ture studies, the time course of MIF and LIF production in relation to each other, as well as clinical phenomena, must be studied serially in selected young patients. The present preliminary study provides further evidence that cellular hypersensitization to a potent encephalitogenic antigen (myelin basic protein) is present during exacerbations of MS, and suggests that it may, in combination with other unrecognized factors, be of pathogenetic significance. References 1. BARTFELD H, ATOYNATAN T, DONNENFELD H:
2.
In vitro cellular immunity to nervous system antigens in multiple sclerosis, in Multiple Sclerosis: Immunology, Virology and UltraGW, WOLFORAM 5, ELLIsoN structure, STEVENS JA, et al (eds), New York, Acad Pr, 1972, pp 333-64 RE, SHEREMATA WA, FELDMAN ROCKLIN RG, et al: Gui1lain-Barr. and multiple sclerosis: in vitro cellular responses to nervous tissue antigens. N Engl I Med 284: 803, 1971
3. SHEREMATA W, COLBY 5, KARKHANIS Y, et al:
Cellular hypersensitivity to P2 myelin protein in Guillain-Barr& Can I Neurol Sci 2: 87, 1975 4. SHEREMATA W, Cosuitova JBR, EYLAR EH: Cellular hypersensitivity to basic myelin (As) protein and clinical multiple sclerosis N
Engi J Med 291: 14, 1974 5. idem: Hypersensitization to basic myelin protein preceding attacks of multiple sclerosis. Trans Am Neurol Assoc 99: 55, 1974 6. PEKAREK J, JEDLICKA P,
SVEJCAR J, et al:
Production of migration inhibitory factor by lymphocytes of patients with multiple sclerosis, to brain antigens. Clin hnmunol Immunopathol (in press) 7. S9.sERo M, BENDIXEN 6: Human lymphocyte migration as a parameter of hypersensitivity. Acta Med Scand 181: 247, 1967 8. MYERS t.W: Leukocyte migration inhibition in multiple sclerosis, in Multiple Sclerosis: Immunology, Virology and Ulm'raslructure, op cit, pp 389-99 9. STRANDSCAARD
W,
J.RGENSEN
PN:
Delayed
hypersensitivity to myelin antigen in multiple sclerosis investigated with the leukocyte migration method. Acia Neurol Scand [Suppl] 48: 243, 1972 10. ARMSTRONG R: Inhibition of lymphocyte migration in multiple sclerosis (abstr). Can J Neurol Sci 2: 324, 1975 11. ALvORD EC, Hsu PC, THOR R: Leukocyte sensitivity to brain fractions in neurological
diseases. Arch Neurol 30: 296, 1974 12. BEROSTRAND H, KALLEN B, NILSsON 0: Ef-
of delayed hypersensitivity in man: ambiguity of polymorphonuclear neutrophils as indicator cells in leukocyte migration test. Inject bnnmn 3: 370, 1973 16. ROSENBERG
SA,
DAVID
JR:
Inhibition
at
leukocyte migration: an evaluation of this in vitro assay of delayed hypersensitivity in man to solLible antigen. J lininunol 105: 1447, 1970
17. READ SE, ZABRISKIE JB: Cellular interactions
in the leukocyte migration inhibition system. Trans Proc 4: 247, 1972
18. MOOKERJEE B, ACKMAN GFD, DossEToR JB: Delayed hypersensitivity in vitro using human peripheral leukocytes. Transplantation 8: 745, 1969 19. KALTREIDER BJ, SOGHOR D, TAYLOR JB, et al: Capillary tube migration for dctection of human delayed hypersensitivity: difficulties encountered with "buffy coat" cells and tuberculin antigen. J Inanmnol 103: 179, 1969 20. Fox RA, GREGOY DS, FELDMAN JD: Macrophage receptors for migration inhibitory factor (MIF), migration stimulator factor (MSF) and agglutinating factor. J linmunol 112: 1867, 1974 21. COHEN 5, FISHER B, YOSHIDA T, et at:
Serum migration inhibition activity in patients with lymphoproliferative diseases. N
fect of basic encephalitogenic protein and some peptides derived from it on the migration in agarose gel of leukocytes from patients with multiple sclerosis, other neurological diseases or carcinoma. Acta Neurol Scand 50: 227, 1974
22. MYERS LW, ELLISON GW: Effect of humoral factors on cellular hypersensitivity to nervous tissue in multiple sclerosis. Fed Proc 32:
13. EYLAR EH, BROSTOFF S, HASHIM G, et al: Basic As protein of the myelin membrane;
23. RAOCH HG, EINSTEIN ER, C5EJTEY I: Enzymatic degradation of myelin basic protein in central nervous system lesions of monkeys
the complete amino acid sequence. I Biol Chem 246: 5770, 1971 14. ROCKLIN RE: Products of activated lymphocytes: leukocyte inhibitory factor (LIF) distinct from migration inhibitory factor
(MIF). I Immunol 112: 1461, 1974 15. SENYK 6, HADLEY WK:
In vitro correlates
Engi J Med 290: 882, 1974 832, 1973
with experimental allergic encephalomyelitis. Neurobiology 3: 195, 1973
24. BROSTOFF SW, REUTER W, HIGHENS M, et al: Specific cleavage of the A1 protein from
myelin with cathepsin D. J Biol Chem 249: 559, 1974
Exogenous bacterial contamination of donor blood G. RocK,* MD, PH D J.C.N. WESTWOOD,t MD
