Fredrick L. Kiechle, MD, PhD
Q. What is the recommended tube for collecting joint fluids for crystal analysis? Has a comparison study been done on joint fluid stored in different types of tubes? We have been encouraged to send joint fluid specimens for crystal analysis in EDTA tubes; empty sterile tubes or containers have been used until now. Will EDTA dissolve the calcium pyrophosphate crystal?
A. Synovial fluid from inflamed joints can clot when collected and therefore should be collected into an anticoagulant for accurate cell counts, differential counts, and crystal analysis.1 It can be transported and stored at room temperature but should be examined as soon as possible, and definitely within 24 hours, because cellular degeneration will occur. There is some disagreement about the best anticoagulant to use. Sodium heparin is a safe choice2,3 and has not been reported to cause crystalline artefacts that can interfere with crystal analysis. In addition, many laboratories use EDTA with good success. However, as noted in the CLSI document on the examination of body fluids: “Some texts indicate that lithium heparin and EDTA should not be used as anticoagulants, because they produce crystalline material that can be confused with pathologic crystals. However, others have used lithium heparin and EDTA without difficulty.”4 Lithium heparin has been reported to form rhomboid crystals that can mimic calcium pyrophosphate dihydrate crystals, which are seen in pseudogout.2 Oxalate should clearly not be used, as it will cause the formation of calcium oxalate crystals. These crystals can be identified in the synovial fluid of patients with primary oxa-losis or chronic renal failure.2,4
Sodium heparin and EDTA are used most commonly in laboratories for cell counts, differential counts, and crystal analysis. Only a small amount of synovial fluid is required. If there is sufficient volume, other chemistry studies, such as glucose, protein, CH50, C3, and C4, can be performed on tubes without anticoagulant, or in the case of C3 or C4, with EDTA. A fluoride tube can also be used for glucose. Synovial fluid for culture should be collected into SPS (sodium polyanethol sulfate) tubes without anticoagulant, or at least with an anticoagulant that does not have a suppressive effect on bacterial growth.2,4
1. Freemont AJ, Denton J, Chuck A, et al. Diagnostic value of synovial fluid microscopy: a reassessment and rationalization. Ann Rheum Dis. 1991;50:101–107.
2. Galagan KA, Blomberg D, Glassy EF, et al., eds. Color Atlas of Body Fluids: An Illustrated Field Guide Based on Proficiency Testing. Northfield, Ill.: College of American Pathologists; 2006.
3. Smith GP and Kjeldsberg CR. Cerebrospinal, Synovial and Serous Body Fluids. In: Henry JB, ed. Clinical Diagnosis and Management by Laboratory Methods. 20th ed. Philadelphia, Pa.: WB Saunders Co.; 2001.
4. Clinical and Laboratory Standards Institute. Body Fluid Analysis for Cellular Composition; Approved Guideline. CLSI document H56-A. Wayne, Pa.: Clinical and Laboratory Standards Institute; 2006.
Katherine A. Galagan, MD
Director of Clinical Laboratories
Virginia Mason Medical Center
Is it considered a best laboratory practice to perform slide microscopy to identify white blood cells in stool?
A. Microscopic examination of a nonconcentrated wet or stained preparation is the traditional means for identifying and quantifying WBCs in fresh or fixed stool. This information can be useful to physicians since the presence of WBCs is associated with certain causes of inflammatory diarrhea, such as invasive bacterial infections and inflammatory bowel disease.1 During microscopic examination, red blood cells, yeasts, and intestinal parasites can also be identified.
Despite the utility of this test, microscopic examination requires expertise and can produce false-negative results if the WBCs have degenerated. Prolonged transportation time, collection of stool on a swab, and storage at room temperature can all negatively affect morphology. WBCs can also be lysed by toxin-producing organisms such as Clostridium difficile, an important cause of diarrhea in certain patient populations.
Because of the limitations of microscopy, some have advocated using fecal lactoferrin tests in place of, or in addition to, the traditional slide exam for WBC detection. Fecal lactoferrin is an iron-binding glycoprotein found in the secondary granules of neutrophils and is therefore an alternative marker of fecal polymorphonuclear leukocytes. This compound remains stable in feces despite leukocyte deterioration and can therefore provide additional positive results when compared with morphologic identification.2,3 Commercially available fecal lactoferrin antigen detection methods are rapid, accurate, and relatively easy to perform.
However, the antigen detection methods are not a complete substitution for the slide exam. Available tests are not quantitative and thus microscopic examination is still required to quantify fecal leukocytes. Microscopy is also useful for detecting RBCs and parasites. Finally, lactoferrin is present in mother’s milk and the test can be positive in nursing infants who do not have intestinal inflammation.
Given the pros and cons of the different methods for WBC and fecal lactoferrin detection, it is up to the laboratory to decide which method to implement as a marker for intestinal inflammation. Availability of skilled microscopists and satisfaction with existing processes are two factors that can contribute to the deci-sion.
1. Harris JC, DuPont HL, Hornick BR. Fecal leukocytes in diarrheal illness. Ann Intern Med. 1971;76:697–703.
2. Guerrant RL, Arujo V, Soares E, et al. Measurement of fecal lactoferrin as a marker of fecal leukocytes. J Clin Microbiol. 1992;30:1238–1242.
3. Yong WH, Mattia AR, Ferraro MJ. Com-parison of fecal lactoferrin latex agglutination assay and methylene blue microscopy for detection of fecal leukocytes in Clostridium difficile-associated disease. J Clin Microbiol. 1994;32(5):1360–1361.
Bobbi S. Pritt, MD, MSc
Director of Clinical Virology and Parasitology
Division of Clinical Microbiology
Mayo Clinic, Rochester, Minn.
Member, CAP Microbiology
Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla.