Key terms: flow cytometry; reagents; reporting; consensus; optimal

Transcription

1 Cytometry Part B (Clinical Cytometry) 72B:S14 S22 (2007) 2006 Bethesda International Consensus Recommendations on the Immunophenotypic Analysis of Hematolymphoid Neoplasia by Flow Cytometry: Optimal Reagents and Reporting for the Flow Cytometric Diagnosis of Hematopoietic Neoplasia Brent L. Wood, 1 * Maria Arroz, 2 David Barnett, 3 Joseph DiGiuseppe, 4 Bruce Greig, 5 Steven J. Kussick, 6 Teri Oldaker, 7 Mark Shenkin, 8 Elizabeth Stone, 9 and Paul Wallace 10 1 Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington 2 Department of Clinical Pathology, CHLO, Egas Moniz Hospital, Lisbon, Portugal 3 Department of Haematology, UK NEQAS for Leukocyte Immunophenotyping, Royal Hallamshire Hospital, Sheffield, England 4 Department of Pathology and Laboratory Medicine, Hartford Hospital, Hartford, Connecticut 5 Department of Pathology, Vanderbilt University Medical Center, Nashville, Tennessee 6 Flow Cytometry, PhenoPath Laboratories, and Department of Laboratory Medicine, University of Washington Medical Center, Seattle, Washington 7 Department of Flow Cytometry, Genzyme Genetics Corp., Los Angeles, California 8 Flow Cytometry, Ameripath, Orlando, Florida 9 Molecular Pathology Laboratory Network, Maryville, Tennessee 10 Roswell Park Cancer Institute, Flow and Image Cytometry, Department of Pathology and Laboratory Medicine, Buffalo, New York Immunophenotyping by flow cytometry has become standard practice in the evaluation and monitoring of patients with hematopoietic neoplasia. However, despite its widespread use, considerable variability continues to exist in the reagents used for evaluation and the format in which results are reported. As part of the 2006 Bethesda Consensus conference, a committee was formed to attempt to define a consensus set of reagents suitable for general use in the diagnosis and monitoring of hematopoietic neoplasms. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. A survey of participants successfully identified the cell lineage(s) to be evaluated for each of a variety of specific medical indications and defined a set of consensus reagents suitable for the initial evaluation of each cell lineage. Elements to be included in the reporting of clinical flow cytometric results for leukemia and lymphoma evaluation were also refined and are comprehensively listed. The 2006 Bethesda Consensus conference represents the first successful attempt to define a set of consensus reagents suitable for the initial evaluation of hematopoietic neoplasia. q 2007 Clinical Cytometry Society Key terms: flow cytometry; reagents; reporting; consensus; optimal The Cytometry Part B: Clinical Cytometry supplement (72B, Supplement 1, 2007) titled 2006 Bethesda International Consensus Conference on Flow Cytometric Immunophenotyping of Hematolymphoid Neoplasia is sponsored by the Clinical Cytometry Society and the Clinical Cytometry Foundation. The Conference was supported by AmeriPath, ARUP, Beckman Coulter, BD Biosciences, CLARIENT, Clinical Cytometry Foundation, Clinical Cytometry Society, GENOPTIX, Genzyme, Laboratory Corporation of America, Trillium Diagnostics, National Cancer Institute, and Wallace H. Coulter Foundation. The contributing authors to this article have declared the following conflicts of interest: Brent Lee Wood has acted as a paid consultant and/or accepted speaker s fees from BD Biosciences and Beckman Coulter in the past, but has received no funding relevant to the current work. Teri Oldaker is employed by and holds stock in Genzyme Corporation, who is a sponsor for the event. The remaining authors have declared no conflicts of interest. *Correspondence to: Brent L. Wood, Department of Laboratory Medicine, University of Washington Medical Center, Box # , 1959 NE Pacific St., Seattle, WA 98195, USA. woodbl@u.washington.edu Received 20 June 2007; Accepted 20 June 2007 Published online in Wiley InterScience ( com). DOI: /cyto.b q 2007 Clinical Cytometry Society

2 OPTIMAL REAGENTS AND REPORTING FOR FLOW CYTOMETRIC DIAGNOSIS OF HEMATOPOIETIC NEOPLASIA BACKGROUND The determination of which reagents will be used to evaluate a given sample typically depends on a combination of morphologic evaluation of the sample, sample type (peripheral blood, bone marrow, tissue, etc.), medical indication as documented on the requisition supplied with the specimen, history of prior testing in the laboratory, and clinical history obtained from the requisition, patient s medical record, or verbal discussion with the clinical team. The particular combination of factors used depends to a large extent on the clinical environment in which the testing is performed. Laboratories that rely on initial evaluation by morphology and/or history prior to panel selection tend to be those in hospital settings where the specimens are received rapidly so that informative morphology is possible and where access to the patient s chart and clinicians is likely to be available. Although this approach can focus the analysis and reduce cost in some