College of American Pathologists
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  Q & A





cap today

July 2001

Q.  In assessing chronic gastritis on mucosal biopsies, the quantity of chronic inflammatory cells in the lamina propria is used to discriminate between gastritis and normal. I have learned from texts and lectures that the normal number of chronic inflammatory cells varies and can include zero, few, and less than five per HPF. Is there a consensus on the number of chronic inflammatory cells in normal patients?

A.  The question asks about the minimum number of mononuclear cells needed to call a gastric biopsy abnormal, but because of similar queries regarding minimal criteria for abnormality in other GI sites, this response is generalized.

The issue of abnormal inflammation in glandular GI tract mucosa can be divided into a three-part algorithm based on qualitative and quantitative assessments:

  1. Qualitative assessment: Any neutrophils, increased eosinophils or abnormal histiocytes (either foamy or clustered) are abnormal. Neutrophils will normally be noted traversing capillaries or larger vessels within the lamina propria but none should be seen within the lamina propria stroma or epithelial compartment. It is exceptional to have pathological neutrophil infiltrates within the lamina propria without also noting them in the epithelium, making the observation of intraepithelial neutrophils a sensitive and specific marker for an acute inflammatory state. Eosinophils are normally present in small numbers throughout the GI tract mucosa. Only if they are present in high numbers and without neutrophils should the diagnosis of eosinophilic enteritis be entertained. A subjective mild eosinophilia is common, especially in the small bowel and right colon. This is a frustrating finding, as it catches the pathologist’s eye but rarely correlates with a clinically relevant etiologic diagnosis. Foamy histiocytes and granulomas are associated with well characterized disease states, which don’t require further comment.
  2. Quantitative assessment of intraepithelial lymphocytes: Inflammatory conditions exist in the GI tract that are mediated by mononuclear inflammation and, therefore, do not differ from normal on the type of cells present—only quantity. Most such conditions have increased numbers of lymphocytes within the surface epithelial compartment. This is helpful for objectifying the presence of a pathological process as (1) quantitative normal ranges are established for lymphocytes within the surface epithelium, and (2) the number of lymphocytes can be standardized to the number of epithelial cells. Normal ranges vary among the discrete sites of the GI tract and are expressed per 100 epithelial cells: stomach, 10 to 20; small bowel, 20 to 40; and colon, five to 10. More intraepithelial lymphocytes will normally be present over lymphoid aggregates. Otherwise, lymphocytes present within the surface epithelium at quantities greater than these should be reported as abnormal (e.g. suggestive of celiac disease in the small bowel).
  3. Quantitative assessment of lamina propria lymphocytes and plasma cells: In the absence of neutrophils, marked eosinophilia, abnormal histiocytes or intraepithelial lymphocytosis, an important inflammatory condition is unlikely in a GI endoscopic biopsy. Possible subtleties are best discussed individually for each site, but pathologists should not fear making the diagnosis of normal mucosa. The stomach is the most likely to be abnormal at this point in the algorithm and is also the most subjective. Most GI pathologists assess this issue at low power only but those who wish to be analytical will usually settle on criteria such as fewer than five mononuclear cells per HPF, fewer than three mononuclear cells between crypts, or no clusters of three to five plasma cells. The antrum probably deserves slightly more leeway than the body. Mild inflammation will commonly accompany foveolar hyperplasia in the reactive gastropathy typical of nonsteroidal anti-inflammatory drugs or bile reflux. No data exist that critically compare modest amounts of chronic inflammation in the stomach to symptoms, etiologic agent, or outcome.

The small bowel will essentially never be judged abnormal based only on the quantity of lamina propria mononuclear cells. The lamina propria is normally rich in lymphocytes and plasma cells with a few eosinophils. "Flat" mucosa is typically artifactual in the absence of intraepithelial lymphocytosis. The only conceivable abnormal situation based on too many lamina propria mononuclear cells is one where the small bowel crypts are widely separated to such a degree that lymphoma or mast cell disease is suspected.

Normal colon mucosa normally contains lymphocytes and plasma cells but they are confined to the luminal half of the lamina propria (except in the cecum where they are present throughout the lamina propria). Abnormal lamina propria mononuclear inflammation should always be accompanied by either intraepithelial lymphocytosis (microscopic colitis) or architectural derangement (idiopathic inflammatory bowel disease). Lacking either of these associations, one should be very critical about calling the biopsy abnormal.

Don’t hesitate to call those biopsies normal. Remember, there is no such thing as "nonspecific chronic gastroenterocolitis."

