reducing the risk of transfusion-transmitted rickettsial diseaseby wbc filtration, using orientia...

METTILLE ET AL.

290 TRANSFUSION Volume 40, March 2000

Increasingly stringent donor-screening practices, withspecific questions targeted to defer persons identifiedas belonging to high-risk groups, along with infectiousdisease marker testing, have significantly reduced the

transfusion transmission of disease.1,2 Methods for the in-activation of most viruses in plasma have been accomplishedwith the introduction of S/D treatment.1,2 The inactivationof viruses and bacteria in platelet concentrates by psoralenand long-wavelength UV light is still in the early stages ofdevelopment and is not yet approved by the FDA, but itlooks promising.3 Unfortunately, neither virus inactivationnor psoralen treatment for RBC components has been ap-proved at this time. The safety of RBC components is reli-ant on infectious-disease marker testing and prevention bydonor screening. Neither method provides a safeguardagainst an asymptomatic but infectious donor whose dis-ease state has not progressed to the stage of seroconversion.

Reducing the risk of transfusion-transmitted rickettsial diseaseby WBC filtration, using Orientia tsutsugamushi

in a model system

F.C. Mettille, K.F. Salata, K.J. Belanger, B.G. Casleton, and D.J. Kelly

BACKGROUND: Careful donor screening and infectiousdisease marker testing have significantly reduced the in-cidence of transfusion-transmitted diseases and im-proved the safety of the blood supply. However, transfu-sion-transmitted diseases resulting from the use ofasymptomatic yet infectious donors continue to put pa-tients at risk. This study was undertaken to determine ifthird-generation WBC filters could remove Orientia tsut-sugamushi-infected cells from contaminated blood.STUDY DESIGN AND METHODS: Packed RBCs wereinoculated with human MNCs infected with O. tsutsuga-mushi at levels estimated to occur in asymptomatic in-fectious donors. WBC reduction was accomplished witha third-generation WBC filter. Prefiltration andpostfiltration specimens were collected, serially diluted,and injected into mice to determine the infectivity of thesamples.RESULTS: Mice receiving WBC-reduced packed RBCsshowed no signs of illness or markers of infectivity,which suggested that a reduction of as much as 105 in-fectious rickettsiae could be achieved by filtration.CONCLUSION: The high-efficiency, third-generation,WBC-reduction filters that were tested may provide pro-tection against the transfusion transmission of scrub ty-phus rickettsiae by removing from contaminated bloodcells that contain intracellular bacteria.

ABBREVIATIONS: DFA = direct fluorescent antibody; IFA = in-

direct fluorescent antibody.

From the Department of Clinical Investigation, Walter Reed

Army Medical Center, and the Department of Rickettsial Dis-

eases, Walter Reed Army Institute of Research, Washington DC;

the Viral and Rickettsial Diseases Program, Naval Medical Re-

search Center, Bethesda, Maryland; and the Department of Biol-

ogy, Bowling Green State University, Bowling Green, Ohio.

Address reprint requests to: CDR Frank Mettille, USJFC,

1562 Mitscher Avenue, Suite 200, Norfolk, VA 23551; e-mail:

[email protected].

The opinions and assertions contained herein are the pri-

vate views of the authors and are not to be construed as official

nor as reflecting the views of the Department of the Army, the

US Department of the Navy, the US Naval Service at large, or the

Department of Defense.

Supported in part by the Department of Clinical Investiga-

tion, Walter Reed Army Medical Center (Work Unit #4827), and

the Naval Medical Research Center (Work Unit #62787

A.001.01.EJX.1295).

Received for publication July 6, 1998; revision received June

6, 1999, and accepted June 16, 1999.

TRANSFUSION 2000;40:290-296.

T R A N S F U S I O N C O M P L I C A T I O N S

REMOVAL OF RICKETTSIAE FROM BLOOD BY FILTRATION

Volume 40, March 2000 TRANSFUSION 291

Nonimmune asymptomatic donors may therefore transmitviral, bacterial, or parasitic infections to blood recipients.Both Rickettsia rickettsii and Orientia tsutsugamushi havebeen implicated in cases of transfusion transmission of dis-ease.4,5 In July 1997, over 700 units of blood componentscollected from military personnel who had trained at FortChaffee, AR, were recalled because of the potential for transfu-sion transmission of tick-borne illnesses.6,7 This incidenthas heightened awareness of the potential threat of rickett-sial agents to the blood supply.

