then dispersed, centrifuged, resuspended. counted, adjusted toa concentration of 2 X 10' to 3 X IO' cells per m illiliter, and thenseeded in 100-pL volumes into the wells of the microtiter plate.Plates were incubated at 37C andmonitored for activity on days 2 to4, but the assay was typically scored on the fifth day by m icroscopicexamination for wells containing evidence of cytopathic effects;specifically, the presence of readily identifiable syncytial cells.TCIDso calculations were performed by the method of K a h x z s

Viral inacrivarion experiments. For V SV, SIN V, PRV, and HIVinactivation studies, I-mL stock suspensions of virus were diluted150 in a human AT-IIVPBS mixture (10.5 mg/mL AT-Ill) to yieldtiters of 6 to 8 log,dm L for VSV and SINV or 4 to 5 log,dmL forHIVandPRV. Subsequently, the AT-Ill plus virus mixture wasdiluted 1 5 with PBS ( I X PBS, Life Technologies Inc), resultingin a protein concentration of2.1 mg/mL. For EMCV inactivationaspiration. An overlay was prepared by mixing 2X minimal essential studies, 1 mL of a stock suspension of EMCV was diluted 1: 0 0 inmedium (MEM) without phenol red (GIBCOIBRL) and molten 2% AT-I11 to obtain a final titer of approximately TCIDso units ofBRL regular temperature agarose (cooled to approximately 42OC). EMC V and a final AT-111 concentration of2.1 mg/m L. A stock

1 : l by volume.Eachwell was overlaid with 2 to 3 mL of this solution of 2.5% albumin prepared immediately before use rommixture and allowed to harden at room temperature. Plates were albumin (human) US Pharmacopeia (Baxter Healthcare Corp) di-


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IODINE-MEDIATED VIRAL INACTIVATION 793

Table 1. Effect of Iodine Treatment on AT-Ill ActivityAlbuminscorbiccid AT-Ill ActivityIodine (%) 1%) (mmolR) (% )

0.01

0.015

0.0, controls 0.0 0.0 1000.05 0.0 1000.1 0.0 980.0 0.394 950.0 0.78820.0 0.0 570.03 0.394 780.03 0.394' 930.04.394 830.04 0.394* 940.0 0.0 220.04 0.591 560.05 0.591 640.05 0.591 930.1 0.591 860.1 0.591* 94

0.02 0.0 0.0 60.05 0.788 400.08 0.788 53

* Ascorbic acid was added before iodine, followed by a 30-minuteincubation.

luted n PBS was added as applicable, and all samples were thor-oughly mixed.A stock solution of 1% iodine/lO% potassium iodide was freshlyprepared from elemental iodine 99.8% , ACS grade, and potassiumiodide 9 9% , ACS grade, obtained from Aldrich Chemical CO (Mil-waukee, WI). After addition of the iodine solution, samples wereincubated for 30 minutes at ambient temperature (24C). At the endof 30 minutes, the samples were treated with .394 mmoVL ascorbicacid SigmaChemicalCO ,St Louis, MO)per 0.01% odine, thestoichiometricratio.Theascorbicacidwasaddedasa 0.1-moVLsolution of I-ascorbic acid, sodium salt (Sigma Chemical CO ),alsofreshly prepared. Additionally,stop controls wererun in which, afterthe 30-minute incubation and subsequent addition of ascorbic acid,virus was spiked into the samples.

RESULTSEffect of iodine on AT-III. Table 1 shows the effect of

iodine on AT-I11 activity, alone and in the presence of albu-min used as a stabilizer and ascorbic acid used to quenchthe iodine reaction. When AT-I11 was incubated with iodinealone, without albumin or ascorbic acid, at least 42% of theactivity was lost, with the loss increasing with increasingiodine concentration. As shown, the addition of albumin andascorbic acid serves to protect AT-I11 from inactivation, withthe amount of protection increasing with increasing albuminconcentration.

Figure 1 shows SDS-PAGE gels of various AT-III sam-ples under nonreduced (Fig 1A) and reduced (Fig 1B) condi-tions. In lane 6 , which shows AT-I11 treated with 0.02%iodine and quenched after 30 minutes with ascorbic acid,two new low-molecular-weight bands at 31 and 45 kD areobserved under both conditions. Under reduced conditions,an additional new band at 35 kD is also observed. Figure 2shows the corresponding Western blots with either AT-III-(Fig 2A) or albumin- (Fig 2B ) detecting antibodies. The AT-

