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The development and use of sterile container sys-tems for blood collection and processing havegreatly reduced bacteria contamination of bloodcomponents. Despite these measures, clinically

apparent transfusion-associated bacterial sepsis persists ata low frequency. Morrow and coworkers1 carried out an in-vestigation of febrile reactions in which 1 of 19,519 units ofapheresis platelets and 1 of 1,700 pools of whole blood-derived platelets (representing 10,219 units) were culturepositive with the same strain of organism isolated from therecipient. Similar results were obtained by Barrett and co-workers.2 Although a large number of tests for the detec-tion of bacteria in whole blood or blood components havebeen studied,13-16 no simple, rapid, affordable, and adequatelysensitive method has been developed that would preventthe majority of septic episodes in recipients of transfusion.

In most cases of transfusion-associated bacterial sep-sis, it is difficult to be certain about the source of contami-nation. Sources may include donor skin, circulating donorblood, processing devices, and the environment. However,the source may often be inferred from the species of organ-ism isolated from the implicated unit. For example, roughlyone-third of reported septic episodes following the trans-fusion of platelet components involved a Staphylococcusspecies.17 These bacteria are typically considered skin or-ganisms, whose optimal growth conditions are similar tothe temperature and oxygen conditions utilized for the stor-age of platelet components (i.e., 20-24°C in an aerobic en-vironment). Investigators have speculated that skin “plugs,”which might form from needle coring during phlebotomy,are responsible for the introduction of skin organisms intowhole blood.18 Other workers believe that modern needlesform skin flaps rather than skin plugs. If skin flaps are in-deed formed, skin-associated bacteria could still enter thecollection system by eluting from a skin flap as a result ofblood flow.

We investigated whether the diversion from the pri-mary collection container of the first few milliliters of wholeblood collected from the “donor” would reduce the bacte-rial load of organisms introduced by needle puncture. In amodel system with either saline or whole blood as a vehicle,

Diversion of initial blood flow to prevent whole-bloodcontamination by skin surface bacteria: an in vitro model

S.J. Wagner, D. Robinette, L.I. Friedman, and J. Miripol

BACKGROUND: Sepsis arising from the transfusion ofbacterially contaminated platelet components continuesto be an infrequent, yet serious transfusion complication.Skin organisms are implicated in a number of these sep-tic episodes. A model system was used to investigate ifa skin organism’s bioburden in blood components couldbe reduced by diverting the first few mL of whole bloodaway from the primary container.STUDY DESIGN AND METHODS: A sterile medicationsite was inserted into a bag containing sterile saline orwhole blood; the site was deliberately contaminated withStaphylococcus aureus and allowed to dry. After needlepuncture of the contaminated medication site, bacterialevels were measured 1) in successive 7-mL tubes ofblood or saline drawn through a diversion arm, 2) in 40mL of a connected transfer pack, and 3) in blood or sa-line from a needle puncture of the original container viaanother sterile medication port.RESULTS: Diverting the first 21 to 42 mL of saline or wholeblood reduces the downstream bioburden of deliberatelyintroduced surface S. aureus by approximately 1 log.CONCLUSION: Development of a diversion system forcollection of whole blood in sample tubes before fillingthe primary container may reduce the bioburden of sub-sequently prepared components and thereby the fre-quency of sepsis due to skin organisms.

From the Product Development Department, Holland Labora-

tory for the Biomedical Sciences, American Red Cross Biomedi-

cal Services, Rockville, Maryland; and Terumo Medical Corpora-

tion, Elkton, Maryland.

Address reprint requests to: Stephen J. Wagner, PhD, Jerome

H. Holland Laboratory, American Red Cross, 15601 Crabbs

Branch Way, Rockville, MD 20855; e-mail:

wagners@usa.redcross.org.

Supported by the American Red Cross and the Terumo

Medical Corporation.

Received for publication July 9, 1999; revision received Au-

gust 11, 1999, and accepted August 16, 1999.

TRANSFUSION 2000;40:335-338.

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336 TRANSFUSION Volume 40, March 2000

the reduction of bacteria load attributed to diversion wasquantified.

MATERIALS AND METHODSSource of materialsWhole blood (500 ± 50 mL) was collected into 70 mL of CPD intriple-pack container systems (primary container, PL-146, BaxterHealthcare, Deerfield IL) by the American Red Cross Biomedi-cal Services’ Holland Laboratory Research Blood Program. Sta-phylococcus aureus was obtained from the American Type Cul-ture Collection (#27217, ATCC, Manassas, VA).

