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 incorporated into the laboratory’s quality management system, where applicable to the scope of services provided. If the laboratory reports values dependent on absolute number of any leukocyte population assessed by volume or conductivity methodology, a hematology permit is required. CI1 Cellular Immunology Standard 1 The laboratory shall have written criteria for the establishment of normal ranges. Ranges should be updated biannually and monitored for statistical changes. 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
Transcript
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