111antibody picked up the two low molecular weight bandsat 31 and 45 kD in lane 5, the same sample that showed thenew bands on SDS-PAGE. This suggests that the new bandsare fragments of AT-111. This degradation is not seen whenAT-111 is treated under the same conditions but protectedwith albumin, as shown in lanes 8 and 9 of the SDS-PAGEgels or lanes 2 and 3 of the Western blots.Viral inactivation. The effects of various concentrationsof iodine on the inactivation of EMCV, PRV, SINV, VSV,and HIV in AT-111 with and without albumin and ascorbicacid are shown in Table 2. With iodine alone, completeinactivation ( 2 6 . 2 logs VSV and EMCV, 26 .9 logs SINV,24.4 logs PRV, and 2 3 . 3 logs HIV) of all viruses wasobserved. With iodine in combination with albumin andascorbic acid, allviruses except SINV were again completelyinactivated. Inactivation of SINV appears to be attenuatedby the presence of albumin. Neither AT-111, albumin, orascorbic acid alone caused any viral inactivation.

DISCUSSIONIodine exists in a number of different forms in aqueoussolution: elemental iodine (I2).hydroiodic acid (HOI), iodinecation (H201+), riiodide ion (IF), iodide ion (I), hypoioditeion (01-), and the iodate ion (IO;), all of which are in pH-dependent equilibrium. However, only elemental iodine (Iz)is believed to play a vital role in viral inactivation. Thesolubility of iodine in water at pH 7.5 is approximately 33 0mg/L at 25"C.26To increase the Iz content of solutions, potas-sium iodide (KI) is added. Iodide (I-) combines with I2 toyield triiodide, I;, which acts as a reservoir for the supplyof I2 through the equilibrium reactions. Nevertheless, theconcentration of the free 12, which is the active form, cannotexceed 33 0 m& at 25C. To quench the iodine reaction, 1-ascorbic acid (vitamin C) is used in these experiments. Thel-ascorbic acid reduces I2 to I- , which is not reactive.27 odineis known to react with various functional groups on proteinsand viruses. These include carbon-carbon double bonds onunsaturated fatty acids, amino groups on certain amino acids(histidine, arginine, lysine), oxidation of sulfhydryl groups(cysteine), reaction with phenol groups (tyrosine), and reac-tionwith bases on nucleotides (cytosine, adenine, gua-nine).",28 In reference to carbon-carbon double bonds, it isproposed that unsaturated fatty acids add to olefinic doublebonds, which might result in changes in physical propertiesof the lipids and a decrease in fluidity of the cell membrane.With respect to amino groups and nucleotide bases, there isa tendency of iodine to bind and block potential hydrogenbinding sites, which could lead to changes in protein struc-ture. In the case of cysteine becoming oxidized, it loses itsability to form disulfide bonds, which is known to play animportant role in protein synthesis. In the case of phenolgroups, it is known that iodine can bind in he ortho positionand sterically hinder hydrogen bond formation. However,the reaction of iodine with proteins or peptides usually differsfrom that of free amino acids as a result of electronic orsteric environments of the reactive centers. Thus, this leadsto considerable variation in the susceptibility of individualresidues to iodination."

Both crosslinked starch-iodine (XL-starch-iodine) and cross-


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794 HIGHSMITH ET AL

ATlll -45 kD-31 kD- h Albumin -

Fig 2. W d e r n blots for AT-Ill (A) and albumin (B) under nonreduced conditions: lane 1, low-molecular-weight markers; lane 2, AT-Ill +0.05% albumin + 0.02% iodine + 0.788 mmol/L ascorbic acid; lane 3, AT-Ill + 0.08% albumin + 0.02% iodine + 0.788 mmol/L ascorbic acid;lane 4,0.08% albumin + 0.02% iodine + 0.788 mmol/L ascorbic acid; lane 5, AT-Ill + 0.02% iodine + 0.788 mmol/L ascorbic acid; lane 6, AT-Ill+ 0.788 mmol/L ascorbic acid; lane 7, 0.08% albumin + 0.788 mmol/L ascorbic acid; lane 8, 0.08% albumin; lane 9, AT-Ill + 0.08% albumin;and lane 10, AT-Ill. Lane 2 contains l68 ng AT-Ill plus 72 ng albumin; lanes 3 and 9 contain 194 ng AT-Ill plus 72 ng albumin; lanes 47 and 8contain 248 ng albumin; and lanes 5, 6 and 10 contain 225 ng AT-Ill.

