MLO1006 Tips from the clinical experts
BD Biosciences flow cytometry systems
Since launching the first IVD-cleared benchtop
flow cytometers, we’ve continued to bring color
and workflow improvements to your clinical lab,
including the 4-color BD FACSCalibur™ and the
BD FACSCanto™ II with 8-color capability.
Today, over 10,000 clinical labs worldwide depend
on BD Biosciences to continually improve sensitivity
and reliability of results, and to simplify workflow.
Innovation. To be continued.
The BD FACSCanto system was the first
IVD-cleared 6-color flow cytometer.
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Edited by Brad S. Karon, MD, PhD
Answering your questions
Culturing vaginal/cervical specimens
QWhat are the guidelines about what organisms to work up from routine cultures from vaginal/cervical swabs?
AVaginal infections (vaginitis/vaginosis) are caused by a limited number of infectious organisms; namely Trichomonas vaginalis and Candida spp. Bacterial vaginosis is known to be caused by a synergistic interaction between multiple bacterial species, both aerobic and anaerobic1 but is not diagnosed by bacterial culture. Group A Streptococcus vulvovaginitis is a symptomatic but benign infection of children (not adults).2 The role of Group B streptococci (GBS) as an etiologic agent of vaginitis in adults has not been established; it is considered a normal inhabitant of the adult vaginal flora and is not associated with an inflammatory response.2 Staphylococcus aureus, the enterococci, and enteric Gram-negative bacilli are also considered part of normal or contaminating flora and have not been associated with adult vaginitis/vaginosis.
—Susan E. Sharp, PhD, D(ABMM)
Director of Microbiology
Kaiser Permanente Pathology
Winn WC, Allen S, Janda W, Koneman E, Procop G, et al. Infections of the Genital Tract. In: Koneman’s color atlas and textbook of diagnostic microbiology. 6th ed. 2006. Philadelphia, PA: Lippincott Williams & Wilkins. 2006:88-89.
McCormack WM. Vulvovaginitis and Cervicitis. In: Principles and Practice of Infectious Diseases. 6th ed. New York: Churchill Livingstone. 2005:1357-1372.
CSF cell count on clear fluid
QOur emergency department insists we use tube #1 and tube #4 for cerebrospinal fluid (CSF) cell count, even if the CSF is colorless. The ER physicians say they want to differentiate subarachnoid hemorrhage from traumatic taps. In the case of concurrent of traumatic taps and recent subarachnoid hemorrhage, what we should do?
AMost cases of subarachnoid hemorrhage are diagnosed by computed tomographic (CT) scanning of the brain. When the CT result is negative, however, most physicians recommend a lumbar puncture to examine CSF. There are several parameters measurable by obtaining a lumbar puncture that can aid in diagnosis of a subarachnoid hemorrhage. These include elevated opening pressure of the lumbar puncture, the presence of red blood cells (RBCs) in the CSF, and the presence of xanthochromia in CSF.1 A traumatic tap, however, can complicate the interpretation of the CSF findings since it has features overlapping with those of a subarachnoid hemorrhage. Since traumatic lumbar punctures are fairly common with an estimated incidence of about 10% to 20%,2 the clinical laboratory routinely needs to try to differentiate a subarachnoid hemorrhage from a traumatic tap.
There is no specific threshold for the number of RBCs in the CSF used to diagnose subarachnoid hemorrhage or to differentiate subarachnoid hemorrhage from traumatic tap. Subarachnoid hemorrhage has been reported with “only a couple of hundred erythrocytes” in the CSF. CSF with a RBC concentration of greater than 6,000 has been described as grossly bloody, whereas a cell count from 500 to 6,000 yields cloudy CSF. CSF with an RBC concentration of less than 400 to 500 appears colorless.3 For these reasons, a colorless tube does not entirely rule out a subarachnoid hemorrhage.
In order to distinguish a traumatic tap from a true subarachnoid hemorrhage using the number of RBCs in the CSF, the trend has been to perform a RBC count on both the first and last CSF tubes collected (tube #1 and tube #4). CSF samples from a traumatic tap generally show clearing of RBCs with successive tubes, whereas those from a true hemorrhage show a more stable RBC count. When the RBC count decreases to zero or close to zero in tube #4, this is strong evidence for a traumatic tap. Sometimes, the specimen shows incomplete clearing in tube #4, however, representing a likely traumatic tap that may or may not be superimposed on a subarachnoid hemorrhage.