cases, morphologic evaluation may delay processing of the sample, require an experienced morphologist to be readily available, and may not be sufficiently sensitive or specific to detect either low levels of disease or patchy involvement by disease (see discussion). In addition, panel selection using submitted clinical information assumes that the history is accurate and complete enough for correct decision making, and may not allow for the detection of unanticipated clinical conditions. On the other hand, the use of more extensive reagent panels without morphologic or clinical evaluation often occurs where limited information is available, as is often the case in a reference lab environment. While allowing more comprehensive evaluation of the sample and providing somewhat improved laboratory efficiency, it requires more reagents, instrument, and technologist s time, which results in increased expense to the patient and medical system, and may generate information and testing that is not directed toward the medical indication for which the sample was submitted. An analogy is often invoked to support more comprehensive testing of this type, the comparison with a pathologist morphologically evaluating a surgical or cytology specimen for the presence of identifiable disease. Although this analogy is true to a point, the initial technique used by a pathologist, e.g., H&E, Pap, MGG, or Wright stained smears or sections, is best regarded as a moderately comprehensive screening technique in that it permits the evaluation of basic cells types and architectural patterns, allowing some diagnoses to be made without additional studies. However, an increasing number of surgical pathology diagnoses requires additional histochemical, immunohistochemical, cytogenetic, or molecular studies for specific diagnosis and classification. These ancillary studies are not all required on every specimen received, only that subset where specific concerns are identified after initial morphologic evaluation. An additional consideration is that the total number of hematopoietic disorders that require potential evaluation S15 is quite large and ever expanding. New markers with greater specificity for classification and prognosis will continue to be described, so any primary reagent panel needs to have the flexibility of encompassing new reagents as they become available. In particular, prognostic markers are increasingly being described that are unique to specific subsets of lymphoma, e.g., Zap-70 expression in the context of CLL/SLL. It is unrealistic to expect that a primary reagent panel will encompass all of these new markers without becoming excessively large. Finally, it is the general experience of many who practice clinical flow cytometry that a significant percentage of specimens evaluated for an appropriate medical indication, up to 50%, have no detectable evidence of hematopoietic neoplasia. In the interest of reducing the financial impact of flow cytometry testing on patients and the health care system, it seems desirable to minimize the degree of initial work performed on all specimens, reserving the more extensive evaluation for those cases where a greater degree of concern for neoplasia is identified by initial evaluation. The conclusion is that the primary purpose of any reagent panel used for specimen evaluation should be sufficient to detect the presence or demonstrate the absence of hematopoietic neoplasia, and that the panel should be as efficient as reasonably possible. This may or may not provide complete characterization and some cases must be followed up with additional markers. BASIC PRINCIPLES The earlier discussion leads to the generation of the following three basic principles: 1. The primary purpose of performing clinical flow cytometry is to address the medical indication(s) as identified by the referring physician, i.e., the testing must be medically necessary and appropriate. 2. The panel should allow the analysis to account for all major cell populations present in the specimen, but does not need to identify all hematopoietic cell types. 3. The flow cytometric testing performed should be comprehensive enough to identify all major categories of hematopoietic neoplasia relevant to the clinical circumstances, including, but not limited to, the submitted medical indication(s). This does not suggest that the initial panel contain all reagents necessary to completely characterize all diagnostic possibilities, but that it be capable of detecting their presence with an appropriate degree of sensitivity so selected additional studies may be performed for definitive diagnosis, classification, and prognostication. PANEL SELECTION A variety of factors are commonly used to determine the panel of reagents to be used in specimen evaluation, as described earlier. A few require additional discussion. Morphology and immunophenotyping are essential and complementary techniques for the appropriate diag-

