Articles on Medical Diseases and Conditions

Tag: Flow Cytometry

  • Flow Cytometry in Cancer

    FCM has until recently been predominantly used to phenotype leukemias and lymphomas and to aid in prognosis of nonhematologic tumors.

    Nonhematologic tumors

    In nonhematologic tumors, predominately aneuploid neoplasms (especially if the S-phase value is increased) generally are more aggressive and have shorter survival time than tumors that are predominantly diploid and have normal S-phase values. However, this varies considerably between different tumor types, and there is often variation between tumors in different patients with the same tumor type. Added to this are various technical problems, such as mixtures of diploid and aneuploid tumor cells, mixtures of normal cells and tumor cells, differences in degree of tumor anaplasia in different areas, whether the tissue is fresh or formalin-fixed, proper adjustment of the instrumentation, and experience in avoiding or interpreting variant DNA peaks. S-phase work in nonhematologic tumors is more difficult than standard ploidy determination and sometimes cannot be done adequately.

    The most intensively studied (or reported) nonhematologic malignancies have been breast, colorectal, prostate, urinary bladder, ovary, and uterus.

    In breast carcinoma, there has been considerable disagreement between various studies, but overall suggestion that DNA ploidy is not a reliable independent factor in predicting likelihood of lymph node metastasis or length of survival. S-phase analysis is much more difficult to perform adequately but appears to have some predictive value regarding lymph node metastasis, degree of tumor differentiation, and presence of estrogen receptors. In colorectal cancer, the majority of studies have found that aneuploid tumors usually have shorter survival than diploid tumors and there is some correlation with probable Duke’s tumor stage (except for stage D) and therefore overall survival. In early superficial (noninvasive) transitional cell carcinoma of the urinary bladder, degree of aneuploidy has predictive value for invasiveness and tumor grade. FCM on bladder washings has additive value to biopsy of early superficial lesions. If the DNA index (measuring aneuploidy) of the biopsy differs from that of bladder washings before treatment, this suggests higher risk of tumor invasion. If the bladder washing DNA index is aneuploid and that of the biopsy is diploid, this suggests carcinoma in situ. However, if both are aneuploid, there is no prognostic assistance. Bladder washing FCM is considered the best test to follow up a patient after tumor surgery. If intravesical chemotherapy is given, it is necessary to wait 6 months after the last chemotherapy dose to resume bladder washing surveillance (because of chemotherapy-induced abnormalities in normal epithelial cells). Fresh urine specimens are much better than preserved specimens; the fresh specimen should be refrigerated immediately and analyzed as soon as possible, but no more than 12 hours later. If that is not possible, appropriate fixative must be added. In prostate carcinoma, DNA diploid tumors tend to be better differentiated (lower grade), respond better to radiation therapy, and have longer survival time; aneuploid tumors tend to be less differentiated (higher grade) with a worse prognosis and usually have less response to estrogen therapy. In ovarian carcinoma, diploid carcinomas in stage III or less have a much better prognosis than aneuploid carcinomas. In melanoma and in renal, endometrial, bone and cervix carcinomas, diploid state has some chance of a better prognosis.

    Hematopoietic malignancies

    In hematopoietic malignancies, malignant lymphomas that are diploid are usually lower grade and less aggressive, with the opposite generally true for aneuploid lymphomas. However, not all reports agree. S-phase analysis is also said to have prognostic value. Burkitt’s lymphoma has an interesting FCM profile, since it is usually diploid but has a high S-phase value and behaves like a typical aneuploid tumor. Childhood acute lymphocytic leukemia (ALL) that is aneuploid has a better response to chemotherapy. This has not been shown with adult ALL or with acute myelogenous leukemia.