Rickettsiaceae are obligate intracellular parasites thatstain as small, pleomorphic, gram-negative coccobacillithat are easily visualized in the cytoplasm of macrophagesand other MNCs.8 They are usually transmitted to humansvia arthropod vectors including fleas, ticks, body lice, andmites; some are maintained in wild-animal reservoirs suchas mice, rats, and other rodents. Rickettsiae are the etiologicagents for many diseases, including scrub typhus (O. tsu-tsugamushi), Rocky Mountain spotted fever (R. rickettsii),and epidemic typhus (R. prowazekii). Most rickettsial spe-cies are easily killed by disinfectant chemicals, but, like vi-ruses, they are best preserved by quick freezing and stor-age at temperatures below –75°C. R. prowazekii and R.rickettsii have been preserved for more than 40 years in adry-ice chamber (Dasch GA written communication, April1997), and O. tsutsugamushi has been shown to survive nor-mal blood banking procedures.9

On July 17, 1998, the British government announcedthat it was introducing WBC reduction for all blood andblood components derived from whole blood destined fortransfusion. Acting on recommendations of the SpongiformEncephalopathy Advisory Committee, the government saidthat the National Blood Service would move to WBC reduc-tion to reduce the risk of transmission of new variant CJD.10

Although the United States government does not yet requireWBC reduction, the FDA’s Blood Products Advisory Com-mittee recommends the universal WBC reduction of all non-WBC cellular blood components for transfusion.11 Filtrationwith third-generation high-efficiency filters is generally viewedas the most efficient and effective method of WBC reduction.1

The efficiency of WBC removal by filtration can be ex-pressed in terms of log reduction value.12 This is the nega-tive logarithm of the ratio of WBCs in the effluent to theWBCs in the original unit. On the basis of design and con-struction, manufacturers of third-generation filters claim toachieve a log reduction value of at least 3 (the removal of99.9% of WBCs, leaving on average 105-106 residual WBCs).Actual reduction is dependent on many factors, includingthe age of the unit, the total time of filtration, and the tem-perature. The mechanism of filtration incorporates suchfactors as entrapment, surface charge and energy, and theinteractions of receptors of the WBCs with the filter material.

No study has yet been conducted to determine if thetransmission of rickettsia-infected packed RBCs can be pre-

vented by WBC reduction using third-generation WBC fil-ters. Laboratory mouse models have been used effectivelyin a variety of O. tsutsugamushi research investigations, includ-ing those on antibiotic efficacy, pathogenesis, and virulence.13,14

While the sensitivity of the mouse system seems quite highand is not a direct measure of human infectivity, previousresearch has shown that as few as one or two organisms cankill or immunize a mouse,13-15 thus providing adequate sen-sitivity for this work. In the present study, the effect of WBCreduction on the transmission and infectivity of O. tsutsuga-mushi was assessed by using the established mouse model.The results of these studies may have implications for safe-guarding the blood supply from rickettsial diseases.

MATERIALS AND METHODSCollection, preparation, and infection of wholebloodThe research protocol employing human subjects in thisstudy was reviewed and approved by the Institutional Re-view Board of the Walter Reed Army Medical Center and theWalter Reed Army Institute of Research. Whole blood wascollected from volunteer donors and separated into com-ponents. Except as noted, the materials and methods usedhave been described previously.9 Briefly, RBCs werealiquoted at the required concentrations by using a qua-druple-bag transfer container (Baxter Fenwal, Deerfield, IL)and were stored at 1 to 10°C before infection. MNCs wereisolated from whole blood, infected with O. tsutsugamushi(Karp strain), and incubated in 75-cm2 cell culture flasks for2 to 3 days at 35°C with 5-percent CO2.9,15 The infected MNCswere harvested and counted, and the number of rickettsiaeper infected cell was determined by Giemsa staining. Foreach trial, 200 MNCs were scored to determine the percent-age of cells infected, and rickettsiae were counted in 50 ofthe infected cells to determine the average number of or-ganisms per infected cell. Rickettsiae-infected MNC sus-pensions were inoculated into the RBC aliquots at roomtemperature using an 18-gauge needle and syringe and aninserted sample-site coupler (Baxter). Aliquots were re-moved for serial dilution and mouse inoculation.

