(269 days)
Not Found
No
The summary describes reagents and accessory reagents for flow cytometry, with interpretation performed by human experts. There is no mention of automated analysis or algorithms that would suggest AI/ML.
No
The device is described as an in vitro diagnostic (IVD) tool used for qualitative identification of cell populations to aid in the differential diagnosis of hematological disorders. It does not provide any form of treatment or therapy.
Yes
The "Intended Use / Indications for Use" section explicitly states that "The ClearLLab reagents are intended for in vitro diagnostic use" and "These reagents are used as an aid in the differential diagnosis of hematologically abnormal patients".
No
The device description explicitly details physical reagents (monoclonal and polyclonal antibodies, fluorospheres, fixative solution, lysing solution, control cells) which are hardware components, not software.
Yes, this device is an IVD (In Vitro Diagnostic).
Here's why:
- Intended Use Statement: The very first sentence explicitly states: "The ClearLLab reagents are intended for in vitro diagnostic use as a panel for qualitative identification of cell populations by multiparameter immunophenotyping on an FC 500 flow cytometer."
- Purpose: The reagents are used "as an aid in the differential diagnosis of hematologically abnormal patients having, or suspected of having the following hematopoietic neoplasms..." This clearly indicates the device is used to analyze samples outside the body to help diagnose a disease.
- Sample Types: The device is used with "peripheral whole blood, bone marrow, and lymph node specimens," which are biological samples taken from a patient.
- Device Description: The description details reagents used to identify specific cell surface markers in these biological samples, which is a common method in in vitro diagnostics.
- Performance Studies: The performance studies evaluate the device's ability to detect abnormal cell populations in patient samples and compare the results to clinical diagnoses, further supporting its use in diagnosis.
All these points align with the definition of an In Vitro Diagnostic device.
N/A
Intended Use / Indications for Use
The ClearLLab reagents are intended for in vitro diagnostic use as a panel for qualitative identification of cell populations by multiparameter immunophenotyping on an FC 500 flow cytometer. These reagents are used as an aid in the differential diagnosis of hematologically abnormal patients having, or suspected of having the following hematopoietic neoplasms; chronic leukemia, acute leukemia, non-Hodgkin lymphoma, myeloma, myelodysplastic syndrome (MDS), and/or myeloproliferative neoplasms (MPN). The reagents can be used with peripheral whole blood collected in EDTA (K2 and K3EDTA), Acid Citrate Dextrose or Heparin, bone marrow collected in K2EDTA, ACD, or Heparin, and lymph node specimens. Interpretation of the results should be confirmed by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings.
These reagents provide multiparameter, qualitative results for the Cluster of Differentiation (CD) parameters listed below:
- ClearLLab T1: CD2, CD56, CD7, CD5, CD7, CD5, CD45
- ClearLLab T2: CD8, CD4, CD3, CD45
- ClearLLab B1: Kappa, Lambda, CD19, CD5, CD45
- ClearLLab B2: CD20, CD10, CD10, CD19, CD38, CD45
- ClearLLab M: CD7, CD13, CD34, CD33, CD45
Product codes
PWD
Device Description
The Clear LLab Reagents panel is used with the following reagents with their respective indicated uses. A description of the reagents provided is described below in Table 1.
The T-Cell Lineage panels: Comprising T1 CD2-FITC/CD56-PE/CD7-ECD/CD5-PC5.5/CD45-PC7 and T2 CD8-FITC/CD4-PE/CD3-PC5.5/CD45-PC7 Monoclonal Antibody Reagents. For use to identify lymphocytes that contain the specific cell surface markers associated with the T-cell lineage.
The B-Cell Lineage panels: Comprising B1 Kappa-FITC/Lambda-PE/CD19-ECD/CD5-PC5.5/CD45-PC7 and B2 CD20-FITC/CD10-PE/CD19-ECD/CD38-PC5.5/CD45-PC7 Monoclonal and Polyclonal Antibody Reagents. For use to identify lymphocytes that contain the specific cell surface markers associated with the B-cell lineage.
The Myeloid Lineage panel: Comprising M CD7-FITC/CD13-PE/CD34-ECD//CD33-PC5.5/CD45-PC7 Monoclonal Antibody Reagents. For use to identify lymphocytes that contain the specific cell surface markers associated with the Myeloid lineage.
Accessory Reagents Required:
- Flow-Check Pro Fluorospheres: Are a suspension of fluorescent microspheres used for daily verification of the optical alignment and fluidics for Forward Scatter (FS) and FL1-FL4 fluorescence parameters. Instrument Alignment Quality Control Reagent to provide instrument alignment Quality Control instructions.
- Flow-Set Pro Fluorospheres: Are a suspension of fluorescent microspheres used as an aid in standardizing forward scatter, side scatter, and fluorescence detectors on the FC 500 (FL1-FL5). Auto Setup Reagent for standardization of flow cytometer light scatter and fluorescence intensity instrument settings to provide application-specific instrument target ranges for standardization.
- QuickComp 4 Kit: Consists of four single-color fluorescent reagents comprised of one monoclonal antibody each. Each antibody is labeled with one of four fluorochromes, three are utilized for this test: CD45-FITC, CD45-PE, and CD45-ECD. The QuickComp 4 Kit is used to adjust color compensation settings on a flow cytometer equipped with AutoSetup software, prior to multi-color analysis with FITC, PE, and ECD conjugated monoclonal antibody reagents.
- Color Compensation Reagents: Compensation reagents CD45-PC5.5 and CD45-PC7. Used to adjust color compensation settings on a flow cytometer with AutoSetup software. Note: Color Compensation Reagents are required. It is recommended to use the reagents with normal whole blood specimens to adjust color compensation settings on a flow cytometer, prior to multi-color analysis.
IOTest 3 Fixative Solution: The IOTest 3 Fixative Solution, which has a formaldehyde base, specially developed for the fixing of leukocytes. Leukocyte fixative solution used on all leukocytic preparations to enable leukocytic preparations to be stored for several hours without deterioration after staining with a fluorescent antibody. It is used to fix leukocytes following immunofluorescent staining with the fluorochrome-conjugated antibodies and lysis of the red blood cells.
Accessory Reagents Recommended but Not Provided:
- VersaLyse Lysing Solution: Red cell lysing reagent intended for the lysis of red blood cells in the preparation of biological samples for flow cytometry analysis. VersaLyse is intended for the lysis of red blood cells in the preparation of red blood cells in the preparation of biological samples for flow cytometry analysis.
- Immutrol Control Cells: Process controls for flow cytometry. Quality control material assayed for lymphocyte, granulocyte and monocyte specific antigens and single platform absolute counts. The light scatter, population distribution, fluorescence intensity, and antigen density mimic those of normal whole blood.
- StemTrol Control cells: An antibody-to-antigen positive control for CD34 and CD45 staining in flow cytometry studies, consisting of a preserved cell line, BK010044. Quality control material for CD34 and CD45.
ClearLLab Immunophenotyping Panel:
- T-cell tube 1 (T1): FITC-CD2, PE-CD56, ECD-CD7, PC5.5-CD5, PC7-CD45
- T-cell tube 2 (T2): FITC-CD8, PE-CD4, PC5.5-CD3, PC7-CD45
- B-cell tube 1 (B1): FITC-Kappa, PE-Lambda, ECD-CD19, PC5.5-CD5, PC7-CD45
- B-cell tube 2 (B2): FITC-CD20, PE-CD10, ECD-CD19, PC5.5-CD38, PC7-CD45
- Myeloid (M): FITC-CD7, PE-CD13, ECD-CD34, PC5.5-CD33, PC7-CD45
Mentions image processing
Not Found
Mentions AI, DNN, or ML
Not Found
Input Imaging Modality
Flow Cytometry
Anatomical Site
Peripheral whole blood, bone marrow, and lymph node specimens.
Indicated Patient Age Range
Not Found
Intended User / Care Setting
For prescription use only. Interpretation of the results should be confirmed by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings.
Description of the training set, sample size, data source, and annotation protocol
Not Found
Description of the test set, sample size, data source, and annotation protocol
A total of 279 abnormal hematologic specimens were enrolled from four sites (Site 1, Site 2, Site 3, Site 4). Data source unknown. The specimens included 137 hematologically abnormal but with no malignancy and 142 with hematolymphoid malignancy, per site's final diagnosis.
The specimen mix consisted of approximately 60% peripheral blood, 30% bone marrow, and 10% lymph nodes for both hematopoietic malignant and nonmalignant categories.
Two qualified flow experts independently evaluated the ClearLLab flow cytometry results. Each expert was blinded to the final diagnosis. They were asked to evaluate the ClearLLab results for the presence or absence of abnormal cell populations and provide the percentage of abnormal population and its associated phenotype. The result was designated as "malignant" if an abnormal population was identified, and "non-malignant" if an abnormal population was not identified.
Summary of Performance Studies (study type, sample size, AUC, MRMC, standalone performance, key results)
Clinical Performance:
A multi-center, retrospective study was conducted at four sites comparing the diagnostic accuracy of the ClearLLab reagents to detect the presence or absence of an abnormal phenotype to the clinical outcome of "malignant" or "non-malignant" based on the clinical sites' final patient diagnosis.
- Sample Size: A total of 279 abnormal hematologic specimens were enrolled. This included 137 hematologically abnormal but no malignancy and 142 with hematolymphoid malignancy.
- Key Results:
- Flow Expert #1:
- Sensitivity: 82.4% (95% CI: 75.3%-87.8%)
- Specificity: 94.2% (95% CI: 88.9%-97.0%)
- PPV: 93.6% (95% CI: 87.9%-96.7%)
- NPV: 83.8% (95% CI: 77.1%-88.8%)
- Flow Expert #2:
- Sensitivity: 85.9% (95% CI: 79.2%-90.7%)
- Specificity: 92.7% (95% CI: 87.1%-96.0%)
- PPV: 92.4% (95% CI: 86.6%-95.8%)
- NPV: 86.4% (95% CI: 79.9%-91.0%)
- Combined Evaluations for Flow Experts #1 and #2:
- Sensitivity: 84.2% (95% CI: 79.5%-87.9%)
- Specificity: 93.4% (95% CI: 89.9%-95.8%)
- Agreement between Flow Experts:
- Positive: 98.4% (95% CI: 94.4%-99.6%)
- Negative: 94.2% (95% CI: 89.3%-96.9%)
- Flow Expert #1:
Whole Blood Specimens (Additional Analysis):
- Sample Size: 160 hematologically abnormal whole blood specimens (78 with no malignancy, 82 with hematolymphoid malignancy).
- Key Results:
- Flow Expert #1:
- Sensitivity: 89.0% (95% CI: 80.4%-94.1%)
- Specificity: 91.0% (95% CI: 82.6%-95.6%)
- PPV: 91.3% (95% CI: 83.0%-95.7%)
- NPV: 88.8% (95% CI: 80.0%-94.0%)
- Flow Expert #2:
- Sensitivity: 92.7% (95% CI: 84.9%-96.6%)
- Specificity: 89.7% (95% CI: 81.0%-94.7%)
- PPV: 90.5% (95% CI: 82.3%-95.1%)
- NPV: 92.1% (95% CI: 83.8%-96.3%)
- Agreement between Flow Experts:
- Positive: 98.4% (95% CI: 94.4%-99.6%)
- Negative: 94.2% (95% CI: 89.3%-96.9%)
- Flow Expert #1:
Key Metrics (Sensitivity, Specificity, PPV, NPV, etc.)
- Clinical Performance (All Specimen Types) - Flow Expert #1:
- Sensitivity: 82.4%
- Specificity: 94.2%
- PPV: 93.6%
- NPV: 83.8%
- Clinical Performance (All Specimen Types) - Flow Expert #2:
- Sensitivity: 85.9%
- Specificity: 92.7%
- PPV: 92.4%
- NPV: 86.4%
- Clinical Performance (All Specimen Types) - Combined Experts:
- Sensitivity: 84.2%
- Specificity: 93.4%
- Agreement between Flow Experts (All Specimen Types):
- Positive: 98.4%
- Negative: 94.2%
- Clinical Performance (Whole Blood Specimen Types) - Flow Expert #1:
- Sensitivity: 89.0%
- Specificity: 91.0%
- PPV: 91.3%
- NPV: 88.8%
- Clinical Performance (Whole Blood Specimen Types) - Flow Expert #2:
- Sensitivity: 92.7%
- Specificity: 89.7%
- PPV: 90.5%
- NPV: 92.1%
- Agreement between Flow Experts (Whole Blood Specimen Types):
- Positive: 98.4%
- Negative: 94.2%
Analytical Performance:
- Repeatability (Qualitative): 100% agreement for all sites and anticoagulants for expected and actual results with respect to the presence or absence of an abnormal phenotype.