linked polyvinylpyrrolidone-iodineXLPVPI) have advantagesas viral inactivating agents. For example, XL-starch-iodine andXLPVPI are easy and inexpensive o manufacture, have a widem g e of part icle sizes, and, consequently, can be tailored to avarietyofchromatographicprocedures.Both are easily re-moved, thus minimizing the risk of producing an adulteratedproduct.Additionally,othightlyindodine,lthoughXLPVP binds iodine less tightly than XL-starch.' Conversely,not all situations require iodineo be tightly bound to a canier.In fact, a liquid iodine solution can be more advantageous forinactivating viruses in protein concentrates. The use of liquid

iodine eliminates the need to crosslink theanier, thereby elimi-nating a very time-consuming step. Another advantage of liquidiodine is the higher initial concentrationf free odine for viralinactivation. Although it can be argued that higher iodine con-centrations lead to higher losses of protein activity, we haveshown that in the presence of albumin, greater than 75% ofAT-III activity can be preserved.Iodine sensitivity (eg, hyperthyroidism, hypothyroidism,thyroiditis, and goiter) is mentioned in the literature as fa rback as the late 1800s. In most cases, the sensitivity resultedfrom excess iodine in foods, dietary supplements, topical

Table 2. Effect of IodineTreatment on Viral InactivationAlbumin

Log Virus Titer Reduction (n = 3)Ascorbic Acid

Iodine %l I%) (mmolR) vsv EMCV SlNV PRVIV0.0, controls 0.0

0.050.10.00.00.0

0.01 0.00.030.030.040.04

0.015 0.00.040.050.10.1

0.02 0.00.050.08

0.00.00.00.3940.394'0.1880.00.3940.394'0.3940.394'0.00.5910.5910.5910.591'0.00.7880.188

000000

26.226.2ND

26.2ND

26.226.226.226.2ND

~ 6 . 226.226.2

0ND0

ND00

26.226.20

26.20

26.226.226.226.20

26.226.226.2

000000

26.96.506.30

26.96.8

26.96.10

26.926.9

6.8

0000

ND0

24.324.3ND

24.3ND

24.324.324.324.3ND

24.324.324.3

0ND0

NDND0

ND23.3ND

23.3NDND

23.3ND

23.3ND

23.3ND

23.3Abbreviation: ND, not determined.

Virus was added immediately after the 30-minute incubation and ascorbic acid neutralization.


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IODINE-MEDIATED VIRAL INACTIVATIO N 795

medications, and/or iodinated contrast medias. It should benoted, however, that the majority of people are not affectedby excess iodine and that the quantity of iodine that is consid-ered excess varies from person to person. Many studies doneto determine population susceptibility to excess iodine maynot be accurate because they deduced their results from apatient population already immunocompromised by disease.Additional information concerning iodine dose and responsecan be divided into four components: route of intake, bio-availability of iodine, duration of intake, and physiologicstatus of subject. Additionally, few controlled toxicity stud-ies have been done, but are necessary to scientifically deter-mine safe limits for iodine.29 On the otherhand, it has beenreported that iodide, the product from iodine conversion byascorbic acid, is relatively

The data show that treatment with 0.01% to 0.02% iodinefor 30 minutes at 24C can cause complete inactivation ofa number of model viruses in an AT-111 concentrate. Iodinecan also cause a significant loss in AT-111 activity, as wellas fragmentation of AT-111. This activity loss and fragmenta-tion are mitigated significantly by the addition of albuminas a stabilizer and the use of ascorbic acid to quench theiodine reaction. Albumin and ascorbic acid also preserved asmall amount of SINV activity. The mechanism for protec-tion by albumidascorbic acid is not currently known. How-ever, it is likely that albumin acts as another target for iodineand thus reduces the effective concentration of iodine at-tacking AT-111 and viruses.

These results are encouraging with respect to the use ofiodine for viral inactivation in plasma products, but alsoraise several issues that must firstbe addressed. Activity lossis undesirable, but is also a characteristic of many otherviral inactivation methods. For instance, AT-111 is typicallypasteurized for 10 hours at 60Cwith 0.5 m o m sodiumcitrate as a stabilizer, with a resulting activity loss of 8% to30%.8,3he resulting activity loss becomes part of the costof the production process and is offset by the increased valueof a product with reduced viral infectivity. Of more concernis whether the inactivated species remaining in the producthave any detrimental effect to the patient. The heat-denaturedAT-111 remaining in pasteurized AT-I11 has never been ob-served to cause any untoward reaction in patient^.^' Theactual effect of iodine on AT-111 and albumin will be thefocus of future studies.

In summary, we report conditions in which greater than6 logs of SINV, VSV, and EMCV, greater than 4 ogs ofPRV, and greater than 3 logs of HI V can be inactivatedwhile maintaining greater than 75% of the AT-111 activity.Additional research is needed to determine the efficacy ofiodine on actual human pathogenic viruses, such as hepatitisA and human parvovirus B 19,as well as the effects of iodineon the product proteins.

ACKNOWLEDGMENTWe are grateful to Dr David B. Clark for his contributions to thepreparation of this manuscript.

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