3 S16 WOOD ET AL. nosis and classification of hematopoietic neoplasms. In particular, morphology is one of the primary screening tools that results in the ordering of flow cytometric testing and plays an important role in quality assurance for flow cytometry (see later). However, its use as a primary method for driving flow cytometric panel selection has certain limitations. It can be inefficient in that it requires a person with sufficient morphologic skills, often a pathologist, to be available to review the material in a timely manner before analysis can proceed. Morphology has a limited sensitivity for the detection of low level involvement by hematopoietic neoplasms, as occurs in situations of marrow staging for lymphoma, monoclonal gammopathy of uncertain significance, and minimal residual disease detection following therapy. In some of these circumstances, the morphologic material required for adequate evaluation is not available at the time of decision making, e.g., marrow biopsy for assessment of lymphoma. Morphology is also inherently subjective and can suggest diagnoses that lead to suboptimal panel selection in some cases, e.g., lymphoma mistaken for acute leukemia or vice versa. This problem may be compounded by the often-compromised morphologic quality available for aged samples in a reference lab setting. Consequently, while morphology has an important role as the initial impetus for flow cytometric testing, in other situations clinical history or laboratory findings provide better guidance, and morphology cannot be advocated as the sole determinant driving panel selection. Nevertheless, some laboratories may elect to incorporate morphologic review into their panel selection process, but it should not be a requirement. The type of specimen received itself often narrows the likely diagnostic possibilities and can be used for initial panel selection. For instance, soft tissue involvement by acute leukemia or a plasma cell neoplasm is unusual and so an initial more limited evaluation for lymphoma may be more appropriate, while bone marrow may contain any variety of hematopoietic neoplasms and a more comprehensive approach may be preferred. Medical indication, as presented on the accompanying requisition, results from prior testing in the laboratory, or clinical information obtained from other sources are important indicators of the type of testing that should be performed. This information can be used to narrow the evaluation, provided sufficient reagents are included to allow the detection of other significant unexpected abnormalities. For patients having an appropriately characterized recent diagnosis of a hematopoietic neoplasm, a more limited panel focused on the detection of that neoplasm for purposes of staging or evaluation of residual disease may be used unless there are extenuating circumstances. In summary, panel selection should be based primarily on specimen type with consideration of information provided from morphology, medical indication, and/or clinical history. Any reduction from the recommended panel for that specimen type is to be avoided, unless there is a specific medical indication, focused morphologic question, recent diagnosis of hematopoietic neoplasia, limited specimen quantity, or other extenuating circumstance. The increased reliance on medical indication for reagent selection distinguishes the current approach from the disease focused panels discussed in prior consensus conferences (1,2), and represents a more realistic way to address the challenges faced in the clinical laboratory. PROPOSED REAGENTS Reagents to be included in an initial panel for the diagnosis of hematopoetic neoplasia were discussed at length by conference participants, but a single set of reagents suitable for use in all medical indications was not identified. Consequently, a survey of participants was conducted to facilitate the recognition of common reagents that laboratories would be likely to use in the evaluation of a specimen submitted for each medical indication. A list of medical indications was provided along with a suggested list of reagents for each major hematopoietic cell lineage: B cell, T cell, myelomonocytic, and plasma cell. The suggested reagents were those identified by a subcommittee as being in common clinical use. To minimize the impact of confounding information from clinical history, morphologic, or other clinical laboratory findings, it was assumed that the laboratory had received an appropriate sample for flow cytometric testing with the provided medical indication and that no additional information was available at the time of panel selection. It was also assumed for samples submitted for minimal residual disease testing that the diagnostic immunophenotype was not known. For each medical indication, the conference participant was asked to indicate whether each reagent would be included in a panel that was both necessary and sufficient for the identification of hematopoietic neoplasia. An opportunity was also provided for the participant to suggest reagents not in the survey list. In an attempt to understand better the performance criteria driving panel selection, each participant was also asked to estimate