淋巴细胞亚群分析--英文

3. Flow Cytometric Analysis of Lymphocyte Subsets (Immunophenotyping) Raquel Cabana rcabana@blueoceanbio.com Introduction The objective of immunophenotyping is to enumerate the antigenically defined three major lymphocyte populations of T lymphocytes (CD3+), B lymphocytes (CD19+) and Natural Killer lymphocytes (CD56+/CD3-). T lymphocytes can be subdivided into CD3+CD4+ T helper lymphocytes and CD3+CD8+ T cytotoxic lymphocytes. It is important to determine the relative and absolute numbers of these subsets to determine if a sample is abnormal (Clinical and Laboratory Standards Institute). Changes in lymphocyte subsets, such as T or B cell populations, can be indicative of immunological changes related to various diseases. The importance of monitoring the immune system became especially important in monitoring of human immunodeficiency virus (HIV) infection where alterations in peripheral blood CD4+ T cell content can be used for diagnosis and prognosis of HIV infection and the management of patients receiving antiretroviral therapy (Bergeron et al., 2002). Preparation of samples for phenotype analysis is dependent on the type of flow instrument available, the antibodies and the fluorescent conjugates selected and the use of lysing agents. Sample preparation can be performed manually by pipetting or automatically in a sample prep station when a large number of samples have to be processed and analyzed. Recently, Blue Ocean Biomedical, Inc. has announced availability of an integrated system which performs automatic sample preparation, data acquisition and analysis on a single platform. Methods for Cells Enumeration Historically, lymphocytes analysis was performed on a white blood cell buffy coat sample collected by density sedimentation. However, the need for accurate enumeration has replaced the density gradient method by the whole blood lysis procedure. The absolute cell concentration is defined as the number of cells of interest in a known volume of the sample using either the Single or the Dual Platform methods (Clinical and Laboratory Standards Institute, Bergeron et al., 2003). The Single Platform bead method is based upon the addition of a known number of fluorescent microspheres to a known volume of sample, either using a tube containing a known number of fluorescent microspheres to which sample is added or by the addition of a specific volume of a calibrated bead suspension to the sample tube. Fluorescent beads of a known concentration can be obtained from a number of different manufacturers. The use of this counting option has significantly reduced the variability of data between labs. In the Single Platform volumetric method, an accurate and reproducible volume of stained sample is passed through the flow cell, and the number of cells analyzed can be directly related to the known volume of the sample analyzed. The sample volume can be measured by using either precision syringes or sensing electrodes in the flow analyzer. The pipetting steps in volumetric systems must be performed with great precision. The final dilution of the sample must also be calculated and taken into account. In the Dual Platform method, two instruments, a hematology analyzer and a flow cytometer are used to count the cells of interest as a percentage of a reference population (e.g. CD3+/CD4+ cells as a percentage of the total lymphocyte population). Then an absolute WBC count provided by a hematology analyzer is used to calculate the percentage of lymphocytes. A disadvantage of the dual platform method is that errors could be compounded by the use of two different instruments. General Specimen Information • Instrument QC - Instrument setup and performance qualification are instrument specific. CLSI recommends daily performance monitoring of fluorescence intensity, color compensation and verification of system performance using QC controls. For this purpose, one can use commercially available preserved cell preparations which express most of the phenotypic markers and can be processed along with the blood sample to be analyzed. • Specimen Collection - Samples must be collected in Vacutainer™ tubes containing either EDTA (lavender top) or sodium heparin (green top). If WBC counts and differential are obtained from the same specimen, the anticoagulant of choice is EDTA. Samples In sodium heparin are stable for up to 72 hrs while in EDTA they are stable up to 48 hrs. EDTA samples will become depleted of granulocytes after 24 hrs; however CD4 counts are still possible up to 120 hrs when using optimized lymph gating strategy. • Specimen Rejection - Blood samples with any of the following characteristics may not yield reliable data and thus should not be processed: 1. Clotted Samples 2. Samples collected in an improper tube 3. Partial blood draw 4. Insufficient number of viable cells (a minimum of 75% viability is recommended). 