Bacterial growth and quantitationWe prepared fresh overnight cultures of S. aureus by inocu-lating single-colony isolates into trypticase soy broth(Becton Dickinson, Cockeysville, MD) and then incubatingthe broth at 28 to 30°C under aerobic conditions. Bacterialevels in samples were quantified by 1-in-10 serial dilutionof fully mixed samples in unbuffered saline, the addition ofeither 1.0 or 0.1 mL of the diluted suspension to 3 mL of 0.8-percent molten agar (43°C), and the pouring of the moltenagar over trypticase soy agar plates. We counted coloniesafter incubation for 16 to 24 hours at 28 to 30°C. Coloniesfrom all plates that contained between 1 and 1000 colonieswere enumerated.

Experimental designSaline experiments. We performed initial experiments (n= 3) in a model system with unbuffered saline. A schematicis shown in Fig. 1. Two hundred mL of sterile saline wasinjected through a sterile medication site (Baxter) into aspecially designed two-port container (Terumo Corp., To-kyo, Japan). This medication site was then painted with 10µL of a fresh overnight culture of S. aureus and allowed todry. The procedure deposited approximately 106 to 107 or-ganisms onto a 0.5-cm2 surface of the medication site. Thisbacterial “spike” is many orders of magnitude greater thanthat which might be present on donor skin, but it allowedaccurate quantitation of bacteria in the model system. Wepierced the contaminated surface of the medication site byusing a 16-gauge needle of an experimental collection setwith a diversion arm. With the clamp leading to the trans-fer pack (representing the primary collection container)closed, saline began to enter the supplemental container(which is needed to purge the air in the tubing). The clampleading to the supplemental container was then closed, andsix successive 7-mL samples of saline were collectedthrough a Luer-Lok syringe fitting (Fig. 1) by using anadapter and needle guard (Terumo) into 13 × 100-mm ster-ile blood-collection tubes (Sherwood Medical, St. Louis,MO) in approximately 1 minute. After obtaining thesesamples, we collected an additional 40 mL of saline by clos-ing the clamp proximal to the Luer-Lok syringe adapter and

opening the clamp leading to the transfer pack. Finally, asample from the original container was collected throughan uncontaminated sterile medication site placed into thesecond port.

Whole-blood experiments. A set of identical experi-ments (n = 3) was performed by substituting 200 mL offreshly collected, CPD whole blood for saline.

Calculations and statisticsTotal CFUs in the collection tubes, transfer pack, and re-maining fluid in the two-port container were calculatedfrom bacteria counts measured in CFUs per mL multipliedby the sample volume. The percentage of capture of sur-face-associated S. aureus was determined by summing thetotal CFUs in each specified tube, dividing by the sum oftotal CFUs in the transfer pack and in all collection tubes,and multiplying by 100 percent. The average and the SD ofthe percentage of capture of surface-associated bacteriawere determined, and comparisons between saline andwhole blood experiments were analyzed by using the alter-

Fig. 1. Schematic of the diversion system.

DECREASING SKIN CONTAMINANTS BY BLOOD DIVERSION

Volume 40, March 2000 TRANSFUSION 337

native t test (Instat, GraphPad Software, San Diego, CA).Two-tailed p values were reported. Differences were con-sidered significant when p<0.05.

RESULTSSaline experimentsTable 1 shows the total calculated CFUs in each tube, thetransfer pack, and the original two-port container. In allthree experiments, most of the surface-contaminating or-ganisms that entered the outflowing saline were recoveredin the first 7-mL tube. In general, subsequent tubes eachcontained fewer organisms than the preceding tube, andthere was less bacteria load in the residual saline of the two-port container than in any tube. The average cumulativepercentage of capture of S. aureus was 95.8 ± 4.1 percentthrough the third tube and 98.8 ± 1.6 percent through thesixth tube.

Whole blood experimentsTable 2 shows the total calculated CFUs in each tube, thetransfer pack, and the original two-port container. Much asin the saline experiments, most of the surface-contaminat-ing organisms that entered the outflowing whole bloodwere recovered in the first 7-mL tube. In general, subse-quent tubes each contained fewer organisms than the pre-ceding tube, and there was less bacteria load in the wholeblood of the two-port container than in any tube. The av-erage cumulative percentage of capture of S. aureus was88.2 ± 5.5 percent through the third tube and 95.3 ± 2.3percent through the sixth tube. Although these averagevalues were less than those obtained from saline experi-ments, the differences did not reach significance (p>0.05).