Sample preparation and mouse inoculationAll animals used in this study were maintained and handledin accordance with the Institute of Laboratory Animal Re-sources’ Commission on Life Sciences, National ResearchCouncil guidelines.16 The inoculated RBC aliquots wereWBC reduced by use of a high-efficiency, rapid-flow WBCfilter (RC400, Pall Biomedical Products, East Hills, NJ). Theprocedure was performed at room temperature in accor-dance with the manufacturer’s instructions, within approxi-mately 30 to 70 minutes, depending on the volume filtered.The filtered RBCs were collected in Baxter transfer bags.Two- to 5-mL aliquots of the nonfiltered and filtered RBCs

METTILLE ET AL.


were removed from the transfer bags by using an 18-gaugeneedle and syringe and an inserted sample-site coupler.

The samples were used to make 1-in-10 serial dilu-tions, using sterile PBS at pH 7.4 (Sigma Chemical Co., St.Louis, MO). The infected aliquots were blended for 5 min-utes, using a 10-mL glass tissue grinder (Kontes; obtainedfrom PGC Scientific, Frederick, MD) on crushed ice. One-half mL of each of the serially diluted samples was intrap-eritoneally inoculated into each of three (Trial I), five (TrialII), or two (Trial III) CD-1 mice (Charles River Laboratories,Wilmington, MA).9

Detection of O. tsutsugamushiThe mice were monitored daily for signs of sickness andinfection, including ruffled coats and morbidity, as de-scribed previously.17 When mice displayed signs of infectiv-ity, one mouse from the set was sacrificed for evaluation.Mice remaining asymptomatic for the entire 17- to 21-dayobservation period were also sacrificed and similar samplescollected and tested.9 A 25-µL sample of blood for assay ofOrientia DNA by PCR was collected from surviving mice byretro-orbital bleeding (Trial III). The blood samples wereabsorbed onto a 2-cm2 piece of filter paper (#16030,Schleicher & Schuell, Keene, NH) that was air-dried andstored at 4°C for later analysis. Necropsies were performed,and samples of the liver and spleen were retained andstored, frozen at –60°C. Two impression smears were pre-pared from the peritoneal exudate, one fixed in absolutemethanol at room temperature for 5 minutes for Giemsastaining and the other fixed in cold acetone (1 to 10°C) for5 minutes for specific direct fluorescent antibody (DFA)staining as described by Casleton et al.9 and Dohany et al.18

For the indirect fluorescent antibody (IFA) test for specificantibody, whole blood obtained by cardiac puncture was al-lowed to coagulate for 12 to 18 hours at 1 to 10°C. Serum wasrecovered by centrifugation and stored, frozen at –20°C,until tested as described by Robinson et al.19 using culturedmouse L-929 cells infected with Karp strain O. tsutsuga-mushi. Genomic DNA of O. tsutsugamushi was extractedfrom the blood spots on the filter paper by boiling the pa-per in 300 µL of 5-percent resin (Chelex-100, Bio-Rad, Rich-mond, CA) in sterile-distilled water for 10 minutes.20

The PCR assay employed first amplifies a 1412-bp re-gion of the groESL operon of O. tsutsugamushi. The DNAamplification reagents (GenAmp N801-055, Perkin-Elmer,Norwalk, CT) were employed by using a programmable thermalcycler (Model PTC-100, MJ Research, Watertown, MA). The47-µL amplification mixtures contained the following (finalconcentration): 3.5 µL of 10× buffer (1.5 mM MgCl2, 50 mMKCl, 10 mM Tris [pH 8.3], 0.001% gelatin), 8 µL of dNTP mix-ture (0.2 mM of each dNTP), 5 µL of each primer(786F 5´-TACCAACCACTGTATGATCG-3´ and 2197R 5´-TCACGGATCTGTTCACAACG-3´ [0.8 µM], Midland, Midland,TX), and 25.5 µL of filtered, distilled water. PCR employed

a hot-start procedure.21 The reaction mixture was heated to80°C for 10 minutes, and 2 µL of DNA extract and 1 unit ofTaq DNA polymerase were added. Amplification proceededafter the reaction was heated to 94°C for 4 minutes, by 35cycles of 94°C for 1 minute, 50°C for 3 minutes, and 70°C for2 minutes, followed by overnight incubation at 4°C.

