| 这个章节我觉得比较重要。目前我们国内也是如此，大部分都是用表面抗原表达率>20%作为阳性标准，胞内抗原>10%作为阳性标准，但实际上这个标准比较武断，而且谁也不知道从哪篇文献中出来的。其实最好的方法，就是仔细分析细胞的抗原表达强度和是否存在其它来源的细胞干扰等，最后用negative、weak、dim、moderate、positive、strong等模糊表示法表达目标细胞的表达谱，确定其性质。请大家仔细阅读下面的内容，尤其是其中的例子。 1.1.1 The pitfalls of the FCM data format of “percent positive” per antibody tested In the context of a leukemia–lymphoma workup, it is important to express the immunophenotyping data in ways that avoid ambiguity and offer the optimal information for correlation with other clinical and laboratory data. Expressing the FCM data as “percent positive” per antibody tested is rarely relevant and may even be misleading. Many institutions have used the 20% level as an arbitrary cutoff value for a marker to be considered positive. None of the publications has described how this number became established, however. The following real-life examples (obtained in 1997 in London, UK) illustrate why the approach of reporting FCM results as percent positive and omitting the fluorescence data is inappropriate in leukemia-lymphoma immunophenotyping, and can lead to erroneous interpretations. Flow cytometry results on a bone marrow from a patient with suspected chronic myeloid leukemia in blast crisis (CML-BC), from an institution where the FCM laboratory is not part of the hematopathology laboratory, are shown below. The specimen was processed by Ficoll-Hypaque. Other procedure-related information was not made available. CD19 31% CD34 30% CD2 11% CD20 20% CD14 7% CD3 13% CD10 29% Kappa 8% CD7 16% CD13 32% Lambda 10% CD5 19% CD33 26% HLA-DR 29% Based on this format of data reporting and the 20% threshold, the case was interpreted as a biphenotypic blast crisis of CML (positive for CD19, CD10, CD13, CD33). However, when the list mode data was visualized on dual parameter dot plots, correlating the forward scatter (FSC) and antibody fluorescence, it became clear that (1) the neoplastic cells constituted 30% of the cell population in the FCM sample and (2) they were of medium cell size and had the following phenotype: CD19 moderate, CD20 dim, CD10 moderate, CD34 weak, HLA-DR moderate. Other antigens, (i.e., CD13, CD33, CD14, CD2, CD3, CD5, CD7, kappa, and lambda) were not expressed by the tumor cells. The CD13 (32%) and CD33 (26%) were present on mononuclear myeloid precursors (promyelocytes, myelocytes, and metamyelocytes) and not on the neoplastic population. The correct phenotype is that of a precursor B-cell acute lymphoblastic leukemia (ALL) and not biphenotypic leukemia. Correlation with the bone marrow aspirate morphology further confirmed a lymphoid blast crisis of CML. A limited (follow-up) panel was performed on the peripheral blood of a patient with known chronic lymphocytic leukemia (CLL), to assess the efficacy of anti-CD20 therapy as part of a clinical trial. The lymphocyte count was 3.1 × 10[sup]7[/sup]/L. The blood film was unremarkable except for a mild increase in large granular lymphocytes. The FCM data were reported as follows: CD2 75% CD19 23% Kappa 17% CD3 62% CD20 18% Lambda 8% Based on these results, it was concluded that there was no residual CLL in the patient’s peripheral blood, especially as the kappa:lambda ratio was within the normal range. However, subsequent reevaluation of the list mode data, using simple correlated displays of FSC and antibody fluorescence, was sufficient to demonstrate the presence of a small population of monoclonal B-cells (CD19 moderate, CD20 weak) with weak kappa expression, in a background of benign T-cells and polyclonal B-cells. Contrary to the initial interpretation, residual CLL was present in the patient’s peripheral blood. It is apparent from the above examples that reporting FCM data as percent positive per antibody tested can negate the usefulness of FCM and easily lead to confusing or erroneous interpretations, which may impact therapeutic decisions. Some laboratories do include fluorescence data in the FCM reports. However, the data may still be expressed in a suboptimal (and, therefore, inappropriate) manner, as shown in the following case example. Below are the FCM results on a peripheral blood specimen studied (in the mid-1990s) at a teaching hospital: CD2 48% moderate CD19 47% moderate CD3 45% moderate CD20 26% moderate CD4 21% moderate CD22 47% moderate CD7 47% moderate sIgM 48% moderate CD8 20% moderate Kappa 3% moderate CD13 3% moderate Lambda 2% moderate CD33 1% moderate CD10 36% moderate CD34 1% weak CD45 100% strong TdT 55% moderate HLA-DR 55% moderate The results indicated a proliferation of immature cells (TdT+). The case was interpreted as ALL with a mixed (B-cell and T-cell) lineage. Because of the data-reporting format, it is unclear whether the immature cells are of B- or T-cell lineage, however. Although fluorescence intensities were mentioned, data interpretation in this particular laboratory was actually based on percent positive with an arbitrary 20% cutoff. When proper visual data analysis was subsequently applied to the raw data, it became apparent that the blood sample contained a clearly identifiable neoplastic population of precursor B-ALL, admixed with a high number of normal T-cells. |
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