7/13/09 2156 2M Page 189 4 Optics “The various signals (light scatter and fluorescence) gener ated by the cells interaction with the laser are detected by photomultiplier tubes and detectors. The number of feo rochromes capable of being measured simultaneously is lint by the number of photodetectors inthe flow cytome- ter. The specificity ofeach photodetector for a given band length of wavelengths is achieved by the arrangement of series of optical filters that are designed to maximize collec~ tion of light derived from a specific fluorochrome while ‘minimizing collection of light from other fluorochromes used to stain the cells. The newer flow cytometers actually use fiber-optic cables to direct light to the detectors. Most clinical flow cytomerers in use today are capable of three to six-color detection using one to two lasers When fluorescent light reaches the photomultiplier tubes, it creates an elecrical current that is converted into a voltage pulse, The voltage pulse is then converted (wsing various methods depending on the manufacturer) into a dig ital signal, The digital signals are proportional to the intensity of light detected. The intensity ofthese converted signals is measured on a relative scale that is generally set into 1 to 256 channels, from lowest energy level or pulse to the highest level. Data Acquisition and Analysis ‘Once the intrinsic and extrinsic cellular properties of many cells (typically 10,000 to 20,000 “events” are collected for cach sample) have been collected and the data digitalized, iis ready for analysis by the operator. Each parameter can be analyzed incependently or in any combination. Graphics of the data can be represented in multiple ways. The first, level of representation isthe single-parameter histogram, which plots chosen parameter (generally fluorescence) on the x-axis versus the number of events on the y-axis, so only a single parameter is analyzed using this type of graph (Fig. 12-3). The operator can then set a marker to differ- tentiate between cells that have low levels of fluorescence (negative) from cells that have high levels of fluorescence (positive) fora particular fluorochrome labeled antibody. ‘The computer will then calculate the percentage of *neg:~ tive" and “positive” events from the total number of events collected, “The next level of representation isthe bivariant histogram, or dual-parameter dot plot, where each dot represents an individual cell or event. Two parameters, one on each axis, are plotted against each other. Each parameter to be ana lyzed is determined by the operator. Using dual-parameter dot plots, the operator can then draw a gate" around a pop- ‘lation of interest and analyze various parameters (extrinsic and intrinsic) ofthe cells contained within the gated region (Fig. 12-4). This allows the operator to sereen out debris and isolate subpopulations of eels of incerest. When analyzing a population of cells using a dual- parameter dot plo, the operator chooses which parameters GHBTER 12 ow Cyomety and aboatoy Aviation 189 70 aria dts cols ee d [a TC Florescentintonsty FIGURE 12-3, Example ofa singe parameter fw histogram. The Yy-anis consis ofthe numberof events, The xan te parameter tobe analyzed, which s chosen by the operator, usually an extrinsic parameter such a2 fuorescet labeled antibody. The operate can ther se a marke Io oat the postive events The compute wll then calculate the percent positive evens within the gesgrated markers & 600] i cate craw around 8 soo ‘ele of erect, °° FSC Height FIGURE 12-4. Adual-parameter dot plot, Both parameters an the xe and y-aees are chosen by the operator. In thiscase, sed whole ood is analyed on FSC Gans) and SSC (y-ani). The operator then rows agate” or slates the population of interest fe, mpho- ‘ft for further ara to analyze on both the x- and y-axes. He or she then divides the dot plot into four quadrants, separating the positives from the negatives in each axis Fig. 12-8). Quadrant 1 con- sists of cells that are positive for orescence on the y-axis and negative for fuorescence on the x-axis. Quadrant 2 con- sists of cells that ate positive for fluorescence on both the sand y-axes. Quadrant 3 consists of cells that are negative for fluorescence on both the x- and y-axes. And quadrant 4 consists of cells that are positive for Buorescence om the axaxis and negative for fluorescence on the y-axis. The ‘computer will then calculate the percentage of cells in each «quadrant based on the total number of events counted (37 {cally 10,000 to 20,000 events per sample). For example, a gate can be drawn around @ population of cells based fn their FSC versus SSC characteristics, and the extrinsic +1814 Cm12_186-200.q44 7/13/09 2956 BM Page 190 2 m2 ———— A tot # pes T cots 3” co rire toro FIGURE 12-5, Quadrant anayss of = dual-paameter dot plot The operator chooses which parameters to ana- Iyze on each axis. (A On eat axis thee are positive (Muarescence postive) and negative (rescence negative) eis (8 fap of 2 duat-parameter