- Operator-to-operator and instrument-to-instrument imprecision (Qualitative): 100% agreement for all operators and instruments for expected and actual results with respect to the presence or absence of an abnormal phenotype.
- Limit of Blank (LoB): 0.27% population of abnormal cells.
- Limit of Detection (LoD): 1% population of abnormal cells.
- Specimen Carryover: Less than 1% for all markers.
- Reagent Carryover: Less than 1% for all markers.
Predicate Device(s)
Not Found
Reference Device(s)
Not Found
Predetermined Change Control Plan (PCCP) - All Relevant Information
Not Found
§ 864.7010 Flow cytometric test system for hematopoietic neoplasms.
(a)
Identification. A flow cytometric test for hematopoietic neoplasms is a device that consists of reagents for immunophenotyping of human cells in relation to the level of expression, antigen density, and distribution of specific cellular markers. These reagents are used as an aid in the differential diagnosis or monitoring of hematologically abnormal patients having or suspected of having hematopoietic neoplasms. The results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings.(b)
Classification. Class II (special controls). The special controls for this device are:(1) Premarket notification submissions must include the following information:
(i) The indications for use must indicate the clinical hematopoietic neoplasms for which the assay was designed and validated, for example, chronic leukemia or lymphoma.
(ii) A detailed device description including the following:
(A) A detailed description of all test components, all required reagents, and all instrumentation and equipment, including illustrations or photographs of nonstandard equipment or methods.
(B) Detailed documentation of the device software including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
(C) A detailed description of methodology and assay procedure.
(D) A description of appropriate internal and external quality control materials that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure, if applicable.
(E) Detailed specifications for sample collection, processing, and storage.
(F) Detailed specification of the criteria for test results interpretation and reporting including pre-established templates.
(G) If applicable, based on the output of the results, a description of the specific number of events to collect, result outputs, and analytical sensitivity of the assay that will be reported.
(iii) Information that demonstrates the performance characteristics of the test, including:
(A) Device performance data from either a method comparison study comparing the specific lymphocyte cell markers to a predicate device or data collected through a clinical study demonstrating clinical validity using well-characterized clinical specimens. Samples must be representative of the intended use population of the device including hematologic neoplasms and the specific sample types for which the test is indicated for use.
(B) If applicable, device performance data from a clinical study demonstrating clinical validity for parameters not established in a predicate device of this generic type using well-characterized prospectively obtained clinical specimens including all hematologic neoplasms and the specific sample types for which the device is indicated for use.
(C) Device precision data using clinical samples to evaluate the within-lot, between-lot, within-run, between run, site-to-site and total variation using a minimum of three sites, of which at least two sites must be external sites. Results shall be reported as the standard deviation and percentage coefficient of variation for each level tested.
(D) Reproducibility data generated using a minimum of three lots of reagents to evaluate mean fluorescence intensity and variability of the recovery of the different markers and/or cell populations.
(E) Data from specimen and reagent carryover testing performed using well-established methods (
e.g., CLSI H26-A2).(F) Specimen and prepared sample stability data established for each specimen matrix in the anticoagulant combinations and storage/use conditions that will be indicated.
(G) A study testing anticoagulant equivalency in all claimed specimen type/anticoagulant combinations using clinical specimens that are representative of the intended use population of the device.
(H) Analytic sensitivity data using a dilution panel created from clinical samples.
(I) Analytical specificity data, including interference and cross-contamination.
(J) Device stability data, including real-time stability of reagents under various storage times and temperatures.
(K) For devices that include polyclonal antibodies, Fluorescence Minus One (FMO) studies to evaluate non-specific binding for all polyclonal antibodies. Each FMO tube is compared to reagent reference to demonstrate that no additional population appears when one marker is absent. Pre-specified acceptance criteria must be provided and followed.
(L) For devices indicated for use as a semi-quantitative test, linearity data using a dilution panel created from clinical samples.
(M) For devices indicated for use as a semi-quantitative test, clinically relevant analytical sensitivity data, including limit of blank, limit of detection, and limit of quantification.
(iv) Identification of risk mitigation elements used by the device, including a detailed description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing the device.
(2) The 21 CFR 809.10 compliant labeling must include the following:
(i) The intended use statement in the 21 CFR 809.10(a)(2) and (b)(2) compliant labeling must include a statement that the results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings. The intended use statement must also include information on what the device detects and measures, whether the device is qualitative, semi-quantitative, and/or quantitative, the clinical indications for which the device is to be used, and the specific population(s) for which the device is intended.
(ii) A detailed description of the performance studies conducted to comply with paragraph (b)(1)(iii) of this section and a summary of the results.
(3) As part of the risk management activities performed under 21 CFR 820.30 design controls, product labeling and instruction manuals must include clear examples of all expected phenotypic patterns and gating strategies using well-defined clinical samples representative of both abnormal and normal cellular populations. These samples must be selected based upon the indications described in paragraph (b)(1)(i) of this section.
0
EVALUATION OF AUTOMATIC CLASS III DESIGNATION FOR CLEARLLAB REAGENTS DECISION SUMMARY
A. DEN Number:
B. Purpose for Submission:
De novo request for evaluation of automatic class III designation of the ClearLLab Reagents
C. Measurand:
Cluster of Differentiation (CD) Surface Markers on white blood cells
D. Type of Test:
Immunophenotyping, qualitative, flow cytometric assay
E. Applicant:
Beckman Coulter Inc.
F. Proprietary and Established Names:
Trade Name: ClearLLab T1, T2, B1, B2, M Common Name: ClearLLab
G. Regulatory Information:
-
- Regulation section:
21 CFR 864.7010
- Regulation section:
-
- Classification:
Class II (Special Controls)
- Classification:
-
- Product code:
PWD
- Product code:
-
- Panel:
81-Hematology
- Panel:
1
H. Indications For Use:
1. Indications for Use
The ClearLLab reagents are intended for in vitro diagnostic use as a panel for qualitative identification of cell populations by multiparameter immunophenotyping on an FC 500 flow cytometer. These reagents are used as an aid in the differential diagnosis of hematologically abnormal patients having, or suspected of having the following hematopoietic neoplasms; chronic leukemia, acute leukemia, non-Hodgkin lymphoma, myeloma, myelodysplastic syndrome (MDS), and/or myeloproliferative neoplasms (MPN). The reagents can be used with peripheral whole blood collected in EDTA (K2 and K3EDTA), Acid Citrate Dextrose or Heparin, bone marrow collected in K2EDTA, ACD, or Heparin, and lymph node specimens. Interpretation of the results should be confirmed by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings.
These reagents provide multiparameter, qualitative results for the Cluster of Differentiation (CD) parameters listed below:
- ClearLLab T1: CD2, CD56, CD7, CD5, CD7, CD5, CD45
- ClearLLab T2: CD8, CD4, CD3, CD45
- ClearLLab B1: Kappa, Lambda, CD19, CD5, CD45
- ClearLLab B2: CD20, CD10, CD10, CD19, CD38, CD45
- ClearLLab M: CD7, CD13, CD34, CD33, CD45
-
- Special conditions for use statement(s):
For prescription use only.
For in vitro diagnostic use.
-
- Special instrument requirements:
Beckman Coulter FC500 flow cytometer, CXP Software
- Special instrument requirements:
Doggintian
I. Device Description:
Itom
The Clear LLab Reagents panel is used with the following reagents with their respective indicated uses. A description of the reagents provided is described below in Table 1.
| Item | Description | Use |
|---|---|---|
| The T-Cell Lineage panels | Comprising T1 CD2- | |
| FITC/CD56-PE/CD7- | ||
| ECD/CD5-PC5.5/CD45-PC7 | ||
| and T2 CD8-FITC/CD4- | ||
| PE/CD3-PC5.5/CD45-PC7 | ||
| Monoclonal Antibody Reagents. | For use to identify lymphocytes | |
| that contain the specific cell | ||
| surface markers associated with | ||
| the T-cell lineage. | ||
| The B-Cell Lineage panels | Comprising B1 Kappa- | For use to identify lymphocytes |
| FITC/Lambda-PE/CD19- | ||
| ECD/CD5-PC5.5/CD45-PC7 | ||
| and B2 CD20-FITC/CD10- | ||
| PE/CD19-ECD/CD38- | ||
| PC5.5/CD45-PC7 Monoclonal | ||
| and Polyclonal Antibody | ||
| Reagents. | that contain the specific cell | |
| surface markers associated with | ||
| the B-cell lineage. | ||
| The Myeloid Lineage panel | Comprising M CD7-FITC/ | |
| CD13-PE/CD34-ECD//CD33- | ||
| PC5.5/CD45-PC7 Monoclonal | ||
| Antibody Reagents. | For use to identify lymphocytes | |
| that contain the specific cell | ||
| surface markers associated with | ||
| the Myeloid lineage. | ||
| Accessory Reagents Required | ||
| Flow-Check Pro Fluorospheres | Flow-Check Pro Fluorospheres | |
| are a suspension of fluorescent | ||
| microspheres used for daily | ||
| verification of the optical | ||
| alignment and fluidics for | ||
| Forward Scatter (FS) and FL1- | ||
| FL4 fluorescence parameters. | Instrument Alignment Quality | |
| Control Reagent to provide | ||
| instrument alignment Quality | ||
| Control instructions. | ||
| Flow-Set Pro Fluorospheres | Flow-Set Pro Fluorospheres are | |
| a suspension of fluorescent | ||
| microspheres used as an aid in | ||
| standardizing forward scatter, | ||
| side scatter, and fluorescence | ||
| detectors on the FC 500 (FL1- | ||
| FL5). | Auto Setup Reagent for | |
| standardization of flow | ||
| cytometer light scatter and | ||
| fluorescence intensity | ||
| instrument settings to provide | ||
| application-specific instrument | ||
| target ranges for | ||
| standardization. | ||
| QuickComp 4 Kit | The QuickComp 4 Kit consists | |
| of four single-color fluorescent | ||
| reagents comprised of one | ||
| monoclonal antibody each. Each | ||
| antibody is labeled with one of | ||
| four fluorochromes, three are | ||
| utilized for this test: CD45- | ||
| FITC, CD45-PE, and CD45- | ||
| ECD. | The QuickComp 4 Kit is used to | |
| adjust color compensation | ||
| settings on a flow cytometer | ||
| equipped with AutoSetup | ||
| software, prior to multi-color | ||
| analysis with FITC, PE, and | ||
| ECD conjugated monoclonal | ||
| antibody reagents. | ||
| Color Compensation Reagents | Compensation reagents CD45- | |
| PC5.5 and CD45-PC7. | Used to adjust color | |
| compensation settings on a flow | ||
| cytometer with AutoSetup | ||
| software. | ||
| Note: Color Compensation | ||
| Reagents are required. It is | ||
| recommended to use the | ||
| reagents with normal whole | ||
| blood specimens to adjust color | ||
| compensation settings on a flow | ||
| cytometer, prior to multi-color | ||
| analysis. |
Table 1: Components of the ClearLLab Reagents
2
3
| IOTest 3 Fixative Solution | The IOTest 3 Fixative Solution, which has a formaldehyde base, specially developed for the fixing of leukocytes. | Leukocyte fixative solution used on all leukocytic preparations to enable leukocytic preparations to be stored for several hours without deterioration after staining with a fluorescent antibody. It is used to fix leukocytes following immunofluorescent staining with the fluorochrome-conjugated antibodies and lysis of the red blood cells. |
|---|---|---|
| Accessory Reagents Recommended but Not Provided | ||
| VersaLyse Lysing Solution | Red cell lysing reagent intended for the lysis of red blood cells in the preparation of biological samples for flow cytometry analysis. | VersaLyse is intended for the lysis of red blood cells in the preparation of biological samples for flow cytometry analysis. |
| Immutrol Control Cells | Process controls for flow cytometry. | Quality control material assayed for lymphocyte, granulocyte and monocyte specific antigens and single platform absolute counts. The light scatter, population distribution, fluorescence intensity, and antigen density mimic those of normal whole blood. |
| StemTrol Control cells | An antibody-to-antigen positive control for CD34 and CD45 staining in flow cytometry studies, consisting of a preserved cell line, BK010044, | Quality control material for CD34 and CD45. |
ClearLLab Immunophenotyping Panel
| Lineage | FITC | PE | ECD | PC5.5 | PC7 |
|---|---|---|---|---|---|
| T-cell tube 1 (T1) | CD2 | CD56 | CD7 | CD5 | CD45 |
| T-cell tube 2 (T2) | CD8 | CD4 | CD3 | CD45 | |
| B-cell tube 1 (B1) | Kappa | Lambda | CD19 | CD5 | CD45 |
| B-cell tube 2 (B2) | CD20 | CD10 | CD19 | CD38 | CD45 |
| Myeloid (M) | CD7 | CD13 | CD34 | CD33 | CD45 |
J. Standards/Guidance Documents Referenced:
CLSI EP6-A, Evaluation of the Linearity of Quantitative Measurement Procedures, A Statistical Approach
CLSI EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Methods
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CLSI EP28-A3c, Defining, Establishing, and Verifying Reference Intervals in the Clinical Laboratory
CLSI H26-A2, Validation, Verification, and Quality Assurance of Automated Hematology Analyzers
CLSI EP17-A2, Protocols for Determination of Limits of Detection and Limits of Quantitation
CLSI EP25-A, Evaluation of Stability on In Vitro Diagnostic Reagents
CLSI EP09-A3 Measurement Procedure Comparison and Bias Estimation Using Patient Samples
CLSI EP12-A2, User Protocol for Evaluation of Qualitative Test Performance
CLSI H43-A2, Clinical Flow Cytometric Analysis of Neoplastic Hematolymphoid Cells
K. Test Principle:
Beckman Coulter's ClearLLab product is comprised of pre-cocktailed, lineage-driven combinations of the consensus Cluster of Differentiation (CD) reagents for evaluation of B and T cell neoplasia and the consensus limited CD reagents for myelomonocytic neoplasia as described in Table 3 of "2006 Bethesda International Consensus Recommendations on the Immunophenotypic Analysis of Hematolymphoid Neoplasia by Flow Cytometry: Optimal Reagents and Reporting for the Flow Cytometric Diagnosis of Hematopoietic Neoplasia" ! The ClearLLab product is intended for use on the FC 500 flow cytometer with instrument set-up performed with Flow-Check Pro Fluorospheres, Flow-Set Pro Fluorospheres, and QuickComp color compensation reagents for alignment, voltage standardization, and compensation.