The Canadian Red Cross blood transfusion service has followed a set protocol for phlebotomy and collection of a unit of blood. Recent requirements for automated testing have necessitated that a second tube of blood be obtained from the blood line following collection of the unit. Evaluation of the techniques used, however, has indicated the possibility of bacterial contamination from the skin of donors, from insertion of the needle through an unsterile rubber stopper, and through backflow from a nonsterile vacuum tube. To test these possibilities swabs were taken from skin and stoppers of vacuum tubes. Further, vacuum tubes were deliberately contaminated with Escherichia coil. The normal sampling procedure, which involves stripping the donor line to refill and mix the blood, was then followed. This resulted in contamination of the segments and even the blood bag. From the department of microbiology and immunology, University of Ottawa *Senior lecturer tProfessor and head Reprint requests to: Dr. G. Rock, Medical director, Canadian Red Cross blood transfusion service, 85 Plymouth St., Ottawa, Ont. KIS 3E2
These findings led to modification of the standard bleeding technique, whereby stripping was eliminated and sterile vacuum tubes were to be used at all times. Le service de transfusion de Ia Croix-Rouge canadienne a suivi un protocole defini pour Ia phlebotomie et Ia collecte d'une unite de sang. Des exigences recentes visant a automatiser les epreuves d'analyse ont necessite l'obtention d'un deuxieme prelevement sur Ia tubulure de perfusion apres Ia collecte de sang. Levaluation des techniques utilisees ont toutefois indique Ia possibilite de contamination bacterienne a partir de Ia peau du donneur, de l'insertion de l'aiguille a travers un bouchon non-sterile, ou a Ia suite d'un reflux venant d'un tube a vide non-sterile. Af in de verifier ces possibilites des prelevements ont ete faits sur Ia peau et sur les bouchons des tubes a vide. De plus, des tubes a vide ont .t6 d6lib6r6ment contamin6s avec de l'Escherichia coil. Le proc6d6 normal d'echantillonage, qui implique l'egoutteur de Ia tubulure de perfusion pour remplir et melanger le sang,
988 CMA JOURNAL/MAY 7, 1977/VOL. 116
a ete suivi alors. Ceci a resulte en une contamination des bouts du tube et mime du sac de sang Iui-m.me. Ces observations ont entraine une modification de Ia technique normale de saignee; on n'egoutte plus Ia tubulure et des tubes a vide steriles sont maintenant utilises en tout temps.
For years the Canadian Red Cross blood transfusion service has followed a standard procedure for drawing blood from donors, all of whom are volunteers. This procedure (Appendix I) is essentially the same as that outlined in the Fenwal manual.1 Changes in automation techniques, however, have made it necessary to obtain an additional tube of blood from the bag. The steps taken to permit the subsampling (Appendix I) basically involve refilling and stripping the tube a second time. This requirement to subsample prompted a reassessment of the entire bleeding procedure. A primary consideration was the possibility of bacterial contamination since subsampling and, indeed, initial sampling effectively result in an open system. Of particular concern was the possibility of backflow from the evacu-
ated tubes into the blood bag since the use of such nonsterile tubes has previously been reported to cause nosocomial infection in patients.2'3 The experiments reported in this paper were designed to evaluate the extent of the hazard at each point of the collection procedure and to indicate appropriate modifications. Background: points of contamination risk in the standard bleeding procedure Examination of the standard bleeding procedure as described in Appendix I indicated three sources of exogenous bacterial contamination of collected blood: 1. Donors' skin Because the degree of cleanliness of the skin of individual donors varies, residual bacterial contamination after cleansing could constitute a source of contamination of collected blood. Skin cleansing was carried out by three simple wipes repeated thrice using cotton-wool balls held in an open bowl of alcoholic benzalkonium chloride (Zephiran). The balls were squeezed out with the ungloved hand before a donor's skin was wiped; after that the skin was dried with a square of sterile gauze. The selected vein was then located with the finger before insertion of the needle. 2. Vacuum tube stoppers Insertion of the needle through the stopper at the time of sampling is likely to cause contamination of the needle unless the unsterile stopper is first sterilized. 3. Vacuum tube interiors The interiors of purple-stoppered vacuum tubes have been shown to be contaminated with Pseudomonas or Serratia species, or both, in concentrations of 1O. organisms per tube (J.C.N. Westwood, M.A. Mitchell: unpublished data). Regurgitation during sampling from the filled collecting kit could cause contamination of the blood in the collecting tube and thus the bag. Stripping of the blood contained in the tubing back into the bag after the vacuum tube is filled (step 15 of Appendix I) appears especially dangerous. The first two sources would cause contamination principally of the exterior of the needle but, unless their contamination was gross, it is unlikely the blood in the bag would become contaminated. The third source, however, would cause contamination of the blood within the vacuum tube and, if reflux occurred, contamination of the blood within the tubing leading to the bag. Under conditions of single sampling, reflux would be unlikely to carry any organisms far enough down the collecting tubing to jeopardize the sterile
