Fundamentals of Laser Technology In 1917, Einstein speculated that under certain conditions, atoms or molecules could absorb light or other radiation and then be stimulated to shed this gained energy. Lasers have been developed with numerous medical and industrial applications.

The electromagnetic spectrum ranges from long radio waves to short, powerful gamma rays (Fig. 1). Within this spectrum is a narrow band of visible or white light, composed of red, orange, yellow, green, blue, and violet light. Laser (light amplification by stimulated emission of radiation) light ranges from the ultraviolet (UV) and infrared (IR) spectrum through all the colors of the rainbow. In contrast to other diffuse forms of radiation, laser light is concentrated. It is almost exclusively of one wavelength or color, and its parallel waves travel in one direction. Through the use of fluorescent dyes, laser light can occur in numerous wavelengths. Types of lasers include glass-filled tubes of helium and neon (most common), yttrium-aluminum-garnet (YAG; an imitation diamond), argon, and krypton.

Fig1. The electromagnetic spectrum. YAG, Yttrium-aluminum-garnet. (From Turgeon ML: Clinical hematology: theory and procedures, ed 5, Philadelphia, 2012, Lippincott, Williams & Wilkins.)

Lasers sort the energy in atoms and molecules, concentrate it, and release it in powerful waves. In most lasers, a medium of gas, liquid, or crystal is energized by high-intensity light, an electrical discharge, or even nuclear radiation. When an atom extends beyond the orbits of its electrons or when a molecule vibrates or changes its shape, it instantly snaps back, shedding energy in the form of a photon. The photon is the basic unit of all radiation. When a photon reaches an atom of the medium, the energy exchange stimulates the emission of another photon in the same wavelength and direction. This process continues until a cascade of growing energy sweeps through the medium.

Photons travel the length of the laser and bounce off mirrors. First, a few and eventually countless photons synchronize themselves until an avalanche of light streaks between the mirrors. In some gas lasers, transparent disks referred to as Brewster windows are slanted at a precise angle, which polarizes the laser’s light. The photons, which are reflected back and forth, finally gain so much energy that they exit as a powerful beam. The power of lasers to transmit energy and information is rated in watts.

Principles of Cell Cytometry

Flow cell cytometry, developed in the 1960s, combines fluid dynamics, optics, laser science, high-speed computers, and fluorochrome-conjugated monoclonal antibodies (MAbs) that rapidly classify groups of cells in heterogeneous mixtures. The principle of flow cytometry is based on cells being stained in suspension with an appropriate fluorochrome—an immunologic reagent, a dye that stains a specific component, or some other marker with specific reactivity. Fluorescent dyes used in flow cytometry must bind or react specifically with the cellular component of interest (e.g., reticulocytes, peroxidase enzyme, DNA content). Fluorescent dyes include acridine orange and thioflavin T. Pygon is preferred for fluorescein isothiocyanate (FITC) labeling. Krypton is often used as a second laser in dual-analysis systems and serves as a better light source for compounds labeled by tetramethyl-rhodamine isothiocyanate and tetramethylcyclopropyl-rhodamine isothiocyanate.

A suspension of stained cells is pressurized using gas and transported through plastic tubing to a flow chamber within the instrument (Fig. 2). In the flow chamber, the specimen is injected through a needle into a stream of physiologic saline called the sheath. The sheath and specimen both exit the flow chamber through a 75-µm orifice. This laminar flow design confines the cells to the center of the saline sheath, with the cells moving in single file.

Fig2. Laser flow cytometry. (Courtesy Ortho Diagnostics,  Raritan, NJ.)

The stained cells then pass through the laser beam. The laser activates the dye and the cell fluoresces. Although the fluorescence is emitted throughout a 360-degree circle, it is usually collected by optical sensors located 90 degrees relative to the laser beam. The fluorescence information is then transmitted to a computer, which controls all decisions regarding data collection, analysis, and cell sorting.

Flow cytometry performs fluorescence analysis on single cells. The major applications of this technology are as follows:

 • Identification of cells

• Cell sorting before further analysis

Immunophenotyping

 Monoclonal antibodies, identified by a cluster designation (CD), are used in most flow cytometry immunophenotyping (Table 1). Cell surface molecules recognized by monoclonal antibodies are called antigens because antibodies can be produced against them or are called markers because they identify and discriminate between (mark) different cell populations. Markers can be grouped into several categories. Some are specific for cells of a particular lineage (e.g., CD4+ lymphocytes) or maturational pathway (e.g., CD34+ progenitor stem cells); the expression of others can vary, according to the state of activation or differentiation of the same cells.

Table1. Commonly Used Monoclonal Antibodies in Flow Cytometry

In flow cytometry, cells can be sorted from the main cellular population into subpopulations for further analysis (Fig. 3). Any fresh specimen that can be placed into a single-cell sus pension is a valid candidate for immunophenotyping (e.g., T cells, B cells, CD34+ stem cells; detection of minimal residual disease in leukemia). Sorting is accomplished using stored computer information.

Fig3. Laser and cell-sorting schematic.

When the laser strikes a stained cell, the dye creates distinctive colored light that the cytometer recognizes. This fluorescent intensity is recorded and analyzed by the computer and cells are sorted according to a preprogrammed selection. If the particular cell in the laser beam is of interest, the computer waits the appropriate time for the cell to reach the droplet break-off point within the charging collar. At that point, the computer signals the charging collar to administer an electro statically positive or negative charge to the stream containing the target cell. A droplet containing this cell is then removed from the main stream before the charge has time to redistribute.

This action produces the cell of interest within a liquid drop that has an electrostatic charge on its surface (only the droplet is charged). The droplet falls between a set of deflection plates, which creates an electrical field. The charged droplets are deflected to the left or right, depending on their polarity, and collected for further analysis.

Multicolor Immunofluorescence

 Current fluorescent methods (e.g., BD FACSCanto II flow cytometer; BD, Franklin Lakes, NJ) can perform up to eight color analysis. The BD LSRII flow cytometer, with up to four lasers, can measure up to 16 colors. It can use four MAbs, each directly conjugated to a distinct fluorochrome, per tube of patient cell suspension. The four most common fluorochromes are FITC, phycoerythrin (PE), peridinin chlorophyll protein (PerCP), and allophycocyanin (APC). The first three fluorochromes are excited by the 488-nm line of an argon laser; the fourth fluorochrome is excited by the 633-nm line of a helium neon or diode laser.

Eight-color immunofluorescence offers the advantages of greater sensitivity and specificity, with increased ability to identify and subclassify individual cells. Improvements in methods and probes may lead to fluorescence in situ hybridization (FISH) in suspension as a routine protocol and enable flow cytometry to operate on a molecular level simultaneously to identify chromosomal abnormalities.

A system that uses a flow cytometer, specific data analysis software, and fluorescent latex particles, the Luminex 100 Total System, has been developed by Luminex Technology (Austin, Texas). This system combines advances in computing and optics with a new concept in color coding to create a simple, cost-effective analysis system (Fig. 4). Latex beads are coupled to various amounts of two different fluorescent dyes, which are analyzed by the flow cytometer and software to allow the distinct separation of up to 64 slightly different colored bead sets. The color-coded microspheres identify each unique reaction. Hundreds of microsphere sets can be identified at once in a single sample. Optical technology recognizes each microsphere and provides a precise, quantitative measure sim ply and in real time.

Fig4. Fluorescent microsphere–based immunoassay for antibodies to hepatitis virus (Luminex xMAP technology). This approach is especially valuable when multiple tests must be done. It  uses aspects of enzyme-linked immunosorbent  assay (ELISA) and flow cytometry. A small amount of  sample is known. Polystyrene microspheres are  internally color-coded with two fluorescent dyes  that can be detected after laser illumination. (From Nairn R, Helbert M: Immunology for medical students, ed 2, St Louis, 2007, Mosby.)

Currently, up to 64 microsphere sets are recognized. The current FlowMetrix system is compatible with the BD FACS Vantage SE System and BD FACSCalibur, the most widely used flow cytometers for cellular analysis. Because Luminex technology requires fewer steps to assess multiple parameters, with a high level of sensitivity and accuracy, it is significantly more cost-effective than current methods of analysis. Some immunologic applications already demonstrated with Flow Metrix are human immunodeficiency virus (HIV) and hepatitis B seroconversion, multicytokine measurement, multiplexed allergy testing, DNA-based tissue typing, herpes simplex viral load, IgG, IgA, and IgM assays, IgG subclassification, autoimmunity panel, epitope mapping, human chorionic gonadotropin (hCG) and α-fetoprotein, HIV viral load, and the TORCHS (toxoplasmosis, other [viruses], rubella, cytomegalovirus, herpesviruses, syphilis) panel.

Sample Preparation

 Specimens that can be used for flow cell analysis include whole blood, bone marrow, and aspirates of body fluids. Whole blood, collected in ethylenediaminetetraacetic acid (EDTA), is the preferred anticoagulant if specimens are processed within 30 hours of collection. Heparin is an alternative anticoagulant for whole blood and bone marrow and can provide stability of specimens more than 24 hours old.

Blood specimens should be stored at room temperature (20° C to 25° C [68° F to 77° F]) before processing. Specimens need to be well mixed prior to delivery into staining tubes. Unsuitable specimens included hemolyzed or clotted samples. For the efficient analysis of white blood cells, whole blood, bone marrow, or aspirates should have the bulk of red blood cells removed prior to analysis. Tissue specimens (e.g., lymph nodes) should be collected and transported in a tissue culture medium at room temperature or at 4° C (39° F) if analysis is delayed. Such a specimen requires disaggregation by enzymatic or mechanical methods to form a single-cell suspension. After proper specimen processing, antibodies are added to the cellular preparation and analyzed. MAbs, tagged with different fluorescent tags, are used for analysis.

Clinical Immunology Applications

Lymphocyte Subsets A six-color flow cytometry diagnostic application uses the BD FACSCanto II flow cytometer and BD Multitest six-color TBNK with BD Trucount tubes to determine the absolute counts of mature T, B, and natural killer (NK) lymphocytes (Fig. 5), as well as CD4+ and CD8+ T cell subsets in human peripheral blood, in a single tube.

Fig5. Flow cell cytometry dot plots. Panel A, Cells stained with the red CD4 antibody account for 59% of all lymphocytes; this is a normal  sample. Panel B, There is a reduction in the number of red-staining CD4+ T cells; this sample is from a patient with HIV infection. FITC, Fluorescein  isothiocyanate (emits green light); PE, phycoerythrin (emits red light); (From Nairn R, Helbert M: Immunology for medical students, ed 2, St Louis, 2007, Mosby.)

Other Cellular Applications

Measuring T Cells for Acquired Immunodeficiency Syn drome Analysis. The quantitation of T and B cells using monoclonal surface markers can be performed using flow cytometry. With the flow cytometer, 10,000 cells can be assayed into subsets in 1 minute with multiparameter analysis. Using MAbs, T and B cell populations can be divided into subpopulations with specific functions. T cells are divided into two functional subpopulations, helper T (Th) cells and suppressor T (Ts) cells.

Normal individuals have a TH/TS ratio of 2:1 to 3:1. This ratio is inverted in certain disorders and diseases, including the acute phase of cytomegalovirus (CMV) mononucleosis, following bone marrow transplantation, and acquired immunodeficiency syndrome (AIDS).

CD4/CD8 Ratio. The CD4 (helper subset) T lymphocyte cell count is one of the standard measures for diagnosing AIDS and the management of disease progress in patients with HIV infection. The analysis of the T cell and B cell ratio is clinically useful in evaluating the immune system status of patients who may be at an increased risk of opportunistic infections. In addition, the absolute number of CD4+ lymphocytes is reflective of the degree of immunodeficiency in HIV-infected individuals and may be used as a guide for initiating antiretroviral therapy and monitoring therapy.

In these cases, two cell surface antigens—CD3, which is present on mature T lymphocytes, and CD4, which is only present on the helper subset of T lymphocytes—are used. The percentage of CD4 lymphocytes is determined by using a fluorochrome-conjugated CD3 antibody (e.g., FITC-CD3) together with a CD4 antibody conjugated to a second fluorochrome (e.g., PE-CD4). The absolute CD4 count can be determined. The absolute number of CD4 lymphocytes is reflective of the degree of immunodeficiency in HIV-infected patients and may be used as a guide for timing the administration of antiretroviral therapy and for monitoring the level of immune reconstitution following the initiation of therapy.

Basic Lymphocyte Screening Panel. A basic immune screening panel typically consists of the detection and quantitation of CD3, CD4, CD8, CD19, and CD16/56. Anti–CD45/CD14 is included to assist in distinguishing lymphocytes from monocytes. This panel reveals the frequency of T cells (CD3+), B cells (CD19+), and natural killer cells (CD3−, CD16+, CD56+). It also provides the frequency of Th inducer cells (CD3+, CD4+) and T suppressor or cytotoxic cells (CD3+, CD8+). Typical percentage ranges for lymphocyte subsets in adult donors are as follows: CD3, 56% to 86%; CD4, 33% to 58%; CD8, 13% to 39%; CD16+ CD56, 5% to 26%; and CD19, 5% to 22%.

However, this panel does not provide information on cell activation or signaling pathway receptors, frequency of T sub sets (e.g., Th1 or Th2), stem or blast cells, B lymphocytes (e.g., immunoblasts or plasma cells), or nonlymphoid elements. HLA-B27 Antigen. The automated BD FACSCanto, BD FACSCalibur, BD FACSort, and BD FACScan flow cytometers can rapidly detect HLA-B27 antigen expression in erythrocyte lysed whole blood (LWB) using a qualitative two-color direct immunofluorescence method. This technology compares the intensity of T lymphocytes stained with anti–HLA-B27 FITC to a predetermined decision marker during analysis. When anti HLA-B27 FITC/CD3 PE MAb reagent is added to human whole blood, the fluorochrome-labeled antibodies bind specifically to leukocyte surface antigens. The stained samples are treated with BD FACS lysing solution to lyse erythrocytes and are then washed and fixed before flow cytometric analysis.

This application of flow cytometry is clinically relevant to the evaluation of seronegative spondyloarthropathies.

اخر الاخبار

اخبار العتبة العباسية المقدسة

شعبة الحرم الشريف تكثف استعداداتها لتقديم خدمات متنوعة لزائري الأربعين

قسم الشؤون الفكرية يفتتح محطتين ثقافيتين في ذي قار لخدمة زائري الأربعين

العتبة العباسية المقدسة تعقد ملتقى خاصًا لمنتسبيها العاملين في مراكز إرشاد التائهين خلال زيارة الأربعين

زيارة الأربعين في ذروة الصيف.. العتبة العباسية المقدسة تواجه التحديات بخطة شاملة

المزيد

الآخبار الصحية

علامات فارقة بين النوبة أو السكتة القلبية.. ماهي؟

الفلفل الأحمر.. سعرات حرارية منخفضة وفوائد صحية عديدة

8 أعراض إذا شعرت بها.. اذهب للطوارئ فورا !

تعرف على أكثر 8 أماكن اتساخا في المنزل

المزيد

الآخبار التكنلوجية

إنتاج الهيدروجين الأخضر في البحر.. هذه أحدث الدراسات

"ناسا" تخطط لبناء مفاعل نووي على سطح القمر !

ابتكار ومواهب وأموال.. مارك زوكربيرغ يعلن الحرب على أبل

عودة التعاون الفضائي بين روسيا وأميركا.. وبحث ملفات "هامة"

المزيد

مواضيع ذات صلة

Future Directions of Molecular Diagnostic Testing

Next Generation Sequencing Technology

Analysis of Amplification Products

platelet aggregation test (Ristocetin test)

plasminogen activator inhibitor 1 (PAI-1)

phospholipase A2 receptor antibodies (anti-pla2r)

phosphate (PO4 , Phosphorus [P])

pheochromocytoma suppression and provocative testing (Clonidine suppression test [CST], Glucagon stimulation test)

parvovirus B19 antibody

Applications of Molecular Biology to Erythrocyte Immunohematology

Direct Antiglobulin Test (Direct Coombs Test)

Indirect Antiglobulin Test (Indirect Coombs Test)