dot plo to densify CDA Tels: CDS onthe axis and CDA on he V-3e. She ceils in quadrant 2 that ae oth postive Tor CD3 and CDs ae rue COA Teele characteristics ofthe gated population can be analyzed— thats ymphocytes canbe gated and then the subpopulations of T call (CD34 and CD4+ oF CD2+) and B calls CD2—, CD19+) can be analyzed (Fig. 12-6). The absolute count of @ particular cell eype-—for instance, CD4+ T Iymphocytes—is obtained by maleplying the absolute cell count of the population of interest (ee, lymphocytes) derived from a hematology analyzer by che percentage of ‘the population of interest in the sample (CD3++ and CD+> lymphocytes)? Detailed phenotypic analysis can determine the lineage and clonality as well as the degree of differentiation and activation of cell population hiss useful for diferenial diagnosis or clareaton of closely related Iymphoproifer- ative disorders. ‘Sample Preparation Samples commonly used for analysis include whole blood, bone marrow, and fluid aspirates. Whole blood should be collected into ethylenediaminetetraaceti acid (EDTA), the anticoagulant of choice for samples processed within 30 hours of collection, Heparin can also be used for whole blood and bone marrow and can provide improved stabil- ity in samples over 24 hours old. Blood should be stored at root temperature 20°C to 25°C) prior to processing and should be well mixed before being pipetted into staining tubes.” Hemolyzed or clotted specimens should be rejected, Peripheral blood, bone marrow, and other samples with large numbers of red cells require erythrocyte removal to allove for efficient analysis of white cells, Historically, density gradient centrifugation with Ficoll- ‘Hypaque (Sigma, St. Louis, MO) was used to generate a cell suspension enriched for lymphocytes or lasts. However, this method results in selective loss of some cell popla- tions Alternatively, there are numerous erythrocyte Isis techniques available, both commercial and noncommercial “issue specimens are best collected and transported in sissu culture medium (RPM 1640) at ether room temper azure oF #C, if analysis willbe delayed. The specimen is then disaggregated to form a single cll suspension, ether by mechanical dissociation or by enzymatic digestion “Mechanical disaggregation, of “teasing” is prefered andis accomplished by the use of either a scalpel snd forceps, a needle and syringe, or wire mesh sereen.® Antibodies are then added to the resulting cellular preparation and processed for analysis. The ansibodies used are typically ‘monoclonal, each with a different Huorescent tag. Clinical Ap Routine applications of flow cytometry in the clinical laboratory include immunophenotyping of peripheral blood lymphocytes, enumeration of CD34+ stem cells in peripheral blood and bone marrow for use in stem cell rans- plantation, and immunophenotypic characterization of acute leukemias, non-Hodgkin’ lymphomas, and other lympho- proliferative disorders, Tmmunophenotyping by flow cytometry has become an important component of inital evaluation and subsequent post-therapeutic monitoring in leukemia and lymphoma ‘management. Flow cytometric findings have been incorpo rated into current leukemia and lymphoma clasifications, beginning with the Revised European-American Lymphoma classification in 19% and, more recently, in the proposed World Health Organization (WHO) classifications.” One of the mos important components of low cytometric analy sis isthe stratification of hematopoietic malignancies by +1014 cn12_106-200.and 7/13/09 2:56 OM Page 192 4 (GARTER 12 Pow Cvomety and laboaton Automaton 191 oto 00d B08 ‘ a ~N ot Le : wie Pa gt FIGURE 12-5. Gating strategy to aalyze lymphocyte sabsets ina sample af whole blood, Whole Bood is ted with Nuorescent-iabeled antibodies pei for Cb2,CD3, CDS, and CDT. Ine sample is washes BCS are yea, an the sample is analyzed onthe flow cytometer. To aalyze using gating strateies, the les ist plotted on FSC versus SSC. (A)A gate o” region, raw around the ymphocye population. {a} On the sutequert pots of Puorescent markers only the mphocye population analyzed, The dat plots divides into Tour quadrants to Saate postive Fam negative populations. The computer calculates the percent postive els in each quadrant ample ofa flow pattern analying two diferent cll surface makers CD3 identifies the T-cell population. CD iene the percentage ofthe Tse population that are helper els (D4) Tree dstinet populations are denied: COs + C4 inthe upper right quadrant; 034 CO4— In the ower right quadrant; and CD3— CDA inthe lower left quadrant their lineage (i.c., B cell, T cell, or myeloid) and the gree of differentiation, Some of the more common cell= dillerensiation antigens are listed in Table 12-2 and include €D2, CD3, CDs, CD7, CDS found on T cells, CD19, Immunophenotyping of white blood cell populations is essential when an immunodeficiency is suspected Enumeration of peripheral blood CD4+ T cells in HIV: infected patients remains the highest volume test performed