condition of the blood in the bag, except when blood from the tubing is stripped back into the bag. Under conditions of multiple sampling, however, in which the tubing is allowed to refill from the bag, the danger of contamination of the blood in the tubing would increase with each sample taken. The degree of danger ot contamination of the bag would depend on whether blood was stripped from the tubing back into the bag. Methods and results
Experiments, series 1 Methods: In this series of experiments donors' skin and vacuum tube stoppers were tested for contamination and a simulated bleeding procedure was designed. The possibility of contamination by reflux from nonsterile vacuum tubes was also investigated. * Skin cultures: Cultures were performed using Rodac contact plates filled with trypticase-soy agar (TSA, Difco) and 5% sheep's blood. The cultures were prepared by contact with the venipuncture sites of 12 donors before the sites were cleansed and after they had been cleansed according to the procedure described above. The plates were incubated aerobically for 48 hours, after which developed colonies were counted. * Vacuum tube-stopper cultures: Stoppers also were examined for contamination by rubbing 40 of them thoroughly with sterile cotton swabs moistened with TS broth. Each swab was immediately inoculated onto the surface of a blood-agar plate (5%
B
FIG. 1-Apparatus simulating donor. A: 2-i bottle with filtered air-inlet tube and rubber outlet tube. B: "Bleeding" procedure: foil has been opened and needles inserted. During tests "bleeding" site was covered with sterile gauze.
sheep's blood in TSA) and incubated aerobically for 48 hours. * Contamination of vacuum tube interiors: Vacuum tubes (Becton-Dickinson red-stoppered) were contaminated by introducing into each, 0.5 ml of an overnight culture of Escherichia cali containing 10. organisms by viable plate count. The contaminating inoculum was introduced with a Cornwall syringe tipped with a 21-gauge needle. Resealing of the stopper after the needle was removed was adequate to retain a vacuum within the tube. * Simulated bleeding procedure: In order to permit the standard bleeding procedure to be carried out under laboratory conditions, simulated donors were prepared that could be "bled" by inserting the needle of the blood-collecting kit into a segment of rubber tubing simulating the donor's vein (Fig. 1). Each of a series of 2-i bottles (standard glucose-saline bottles) was fitted with a filtered air-inlet tube and an outlet tube to which was attached 4 cm of rubber tubing (internal diameter, 0.6 cm). Into each bottle was introduced 1800 ml of dextrose broth adjusted to pH 9.0 and containing 0.002% of phenol red indicator. When 450 ml of this broth had been run into the collecting bag and mixed with the acid anticoagulant a final pH of 7.4 was obtained. The whole length of the outlet tube together with the stopper end of the bottle was then sheathed in aluminum foil and the entire apparatus autoclaved at 104 kPa (15 lb/in2) for 15 minutes with the distal end of the outlet tube open. Before use, the distal end of the outlet tube was clamped and the bottle inverted and fixed on a retort stand at a convenient height above the bench. Trapped air was expelled from the outlet tube by gentle squeezing to ensure complete filling. For bleeding of the "donor", the foil over a segment of the rubber tubing was carefully opened without contaminating the underlying tubing and the intravenous needle of the blood-collecting kit was inserted. When a free flow of broth was established, the site of puncture was covered with a sterile gauze pad. Three collecting bags were filled simultaneously from each "donor" (Fig. 2). When each bag was full to the 450-ml mark it was clamped, sampled by the standard technique using vacuum tubes contaminated with E. coli and sealed. The filled bags with their attached segmented tubing were incubated at 370C for 48 hours. The entire "bleeding" and sampling procedure was carried out by Red Cross nurses skilled in these techniques. In order to test for the possibility of contamination by reflux from non-
CMA JOURNAL/MAY 7, 1977/VOL. 116 989
Table I-Colony counts obtained on Rodac contact plates from skin at venipuncture site Colony count Donor
Before cleansing
A B C D E F G H J K 1
18 19 >300 43 33 Confluent >300 22 50 18 51 22
After cleansing 0 0 1 0 0 102 0 0 0 0 3 0
Table Ill-Contact plate counts from donors: overall counts Before After Datum cleansing cleansing Total count > 3051 130 Average count per donor > 31.1 1.3 Reduction in count, >95.7
Table Il-Contamination of blood in bags with Eschenchia coli following use of standard sampling procedure Growth of E. coli* Segments of tube
No. of bags
Bag
8
7
6
5
4
3
2
1
3
-
-
-
-
-
-
-
-
-
Single
8 1
+
+
+
+
+
+
+
+
+
Double
7 1
-
-
-
-
+
+
+
+
±
1
-
-
-
-
-
-
-
-
+
8
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
+
Sampling Control (notsampled)
Triple
*Gro.h of E. coli (+) indicated by turbidity and acid pH and confirmed by subculture.
