Point-of-Care Testing, Semi-Automated and Automated Blood Cell Analysis PDF

Point-of-care Testing, Semi-Automated and Automated Blood Cell Analysis Mirabele A. Camoro, RMT, MLS (ASCPi) Shelley N. Lee, RMT At the end of the session, the students with 75% accuracy will be able to: 1. Describe correctly the aspects of establishing a point-of-care testing program, including...

Point-of-care Testing, Semi-Automated and Automated Blood Cell Analysis Mirabele A. Camoro, RMT, MLS (ASCPi) Shelley N. Lee, RMT At the end of the session, the students with 75% accuracy will be able to: 1. Describe correctly the aspects of establishing a point-of-care testing program, including quality management and selection of instrumentation. 2. Discuss adequately and correctly the advantages and disadvantages of point-of-care testing as they apply to hematology tests. 3. Describe correctly the principles of common instruments used for POCT for hemoglobin level, hematocrit, WBC counts, and platelet counts At the end of the session, the students with 75% accuracy will be able to: 4. Explain thoroughly the different principles of automated blood cell counting and analysis. 5. Identify adequately and accurately the parameters measured on the analyzers discussed. 6. Explain correctly the derivation of calculated or indirectly measured parameters and the derivation of the WBC differential count on the different instruments discussed. At the end of the session, the students with 75% accuracy will be able to: 7. List adequately and correctly the sources of error in automated cell counting and determine appropriate corrective action. Table of Contents 1. Point-of-Care Testing and Disposable Blood Test Systems 2. General Principles of Semi-Automated and Automated Blood Cell Analysis 3. Principal Instruments 4. Automated Reticulocyte Counting 5. Limitations and Interferences 6. Clinical Utility of Blood Cell Analysis POCT and Disposable Blood Test Systems Point-of-Care Testing (POCT) offers the ability to produce rapid an accurate results that facilitate faster treatment, which can decrease hospital length stay. Rarely performed by trained laboratory personnel; most often by nurses Is defined as a diagnostic testing at or near the site of patient care Point-of-Care Testing (POCT) CLIA of 1988: ○ intorduced the concept of “testing site neutrality” Is classified as “waived” or “moderately complex” Waived: simple test with an insignificant risk of an erroneous result Key Elements for a Succesful POC Program: 1. Appointing a loboratory POC testing coordinator. 2. Create a multidisciplinary team with authority to affect all aspects of the POC program 3. Administrative support Written Policy Defines the program, outlines who is responsible for each part of the program Indicates where the testing is performed and who performs the testing Selection of Instrument POC operators need handheld analyzers that are: ○ lightweight ○ accurate ○ fast ○ require little specimen material Selection of Instrument The POC testing system should address the following concerns: ○ What is the range of measurement? ○ How well does the test system correlate with laboratory instrumentaion? ○ Can it be interfaced to the laboratory information system? ○ Does it provide reliable results? ○ Does the company supply excellent technical support? ○ Is it affordable? POCT Advantages: 1. Workflow provides mor effective health-care provider-patient interaction 2. The immediate availability of the results provides convenience to both the patient and the health-care provider POCT Disadvantages: 1. Quality control and documentation problems 2. Operator’s competency LeukoChek Blood Diluting System for Manual White Blood Cell and Platelet Counts Consists of a 20 uL capillary pipette and plastic reservoir containing 1.98 mL of 1% buffered ammonium oxalate that makes a 1:100 dilution of whole blood. The HemoCue Hb 2011 System for Measuring Hemoglobin Hemoglobin is converted to azidemethemoglobin and is read photometrically at two wavelengths (570 nm and 880 nm). This method avoids the necessity of specimen dilution and interference from turbidity READACRIT Centrifuge with Built-in Capillary Tube Compartments and Scales to read Hematocrit SUREPREP Capillary Tubes (Becton, Dickinson) Eliminates the use of sealants Have a factory-inserted plug that seals automatically when the blood touches the plug Sediplast (Polymedco) Disposable Sedimentation Rate System Hemastat II Separation Technology POCT microhematocrit centrifuge-based device I-STAT Instrument for Measuring Hematocrit Use the conductivity method to determine the hematocrit Plasma conducts electrical current, whereas WBCs act as insulators Sources of Error Conductivity of a whole blood specimen is dependent on the amount of electrolytes in the plasma portion Conductivity does not distinguish RBCs from other non-conductive elements such as proteins, lipids, and WBCs that may be present in the specimen Other instruments that measure hematocrit include the following: ABL 77 (Radiometer) IRMA (Accriva, a subsidiary of LifeHealth) Gem Premier (Instrumentation Laboratory Company) AVOXimeter 1000E (Instrumentation Laboratory) Measures total hemoglobin by a spectrophotometric method Ichor Hematology Analyzer (Helena Laboratories) Performs a CBC along with platelet aggregation Quantitative buffy coat analysis (WBC STAR, manufactured by QBC Diagnostics, Inc., Philipsburg, PA) Involves centrifugation in specialized capillary tubes designed to expand the buffy coat layer The components (platelets, mononuclear cells, and granulocytes) can be measured with the assistance of fluorescent dyes and a measuring device General Principles of Semi-Automated and Automated Blood Cell Analysis Two basic principles of Hematology Analyzers: Electronic impedance (resistance) Optical scatter Electronic Impedance (Resistance) Electronic impedance, or low-voltage direct current (DC) resistance, was developed by Coulter in the 1950s and is the most common methodology used Electronic Impedance (Resistance) Based on the detection and measurement of changes in electrical resistance produced by cells as they traverse a small aperture Electronic Impedance (Resistance) The number of pulses is proportional to the number of cells counted The height of the voltage pulse is directly proportional to the volume of the cell Electronic Impedance (Resistance) The data are plotted on a frequency distribution graph, or volume distribution histogram, with relative number on the y-axis and volume (cha-nnel number equivalent to a specific volume) on the x-axis Radiofrequency (RF) Low-voltage DC impedance, as described previously, may be used in conjunction with RF resistance, or resistance to a high voltage electromagnetic current flowing between both electrodes simultaneously Radiofrequency (RF) Conductivity, as measured by this high-frequency electromagnetic probe, is attenuated by nucleus-to-cytoplasm ratio, nuclear density, and cytoplasmic granulation. Radiofrequency (RF) Two different cell properties, such as low-voltage DC impedance and RF resistance, can be plotted against each other to create a two-dimensional distribution cytogram or scatterplot (e.g. Sysmex SE-9500) Optical Scatter A hydrodynamically focused sample stream is directed through a quartz flow cell past a focused light source The light source is generally a tungsten-halogen lamp or a helium-neon laser Laser light, termed monochromatic light because it is emitted at a single wavelength Optical Scatter These characteristics allow for the detection of interference in the laser beam and enable enumeration and differentiation of cell types Optical scatter may be used to study RBCs, WBCs, and platelets Optical Scatter As cells pass through the sensing zone and interrupt the beam, light scattered in all directions The detection of scattered rays and their conversion into electrical signals is accomplished by photodetectors (photodiodes and photomultiplier tubes) at specific angles Optical Scatter Analog-to-digital converters change the electronic pulses to digital signals for computer analysis Optical Scatter Forward-angle light scatter (0 degrees) correlates with cell volume or size, primarily because of diffraction of light Orthogonal light scatter (90 degrees), or side scatter, results from refraction and reflection of light from larger structures inside the cell and correlates with the degree of internal complexity or cell granularity Principal Instruments Hematology Analyzer Common Basic Components: Hydraulics Pneumatics Electrical systems Hydraulics Includes an aspirating unit, dispensers, diluters, mixing chambers, aperture baths or flow cells or both, and a hemoglobinometer Pneumatics Generates the vacuums and pressures required for operating the valves and moving the sample through the hydraulics system Electrical Systems Controls operational sequences of the total system and includes electronic analyzers and computing circuitry for processing the data generated Hematology Analyzer Hematology blood cell analyzers are produced by multiple manufacturers including but not limited to: ○ Abbott Laboratories ○ HORIBA Medical ○ Siemens Healthcare Diagnostics ○ Beckman Coulter ○ Sysmex Corpor BECKMAN COULTER INSTRUMENTATION RBC and WBC counts and hemoglobin are considered to be measured directly. The aspirated whole-blood sample is divided into two aliquots, and each is mixed with an isotonic diluent. The first dilution is delivered to the RBC aperture chamber, and the second is delivered to the WBC aperture chamber BECKMAN COULTER INSTRUMENTATION In the RBC chamber, RBCs and platelets are counted and discriminated by electrical impedance as the cells are pulled through each of three sensing apertures (50 mm in diameter, 60 mm in length). 2 to 20 fL: counted as platelets > 36 fL: counted as RBCs BECKMAN COULTER INSTRUMENTATION In the WBC chamber, a reagent to lyse RBCs and release hemoglobin is added before WBCs are counted simultaneously by impedance in each of three sensing apertures (100 mm in diameter, 75 mm in length). BECKMAN COULTER INSTRUMENTATION WBC dilution is passed to the hemoglobinometer for determination of hemoglobin concentration (light transmittance read at a wavelength of 525 nm). Three-part Leukocyte Subpopulation Analysis: ○ 35- 90 fL: are considered lymphocytes ○ 90- 160 fL: mononuclears (monocytes, blasts, immature granulocytes, and reactive lymphocytes) ○ 160- 450 fL: granulocytes BECKMAN COULTER INSTRUMENTATION R1 Flag: ○ when cell populations overlap or a distinct separation of populations does ○ represents excess signals at the lower threshold region of the WBC histogram and a questionable WBC count. ○ may indicate the presence of nucleated RBCs, clumped platelets, unlysed RBCs, or electronic noise. R2 Flag: ○ indicates interference and loss of valley owing to overlap or insufficient separation between the lymphocyte and mononuclear populations. Sysmex Instrumentation The WBC, RBC, platelet counts, hemoglobin, and HCT are considered to be measured directly. Three hydraulic subsystems are used for determining the hemogram: ○ the WBC channel, ○ the RBC/platelet channel, and ○ a separate hemoglobin channel. Sysmex Instrumentation In the WBC and RBC transducer chambers, diluted WBC and RBC samples are aspirated through the different apertures and counted using the impedance (DC detection) method for counting and volumetrically sizing cells. In the hemoglobin flow cell, hemoglobin is oxidized and binds to sodium lauryl sulfate (SLS), forming a stable SLS–hemoglobin complex, which is measured photometrically at 555 nm. Sysmex Instrumentation Flags are triggered for the possible presence of morphologic abnormalities. A POSITIVE or NEGATIVE interpretive message is displayed. Abbott Instrumentation The WBC, RBC, hemoglobin, and platelet parameters are considered to be measured directly. A 60- to 70-mm aperture is used in the RBC/platelet transducer assembly for counting and volumetrically sizing of RBCs and platelets by the electronic impedance method. Abbott Instrumentation A unique von Behrens plate is located in the RBC/platelet counting chamber minimize the effect of recirculating cells ○ 1- 35 fL: considered platelets ○ >35 fL: considered RBCs Hemoglobin is measured directly using a modified hemiglobincyanide method that measures absorbance at 540 nm. Abbott Instrumentation The WBC count and differential are derived from the optical channel using CELL- DYN’s patented multi angle polarized scatter separation (MAPSS) technology with three-color fluorescent technology. (Optical scatter) Siemens Healthcare Diagnostics Instrumentation Simplified the hydraulics and operations of the analyzer by replacing multiple complex hydraulic systems with a unified fluids circuit assembly, or Unifluidics technology. Four independent measurement channels are used in determining the hemogram and differential: RBC/platelet channel, hemoglobin channel, and peroxidase (PEROX) and basophil lobularity (BASO) channels for WBC and differential data. Siemens Healthcare Diagnostics Instrumentation WBC, RBC, hemoglobin, and platelets are measured directly. Hemoglobin is determined using a modified cyanmethemoglobin method that measures absorbance in a colorimeter flow cuvette at approximately 546 nm. The RBC/platelet method uses flow cytometric light scattering measurements (optical scatter). Automated Reticulocyte Counting AUTOMATED RETICULOCYTE COUNTING Reticulocyte counting is the last of the manual cell-counting procedures to be automated and has been a primary focus of hematology analyzer advancement in recent years. The imprecision and inaccuracy in manual reticulocyte counting are due to multiple factors, including stain variability, slide distribution error, statistical sampling error, and interobserver error. AUTOMATED RETICULOCYTE COUNTING All these potential errors, with the possible exception of stain variability, are correctable with automated reticulocyte counting Increasing the number of RBCs counted produces increased precision. AUTOMATED RETICULOCYTE COUNTING Sysmex R-3000/3500: a stand-alone reticulocyte analyzer that uses auramine O, a supravital fluorescent dye, and measures forward scatter and side fluorescence as the cells, in a sheath stream, pass through a flow cell by an argon laser. signals are plotted on a scattergram with forward scatter intensity, which correlates with volume, plotted against fluorescence intensity, which is proportional to RNA content. AUTOMATED RETICULOCYTE COUNTING reticulocytes fall into low-fluorescence, middle-fluorescence, or high-fluorescence regions, with the less mature reticulocytes showing higher fluorescence. AUTOMATED RETICULOCYTE COUNTING Immature reticulocyte fraction (IRF): sum of the middle-fluorescence and high-fluorescence ratios and indicates the ratio of immature reticulocytes to total reticulocytes in a specimen ○ High total reticulocyte count and increased IRF: e.g. anemias with increased marrow erythropoiesis, such as hemolytic anemia ○ Decreased absolute reticulocyte count & IRF: e.g. chronic renal disease ○ Normal to decreased absolute reticulocyte count & increased IRF: e.g. early response to therapy in nutritional anemias Limitations and Interferences Clinical and Laboratory Standards Institute (CLSI) Approved a standard for validation, verification, and quality assurance of automated hematology analyzers. Provides guidelines for instrument calibration and assessment of performance criteria, including accuracy, precision, linearity, sensitivity, and specificity. Quality control systems should reflect the laboratory’s established performance goals and provide a high level of assurance that the instrument is working within its specified limits. LIMITATIONS & INTERFERENCES Calibration: process of electronically correcting an instrument for analytical bias (numerical difference from the “true” value), may be accomplished by appropriate use of reference methods, reference materials, or commercially prepared calibrators. LIMITATIONS & INTERFERENCES Instrument limitations: has limitations related to methodology that are defined in instrument operation manuals and in the literature. A common limitation of impedance methods is an instrument’s inability to distinguish cells reliably from other particles or cell fragments of the same volume. LIMITATIONS & INTERFERENCES Specimen limitations: resulting from inherent specimen problems include those related to the presence of cold agglutinins, icterus, and lipemia. ○ MCV (often greater than 130 fL), markedly decreased RBC count, and increased MCHC (often greater than 40 g/dL). Clinical Utility of Blood Cell Analysis  Automated hematology analyzers have had a significant impact on laboratory workflow, particularly automation of the WBC differential. In addition, newer parameters that can now be measured, such as the immature reticulocyte fraction (IRF) and the reticulocyte hemoglobin concentration (RET-He and CHr), have documented clinical utility. - SEE YOU NEXT SEM! -