Effect of Anticoagulants on Microbial Viability

(Please note that this report is formatted for submission to the journal of Clinical Laboratory Sciences)

The Effect of Anticoagulants on Microbial Viability

Project Abstract:

OBJECTIVE: To determine if the anticoagulants in specimen collection tubes, which are occasionally received in the microbiology laboratory as suboptimal specimens, have an inhibitory effect on the viability of four different microorganisms over time.

DESIGN: Anticoagulated and non-anticoagulated specimen collection tubes containing one milliliter of serum were inoculated with bacteria. The tubes were left at room temperature for varying time intervals (3, 6, 24, and 48 hours), streaked on plates, and observed for growth. The colony counts of the plates inoculated from the anticoagulated tubes were compared to the colony counts from the non-anticoagulated tubes to determine if there was a drop in the viability of the organisms. 

RESULTS: Of the four organisms tested, S. aureus and C. albicans showed similar results at each of the various time intervals.  Each exhibited an increase in colony count in the sodium heparin and control tubes, however, in the EDTA tube the viability did not increase, but remained constant.  The results for E. coli were opposite from those of S. aureus and C. albicans.  EDTA stimulated the growth of E. coli, whereas, the sodium heparin and control tubes showed a dramatic decrease in the viability over time.  The fourth organism, H. influenzae, appeared to exhibit a decrease in viability in all three specimen collection tubes over time, however, several data points were somewhat inconsistent.

CONCLUSION: Based on these results, if a specimen is collected in the specimen collection tubes tested in this study, and then received and plated in the microbiology laboratory within 6 hours of inoculation, there was no significant loss in colony count with the four microorganisms tested. However, if the specimens were plated at 24 or 48 hours, there was a significant decrease in the viability of some of the organisms in certain collection tubes, making the recovery of these organisms uncertain.

SETTING: Department of Clinical Laboratory Sciences, Weber State University, Ogden, Utah.

PARTICIPANTS: Healthy, volunteer blood donors currently not taking antimicrobial drugs.

ABBREVIATIONS: EDTA = Ethylenediaminetetra-acetic acid, SST = serum separator tube, CFU = colony forming units, CO2 = carbon dioxide, S. aureus = Staphylococcus aureus, E. coli = Escherichia coli, H. influenzae = Haemophilus influenzae, and C. albicans = Candida albicans.

INDEX TERMS: microbiology; anticoagulants; specimen collection tubes.

Jamie Kulbacki BS CLS(ASCP) is recent graduate and now is employed at LDS Hospital, Salt Lake City, UT.
Cicily Breinholt BS CLS(ASCP) is a recent graduate and is now employed at McKay Dee Hospital, Ogden, UT.
Jennifer Marquardt BS CLS(ASCP) is a recent graduate and is now employed at LDS Hospital, Salt Lake City, UT.
Scott Wright MS is an Associate Professor in the Department of Clinical Laboratory Sciences, Weber State University, Ogden, UT.

Address for correspondence:
Scott Wright
Clinical Laboratory Sciences
Weber State University
3905 University Circle
Ogden UT 84408-3905

Body fluids, which need to be cultured, occasionally arrive at clinical microbiology laboratories as suboptimal specimens that have been collected in tubes that contain anticoagulants. Most laboratory policies dictate that these specimens are to be rejected due to the concept that anticoagulants inhibit microbial viability, even though there are occasions when a proper culture specimen cannot be obtained.  Documentation for the basis of this rejection could not be found in the literature reviewed.  The objective of this research was to prove or disprove the concept that these suboptimal samples should be rejected by the microbiology laboratory.
       
The project involved specimen collection anticoagulant tubes and  non-anticoagulant tubes in which serum was added and then inoculated with one of four microorganisms.  After the specimens were inoculated, they were left at room temperature for various time intervals, simulating the time it takes for a specimen, after collection, to arrive in the clinical microbiology laboratory.  Times were selected to represent transportation of specimens from both urban and rural hospitals to a central laboratory.  The specimens were then streaked onto chocolate agar plates at each time interval and incubated overnight in a 37oC CO2 incubator.  The colony counts from the tubes containing the anticoagulants were compared to the colony counts from the non-anticoagulated tubes.

MATERIALS AND METHODS
Three types of standard specimen collection tubes were used, purple top tubes containing the anticoagulant ethylenediaminetetra-acetic acid (EDTA) and green top tubes containing the anticoagulant sodium heparin, and red top tubes containing no anticoagulant. 

Four bacterial species were selected to represent common clinical pathogens with various fastidious and non-fastidious growth requirements.  These organisms were: S. aureus, E. coli, C. albicans (all three of which are considered non-fastidious), and H. influenzae (fastidious).

Blood was collected in nine ml serum separator tubes (SST’s) from healthy volunteer donors who were not currently taking antimicrobial drugs.  The blood was centrifuged for ten minutes at 3,000 rpm, and the serum was removed.  For each microorganism tested, one ml of serum from a donor was then added to each of the following tubes:  one purple top tube containing EDTA, one green top tube containing sodium heparin, and two red top tubes containing no anticoagulant (control and blank).

A bacterial suspension was prepared from each of the four organisms in sterile saline and the concentration was standardized using a Crystal Spec Nephelometer (Table 1).  Each organism was then diluted to obtain a standardized inoculate (Table 1) and added to the three tubes described above.  Sterile saline was added to a second red top tube in place of the inoculate.  The tubes were then left at room temperature for 3, 6, 24, and 48 hours.  At each time interval, a 10 µl calibrated loop was used to inoculate a chocolate agar plate from each of the tubes (no further dilutions or adjustments were made).  The plates were then incubated at 37°C overnight in a CO2 incubator.  The number of colonies on each plate were counted (up to a maximum of 300 colonies). This procedure was repeated ten times during the course of the project.

RESULTS
The mean of the colony forming units (CFU) for each organism, time intervals, and respective tubes, of the ten repetitions are shown in Tables 2, 3, 4, and 6, along with the correlation coefficient for the control verses sodium heparin and control verses EDTA.  The means were then graphed in Figures 1, 2, 3, and 4.  It is important to note that colony counts of zero represent no colonies from the specimen collection tube using a 10 µl calibrated loop.
S. aureus
The correlation between the control tube and the sodium heparin tube exhibited a strong positive correlation, with an increase in growth in both tubes overtime.  Yet, the correlation between the control tube and the EDTA tube exhibited a strong negative correlation, as the CFU in the control tube increased, the CFU in the EDTA tube decreased.  The overall CFU in the EDTA tube dropped only slightly over the 48 hours. 
E. coli
The correlation between the control tube and the sodium heparin tube demonstrated a positive correlation, with a decrease in growth in both tubes at 24 and 48 hours.  The correlation between the control tube and the EDTA tube demonstrated a strong negative correlation, as the control tube decreased in growth the EDTA tube increased. 
C. albicans
A strong positive correlation was seen between the control tube and the sodium heparin tube, with an increase in growth in both tubes observed overtime.  There was no significant correlation between the control tube and the EDTA tube.  The control tube increased in growth, whereas growth in the EDTA tube remained almost steady.
H. influenzae
The initial colony counts at 3 hours showed a significant variation between the three tubes as compared to the other three organisms.  There was not a significant correlation between the control tube and the sodium heparin tube and only a slight negative correlation between the control tube and the EDTA tube.  Over the ten plating replicates, the colony counts at 24 and 48 hours for all tubes were zero, except for those entries listed in Table 5.

DISCUSSION
For the purpose of transporting patient specimens containing S. aureus and C. albicans, each of the specimen collection tubes containing anticoagulants proved to be acceptable at 3, 6, 12, and 24 hours.  Both organisms grew in the sodium heparin and control tubes over time.  In the EDTA tubes, although there was no actual growth, the colony count remained relatively constant over the time intervals and viability of the organisms decreased only slightly.

The results obtained for E. coli were opposite to those of S. aureus and C. albicans.  There was a significant drop in the viability of the organism in both the control tube and sodium heparin between 6 and 24 hours.  Interestingly, the organism grew significantly in the presence of EDTA during that time period.  Overall, the results indicate that for E. coli, up to 6 hours in any of the specimen collection tubes would be acceptable, however, at 24 or 48 hours, recovery of the organism would be uncertain.

The results for H. influenzae, the only fastidious organism used in the study, exhibited sporadic results that are difficult to explain.  The initial 3 hour colony count varied significantly between the control, sodium heparin, and EDTA tubes (which was not observed with the other test organisms).  At 6 hours, all tubes showed a decrease in colony count, while at 24 hours, the count from the sodium heparin and control tubes showed an increase.  This increase is due to the effect of a few aberrant data points (Table 5) on the mean of the data seen in Graph 4, especially in the case of the control tube.  All other replicates at 24 and 48 hours for the control and sodium heparin tubes had zero colonies for H. influenzae (data not shown in Table 5).  It can therefore be concluded, that possibly with the exception of the sodium heparin tubes, all the specimen collection tubes decreased the viability of H. influenzae at 24 and 48 hours.

In conclusion, the data from this study shows that if a specimen is collected in any of the three specimen collection tubes, and then received and plated in the microbiology laboratory within 6 hours, the organisms that were tested would be viable, and demonstrate only a slight decrease in concentration.  H. influenzae may be the one exception, although the data is inconclusive.  If the collection tubes are received at 24 or 48 hours, recovery of the organisms that were tested becomes unlikely, and the specimens should be rejected. 
It is important to note that serum was the only specimen fluid examined in this study, and the effects of the specimen collection tubes on other common fluids, i.e. synovial fluid and cerebral spinal fluid, is unknown.  The recovery of microorganisms from these types of fluids in specimen collection tubes should be examined in future studies, if they are to be considered as viable culture transport mechanisms to the microbiology laboratory.

HUMAN SUBJECT REVIEW
This project was approved by the Institutional Review Board of Weber State University.  Each blood donor signed a consent form which stated the purpose of the experiment and the risks of blood donation.

ACKNOWLEDGEMENTS
This undergraduate student research project was funded through a grant provided by the Dumke College of Health Professions Marriott Endowment.   The authors would like to thank Lloyd Burton, Department of Health Administrative Services, Weber State University for his advice on the statistical analysis of the data.

The project results were presented as a poster presentation at the Weber State University Symposium for Undergraduate Student Research (2004) and awarded the Outstanding Undergraduate Research Project for Dumke College of Health Professions.  The project results were also presented as a poster presentation at the Utah Society of Clinical Laboratory Science Spring Seminar (2004) and the National Conference for Undergraduate Research in Indianapolis, Indiana (2004).