  • Flow Cytometry (FCM)

    Considerably simplified, flow cytometry (FCM) counts and analyzes certain aspects of cells using instrumentation similar in principal to many current hematology cell-counting machines. If the cells to be analyzed come from solid tissue, the cells or cell nuclei must first be extracted from the tissue and suspended in fluid. Next, the cell nuclei are stained with a fluorescent dye that stains nucleic acids in order to assay the amount of nuclear deoxyribonucleic acid (DNA); in addition, antibodies with a fluorescent molecule attached can be reacted with cell antigens in order to identify or characterize (phenotype) the cells. The cells or cell nuclei are first suspended in fluid and then forced through an adjustable hole (aperture) whose size permits only one cell at a time to pass. As the cells pass through the aperture each cell also passes through a light beam (usually produced by a laser) that activates (“excites”) the fluorescent molecules and also strikes the cell, resulting in light scatter. A system of mirrors and phototubes detects the pattern of light scatter and the fluorescent wavelengths produced (if a fluorescent dye is used), then records and quantitates the information. The pattern of light scatter can reveal cell size, shape, cytoplasm granules (in complete cells), and other cell characteristics. The electronic equipment can sort and analyze the different pattern. This information is most often collected as a bar-graph histogram, which is then displayed visually as a densitometer tracing of the bar graph; the concentration of cells in each bar appears as a separate peak for each cell category, with a peak height proportional to the number of cells in each bar of the bar graph. If all cells had similar characteristics (according to the parameters set into and recorded by the detection equipment) there would be a single narrow spikelike peak. One great advantage of cell counting by FCM rather than counting manually is that many thousand (typically, 10,000) cells are counted in FCM compared to 100 (possibly 200) in manual counts. Therefore, FCM greatly reduces the statistical error that is a part of manual cell counts.

    At present, flow cytometry is most often used for the following functions:

    1. Cell identification and phenotyping. Use of fluorescent-tagged antibodies, especially monoclonal antibodies specific for a single antigen, helps to identify various normal and abnormal cells and also subgroups of the same cell type. For example, lymphocytes can be separated into B- and T-cell categories; the T-cells can be further phenotyped as helper/ inducer, suppressor/cytoxic, or natural killer cell types. This technique may also be used to identify subgroups of certain malignancies, most often of hematologic origin (leukemias and lymphomas; see also the discussion of cell lineage studies in Chapter 7).
    2. Analysis of nuclear DNA content. Cell nuclear chromatin is stained (usually with a reagent that also contains a fluorescent molecule), and the fluorescence corresponding to total nuclear chromatin content is measured by the flow cytometer. The major determinant of total nuclear chromatin content when cells are in their resting stage is chromosome material, which in turn is related to the number of chromosomes (“ploidy”). Normally, there are 2 sets of chromosomes (“diploid”). As described previously, the FCM produces a visual representation of the DNA content of various cell groups present that in diploid cells from a resting state would be displayed as a single homogeneous spikelike peak appearing in a certain expected location of the FCM densitometer graph. A sufficient number of cell nuclei with more or less DNA than normal (an arbitrary cutoff point most often set at 5 percent deviance from diploid state) is called aneuploidy and often is seen as a separate peak from the usual diploid peak. Another way of demonstrating this is to obtain a DNA index in which the patient DNA content is compared to DNA of a (normal) control cell specimen. Ordinarily, the patient and control DNA patterns would be identical, providing a DNA index of 1.0. A DNA index sufficiently more than or less than 1.0 indicates aneuploidy. Aneuploidy is most often associated with malignancy, and if found in certain tumors may predict a more aggressive behavior. However, it should be noted that aneuploid DNA can be found in some nonneoplastic cells as part of the reaction to or regeneration after inflammation or tissue destruction and has also been reported in some benign tumors.
    3. Cell proliferation status. In standard FCM, this is done by determination of the S-phase fraction (percent S-phase; percent S; SPF). A normal cell activity cycle consists of five time periods. The cell normally spends most of its time in the G0 (“gap zero”) resting phase. It may be stimulated to enter the G1 (gap 1) stage in which ribonucleic acid (RNA) and some protein is synthesized. Next comes the S (synthesis) phase when DNA content increases to twice the resting amount in preparation for mitosis. Then comes the G2 (gap 2) period when additional RNA is produced and a very short M (mitosis) period, which is difficult to separate from G2, when mitosis takes place. After this the cell returns to G0. The S-phase (proliferation phase) features doubling of the nuclear DNA content, which is detected on the FCM histogram. The number of cells in the S-phase, if considerably increased, is shown as a small peak or elevation midway between the G0/G1 and the G2M peaks in the FCM histogram. Increase in the S-phase fraction (SPF) area suggests an unusual degree of cell proliferative activity. Since tumor cells tend to replicate more readily than normal cells, increased SPF activity can therefore raise the question of malignancy. In a considerable number of tumors, the degree of SPF activity correlates roughly to the degree of aggressiveness in tumor spread, which has prognostic significance. SPF has become one of the better and more reliable overall tumor prognostic markers (indicators). However, there are certain problems. Not all tumors with increased SPF are malignant; not all malignant tumors with increased SPF metastasize; and not all malignant tumors with relatively small SPF fail to metastasize. In addition, the S-phase peak is usually not large, even when considerable S-activity is occurring. The S-phase peak can be interfered with by cell debris or by poor separation of the G0/G1 and G2/M peaks. SPF can be falsely increased to variable degrees by reactive normal tissue cells (e.g., fibroblasts, endothelial cells, inflammatory cells, or residual areas of nontumor epithelial cells) intermixed with or included with tumor cells.
    4. To provide helpful information in patients with cancer. This includes use of information from the previous two categories (analysis of nuclear DNA content and cell proliferation status) to help determine prognosis; to help diagnose cancer in effusions, urine, or other fluids in which cancer cells may be few or mixed with benign cells such as activated histiocytes or mesothelial cells; to detect metastases in lymph nodes or bone marrow when only small numbers of tumor cells are present; or to supplement cytologic examination in fine-needle aspirates.