Table Ill--Contact plate counts from donors: degree of initial contamination Before cleansing Colony count, range 0-20 21-50 51-100 >100 Total
No. of donors 58 26 7 7 98
Total count 442 1043 516 >1050 > 3051
Average count per donor 7.6 40.1 73.7 >150 >31.1
After cleansing No. of donors in colony count ranges
0 44 13 5 3 65
Average count per donor 0.6 2.2 0.3 5.6 -
Total count 33 56 2 39 130
1-5 6-10 > 10 11 3 10 2 1 2 - 3 1 26 5 2
Table V-Contact plate counts from donors: percentage reduction at different initial contamination levels
Colony count, No. of range donors 0-20 21-50 51-100 >100 Total
58 26 7 7 98
Before cleansing Average Total count count per donor 442 1043 516 >1050 > 3051
7.6 40.1 73.7 >150 > 31.1
After cleansing Average Total count count per donor 33 56 2 39 130
0.6 2.2 0.3 5.6 1.3
reduction 92.5 94.6 98.6 96.3 95.7
Appendix I-Steps in standard bleeding
tion of the sampling procedure, designed to prevent direct contact of the sampling needle with the contaminated contents of the vacuum tube, will permit even multiple sampling to be carried out without reflux contamination. In view of the heavy bacterial challenge used in these experiments (1O. organisms per tube as opposed to the peak loading of lO. per tube found in the purple-stoppered vacuum tubes as supplied), the margin of safety demonstrated is considerable, but a hazard remains - only a moment's carelessness or inattention would permit the needle to contact the blood in the vacuum tube. This last element of risk can only be eliminated by the use of sterile vacuum tubes. The elimination of the other two demonstrable sources of contamination, the donors' skin and the vacuum tube stoppers, requires no more than a simple modification of technique. We believe that the modified procedure we have developed provides the maximum degree of safety available with the present system of single-line donation. We acknowledge the technical assistance of C. McLennan, P. Gay, Miss Fran Brisson and the nursing staff of the Ottawa centre of the blood transfusion service in carrying out this study. References 1. Fenwal Technical Manual, Fenwal Laboratories, 1962, p 6 Evacuated blood-collection tubes - the backflow hazard. Can Med Assoc / 113: 208 1975 3. MCLEISH WA, CORRIGAN EN, ELDER RH, et al: Contaminated vacuum tubes (C). Can Med Assoc 1 112: 682, 1975
procedure A.
Bleeding
1. Bare arm and apply cuff; inflate to 40 mm Hg. 2. Cleanse skin with alcoholic Zephiran - once down middle of arm then once on each side, repeating this step three times. 3. Inflate cuff to 80 to 100 mm Hg and locate vein with finger. 4. Insert needle. 5. Bleed. 6. Release cuff. 7. Clamp tubing at bag end. 8. Pick up vacuum tube no. 1. 9. Remove needle and control bleeding. 10. Insert needle into vacuum tube, which is then filled from tubing, and mix by inverting three times. 11. Strip tubing into vacuum tube. B.
For single sampling
12. Heat-seal needle end of tubing. 13. Release clamp at bag end of tubing, which refills tubing from bag. 14. Separate and discard needle and attached fragment of tubing. 15. Strip tubing back into bag, mix and release, which refills tubing from bag. 16. Heat-seal tubing in sections starting from needle end.
2. ENG LK, JOHNSON DL, NEUFELD PD, Ct al:
C.
For double sampling 12. Clamp needle end of tubing.
13. Remove needle from vacuum tube no. 1 and insert in vacuum tube no. 2. 14. Release clamp at bag end of tubing, which refills tubing from bag. 15. Release clamp at needle end of tubing to fill vacuum tube from tubing and bag. 16. Clamp needle end of tubing. 17. Heat-seal needle end of tubing; separate and discard needle and attached fragment of tubing. 18. Strip tubing back into bag and mix. 19. Heat-seal tubing in sections starting from needle end. Appendix 11-Modified bleeding procedure A. Bleeding 1. Bare arm and apply cuff; inflate to 40 mm Hg. 2. Cleanse skin with alcohol and cetrimide (Savlon) swabs using a circular motion and then cleanse with additional individually packaged swab of 70% isopropyl alcohol. 3. Inflate cuff to 80 to 100 mm Hg if necessary to locate vein with finger; swab area again with 70% isopropyl alcohol. 4. Insert needle. 5. Bleed. 6. Release cuff. 7. Clamp tubing at bag end. 8. Pick up vacuum tube no. 1. 9. Remove needle and control bleeding. 10. Swab top of vacuum tube with an individually packaged swab of 70% isopropyl alcohol, insert needle into vacuum tube, release clamp to fill vacuum tube from tubing and bag and immediately replace clamp about 2.5 cm from needle. 11. Do not strip the blood back into the bag. B. For single sampling 12. Heat-seal needle end of tubing. 13. Separate and discard needle and attached fragment of tubing. 14. Heat-seal tubing in sections starting from bag end. C. For double sampling 12. Clamp needle end of tubing. 13. Remove needle from vacuum tube no. 1 and insert in vacuum tube no. 2 - again after swabbing the stopper. 14. Release clamp at needle end of tubing to fill vacuum tube from tubing and bag. 15. Clamp needle end of tubing. 16. Heat-seal needle end of tubing; separate and discard needle and attached fragment of tubing. 17. Heat-seal tubing in sections starting from bag end.E
CMA JOURNAL/MAY 7, 1977/VOL. 116 991
2.