In the case where there is suspicion of both a traumatic tap and a subarachnoid hemorrhage, other CSF parameters can help discern whether a subarachnoid hemorrhage is present, such as the opening pressure, which is elevated in many cases of subarachnoid hemorrhage but not in traumatic tap. Furthermore, presence of xanthochromia, microscopic evidence of erythrophagocytosis, and hemosiderin-laden macrophages are findings indicating subarachnoid hemorrhage, as long as there has not been a prior traumatic tap. Xanthochromia is yellow-tinged supernatant of CSF, which results from a breakdown hemoglobin that follows a hemorrhage. Three hemoglobin breakdown products include oxyhemoglobin, bilirubin, and methemoglobin. Oxyhemoglobin (with absorption at 416 nm, 540 nm, and 578 nm) appears in CSF within two hours of onset of subarachnoid hemorrhage and peaks within 24 hours to 36 hours. Bilirubin (with an absorption curve between 400 nm to 500 nm) appears approximately 10 hours after onset of bleeding. Methemoglobin (with absorption at 540 nm, 575 nm, and 630 nm) can appear at any time after oxyhemoglobin but is only seen with encapsulated CNS bleeding.4 Xanthochromia can be accurately measured by spectrophotometer based on the specific absorption pattern.5
Because RBC lysis begins as early as one to two hours after a traumatic tap, the CSF must be evaluated as soon as possible to avoid false-positives. There is also a latex agglutination immunoassay for cross-linked fibrin derivative D-dimer, which is specific for fibrin degradation and is negative in traumatic tap.6 False-positive results, however, are sometimes seen in cases with DIC, fibrinolysis, and trauma from repeated lumbar punctures. Occasionally, a clinician may repeat the lumbar puncture at the next higher vertebral interspace in an attempt to eliminate confounding data from a traumatic tap.
Perform the RBC differential counts in tube #1 and tube #4, even when blood is not visible in the CSF, in order to help rule out a subarachnoid hemorrhage and/or a traumatic tap. In cases where the differential is equivocal, other CSF parameters may aid in the diagnosis.
—Karen MacDonell, MD
—Guang Fan, MD, PhD
Department of Pathology
Oregon Health and Science University
Shah KH, Edlow JA. Distinguishing traumatic lumbar puncture from true subarachnoid hemorrhage. J Emerg Med. 2002;23(1):67-74.
Shah KH, et al., Incidence of traumatic lumbar puncture. Acad Emerg Med. 2003;10(2):151-154.
Wallach J ed. Interpretation of diagnostic tests. 7th ed. Baltimore, MD: Lippincott Williams & Wilkins, 2000; 263-292.
Graves P, Sidman R. Xanthochromia is not pathognomonic for subarachnoid hemorrhage. Acad Emerg Med. 2004;11(2):131-135.
Edlow JA, Bruner KS, Horowitz GL. Xanthochromia.Arch Pathol Lab Med. 2002;126(4):413-415.
Smith GP, Kjedsberg CR. Cerebrospinal, synovial and serous body fluids. In: Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods. 20th ed. Philadelphia, PA: Saunders. 2001:403-411.
Propoxyphene in urine
Q Will any other drug cause a positive propoxyphene result in a urine drug-screen test?
APropoxyphene (Darvon) is a synthetic narcotic, similar in structure to methadone. It has a similar analgesic effect as other morphine-like opioids. It is less potent than codeine and is sometimes prescribed in combination with aspirin or acetaminophen. The drug is metabolized by the liver and most of it is excreted in the urine as N-norpropoxyphene.
Because of its structural similarity to methadone, some screening tests will cross react. For instance, in the Roche Integra microparticle screening test, 0.9 gm/mL of methadone will give the same reaction as 300 ng/mL of propoxyphene. Other drugs were found not to crossreact.1 In gas chromatography-mass spectrometry, or GC-MS, confirmatory tests, propoxyphene is distinct. Although it has the same retention time as methadone, it has a different ionic pattern.
Propoxyphene is not commonly seen as an abused drug, and most people using it have prescriptions for it.2
—edited by Brad S. Karon, MD, PhD
on behalf of Daniel M. Baer, MD
Cobas Integra 400/700/800, Propoxyphene Data Sheet, 2004-2005.
Shults TF. Medical Review Officer Handbook, 7th ed. Research Triangle Park, NC, Quadrangle Research, LLC, 1999.
Transfusion-testing protocol sought
QMicrobiology 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?
ASeptic-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:
fever associated with transfusion (39°C or greater or an increase of 2°C or more);
chills and rigors;
tachycardia ( >120 bpm or an increase of 40 bpm or greater over baseline);
hypotension or hypertension with rise or fall of systolic blood pressure of 30 mm of Hg;
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 to150 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°C in CO2 and at 25°C. Red-cell unit cultures should also be done at 1°C to 6°C 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 SOP.
—Krishna Oza, MD
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.
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.
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.
Isenberg HD, ed. Culture of Blood Bank Products. In: Clinical Microbiology Procedure Handbook. 2nd ed. American Society for Microbiology, Washington, DC. 2004;§13.13.
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.
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.
Specimen requirements for direct-measure LDL
QI have seen conflicting information about the specimen requirements for direct-measure low-density lipoprotein (LDL). Is there any reason to require fasting specimens for direct measure LDL?
AThere is very little biological variation (<5%) between LDL-C values between fasting and non-fasting conditions. If a patient has hypertriglyceridemia (above 500 mg/dL to 700 mg/dL), however, there can be an analytical interference with some direct LDL-C measurements. This is especially true of hypertriglyceridemia expressed as hyperchylomicronemia as compared to hyperpre-betalipoproteinemia (elevated VLDL). Some direct LDL-C analytical methods are more robust than others to handle the marked elevations in triglycerides. Be sure to ask the manufacturer of the method you select about this issue.
—Herbert K. Naito, PhD, MBA
NorthStar Consulting Service
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.