  • White Blood Cell Identification and Phenotyping

    WBC identification is usually done by Wright-stained peripheral blood smear examination. However, this approach creates problems due to the statistically small number of cells counted (usually 100), nonuniform cell distribution on the smear, and the need for subjective interpretation that can produce differences in cell counts in the same smear by the same technologist or between different technologists. Automated cell differential machines can improve the situation somewhat but currently still have problems with individual cells that are transitional between classification categories, atypical, or abnormal. In addition, neither a manual or machine differential can subtype normal or abnormal cells.

    Flow Cytometry

    Another approach to WBC counting is flow cytometry. Various WBC types and subtypes contain one or more antigens that are unique or are shared by a limited number of other cells. These antigens can be detected by specific monoclonal antibodies that can be tagged with a fluorescent molecule. A flow cytometer is able to activate the fluorescent molecule and detect, differentiate, and identify light wavelengths being produced. This permits detection, identification, and quantitation of the cells that possess the antigens being searched for. Usually an algorithmic approach is used in which one or two antibodies are tried, followed by one or two others depending on the initial results, and so on until final identification.

    One problem (still present to some extent) was confusion because different manufacturers developed antibodies against the same or closely related cell antigens, but used different names for their antibody. Therefore, a standard nomenclature called cluster designation (CD) was developed in which each WBC antigen was given a CD number and the various antibodies (antibody “cluster”) that reacted with the same WBC antigen were assigned the corresponding CD number or numbers. That way, antibodies from various manufacturers, beside the proprietary brand name, could also be given a CD number that would indicate what antigen the antibody reacts with. Each antigen corresponds to a WBC category or subgroup. However, more than one CD antigen may be present on cells of the same WBC category or subgroup. For example, CD-4 antigen is found on the lymphocyte T-cell helper subgroup and CD-8 antigen on the lymphocyte T-cell suppressor subgroup. However, both CD-2 and CD-7 antigen are found on the lymphocyte T-cell natural killer subgroup. Certain platelet and megakaryocyte antigens are also included in the CD system.