CD20, CD22 found on B cells, CD1b, CD13, CD1S, ‘DIG on myeloid cells, and CD11b, CD14, HLA-DR (MHC chss 1) on monocytoid eels? (CD45 isa pun-leukoeyte marker present on all white cells but with varying levels of expression; this results in varying levels of fluorescence, This variance in expression is based on the cells maturity. Blasts express lower levels of CD45 (low uorescence) but show an inerease of CD45 expression as the cell matures, so mature white cells have much brighter fluorescence compared to their earlier progenitor stages. This varying level of CD45 expression is useful in differen siating various populations of white cells. in the flow cytometry laboratory, because itis used in chs sifying stages of HIV disease and determining treatment ‘options. HIV type 1 infections cause a rapid, profound decrease in CD4+ cell numbers and an expansion of CD8+ T-cell levels during the carly course (12 to 18 months) of the illness" Some individuals continue to rapidly lose CD4+T cells and progress to AIDS, while oth- cers maintain relatively stable CD4+ T-cell counts and remain AIDS-free for years. During this ehronie phase of HIV-1 disease, the decline in CD4+T cell can be slow over many years due to maintenance of homeostatic mechanisms, However, as these homeostatic mechanisms start to fal, +184 m2. 106-200.q4d 7/13/09 2956 BM Page 192 4 192 SECTON 7 Bae marae racers TLE ANTIGEN CELL TYPE FUNCTION 2 Thymocytes, Tell NK els Involved in Tel atvation con Thymocytes, Tells ‘Associated with T-cell antigen receptor role in TCR signal transduction cos Helper Teall, monoeytes, macrophages Coreceptor for MC cs I receptor for HN cos Mature Tel thymocytes, subset of 8 cls (81) Postve or negative modulation ofT- and Bel eceporsonling coe Trymocyte subsets cytotec Tels Corecepor for MHC css wo 'B- and Tell recursos, bone marrow Protease; marker for pre-B CALLA stromal eels one Myeloid an NK els ‘4M subunit of integrin CRG, binds complement component iC, on Myelomonceytic cells Zine metalloprotease ors Monocyte es Upopolsaecharie receptor ois ‘Neutophis eosinophils, monocytes “Terminal wischarde expressed on lyolipids oe Macrophages, NK cls, neutrophils Low anit Fe eceptay, mediates phagocytosis and ADC cos 'B cal, flicular dente cels Part of B-cell coreceptor signal transduction molecule that regulates B-cell evelopment and activation con Bel folicular dendrite cel subst of Receptor for complement component C36; part of &-cell rnmature thymocytes coreceptor with €D 19 on Bal, monocytes, folicular dendritic els Regulation of gF synthesis: Wiggers release of I-16, and (GM-CSF trom manocytes cons Activated T el, Bes, monocytes Receptor for 2 on Most leukoeyts ‘Aahesion molecule mediating haming to peripheral lymphoid gars ors ‘All hematopoietic els Tyrosine phosphatase augments signaling case Different forms onal hematopoietic els sel in - and 8-ell antigen stimulated activation se NK cel, subse of Fels Not known or NK ces, subsets of Tels Subunit of NKGZ-A complex involved in inhibition of NK cell cytotoxicity c= at ae TER = 3-99 rope AE =m song as Y= har eral is CAA = commer ae plone niente = Foam cpa AE = ansehen ea k= ranialn OMS = peevoneomephae cserpsimsoey there isa further decline in CD4+ T and CD8+ T cells, which eventually leads to the development of AIDS." (CD 4+ T-cell levels are used to stage HIV disease progres- sion, are prognostic for the development of AIDS, and are used to monitor response to antiretroviral therapy. The ‘Centers for Disease Control and Prevention (CDC) guide lines stage HIV-1 disease by CD4+ T-cell level into three groups: >500 CD4 eellsmm, or >28 percent CD4 cells within lymphocytes; 200 to $00 CD4 cells/mnm3, or 14 to 28 percent CD§ cells; and <200 CD4 eells/ram3, or <4 percent CD4 cells. Additional examples of flow cytometry use inchude the determination of DNA content, or ploidy status of tumor cells. This ean provide the physician with important prog- nostic information.¥ Monitoring patients who have been treated for leukemia or lymphoma for "minimal residual disease” has also become another important function of the flow cytometer, since statistically significant rare cell events ‘ean be easily detected, In the case ofa fetal-maternal hem- orthage, using flow cytometry to detect hemoglobin F postive cells is much more sensitive than the traditional Kleihauer-Betke method.” Flow cytometry is also being. used in human leukocyte antigen typing and cross-matching for solid organ transplantation.) Immunophenotyping by flow cytometry, in whatever ‘capacity that it is used, is not possible without the use of ffuorescent-labeled monoclonal and polyclonal antibodies. “Monoclonal antibodies specific for various surface antigens are preferable to wsing polyclonal antibodies. The ability to produce monoclonal antibodies through hybridoma and. recombinant DNA techniques has contributed greatly to the accuracy of flow eytometty and has widened its use. (Gee Chapter 4 for a discussion of monoclonal antibody production.) +