L04 - Instrumentation In Hematology PDF

6/28/2024 INSTRUMENTATION IN HEMATOLOGY Chapter 30 Preamble ◦ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. ◦ PowerPoints DO NOT cover the details needed for the Unit exam ◦ Each student is responsible for...

6/28/2024 INSTRUMENTATION IN HEMATOLOGY Chapter 30 Preamble ◦ PowerPoints are a general overview and are provided to help students take notes over the video lecture ONLY. ◦ PowerPoints DO NOT cover the details needed for the Unit exam ◦ Each student is responsible for READING the TEXTBOOK for details to answer the UNIT OBJECTIVES ◦ Unit Objectives are your study guide (not this PowerPoint) ◦ Test questions cover the details of UNIT OBJECTIVES found only in your Textbook! 1  6/28/2024 Objectives 1. Describe the basic theory of the following principles of cell counting and sizing: 1) Electrical impedance and 2 ) Optical detection 2. Explain the fundamental concepts of laser technology 3. Describe the principles of flow-cell cytometry and two basic uses in hematology. 4. List the parameters measured and the methods used to measure these paraments in basic benchtop hematology analyzers 5. Describe the process and output of total cell and histogram electrical impedance systems 6. Describe the process and output of a laser scatter technology system. 7. Describe the appearance of microcytic and macrocytic rbc’s on a histogram 8. Define RDW, how it is calculated, the normal range and how it is relations to MCV 9. Describe the appearance of a leukocyte histogram by both electrical and optical detection methods. 10. Describe the construction of a platelet histogram. 11. Define MPV, it’s relationship with platelets and four disorders associated with it. 12. Describe the generation, by laser technology, of a histogram of: RBC, WBC and Platelets 13. Describe the general functions that flow cytometry can provide and the hematological applications. 14. Interpret histogram and scatter plot data Instrumental Principles ◦ Basic cell counting principles ◦ Fundamentals of laser technology ◦ Principles of flow cytometry 2  6/28/2024 Basic Cell Counting Principles ◦ Instrumentation and the automation of procedures continue to increase in the clinical hematology laboratory. ◦ The counting of the cellular elements of the blood (erythrocytes, leukocytes, and platelets) can be based on one of two classic methods: ◦ Electrical impedance ◦ Optical detection Electrical Impedance #1 ◦ Referred to as the Coulter principle. ◦ Cell counting and sizing are based on the detection and measurement of changes in electrical impedance (resistance) produced by a particle as it passes through a small aperture. ◦ The number of pulse generated during a specific period is proportional to the number of particles or cells, recorded by a scattergram. ◦ The amplitude (magnitude) of the electrical pulse produced indicates the cell’s volume. ◦ The output histogram is a display of the distribution of cell volume and frequency. 3  6/28/2024 Optical Detection Principle ◦ In the optical or hydrodynamic focusing method of cell counting and cell sizing, laser light is used. ◦ A diluted blood specimen passes in a steady stream through which a beam of laser light is focused. ◦ As each cell passes through the sensing zone of the flow cell, it scatters the focused light. ◦ Scattered light is detected by a photodetector and converted to an electrical pulse. ◦ The number of pulses generated is directly proportional to the number of cells passing through the sensing zone in a specific period. Characteristics of Light Scatter #1 ◦ Optical light scatter ◦ In this category, light amplification is generated by stimulated emission of radiation. Three independent processes are operational. These are as follows: 1. Diffraction and the bending of light around corners with the use of small angles 2. Refraction and the bending of light because of a change in speed with the use of intermediate angles 3. Reflection and light rays turned back by the surface or an obstruction with the use of large angles 4  6/28/2024 Characteristics of Light Scatter #2 ◦ Angles of light scatter: Various angles of light scatter can aid in cellular analysis. These are as follows: ◦ Forward light scatter 0°. This is diffracted light, which relates to the volume of the cell. ◦ Forward low-angle light scatter 2° to 3°. This characteristic can relate to size or volume. ◦ Forward high angle 5° to 15°. This type of measurement allows for description of the refractive index of cellular components. ◦ Orthogonal light scatter 90°. The result of this application of light scatter is the production of data based on reflection and refraction of internal components, which correlates with internal complexity. Radio Frequency (RF) ◦ In this newer application, high-voltage electromagnetic current is used to detect cell size, based on the cellular density. ◦ The RF pulse is directly proportional to the nuclear size and density of a cell. RF or conductivity is related to the nuclear:cytoplasmic ratio, nuclear density, and cytoplasmic granulation. 5  6/28/2024 Fundamentals of Laser Technology #1 ◦ The electromagnetic spectrum ranges from long radio waves to short, powerful gamma rays. ◦ Within this spectrum is a narrow band of visible or white light, which is composed of red, orange, yellow, green, blue, and violet light. ◦ Light amplified by stimulated emission of radiation (laser) light ranges from the ultraviolet and infrared spectrum through all the colors of the rainbow. Fundamentals of Laser Technology #2 ◦ LASER = Light Amplified by Stimulated Emission of Radiation ◦ Its emission ranges from ultraviolet to infrared through all the colors of the rainbow ◦ In contrast to other diffuse forms of radiation, laser light is: ◦ concentrated, ◦ almost exclusively of one wavelength or color, ◦ Its parallel waves travel in one direction. 6  6/28/2024 Principles of Flow Cytometry #1 ◦ Laser light is the most common light source used in flow cytometers because of the properties of intensity, stability, and monochromatism. ◦ Flow cytometry is defined as the simultaneous measurement of multiple physical characteristics of a single cell, as the cell flows in suspension through a measuring device. ◦ Flow cytometry combines the technologies of fluid dynamics, optics, lasers, computers, and fluorochrome-conjugated monoclonal antibodies that rapidly classify groups of cells with heterogeneous mixtures. ◦ The principle of flow cytometry is based on the fact that cells are stained in suspension. Flow cytometry has specifically come to denote the use of fluorescence measurement, usually with a laser light source. Principles of Flow Cytometry #2 7  6/28/2024 Principles of Flow Cytometry #3 ◦ The cellular light scatter patterns can be used to identify cells. ◦ Both intrinsic and extrinsic properties of cells can be analyzed by flow cytometry. ◦ Intrinsic properties include forward- and right-angle light scatter, which correlate with size and granularity of a cell, respectively. This data output does not require addition of dyes or stains for detection. ◦ By contrast, extrinsic properties rely on the binding of various probes to the cells. The scattered light passes through a variety of filters and lenses and is then measured by photomultiplier tubes, which convert the light signals into electronic signals for computer analysis. Principles of Flow Cytometry #4 ◦ Light scattered along the axis of the laser beam is “forward scatter.” ◦ Light scattered perpendicular to the axis is “side scatter” or “orthogonal scatter.” ◦ Forward scatter is roughly proportional to cell size; side scatter is roughly proportional to cytoplasmic granularity. ◦ Granulocytes have a much larger side-scattered light signal than do lymphocytes. ◦ Data are plotted on histograms. ◦ Populations of similar cells form discrete and characteristic two-dimensional “clusters” of scatter when the forward and side scatters are plotted against each other. 8  6/28/2024 Principles of Flow Cytometry #5 Hematology Analyzer Video 9  6/28/2024 Whole Blood Cell Analysis ◦ Degree of instrument sophistication is frequently described by the number of parameters that the instrument generates. ◦ Parameter is not the same as sample, which is a subset of a population measured by a statistic. ◦ Recent innovations of hematology instrumentation include: ◦ Quantitation of nRBC counts ◦ A channel for enumeration of immature granulocytes (IGs) ◦ Random access CD4 lymphocyte counting ◦ Analysis of CD34, CD38, and CD61 cell markers ◦ Measurement of reticulocyte hemoglobin ◦ Enumeration of hematopoietic progenitor cells (HPCs) ◦ Counting of IGs Types of Automated Cell Counting Instruments ◦ Major types of automation are representative of the ways that blood cells can be counted, leukocytes differentiated, and other components (e.g., MCH and MCHC) calculated. ◦ Hemoglobin is measured by the traditional cyanmethemoglobin flow-cell method at 525 and 546 nm, depending on the instrument manufacturer. 10  6/28/2024 General Histogram Characteristics ◦ Histograms are graphic representations of cell frequencies versus sizes. ◦ In a homogeneous cell population, the curve assumes a symmetrical bell- shaped or Gaussian distribution. A wide or more flattened curve is seen when the standard deviation from the mean is increased. ◦ Histograms not only provide information about erythrocyte, leukocyte, and platelet frequency and their distribution about the mean but also depict the presence of subpopulations. ◦ Histograms provide a means of comparing the sizes of a patient’s cells with normal populations. Shifts in one direction or the other can be of diagnostic importance. The position of the curve on the x-axis reflects cell size. In the Coulter system, the size (volume in femtoliters) is represented on the x-axis. Analysis of Instrumental Data Output ◦ The erythrocyte histogram ◦ Quantitative descriptors of erythrocytes ◦ The leukocyte histograms ◦ Platelet histograms ◦ Derived platelet parameters 11  6/28/2024 The Erythrocyte Histogram ◦ The erythrocyte histogram reflects the native size of erythrocytes or any other particles in the erythrocyte size range. ◦ The erythrocyte histogram in the Coulter system displays cells as small as 24 fL, but only those greater than 36 fL are counted as erythrocytes. ◦ The extension of the lower end of the scale from 36 to 24 fL allows for the detection of erythrocyte fragments, leukocyte fragments, and large platelets. Quantitative Descriptors of Erythrocytes #1 ◦ An expression of erythrocyte size is the RDW in the Coulter series. This term refers to variation in erythrocyte size. ◦ Correlations between the RDW and the MCV exist for various types of anemia. 12  6/28/2024 The Leukocyte Histograms #1 ◦ Size-referenced leukocyte histograms display the classification of leukocytes according to size following lysis. It does not display the native cell size. ◦ The histogram of leukocyte subpopulations reflects the sorting of these cells by their relative size, which is primarily related to their nuclear size. ◦ As the leukocyte passes through the aperture in the electrical impedance system, they displace their volume in a conductive fluid, which causes a change in electrical resistance as each cell passes through the aperture. Platelet Histograms ◦ Platelet counting and sizing in both the electrical impedance and the optical systems reflect the native cell size. ◦ In the electrical impedance method, counting and sizing take place in the RBC aperture. ◦ In the optical system, forward light scatter pattern discrimination between erythrocytes and platelets in the flow cell determines the platelet count and frequency distribution. 13  6/28/2024 Derived Platelet Parameters ◦ Mean platelet volume calculation ◦ The MPV is a measure of the average volume of platelets in a sample. The MPV is analogous to the erythrocytic MCV. ◦ It is derived from the same data as the platelet count. ◦ In healthy patients, there is an inverse relationship between platelet count and size. Laser Technology #1 ◦ Principle of flow cytometry relies on three distinct steps: ◦ Cytochemical reactions prepare the blood cells for analysis. ◦ A cytometer measures specific cell properties. ◦ Algorithms convert these measurements into familiar results for cell classification, cell count, cell size, and hemoglobinization. 14  6/28/2024 Laser Technology #2 ◦ The instrument’s sampling mechanism divides blood samples into aliquots that are treated in four separate reaction chambers: ◦ Hemoglobin ◦ Red blood cells/platelets ◦ Peroxidase ◦ Basophil/lobularity (nuclear) channel Red Blood Cells/Platelets ◦ The RBC/platelet channel uses a laser-based optical assembly that is shared with the basophil/lobularity channel. ◦ The light scattered at low and high angles simultaneously measures RBC volume (size) and optical density (hemoglobin concentration) of each cell. ◦ Additional parameters obtained from the histogram are the MCV and RDW. ◦ The platelet histogram is derived from measurements made with the high- angle detector. 15  6/28/2024 Applications of Flow Cytometry ◦ General properties of flow cytometry ◦ Hematological applications ◦ Clinical applications of flow cytometry ◦ Other cellular applications General Properties of Flow Cytometry ◦ Major advances in this technology are owing to several factors: ◦ The ability to produce monoclonal antibodies resulted in the subsequent development of specific surface markers for various subpopulations of cells. ◦ The development of new fluorescent probes for DNA, RNA, and other cellular components increased the variety of possible applications at the molecular and cellular levels. ◦ The expansion of computer applications has improved the instrumentation technology, making it easier to operate and more practical for use in clinical as well as research laboratories. 16  6/28/2024 Hematological Applications ◦ Automated differentials can be based on a variety of principles. ◦ These include determination of cell volume by electrical impedance or forward light scatter, cytochemistry or peroxidase staining, and VCS technology. ◦ Flow cytometry can be applied practically to several techniques in the clinical hematology laboratory. Automated Differentials ◦ Evaluation of internal cellular organelles and nuclear characteristics can be by: ◦ 90-degree laser scatter ◦ Polarizing laser light ◦ RF ◦ Separate measurements can be made of individual measurements of volume, conductivity, and light scatter. 17  6/28/2024 Clinical Applications of Flow Cytometry ◦ Reticulocytes: enumeration of reticulocytes by flow cytometry is more accurate, precise, and cost-effective. ◦ Platelets counts: can provide an estimate of young, reticulated platelets by counting platelets that stain with an RNA dye. ◦ Immunophenotyping: monoclonal antibodies, identified by a cluster designation (CD), are used in most flow cytometry immunophenotyping. ◦ Hematological malignancy ◦ Flow cytometry has become an important tool in the diagnosis and classification of hematologic neoplasia by immunophenotyping. Flow cytometry techniques with bone marrow cells are applicable to DNA cell cycle analysis. Other Cellular Applications ◦ Solid organ transplantation ◦ Stem cell transplantation ◦ Monitoring monoclonal antibody therapy ◦ Paroxysmal nocturnal hemoglobinemia testing ◦ Fetal hemoglobin ◦ Blood parasites ◦ Cell functioning analysis ◦ Chromosomal analysis ◦ Cell sorting 18  6/28/2024 Digital Microscopy ◦ This new technology, referred to as automated digital cell morphology, provides an unprecedented level of efficiency and consistency. ◦ In its simplest form, automated digital cell morphology is a process where blood cells are automatically located and preclassified into categories of blood cells. Instruments in Coagulation Studies ◦ Electromechanical methods ◦ Photo-optical methods ◦ Platelet agglutination ◦ Platelet aggregation ◦ New automation 19  6/28/2024 Electromechanical Methods ◦ The earliest instruments to detect blood clotting. The principle of electromechanical methodology is the measurement of conduction or impedance of an electrical current by the formation of fibrin. An example of such a semiautomated instrument is the fibrometer. This system consists of a 37°C heat block, an automatic pipette, and a mechanical mixer and timer block. ◦ After the appropriate containers are filled and plasma samples and thromboplastin substrate are incubated, plasma is added to the substrate to initiate the timing mechanism. This timing mechanism triggers a digital readout time and the probe unit. Photo-Optical Methods ◦ The principle of photo-optical measurement is that a change in light transmission measured as optical density (absorbance) versus time can be used to quantitatively determine the activity of various coagulation stages or factors. ◦ Photo-optical clot detection systems can be used for the determination of a wide variety of assays (e.g., aPTT, PT, fibrinogen levels, and thrombin time). ◦ Quantitative factor assays based on the aPTT (factors VIII, IX, XI, and XII) and quantitative factors assays based on the PT (factors V, VII, and X) are examples of available assays. 20  6/28/2024 Viscosity-Based Detection System (VDS) ◦ Viscosity is defined as the resistance that a material has to a change in its form. ◦ If this principle is used as a mechanism for clot detection, the natural thickening (viscosity) is monitored by the motion (amplitude of an oscillating steel ball in a specially designed cuvette), as a change in form takes place. ◦ The final result is accurate and is insensitive to colored plasma, lipemic plasma, bilirubin, or turbid reagents, and it is a reliable measurement for the hemostasis laboratory. Platelet Agglutination ◦ The ristocetin cofactor assay measures the ability of a patient’s plasma to agglutinate formalin-fixed platelets in the presence of ristocetin. ◦ The rate of ristocetin-induced agglutination is related to the concentration of von Willebrand factor, and the percent normal activity can be obtained from the standard curve. ◦ Patient values are determined by comparison to a standard curve, allowing the quantitation of percent ristocetin cofactor activity. 21  6/28/2024 Platelet Aggregation ◦ The principle of the test is that platelet-rich plasma is treated with a known aggregating agent. ◦ If aggregated, cloudiness or turbidity patterns are determined by photometrically comparing the light transmitted through a suspension of aggregated platelets with that of a suspension of nonaggregated platelets using an aggregometer. ◦ Depending on the agonist, the response may be only primary or both primary and secondary ◦ Primary response: reversible aggregation of platelets ◦ Secondary response: irreversible aggregation of platelets Newer Automation ◦ The Siemens PFA-100 is an automated system that incorporates a high shear flow system to simulate the in vivo hemodynamic conditions of platelet adhesion and aggregation as encountered at a vascular lesion. ◦ This system evaluates the ability of platelets to occlude an aperture in a biochemically active membrane. ◦ Results are reported as closure time (CT). ◦ Assesses multiple facets of primary hemostasis, namely adherence, activation, and aggregation. 22  6/28/2024 Postamble ◦ READ the TEXTBOOK for the details to answer the UNIT OBJECTIVES. ◦ USE THE UNIT OBJECTIVES AS A STUDY GUIDE ◦ All test questions come from detailed material found in the TEXTBOOK (Not this PowerPoint) and relate back to the Unit Objectives 23