1. Dixon MF, Genta RM, Yardley JH, et al. Classification and grading of gastritis. The updated Sydney System. International Workshop on the Histopathology of Gastritis, Houston 1994. Am J Surg Pathol. 1996;10: 1161-1181.
2. Dobbins WO. Human intestinal intraepithelial lymphocytes. Gut. 1986;27:
3. Owen DA, Segal GH, Petras RE, et al. Stomach, small intestine and colon. In: Sternberg SS, ed. Histology for Pathologists. New York, NY: Raven Press; 1992;533-592.

Jerry Dayharsh, MD
Larry Burgart, MD
Department of Pathology
Mayo Clinic
Rochester, Minn.

Dr. Burgart is a member of the Surgical Pathology Committee.

Q.  Is there an established or accepted level for WBCs in peritoneal dialysate fluid at which point a differential is not indicated? We are trying to provide a 100-cell differential, regardless of the WBC count.

A.  The laboratory medicine literature on peritoneal dialysate fluid is almost nonexistent. Standard reference texts1-4 refer peripherally to dialysate fluids in the course of discussing ascitic fluid in general. They suggest that total leukocyte counts of greater than 300-500/µL in ascitic fluid are abnormal and that, if greater than 25 to 50 percent of these cells are neutrophils, the diagnosis of peritonitis should be considered. McBride indicates that an absolute neutrophil count of 240-500/µL has a sensitivity and specificity for bacterial peritonitis in excess of 90 percent.4

According to literature related specifically to nephrology and dialysis,5,6 normal peritoneal dialysis fluid should contain less than 50 WBC/µL,7 less than 15 percent of which should be neutrophils. If the percentage of neutrophils exceeds 35 percent, the fluid should be considered suspicious for bacterial infection. Common nonbacterial causes of neutrophilia in dialysate fluids include infectious diarrhea or active colitis, menstruation or ovulation, and pelvic inflammatory disease. One report even attributed neutrophilia to travel over bumpy mountain roads en route to a dialysis center.

Bacterial peritonitis should be seriously considered when any two of the following three criteria are present:

  1. Symptoms and signs of peritoneal irritation
  2. Cloudy peritoneal fluid with a total leukocyte count greater than 100/µL, of which at least 50 percent are neutrophils
  3. Presence of bacteria on Gram stain or culture

This last criterion is considered to be a minor one for purposes of initiating treatment as the sensitivity is only about 50 percent and results may not be available in less than two hours. However, a positive Gram stain is predictive of culture results in 85 percent of cases and should always be performed when cultures are set up.

None of the above information specifically addresses the question of when to perform a WBC differential count on dialysis fluid. Assuming that antibiotic administration is based on a combination of clinical and laboratory criteria, and that the specimen would not have been submitted to the laboratory unless there was clinical evidence suggestive of sepsis, a conservative approach would be to perform a differential count on all cloudy dialysates, regardless of the total leukocyte count. The problem with this approach is that there will be times when a differential WBC would be performed on fluids containing very few cells, definitely a laborious task. Other sets of criteria may be equally valid. Probably the best approach is to arrive at criteria for your institution in consultation with your medical staff.

1. Kjeldsberg CR, Knight JA. Body Fluids: Laboratory Examination of Amniotic, Cerebrospinal, Seminal, Serous and Synovial Fluids; A Textbook Atlas. 2nd ed. Chicago, Ill: ASCP Press; 1986:107-109.
2. Ringsrud K, Linne J. Urinalysis and Body Fluids: A Color Text and Atlas. St. Louis, Mo: Mosby; 1995:204-206.
3. Strasinger S. Urinalysis and Body Fluids. 3rd ed. FA Davis & Co: Philadelphia, Pa; 1994:177.
4. McBride L. Textbook of Urinalysis and Body Fluids: A Clinical Approach. Philadelphia, Pa: Lippincott; 1998:244-245.
5. Daugirdas H, Ing T, eds. Handbook of Dialysis. 2nd ed. Toronto, Ontario: Little Brown; 1994:338-344.
6. Keane WF. ISPD guidelines/recommendations: adult peritoneal dialysis-related peritonitis treatment recommendations: 2000 update. Perit Dial Int. 2000;20:
7. Sulowicz W. Usefulness of light microscopy and cytur test estimation of peritoneal dialysate in early diagnosis of peritonitis. Int Urol Nephrol. 1990;22: 189-197.

David Blomberg, MD
Department of Pathology
Miller-Dwan Medical Center
Duluth, Minn.

Hematology/Clinical Microscopy
Resource Committee

Q.  What kind of sterilization procedure do you recommend and how long should the cycle be for Creutzfeldt-Jakob disease postmortem cases? What is your opinion about the real situation with iatrogenic CJD in the United States and the necessity to use specific decontamination procedures to prevent transmission of prions in real practice?