5. Samples exposed to temperature extremes 6. Samples Improperly labeled 7. Loss of specimen integrity (e.g., Lipemia) • Selection of Lysing Agent - After immunostaining of the cells, RBCs lysis is used to remove erythrocytes from the sample. Several lysing methods are available for whole blood lysis and the mechanism of action for red blood cell lysis depends on the nature of the reagent. Ammonium chloride is the most popular lysing reagent which increases the osmotic pressure resulting in a rupture of the red cell membrane. Other lysing mechanisms include incubation of cells with formic acid or other acidic solutions followed by addition of a basic agent to neutralize pH or the use of diethylene glycol or detergents such as saponin (Bossuyt et al., 1997). • Immunostaining Panel - The ability to measure multiple cell surface markers is limited by the number of fluorochromes that can be simultaneously detected in a particular instrument. The fluorochromes selected are determined by excitation source and the wavelengths for excitation and the emission filters available. When building a marker panel, it is important to choose the brightest fluorochrome for the least expressed protein (e.g. PE for CD56) and the dimmest fluorochrome for the most expressed protein (e.g. FITC for CD45). It is also essential to choose fluorochromes with the least possible spectral overlap to minimize the need for color compensation. Tandem dyes should be used with caution as they may suffer from uncoupling and photo-bleaching. Color compensation should be performed as part of the instrument set up before sample analysis to avoid spectral overlap between the different fluorochromes. The optimal method for setting up compensation matrix is to use cells stained with a single color immunofluorescent marker (Cabana et al.) • Principles of the procedure - The fluorochrome-labeled antibodies are mixed with the sample where they bind to the cell surface antigens. The stained samples are then incubated with an erythrocyte lysing solution to remove the RBCs before analysis. If samples cannot be analyzed immediately, a fixative can be added to preserve the stained and lysed samples. • Reference Intervals - Reference intervals for immunophenotyping test results must be determined for each laboratory. Separate reference intervals must be established for adults and children (CLSI guideline C28). The following table summarizes the mean ± SD, median and 95% reference range of percentages and absolute values of lymphocyte subsets in 220 healthy adults (Yaman et al., 2005). Lymphocyte Subsets Mean ±SD Median 95% reference range CD3 % 72.70±8.44 73.20 52.30-84.64 cells/µL 1680±528 1637 725-2960 CD4 % 47.37±9.10 49.35 30.00-60.34 cells/µL 1096±391 1055 437-2072 CD8 % 28.99±5.99 29.65 17.76-39.94 cells/µL 669±239 630 307-1184 CD19 % 10.96±4.44 10.85 3.90-20.79 cells/µL 254±122 240 74-586 CD56 % 7.03±3.26 7.15 0.10-13.20 cells/µL 161±92 155 3.08-367 CD4/CD8 1.68±0.43 1.65 1.06-2.76 Lymphocyte Subset Analysis The following figures show a “Four-Color, Dual Anchor” gating strategy to identify the major lymph subsets (T, B and NK) while providing several internal quality controls. Tube 1: CD45- CD4-CD8-CD3. (Count helper/cytotoxic T cells) Tube 2: CF45-CD56- CD19-CD3. (Count total TBNK) In the examples, the Blue Ocean Biomedical LSA panels include the following antibodies: LSA1: CD45 FITC, CD4 PE, CD8 PE DL594, CD3 PE DL649. LSA2: CD45 FITC, CD56 CD16 PE, CD19 PE DL594M CD3 PE DL649. The advantages of this panel are: 1. lymphocytes are easily distinguished based on CD45 fluorescence and 90’ (side) scatter 2. Replicate CD3 determinations ensure reproducibility between tubes. Four Color; Two tube Lymphocyte Subset Enumeration Assay Figure 1. Whole Blood Stained with LSA1 CD45/CD4/CD8/CD3 and Analyzed on a Blue Ocean Biomedical CR150 flow cytometer. Figure 2. Whole Blood Stained with LSA2 CD45/CD56CD16/CD19/CD3 and Analyzed on a Blue Ocean Biomedical CR150 flow cytometer. Acronyms, Definitions, and Abbreviations: Ethlylene diaminetetraacetic acid EDTA Dual Platform DP Single Platform SP RBCs Red Blood Cells HIV Human Immunodeficiency Virus MABs Monoclonal Antibodies LSA Lymphocyte Subset Analysis WBCs White Blood Cells Hrs Hours CD Cluster of differentiation. FITC Fluorescein isothiocyanate PE Phycoerythrin QC Quality Control PE DL Phycoerythrin Dy Light CLSI Clinical and Laboratory Standards Institute References Bergeron M, Lustyik G, Ding T, Nicholson J, Janossy G, Shapiro H, et al. When do non volumetric flow cytomters become volumetric? Time can tell .how absolute your instrument is about absolute counts. Cytometry 52B:37-39 (2003). Bergeron M, Nicholson J, Phaneuf S,Ding T, Soucy N, Badley A, Hawley Foss C, Mandy F. Selection of Lymphocyte Gating Protocol Has an Impact on the Level of Reliability of T-Cell Subsets in Aging Specimens. Cytometry (Clinical Cytometry) 50:53– 61 (2002). Bossuyt X, Marti G, Fleisher T. Comparative Analysis of Whole Blood Lysis Methods for Flow Cytometry. Cytometry (Communications in Clinical Cytometry) 30:124–133 (1997). Cabana R, Cheetham M, Enten J, Song Y, Thomas M and Brendan SY. 3-Color Compensation. Beckman Coulter Application Information. A2045A. Clinical and Laboratory Standards Institute - Enumeration of Immunologically Defined Cell Populations by Flow Cytometry; Approved Guideline – Second Edition, document H42-A2. Yaman A, Cetiner S, Kibar F, Tasova Y, Seydaglu G, Dunbar I. Reference Ranges of Lymphocyte Subsets of Healthy Adults in Turkey. Med Princ Pract 14:189–193 (2005).