the likely sensitivity of detection for neoplasia in each cell lineage and for each medical indication as <0.1, , 1 5, 6 20%, or >20% of total white cells. It should be emphasized that the testing algorithm used in this survey and the reagent combinations elicited in many cases do not reflect current practice in participant laboratories, but rather are an educated estimate by individuals having extensive experience in clinical flow cytometric immunophenotying. The inclusion of a reagent by greater than two thirds of the participants was used to define consensus for each medical indication and cell lineage. The survey results are provided in Table 1. For any particular medical indication, consensus was achieved on whether a particular lineage required evaluation and this is summarized in Table 2. Evaluation of the myelomonocytic lineage showed the greatest degree of variability across medical indications, likely reflecting the more varied experience of the participants with evaluation of that lineage. In particular, the indications of

4 Consensus cutoff is 66% (red) with other potential cutoffs at 60% (blue) and 50% (green) for comparison. N ¼ 35. Table 1 Results of Consensus Survey of Reagents Useful for Initial Evaluation for Hematopoietic Neoplasia OPTIMAL REAGENTS AND REPORTING FOR FLOW CYTOMETRIC DIAGNOSIS OF HEMATOPOIETIC NEOPLASIA S17

5 S18 WOOD ET AL. Table 2 Cell Lineages to be Evaluated for Each Medical Indication Lineage to Medical indication be evaluated Anemia B, T, M, P Leukopenia B, T, M, P Thrombocytopenia B, T, M, P Pancytopenia B, T, M, P Neutrophilia M (limited) Monocytosis M Lymphocytosis B, T Eosinophilia T, M Erythrocytosis M (limited) Thrombocytosis M (limited) Blasts in blood or marrow B, T, M Lymphadenopathy B, T Extranodal masses B, T Splenomegaly B, T, M (limited) Transformation of chronic leukemia B cell B Transformation of chronic leukemia T or NK cell T Staging for non-hodgkin lymphoma B cell B Staging for non-hodgkin lymphoma T/NK cell T Skin rash B, T Atypical cells in body fluids (CSF, serous, ocular, etc.) B, T, M (limited) Monoclonal gammopathy B, P Unexplained Plasmacytosis of bone marrow B, P Monitoring of Rx response (unknown diagnostic immunophenotype) Mature B cell neoplasm B Mature T or NK cell neoplasm T Acute lymphoid leukemia B cell B Acute lymphoid leukemia T cell T Acute myeloid leukemia M MDS/MPD/Overlap Syndrome M Plasma cell neoplasm P B, B cell; T, T cell; M, myeloid; P, plasma cell. neutrophilia, thrombocytosis, and erythrocytosis yielded the greatest degree of variability, with some participants suggesting that evaluation by flow cytometry was not indicated. For these indications, a more limited set of reagents reached consensus that consisted of CD13, CD33, CD34, and CD45. A similar limited set of myelomonocytic reagents for evaluation of splenomegaly and atypical cells in body fluids did not quite reach complete consensus. Overall, the findings suggest that the greater source of variability in reagent usage is in whether a particular lineage is evaluated for each medical indication rather than the reagents used for its evaluation. The findings suggest that the consensus reagents listed in Table 3, when evaluated in appropriate combinations, represent the minimum to allow efficient evaluation of the indicated cell lineages for hematopoietic neoplasia with an acceptable degree of sensitivity. For example, if the B cell lineage needs to be evaluated, the combination of CD5, CD10, CD19, CD20, CD45, kappa, and lambda was advocated, whereas if a B cell neoplasm was not a primary concern for that medical indication then Lineage B cells Table 3 Consensus Reagents for Initial Evaluation for Hematopoietic Neoplasia T cells and NK cells Myelomonocytic cells Myelomonocytic cells (limited) Plasma cells Primary reagents CD5, CD10, CD19, CD20, CD45, Kappa, Lambda CD2, CD3, CD4, CD5, CD7, CD8, CD45, CD56 CD7, CD11b, CD13, CD14, CD15, CD16, CD33, CD34, CD45, CD56, CD117, HLA-DR CD13, CD33, CD34, CD45 CD19, CD38, CD45, CD56 no reagents consistently achieved consensus. However, use of CD45 and CD19 for a more limited evaluation was commonly suggested for indications having increased peripheral blood counts. Of the T cell reagents, CD2 was the most varied, reaching consensus in just less than half of the medical indications where T cell evaluation was advocated, but approached consensus in the remainder. Consequently, inclusion of CD2 in the consensus panel seems reasonable. CD64 and CD38 were commonly included in evaluation of the myelomonocytic lineages, but did not reach sufficient consensus to be included in the panel. For the plasma cell lineage, CD19 and CD56 achieved consensus in a subset of medical indications where evaluation of plasma cells was advocated, but did not achieve consensus for others. Overall, inclusion of CD19 and CD56 in the consensus panel seems reasonable. A few participants suggested that additional reagents should be included in the survey, but none were remotely close to achieving consensus. The most commonly suggested were CD16 and CD57 to evaluate