The second amplification was performed similarly,except that 5 µL each of two primers internal to the first pair,1149F 5´-ATTGTACATGGTGATCAATG-3´ and 1667R 5´-TTCAACAGTTATCACTCCTT-3´ (Cruachem, Sterling, VA),and 1 µL of the primary PCR component in place of ex-tracted DNA template were used, and annealing was at57°C, rather than 50°C. The internal primer sequences usedwere provided by G.A. Dasch (Naval Medical Research In-stitute, Bethesda, MD). Amplification of the extracted DNAwas determined by visual detection of the ethidium bro-mide-stained bands (Sigma) as described previously.9

Statistical analysis. All statistical analysis was per-formed by using statistical software (SPSS 8.0, SPSS, Chi-cago, IL). Comparisons between groups were made by us-ing chi-square tests with Yates’s continuity coefficient toreduce the possibility of Type I error.

RESULTSThis study was conducted to determine if the risk of trans-fusion of the intracellular pathogen O. tsutsugamushi couldbe reduced by WBC reduction using a third-generation,high-efficiency WBC filter. Three trials were performed,each differing in the amount of RBCs filtered (50 mL in TrialI, 200 mL in Trials II and III), the infectivity of the inoculum,the number of mice tested, and the serial dilutions tested.

Recovery of infected MNCs and degree ofinfectivity after incubationExamination of Giemsa-stained cytospin smears revealedthat the percentage of infected MNCs ranged from 7.75 to14.5 (Table 1). Rickettsiae stained as deep blue inclusionsin the cytoplasm. The number of rickettsiae counted perinfected cell ranged from 1 to 30. Although Trials I and II hada greater percentage of infected cells than Trial III, thosecells infected in the latter trial averaged greater numbers ofrickettsiae per cell.

Inoculation of RBC aliquotsThe number of rickettsiae per mL of inoculum used to seedthe RBCs varied with each trial, because of the variabilityin the three factors determining the inoculum: the numberof MNCs per mL in the initial suspension, the percentageof cells infected, and the average number of rickettsiae perinfected cell. Approximately 106 rickettsiae per mL of MNCinoculum were used in Trials I and II. The Trial III MNC in-oculum contained a concentration of rickettsiae about 10-fold that in Trials I and II.



Serially diluted inoculum concentrationsFor each trial, 1-in-10 serial dilutions of filtered and unfil-tered seeded RBCs were inoculated into mice. The esti-mated number of rickettsiae released from the seeded RBCsafter disruption by blending with the tissue grinder variedin each trial (Table 2). The initial, undiluted, seeded RBCsin Trials I and II represented about 6 × 104 (57,500 and72,000, respectively) rickettsiae, with Trial III increasing thequantity to about 7 × 105 rickettsiae.

Mouse evaluation of O. tsutsugamushi infectivityMouse infectivity data on the detection of O. tsutsugamushiin the three trials compared favorably (Table 3). In each ofthe three trials, only mice that received nonfiltered, in-fected, serially diluted inocula displayed evidence of O. tsu-tsugamushi infectivity, such as illness, death, rickettsiae byDFA, or Giemsa stain: specific antibody by IFA; and, for TrialIII, specific DNA by PCR (Fig. 1). Mice that became ill didso 9 to 16 days after the rickettsial inoculation. Mice thatdied did so 15 to 17 days after inoculation, approximately 6to 7 days after the first signs of illness.

Nested PCR for DNA analysis of inoculated micePCR analysis was performed on DNA extracted from whole-blood spots obtained by the retro-orbital bleeding of allsurviving Trial III mice on Day 21, before sacrifice. PCR wasalso performed on a liver and spleen homogenate obtainedfrom the only mouse that died in any of the trials, after in-oculation with filtered blood. This mouse, which had beeninoculated with 0.5 mL of filtered blood (100), died on Day15 without showing typical signs of illness. Nested PCRanalysis identified bands approximating those of the posi-tive control on specimens obtained from the mice inocu-lated with nonfiltered dilutions (10–2 and 10–3) (Fig. 1). Noneof the mice inoculated with filtered dilutions, including themouse from Trial III that died on Day 15 after inoculation,had detectable O. tsutsugamushi DNA in PCR (Table 3).

