green light, between 515 and 535 nm). The analog output from the PMT, a voltage pulse lasting for the duration of time that it takes the cell to transit the laser line, is converted into one or more numbers that quantify the pulse (area, peak, or duration of the pulse). Precisely, how this is accomplished differs from cytometer to cytometer. The digitized data, often corrected for spectral overlap (also called color compensation) are stored in a data file called a list-mode file [50]. Conventionally, these are given the extension fcs or .lmd. In some cytometers, both compensated and uncompensated data can be stored in the same file. A typical list-mode file can be likened to a large data table in which the columns are the parameters (e.g., FSc, SSe, green, orange, and red fluorescence intensity), and each row represents a distinct event. In the parlance of flow cytometrists, events are signals that may represent cells but can also be debris or other sources of noise. The process of data collection (f) (f) Z35026.1md FS (f) LL. [lJ., (f) CD45 FITe· CD45 FITC FIGURE 6.2 Discrimination of lymphocyte, monocyte, and granulocyte populations by light scatter. This fixed control cell preparation (CD Chex CD34, Streck Laboratories) was also stained with anti-CD45, a marker expressed to varying degrees on all leukocytes. Color eventing was used to mark the lymphocyte (green), monocyte (red), and granulocyte (blue) populations identified by light scatter alone. Subcellular debris can be seen on the left-hand side of the lymphocyte population. The populations visualized by light scatter also form discrete populations based on CD45 and light scatter (right panel and inset). Note that debris and red cells can be identified by light scatter but are better resolved with anti-CD45. 188 Handbook of Human Immunology, Second edition T in which celb arc run through the cytometer and parameters describing individual events are stored in data files. is known as (/cqllisilioll. Depending on the complexity of the data. they can either be analyzed in real time during sample acquisition or o.lfline by recalling the stored list-mode data files. 6.5 SAMPLES Flow cytometric assays can be performed using anticoagulated blood or bone marrow. isolated cell suspensions, cell cultures, or disaggregated tissue. In peripheral blood, where erythrocytes outnu mber wh ite cells by 1000 to I, it is usually necessary to remove them from the sample. It is not necessary to physically remove platelets and small debris, because these can be thresholded out during acquisition (in this case, the cytometer does not count them), live gated during acquisition (events are counted but not saved in the list-mode fi Ie), or gated out on the basis of their low light scatter during analysis. Similarly, cell doublets and clumps can be recognized by their light-scatter properties, and gated from the analysis (Figure 6.3). As mentioned earlier, normal leukocytes can be segregated into three distinct groups, corresponding to lymphocytes, monocytes, and granulocytes, on the basis of light scatter. A typical three-part differential was illustrated in Figure 6.2. Lymphocytes comprise the leukocyte population with the smallest cell diameter (FSc) and the least internal complexity (SSe). Monocytes are larger cells and have greater internal complexity; neutrophils are cells of approximately the same size as monocytes but ... ~37634.1md, Z37639.lmd I I I I "j I I " I I 200 400 600 800 1000 600 800 1000 o 200 400 600 800 1000 0 Forward scatter ) 400 ~~'.....>~r-. 200 . ..."-.-- ", 1000 800 co u 600 Cf) Q) l:l Ui 400 200 0 0 Understanding Clinic.al rlow Cytornetry 1 40 30 a.. 20 10 0 300 400 500 Volts FIGURE 6.10 Determination of the optimal PMT voltage on unstained peripheral blood lymphocytes. Unstained peripheral blood cells were acquired within a lymphocyte light scatter gate. For each fluorescence parameter, the PMT voltage was gradually increased and the percent of events falling on scale and the CY of the peak were determined at each voltage setting. The results were plotted, and negative exponentially weighted curve smoothes were applied to the points. The example shown here is for the FLJ PMT, which was set up to measure green fluorescence. At the lowest voltage tested, most of the