A.  Creutzfeldt-Jakob disease and other transmissible spongiform encephalopathies are caused by infectious agents that are not inactivated by standard sterilization methods. Thus, special procedures are required to treat contaminated instruments, glassware, and other hard surfaces. Any visible blood should be removed by soaking or washing. Surgical instruments and glassware may then be decontaminated chemically or by autoclaving. Chemical decontamination may be accomplished by soaking for one hour in either sodium hypochlorite (freshly opened Clorox or similar chlorine bleach) or 2N sodium hydroxide. Gravity-displacement autoclaving should be performed at 132°C for at least one hour.1-3 As an additional precaution, one may combine the chemical and autoclaving procedures, either by using chemical treatment followed by autoclaving or, best of all, by autoclaving in sodium hydroxide.1,3

After chemical or combined autoclaving/chemical treatment, the instruments and glassware must be rinsed thoroughly with water. Unless this rinse is sterile, surgical instruments will then require standard autoclaving prior to reuse. Instruments used in neurosurgery or ophthalmologic surgery on known or suspected cases of CJD are frequently discarded. Any tissue samples should be fixed in formalin, exposed to 96 to 100 percent formic acid for one hour, and then returned to formalin for additional fixation prior to processing.1

While the preceding procedures should be used whenever prion disease is suspected in a specific patient, there is no need for their routine use. At least 98 percent of all CJD cases are sporadic or familial; only one to two percent are iatrogenic. To date, all iatrogenic cases have involved exposure to CNS or closely related tissues. Most have been in recipients of cadaveric human growth hormone or dural grafts; a few cases due to corneal transplants, depth electrodes, and contaminated surgical instruments have also occurred.4

No cases have been documented of iatrogenically transmitted CJD following surgery at other body sites, presumably because prions are found less often and at lower titers outside the CNS.4-6 However, prudence would suggest using CJD precautions for any invasive procedure in an affected patient.

Issues of transmissibility from exposure to non-neural tissue may be somewhat different for the new variant CJD, or vCJD, the form related to bovine spongiform encephalopathy. In vCJD, the agent is frequently present in lymphoid tissues, including lymphoid tissues associated with the gastrointestinal tract.7 One British patient with vCJD had an appendectomy within a year prior to the onset of neurologic symptoms. On review, prions were detected in the paraffin block of his appendix.8 Subsequently, over 3,000 tonsil and appendectomy specimens were re-examined. None contained prions, however,9 and CJD precautions have not been instituted in routine medical practice in the United Kingdom. No cases of vCJD have been reported in the United States; the most recent data on this topic are available on the Web site of the National Prion Disease Pathology Surveillance Center (

1. Brown P. Guidelines for high risk autopsy cases: special precautions for Creutzfeldt-Jakob disease. In: Hutchins G, ed. Autopsy Performance and Reporting. Northfield, Ill: College of American Pathologists; 1990:68-74.
2. Rosenberg RN, White CL, Brown P, et al. Precautions in handling tissues, fluids, and other contaminated materials from patients with documented or suspected Creutzfeldt-Jakob disease. Ann Neurol. 1986;19:75-77.
3. Taylor DM. Inactivation of transmissible degenerative encephalopathy agents: a review. Vet J. 2000;159:10-17.
4. Brown P, Preece M, Brandel J-P, et al. Iatrogenic Creutzfeldt-Jakob disease at the millennium. Neurology. 2000;55: 1075-1081.
5. Brown P, Cervenáková L, McShane LM, et al. Further studies of blood infectivity in an experimental model of transmissible spongiform encephalopathy, with an explanation of why blood components do not transmit Creutzfeldt-Jakob disease in humans. Transfusion. 1999;39:1169-1178.
6. Ironside JW, Head MW, Bell JE, et al. Laboratory diagnosis of variant Creutzfeldt-Jakob disease. Histopathology. 2000a;37:1-9.
7. Hill AF, Butterworth RJ, Joiner S, et al. Investigation of variant Creutzfeldt-Jakob disease and other human prion diseases with tonsil biopsy samples. Lancet. 1999;353:183-189.
8. Hilton DA, Fathers E, Edwards P, et al. Prion immunoreactivity in appendix before clinical onset of variant Creutzfeldt-Jakob disease. Lancet. 1998; 352:703-704.
9. Ironside JW, Hilton DA, Ghani A, et al. Retrospective study of prion-protein accumulation in tonsil and appendix tissues. Lancet. 2000b;355:1693-1694.