Statistical analysis. Significantdifferences were found between filteredand nonfiltered dilutions with regard toillness (p = 0.0001), Giemsa staining (p =0.04), IFA (p = 0.0001), DFA (p = 0.02), andPCR (p = 0.005), but not death (p = 0.35).One goal of this study was to test the nullhypothesis that the proportion positive(infected mice) was identical in the twopopulations. The criterion for signifi-cance (alpha) was set at 0.05. The testwas one-tailed, which means that onlyan effect in the expected direction wouldbe interpreted. A one-tailed test wasused because there was no biologicallyplausible reason to believe that filtering

would lead to an increase in illness, death, or other out-comes of interest. With a sample size of 62 for each of thetwo groups (filtered and nonfiltered), the study had a powerof 80 percent to yield a significant result. This computationassumes that the difference in proportions is 22 percent. Asmaller difference in rates could not be detected with ac-ceptable power. It is also assumed that the magnitude ofthis effect size is reasonable, in the sense that an effect ofthis magnitude could be anticipated in this field of research.

DISCUSSIONThe results of this study suggest that high-efficiency, third-generation, WBC-reduction filters from at least one com-pany may provide protection against the transfusion trans-mission of scrub typhus by removing intracellular bacteriafrom contaminated blood. Human MNCs infected with O.tsutsugamushi were chosen, because sensitive isolation anddetection methods were available for that organism.Casleton et al.9 showed that an inoculum of infected MNCswith as few as 18 rickettsiae stored in units of RBCs for upto 10 days at 4°C was sufficient to infect mice. Their datasupport the reported case of the transfusion transmission

TABLE 1. Intracellular infection with O. tsutsugamushi of MNCs used asinoculum

Average Rickettsiae TreatedPercentage of number of per mL of RBCs Rickettsiae

MNCs MNCs rickettsiae per infected plus MNC per mLTrial (/mL) infected infected MNC* MNCs† inoculum of RBCs‡

I 6.67 × 106 14.50 5.95 5.75 × 106 049 + 1 1.16 × 105

(50)II 6.67 × 106 12.75 8.50 7.23 × 106 196 + 4 1.46 × 105

(200)III 1.00 × 108 07.75 8.95 6.94 × 107 196 + 4 1.38 × 106

(200)

* Number of individual rickettsiae counted in Giemsa-stained cells observed under 1000×magnification.

† Total MNCs per mL of inoculum × number infected cells per 100 cells x average numberof rickettsiae per infected cell = number of rickettsiae per mL of inoculum.

‡ Each mouse inoculated with 0.5 mL of contaminated packed RBCs, undiluted or diluted.

TABLE 2. Estimated number of rickettsiae (O.tsutsugamushi) per mL of packed RBCs after serial

dilution of inoculaNumber of rickettsiae(/mL)

Dilution Trial I Trial II Trial III

Undiluted 57,500* 72,000 695,0001-in-10 05,700* 07,250 069,5001-in-100 0,575 0,0725 006,9501-in-1,000 00,057.5 000,0,72.5 NT1-in-10,000 00000,5.75 00000007.25 NT1-in-100,000 000000,0.575 000000000.727 NT1-in-1,000,000 000000,0.058 000000000.073 NT

* Number of rickettsiae per mL of inoculum (based on direct count)× correction factor for dilution (inoculum + RBCs) × correctionfactor for amount inoculated (0.5 mL) × correction factor for se-rial dilution = number of rickettsiae in infected diluted inoculum.

METTILLE ET AL.


of Rocky Mountain spotted fever from a unit of blood thathad been stored for 9 days at 4°C and at room temperaturefor less than 1 hour.5 Our data suggest that the risk of trans-fusion transmission of O. tsutsugamushi, implicated in acase of transfusion-transmitted rickettsiosis, might be re-duced by WBC filtration.4

Transfusion-transmitted rickettsial disease has beendocumented in only a few cases. Rickettsial disease in oth-erwise healthy individuals causes symptoms that includesevere headache, myalgia, anorexia, fever, chills, and rash.Unchecked by antibiotic therapy, the disease may progressto anuria, deafness, pulmonary edema, or cardiac fail-ure.5,22-25 Although reports vary, the rate of mortality in un-treated scrub typhus may vary between 0 and 60 percent,depending on the strain of Orientia involved and on thehost factors.22-24 It is likely that patients with compromisedmedical conditions requiring transfusions would incur ahigher rate of morbidity and mortality.

Transfusion transmission of rickettsial disease, whilenot posing a major concern in the United States, may wellbe a potential threat in countries where the disease is en-demic (in Asia and the Southwest Pacific) and for the USmilitary units deployed to these and other regions.22,26-29 Infact, there is cause for greater concern, because of recentevidence of antibiotic-resistant scrub typhus in Thailand,a country of historic, strategic military interest to the UnitedStates.30 Currently, the US military has a significant pres-ence in that part of the world, with troops stationed in Ko-

Fig. 1. Agarose gel electrophoresis of nested PCR-amplified

DNA using 519-bp nested primer set. DNA template consisted

of primary PCR DNA component (Trial III). Lane 1, 1-kb DNA

marker ladder; Lane 2, kit template control; Lane 3, positive

Karp O. tsutsugamushi DNA control (519 bp); Lane 4, filtered,

undiluted inoculum; Lane 5, filtered inoculum diluted 1-in-

10; Lane 6, filtered inoculum diluted 1-in-100; Lane 7,

nonfiltered inoculum diluted 1-in-10; Lane 8, nonfiltered in-

oculum diluted 1-in-100; Lane 9, negative control (nonin-

fected mouse).

TABLE 3. Infection of mice after inoculation of filtered and unfiltered blood (as RBCs) seeded with dilutions ofO. tsutsugamushi-infected MNCs

Number of mice showing infectivity

Treatment RBC aliquot Illness Death Giemsa stain IFA DFA PCR

Trial I Undiluted 3/3 2/3 1/1† 0/1 1/1 NT Nonfiltered 1-in-10 3/3 0/3 0/1 3/3 0/1 NT

1-in-100 0/3 0/3 0/1 0/3 0/1 NT1-in-1000 0/3 0/3 0/1 0/3 0/1 NT

Filtered Undiluted 0/3 0/3 0/1 0/3 0/1 NT1-in-10 0/3 0/3 0/1 0/3 0/1 NT1-in-100 0/3 0/3 0/1 0/3 0/1 NT1-in-1000 0/3 0/3 0/1 0/3 0/1 NT

Trial II Undiluted 5/5 0/5 1/1 5/5 1/1 NT Nonfiltered 1-in-10 0/5 0/5 0/1 0/5 0/1 NT

1-in-100 0/5 0/5 0/1 0/5 0/1 NT1-in-1000 0/5 0/5 0/1 0/5 0/1 NT

Filtered Undiluted 0/5 0/5 0/1 0/5 0/1 NT1-in-10 0/5 0/5 0/1 0/5 0/1 NT1-in-100 0/5 0/5 0/1 0/5 0/1 NT1-in-1000 0/5 0/5 0/1 0/5 0/1 NT

Trial III Undiluted 2/2 2/2 NT NT NT NT Nonfiltered 1-in-10 2/2 0/2 1/1† 2/2 1/1 2/2

1-in-100 2/2 0/2 1/1† 2/2 1/1 2/2 Filtered Undiluted 0/2 1/2‡ 0/2 0/2 0/2 0/2‡

1-in-10 0/2 0/2 0/2 0/2 0/2 0/21-in-100 0/2 0/2 0/2 0/2 0/2 0/2

* For Trials I and II, blood for inoculation was diluted 1-in 10, 100 to 10–6, and for Trial III, 100 to 10–2. No mouse receiving filtered or nonfilteredblood at dilutions >10–2 (10–2-10–6) showed evidence of infection; data are not presented for dilutions >10–4. All survivors were sacrificed onDay 21.

† Test not performed for remaining mice of set.‡ One animal died on Day 15 after inoculation; PCR of liver and spleen homogenates were negative.



rea and Okinawa, as well as continued training exercisesconducted in Australia, Thailand, the Philippines, Indone-sia, Singapore, and Malaysia and renewed cooperative in-terests with Vietnam. These soldiers and Marines representthe donor population from which the transfusion facilitiessupporting them recruit and who by the vary nature of theirduties (training and living in the field) have direct exposureto the vectors capable of transmitting the rickettsial agents.

The use of WBC-reduced cellular blood componentshas been advocated as a means of lowering the risk of non-hemolytic transfusion reactions, HLA alloimmunization,infectious disease transmission (particularly CMV), andimmunomodulation.1 Filtration using third-generation high-efficiency filters is generally viewed as the most efficient andeffective method of WBC reduction. Frozen-deglycerolized andsaline-washed RBCs are also WBC reduced to various de-grees, but their processing is more labor-intensive.1 WBCfiltration can be accomplished in the blood bank laboratoryor at the patient’s bedside. Many transfusion specialists pre-fer laboratory filtration, believing that quality assurancewould be easier and more reliable under such conditions;others advocate WBC filtration at the patient’s bedside, be-cause that procedure is simpler and less expensive.31

Regardless of where the filtration takes place, actualreduction is dependent on many external factors, includ-ing the age of the unit, the temperature during the proce-dure, and the total time of the filtration. In addition, theexperience of the transfusion practitioners and the degreeof adherence to the manufacturers’ instructions influencethe procedure’s effectiveness. A detailed examination ofthese external factors was beyond the scope of this study,as we examined only filtration performed at room tempera-ture, in accordance with the manufacturer’s instructions,and within 70 minutes of seeding with infected MNCs. Inaddition, we examined the product of a single manufacturerand the filtration of freshly seeded blood, rather than test-ing various, extended holding times. Nevertheless, the re-sults of this study may indicate an additional clinical appli-cation for third-generation filters.

The prodromal period and the level of rickettsemia inscrub typhus can vary from 3 days to 14,22-25 and Shirai etal.32 showed that individuals become rickettsemic approxi-mately 4 to 7 days after chigger attachment and 1 to 3 daysbefore they become symptomatic. In extrapolating thesedata, Casleton et al.9 reported that the level of rickettsiaewould approximate 15 to 50 per mL of whole blood beforethe development of signs and symptoms. Given the uncer-tainty of rickettsial levels in asymptomatic patients, a con-centration of 75 organisms per mL was selected in this studyas the concentration differentiating symptomatic and as-ymptomatic levels of rickettsemia. Because symptomaticdonors would be deferred during the donation process, tobe effective from a purely qualitative standpoint, WBC fil-tration would have to protect against a 75-organism-per-

mL challenge. In Trial II, therefore, the 10–3 dilution that ap-proximated the rickettsial load that might be found in anasymptomatic but infectious donor, as well as three more-concentrated levels (100, 10–1, and 10–2 dilutions), was tested.Thus, when dilution levels that we estimated may be present inan asymptomatic blood donor were used, WBC filtration of200 mL of freshly inoculated blood was effective in prevent-ing the transmission of disease to susceptible animals. ForTrial II, 200 mL rather than 50 mL was used, to more closelyapproximate a normal packed RBC transfusion. Trial III wasperformed to duplicate trial II and to expand the filtrationchallenge by at least 10-fold. Results of that trial suggest ani-mal protection at a level of approximately 700,000 organ-isms, or in excess of 10,000-fold the amount anticipated inan infectious asymptomatic donor (Table 2).

Transfusion medicine has made tremendous stridesover the last several decades in the prevention of transfu-sion transmission of disease. Novel advances in virus andbacteria inactivation have lead to noncellular components,which have significantly reduced patient risks. Until totallyvirus-, bacteria-, and parasite-free components are avail-able, transfusion medicine practitioners should remain awareof the threats posed by diseases such as scrub typhus andthose caused by other rickettsial agents. These data weredeveloped from a small series of experiments using the ar-tificial model of seeded human blood, rather than bloodfrom an actual patient with a subclinical infection. Thus,extrapolation to actual cases of transfusion transmission isnot possible. Nevertheless, results of this study suggest thatthe use of high-efficiency, third-generation WBC-reductionfilters reduces the risk of the transmission of O. tsutsuga-mushi at rickettsial levels far in excess of those found inasymptomatic donors. Additional studies using filters fromother companies or treating contaminated blood compo-nents stored for various prefiltration periods, rather thanusing filtration of fresh blood as accomplished in this study,are needed. Such studies might further strengthen the casefor pretransfusion filtration as one means of reducing the riskof transfusion transmission of infectious agents.

ACKNOWLEDGMENTS

The authors thank Gregory A. Dasch, PhD, for providing primer

information and manuscript review, Teik Chye Chan for guid-

ance and technical assistance in the laboratory, and R.A.

Frommelt for statistical assistance.

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reducing the risk of transfusion-transmitted rickettsial diseaseby wbc filtration, using orientia...

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