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  Q & A





December 2011

Fredrick L. Kiechle, MD, PhD

Question Q. Our lab occasionally receives a formalin-fixed specimen from a patient who has been treated with rituximab for a B-cell malignancy. Since the drug targets cells that express CD20, should we be performing immunohistochemistry for other B-cell markers to look for residual disease?

A. Rituximab, sold under the trade name Rituxan (Genentech), is a chimeric monoclonal IgG1 kappa antibody directed against the CD20 antigen, which is normally present on the surfaces of pre-B and mature B lymphocytes. Rituximab is currently indicated as single-agent therapy or in combination with chemotherapy for most patients with non-Hodgkin lymphomas that express CD20, particularly for those patients with follicular lymphoma or diffuse large B-cell lymphoma. Other indications for rituximab therapy are chronic lymphocytic leukemia and the non-neoplastic conditions rheumatoid arthritis, Wegener’s granulomatosis, and microscopic polyangiitis.

Rituximab acts by binding to the CD20 antigen expressed on malignant cells and initiating cell lysis via one of several possible mechanisms. Downregulation of CD20 by B-cell non-Hodgkin lymphoma cells has been described as a potential molecular mechanism for developing resistance to rituximab.1 In fact, over the past few years there have been reports of loss of CD20 expression in CD20-positive B-cell lymphomas treated with rituximab. Consequently, it is important for pathologists to be aware of this problem so that an appropriate workup can be performed on post-treatment specimens.

When immunohistochemically evaluating a specimen for a B-cell non-Hodgkin lymphoma, it is prudent to order one or more additional B-cell markers if there has been a history of anti-CD20 therapy. Anti-CD79a is useful because it has high specificity for the B-cell lineage,2 but one must keep in mind that the CD79a antigen is expressed on a wider range of cells than CD20—in particular, on plasma cells. Another immunohistochemical marker, CD19, is sensitive for B lymphocytes but may also be positive in a small subset of T-cell lymphomas.2 PAX5 is another very useful marker of B cells in this setting.

Finally, it is important to document loss of expression of CD20 by immunohistochemistry because it will have important therapeutic, and potentially prognostic, significance. It must be noted, however, that evaluating loss of CD20 in chronic lymphocytic leukemia/small lymphocytic lymphoma by immunohistochemistry is not straightforward since the antigen is normally expressed dimly and may appear negative in even pretreatment cases.


1. Hiraga J, Tomita A, Sugimoto T, et al. Down-regulation of CD20 expression in B-cell lymphoma cells after treatment with rituximab-containing combination chemotherapies: its prevalence and clinical significance. Blood. 2009;113(20): 4885–4893.

2.   Adams H, Liebisch P, Schmid P, et al. Diagnostic utility of the B-cell lineage markers CD20, CD79a, PAX5, and CD19 in paraffin-embedded tissues from lymphoid neoplasms. Appl Immunohistochem Mol Morphol. 2009;17(2):96–101.

Jennifer G. Pryor, MD
Williamsport Pathology Association
Williamsport, Pa.

Member, CAP
Immunohistochemistry Committee

Question Q. We have an Iris Diagnostics urine analyzer in our lab. When the pH of a urine goes over 8.0, is it necessary to do a sulfosalicylic acid test to prove false results?

A. The CAP checklist question URN.31250 says, “There are criteria for identifying urine samples that may give erroneous results by the dipstick reader, and thus require evaluation by alternate means (visual examination or other confirmatory method).”

Reagent strips use the protein error method for detection and are most specific for albumin. Reagent strips may be falsely positive with highly buffered or alkaline urines (pH >8.0), urines with a large amount of hemoglobin, urines contaminated with ammonium compounds (disinfectants), contrast agents, or chlorhexadine, or by delayed reading of the strip. False-negative reagent strip readings occur at a pH of less than 3.0 or when proteins other than albumin are present (for example, Bence-Jones, or other abnormal immunoglobulins). These limitations are listed in the package insert for the Aution sticks commonly used in the Iris. The Iris literature claims an overall sensitivity of 92.1 percent for protein and a specificity of 96.8 percent. The instrument has an analytical range of 5.0 to 9.0 for pH and 10 to 600 mg/dL for protein (albumin).

A confirmatory test is defined as a test that detects the same substance as the reagent strip with greater sensitivity or specificity by using a different reaction.

Sulfosalicylic acid (SSA) detects all proteins by precipitation and is sensitive to 5 to 10 mg/dL. A common procedure for performing an SSA test involves centrifuging 12 mLs of urine and decanting the supernatant (about 11 mLs) into a second clean 16- × 125-mL tube. Three milliliters of seven percent sulfosalicylic acid is added, and the tube is inverted twice. The mixture is allowed to stand for 10 minutes and then observed for precipitation.

Each laboratory determines and validates the range of reportable values for each testing method or instrument it uses. If results in the reportable range may be falsely positive or negative because of sample characteristics, a confirmatory method such as an SSA test should be performed.