In vitro cellular immunity to nervous system antigens in multiple sclerosis, in Multiple Sclerosis: Immunology, Virology and UltraGW, WOLFORAM 5, ELLIsoN structure, STEVENS JA, et al (eds), New York, Acad Pr, 1972, pp 333-64 RE, SHEREMATA WA, FELDMAN ROCKLIN RG, et al: Gui1lain-Barr. and multiple sclerosis: in vitro cellular responses to nervous tissue antigens. N Engl I Med 284: 803, 1971
3. SHEREMATA W, COLBY 5, KARKHANIS Y, et al:
Cellular hypersensitivity to P2 myelin protein in Guillain-Barr& Can I Neurol Sci 2: 87, 1975 4. SHEREMATA W, Cosuitova JBR, EYLAR EH: Cellular hypersensitivity to basic myelin (As) protein and clinical multiple sclerosis N
Engi J Med 291: 14, 1974 5. idem: Hypersensitization to basic myelin protein preceding attacks of multiple sclerosis. Trans Am Neurol Assoc 99: 55, 1974 6. PEKAREK J, JEDLICKA P,
SVEJCAR J, et al:
Production of migration inhibitory factor by lymphocytes of patients with multiple sclerosis, to brain antigens. Clin hnmunol Immunopathol (in press) 7. S9.sERo M, BENDIXEN 6: Human lymphocyte migration as a parameter of hypersensitivity. Acta Med Scand 181: 247, 1967 8. MYERS t.W: Leukocyte migration inhibition in multiple sclerosis, in Multiple Sclerosis: Immunology, Virology and Ulm'raslructure, op cit, pp 389-99 9. STRANDSCAARD
W,
J.RGENSEN
PN:
Delayed
hypersensitivity to myelin antigen in multiple sclerosis investigated with the leukocyte migration method. Acia Neurol Scand [Suppl] 48: 243, 1972 10. ARMSTRONG R: Inhibition of lymphocyte migration in multiple sclerosis (abstr). Can J Neurol Sci 2: 324, 1975 11. ALvORD EC, Hsu PC, THOR R: Leukocyte sensitivity to brain fractions in neurological
diseases. Arch Neurol 30: 296, 1974 12. BEROSTRAND H, KALLEN B, NILSsON 0: Ef-
of delayed hypersensitivity in man: ambiguity of polymorphonuclear neutrophils as indicator cells in leukocyte migration test. Inject bnnmn 3: 370, 1973 16. ROSENBERG
SA,
DAVID
JR:
Inhibition
at
leukocyte migration: an evaluation of this in vitro assay of delayed hypersensitivity in man to solLible antigen. J lininunol 105: 1447, 1970
17. READ SE, ZABRISKIE JB: Cellular interactions
in the leukocyte migration inhibition system. Trans Proc 4: 247, 1972
18. MOOKERJEE B, ACKMAN GFD, DossEToR JB: Delayed hypersensitivity in vitro using human peripheral leukocytes. Transplantation 8: 745, 1969 19. KALTREIDER BJ, SOGHOR D, TAYLOR JB, et al: Capillary tube migration for dctection of human delayed hypersensitivity: difficulties encountered with "buffy coat" cells and tuberculin antigen. J Inanmnol 103: 179, 1969 20. Fox RA, GREGOY DS, FELDMAN JD: Macrophage receptors for migration inhibitory factor (MIF), migration stimulator factor (MSF) and agglutinating factor. J linmunol 112: 1867, 1974 21. COHEN 5, FISHER B, YOSHIDA T, et at:
Serum migration inhibition activity in patients with lymphoproliferative diseases. N
fect of basic encephalitogenic protein and some peptides derived from it on the migration in agarose gel of leukocytes from patients with multiple sclerosis, other neurological diseases or carcinoma. Acta Neurol Scand 50: 227, 1974
22. MYERS LW, ELLISON GW: Effect of humoral factors on cellular hypersensitivity to nervous tissue in multiple sclerosis. Fed Proc 32:
13. EYLAR EH, BROSTOFF S, HASHIM G, et al: Basic As protein of the myelin membrane;
23. RAOCH HG, EINSTEIN ER, C5EJTEY I: Enzymatic degradation of myelin basic protein in central nervous system lesions of monkeys
the complete amino acid sequence. I Biol Chem 246: 5770, 1971 14. ROCKLIN RE: Products of activated lymphocytes: leukocyte inhibitory factor (LIF) distinct from migration inhibitory factor
(MIF). I Immunol 112: 1461, 1974 15. SENYK 6, HADLEY WK:
In vitro correlates
Engi J Med 290: 882, 1974 832, 1973
with experimental allergic encephalomyelitis. Neurobiology 3: 195, 1973
24. BROSTOFF SW, REUTER W, HIGHENS M, et al: Specific cleavage of the A1 protein from
myelin with cathepsin D. J Biol Chem 249: 559, 1974
Exogenous bacterial contamination of donor blood G. RocK,* MD, PH D J.C.N. WESTWOOD,t MD
The Canadian Red Cross blood transfusion service has followed a set protocol for phlebotomy and collection of a unit of blood. Recent requirements for automated testing have necessitated that a second tube of blood be obtained from the blood line following collection of the unit. Evaluation of the techniques used, however, has indicated the possibility of bacterial contamination from the skin of donors, from insertion of the needle through an unsterile rubber stopper, and through backflow from a nonsterile vacuum tube. To test these possibilities swabs were taken from skin and stoppers of vacuum tubes. Further, vacuum tubes were deliberately contaminated with Escherichia coil. The normal sampling procedure, which involves stripping the donor line to refill and mix the blood, was then followed. This resulted in contamination of the segments and even the blood bag. From the department of microbiology and immunology, University of Ottawa *Senior lecturer tProfessor and head Reprint requests to: Dr. G. Rock, Medical director, Canadian Red Cross blood transfusion service, 85 Plymouth St., Ottawa, Ont. KIS 3E2