DISCUSSIONA preliminary clinical study has been reported on the useof additional satellite containers to collect the first few mil-liliters of whole blood.19 In thatstudy, 15 mL of whole blood wasdiverted into each of two satellitecontainers before the primarycontainer was filled. One hundredsixteen (3.4%) of 3440 collectedunits of whole blood containedbacteria in at least one of the twosatellite containers, whereas only7 of the components preparedfrom those 116 implicated unitswere culture positive.

Analysis of this study is diffi-cult, because the culture-positiverate is higher than predicted fromthe literature,20 the study did not

compare the rates of culture-positive whole blood unitsbefore and after diversion, and there was no quantitativemeasure of bacteria level to indicate the actual reductionin it when diversion was employed. We employed a modelsystem to study the reduction in bacteria load when 7 to 42mL of saline or whole blood is diverted after needle-punc-ture of a deliberately contaminated surface. Results suggestthat the bacteria load is reduced with the diversion maneu-ver by approximately 1 log. It is not known if comparableresults would be obtained by using human skin in a modelsystem. It is unlikely that a similar experiment with delib-erate contamination could be conducted in vivo.

Results demonstrating reduction in bacteria load by us-ing a diversion system have also been obtained by Figueroaand coworkers.21 In that study, a small liquid inoculum wasintroduced into the lumen of a collection needle and experi-ments were conducted either before or after the inoculumdried, rather than the organisms’ being introduced throughpiercing of a contaminated, dried surface. Therefore, com-parison of their study to ours is complicated by differencesin method.

It is difficult to predict the outcome of removing 1 logof skin organisms from donor blood, because there is littleinformation about the initial bacteria load introduced intowhole blood from skin sources and the ways in which thatload relates to the potential for outgrowth and sepsis. Onewould expect bacteria loads on the skin to be small, becauseof repeated cleansing procedures. In addition, there may befewer bacteria present in platelet-rich plasma than in RBCs,because many organisms, through their interaction withmonocytes and granulocytes during the room-temperaturewhole blood hold, sediment with the RBC fraction as a buffycoat during the first centrifugation.22,23 A similar reductionin bacteria load might be expected in platelets preparedfrom buffy coats, because monocyte and granulocyte lev-els are lower than those in whole blood.24 It is conceivablethat the reduction of 1 log of initial bacteria load by diver-sion of the first few milliliters of whole blood would prevent

TABLE 1. Saline experiments: total CFUs in transfer container, two-portcontainer, and 7-mL sample tubes

Number Initial 40-mLof number Tube transfer Two-port

experiments of CFUs 1 2 3 4 5 6 pack container

1 0 004,060 ,00455 ,0175 ,0182 ,0098 063 120 0002 0 015,890 ,00343 ,0161 ,0105 ,0070 021 068 0303 0 330,600 10,430 4,900 2,730 1,057 644 574 200

TABLE 2. Whole blood experiments: total CFUs in transfer container, two-portcontainer, and 7-mL sample tubes

Number Initial 40-mLof number Tube transfer Two-port

experiments of CFUs 1 2 3 4 5 6 pack container

1 0 164,500 21,840 24,920 19,320 10,080 2,912 19,000 2,0002 0 476,000 31,360 21,560 15,260 08,750 4,032 09,560 2,0003 0 207,200 12,880 11,830 06,090 02,520 2,310 13,200 3,300

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338 TRANSFUSION Volume 40, March 2000

the subsequent outgrowth of skin organisms in some frac-tion of platelet components that might otherwise causesepsis upon transfusion.

It is not possible to implement the model system, be-cause it is configured as an “open” system. A similar, closed-diversion system may be feasible to manufacture and couldbe used to collect samples for infectious disease and sero-logic testing. Such a system might reduce the frequency ofsepsis due to skin-associated bacteria as well as prevent theunnecessary destruction of some whole blood units whensamples cannot be collected at the end of the procedure.

REFERENCES01. Morrow JF, Braine HG, Kickler TS, et al. Septic reactions to

platelet transfusions. A persistent problem. JAMA

1991;266:555-8.