the T and NK cell lineages for large granular lymphocytic leukemia and NK cell neoplasms, and cytoplasmic light chains to assess clonality when evaluating the plasma cell lineage. One additional important finding is the inclusion of CD45 at least once in every combination of reagents, regardless of medical indication or lineage being evaluated. Even when a lineage is not the primary focus of evaluation for a particular medical indication, CD45 was included by roughly 50% of the participants in evaluation of each of the other lineages. This reflects the general utility of CD45 in combination with side scatter for the evaluation of each of the hematopoietic lineages, and suggests that CD45 should be included at least once in any reagent combination. A secondary set of reagents suitable for further characterization in specific situations is also provided with the anticipation that only a limited number would be used on any particular specimen. The secondary reagents are summarized in Table 4. It is recognized that specific practice environments may require the exchange of certain primary and secondary reagents in these panels to accommodate their particular patient population and/or disease prevalence, but that the overall number of

6 Table 4 Reagents for Secondary Evaluation of Specific Hematopoetic Cell Lineages Lineage B cells T cells and NK cells Myelomonocytic cells Plasma cells OPTIMAL REAGENTS AND REPORTING FOR FLOW CYTOMETRIC DIAGNOSIS OF HEMATOPOIETIC NEOPLASIA Secondary reagents CD9, CD11c, CD15, CD22, ccd22, CD23, CD25, CD13, CD33, CD34, CD38, CD43, CD58, ccd79a, CD79b, CD103, FMC7, Bcl-2, ckappa, clambda, TdT, Zap-70, cigm CD1a, ccd3, CD10, CD16, CD25, CD26, CD30, CD34, CD45RA, CD45RO, CD57, ab-tcr, gd-tcr, ctia-1, T-beta chain isoforms, TdT CD2, CD4, CD25, CD36, CD38, CD41, CD61, ccd61, CD64, CD71, cmpo, CD123, CD163, CD235a CD10, CD117, CD138, ckappa, clambda reagents used should remain similar. An example would be a children s hospital where precursor B cell leukemia/lymphoma is common and lymphoma is rare, so the exclusion of kappa and lambda with the incorporation of CD34 and CD38 in the B cell lineage evaluation might be more appropriate. The levels of involvement (sensitivity) targeted for detection by the participants showed remarkable consistency for a given cell lineage, regardless of medical indication. In particular, no observable difference in desired sensitivity was noted between indications for diagnostic versus monitoring of therapeutic response, suggesting that in the absence of prior immunophenotypic information it is desirable to evaluate all samples with a moderately sensitive reagent combination for the given lineage. Additionally, this response suggests that a single set of reagents might be suitable for both types of applications, at least at the level of sensitivity suggested. The levels of detection advocated for each of the cell lineages suggest these target sensitivities: B cell, 0.1%; T cell, 1%; Myelomonocytic, 0.5%; and Plasma cell, 0.1%. Whether these differences in sensitivity reflect the optimal level of detection desired by the participants for each of the lineages or rather limitations imposed by the selected reagents or the participants own experience cannot be determined from this survey. It is also possible that there was some lack of uniformity in the way this question was interpreted, and further discussion of an optimal level of sensitivity for initial evaluation is warranted. S19 PRINCIPLES OF PANEL CONSTRUCTION Construction of complete reagent panels is beyond the scope of this proposal, as it is dependent on laboratory-specific constraints such as the available instrumentation, the number and type of simultaneous fluorochromes to be used, instrument performance characteristics, and availability of suitable specific antibody fluorochrome conjugates. However, there are a number of basic principles that should be followed in the construction of panels using the aforementioned reagents for initial sample evaluation. 1. Highly expressed antigens should be coupled with dim fluorochromes and dimly expressed antigens should be coupled with bright fluorochromes. The diagnostically useful range of intensities for each antigen as a result of aberrant expression during hematopoietic neoplasia should also be taken into consideration. This principle ensures reasonable sensitivity for each antigen and avoids compensation problems due to excessively bright fluorescence. 