These findings led to modification of the standard bleeding technique, whereby stripping was eliminated and sterile vacuum tubes were to be used at all times. Le service de transfusion de Ia Croix-Rouge canadienne a suivi un protocole defini pour Ia phlebotomie et Ia collecte d'une unite de sang. Des exigences recentes visant a automatiser les epreuves d'analyse ont necessite l'obtention d'un deuxieme prelevement sur Ia tubulure de perfusion apres Ia collecte de sang. Levaluation des techniques utilisees ont toutefois indique Ia possibilite de contamination bacterienne a partir de Ia peau du donneur, de l'insertion de l'aiguille a travers un bouchon non-sterile, ou a Ia suite d'un reflux venant d'un tube a vide non-sterile. Af in de verifier ces possibilites des prelevements ont ete faits sur Ia peau et sur les bouchons des tubes a vide. De plus, des tubes a vide ont .t6 d6lib6r6ment contamin6s avec de l'Escherichia coil. Le proc6d6 normal d'echantillonage, qui implique l'egoutteur de Ia tubulure de perfusion pour remplir et melanger le sang,
988 CMA JOURNAL/MAY 7, 1977/VOL. 116
a ete suivi alors. Ceci a resulte en une contamination des bouts du tube et mime du sac de sang Iui-m.me. Ces observations ont entraine une modification de Ia technique normale de saignee; on n'egoutte plus Ia tubulure et des tubes a vide steriles sont maintenant utilises en tout temps.
For years the Canadian Red Cross blood transfusion service has followed a standard procedure for drawing blood from donors, all of whom are volunteers. This procedure (Appendix I) is essentially the same as that outlined in the Fenwal manual.1 Changes in automation techniques, however, have made it necessary to obtain an additional tube of blood from the bag. The steps taken to permit the subsampling (Appendix I) basically involve refilling and stripping the tube a second time. This requirement to subsample prompted a reassessment of the entire bleeding procedure. A primary consideration was the possibility of bacterial contamination since subsampling and, indeed, initial sampling effectively result in an open system. Of particular concern was the possibility of backflow from the evacu-
ated tubes into the blood bag since the use of such nonsterile tubes has previously been reported to cause nosocomial infection in patients.2'3 The experiments reported in this paper were designed to evaluate the extent of the hazard at each point of the collection procedure and to indicate appropriate modifications. Background: points of contamination risk in the standard bleeding procedure Examination of the standard bleeding procedure as described in Appendix I indicated three sources of exogenous bacterial contamination of collected blood: 1. Donors' skin Because the degree of cleanliness of the skin of individual donors varies, residual bacterial contamination after cleansing could constitute a source of contamination of collected blood. Skin cleansing was carried out by three simple wipes repeated thrice using cotton-wool balls held in an open bowl of alcoholic benzalkonium chloride (Zephiran). The balls were squeezed out with the ungloved hand before a donor's skin was wiped; after that the skin was dried with a square of sterile gauze. The selected vein was then located with the finger before insertion of the needle. 2. Vacuum tube stoppers Insertion of the needle through the stopper at the time of sampling is likely to cause contamination of the needle unless the unsterile stopper is first sterilized. 3. Vacuum tube interiors The interiors of purple-stoppered vacuum tubes have been shown to be contaminated with Pseudomonas or Serratia species, or both, in concentrations of 1O. organisms per tube (J.C.N. Westwood, M.A. Mitchell: unpublished data). Regurgitation during sampling from the filled collecting kit could cause contamination of the blood in the collecting tube and thus the bag. Stripping of the blood contained in the tubing back into the bag after the vacuum tube is filled (step 15 of Appendix I) appears especially dangerous. The first two sources would cause contamination principally of the exterior of the needle but, unless their contamination was gross, it is unlikely the blood in the bag would become contaminated. The third source, however, would cause contamination of the blood within the vacuum tube and, if reflux occurred, contamination of the blood within the tubing leading to the bag. Under conditions of single sampling, reflux would be unlikely to carry any organisms far enough down the collecting tubing to jeopardize the sterile
condition of the blood in the bag, except when blood from the tubing is stripped back into the bag. Under conditions of multiple sampling, however, in which the tubing is allowed to refill from the bag, the danger of contamination of the blood in the tubing would increase with each sample taken. The degree of danger ot contamination of the bag would depend on whether blood was stripped from the tubing back into the bag. Methods and results
Experiments, series 1 Methods: In this series of experiments donors' skin and vacuum tube stoppers were tested for contamination and a simulated bleeding procedure was designed. The possibility of contamination by reflux from nonsterile vacuum tubes was also investigated. * Skin cultures: Cultures were performed using Rodac contact plates filled with trypticase-soy agar (TSA, Difco) and 5% sheep's blood. The cultures were prepared by contact with the venipuncture sites of 12 donors before the sites were cleansed and after they had been cleansed according to the procedure described above. The plates were incubated aerobically for 48 hours, after which developed colonies were counted. * Vacuum tube-stopper cultures: Stoppers also were examined for contamination by rubbing 40 of them thoroughly with sterile cotton swabs moistened with TS broth. Each swab was immediately inoculated onto the surface of a blood-agar plate (5%
B
FIG. 1-Apparatus simulating donor. A: 2-i bottle with filtered air-inlet tube and rubber outlet tube. B: "Bleeding" procedure: foil has been opened and needles inserted. During tests "bleeding" site was covered with sterile gauze.