02. Barrett BB, Andersen JW, Anderson KC. Strategies for the

avoidance of bacterial contamination of blood compo-

nents. Transfusion 1993;33:228-34.

03. Gong J, Högman CF, Lundholm M, Gustafsson I. Novel au-

tomated microbial screening of platelet concentrates.

APMIS 1994;102:72-8.

04. Blajchman MA, Ali AM. Bacteria in the blood supply: an

overlooked issue in transfusion medicine. In: Nance ST, ed.

Blood safety: current challenges. Bethesda: American Asso-

ciation of Blood Banks, 1992:213-28.

05. Wagner SJ, Robinette D. Evaluation of an automated micro-

biologic blood culture device for detection of bacteria in

platelet components. Transfusion 1998;38:674-9.

06. Burstain JM, Brecher ME, Workman K, et al. Rapid identifi-

cation of bacterially contaminated platelets using reagent

strips: glucose and pH analysis as markers of bacterial me-

tabolism. Transfusion 1997;37:255-8.

07. Wagner SJ, Robinette D. Evaluation of swirling, pH, and

glucose tests for the detection of bacterial contamination in

platelet concentrates. Transfusion 1996;36:989-93.

08. Myhre BA, Demianew SH, Yoshimori RN, et al. pH changes

caused by bacterial growth in contaminated platelet con-

centrates. Ann Clin Lab Sci 1985:509-14.

09. Brecher ME, Boothe G, Kerr A. The use of a chemilumines-

cence-linked universal bacterial ribosomal RNA gene probe

and blood gas analysis for the rapid detection of bacterial

contamination in white cell-reduced and nonreduced

platelets. Transfusion 1993;33:450-7.

10. Kim DM, Brecher ME, Bland LA, et al. Prestorage removal

of Yersinia enterocolitica from red cells with white cell-re-

duction filters. Transfusion 1992;32:658-62.

11. Yomtovian R, Lazarus HM, Goodnough LT, et al. A prospec-

tive microbiologic surveillance program to detect and pre-

vent the transfusion of bacterially contaminated platelets.

Transfusion 1993;33:902-9.

12. Reik H, Rubin SJ. Evaluation of the buffy-coat smear for

rapid detection of bacteremia. JAMA 1981;245:357-9.

13. Anderson KC, Lew MA, Gorgone BC, et al. Transfusion-re-

lated sepsis after prolonged platelet storage. Am J Med

1986;81:405-11.

14. Chiu EKW, Yuen KY, Lie AKW, et al. A prospective study of

symptomatic bacteremia following platelet transfusion and

of its management. Transfusion 1994;34:950-4.

15. Chongokolwatana V, Morgan M, Feagin JC, et al. Compari-

son of microscopy and a bacterial DNA probe for detecting

bacterially contaminated platelets (abstract). Transfusion

1993;33(Suppl):50S.

16. Feng P, Keasler SP, Hill WE. Direct identification of Yersinia

enterocolitica in blood by polymerase chain reaction ampli-

fication. Transfusion 1992;32:850-4.

17. Wagner SJ, Friedman LI, Dodd RY. Transfusion-associated

bacterial sepsis. Clin Microbiol Rev 1994;7:290-302.

18. Gibson T, Norris W. Skin fragments removed by injection

needles. Lancet 1958;2:983-5.

19. Vassort-Bruneau C, Perez P, Janus G, et al. New collection

system to prevent contamination with skin bacteria (ab-

stract). Vox Sang 1998;74S1.

20. Leiby DA, Kerr KL, Campos JM, Dodd RY. A retrospective

analysis of microbial contaminants in outdated random-

donor platelets from multiple sites. Transfusion

1997;37:259-63.

21. Figueroa PI, Yoshimori R, Nelson G, et al. Distribution of

bacteria in fluid passing an inoculated collection needle

(abstract). Transfusion 1995;35;(Suppl):11S.

22. Wagner SJ, Robinette D, Nazario M, Moroff G. Bacteria lev-

els in components prepared from deliberately inoculated

whole blood held for 8 or 24 hours at 20 to 24°C. Transfu-

sion 1995;35:911-6.

23. Högman CF, Gong J, Eriksson L, et al. White cells protect

donor blood against bacterial contamination. Transfusion

1991;31:620-6.

24. Hirosue A, Yamamoto K, Shiraki H, et al. Preparation of

white-cell-poor blood components using a quadruple bag

system. Transfusion 1988;28:261-4. ��