Barbara J. Crain, MD, PhD
Associate professor of pathology
The Johns Hopkins University
School of Medicine

Chair, Neuropathology Committee

Q.  Are there any methods that increase the chances of seeing positive crystals in synovial fluids (for example, placing a sample in the refrigerator prior to observation)?

A.  To my knowledge, there is no described method for enhancing the detection of monosodium urate crystals in joint fluid. Several authors have described the appearance of MSU crystals in refrigerated samples that were not present on initial examination.1,2 However, the significance of this finding is unknown. Furthermore, other authors have not observed this phenomenon.3-5

Calcium pyrophosphate crystals normally decrease in number over a period of several days, and refrigeration does not prevent this phenomenon. Alizarin red S staining of wet mounts or fixed preparations has been advocated to enhance detection of CPP crystals.6,7 This method stains a variety of calcium-containing compounds in synovial fluid, thus limiting its specificity.7,8 However, the method described by Lazcano et al, in which fixed cytology preparations are stained, permits evaluation of morphology and intra- versus extra-cellular localization of alizarin red S-staining material, thus improving specificity.7

1. Font F, Goldman J, Toro R. Monosodium urate crystals and spherulites in asymptomatic metatarsophalangeal joints [abstract]. Arthritis Rheum. 1982;25 (suppl):S53.
2. Bluhm GB, Riddle JM, Barnhart MI, et al. Crystal dynamics in gout and pseudogout. Med Times. 1969;97:135-144.
3. Kerolus G, Clayburne G, Schumacher HR, Jr. Is it mandatory to examine synovial fluids promptly after arthrocentesis? Arthritis Rheum. 1989;32:271-278.
4. Schumacher H, Jimenez S, Gibson T, et al. Acute gouty arthritis without urate crystals identified on initial examination of synovial fluid. Report on nine patients. Arthritis Rheum. 1975;18:603-612.
5. Bible MW, Pinals RS. Late precipitation of monosodium urate crystals [letter]. J Rheumatol. 1982;9:480.
6. Paul H, Reginato AJ, Schumacher HR. Alizarin red S staining as a screening test to detect calcium compounds in synovial fluid. Arthritis Rheum. 1983;26:191-200.
7. Lazcano O, Li CY, Pierre RV, et al. Clinical utility of the alizarin red S stain on permanent preparations to detect calcium-containing compounds in synovial fluid. Am J Clin Pathol. 1993;99:90-96
8. McCarty DJ. Crystal identification in human synovial fluids. Methods and interpretation. Rheum Dis Clin North Am. 1988;14:253-267.

Steven H. Kroft, MD
University of Texas-Dallas
Southwestern Medical Center

Hematology/Clinical Microscopy Resource Committee

Q.  What is the best diagnostic test for Crohn’s disease?

A.  No definitive diagnostic test for Crohn’s disease—other than bowel biopsy showing typical histopathology—exists. A serological test called anti-Saccharomyces cerevisiae antibody, or ASCA, which suggests Crohn’s disease over irritable bowel syndrome and ulcerative colitis, is available from at least one reference laboratory (Prometheus, San Diego). Reagents are available from Inova Diagnostics (San Diego).

The maximum test sensitivity for Crohn’s disease is obtained when IgG and IgA class ASCA tests are performed. This yields a combined sensitivity of approximately 52 percent. The low sensitivity of the test implies it will not be useful in screening unselected patients for that diagnosis. Patients with Crohn’s limited to the large bowel (Crohn’s colitis) are generally not positive for ASCA but instead are often positive for a form of antineutrophil cytoplasmic antibody, which is called atypical P-ANCA to distinguish it from typical P-ANCA that is associated with some systemic vasculitides. I would recommend that both ASCA and ANCA be ordered if one is attempting to find a test to increase the probability of Crohn’s disease in the differential diagnosis. For a review of current medical opinion concerning these tests, please see "Shades of gray in gastrointestinal testing" by William Check, PhD, in CAP TODAY (August 2000).

Another recommendation in evaluating a patient with the differential diagnosis of irritable bowel syndrome versus inflammatory bowel disease is that the patient also be tested for celiac disease. Celiac disease (gluten-sensitive enteropathy) causes intestinal cramping and urgency similar to IBS and IBD. It occurs at a frequency of about three to four per 1,000 people in the U.S. population, but only 30 to 40 percent of patients have typical clinical symptoms. I recommend IgA antiendomysial antibody and antitissue transglutaminase ELISA for screening. Many gastroenterologists also screen using antigliadin antibody; however, in my experience, this test is neither specific nor sensitive for celiac disease.

James A. Goeken, MD
Director, Immunopathology Laboratory
University of Iowa Health Care
Iowa City

Diagnostic Immunology
Resource Committee