Answering your questions

Transfusion-testing protocol sought

Q Microbiology often has requests from blood banks to culture units from transfusion reactions. Is there a protocol to cover this? What media and incubation temperature are recommended?

A Septic-transfusion reaction resulting from bacterial contamination of blood products can be a serious and fatal complication of transfusion therapy. Of the more than 9 million platelet-unit concentrates transfused each year in United States, the estimated rate of bacterial contamination is one in 1,000 to 3,000 units — resulting in transfusion-associated sepsis in many recipients. To reduce this risk, on March 1, 2004, AABB adopted a new standard requiring measures to detect and limit the bacterial contamination in all platelet components. Even when testing complies with the new standard, false-negatives can occur and cause fatal bacterial sepsis in the recipients.

In the completed survey of Infectious Disease Society of America, Emerging Infections Network, or IDSA EIN, only 36% (143 of 399) of members were aware that the bacterial contamination of platelets is the most common infectious risk of transfusion therapy, and only 20% were aware of the new AABB standard. The survey results indicated huge gap in the clinician's knowledge of the infectious complication of transfusion therapy. Thus, the first and most important step in the culturing of blood components is increasing the clinician's awareness for the infectious complication, and recognition of clinical symptoms and signs in recipients of transfusion. Since these reactions happen within four hours to six hours of transfusion, all the bags of transfused units should be kept for that period of time and should be submitted for cultures before discarding. A blood sample from the recipient should be also sent to laboratory for cultures. Following is the list of symptoms and signs that should lead to culturing blood components:

• tachycardia ( >120 bpm or an increase of 40 bpm or greater over baseline);
• fever associated with transfusion (39^0C or greater or an increase of 2^0C or more);
• chills and rigors;
• hypotension or hypertension with rise or fall of systolic blood pressure of 30 mm of Hg;
• nausea/vomiting;
• dyspnea;
• septic shock; and
• disseminated intravascular coagulopathy, or DIC.

As the above symptoms and signs are non-specific and can be seen with other associated clinical conditions, septic reaction from transfusion is underreported.

Platelets are particularly vulnerable to bacterial growth due to room temperature storage, whereas the other blood components are refrigerated or frozen, reducing the potential proliferation of contaminating bacteria. Gram-positive bacteria from donor skin (e.g., Staphylococcus spp. and Streptococcus spp.) are the most commonly recognized contaminants in platelets and less frequently followed by Gram-negative bacteria (e.g., Serratia, Enterobacter, Salmonella spp. Providentia retteri and Klebsiella pneumoniae), which account for more severe and fatal infection attributed to donor bacteremia or contamination during product processing. Gram-negative cold-loving bacteria (e.g., Yersinia enterocolitica and Pseudomonas spp.) are the common contaminants seen in packed red blood cells, with the prevalence rate estimated at 1 in 500,000 units. In the U.S., bacterial contamination is the second most common cause of death from transfusion, following clerical errors. About 100 to 150 transfused individuals suffer severe morbidity and mortality from bacterial contamination of blood components.

Management of septic-transfusion reactions from bacterial contamination:
It is essential to have a protocol to recognize and manage septic reaction, and culture the suspected component unit with corresponding recipient samples. A protocol for this should be developed in collaboration with clinicians (infectious disease), nursing units, transfusion-medicine specialists, and microbiology-laboratory services. Following are the important constituents of such a protocol or algorithm including culturing of the blood component:

• When septic reaction is suspected, stop the transfusion, report the reaction, and submit the unit with all the attached tubing to the blood bank or transfusion service for culturing in a sealed bag.
• The IV-access site for the recipient should be kept open for the further therapy. A sample for blood cultures of the recipient should be drawn prior to antibiotic therapy. Also samples from catheters for IV therapy and fluids can be submitted for cultures.
• Blood bank will visually inspect the remaining components, work up the transfusion reaction, and submit samples to the microbiology laboratory for cultures. Micro will also inform the transfusion-medicine specialist and the blood center which collected the unit to prevent by quarantine the potential further reactions from the other components of donation.
• Microbiology laboratory will perform Gram stain and will inoculate sets of broth (aerobic and anaerobic blood-culture bottles) and plate cultures similar to blood-culture protocol for both aerobic and anaerobic bacteria, depending on the quantity of sample available and results of Gram stain (if positive, plate cultures on blood and chocolate agar can be initiated). Samples should be directly obtained from inside the blood bag. It is important that segments should not be used for culture because they can be falsely negative.
• A simultaneous set of blood cultures from recipient for both aerobic and anaerobic bacteria will be performed. Other related specimens will be also cultured similarly.
• Incubation will be done at 35^0C in CO2 and at 25^0C. Red-cell unit cultures should also be done at 1^0C to 6^0C for isolation of psychrophillic bacteria.
• Depending on the blood-culture system, manual or automated cultures can be performed. For automated systems, one should follow the manufacturer's instructions.
• Subcultures should be performed for the identification of isolates when positive growth (turbidity, hemolysis, gas, and so forth) is indicated. In the manual method, blind subcultures can be performed at 18 hours and 48 hours. Blood and chocolate agar can be used for subcultures. Other solid media can also be used for identification purposes if a particular organism is suspected from Gram stain of positive cultures or clinical suspicion. Antibiotic-susceptibility testing should be performed promptly.
• Results of positive cultures should be reported to the transfusion-medicine specialist, blood center (collection facility) medical director, and patient's clinician.
• If bacteria are isolated from both the recipient and blood component, further characterization to establish relatedness of the strains may be indicated (e.g., molecular typing, serotyping).
• Isolates from both the residual blood products and recipients should be retained.
• Notify appropriate state and local health department if any organism with public-health significance is identified in either residual component or recipients.

The above information pertains mostly to hospital transfusion services. The donor-related microbiological studies performed by the collection facilities are beyond the scope of this answer. The above guidelines and the following reading material can be helpful in the development and adaptation of a protocol for management of septic-transfusion reaction and microbiology culture standard operating procedure (SOP).

—Krishna Oza, MD
Hematopathology
US LABS
Brentwood, TN

Further reading

1. Rao PL, Strausbaugh LJ, Liedtke LA, Srinivasan A, Kuehnert MJ; Bacterial infections associated with blood transfusion: experience and perspective of infectious diseases consultants. Transfusion. 2007;47(7):1206-1211.
2. Hillyer CD, Josephson CD, Blajchman MA, Vostal JG, et al. Bacterial contamination of blood components: risks, strategies, and regulation: Hematology. Am Soc Hematol Educ Program. 2003;575-589.
3. Shulman IA. College of American Pathologist Laboratory Accreditation Checklist Item TRM.44955. Phase I Requirement on Bacterial Detection in platelets. Arch Pathol Lab Med. 2004;128(9):958-963.
4. Isenberg HD, ed. Culture of Blood Bank Products. In: Clinical Microbiology Procedure Handbook. 2nd ed. American Society for Microbiology, Washington, DC. 2004;SS13.13.
5. Delage G, Goldman M, Heddle N, McCombie N, Robillard P. Guideline for Investigation of Suspected Transfusion Transmitted Bacterial Contamination. http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/08vol34/34s1/34s1-eng.php. Accessed December 6, 2009.
6. American Association of Blood Banks. Bacterial Contamination of Platelets: Summary for Clinicians on Potential Management Issues; Related to Transfusion Recipients and Blood Donors.
   AABB Bacterial Contamination Task Force. http://www.aabb.org/Content/News_and_Media/Topics_of_Interest/bactcontplat022305. Accessed December 6, 2009.

MLO's “Tips from the Clinical Experts” provides practical, up-to-date solutions to readers' technical and clinical issues from a panel of experts in various fi elds. Readers may send questions to Brad S. Karon, MD, PhD, by e-mail at [email protected].

Brad S. Karon, MD, PhD, is assistant professor of laboratory medicine and pathology, and director of the Hospital Clinical Laboratories, point-of-care testing, and phlebotomy services at Mayo Clinic in Rochester, MN.