1. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance The following specialty sustaining standards of practices shall be If the laboratory reports values dependent on absolute number incorporated into the laboratorys quality management system, where of any leukocyte population assessed by volume or conductivity applicable to the scope of services provided. methodology, a hematology permit is required. Cellular Immunology Standard 1 Ranges should be updated biannually and monitored for statistical changes. CI1 The laboratory shall have written criteria for the establishment of normal ranges. At a minimum, criteria should include gender, age, and race. Normal ranges should be divided into pediatric and adult ranges. Normal ranges should be determined from specimens that are the average age of the specimens routinely tested in the laboratory. All reporting requirements (including normal values) noted in Section 58-1.11 are applicable. January 2008 Page 166 of 197

2. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 2 A freshly prepared whole blood specimen from a healthy donor shall be included as a positive control: CI2a a) on each assay plate or run for a function assay; CI2b b) in the first run of each day of testing for non-malignant leukocyte immunophenotyping; CI2c c) at least monthly for malignant leukocyte immunophenotyping; and, b) Validated whole blood commercial controls may be substituted for freshly drawn blood. CI2d d) if sample preparation problems occur, additional control bloods shall be used. Cellular Immunology Standard 3 CI3 The whole blood normal control for each assay shall be collected and Validated commercial controls should be stored according to stored under conditions as similar to those of the test specimens as manufacturers recommendations. possible. Cellular Immunology Standard 4 CI4 For longitudinal studies of function involving serial monitoring, the laboratory shall issue instructions to clients that specimen collections shall be performed at the same time of day. January 2008 Page 167 of 197

3. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 5 Information used to generate results may include, but is not limited to, raw data, worksheets, instrument readings, and CI5 Results for each test and information used to generate those results personal observations. shall be reviewed by a qualified person prior to reporting and such review shall be documented. For purposes of this standard, a qualified person is an individual holding a certificate of qualification in the appropriate Cellular Immunology subcategory tested. A person qualified as a supervisor pursuant to Section 58-1.4(a), (b) (c) or (e) of 10 NYCRR may review results during a temporary absence of the CQ holder, not to exceed 21 days, in accordance with a protocol approved by the CQ holder. Supervisor-reviewed results should be reviewed by the CQ holder upon his or her return and this review should be documented. If the reported results are directly provided by the instrumentation for a particular subset and no analyst intervention is possible and no interpretation of the analysis is required, review by a qualified supervisor is sufficient. LYMPHOID FUNCTION ASSAYS (formerly LIMITED I) Cellular Immunology Standard 6 CI6 The laboratory shall provide specimen transport instructions to clients indicating that blood samples shall be maintained at 18 23 degrees C and transported within appropriate time frames. Cellular Immunology Standard 7 The laboratory shall not test: CI7a a) blood specimens that are older than 24 hours post-collection; and, CI7b b) whole blood in culture assays except when heparin is used as the anticoagulant. January 2008 Page 168 of 197

4. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 8 CI8 The laboratory shall establish criteria for acceptable lot to lot variations The laboratory may accept the manufacturers specifications of of culture materials. endotoxin levels, which must be less than or equal to 50 EU/mL Cellular Immunology Standard 9 Viability assessment should be performed from an aliquot of the whole blood specimen before the cell culture is started. CI9 Cell viability shall be greater than 80% at the initiation of the cell culture and viability percentage shall be included in the laboratory report. Specimens with viability of less than 80% should be rejected and a new specimen requested. Cellular Immunology Standard 10 CI10 All control and patient tests shall be run in triplicate. Cellular Immunology Standard 11 For proliferation assays: CI11a a) negative control wells containing medium without a stimulant shall be used for each harvest; CI11b b) the laboratory shall determine the day of peak cell response for each stimulant to ensure the appropriate harvesting time; and, b) The laboratory should document the data used to support the harvesting schedule for assessing peak response. CI11c c) the laboratory shall perform the assay on the pre-determined peak day of proliferation or activation. c) Harvesting should occur one day before and one day after the pre-determined peak day of proliferation to ensure that the assay is performed on the peak day of cell response. Cellular Immunology Standard 12 CI12 For protocols that use tritiated thymidine to assess lymphocyte proliferation or stimulation, the minimum response and highest background unstimulated values shall be defined. January 2008 Page 169 of 197

5. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 13 CI13 For mitogen-induced proliferation assays, one to three mitogen Lot titration is performed to determine the optimal concentration concentrations determined to be in the optimal range by previous for response before each mitogen is used for testing. Two titration shall be used for each assay. additional concentrations, above and below optimal concentration, to cover individual response levels should be included. Cellular Immunology Standard 14 For antigen-induced proliferation assays: CI14a a) for each patient and control sample in Negative Response-type ii) Assays that demonstrate a higher value should be rejected. assays, where the normal response is a lack of proliferation due to lack of previous exposure; iii) A positive response is delineated by a proliferation response i) a limit of acceptable day to day variance observed with the higher than the established response of unexposed normal control cells shall be determined; donors and/or the stimulation index is not within one or two standard deviations of the mean value for the healthy control ii) the maximum response or background value for unstimulated population. samples shall be established; and, iii) results shall be reported only as positive/negative response; CI14b b) in Positive Response-type assays, where challenge with an antigen is expected to normally induce a positive stimulation response due to a previous exposure, results should be reported as positive if values fall within the normal range and background responses are negative. January 2008 Page 170 of 197

6. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 15 For alloantigen-stimulated proliferation: Inactivation by irradiation using 30 Gray is recommended. Inactivation should be checked by stimulating stimulant cells CI15a a) all stimulant cells shall be inactivated to prevent proliferation; with mitogen (post inactivation) in the absence of responder patient cells. CI15b b) a positive control of pooled stimulant cells from five individuals inactivated to proliferate shall be included; CI15c c) patient (responder) cells alone to measure unstimulated background level of the specimen shall be included; and, CI15d d) donor (stimulator) cells alone with and without mitogen shall be included. Cellular Immunology Standard 16 b) The cryopreserved control cells should demonstrate reliable For Cytolytic assays: activity levels upon thawing and appropriate responses to cytokines and/or specific antigens. CI16a a) selection and preparation of the target cells and the effector cells for each type of cytolytic assay used shall be appropriate for the analysis; f) Optimally the assay should be set up within 8 hours of collection. When possible, blood samples should be assayed on CI16b b) in addition to a fresh normal control, each assay shall include a set of the day of collection. If this is not possible: cryopreserved cells demonstrating low, intermediate, and high cytolytic activity levels for each type of cytolytic assay used; i) PBMCs (peripheral blood mononuclear cells) should be isolated immediately and stored overnight at 4 degrees C in CI16c c) target cells shall be labeled and shall maintain a low spontaneous 10% type AB serum; release of label; ii) If the PBMCs cannot be isolated on the same day or the CI16d d) the range of Effector:Target ratios (four or more) shall be appropriate assay is being performed using a whole blood format, for each type of cytolytic assay; specimens should be collected in heparin and stored at room temperature overnight. CI16e e) positive and negative controls for each specimen and a normal control shall be included in each run; and, CI16f f) maximum time from specimen collection to assay is 24 hours. January 2008 Page 171 of 197

7. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 17 For Cytokine assays: b) The laboratory should document the data used to support CI17a a) a positive and a negative control shall be included with each test run; the harvesting schedule for assessing peak response. and, CI17b b) the laboratory shall determine the day of peak cell response for each cytokine to ensure the appropriate harvesting time. Cellular Immunology Standard 18 Detection assays using ELISAs shall include: c) Potential matrix effects should be assessed using an appropriate dilution series. Results should be determined from CI18a a) the capture and detection antibodies to react on different epitopes of the most linear portion of the standard curve. the antigen to be measured in a non-inhibitory fashion; d) The interassay variability should have a coefficient of CI18b b) analysis in the presence and absence of stimulator for in vitro assays variance of less than 20%. to compare the patients background levels to activated levels; CI18c c) minimally, two dilutions of the patients specimen for quantification; CI18d d) an internal control (assayed aliquots of frozen supernatant) used for each cytokine assay to determine interassay variability; f) Duplicate wells should have a coefficient of variation of less than 20%. CI18e e) cytokine assay standards calibrated against WHO reference standards; and, CI18f f) duplicate wells for each patient sample and standards. FLOW CYTOMETRY CALIBRATION (formerly LIMITED II, III, AND IV) January 2008 Page 172 of 197

8. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 19 CI19 For those instruments (stream-in-air systems) requiring daily manual optical alignment, a minimum of 5000 counts shall be assessed during instrument calibration. Cellular Immunology Standard 20 The manufacturers recommended procedures should be On each day of use, after maintenance procedures and after the followed. resolution of any instrumentation failures, the following checks shall be performed on the flow cytometer: CI20a a) calibration with stable beads labeled with fluorochromes; b) Electronic compensation can be first adjusted with individually fluorescent-labeled beads. Fine tune adjustments CI20b b) compensation for color spectral overlap for each fluorescent dye that should be completed using cells stained with mutually exclusive is used for testing; antibodies brightly labeled with fluorescent dyes. CI20c c) determination of adequate fluorescent resolution so that there is a c) Each laboratory should establish and measure a minimally measurable difference between the autofluorescent/non-specific peak acceptable separation between fluorescent peaks of different and a dimly positive fluorescent peak for each fluorescent parameter intensities. used for testing; and, d) The instrument is monitored using a stable fluorescent bead CI20d d) standardization to ensure that performance is constant from day to by either using a fixed voltage and measuring the variability of day. fluorescent peak channel or adjusting voltages to place the fluorescent bead signal at the same peak channel and recording the voltage variability. January 2008 Page 173 of 197

9. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 21 For accurate quantification of any marker by flow cytometry, it is necessary to ensure fluorescence linearity. Fluorescein Monthly assessment of PMT fluorescence detection, monitored with (520nm) and R-phycoerythrin (580 nm) and any other multi-level fluorescent beads, is required for laboratories maintaining a fluor routinely used by the laboratory should be included. permit in Lymphoid Function, Non-Lymphoid Immunophenotyping, or Malignant Leukocyte Immunophenotyping. Laboratories holding the a) The correlation coefficient of the Mean Fluorescent Intensity category of Limited II should perform this assessment as needed. This (MFI) versus fluorescent molecules per bead should be equal to analysis evaluates: or greater than 0.98 (1.0 is optimal). CI21a a) linearity at the settings used for clinical measurement; b) Monitoring assesses the PMTs capable range of measurement related to the marker intensity (antigen density on CI21b b) fluorescent sensitivity and resolution at settings used for clinical or in the cell) and ability to resolve populations of different measurement; and, intensities. CI21c c) PMT changes. c) Monitoring provides PMT performance history and large shifts or fluctuations indicate that maintenance may be required. LEUKOCYTE IMMUNOPHENOTYPING (formerly LIMITED II OR IV) Cellular Immunology Standard 22 Specimen age cut-off refers to the maximum acceptable time from specimen collection to processing (stained, lysed, and The laboratory shall establish maximum specimen age cut-offs for whole fixed). blood testing of lymphocyte immunophenotypes and these cut-off points shall not exceed: When establishing the cutoffs, the laboratory should consider staining protocol, anticoagulants and instrumentation. CI22a a) 30 hours if using tri-potassium EDTA anticoagulant; or, This validation shall demonstrate that results at time zero and at CI22b b) 48 hours if using ACD or heparin anticoagulant. the maximum time are the same (+ 3%) using specimens from both normal and infected or diseased individuals. Cellular Immunology Standard 23 The isotype control (the negative control for non-specific antibody binding) should be used for setting analysis cursors CI23 Immunophenotyping negative controls shall be isotype matched that distinguish negative from positive staining cells. The antibody at similar concentrations and F/P ratios with the test antibody. analysis cursors should remain the same throughout a patients testing with matched antibody/isotype controls. January 2008 Page 174 of 197

10. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance NON-MALIGNANT IMMUNOPHENOTYPING (formerly LIMITED II) Cellular Immunology Standard 24 CI24 When implementing single-platform methods, the laboratory shall follow Maximum acceptable age should not exceed 30 hours. the manufacturers recommendations for anti-coagulant and maximum acceptable specimen age. Cellular Immunology Standard 25 CI25 When implementing single-platform methods dependent on accurate volume delivery, the laboratory shall calibrate pipets weekly using either a gravimetric method or control beads. Cellular Immunology Standard 26 CI26 The laboratory shall provide instructions to clients indicating that blood samples shall be transported and maintained at 18 to 23 degrees C. Cellular Immunology Standard 27 CI27 Immunophenotyping of blood lymphocytes by flow cytometry shall be conducted using the whole blood lysis method. Cellular Immunology Standard 28 This validation should demonstrate that results at time zero and CI28 Each laboratory shall establish the maximum time that fixed specimens at their maximum time are the same (within 3%) using may be stored before analysis, and this time shall not exceed 24 hours. specimens from both normal and infected or diseased individuals. January 2008 Page 175 of 197

11. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 29 CI29 Immunophenotyping by flow cytometry shall be conducted using Single color analysis is acceptable for enumerating CD3 T appropriately validated two, three, or four-color immunofluorescence. lymphocytes and CD19 B lymphocytes, but is not acceptable for enumerating CD4 T lymphocytes, CD8 T lymphocytes, and natural killer (NK) lymphocytes. Enumeration of CD4 T lymphocytes, CD8 T lymphocytes and natural killer (NK) lymphocytes should include the use of CD3. LYMPHOID AND T-LYMPHOID IMMUNOPHENOTYPING Cellular Immunology Standard 30 D45 and CD14 antibodies should be used to set lymphocyte gates and to determine lymphocyte purity of the gate and Lymphocyte immmunophenotyping using two color analysis shall lymphocyte recovery. include the following panels: CI30a a) full lymphocyte analysis panel CD45/CD14 b) Suggested abbreviated patient analysis panels: Isotype control CD3/CD4 i) HIV induced immunodeficiency: CD3/CD8 CD45/CD14, isotype control, CD3/CD4 and CD3/CD8. CD3/CD19 CD3/CD56 CD16 ii) General immunodeficiency panel: CD45/CD14, isotype control, CD3/CD19 and CD3/CD56 CI30b b) abbreviated test panels shall contain a minimum of two marker tubes CD16. in addition to the CD45/ CD14 tube and the isotype control tube. Both marker tubes should contain CD3. The second marker for each tube may be chosen at the laboratorys discretion as appropriate for the patients diagnosis. January 2008 Page 176 of 197

12. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 31 Lymphocyte immunophenotyping by two-color analysis shall include: The laboratory should develop a mechanism to distinguish between initial and subsequent samples. If the laboratory CI31a a) a full panel for a patients initial testing; cannot distinguish between initial and subsequent samples, a full panel should be run. CI31b b) an abbreviated panel for subsequent testing performed by the same laboratory for the same patient; and, CI31c c) enumeration of B lymphocytes (CD19+) and NK lymphocytes (CD3- /CD16+ and/or CD56+) when discrepancies occur during subsequent testing with the use of the abbreviated test panel for HIV-induced immunodeficiency. Cellular Immunology Standard 32 Purity is defined as the percentage of cells within the gate that are lymphocytes. Acceptable gate purity is optimally greater For two-color analysis: than or equal to 90%. CI32a a) lymphocyte purity of the gate shall be determined for all specimens; If minimum lymphocyte purity and/or recovery cannot be and, obtained with repeated testing (e.g., immunocompromised patients), testing is permitted provided the report clearly CI32b b) specimens with gate purity less than 85% shall be rejected. indicates the limitations of the results. Under these circumstances, the laboratory should establish limits of recovery and purity beyond which test results will not be reported. Cellular Immunology Standard 33 For two-color analysis: Recovery is defined as the percentage of lymphocytes in the sample that are within the gate. CI33a a) lymphocyte recovery shall be determined for all specimens; and, An acceptable lymphocyte recovery is optimally greater than or CI33b b) specimens with recoveries less than 90% shall be rejected. equal to 95%. January 2008 Page 177 of 197

13. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 34 CI34 For two-color analysis, all reported values shall be corrected for lymphocyte purity. Cellular Immunology Standard 35 CI35 For three- and four-color analysis, CD45/side-scatter gating shall be This is not applicable to flow cytometry analysis performed in a used to gate lymphocytes based on the CD45 bright and low side light- lineage specific manner and in accordance with the scatter population. manufacturer recommendations. January 2008 Page 178 of 197

14. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 36 If a full lymphocyte analysis panel is performed, the following markers should be included: CI36 Lymphocyte immunophenotyping using three color analysis shall include a minimum of two tubes, both of which shall include CD45 and CD3. CD45/CD3/CD4 CD45/CD3/CD8 CD45/CD3/CD19 CD45/CD3/CD56 CD16 The third marker for each tube may be chosen at the laboratorys discretion as appropriate for the patients diagnosis. For example, the following is suggested for test panels for three color analysis: ii) HIV induced immunodeficiency: CD45/CD3/CD4 and CD45/CD3/CD8 ii) General immunodeficiency panel: CD45/CD3/CD19 and CD45/CD3/CD56 CD16 Laboratories analyzing in a lineage specific manner on the flow cytometer are an exception. The staining panel for lineage specific analysis should include the following tubes: isotype control CD4/CD8/CD3 CD16/CD19/CD3 January 2008 Page 179 of 197

15. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 37 If a full lymphocyte analysis panel is performed, the following CI37 markers should be included: Lymphocyte immunophenotyping using four color analysis shall include one or more tubes which utilize CD45 and CD3. CD45/CD3/CD4/CD8 CD45/CD3/CD19/CD56 CD16 The third and fourth markers may be chosen at the laboratory s discretion as appropriate for the patient s diagnosis. For example, the following is suggested for test panels for four color analysis: i) HIV induced immunodeficiency: CD45/CD3/CD4/CD8 ii) General immunodeficiency panel: CD45/CD3/CD19/CD56 CD16 It is highly recommended that a second two, three, or four color tube for confirmation of sample preparation including a replicate CD marker such as CD3 be included. Cellular Immunology Standard 38 In severe late stage AIDS or immunosuppressed patients, cell CI38 At least 2,500 gated lymphocytes shall be counted per sample. counts may be very low. In these extreme cases, events collected may be lower. January 2008 Page 180 of 197

16. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 39 CD3 values shall: The use of different fluorochromes could further affect this assay. The laboratory should document that the specimen was CI39a a) be monitored within a patients lymphocyte immunophenotyping repeated and/or restained. panel; and, CI39b b) not vary more than 3% among all of a patients stained CD3 tubes unless a greater difference is routinely obtained due to the use of different fluorescent reagents. Cellular Immunology Standard 40 Lymphosum refers to the sum of all subsets of lymphocytes. The lymphosum shall be monitored when all the required lymphocyte The sum of the values of CD3+ plus CD19+ plus CD3-/CD16+ CI40 subset tubes have been included in the lymphocyte analysis panel. and/or CD56+ cells (corrected for lymphocyte purity) should optimally be equal to 100% 5%. Cellular Immunology Standard 41 The sum of CD3+/CD4+ and CD3+/CD8+ should be within 10% of the total CD3 mean. If a greater difference is found, the CI41 The T-sum shall be monitored when the required lymphocyte subset laboratory should rephenotype to confirm that no preparation tubes have been included in the lymphocyte analysis panel. problems occurred. A greater variance is acceptable in patients with an increased population of CD4-/CD8-/CD3+ cells (e.g., delta/gamma T cells). Cellular Immunology Standard 42 CI42 Percentages and absolute numbers of lymphocyte subsets shall be Some systems (single platform instrumentation) may provide reported. only absolute values. Absolute values are not mandatory for pediatric specimens. January 2008 Page 181 of 197

17. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 43 CI43 The laboratory performing white blood cell counts and differentials for Absolute number generation by single platform instrumentation the purpose of calculating absolute numbers of lymphocyte/leukocyte may require an additional permit for hematology depending on subsets shall hold a New York State laboratory permit in Hematology. the methodology used: A hematology permit is not required if the laboratory calculates absolute lymphocyte subset numbers by an indirect particle (bead) counting method. A hematology permit is required if a laboratory uses a volume (or conductivity) method for performing absolute lymphocyte counts. MALIGNANT LEUKOCYTE IMMUNOPHENOTYPING (formerly LIMITED IV) Cellular Immunology Standard 44 CI44 The laboratory shall test leukemia/lymphoma specimens within 48 hours Specimens should be assessed by light scatter and viability to of collection. ascertain specimen integrity. Cellular Immunology Standard 45 Specimens for Limited IV analysis shall be: CI45a a) placed into appropriate anticoagulant, saline, or media for transport; CI45b b) maintained at appropriate temperatures; and, CI45c c) processed using sterile techniques. January 2008 Page 182 of 197

18. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 46 CI46 Upon receipt, leukemia/lymphoma specimens shall be visually inspected Specimens which are fixed, frozen, warm, or, in the case of for hemolysis, clots, and evidence of deterioration. peripheral blood or bone marrow, clotted or hemolyzed, should be rejected. Bone marrow specimens with a few small clots may be tested. Cellular Immunology Standard 47 Adjunctive diagnostic testing may include smears, touch prints, immuno-chemistry. CI47 Whenever possible, adjunctive diagnostic testing shall use the same source specimen that was used for flow cytometric analysis. When specimen analysis will be delayed, a stained smear, tissue imprint and/or tissue section should be made directly after specimen collection to provide morphologic assessment of the specimen in the fresh state. Cellular Immunology Standard 48 Leukemia/lymphoma specimens shall: CI48a a) be processed in a manner that maintains viability, removes non- critical events (e.g., RBCs) and does not remove antigens; and, CI48b b) prior to staining, undergo a white blood cell concentration adjustment to optimize cell-to-stain ratio and instrument acquisition cell flow rate. January 2008 Page 183 of 197

19. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 49 All leukemia/lymphoma specimens shall: Specimens with > 80% viability are optimal for flow cytometric analysis. CI49a a) meet the laboratorys pre-established criteria for viability; When specimens with viabilities between 50 and 80% are CI49b b) be assessed for viability after specimen processing (before staining reported, the report should indicate that results may be affected and fixation) on a non-fixed aliquot of the specimens single cell by the lower viability. suspension; and, CI49c c) be rejected when the viability is < 50%. Cellular Immunology Standard 50 CI50 The laboratory shall implement a method to minimize non-specific binding of antibodies to lymphoma and bone marrow specimens. Cellular Immunology Standard 51 CI51 Immunoglobulin analysis shall include a pan B-cell marker in combination with anti-immunoglobulin antibody(ies). Cellular Immunology Standard 52 a) New combinations of antibodies should be tested individually and together before use to ensure that there is no For leukemia/lymphoma testing: blockage/interference. CI52a a) antibody/marker combinations shall be validated for non-inhibitory b) The panels should contain markers with cell type staining; and, redundancy to confirm lack of cell lineage or developmental stage of the aberrant cells lineage. CI52b b) marker panels or antibody combinations shall be designed for optimal determination of disease state(s). Multi-color analysis (two-, three-, or four-color) is recommended. Single color analysis is acceptable, but more information is gained by using multi-color analysis. January 2008 Page 184 of 197

20. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 53 CI53 Matched isotype controls shall be used to determine dim reactivity versus nonspecific binding and to set positive analysis regions. Cellular Immunology Standard 54 Data acquisition in leukemia/lymphoma analysis shall: CI54a a) include the collection of all viable leukocyte populations (no restrictive population gate) into listmode storage format for later a) For each specimen, the laboratory should optimize light analysis; and, scatter for best population separation to allow gates/regions to CI54b be drawn, while reducing debris and non-leukocyte b) minimally include the collection of 10,000 events (5,000 if the contamination. Data analysis should be completed with multiple specimen presents as a single population) unless the specimen has a gates set on the apparent populations with minimal low cellular content (e.g., cerebral spinal fluid and fine needle contamination from other cell populations. aspiration). Cellular Immunology Standard 55 CI55 Analysis of cells with low expression of a particular marker/antigen (cells dimly fluorescent) shall be integrated with the matched isotypic control(s) to determine relative positivity. Cellular Immunology Standard 56 CI56 When abnormal staining reactivities (higher or lower expression than observed on normal cells) are obtained, the report shall include a description of the quality of staining (dim or low intensity and/or bright or high intensity). January 2008 Page 185 of 197

21. New York State Department of Health Clinical Laboratory Standards of Practice Cellular Immunology Tag # Standard Guidance Cellular Immunology Standard 57 CI57 Repeated antibody(ies) within the patients testing panel shall be If results demonstrate inconsistencies, the laboratory should consistent (within 3%) unless a greater difference is routinely obtained review the analysis for procedural error and restain if necessary. due to the use of different fluorescent reagents. Cellular Immunology Standard 58 a) name or unique identifier, gender, age; The leukemia/lymphoma report shall include: b) specimen source, collection date and time, date and time CI58a a) patient identifiers; received in the laboratory, processing date and time, and viability; CI58b b) specimen information; c) descriptions of light scatter characteristics, percent of total CI58c c) marker results; and, leukocytes, and marker expression (with staining intensity when abnormal) for each aberrant population identified. CI58d d) diagnosis or characterization. The flow cytometric results should be correlated with pathology results, when available. January 2008 Page 186 of 197

22. New York State Department of Health Clinical Laboratory Standards of Practice Cytokines Tag # Standard Guidance The following specialty sustaining standards of practices shall be It is recommended that WHO international cytokine standards incorporated into the laboratorys quality management system, where be evaluated as additional inter-assay control when available. applicable to the scope of services provided. Since these assays are not cleared or approved by the FDA, the laboratory must submit copies of package inserts and patient reports before initiating testing as described in the Submission Guideline. These Guidelines can be found at www.wadsworth.org/labcert/clep/clep.html. Cytokine Standard 1 Normal range of values should be established for each matrix (e.g., serum, plasma, or CSF). The effect of diurnal variation on CK1 Laboratories shall establish or verify the reference interval for each cytokine levels should be taken into consideration and should cytokine for each matrix tested. be included in collection instructions. Cytokine Standard 2 CK2 All results that fall outside the reference interval shall be diluted and False positive results may be obtained when the specimen is retested. If the results of the two results do not agree within an run neat due to matrix interference. acceptable degree of variability, a third dilution shall be retested until reliable results are achieved. Cytokine Standard 3 Laboratories should establish an acceptable range of variation for duplicate values (e.g., less than 20 percent coefficient of CK3 All specimens shall be run in duplicate with non-automated methods variation). unless validation studies indicate acceptable precision. January 2008 Page 187 of 197