Getting Started Welcome to ReticFit. Before you attempt to do any analyses we highly suggest that you take a few minutes to read about the various options available to you within this program. If you should have any problem, please don't hesitate to give us a call at (207) 729-6767 or use our free BBS at (207) 729-6420. Our FAX number is (207) 729-5443. At Verity, we value you as a customer and if you should have any suggestions, please communicate them to us. Purpose ReticFit has been designed to provide a simple, accurate, and efficient means of quantifying reticulocytes. The program can be set up to run automatically or manually. In either mode, ReticFit is a tool to assist in the assessment of patient samples; it is not a stand-alone diagnostic. The more standardized your sample prep and data collection procedures are, the more reliable and consistent ReticFit's analysis will be. Therefore, each lab should implement proficiency testing to ensure that technicians can deliver reproducible data to ReticFit and use the program following a prescribed, consistent process. To assist labs in standardizing the results of different operators, we have included a Proficiency test with ReticFit which should be completed by each operator prior to general use of the program. Results can be sent to Verity where they will be databased and evaluated. Evaluation will compare operator results with results gathered by experts, as well as with results of others who have completed the testing. See Proficiency Testing for more on this topic. Data Collection This manual assumes that the data analyzed by ReticFit has been collected using a kit which is certified to be compatible with ReticFit. ReticFit is certified to work with Becton Dickinson's Retic-COUNT kit, to the best of our knowledge the only kit which is FDA approved for reticulocyte analysis on flow cytometers. Instructions in the Retic- COUNT kit must be followed carefully in order to assure consistent, reliable data is collected. The Becton Dickinson part number for Retic-COUNT instruction booklet is #23-1789-00, dated 5/91. When the ReticFit instructions refer to Retic-COUNT, they are referring to this item. As other kits and protocols become available, Verity will test the use of ReticFit with them to determine whether their use is compatible with ReticFit software. Data Collection Prior to analysis you must install ReticFit software on your computer and complete the Proficiency Testing. You should not begin patient analysis with ReticFit until you have satisfactorily completed these steps. Once data has been collected using a certified compatible kit or process, you are ready to use ReticFit software for data analysis. This process is detailed in both Proficiency Testing and Using ReticFit sections of this manual. It involves identifying pairs of files, both controls and tests, creating gates on forward scatter versus side scatter plots to include reticulocytes and exclude other populations, and confirming ReticFit's automatic cursor placement. ReticFit allows you to move rapidly from one test to the next, making batches of files to be analyzed with a minimum of operator time and interaction. Results calculated by ReticFit include percent retics, reticulocyte maturity index (RMI), and mean retics. If the user enters a value for red blood count (RBC) for a sample, the program can also calculate absolute retics. After analysis, results may be printed and saved to a cumulative database. Histograms or Listmode? ReticFit allows you to analyze both listmode and histogram format data files. There are advantages and disadvantages to each. Histograms require less disk space and are quicker to process. You must do your gating prior to running ReticFit, however, and you must assume that it was done correctly. If you do not retain the original listmode file, you have no way to review and revise how a gate was created. Listmode files are larger and slower for ReticFit to process. They have the advantage, though, of allowing you to create the light scatter gate during the analysis stage. The format which works best for you depends on the procedures you outline for data storage and quality control in your setting. Cautions and Limitations Reticulocyte test results are used, along with other data, to determine whether certain treatments may be necessary for a patient. Because of this, the operator and those who evaluate test results should be well aware of the limitations of each step of the analysis. Marginal or questionable results should always be cross-checked with another means of testing. We have listed below some of the limitations of reticulocyte analysis and ReticFit software. This list is not intended to be exhaustive. Its intent is to raise awareness of a few areas where caution is advised. The data collected are only as good as the instrument on which the sample is run. Software will not correct poor data. Regular calibration and maintenance are critical for dependable results. The data collected are only as good as the sample preparation. Preparation steps defined by kit manufacturers must be followed consistently and completely. Many of the limitations defined by kit manufacturers are also limitations inherited by ReticFit. For example, if a kit specifies that the sample must be run within 48 hours of collection from the patient, ReticFit inherits the same limitation. All data collection and sample preparation restrictions defined in the kits affect ReticFit.