Compilation of Rules and Regulations of the State of Georgia
Department 111 - RULES OF DEPARTMENT OF COMMUNITY HEALTH
Chapter 111-8 - HEALTHCARE FACILITY REGULATION
Subject 111-8-10 - LICENSURE OF CLINICAL LABORATORIES
Rule 111-8-10-.22 - Quality Control for Flow Cytometry

(1) A standard, consisting of latex beads or other uniform particles, shall be run to insure proper focusing and alignment of all lenses in the path, and for both the existing light source and signal (light scatter, fluorescence, etc.) detectors. The results of optical focusing/ alignment must be recorded each day.

(2) A threshold value for acceptable optical standardization shall be established for all relevant signals for each instrument and the focusing procedure repeated until these values are achieved or surpassed. If a particular threshold value cannot be attained, a written protocol for instituting corrective action must be available and used.

(3) A fluorescent standard, for each fluorochrome to be used, shall be run to insure adequate amplification of the fluorescent signal(s) each day of use and after any maintenance or adjustment of the instrument. This standard may be incorporated in the beads or other particles used for optical standardization or may be a separate bead of fixed cell preparation. If acceptable fluorescence separation cannot be attained, a written protocol instituting corrective action must be available and used.

(4) When performing analyses using two or more fluorochromes simultaneously, an appropriate procedure must be used to compensate for "spill over" into the other fluorescence detectors.

(5) For laser based instruments, the current input (amps) and laser light output (milliwatts), at the normal operating wavelength measured after the laser is peaked and normal operating power set, must be recorded as part of a daily quality control record.

(6) The use time of instruments with mercury fluorescent lamps must be recorded. Lamps must be replaced when the allowable use limit has been reached.

(7) The laboratory must run a positive and negative control each day of patient testing. The negative controls should include normal serum from a healthy individual. The positive control (using appropriate dilutions) should react with cells representing all HLA types (i.e., pulled high PRA sera).

(8) Each laboratory must establish and document its own threshold with multiple normal sera for discriminating positive crossmatches. For significant change in protocol, instrumentation, or software, the characterization of the positive threshold must be repeated.

(9) For internal labeling, the method used to allow fluorochrome labeled antibodies to penetrate the cell membrane must be documented as effective.

(10) Whether analyzed directly or fixed prior to analysis, labeled cells must be analyzed within a time period demonstrated by the laboratory to avoid significant loss of any cell sub-population or total cell numbers. Test samples must be analyzed within the same period, after staining, as the control samples.

(11) If analysis will be based on a population of cells selected by flow cytometry "gating" on size or density parameters, or selected depletion or enrichment techniques, control stains must be run for each test individual to detect the presence of contaminating cells in the selected population (i.e., monocyte contamination of lymphocytes gated by forward angle or forward angle vs. 90 degree light scatter must be detected with a monocyte specific marker antibody). For cell surface labeling, a method must be used to determine the proportion of viable cells in the population of counted cells (i.e., thidium bromide staining, CD45 staining). If this value falls below an established laboratory threshold, an appropriate reanalysis of specific measurements must be done.

(12) Conclusions about abnormal proportions of abnormal cell surface marker shall only be drawn in comparison with local control data obtained with the same instrument, reagents and techniques. Conclusions about leukemia/lymphoma classification shall be based on local or published reference data. Determination of percent positives must take into consideration the results of the negative control reagent.

(13) The specificity of monoclonal antibodies shall be verified by the published and/or manufacturer's documentation and, whenever possible, verified locally through tests with appropriate control cells prepared and tested by the same analysis. The quantities of reagents used for each test sample must be determined by the manufacturers from published data and, whenever possible, should be verified locally by appropriate titration procedures.

(14) Terminology used must be defined and/or must conform to nomenclature recommended/approved by the most recent International Workshop of Differentiation Antigens of Human Leukocytes or other appropriate scientific organizations.