2. At least one common reagent should be present in each of the tubes used for the evaluation of a particular lineage to allow correlation or tracking of populations between tubes. However, reliance on the expression of any single antigen for population identification assumes expression of that antigen by the neoplastic population, which may not be a valid assumption in some cases. Consequently, the simultaneous use of more than one of these techniques is generally appropriate. Common examples are as follows: a. CD45 versus side scatter. This combination of parameters is a powerful tool for the identification of basic hematopoietic populations, e.g., lymphocytes, monocytes, maturing neutrophils, myeloid blasts, and lymphoid blasts (hematogones), and is commonly used to allow tracking of these populations between tubes in a panel. In addition, these parameters can provide a basic white cell differential, allow identification of abnormal populations to further evaluate, and aid in the assessment of sample preparation quality between tubes. As a result, this method is often used as the primary starting point for panel construction in clinical laboratories. b. Lineage-associated antigens. Antigens that are broadly expressed throughout maturation of a particular lineage can provide identification and tracking of all members of that lineage, particularly when used in conjunction with either forward or side scatter. Commonly used reagents are CD19 for B cells and CD3 for T cells. c. Maturational stage-associated antigens. Antigens that are expressed at a particular maturational stage can be used to identify specific populations. The antigen most commonly used for this purpose is CD34 for the identification of early progenitors or blasts. d. Forward scatter versus side scatter. This most basic method requires no additional reagents, but has a limited ability to discriminate between populations. It is often used to distinguish populations having lymphocyte scatter characteristics (low forward and side scatter) from myeloid populations (larger forward and side scatter), but should not be used as the primary method for population identification in light of the more powerful approaches outlined earlier. One exception is in the recognition of neoplasms composed of large cells where it may provide the best method for tracking populations between tubes.

7 S20 WOOD ET AL. Table 5 Reporting Specifications for Leukemia and Lymphoma 3. Antigens for subpopulation identification within a particular lineage should be combined with a lineage associated antigen. This ensures the identification of subpopulations within the appropriate lineal context and aids in the recognition of nonspecific patterns of antibody binding. A common usage is the evaluation of immunoglobulin light chain expression, i.e., kappa and lambda, in combination with a B cell lineage antigen, e.g., CD19 or CD20. Similarly the identification of CD4- and CD8-positive T cell subpopulations should be combined with CD3 for T cell lineage identification. 4. Antigens that are differentially expressed with maturation within a particular lineage should be combined to accentuate maturational patterns of expression. This is often best accomplished by combining antigens preferentially expressed early in maturation with those expressed later in differentiation. Abnormalities in the patterns of maturational expression are common and are extremely useful for the recognition of hematopoietic neoplasms. For the B cell lineage, the combination of CD10 and CD20 is useful to follow early B cell maturation from early precursors (CD10þ and CD20 ) to naive mature forms (CD10 and CD20þ). Similarly, for T cells the differential expression of CD3, CD4, and CD8 is useful to monitor T cell differentiation from early forms (CD3, CD4, CD8 ) to intermediate forms (surface

8 OPTIMAL REAGENTS AND REPORTING FOR FLOW CYTOMETRIC DIAGNOSIS OF HEMATOPOIETIC NEOPLASIA S21 Table 5 (continued) Reporting Specifications for Leukemia and Lymphoma Each element of the report is indicated as being required or optional. Recommendations from the 1997 consensus conference (Cytometry (1997) 30: ) are indicated by the designation 1997, and those from the 2006 consensus conference by the designation CD3, CD4þ, and CD8þ) to mature forms (CD3þ, CD4þ or CD3þ, CD8þ). For myeloid blasts, the differential expression of CD34, CD117, and CD38 is useful to monitor maturation from stem cells (CD34 bright, CD117 dim, and CD38 negative) to committed progenitors (CD34 variable, CD117þ, CD38 intermediate) to more mature forms (CD34, CD117 /þ, CD38 low to absent). For the neutrophilic lineage, a variety of combinations are commonly used including CD13 and CD16, CD11b and CD16, and CD11b and CD13. For the monocytic lineage, commonly used combinations include CD64 and CD14 and HLA-DR and CD Antigens differently expressed between closely related lineages may be used to allow improved lineage distinction. A common example is the use of CD11b and HLA-DR to distinguish neutrophilic (HLA-DR ) from monocytic differentiation (HLA-DRþ). QUALITY ASSURANCE Continuous improvement in the quality of the information provided by flow cytometric analysis should be a driving principle in every laboratory. Improvement in assay quality requires comparison of the flow cytometric findings with external sources of information obtained from a variety of sources, including the following: 1. Clinical history. The course of a patient s disease can provide important information regarding the clinical significance of flow cytometric findings and provides a primary source of validation for the assay. 2. Previous flow cytometric findings. Awareness of the results from prior flow cytometric studies can be important to provide context for the study, focus analysis on important prior abnormalities, ensure consistency in reporting, and document changes in the relative levels of involvement by disease. 3. Morphologic evaluation. Morphologic evaluation of the sample being tested provides important validation of flow cytometric findings and potential recognition of false negative results that can lead to assay improvement. Ideally, morphologic evaluation should be performed at the time of flow cytometric data interpretation and prior