Brunzel NA. Fundamentals of Urine and Body Fluid Analysis. Philadelphia, Pa.: WB Saunders. 1994.

Schumann JL. Manual of Cytodiagnostic Urinalysis. 2nd ed. Salt Lake City: University of Utah School of Medicine. 1989.

Roberta L. Zimmerman, MD
Grand Itasca Clinic and Hospital
Grand Rapids, Minn.

Member, CAP Hematology/
Clinical Microscopy Resource Committee

Question Q. The absolute percent of neutrophils is important in making decisions about treatment for an infectious disease. We run into a problem with counts of less than 1,000. What is the correct procedure to report the absolute percent of neutrophils in patients with total counts of 800, 600, or 500 or less?

A. An accurate absolute neutrophil count (ANC) is important in determining risk of infection and in therapeutic decisionmaking. The general thresholds for risk of infection are as follows:

  • ANC <0.5 × 109/L: significantly increased risk of serious infection and with fever often requires parenteral antibiotics.
  • ANC 0.5–1.0 × 109/L: some propensity for infection but often can be managed as an outpatient.

Automated hematology analyzers can perform an accurate leukocyte differential in low WBC counts, even as low as 0.1–0.2 × 109/L in a subset of cases. Since automated hematology analyzers assess many more leukocytes than a manual differential (8,000–10,000 automated events compared with 100–200 by manual differential), the precision is better, and thus it is preferable to use the automated differential if accurate.

Based on its international group of 20 laboratorians, the International Society of Laboratory Hematology suggests that smear review be performed for a WBC of <4.0 × 109/L or absolute neutrophil count of <1.0 × 109/L the first time, and again if the WBC delta fails within a three-day period.1 Each laboratory must determine its own criteria for technologist review and release of leukocyte differential results as appropriate for its patient population. For cases requiring technologist review, evaluation of the automated hematology analyzer cytograms may be useful in assessing the accuracy of the differential. Generally, an accurate automated differential will show reasonably discrete cell populations with the neutrophil population located in the expected region of the cytograms. Technologist scanning review of the blood smear may also be sufficient to confirm the accuracy of the automated differential without the need for a manual differential. In these cases, the automated differential can be reported even if the WBC is low.

In cases where the automated differential is deemed not to be accurate as a result of poor instrument cytogram patterns or technologist scanning review in conflict with the instrument results, or both, a manual differential can be performed. Factors contributing to the relatively poor precision of a manual differential include a low number of cells counted (often 100–200), interobserver variability, and leukocyte distribution on the smear, a factor contributing to increasing imprecision with a decreasing leukocyte count.

In one study of manual differentials, the ANC showed a coefficient of variation of five percent at 5.0 × 109/L, 18 percent at 0.50 × 109/L, and 42 percent at 0.05 × 109/L, although a single individual performed the differentials, thus effectively eliminating interobserver variability.2 In a laboratory with many technologists, these coefficients of variation would be expected to be even higher.

The literature does not contain data to support specific guidelines for manual leukocyte differentials in leukopenic patients, and laboratory practice in this area differs significantly. Some laboratories will perform a manual differential for all WBC levels, including very low WBCs (<0.5 × 109/L), although they may define a maximal frequency (such as not more than every 24 hours) for performing a manual differential in this setting. Others choose to perform and report manual differentials down to a defined WBC threshold, such as 0.5 × 109/L, since the precision in this region is poor and a significantly increased propensity for infection occurs at a neutrophil count below that level. In addition, separate criteria can be set for outpatients and inpatients, if appropriate for a specific patient population. For example, some medical centers report a leukocyte differential on all oncology outpatients, regardless of the overall leukocyte count, but may limit inpatient manual leukocyte differentials to certain patient populations or a defined leukocyte threshold. Tracy I. George, MD, addressed a related topic in a question and answer in the July 2009 CAP TODAY, in which a reader asked about repeat differentials in oncology patients with a low WBC count. In cases where a manual differential is performed, an absolute count for each cell type should be obtained and reported by multiplying the percentage of the cell type by the overall WBC count. These absolute numbers are what carry clinical significance.3


1. Barnes PW, McFadden SL, Machin SJ, et al. The international consensus group for hematology review: suggested criteria for action following automated CBC and WBC differential analysis. Lab Hematol. 2005;11:83–90.

2. Ross DW, McMaster K. Neutropenia: the accuracy and precision of the neutrophil count in leukopenic patients. Cytometry. 1983;3:287–291.

3. Etzell JE. For WBC differentials, report in absolute numbers. CAP TODAY. 2010;3:12.

Joan E. Etzell, MD
Director, Clinical
Hematology Laboratory
University of California
San Francisco

Vice Chair, CAP Hematology/
Clinical Microscopy Resource Committee

Dr. Kiechle is medical director of clinical pathology, Memorial Healthcare, Hollywood, Fla.