This test depends on the ability of a monoclonal or polyclonal antibody to bind to the surface of cells expressing discrete antigenic determinants. Specific cell staining is accomplished by incubating specimens prepared for staining with the appropriate antibody reagent. The ClearLLab Reagents are composed of five panels containing four or five monoclonal or polyclonal antibody reagents, each specific for a different cell surface antigen and conjugated to a specific fluorochrome. After sample preparation, the specimens are analyzed on the flow cytometer with manual gating.
The FC 500 flow cytometer running under the control of CXP Software applies the principles of flow cytometry to analyze a whole blood, bone marrow or lymph node sample. Samples are prepared and stained with a monoclonal antibody reagents followed by lysis of red blood cells prior to introduction into the instrument. Cellular populations are identified based on the specific monoclonal and polyclonal antibodies and fluorochromes used in the different panels. Detection of fluorescent antibodies bound to cells utilizes the capability of the FC 500 flow cytometer to
1 2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: optimal reagents and reporting for the flow cytometric diagnosis of hematopoietic neoplasia. Wood BL, Arroz M, Barnett D, DiGiuseppe J, Greig B, Kussick SJ, Oldaker T, Shenkin M, Stone E, Wallace P. Cytometry B Clin Cytom. 2007; 72 Suppl 1:S14-22
5
detect fluorescence with one laser, a blue laser with a 488 nm excitation and five different detection channels (FL1-FL5).
The CXP SYSTEM application software (K030828) provides automated instrument setup of light scatter and fluorescence intensity standardization, color compensation, and verification when used with the quality control reagents. The CXP Software has an Auto-Set Panel which automatically standardizes the cytometer, adjusts compensation settings, passes cytometer settings to designated test protocols, and verifies cytometer setup and antibody performance. Compensation settings are determined using CD45-FITC, CD45-PE, and CD45-ECD supplied in the QuickComp 4 Kit and CD45-PC5.5 and CD45-PC7 single color reagents. The existing FC 500 CXP software will support the ClearLLab reagents for use on the FC 500 flow cytometer without any further modifications from the original clearance in K030828.
Sample Preparation & Analysis
Whole Blood (WB) and Bone Marrow (BM) preparation:
Specimens are prepared manually with three pre-wash steps using phosphate buffered saline (PBS) containing 2% Heat Inactivated Fetal Calf Serum (HI-FCS). The remaining white blood cell pellet is then resuspended in PBS containing HI-FCS. The washed specimen is stained with the ClearLLab 5-Color Reagents. VersaLyse Lysing Solution for lysing red blood cells and the IOTest 3 Fixative Solution are used for red blood cell lysis and fixation prior to analysis.
Lymph Node preparation:
Lymph node specimens require disaggregation of the tissue into a single cell suspension. Single cell suspensions prepared from lymphoid tissues may not require washing prior to staining if the specimen was washed during the disaggregation process. If washing steps were not performed for removal of residual soluble proteins, or if the cells were re-suspended in a buffer containing human serum or serum proteins, then pre-washing is necessary.
Sample analysis:
White blood cells (WBCs) are analyzed on an FC 500 flow cytometer. Data are analyzed using a sequential gating strategy. The total WBC gate is defined in the first histogram (Side Scatter vs. CD45-PC7) by the inclusion of all CD45+ events with low, medium and high Side Scatter (SSC). A second histogram defines specific leukocyte subsets such as lymphocytes, monocytes, and granulocytes. The low SSC/bright CD45+ population identifies lymphocytes. Applicable markers are then displayed in subsequent histograms gated on lymphocytes. This process is repeated for mid SSC/medium CD45+ populations to identify monocytes and for high SSC/medium CD45+ to identify granulocytes.
L. Interpretation of Results
Flow cytometric immunophenotyping is multiparametric and relies on simultaneous identification and characterization of both normal and candidate malignant cell populations. Normal cells, which vary in type and relative numbers depending on the sample type, patient age, and clinical setting, display highly conserved and reproducible patterns of cell surface marker expression and light scatter. These normal internal control patterns may serve as both process controls for sample type. sample degradation, sample preparation and staining, data acquisition, and data analysis as well as references against which candidate malignant populations may be compared. Candidate malignant populations may display aberrant loss of a marker, aberrant gain of a marker, or aberrant over- or under-expression of a marker. Any population that does not conform to the normal internal control pattern is evaluated for
6
malignancy. Interpretation of these patterns requires expert knowledge and training in the technique. It is not possible to predict the phenotype of a particular patient's hematolymphoid malignancy and apply fixed templates for samples for detection of specific phenotypes. Even well-characterized diseases such as chronic lymphocytic leukemia may display case-to-case immunophenotypic variability and it is therefore the role of the expert interpreter to assess the significance of findings and to interpret them together with clinical history, morphological assessment, and other ancillary techniques in order to arrive at a final diagnosis. The device labeling states that interpretation of specimens should be performed by a pathologist or equivalent professional who has the appropriate training. Finally, a small number of rare samples contain no or very few normal cells and require careful evaluation and correlation with other sources of information such as clinical history and other laboratory findings to obtain the appropriate final diagnosis.
M. Performance Characteristics:
All results provided below met the manufacturer's pre-specified acceptance criteria.
-
- Analytical Performance:
- Precision and Reproducibility: Precision studies were conducted according to the a. methodology presented in CLSI EP05-A3, Evaluation of Precision Performance of Quantitative Measurement Procedures; Approved Guideline – Third Edition.
- Precision (repeatability): Thirty-eight specimens across the three specimen types were i. evaluated. For Whole Blood (WB) and Bone Marrow (BM), testing was conducted at three sites, with each of the three sites using a different anticoagulant according to their current clinical testing procedures. For lymph node specimens (LN), testing was conducted at two sites. Since LN specimens are not collected using an anticoagulant, this was not a variable for lymph node specimen analysis.
A minimum of one specimen for each specimen type and lineage were collected at each site. Lineage is defined by the tubes used, where T lineage is comprised of T1 and T2 tubes, B lineage is comprised of B1 and B2 tubes, and M lineage is comprised of Myeloid tube. Specimens were selected to cover the markers in each lineage tube regardless of the disease. The same specimen was analyzed twice each day with one time point in the morning and one time point in the afternoon. Six sample preparations per time point were made for a total of 12 replicates per specimen per lineage tube.
A qualitative approach was applied to repeatability assessment where the presence or absence of an abnormal phenotype was reported. An analysis of the % abnormal population, if present, or pre-selected lineage-specific normal populations was assessed for repeatability and reproducibility. The specimens were tested in 12 replicates. The expected result was to obtain the same number of positive or negatives for all replicates. The total actual number positive or negative was divided by the total expected number positive or negative to calculate the % positive or % negative results. The qualitative results for both abnormal and normal populations met 100% agreement for all sites and anticoagulants for expected and actual results with respect to the presence or absence of an abnormal phenotype.
7
| ClearLLab Lineage Tube | Cells | Markers | Population
of cells
Identified |
|----------------------------------|-----------------------|-----------------------------------------------------------------|--------------------------------------|
| T1 CD2/CD56/CD7/CD5/CD45 | T | CD45+ Bright, Low SS
CD2+CD5+CD7+ (Partial)
CD56- (Few +) | N#1 |
| | NK | CD45+ Bright, Low SS
CD2+CD7+CD56+CD5- | N#2 |
| T2 CD8/CD4/CD3/CD45 | T Helper | CD45+ Bright, Low SS
CD3+CD4+CD8- | N#3 |
| | T
Suppressor | CD45+ Bright, Low SS
CD3+CD4-CD8+ | N#4 |
| B1
KAPPA/LAMBDA/CD19/CD5/CD45 | B | CD45+ Bright, Low SS
CD19+ CD5-Kappa+ and
Lambda+ | N#5 |
| B2 CD20/CD10/CD19/CD38/CD45 | B | CD45+ Bright, Low SS
CD19+CD20+ CD38
Dim/Variable CD10- | N#6 |
| | Mature
Neutrophils | CD45+ Dim, High SS
CD10+ | N#7 |
| | Mature
Neutrophils | CD45+ Dim, High SS
CD7-CD13+CD34-CD33+ | N#8 |
| M CD7/CD13/CD34/CD33/CD45 | Monocytes | CD45+ Med, Med SS
CD7-CD13+ CD34-CD33+
Bright | N#9 |
Pre-selected Lineage-Specific Normal Population
| Total Samples | 38 | ||
|---|---|---|---|
| Gender | Male | 22 | |
| Female | 16 | ||
| Age | Child | N | 1 |
| (2-12) | Mean | 3 | |
| Adolescent | N | 0 | |
| (12-21) | Mean | 0 | |
| Adult | N | 19 | |
| (21-65) | Mean | 53 | |
| Senior | N | 18 | |
| (66-89) | Mean | 77 |
8
| Specimen Type | |||
|---|---|---|---|
| Site | Anticoagulant | WB | BM |
| Site 1 | ACD | 6 | 6 |
| Site 2 | K2EDTA | 4 | 5 |
| Site 3 | Heparin | 6 | 7 |
| Total Samples | 16 | 18 |
Repeatability for Site 1 using the ClearLLab Immunophenotyping Panel
| Site 1 | Population
of Cells
Identified | Specimen
Type | N | Lineage
Tube | Mean
(%) | Repeatability | | Between Runs | | Within
Specimen | |
|--------|--------------------------------------|------------------|----|-----------------|-------------|---------------|------|--------------|-------|--------------------|-------|
| Sample | | | | | | SD | %CV | SD | %CV | SD | %CV |
| 7 | ABN#1 | LN | 12 | B1 | 63.40 | 1.30 | 2.04 | 0.78 | 1.23 | 1.51 | 2.39 |
| 7 | ABN#2 | LN | 12 | B1 | 14.99 | 1.13 | 7.55 | 0.38 | 2.56 | 1.20 | 7.98 |
| 10 | N#5 | WB | 12 | B1 | 2.03 | 0.14 | 6.74 | 0 | 0 | 0.14 | 6.74 |
| 10 | ABN | WB | 12 | B1 | 61.96 | 1.32 | 2.13 | 0 | 0 | 1.32 | 2.13 |
| 13 | ABN#1 | LN | 12 | B1 | 69.32 | 1.25 | 1.80 | 1.96 | 2.83 | 2.33 | 3.36 |
| 13 | ABN#2 | LN | 12 | B2 | 85.97 | 0.70 | 0.81 | 1.82 | 2.1 | 1.95 | 2.26 |
| 14 | N#6 | BM | 12 | B2 | 7.56 | 0.45 | 5.92 | 0.22 | 2.92 | 0.50 | 6.60 |
| 14 | N#7 | BM | 12 | B2 | 29.15 | 0.96 | 3.30 | 0.58 | 2.00 | 1.13 | 3.86 |
| 17 | ABN | WB | 12 | B1 | 87.49 | 0.53 | 0.60 | 0.86 | 0.98 | 1.01 | 1.15 |
| 18 | ABN | WB | 12 | B2 | 98.10 | 0.16 | 0.17 | 0.02 | 0.02 | 0.16 | 0.17 |
| 18 | ABN | WB | 12 | B1 | 86.24 | 0.66 | 0.77 | 0 | 0 | 0.66 | 0.77 |
| 19 | N#6 | BM | 12 | B2 | 83.02 | 0.46 | 0.55 | 0.33 | 0.39 | 0.56 | 0.68 |
| 19 | N#7 | BM | 12 | B2 | 19.56 | 0.85 | 4.32 | 1.08 | 5.51 | 1.37 | 7.01 |
| 11 | N#1 | WB | 12 | T1 | 71.28 | 0.65 | 0.91 | 0.23 | 0.32 | 0.69 | 0.97 |
| 11 | N#2 | | 12 | T1 | 11.17 | 0.51 | 4.59 | 0.74 | 6.64 | 0.90 | 8.07 |
| 11 | N#3 | | 12 | T2 | 35.72 | 0.44 | 1.23 | 0.09 | 0.24 | 0.45 | 1.25 |
| 11 | N#4 | | 12 | T2 | 32.75 | 0.61 | 1.85 | 0 | 0 | 0.61 | 1.85 |
| 12 | N#1 | BM | 12 | T1 | 76.44 | 0.83 | 1.09 | 2.74 | 3.58 | 2.86 | 3.74 |
| 12 | N#2 | | 12 | T1 | 8.84 | 0.70 | 7.90 | 0 | 0 | 0.70 | 7.90 |
| 12 | N#3 | | 12 | T2 | 41.61 | 1.24 | 2.99 | 1.17 | 2.82 | .71 | 4.11 |
| 12 | N#4 | | 12 | T2 | 38.51 | 1.31 | 3.39 | 1.89 | 4.92 | 2.30 | 5.97 |
| 15 | N#1 | BM | 12 | T1 | 81.19 | 0.83 | 1.02 | 0 | 0 | 0.83 | 1.02 |
| 15 | N#2 | | 12 | T1 | 16.92 | 0.67 | 3.96 | 0 | 0 | 0.67 | 3.96 |
| 5 | ABN | WB | 12 | M | 84.26 | 1.01 | 1.20 | 2.82 | 3.35 | 3.00 | 3.56 |
| 8 | ABN | BM | 12 | M | 78.78 | 1.14 | 1.44 | 2.36 | 3.00 | 2.62 | 3.33 |
| 9 | ABN | BM | 12 | M | 7.06 | 0.41 | 5.81 | 0.71 | 10.10 | 0.82 | 11.65 |
| 16 | N#8 | WB | 12 | M | 47.31 | 1.98 | 4.18 | 1.65 | 3.49 | 2.58 | 5.45 |
| 16 | N#9 | | 12 | M | 3.69 | 0.32 | 8.54 | 0.25 | 6.86 | 0.40 | 10.95 |
Repeatability for Site 2 using the ClearLLab Immunophenotyping Panel
| Site 2 | Population
of Cells | Specimen
Type | N | Lineage
Tube | Mean
(%) | Repeatability | | Between Runs | | Within
Specimen | |
|--------|------------------------|------------------|----|-----------------|-------------|---------------|------|--------------|------|--------------------|------|
| Sample | Identified | | | | | SD | %CV | SD | %CV | SD | %CV |
| 7 | N#5 | LN | 12 | B1 | 25.04 | 0.71 | 2.84 | 0.63 | 2.53 | 0.95 | 3.80 |
| | N#6 | | 12 | B2 | 25.99 | 0.50 | 1.92 | 0.91 | 3.49 | 1.04 | 3.99 |
| | N#7 | | 12 | B2 | 17.14 | 1.40 | 8.18 | 0.60 | 3.47 | 1.52 | 8.89 |
| 9 | N#5 | BM | 12 | B1 | 22.40 | 0.72 | 3.21 | 0.19 | 0.83 | 0.74 | 3.31 |
| | N#6 | | 12 | B2 | 4.41 | 0.27 | 6.14 | 0.00 | 0.00 | 0.27 | 6.14 |
| | N#7 | | 12 | B2 | 28.04 | 0.28 | 1.01 | 0.29 | 1.03 | 0.41 | 1.45 |
| 11 | N#5 | WB | 12 | B1 | 17.63 | 0.27 | 1.51 | 0.10 | 0.56 | 0.28 | 1.61 |
| | N#6 | | 12 | B2 | 18.37 | 0.32 | 1.72 | 0.19 | 1.02 | 0.37 | 2.00 |
| | N#7 | | 12 | B2 | 73.47 | 0.69 | 0.93 | 0.30 | 0.41 | 0.75 | 1.02 |
9
| Site 2
Sample | Population
of Cells
Identified | Specimen
Type | N | Lineage
Tube | Mean
(%) | Repeatability | | Between Runs | | Within
Specimen | |
|------------------|--------------------------------------|------------------|----|-----------------|-------------|---------------|------|--------------|------|--------------------|------|
| | | | | | | SD | %CV | SD | %CV | SD | %CV |
| 12 | N#5 | LN | 12 | B1 | 25.91 | 0.49 | 1.90 | 0.76 | 2.95 | 0.91 | 3.51 |
| 12 | N#6 | | 12 | B2 | 26.36 | 0.40 | 1.50 | 0.60 | 2.28 | 0.72 | 2.73 |
| 12 | N#7 | | 12 | B2 | 5.05 | 0.38 | 7.61 | 0 | 0 | 0.38 | 7.61 |
| 1 | N#1 | BM | 12 | T1 | 70.34 | 0.80 | 1.14 | 0.50 | 0.71 | 0.94 | 1.34 |
| 1 | N#2 | | 12 | T1 | 9.83 | 0.33 | 3.38 | 0.55 | 5.60 | 0.64 | 6.54 |
| 1 | N#3 | | 12 | T2 | 33.86 | 0.51 | 1.51 | 0.60 | 1.77 | 0.79 | 2.33 |
| 1 | N#4 | | 12 | T2 | 37.25 | 0.86 | 2.30 | 0 | 0 | 0.86 | 2.30 |
| 4 | N#1 | BM | 12 | T1 | 70.37 | 0.73 | 1.03 | 0.48 | 0.68 | 0.87 | 1.24 |
| 4 | N#2 | | 12 | T1 | 8.66 | 0.57 | 6.59 | 0.62 | 7.15 | 0.84 | 9.72 |
| 4 | N#3 | | 12 | T2 | 46.81 | 0.51 | 1.10 | 0.61 | 1.30 | 0.80 | 1.70 |
| 4 | N#4 | | 12 | T2 | 27.61 | 0.75 | 2.73 | 0 | 0 | 0.75 | 2.73 |
| 10 | ABN | WB | 12 | T1 | 7.38 | 0.16 | 2.12 | 0 | 0 | 0.16 | 2.12 |
| 3 | ABN | BM | 12 | M | 58.53 | 0.40 | 0.69 | 0.17 | 0.30 | 0.44 | 0.75 |
| 5 | ABN | WB | 12 | M | 3.04 | 0.15 | 4.78 | 0.13 | 4.34 | 0.20 | 6.46 |
| 6 | ABN | WB | 12 | M | 62.27 | 0.48 | 0.77 | 0.09 | 0.15 | 0.49 | 0.79 |
| 8 | N#8 | BM | 12 | M | 70.44 | 0.47 | 0.66 | 0.37 | 0.53 | 0.59 | 0.84 |
| 8 | N#9 | | 12 | M | 6.86 | 0.16 | 2.27 | 0.09 | 1.28 | 0.18 | 2.61 |
Repeatability for Site 3 using the ClearLLab Immunophenotyping Panel
| Site 3 | Population
of Cells
Identified | Specimen
Type | N | Lineage
Tube | Mean
(%) | Repeatability | | Between Runs | | Within
Specimen | |
|--------|--------------------------------------|------------------|----|-----------------|-------------|---------------|------|--------------|------|--------------------|------|
| Sample | | | | | | SD | %CV | SD | %CV | SD | %CV |
| 1 | ABN | BM | 12 | B1 | 33.95 | 0.93 | 2.73 | 1.68 | 4.94 | 1.92 | 5.65 |
| 1 | N#6 | BM | 12 | B2 | 98.82 | 0.11 | 0.11 | 0.10 | 0.11 | 0.15 | 0.15 |
| | N#7 | | 12 | B2 | 28.17 | 0.68 | 2.43 | 1.02 | 3.63 | 1.23 | 4.37 |
| 6 | ABN | WB | 12 | B1 | 97.16 | 0.10 | 0.11 | 0.04 | 0.04 | 0.11 | 0.12 |
| 6 | N#6 | WB | 12 | B2 | 99.89 | 0.02 | 0.02 | 0.01 | 0.01 | 0.02 | 0.02 |
| | N#7 | | 12 | B2 | 4.18 | 0.12 | 2.85 | 0 | 0 | 0.12 | 2.85 |
| 11 | N#5 | BM | 12 | B1 | 18.40 | 0.50 | 2.70 | 0.37 | 2.03 | 0.62 | 3.37 |
| 11 | N#6 | BM | 12 | B2 | 78.88 | 1.82 | 2.31 | 4.26 | 5.40 | 4.63 | 5.87 |
| | N#7 | | 12 | B2 | 46.24 | 0.61 | 1.31 | 0.09 | 0.20 | 0.61 | 1.33 |
| 12 | ABN | BM | 12 | B1 | 53.80 | 0.66 | 1.23 | 0.41 | 0.77 | 0.78 | 1.45 |
| 12 | N#6 | BM | 12 | B2 | 91.62 | 0.53 | 0.58 | 0 | 0 | 0.53 | 0.58 |
| | N#7 | | 12 | B2 | 15.42 | 0.37 | 2.43 | 0.18 | 1.16 | 0.42 | 2.69 |
| 15 | N#5 | WB | 12 | B1 | 10.83 | 0.39 | 3.59 | 0 | 0 | 0.39 | 3.59 |
| 15 | N#6 | WB | 12 | B2 | 99.20 | 0.21 | 0.21 | 0.12 | 0.12 | 0.24 | 0.24 |
| | N#7 | | 12 | B2 | 35.81 | 1.28 | 3.58 | 1.80 | 5.02 | 2.21 | 6.17 |
| 2 | ABN | WB | 12 | T1 | 74.75 | 1.46 | 1.95 | 1.61 | 2.16 | 2.18 | 2.91 |
| 3 | N#1 | BM | 12 | T1 | 86.56 | 0.32 | 0.37 | 0 | 0 | 0.32 | 0.37 |
| 3 | N#2 | BM | 12 | T1 | 1.45 | 0.09 | 6.40 | 0 | 0 | 0.09 | 6.40 |
| | N#3 | BM | 12 | T2 | 60.52 | 0.42 | 0.69 | 0 | 0 | 0.42 | 0.69 |
| | N#4 | | 12 | T2 | 19.56 | 0.28 | 1.41 | 0 | 0 | 0.28 | 1.41 |
| | N#1 | | 12 | T1 | 67.56 | 1.49 | 2.20 | 0 | 0 | 1.49 | 2.20 |
| 8 | N#2 | WB | 12 | T1 | 10.00 | 0.17 | 1.69 | 0.13 | 1.30 | 0.21 | 2.13 |
| 8 | N#3 | WB | 12 | T2 | 41.41 | 0.67 | 1.63 | 0.18 | 0.44 | 0.70 | 1.68 |
| | N#4 | | 12 | T2 | 20.77 | 0.49 | 2.38 | 0 | 0 | 0.49 | 2.38 |
| | N#1 | | 12 | T1 | 69.73 | 1.27 | 1.83 | 0.36 | 0.51 | 1.32 | 1.90 |
| | N#2 | | 12 | T1 | 14.46 | 0.54 | 3.74 | 0.49 | 3.39 | 0.73 | 5.05 |
| 9 | N#3 | BM | 12 | T2 | 43.97 | 1.01 | 2.30 | 0.12 | 0.28 | 1.02 | 2.31 |
| 9 | N#4 | BM | 12 | T2 | 28.49 | 0.97 | 3.41 | 0 | 0 | 0.97 | 3.41 |
| | 4 | ABN | WB | 12 | M | 60.71 | 1.36 | 2.24 | 0 | 0 | 1.36 |
| 5 | N#8 | BM | 12 | M | 74.08 | 0.95 | 1.28 | 1.14 | 1.54 | 1.48 | 2.00 |
| 5 | N#9 | BM | 12 | M | 7.98 | 0.17 | 2.10 | 0.11 | 1.38 | 0.20 | 2.52 |
| | 7 | ABN | BM | 12 | M | 34.34 | 0.48 | 1.40 | 2.17 | 6.32 | 2.22 |
10
| Site 3 | Population
of Cells | Specimen | N | Lineage
Tube | Mean
(%) | Repeatability | | Between Runs | | Within
Specimen | |
|--------|------------------------|----------|----|-----------------|-------------|---------------|------|--------------|-----|--------------------|------|
| Sample | Identified | Type | | | | SD | %CV | SD | %CV | SD | %CV |
| 10 | ABN | WB | 12 | M | 42.42 | 1.30 | 3.06 | 0 | 0 | 1.30 | 3.06 |
Population of Cells
ABN: abnormal population identified in each sample; some samples have more than one abnormal population identified
N: normal population identified based on pre-selected lineage-specific populations (see Table above)
| | Positive
(abnormal phenotypes) | Negative
(normal phenotypes) | Total | % positive
(actual/expected) | % negative
(actual/expected) |
|--------|-----------------------------------|---------------------------------|-------|---------------------------------|---------------------------------|
| Site 1 | 11 | 17 | 28 | 100 | 100 |
| Site 2 | 4 | 22 | 26 | 100 | 100 |
| Site 3 | 7 | 26 | 33 | 100 | 100 |
- ii. Operator-to-operator and instrument-to-instrument imprecision: Seven whole blood specimens with or without abnormal phenotypes to cover all CD markers were evaluated. The same specimen was prepared and analyzed twice in one day by three operators. Each operator made three sample preparations per time point and analyzed the samples on two FC 500 flow cytometers that had successfully passed quality control for a total of 12 determinations per operator. Considering three operators, a total of 36 acquisitions per specimen per ClearLLab reagent tube were collected. A qualitative approach was applied to the assessment of imprecision where the presence or absence of an abnormal phenotype was reported. In all cases, the operators were blinded to the patient's diagnosis. The qualitative results for both abnormal and normal populations met 100% agreement for all operators and instruments for expected and actual results with respect to the presence or absence of an abnormal phenotype. The demographics of the seven samples included six men and one woman between the ages of 21-65 years old.
| Site 2 | Mean | Repeatability | Between Runs | Between Instruments | Between Operators | Within Specimen | |||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Sample | Lineage | N | (%) | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| 2 | B1 | 36 | 88.44 | 0.45 | 0.51 | 0 | 0 | 0 | 0 | 0.94 | 1.06 | 1.04 | 1.18 |
| 2 | B2 | 36 | 88.45 | 0.39 | 0.44 | 0.15 | 0.18 | 0 | 0 | 0.82 | 0.92 | 0.92 | 1.04 |
| 4 | B1 | 36 | 9.63 | 0.65 | 6.77 | 0.47 | 4.93 | 0 | 0 | 0.57 | 5.91 | 0.99 | 10.25 |
| 4 | B2 | 36 | 9.93 | 0.39 | 3.93 | 0 | 0 | 0.13 | 1.36 | 0 | 0 | 0.41 | 4.16 |
| 5 | B2 | 36 | 66.59 | 1.76 | 2.64 | 1.18 | 1.78 | 0 | 0 | 0 | 0 | 2.12 | 3.18 |
| 5 | B1 | 36 | 85.30 | 0.30 | 0.35 | 0.17 | 0.20 | 0 | 0 | 0.14 | 0.16 | 0.37 | 0.44 |
| 5 | B2 | 36 | 85.08 | 0.26 | 0.31 | 0.12 | 0.14 | 0.07 | 0.08 | 0 | 0 | 0.29 | 0.35 |
| 7 | B1 | 36 | 10.43 | 0.37 | 3.52 | 0 | 0 | 0 | 0 | 0 | 0 | 0.37 | 3.52 |
| 7 | B2 | 36 | 10.43 | 0.28 | 2.71 | 0.32 | 3.05 | 0 | 0 | 0 | 0 | 0.43 | 4.08 |
| 7 | B2 | 36 | 59.39 | 1.04 | 1.75 | 0.79 | 1.32 | 0 | 0 | 0.71 | 1.20 | 1.49 | 2.50 |
| 1 | T1 | 36 | 96.70 | 0.11 | 0.11 | 0 | 0 | 0 | 0 | 0.12 | 0.12 | 0.16 | 0.16 |
| 1 | T2 | 36 | 97.30 | 0.09 | 0.09 | 0 | 0 | 0.15 | 0.15 | 0 | 0 | 0.17 | 0.18 |
| 3 | T1 | 36 | 73.39 | 0.53 | 0.72 | 0.11 | 0.15 | 0 | 0 | 0.69 | 0.95 | 0.88 | 1.20 |
| 3 | T1 | 36 | 14.18 | 0.51 | 3.61 | 0 | 0 | 0.31 | 2.21 | 0 | 0 | 0.60 | 4.23 |
| 3 | T2 | 36 | 52.38 | 0.62 | 1.18 | 0.11 | 0.20 | 0.43 | 0.82 | 0 | 0 | 0.76 | 1.45 |
| 1 | T2 | 36 | 19.07 | 0.36 | 1.87 | 0 | 0 | 0.11 | 0.59 | 0.36 | 1.86 | 0.52 | 2.71 |
| 1 | M | 36 | 96.38 | 0.12 | 0.12 | 0.09 | 0.09 | 0 | 0 | 0.05 | 0.05 | 0.16 | 0.16 |
| 3 | M | 36 | 68.65 | 0.93 | 1.35 | 0.45 | 0.66 | 0 | 0 | 0.85 | 1.23 | 1.34 | 1.95 |
Results for ClearLLab for all Operators and Instruments Combined
11
| Site 2 | Lineage | N | Mean
(%) | Repeatability | | Between
Runs | | Between
Instruments | | Between
Operators | | Within
Specimen | |
|--------|---------|----|-------------|---------------|------|-----------------|------|------------------------|------|----------------------|------|--------------------|------|
| Sample | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| | M | 36 | 7.81 | 0.27 | 3.45 | 0 | 0 | 0.23 | 2.94 | 0.19 | 2.42 | 0.40 | 5.13 |
| 5 | M | 36 | 64.05 | 0.41 | 0.64 | 0.31 | 0.48 | 0.43 | 0.67 | 0 | 0 | 0.67 | 1.04 |
| | Positive
(abnormal phenotypes) | Negative
(normal phenotypes) | Total | % positive
(actual/expected) | % negative
(actual/expected) |
|-------------|-----------------------------------|---------------------------------|-------|---------------------------------|---------------------------------|
| Site 1 LHSC | 40 | 60 | 100 | 100 | 100 |
- iii. Site-to-Site Reproducibility with Control Material: One level of control material (IMMUNO-TROL Cells (IT)) was prepared in duplicate with the ClearLLab reagents and analyzed twice each day for 20 days at four sites with each site using one FC 500 flow cytometer, generating a total of 80 replicates per ClearLLab reagent tube per site for an overall total of 320 replicates per ClearLLab reagent lineage tube. Eight replicates were excluded due to QC failures. An additional nine replicates were excluded from the CD34 analysis in the Myeloid Lineage tube due to CD34 pipetting and washing error, resulting in a total exclusion of 17 replicates from the overall study. To assess the precision of percent positive parameter measured for CD34-ECD marker the experiments were performed using one level of control product (Immuno-Trol) with Stem-Trol Control Cells (a preserved cell line that expresses CD34, BK010044) added.
| Pop.
Lineage
** | | Marker | N | Mean
(%) | Repeatability | | Between
Runs | | Between
Days | | Between Sites | | Reproducibility | |
|-----------------------------|---------------|-----------------------------|-----|-------------|---------------|------|-----------------|------|-----------------|------|---------------|-------|-----------------|--------|
| | | | | | SD | %CV | SD | %CV | SD | %CV | SD | %CV | SD | %CV |
| B-cell
Lineage
Tube 1 | LY+ | CD19+
Kappa+
CD19+LY | 312 | 57.38 | 1.60 | 2.78 | 1.92 | 3.35 | 1.08 | 1.88 | 1.34 | 2.33 | 3.03 | 5.29 |
| | LY+ | CD19+
Lambda+
CD19+LY | 312 | 40.29 | 1.50 | 3.71 | 1.67 | 4.16 | 0 | 0 | 0.48 | 1.20 | 2.30 | 5.70 |
| B-cell
Lineage
Tube 2 | | CD19+ | 312 | 14.23 | 0.37 | 2.63 | 0.39 | 2.73 | 0 | 0 | 0.67 | 4.74 | 0.86 | 6.07 |
| | | CD5+ | 312 | 71.83 | 0.61 | 0.85 | 0.70 | 0.98 | 0.79 | 1.10 | 2.05 | 2.86 | 2.39 | 3.33 |
| | GR+ | CD10+ | 312 | 99.96 | 0.02 | 0.02 | 0.07 | 0.07 | 0.01 | 0.01 | 0.03 | 0.03 | 0.08 | 0.08 |
| | LY+ | CD19+ | 312 | 14.36 | 0.37 | 2.58 | 0.29 | 2.04 | 0.16 | 1.14 | 0.66 | 4.58 | 0.83 | 5.75 |
| | | CD20+ | 312 | 14.82 | 0.43 | 2.92 | 0.51 | 3.47 | 0.60 | 4.07 | 1.22 | 8.23 | 1.52 | 10.24 |
| | MO+ | CD38+ | 312 | 94.57 | 0.66 | 0.70 | 0.91 | 0.96 | 0.78 | 0.83 | 4.92 | 5.20 | 5.11 | 5.40 |
| T-cell
Lineage
Tube 1 | | CD2+ | 312 | 81.75 | 0.65 | 0.79 | 0.78 | 0.95 | 0.66 | 0.81 | 1.13 | 1.38 | 1.66 | 2.03 |
| | LY+ | CD5+ | 312 | 72.71 | 0.63 | 0.87 | 0.79 | 1.09 | 0.32 | 0.44 | 0.96 | 1.32 | 1.73 | 1.97 |
| | | CD56+ | 312 | 13.76 | 0.46 | 3.32 | 0.97 | 7.07 | 0 | 0 | 0.18 | 1.31 | 1.09 | 7.92 |
| | | CD7+ | 312 | 74.59 | 0.65 | 0.87 | 0.77 | 1.03 | 0.73 | 0.97 | 1.73 | 2.33 | 2.13 | 2.86 |
| T-cell
Lineage
Tube 2 | | CD3+CD4+ | 312 | 45.47 | 0.55 | 1.22 | 0.49 | 1.07 | 0.19 | 0.42 | 0.37 | 0.82 | 0.85 | 1.87 |
| | LY+ | CD3+CD8+ | 312 | 22.67 | 0.43 | 1.89 | 0.26 | 1.16 | 0.19 | 0.82 | 0.29 | 1.27 | 0.61 | 2.68 |
| | | CD3+ | 312 | 71.14 | 0.54 | 0.76 | 0.64 | 0.90 | 0.23 | 0.32 | 0.62 | 0.87 | 1.07 | 1.50 |
| Myeloid
Lineage | GR+ | CD13+ | 312 | 99.92 | 0.40 | 0.40 | 0 | 0 | 0.27 | 0.27 | 0.16 | 0.16 | 0.50 | 0.50 |
| | LY+ | CD7+ | 312 | 73.91 | 0.70 | 0.95 | 1.74 | 2.35 | 2.22 | 3.01 | 4.17 | 5.64 | 5.08 | 6.88 |
| | MO+ | CD33+ | 312 | 95.53 | 0.89 | 0.93 | 0.82 | 0.85 | 2.11 | 2.20 | 3.82 | 4.00 | 4.53 | 4.74 |
| | Stem
Trol+ | CD34+ | 303 | 11.77 | 0.78 | 6.65 | 0.97 | 8.23 | 0.55 | 4.70 | 1.59 | 13.53 | 2.09 | 17.80* |
Reproducibility with Control Material
** Population measured (Pop.)
LY+: Lymphocyte; GR+: Granulocyte; MO+: Monocyte; StemTrol: StemTrol cells
12
*Stemtrol Cells for CD34 measurements: The repeatability of the CD34 measurement included two additional unique variability sources. One of them was the variability contributed by the pipetting of the Stem-Trol reagent needed for staining. The other source was associated with an additional washing and decanting step. These additional sources of variability contribute to the increased imprecision.
- iv. ClearLLab Reagents Lot-to-Lot Reproducibility: Lot-to-lot variability was evaluated using normal whole blood specimens. For the CD34 marker, which is not expressed in normal specimens, a specific cell line (Stem-Trol) was spiked in the specimens. Three normal whole blood specimens were tested in triplicate with three lots of each ClearLLab reagent. Mean Fluorescence Intensity (MFI) differences of the conjugates and percent positive cell recovery for each cell lineage population were evaluated between lots and all demonstrated acceptable lot to lot variability performance.
| ClearLLab Reagents | Cells | Markers |
|---|---|---|
| T1 Lineage Tube | ||
| CD2/CD56/CD7/CD5/CD45 | NK | CD45bright CD2+CD7+ CD5-CD56+ |
| T2 Lineage Tube | ||
| CD8/CD4/-/CD3/CD45 | T | CD45bright CD2+CD7+ CD5+CD56- |
| T2 Lineage Tube | ||
| CD8/CD4/-/CD3/CD45 | T Helper | CD45bright CD3+CD4+ |
| T2 Lineage Tube | ||
| CD8/CD4/-/CD3/CD45 | T Suppressor | CD45bright CD3+CD8+ |
| B1 Lineage Tube | ||
| Kappa/Lambda/CD19/CD5/CD45 | B#1 | CD45bright CD19+CD5-Kappa+ |
| B1 Lineage Tube | ||
| Kappa/Lambda/CD19/CD5/CD45 | B#2 | CD45bright CD19+CD5-Lambda+ |
| B2 Lineage Tube | ||
| CD20/CD10/CD19/CD38/CD45 | B#3 | CD45bright CD19+CD20+ |
| CD38dim/variable CD10- | ||
| B2 Lineage Tube | ||
| CD20/CD10/CD19/CD38/CD45 | Mature Granulocytes | CD10+ |
| M Lineage Tube | ||
| CD7/CD13/CD34/CD33/CD45 | Mature Neutrophils | CD45+ Dim, High SS CD7-CD13+CD34- |
| CD33+ | ||
| M Lineage Tube | ||
| CD7/CD13/CD34/CD33/CD45 | Monocytes | CD45+ Med, Med SS CD7-CD13+ CD34- |
| CD33+ Bright |
Normal population analyzed in each Clear LLab reagent
Normal population % positive recovery on Sample 1
| Sample 1 | N | Mean | Repeatability | Between Lots | Within Sample | |||
|---|---|---|---|---|---|---|---|---|
| SD | %CV | SD | %CV | SD | %CV | |||
| NK | 9 | 2.58 | 0.13 | 5.16 | 0.00 | 0.00 | 0.13 | 5.16 |
| T | 9 | 95.79 | 0.11 | 0.12 | 0.00 | 0.00 | 0.11 | 0.12 |
| T Helper | 9 | 46.68 | 0.25 | 0.54 | 0.00 | 0.00 | 0.25 | 0.54 |
| T Suppressor | 9 | 41.14 | 0.36 | 0.88 | 0.00 | 0.00 | 0.36 | 0.88 |
| B #1 | 9 | 62.91 | 0.46 | 0.73 | 0.50 | 0.79 | 0.68 | 1.08 |
| B #2 | 9 | 37.09 | 0.47 | 1.28 | 0.46 | 1.23 | 0.66 | 1.77 |
| B #3 | 9 | 66.24 | 0.72 | 1.08 | 0.36 | 0.54 | 0.80 | 1.21 |
| Mature Granulocytes | 9 | 56.40 | 1.61 | 2.86 | 0.60 | 1.06 | 1.72 | 3.05 |
| Mature Neutrophils | 9 | 58.70 | 1.35 | 2.30 | 0.00 | 0.00 | 1.35 | 2.30 |
| Monocytes | 9 | 2.49 | 0.05 | 1.84 | 0.04 | 1.72 | 0.06 | 2.52 |
13
| Sample 2 | N | Mean | Repeatability | Between Lots | Within Sample | |||
|---|---|---|---|---|---|---|---|---|
| SD | %CV | SD | %CV | SD | %CV | |||
| NK | 9 | 8.58 | 0.30 | 3.45 | 0.26 | 3.00 | 0.39 | 4.57 |
| T | 9 | 88.26 | 0.29 | 0.33 | 0.00 | 0.00 | 0.29 | 0.33 |
| T Helper | 9 | 70.56 | 0.39 | 0.55 | 0.57 | 0.80 | 0.69 | 0.98 |
| T Suppressor | 9 | 23.59 | 0.48 | 2.02 | 0.51 | 2.18 | 0.70 | 2.97 |
| B #1 | 9 | 64.67 | 2.20 | 3.41 | 0.62 | 0.96 | 2.29 | 3.54 |
| B #2 | 9 | 35.36 | 2.27 | 6.43 | 0.65 | 1.82 | 2.36 | 6.68 |
| B #3 | 9 | 59.28 | 2.24 | 3.78 | 0.76 | 1.29 | 2.37 | 4.00 |
| Mature Granulocytes | 9 | 68.03 | 1.13 | 1.66 | 0.74 | 1.09 | 1.35 | 1.99 |
| Mature Neutrophils | 9 | 70.37 | 2.31 | 3.28 | 0.00 | 0.00 | 2.31 | 3.28 |
| Monocytes | 9 | 4.30 | 0.32 | 7.37 | 0.00 | 0.00 | 0.32 | 7.37 |
Normal population % positive recovery on Sample 2
Normal population % positive recovery on Sample 3
| Sample 3 | N | Mean | Repeatability | Between Lots | Within Sample | |||
|---|---|---|---|---|---|---|---|---|
| SD | %CV | SD | %CV | SD | %CV | |||
| NK | 9 | 13.30 | 0.26 | 1.93 | 0.23 | 1.76 | 0.35 | 2.61 |
| T | 9 | 80.68 | 0.31 | 0.38 | 0.00 | 0.00 | 0.31 | 0.38 |
| T Helper | 9 | 64.99 | 0.35 | 0.54 | 0.50 | 0.77 | 0.61 | 0.94 |
| T Suppressor | 9 | 29.63 | 0.28 | 0.96 | 0.37 | 1.26 | 0.47 | 1.59 |
| B #1 | 9 | 57.65 | 1.64 | 2.85 | 0.00 | 0.00 | 1.64 | 2.85 |
| B #2 | 9 | 42.20 | 1.68 | 3.98 | 0.00 | 0.00 | 1.68 | 3.98 |
| B #3 | 9 | 72.65 | 1.48 | 2.04 | 1.14 | 1.57 | 1.87 | 2.57 |
| Mature Granulocytes | 9 | 63.57 | 1.55 | 2.44 | 0.43 | 0.67 | 1.61 | 2.53 |
| Mature Neutrophils | 9 | 58.29 | 1.05 | 1.80 | 0.00 | 0.00 | 1.05 | 1.80 |
| Monocytes | 9 | 5.02 | 0.21 | 4.19 | 0.05 | 0.97 | 0.22 | 4.30 |
- v. Reagents Required as listed in Table 1 in Section I above: All tests performed for the reagents listed in Table 1 above for reproducibility and stability were acceptable.
- Linearity/Reportable Range: b.
Instrument Linearity : A study was performed for instrument linearity to evaluate the linearity and dynamic range of the FC500 flow cytometer's acquisition system, from the photomultiplier tubes (PMT) to the electronics, independent of the application and gating methodology. The FC 500 flow cytometer's acquisition system demonstrated linear results independent of the application and gating methodology.
- Detection Limit: The study was used to establish the detection capability, i.e., the ability ﻥ to differentiate between abnormal and normal populations, of the ClearLLab reagents.
- i. Limit of Blank (LoB):
The LoB was determined by testing five replicates of three normal specimens run with two lots of each ClearLLab reagent (B1, B2, T1, T2 and M). The highest LoB
14
observed from the two lots was 0.27% population of abnormal cells.
- ii. Limit of Detection (LoD):
The LoD was determined using one representative clinical specimen collected in K2 or K3 EDTA for each ClearLLab reagent. Each clinical specimen was spiked into a normal specimen to target low levels of abnormal population as a percentage of the total percentage of leukocytes in the sample. The percentages used were 2%, 1% and 0.5% of abnormal cells in the total population of white cells measured.
The Limit of Detection defined as 1% population of abnormal cells for the ClearLLab application is therefore confirmed.
- d. Analytical Specificity:
- i. Interfering Substances:
Not applicable. Adequate washing with phosphate buffered saline (PBS) before staining with reagents removes residual plasma and interfering substances.
- ii. Assay Carryover:
Carryover studies were conducted on three FC 500 flow cytometry systems according to the methodology presented in CLSI H26-A2: Validation, Verification, and Ouality Assurance of Automated Hematology analyzers; Approved Standard-Second Edition, Section 5.7 with regard to high or low value sample order. Two studies were conducted to evaluate specimen and reagent carryover.
Specimen Carryover
Three samples with high WBC counts (HTv1, HTv2, HTv3) were prepared from one clinical whole blood specimen containing a high level of WBCs. The samples were prepared to target 20,000 cells /uL WBCs. CD34 marker analysis was performed using a CD34+ clinical whole blood specimen.
Three samples with low WBC counts (LTv1, LTv2, LTv3) were prepared by spiking a diluted (1:10,000) whole blood specimen with a red blood cell pool which has been depleted of white blood cells and diluted 4:1 with Immuno-Trol storage buffer. This resulted in a contrived specimen that had a red blood cell concentration similar to whole blood with very low concentrations of white blood cells.
The reagent selected for specimen carryover was the ClearLLab M reagent. M reagent was chosen as a representative reagent since it contains all the different types of fluorochrome conjugates used in the device (FITC, PE, ECD, PC5.5 and PC7) as well as markers that allow identification and assessment of recovery of all leukocyte populations (lymphocytes, monocytes and granulocytes). Testing with M reagent also assesses the impact of lysing and fixative reagents on integrity of cell membranes for all relevant cell subsets and the affinity of various conjugated antibodies that is representative of all ClearLLab reagents.
Three HTv samples (HTv1, HTv2, HTv3) were analyzed consecutively followed immediately by the three LTv samples (LTv1, LTv2, LTv3). This was repeated three
15
times on three FC 500 flow cytometers for a total of nine specimen carryover runs. No significant carryover was detected as determined by percent carryover for specimen and reagent calculated from the analysis of the averages of the three runs on each flow cytometer for LTv1, LTv3, and HTv3. The specimen carryover is less than 1% for all markers.
Reagent Carryover
High concentration samples (HTv1, HTv2, HTv3): Normal whole blood specimens or clinical specimen (for CD34 measurement) stained with ClearLLab reagents were used as the high concentration samples.
Low concentration samples (LTv1, LTv2, LTv3): The same normal blood specimen or clinical whole blood as in the high concentration sample was processed in triplicate using antibody dilution buffer (RD1), in place of antibody reagent.
All the ClearLLab reagents (B1, B2, T1, T2 and M reagents) were selected for the reagent carryover studies. The potential for carryover of these products and the subsequent impact on performance were assessed for FS, SS, and FL1-FL5, in all seven of the detection channels.
The testing consisted of a high concentration sample analyzed consecutively three times (HTv1. HTv2. HTv3) followed immediately by testing the low concentration sample three times (LTv1, LTv2, LTv3). This run was repeated three times on three FC 500 flow cytometers for a total of nine reagent carryover runs.
The reagent carryover is less than 1% for all markers demonstrating acceptable scatter and fluorescent carryover performance.
- e. Traceability and Stability (Reagent and Specimen):
- i. Traceability:
For monoclonal antibodies. Leukocvte Typing Workshop information for each antibody clone was provided. Each clone identity is controlled and is traced back to the original source. Polyclonal antibodies for identification of Kappa or Lambda cell markers are supported by reference literature.
| # | Marker | Clone/Ig Chain
(species) | Leukocyte
Typing
International
Workshop* | Reactivity |
|----|--------|-----------------------------------|---------------------------------------------------|-------------------------------------------------------------------------------------------------------------------------|
| 1 | CD2 | 39C1.5 IgG2a (rat) | Second | T cells and most of the NK cells |
| 2 | CD3 | UCHT1 IgG1
(mouse) | First | Mature T cell (cytoplasmic
expression in immature T cells) |
| 3 | CD4 | SFCI12T4D11
IgG1 (mouse) | First | Helper / inducer T cells,
monocytes, immature myeloid |
| 4 | CD5 | BL1a IgG2a
(mouse) | Third | Thymocytes, mature T cells,
subpopulation of B cells |
| 5 | CD7 | 8H8.1 IgG2a
(mouse) | Second | T cells, NK cells, subpopulation
of immature myeloid cells |
| 6 | CD8 | SFCI21Thy2D3
(T8) IgG1 (mouse) | Second | Cytotoxic / suppressor T cells,
subpopulation of NK cells |
| 7 | CD10 | ALB1 IgG1
(mouse) | Third | Common acute leukemia antigen
(CALLA), lymphatic precursor
cells, neutrophils, subpopulation
of mature B cells |
| 8 | CD13 | SJ1D1 366 IgG1
(mouse) | Third | Mature and immature myeloid
cells |
| 9 | CD19 | J3-119 IgG1
(mouse) | Fourth | Precursor and mature B cells |
| 10 | CD20 | B9E9 (HRC20)
IgG2a (mouse) | Fifth | B cells and a subpopulation of B
precursor cells |
| 11 | CD33 | D3HL60.251 IgG1
(mouse) | Fifth | Monocytes, myeloid precursor
cells, weak on neutrophils |
| 12 | CD34 | 581 IgG1 (mouse) | Fifth | Myeloid and lymphoid precursor
cells |
| 13 | CD38 | LS198-4-3 IgG1
(mouse) | Fifth | Activated lymphocytes,
subpopulation of B cells,
plasma cells |
| 14 | CD45 | J.33 IgG1 (mouse) | Third | All leukocytes |
| 15 | CD56 | N901/NKH-1 IgG1
(mouse) | Second | NK cell subset and activated T-
cells |
| 16 | Kappa | N/A: Polyclonal | N/A | Subpopulation of immature B
Lymphocytes, subpopulation
of mature B cells |
| 17 | Lambda | N/A: Polyclonal | N/A | Subpopulation of immature B
Lymphocytes, subpopulation
of mature B cells |
16
17
| Workshop | CD2 assigned | Number of CDs assigned | |
|---|---|---|---|
| I First | Paris 19822 | CD1-CDw15 | 15 |
| II Second | Boston 19843 | CD16-CDw26 | 11 |
| III Third | Oxford 19874 | CD27-CD45 | 19 |
| IV Fourth | Vienna 19895 | CD46-CDw78 | 33 |
| V Fifth | Boston 19936 | CD79-CDw109 | 31 |
*Human Leukocyte Differentiation antigens Workshops
ii. Reagent Stability:
Testing of reagent stability was based on CLSI EP-25A (Evaluation of Stability of In vitro Diagnostic Reagents, Approved Guideline. Three lots of each ClearLLab reagent were evaluated for real-time open- and closed-vial stability. This study evaluated recovery and percent positive cells for each marker of the different lineage reagents (T1/T cell Lineage Markers (TL) 1, T2/TL2, B1/B cell Lineage markers (BL) 1, B2/BL2. M/Myeloid Lineage markers). Closed-vial stability testing was performed with stabilized control cells: Immuno-Trol and Immuno-Trol spiked with Stem-Trol to assess CD34 marker expression. Immuno-Trol Control Cells were used as a standardized assay system to enable traceability throughout the length of the study. Open-vial stability testing was performed with normal whole blood samples spiked with Stem-Trol cell line to assess the CD34+ cell population. Open-vial testing was performed at six time points with ClearLLab reagents. Percent difference from Time zero percent positive are measured for each marker of the different lineages tubes. The open- and closed-vial stability claims at refrigerated temperature (2-8℃) conditions are 90 and 365 days respectively.
iii. Whole Blood and Bone Marrow Specimen Stability:
A total of 45 clinical specimens consisting of 10 bone marrow specimens at each of three sites per anticoagulant (n = 30) and five whole blood specimens at each of three sites per anticoagulant (n = 15) were collected with different anticoagulants. Specimens provided from Site 1 were collected in ACD, from Site 2 were collected in Heparin, and from Site 3 were collected in K2EDTA. Each specimen was stained with the ClearLLab reagents for the B-cell Lineage Tube 1 and 2, T-cell Lineage Tube 1 and 2 and Myeloid Lineage reagent panels. Specimens were held at room temperature for the indicated time and prepared and analyzed at the following target time points post specimen collection: T0, T28, and T52 hours. The study examined the combined effect
2 Bernard AR, Boumsell L, Dausset J, et al. eds. Leucocyte Typing: Human Leucocyte Differentiation Antigens Detected by Monoclonal antibodies. Berlin: Springer-Verlag, 1984.
3 Reinherz EL, Haynes BF, Nadler L, Bernstein ID, eds. Leukocyte Typing II. New York: Springer-Verlag, 1985.
4 McMichael AJ, Beverley PCL, Cobbold S, et al. eds. Leucocyte Typing III. White Cell Differentiation Antigens. Oxford: Oxford University Press, 1987.
3 Knapp W, Dorken B, Gilks W et al., eds. Leucocyte Typing IV. Oxford University Press, 1989.
Schlossman SF, Boumsell L, Gilks W, et al. eds. Leucocyte Typing V: White cell differentiation antigens. Oxford: Oxford University Press, 1995.
18
of specimen and prepared sample stability drift of ClearLLab reagents' CD marker panel percent positive recoveries. The results at room temperature (RT, 20-25°C) are presented in the table below:
| Whole Blood | Bone Marrow | ||||
|---|---|---|---|---|---|
| Anticoagulant | Specimen | ||||
| stability | |||||
| C(RT) | Prepared | ||||
| Sample | |||||
| stability | Specimen | ||||
| stability | |||||
| C(RT) | Prepared | ||||
| Sample | |||||
| stability | |||||
| EDTA | 24 hours | 1 Swerdlow, S, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues 4 ** Edition. . Lyon: International Agency for Research on Cancer, 2008. |
20
The sample size was determined based on the approach recommended by Burderer. * For this study the assumption was prevalence =1 (design was not prevalence driven). The target outcome for all specimen types that were identified as positive by flow cytometry was 80%±10% agreement with the clinical diagnosis and for all specimen types that were identified as negative by flow cytometry was 90%±10%. For whole blood specimens only, the target outcomes for both positive and negative was 90%±10%.
A total of 279 abnormal hematologic specimens were enrolled: 137 hematologically abnormal but no malignancy and 142 with hematolymphoid malignancy per site's final diagnosis. The demographic information regarding the 279 subjects is shown in the graphic below.
Image /page/20/Figure/2 description: This image is a flowchart that shows the results of abnormal hematology by CBC/Diff. The total number of cases is 279. These cases are divided into two groups: no malignancy (n=137) and hematopoietic malignancy (n=142). Each of these groups is further divided by specimen mix, and the hematopoietic malignancy group is further divided into acute leukemia, chronic leukemia, lymphomas, plasma cell neoplasm, and others, each with n=23, 16%.
8 Statistical methodology. I. Incorporating the prevalence of disease into the sample size calculation for sensitivity and specificity. NM, Burderer. s.l. : Acad. Emerg. Med, 1996, Vols. 3(9): 895-900.
21
| Category | |||||||
|---|---|---|---|---|---|---|---|
| Sites | Abnormal | ||||||
| Hematology | |||||||
| -No | |||||||
| Malignancy | Acute | ||||||
| Leukemia | Chronic | ||||||
| Leukemia | Lymphoma | Plasma | |||||
| Cell | |||||||
| Neoplasm | Others: | ||||||
| MDS | |||||||
| MPN | Total | ||||||
| Number | |||||||
| of | |||||||
| Samples | |||||||
| per Site | |||||||
| Site 1 | 24 | 4 | 10 | 19 | 4 | 1 | |
| Site 2 | 29 | 6 | 3 | 11 | 3 | 2 | 54 |
| Site 3 | 42 | 6 | 15 | 12 | 0 | 6 | 81 |
| Site 4 | 42 | 7 | 8 | 20 | 1 | 4 | 82 |
| Total Number | |||||||
| per Category | 137 | 23 | 36 | 62 | 8 | 13 | 279 |
Number of Samples in Disease categories from each site
Statistical analysis results for sensitivity, specificity, PPV, and NPV for the two experts are presented below. The agreements between the two experts were also calculated.
Agreement for Presence (Malignant) or Absence (Non-Malignant) of Abnormal Cell Populations for All Specimen Types for Flow Expert #1
| ClearLLab Reagents
| Population | Clinical Outcome | ||
|---|---|---|---|
| Malignant | Non-Malignant | Total | |
| Malignant | 117 | 8 | 124 |
| Non-Malignant | 25 | 129 | 154 |
| Total | 142 | 137 | 279 |
| Agreement Statistics for All Specimen Types- Flow Expert #1 | |||
|---|---|---|---|
| Statistics | Estimate | Lower 95%CI | Upper 95%CI |
|---|---|---|---|
| Sensitivity | 82.4% | 75.3% | 87.8% |
| Specificity | 94.2% | 88.9% | 97.0% |
| PPV | 93.6% | 87.9% | 96.7% |
| NPV | 83.8% | 77.1% | 88.8% |
22
Agreement for Presence (Malignant) or Absence (Non-Malignant) of Abnormal Cell Populations for All Specimen Types for Flow Expert #2
| ClearLLab Reagents
| Population | Clinical Outcome | ||
|---|---|---|---|
| Malignant | Non-Malignant | Total | |
| Malignant | 122 | 10 | 132 |
| Non-Malignant | 20 | 127 | 147 |
| Total | 142 | 137 | 279 |
Agreement Statistics for All Specimen Types- Flow Expert #2
| Statistics | Estimate | Lower 95%CI | Upper 95%CI |
|---|---|---|---|
| Sensitivity | 85.9% | 79.2% | 90.7% |
| Specificity | 92.7% | 87.1% | 96.0% |
| PPV | 92.4% | 86.6% | 95.8% |
| NPV | 86.4% | 79.9% | 91.0% |
Agreement for Presence (Malignant) or Absence (Non-Malignant) of Abnormal Cell Populations for All Specimen Types for Combined Evaluations for Flow Experts #1 And #2
| ClearLLab Reagents
| Population | Clinical Outcome | ||
|---|---|---|---|
| Malignant | Non-Malignant | Total | |
| Malignant | 239 | 18 | 256 |
| Non-Malignant | 45 | 256 | 301 |
| Total | 284 | 274 | 558 |
Agreement Statistics for All Specimen Types- Combined Evaluations for Flow Experts #1 And #2
| Statistics | Estimate | Lower 95%CI | Upper 95%CI |
|---|---|---|---|
| Sensitivity | 84.2% | 79.5% | 87.9% |
| Specificity | 93.4% | 89.9% | 95.8% |
23
| Specimen Types | |||
|---|---|---|---|
| ClearLLab Reagents | Flow Expert #1 | ||
| Flow Expert #2 | Malignant | Non-Malignant | Total |
| Malignant | 123 | 9 | 132 |
| Non-Malignant | 2 | 145 | 147 |
| Total | 125 | 154 | 279 |
Agreement between Flow Experts for Presence (Malignant) or Absence (Non-Malignant) of Abnormal Cell Populations for All Specimen Types
Agreement between Flow Experts for All Specimen Types
| Agreement | Estimate | Lower 95%CI | Upper 95%CI |
|---|---|---|---|
| Positive | 98.4% | 94.4% | 99.6% |
| Negative | 94.2% | 89.3% | 96.9% |
There were a total of 33 discordant specimens by Flow Expert #1 of which 25 specimens were False Negative (FN) and eight were False Positive (FP). Flow Expert #2 had a total of 30 discordant specimens of which 20 were FN specimens and 10 were FP specimens. The sponsor discussed the discordant cases from both experts. The false negative results fell into specimen type limitation, flow cytometry limitation, or ClearLLab reagents limitation. These limitations occur when the phenotypically abnormal population is not found in a particular specimen, the clinical condition is not typically detected through flow cytometry, or the reagents did not have all the antibodies required to detect a particular clinical condition, respectively. For all false positive cases, one or both flow experts identified an abnormal phenotype. False positive cases were primarily due to increases in populations that could represent reactive responses to external stimuli such as viral, bacterial and cytokine stimulation where lymphoid proliferation is common.
An additional analysis was performed for the160 hematologically abnormal whole blood specimens consisting of 78 hematologically abnormal with no malignancy and 82 with hematolymphoid malignancy from all sites combined. Statistical analysis results on sensitivity, specificity, PPV, and NPV for the two experts are presented below.
Agreement for Presence (Malignant) or Absence (Non-Malignant) of Abnormal Cell Populations for Whole Blood Specimen Types for Flow Expert #1
| ClearLLab Reagents
| Population | Clinical Outcome | ||
|---|---|---|---|
| Malignant | Non-Malignant | Total | |
| Malignant | 73 | 7 | 80 |
| Non-Malignant | 9 | 71 | 80 |
| Total | 82 | 78 | 160 |
24
| Statistics | Estimate | Lower 95%CI | Upper 95%CI |
|---|---|---|---|
| Sensitivity | 89.0% | 80.4% | 94.1% |
| Specificity | 91.0% | 82.6% | 95.6% |
| PPV | 91.3% | 83.0% | 95.7% |
| NPV | 88.8% | 80.0% | 94.0% |
Agreement Statistics for Whole Blood Specimen Types- Flow Expert #1
Agreement for Presence (Malignant) or Absence (Non-Malignant) of Abnormal Cell Populations for Whole Blood Specimen Types for Flow Expert #2
| ClearLLab Reagents
| Population | Clinical Outcome | ||
|---|---|---|---|
| Malignant | Non-Malignant | Total | |
| Malignant | 76 | 8 | 84 |
| Non-Malignant | 6 | 70 | 76 |
| Total | 82 | 78 | 160 |
Agreement Statistics for Whole Blood Specimen Types for Flow Expert #2
| Statistics | Estimate | Lower 95%CI | Upper 95%CI |
|---|---|---|---|
| Sensitivity | 92.7% | 84.9% | 96.6% |
| Specificity | 89.7% | 81.0% | 94.7% |
| PPV | 90.5% | 82.3% | 95.1% |
| NPV | 92.1% | 83.8% | 96.3% |
Agreement between Flow Experts for Whole Blood Specimen Types
| Agreement | Estimate | Lower 95%CI | Upper 95%CI |
|---|---|---|---|
| Positive | 98.4% | 94.4% | 99.6% |
| Negative | 94.2% | 89.3% | 96.9% |
N. Instrument Name: Beckman Coulter FC500 flow cytometer
O. System Descriptions:
-
- Modes of Operation:
Flow cytometers combine fluidics, optics, and electronics subsystems to measure and analyze signals emitted when particles in a liguid stream flow through a glass cuvette at which beams of laser light are directed. The scatter and fluorescence light signals from these particles provide information about cell size, internal complexity, and relative fluorescence intensity. The Instructions for Use for Beckman Coulter FC500 flow cytometer instrument includes more details on the system components and theory of operations.
- Modes of Operation:
The previously cleared Instrument is indicated for use with cleared or approved in vitro diagnostic (IVD) assays for the identification and enumeration of human cell subsets that are indicated for use with the instrument.
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2. Software:
FDA has reviewed the applicant's Hazard Analysis and software development processes for this line of product types:
Yes X
-
- Calibration and Quality Controls:
See discussion of Traceability in section N.1.f.i above
- Calibration and Quality Controls:
P. Labeling:
The labeling is sufficient and it satisfies the requirements of 21 CFR Parts 801 and 809, as applicable, and the special controls for this device type.
Q. Patient Perspectives:
This submission did not include specific information on patient perspectives for this device.
R. Identified Risks to Health and Identified Mitigations:
| Identified Risks to Health | Identified Mitigations |
|---|---|
| Incorrect test results (false negatives or false | |
| positives) | General Controls and Special Controls (1) and |
| (2) | |
| Incorrect interpretation of device results by the | |
| end user | General Controls and Special Control (1), (2) |
| and (3) | |
| Patient harm from specimen(s) collection | General Controls and Special Control(1) |
S. Benefit/Risk Analysis:
| Summary | |
|---|---|
| Summary of the Benefit(s) | Flow cytometric immunophenotyping is an integral part of the |
| diagnosis for hematological malignancies. Phenotypically | |
| abnormal cell populations have been documented in many | |
| hematologic neoplasms, including lymphoma, chronic lymphoid | |
| leukemias, plasma cell neoplasms, acute leukemia, mast cell | |
| disease, myelodysplastic syndromes, and myeloproliferative | |
| disorders. The 2008 World Health Organization (WHO) | |
| Classification endorses a multiparametric approach to diagnosis | |
| and outlines the morphologic, immunophenotypic, and genotypic | |
| features characteristic of each disease entity. Immunophenotyping | |
| studies are used in routine diagnosis in the vast majority of | |
| hematologic malignancies both to determine lineage in malignancy | |
| and to distinguish benign from malignant processes. Flow | |
| cytometry may provide diagnostic information, with other ancillary | |
| testing to diagnose hematological neoplasms. | |
| The safety and effectiveness of the ClearLLab reagents was | |
| demonstrated in a multicenter clinical study consisting of 279 total | |
| patient specimens, 137 patients presenting for flow cytometry | |
| immunophenotyping which were found to be non-malignant and | |
| 142 patient specimens found to have hematologic malignancy. The | |
| patients with hematologic malignancy included 16% acute | |
| leukemia, 25% chronic leukemia, 44% lymphoma, 6% plasma cell | |
| neoplasm and 9% Others (includes MDS, MPN and eosinophilic | |
| hyperplasia). As shown in Comparison of ClearLLab Phenotypes | |
| vs. Diagnosis of Malignancy/Non-Malignancy, the clinical trial | |
| demonstrated the performance of this reagent panel to identify | |
| abnormal cell populations indicative of hematologic malignancy | |
| and to aid in demonstrating the absence of disease. The results of | |
| two independent flow cytometry experts demonstrated sensitivity | |
| of 82% (75-88%, 95% CI) for Flow Expert #1 and 86% (79–91%) | |
| for Flow Expert #2 and a specificity of 94% (89–97%) for Flow | |
| Expert #1 and 93% (87–96%) for Flow Expert #2. The PPV of | |
| ClearLLab reagents to identify malignancy was 94% (88–97%) and | |
| 92% (87-96%), respectively for each Flow Expert. Agreement | |
| between experts was 98% for positive specimens and 94% for | |
| negative specimens. | |
| Summary of Risk(s) | There is minimal potential risk associated with use of this device |
| given the combination of required general controls and special | |
| controls. The recognized risks for these assays are 1) possible | |
| erroneous results (false positive or false negative), 2) incorrect | |
| interpretation of device results by the pathologist or equivalent | |
| professional, and 3) related to patient harm from specimen(s) | |
| collection. | |
| Summary of Other Factors | With respect to risk of potential harm to the patient while |
| obtaining specimen(s), all flow cytometry studies are conducted | |
| ex-vivo and are independent of the assay configuration. The only | |
| intervention is blood drawing and/or obtaining tissues (bone | |
| marrow, lymph node) by well-established biopsy procedures. | |
| Conclusions | Yes, the probable benefits of this device outweigh the probable |
| Do the probable benefits | |
| outweigh the probable risks? | risks, given the combination of required general controls and |
| special controls established for this device. |
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T. Conclusion:
The information provided in this de novo submission is sufficient to classify this device into class II under regulation 21 CFR 864.7010. FDA believes that the special controls, along with the applicable general controls, including design controls, provide reasonable assurance of the safety and effectiveness of the device type. The device is classified under the following:
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| Product Code: | PWD |
|---|---|
| Device Type: | Flow Cytometric Test System for Hematopoietic Neoplasm |
| Class: | II (special controls) |
| Regulation: | 21 CFR 864.7010 |
-
a. Identification.
A flow cytometric test system for hematopeietic neoplasms is a device that consists of reagents for immunophenotyping of human cells in relation to the level of expression, antigen density, and distribution of specific cellular markers. These reagents are used as an aid in the differential diagnosis or monitoring of hematologically abnormal patients having or suspected of having hematopoietic neoplasms. The results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings. -
Classification. Class II (special controls). A flow cytometric test system for hematopoietic b. neoplasms must comply with the following special controls:
-
- Premarket notification submissions must include the following information:
- i. The indications for use must indicate the clinical hematopoetic neoplasms for which the assay was designed and validated, for example, chronic leukemia or lymphoma.
- ii. A detailed device description including the following:
- (A) A detailed description of all test components, all required reagents, and all instrumentation and equipment, including illustrations or photographs of nonstandard equipment or methods.
- (B) Detailed documentation of the device software including, but not limited to, standalone software applications and hardware-based devices that incorporate software.
- (C) A detailed description of methodology and assay procedure.
- (D) A description of appropriate internal and external quality control materials that are recommended or provided. The description must identify those control elements that are incorporated into the testing procedure, if applicable.
- (E) Detailed specifications for sample collection, processing, and storage.
- (F) Detailed specification of the criteria for test results interpretation and reporting including pre-established templates.
- (G) If applicable, based on the output of the results, a description of the specific number of events to collect, result outputs, and analytical sensitivity of the assay that will be reported.
- iii. Information that demonstrates the performance characteristics of the test, including:
- (A) Device performance data from either a method comparison study comparing the specific lymphocyte cell markers to a predicate device or data collected through a clinical study demonstrating clinical validity using well-characterized clinical specimens. Samples must be representative of the intended use population of the device including hematologic neoplasms and the specific sample types for which the test is indicated for use.
- (B) If applicable, device performance data from a clinical study demonstrating clinical validity for parameters not established in a predicate device of this generic type using well-characterized prospectively obtained clinical specimens including all hematologic neoplasms and the specific sample types for which the device is indicated for use.
-
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- (C)Device precision data using clinical samples to evaluate the within-lot, between-lot, within-run, between run, site-to-site and total variation using a minimum of three sites, of which at least two sites must be external sites. Results shall be reported as the standard deviation and percentage coefficient of variation for each level tested.
- (D) Reproducibility data generated using a minimum of three lots of reagents to evaluate mean fluorescence intensity and variability of the recovery of the different markers and/or cell populations.
- (E) Data from specimen and reagent carryover testing performed using well-established methods (e.g., CLSI H26-A2).
- (F) Specimen and prepared sample stability data established for each specimen matrix in the anticoagulant combinations and storage/use conditions that will be indicated.
- (G) A study testing anticoagulant equivalency in all claimed specimen type/anticoagulant combinations using clinical specimens that are representative of the intended use population of the device.
- (H) Analytic sensitivity data using a dilution panel created from clinical samples.
- (I) Analytical specificity data, including interference and cross-contamination.
- (J) Device stability data, including real-time stability of reagents under various storage times and temperatures.
- (K) For devices that include polyclonal antibodies, Fluorescence Minus One (FMO) studies to evaluate non-specific binding for all polyclonal antibodies. Each FMO tube is compared to reagent reference to demonstrate that no additional population appears when one marker is absent. Pre-specified acceptance criteria must be provided and followed.
- (L) For devices indicated for use as-a semi-quantitative test, linearity data using a dilution panel created from clinical samples.
- (M) For devices indicated for use as a semi-quantitative test, clinically relevant analytical sensitivity data, including limit of blank, limit of detection, and limit of quantification.
- iv. Identification of risk mitigation elements used by the device, including a detailed description of all additional procedures, methods, and practices incorporated into the instructions for use that mitigate risks associated with testing the device.
-
- The 21 CFR 809.10 compliant labeling must include the following:
- i. The intended use statement in the 21 CFR 809.10(a)(2) and 21 CFR 809.10(b)(2) compliant labeling must include a statement that the results should be interpreted by a pathologist or equivalent professional in conjunction with other clinical and laboratory findings. The intended use statement must also include information on what the device detects and measures whether the device is qualitative, semi-quantitative, and/or quantitative, the clinical indications for which the device is to be used, and the specific population(s) for which the device is intended.
- ii. A detailed description of the performance studies conducted to comply with paragraph (1)(iii) and a summary of the results.
-
- As part of the risk management activities performed under 21 CFR 820.30 design controls, product labeling and instruction manuals should include clear examples of all expected phenotypic patterns and gating strategies using well-defined clinical samples representative of both abnormal and normal cellular populations. These samples must be selected based upon the indications described in paragraph (1)(i) of this section.