sheep's blood in TSA) and incubated aerobically for 48 hours. * Contamination of vacuum tube interiors: Vacuum tubes (Becton-Dickinson red-stoppered) were contaminated by introducing into each, 0.5 ml of an overnight culture of Escherichia cali containing 10. organisms by viable plate count. The contaminating inoculum was introduced with a Cornwall syringe tipped with a 21-gauge needle. Resealing of the stopper after the needle was removed was adequate to retain a vacuum within the tube. * Simulated bleeding procedure: In order to permit the standard bleeding procedure to be carried out under laboratory conditions, simulated donors were prepared that could be "bled" by inserting the needle of the blood-collecting kit into a segment of rubber tubing simulating the donor's vein (Fig. 1). Each of a series of 2-i bottles (standard glucose-saline bottles) was fitted with a filtered air-inlet tube and an outlet tube to which was attached 4 cm of rubber tubing (internal diameter, 0.6 cm). Into each bottle was introduced 1800 ml of dextrose broth adjusted to pH 9.0 and containing 0.002% of phenol red indicator. When 450 ml of this broth had been run into the collecting bag and mixed with the acid anticoagulant a final pH of 7.4 was obtained. The whole length of the outlet tube together with the stopper end of the bottle was then sheathed in aluminum foil and the entire apparatus autoclaved at 104 kPa (15 lb/in2) for 15 minutes with the distal end of the outlet tube open. Before use, the distal end of the outlet tube was clamped and the bottle inverted and fixed on a retort stand at a convenient height above the bench. Trapped air was expelled from the outlet tube by gentle squeezing to ensure complete filling. For bleeding of the "donor", the foil over a segment of the rubber tubing was carefully opened without contaminating the underlying tubing and the intravenous needle of the blood-collecting kit was inserted. When a free flow of broth was established, the site of puncture was covered with a sterile gauze pad. Three collecting bags were filled simultaneously from each "donor" (Fig. 2). When each bag was full to the 450-ml mark it was clamped, sampled by the standard technique using vacuum tubes contaminated with E. coli and sealed. The filled bags with their attached segmented tubing were incubated at 370C for 48 hours. The entire "bleeding" and sampling procedure was carried out by Red Cross nurses skilled in these techniques. In order to test for the possibility of contamination by reflux from non-
CMA JOURNAL/MAY 7, 1977/VOL. 116 989
Table I-Colony counts obtained on Rodac contact plates from skin at venipuncture site Colony count Donor
Before cleansing
A B C D E F G H J K 1
18 19 >300 43 33 Confluent >300 22 50 18 51 22
After cleansing 0 0 1 0 0 102 0 0 0 0 3 0
Table Ill-Contact plate counts from donors: overall counts Before After Datum cleansing cleansing Total count > 3051 130 Average count per donor > 31.1 1.3 Reduction in count, >95.7
Table Il-Contamination of blood in bags with Eschenchia coli following use of standard sampling procedure Growth of E. coli* Segments of tube
No. of bags
Bag
8
7
6
5
4
3
2
1
3
-
-
-
-
-
-
-
-
-
Single
8 1
+
+
+
+
+
+
+
+
+
Double
7 1
-
-
-
-
+
+
+
+
±
1
-
-
-
-
-
-
-
-
+
8
-
-
-
-
-
-
-
-
-
1
-
-
-
-
-
-
-
-
+
Sampling Control (notsampled)
Triple
*Gro.h of E. coli (+) indicated by turbidity and acid pH and confirmed by subculture.
Table Ill--Contact plate counts from donors: degree of initial contamination Before cleansing Colony count, range 0-20 21-50 51-100 >100 Total
No. of donors 58 26 7 7 98
Total count 442 1043 516 >1050 > 3051
Average count per donor 7.6 40.1 73.7 >150 >31.1
After cleansing No. of donors in colony count ranges
0 44 13 5 3 65
Average count per donor 0.6 2.2 0.3 5.6 -
Total count 33 56 2 39 130
1-5 6-10 > 10 11 3 10 2 1 2 - 3 1 26 5 2
Table V-Contact plate counts from donors: percentage reduction at different initial contamination levels
Colony count, No. of range donors 0-20 21-50 51-100 >100 Total
58 26 7 7 98
Before cleansing Average Total count count per donor 442 1043 516 >1050 > 3051
7.6 40.1 73.7 >150 > 31.1
After cleansing Average Total count count per donor 33 56 2 39 130
0.6 2.2 0.3 5.6 1.3
reduction 92.5 94.6 98.6 96.3 95.7
Appendix I-Steps in standard bleeding
tion of the sampling procedure, designed to prevent direct contact of the sampling needle with the contaminated contents of the vacuum tube, will permit even multiple sampling to be carried out without reflux contamination. In view of the heavy bacterial challenge used in these experiments (1O. organisms per tube as opposed to the peak loading of lO. per tube found in the purple-stoppered vacuum tubes as supplied), the margin of safety demonstrated is considerable, but a hazard remains - only a moment's carelessness or inattention would permit the needle to contact the blood in the vacuum tube. This last element of risk can only be eliminated by the use of sterile vacuum tubes. The elimination of the other two demonstrable sources of contamination, the donors' skin and the vacuum tube stoppers, requires no more than a simple modification of technique. We believe that the modified procedure we have developed provides the maximum degree of safety available with the present system of single-line donation. We acknowledge the technical assistance of C. McLennan, P. Gay, Miss Fran Brisson and the nursing staff of the Ottawa centre of the blood transfusion service in carrying out this study. References 1. Fenwal Technical Manual, Fenwal Laboratories, 1962, p 6 Evacuated blood-collection tubes - the backflow hazard. Can Med Assoc / 113: 208 1975 3. MCLEISH WA, CORRIGAN EN, ELDER RH, et al: Contaminated vacuum tubes (C). Can Med Assoc 1 112: 682, 1975
procedure A.
Bleeding
1. Bare arm and apply cuff; inflate to 40 mm Hg. 2. Cleanse skin with alcoholic Zephiran - once down middle of arm then once on each side, repeating this step three times. 3. Inflate cuff to 80 to 100 mm Hg and locate vein with finger. 4. Insert needle. 5. Bleed. 6. Release cuff. 7. Clamp tubing at bag end. 8. Pick up vacuum tube no. 1. 9. Remove needle and control bleeding. 10. Insert needle into vacuum tube, which is then filled from tubing, and mix by inverting three times. 11. Strip tubing into vacuum tube. B.
For single sampling
12. Heat-seal needle end of tubing. 13. Release clamp at bag end of tubing, which refills tubing from bag. 14. Separate and discard needle and attached fragment of tubing. 15. Strip tubing back into bag, mix and release, which refills tubing from bag. 16. Heat-seal tubing in sections starting from needle end.
2. ENG LK, JOHNSON DL, NEUFELD PD, Ct al:
C.
For double sampling 12. Clamp needle end of tubing.
13. Remove needle from vacuum tube no. 1 and insert in vacuum tube no. 2. 14. Release clamp at bag end of tubing, which refills tubing from bag. 15. Release clamp at needle end of tubing to fill vacuum tube from tubing and bag. 16. Clamp needle end of tubing. 17. Heat-seal needle end of tubing; separate and discard needle and attached fragment of tubing. 18. Strip tubing back into bag and mix. 19. Heat-seal tubing in sections starting from needle end. Appendix 11-Modified bleeding procedure A. Bleeding 1. Bare arm and apply cuff; inflate to 40 mm Hg. 2. Cleanse skin with alcohol and cetrimide (Savlon) swabs using a circular motion and then cleanse with additional individually packaged swab of 70% isopropyl alcohol. 3. Inflate cuff to 80 to 100 mm Hg if necessary to locate vein with finger; swab area again with 70% isopropyl alcohol. 4. Insert needle. 5. Bleed. 6. Release cuff. 7. Clamp tubing at bag end. 8. Pick up vacuum tube no. 1. 9. Remove needle and control bleeding. 10. Swab top of vacuum tube with an individually packaged swab of 70% isopropyl alcohol, insert needle into vacuum tube, release clamp to fill vacuum tube from tubing and bag and immediately replace clamp about 2.5 cm from needle. 11. Do not strip the blood back into the bag. B. For single sampling 12. Heat-seal needle end of tubing. 13. Separate and discard needle and attached fragment of tubing. 14. Heat-seal tubing in sections starting from bag end. C. For double sampling 12. Clamp needle end of tubing. 13. Remove needle from vacuum tube no. 1 and insert in vacuum tube no. 2 - again after swabbing the stopper. 14. Release clamp at needle end of tubing to fill vacuum tube from tubing and bag. 15. Clamp needle end of tubing. 16. Heat-seal needle end of tubing; separate and discard needle and attached fragment of tubing. 17. Heat-seal tubing in sections starting from bag end.E
CMA JOURNAL/MAY 7, 1977/VOL. 116 991