9 S22 WOOD ET AL. to the finalization of the report as part of a quality control program. Correlation of the flow cytometric findings to reports providing morphologic evaluation by a pathologist should be performed for each sample. 4. Other assays performed on the sample. Results from hematology analyzers and cytogenetic, molecular genetic, or immunohistochemical studies performed on the same sample can provide useful correlation for the confirmation of flow cytometric findings and identification of false negative results. This may also include results of other flow cytometric testing performed on the same sample at other locations, e.g., at a centralized reference lab as part of a clinical trial. 5. Concurrent laboratory findings for the patient. The documentation of laboratory abnormalities associated with the patient s clinical situation can allow useful correlation with the patient s disease status, e.g., identification of the type and quantity of a serum or urine clonal immunoglobulin. As part of a quality assurance program, the above information should be evaluated for each sample, as appropriate. Discrepancies of external information with flow cytometric findings should be noted, investigation performed as to their likely cause, and the findings documented. REPORTING The accurate and informative communication of clinical flow cytometric findings to physicians involved in patient care is a critical component of the flow cytometric evaluation for hematopoietic neoplasia. The current consensus conference reevaluated the recommendations of the US-Canadian consensus conference from 1997 (3) and updated the required and optional report components as indicated in Table 5. The most significant updates to the 1997 consensus are changes in the Data Analysis and Interpretation sections of the reporting specification. The requirement of inclusion of World Heath Organization (WHO) differential diagnoses, the inclusion of interpretation limitations, and the identity of the interpreting pathologist were added in the Interpretation section. The reporting of the percent of abnormal cells present in the specimen is changed from optional to required. And finally, the requirement is added that when clinically useful, descriptions of antibody fluorescence intensity and distribution should be included in the Data Analysis section. The recommended descriptions of antibody distribution are Negative, Positive, or Partially expressed and are relative to an appropriate negative control population. The recommended descriptions of antibody fluorescence intensity are Dim, Bright, and Heterogeneous, with the intensity relative to the closest normal hematolymphoid population. For example, in a case of precursor B cell lymphoblastic leukemia/lymphoma, the appropriate reference population for fluorescence intensity would be the level of expression seen on normal immature B cell precursors or hematogones. Reporting in this manner provides a clearer picture of the immunophenotypic deviation from normal, which is the method by which hematopoietic neoplasia is identified, and minimizes the impact of variability in reagent and instrument performance between labs. The group strongly affirmed the conclusion from the 1997 consensus conference that the reporting of numerical values for each antibody in a simple tabular form is generally unsatisfactory to indicate the presence of abnormal cells, cannot describe their phenotype in sufficient detail, and limits the ability of the recipient of the report to interpret results. Reporting of results in this manner is to be strongly discouraged. It is recognized that there are several types of flow cytometry laboratories operating in diverse clinical environments around the world. Reports must be customized to meet the local practice environment but still contain the basic elements shown. LITERATURE CITED 1. Stewart CC, Behm FG, Carey JL, Cornbleet J, Duque RE, Hudnall SD, Hurtubise PE, Loken M, Tubbs RR, Wormsley S. U.S. Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: Selection of antibody combinations. Cytometry 1997;30: Braylan RC, Orfao A, Borowitz MJ, Davis BH. Optimal number of reagents required to evaluate hematolymphoid neoplasias: Results of an international consensus meeting. Cytometry 2001;46: Braylan RC, Atwater SK, Diamond L, Hassett JM, Johnson M, Kidd PJ, Leith C, Nguyen D. U.S. Canadian consensus recommendations on the immunophenotypic analysis of hematologic neoplasia by flow cytometry: Data reporting. Cytometry 1997;30: