Prevention of growth of Yersinia enterocolitica in blood by polyester fiber filtration B. WENZ, E.R. BURNS,AND L.F. FREUNDLICH The ability of pol ester white cell-reduction blood filters to revent the growth of Yersinia enterocoitic8 In units of donated blood was studied. &teen units of freshly drawn blood were inoculated with 10, 50, 100, or 150 colony-forming untts (CFU) per mL of a clinical Isolate of Y. enterocolitice (serotype 0:3). The units were subsequently fractionated into red cell concentrate and resuspended In AS-1 or A S 3 solution. One-half of the red cell concentrates In each solution were filtered within 15 hours of phlebotomy and stored for 42 days. The remalnln units sewed as unfiltered controls. Bacterial growth was monitored by weekly cu tures and, on the last stora e day, by the presence of endotoxin and the formation of methem lobin. One hunired twelve primary cultures (560 plates) were performed. Units co ected In AS-1 and filtered remained sterile when initially inoculated with 50 CFU or less. Filtered units spiked with 100 CFU or less and collected in A S 3 remained sterile throughout their shelf life. All unfiltered units supported bacterial growth and the formation of endotoxin and methemogiobin. The filtration of fresh1 donated blood proves to limit the growth of Y. enterocolitice in red cell components.hiNSFUSION

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1992;32:663-666.

Abbrevlatlonr: CFU = colony-formlng unltr; RBC(s) = red cell(r);WBC(r) = whlte cell(a).

ated with products stored > 21 days), culturing or screening older units of blood for the presence of bacteria or endotoxin prior to use, and adding antibiotics such as gentamicin to prevent bacterial growth. The committee's consensus was that these measures would be logistically difficult or ineffective and could create secondary problems equal to or greater than the problem i t ~ e l f Prelim.~ inary data were presented that suggested that polyester and cellulose acetate fiber filtration of relatively fresh blood components prevents the growth of Y. entemcolitica in RBC concentrate. We have conducted subsequent studies to confirm these observations.

THELIKELIHOOD OF BACERIAL contamination of red cell (RBC)concentrates is minimized by the use of sealed plastic bags with integral satellites, aseptic phlebotomy technique, and cold storage. None of these precautions eliminates the potential of drawing contaminated blood from an asymptomatic donor with a transient bacteremia. Most organisms obtained in this manner will not grow at 4"C, but there are notable exceptions.' Yersinia entmcolitica is a ubiquitous gram-negative bacillus of the family Enterobacteriaceae. In man it most frequently causes a self-limited gastroenteritis. More dramatic infections are restricted to immunocompromised patients. Y. entemcolitica produces endotoxin and has been shown to grow at temperatures that range from 4 to 42"C.2 To date, 30 cases of transfusion-associated Y. enterocolitica infection have been documented ~ o r l d w i d e .Thirteen ~ of these cases occurred in the United States and were the proximate cause of seven deaths. In May 1991, the Blood Product Advisory Committee of the Food and Drug Administration met to discuss measures that might be taken to curtail this growing problem. Considerations included reducing the shelf life of RBC concentrates (the majority of cases are associ-

Materials and Methods Sixteen units of blood were analyzed in this study. All blood was obtained from healthy individuals who provided informed consent and met American Association of Blood Banks donor standards. The sequence in which fractions were processed was designed to simulate routine production methods. Units were collected into one of two systems (Adsol, AS-1, BaxterPenwal, Deerfield, n,or Nutricel, AS-3, Cutter Biologicals, Hialeah, FL). Immediately following collection, we inoculated all units with 10,50, 100, or 150 colony-forming units (CFU) per mL of Y. enterucoZitica. Experiments using 10 and 100 CFU per mL were repeated subsequently to confirm that the data were reproducible. The organism used, serotype 0:3, a clinical isolate responsible for a transfusion-associated death,' was obtained as a gift from Lee Bland (Centers for Disease Control, Atlanta, GA). The isolate was subcultured to trypticase soy broth and incubated at 35°C for 48 hours. We prepared the primary inoculum by spectrophotometrically adjusting a saline suspension to a density equivalent to a 0.5 McFarland

From the Department of Laboratory Medicine, Albert Einstein College of Medicine, New York, New York. Supported in part by a grant-from the Pall Biomedical Corporation, Glen Cove, NY. Received for publication November 13, 1991; revision received February 10, 1992, and accepted February 13, 1992.

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turbidity standard. Additional dilutions were prepared in sterile saline to achieve the appropriate final concentrations, which were verified by plate counts. We held the inoculated units at room temperature for 3 hours and then fractionated them to RBC concentrates and platelet-rich plasma. The RBC concentrate was resuspended in AS-1 or AS-3 solution and stored at 4°C for the remainder of the study. After an overnight period of refrigeration, we filtered onehalf (n = 8) of the units in both nutritive solutions through a polyester fiber white cell (WBC)-reduction filter (BPF4, Pall Biomedical Corp., Glen Cove, NY)and returned them to storage. Filters were connected to the unit using a sterile connecting device ( S O , Haemonetics Corporation, Braintree, MA) to prevent inadvertent contamination. All specimens were obtained from the units by using the sterile connecting device as well. We sampled units at zero time and at weekly periods from Day 7 to Day 42 of the study (Fig. 1). We performed the weekly cultures by obtaining 5-mL aliquots from each unit and plating 1 mL of blood onto each of five plates (n = 560) containing trypticase soy agar with 5-percent sheep blood. The plates were incubated at 35°C and checked daily for 5 days. Plates containing colonies were subcultured, and the organism was identified by morphologic and biochemical characteristics. Tests for the latter included the gram-negative biochemical identification and antimicrobial sensitivity system (negative break-point combo type 6, Baxter Microscan, Sacramento, CA) and the enteric identification system (Enterotube, Roche Diagnostic Systems, Belleville, NJ). At the end of each RBC concentrate's shelf life, we performed a qualitative assay for endotoxin (Pymgent, Whittaker Bioproducts, Walkersville, MD) and quantified the percentage of methemoglobin present in

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each unit. The latter variable reflected the change caused by the conversion of nitrate to nitrite in the blood, which is an indirect measure of the presence of nitrate reductase, an enzyme associated with Y. enterocolitica. Blood gas analyses were also performed on Day 42 of blood storage.

Results Figure 2 summarizes the growth of Y. enterocolitica in the filtered and unfiltered units of RBC concentrate. Growth of Y. enterocoliticu could not be detected in any of the unfiltered units before Day 14. All filtered units that were initially inoculated with 10 or 50 CFU per mL remained sterile throughout the 42day storage period. The filtered unit that was initially inoculated with 100 CFU per mL and stored in AS-3 solution remained sterile throughout storage. Growth was detected on Day 42 of storage in the corresponding filtered unit that was resuspended in AS-1 solution; however, this finding is not significant because of the limited data available. The ability of the filter to inhibit growth of Y. enterocoliticu in RBC concentrate is exceeded by a challenge dose of 150 CFU per mL; by storage Day 28, both units in this category supported growth. Figure 2 suggests that the growth of Y. enterocolitica in unfiltered units is better supported by AS-3 than by AS-1 media; however, the amount of data available in this study is insufficient to confirm this observation. Endotoxin was present in all units that had positive cultures at any time during the storage period. No endotoxin was detected by the assay in those filtered units that remained sterile. The concentration of methemoglobin ranged from 7.7 to 11.4 percent in units supporting growth and 1.8 to 3.2 percent in the filtered, sterile units. The blood gas values on Day 42 of storage were consistently abnormal for the units from which Y. enterocolitica was cultured (0,saturation = 16.3-22.5%; PO, = 28-39 torr; pC0, = 55-64 torr) and within normal limits for the filtered units without bacterial growth (0, saturation = 98-98.5%; PO, = 187-220 torr; pC0, = 7.7-9.4 torr).

Discussion

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Fractionate after 3 hours

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Hold for 12 hours at 4°C

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1 Cultwe on day 1 and weeks 1-6 endotoxin assay on day 42 FIG. 1. Diagram of experimental processing protocol.

Until such time as it is practical to sterilize blood components as is currently done for blood derivatives, bacterial contamination of these components may occur. In this respect, the storage of platelet concentrate presents the greatest risk factor.'j In the majority of cases, the contaminant is introduced into the blood during the phlebotomy process. Bacteria present in skin flora readily proliferate at 22°C during the platelet concentrate's storage period. Cold storage retards the growth of these bacteria in other components. However, transient bacteremia caused by organisms such as Y. enferocolificu that are capable of multiplying in the cold proves to be a small but significant threat to the safety of stored RBC concentrate. The potential impact(s) of measures suggested to control this problem has been determined to be unacceptable to the Food and Drug Administration and the professional blood banking c ~ m m u n i t y . ~ 1. Reducing the shelf life of FU3C concentrate from its present 35 to 42 days to 25 days would increase the expiration rate from the current nationwide average

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INHIBITION OF YERSINLA GROWTH BY FILTRATION

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FIG. 2. Growth of Yersiniu entemcolitica in filtered and unfiltered red cell concentrate stored in AS-1 or AS-3 solution at 4'C for 42 days. Maximum growth is arbitrarily depicted at 10,ooO colony-forming units per mL, which represents a confluence of colonies on the plates. A inoculation of 10 CFU per rnL; B: inoculation of 50 CFU per mL, C: inoculation of 100 C N per rnL; D: inoculation of 150 CFU per mL. AS1. 0-0 (n=16); AS-1 plus BPF4 filter, A-A (n= 16); AS-3, @*--@ (n= 16); AS-3 plus BPF4 filter, 0-0 (n-16).

of 3.8 percent to 9.4 percent. This would result in a 12-percent increase in operating costs according to the American Red Cross. The Council of Community Blood Centers points out that this measure would curtail the availability of autologous donations by 25 to 30 percent; furthermore, this measure would prevent only a percentage of cases of contamination, because 5 of the 30 reports involved blood that was no more than 21 days old. 2. Although research performed at the American Red Cross proved that the addition of 1 to 10 pg of gentamicin per mL of blood inhibits the growth of Y. enterocolitica, the risk of atopic reactions and the chance of creating antibiotic-resistant strains are unacceptable to the Blood Product Advisory Committee. 3. The performance of a large number of screening tests prior to issuing units of older RBC concentrate is judged by experts in the field to be tedious and errorprone. Screening tests performed at the time of donation would be largely ineffective because of the low number of contaminating organisms present at that time. The present generation of WBC-reduction filters removes in excess of 99.9 percent of WBCs from units of b l ~ o d Their . ~ mechanism of action is unknown; however, existing data suggest adsorption rather than mechanical sieving.* Our study supports previous

observations that the use of these filters removes Y. enterocolitica from RBC concentrate. Whether this effect is dependent on phagocytosis and WBC removal, or is an independent factor, is currently not known, nor is it known whether other organisms can be removed in this manner. Although this study was confined to testing the ability of polyester fiber to remove Y. enterocolitica, unpublished data confirm the ability of a cellulose acetate filter to retard the growth of Y. entemcolitica in RBC concentrate, as weL4 It is of interest that, in at least one instance, cotton wool filtration failed to prevent the growth of Y. enterocolitica in donor blood, and transfusion of that blood resulted in the recipient's death.s The potential for these filters to remove Yersinia is finite and appears to be limited to 100 CFU per mL (total, 5 x lo4 CFU)of whole blood or less. This number does not detract from the potential clinical effect of filtration. Logarithmic growth in blood follows a lag phase of 10 to 20 days in this and other studies. Control measures must be used prior to the production of endotoxin in the RBC concentrate, that is, at a time shortly after donation when the concentration of organisms is considered to be very low. Y. enterocolitica is not the only organism that has been associated with RBC concentrate transfusion-induced toxemia, but it is the most f r e q ~ e n t .The ~ incidence of this problem is not currently known, but, to

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judge from the cluster of reported cases, it appears either to be on the rise or to be recognized more frequently. The filtration of fresh blood is a simple procedure that can readily be accommodated into the blood-processing routine. The potential benefits derived from W C reduction of blood, such as a decrease in HLA immunization and cytomegalovirus transmission, have been suggested.1° The curbing of transfusion-associated bacteremia and endotoxemia may represent an additional function of these filters. References 1. Arduino MJ, Bland LA, Tipple MA, Aguero SM, Favero MS, Jarvis WR. Growth and endotoxin production of Yersinia enterocolitica and Entembacter agglomemns in packed erythrocytes.

J Clin Microbiol 1989;27:1483-5. 2. Bercovier H, Mollaret HH. Yersinia. In: Krieg NR, ed. Bergey's manual of systematic bacteriology, vol 1. Baltimore: Williams & Wilkins. 1984: 498-506. 3. FDA Committee endorses education and research to combat rare bacterial reactions: rejects operational changes for now. Council of Community Blood Centers Newsletter. May 10, 1991:l. 4. Sayers M, chairman. Minutes to the 32nd meeting of the Blood Product Advisory Committee. Bethesda: Food and Drug - Administration, May 9; 1991. 5. Jacobs J. Jamaer D. Vandeven J. Wouters M. Vermvlen C. Vandepitte J: Yersinin enterocolitica in donor blood: a ca'se report and review. J Clin Microbiol 1989;27:1119-21.

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6. Braine HG, Kickler TS, Charache P, et al. Bacterial sepsis sccondary to platelet transfusion: an adverse effect of extended storage at room temperature. Transfusion 1986;26:391-3. 7. Wenz B, Bums ER, Lee V, Miller WK. A rare-event analysis model for quantifying white cells in white cell-depleted blood. Transfusion 1991;31:156-9. 8. Wenz B. Clinical and laboratory precautions which reduce the adverse reactions, alloimmunization, infectivity and possibly immunomodulationassociated with homologous transfusions. Transfus Med Rev 1990;4:3-7. 9. Tipple MA, Bland LA, Murphy JJ, et al. Sepsis associated with transfusion of red cells contaminated with Yeminia entemolitica. Transfusion 1990;30:207-13. 10. Brozwic B. The role of leucocyte depletion in blood transfusion practice: proceedings of the international workshop, London, 9 July 1988. Oxford: Blackwell, 1989. Barry Wenz, MD, Professor of Laboratory Medicine. Director of Clinical Pathology, Albert Einstein College of Medicine, Room l E l l , Bronx Municipal Hospital Center, Pelham Parkway South, Bronx, NY 10461 [Reprint requests] Edward R. Bums, MD, Associate Professor of Laboratory Medicine, Director of Hematology Laboratory, Albert Einstein College of Medicine. Lawrence F. Freundlich, MS, Principal Associate in Laboratory Medicine, Director of Microbiology, Albert Einstein College of Medicine. Dkclosurc: One of the authors (BW)serves as a medical consultant to the Pall Biomedical Corporation

Prevention of growth of Yersinia enterocolitica in blood by polyester fiber filtration.

The ability of polyester white cell-reduction blood filters to prevent the growth of Yersinia enterocolitica in units of donated blood was studied. Si...
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