The Evolution of White Blood Cell Differential Technologies
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DIAGNOSTICS The Evolution of White Blood Cell Differential Technologies Authors: Donald Wright, Gabriella Lakos Abbott Diagnostics, Hematology, Santa Clara, CA 95054 DIAGNOSTICS INTRODUCTION Hematology analyzers count and characterize blood cells for the screening and monitoring of KEY ACRONYMS disease. Analyzers vary in capabilities, sophistication CBC = Complete Blood Count, also known as and detection technologies. The most common Full Blood Count (FBC) technologies are electrical impedance, radio frequency conductivity, optical light scatter (optical WBC = White Blood Cell flow cytometry), cytochemistry and fluorescence. MAPSSTM = Multi-Angle Polarized Scatter Optimal combinations of these detection methods Separation provide an accurate automated complete blood count (CBC) including white blood cell (WBC) differential IG = Immature Granulocyte in a short turnaround time. RBC = Red Blood Cell Although many other detection methods are still in use, optical technology has represented a key PLT = Platelet innovation in automated hematology analysis since NRBC = Nucleated Red Blood Cell its introduction.1,2,3,4 Light, scattered and detected at specific angles, captures an array of information about cell size, structure, inner complexity, nuclear segmentation and cytoplasmic granulation. As an different types of WBCs (neutrophil, eosinophil and innovative expansion of optical flow cytometry, basophil granulocytes, lymphocytes and monocytes) Multi-Angle Polarized Scatter Separation (MAPSS™) present in normal blood. This provides information technology (Abbott Diagnostics, Hematology, Santa for diagnosis and assessment of infections, immune Clara, CA, USA) uses four different light scatter system or bone marrow disorders, and hemato- signals to characterize distinct cellular features for oncological diseases. Traditionally, the WBC the identification of various blood cells. MAPSS™ differential has been determined by manual counting technology reliably automates WBC differentials, and and classification of 100 or 200 WBCs on a stained continues to be improved and expanded upon. The blood smear.5 This method, although it is highly MAPSS™ and advanced MAPSS™ technology that imprecise,6 is still considered the reference method are integrated in Abbott hematology analyzers enable for the WBC differential,7 and may be performed clinical laboratories to obtain high quality results. as a reflex test after an automated CBC analysis. Today WBC differentials are usually performed THE EVOLUTION OF WBC on automated hematology analyzers. Although the DIFFERENTIAL TECHNOLOGY automated WBC differential is very reliable and accurate, a manual differential is often required to The automated WBC differential provides the confirm the presence of immature or reactive cells, absolute and relative (%) concentrations of the five blasts and other pathological cell types. 1950s 1960s 1970s 1980s 1990s 2000s Prior to 1950 1953 1968 1974 1981 1996 1998 to Present WBC counts were Coulter principle First whole blood First automated First commercially Intoduction of Refining the automated usually performed patented benchtop, automated 5-part dierential successful digital fluorescent analysis dierential manually using the hematology analyzer First routine automated analyzer (Technicon microscopy (Geometric Introduction of advanced hemocytometer (Coulter Model S) NRBC count HEMALOG D) Data Hematrak 590) generations from 1956 • First transistorized First automated various manufacturers First commerically instrument (Coulter fluorescent retic count available counting Model FN) 1985 Introduction of IG count First three-method instrument (Coulter First benchtop analyzer into CBC - a “true” 6-part platelet count (Abbott Model A) with automated CBC dierential and 5-part dierential CELL-DYN 4000) (Technicon H*1) Figure 1. Automated WBC and differential count technology development timeline 2 DIAGNOSTICS Impedance and Cytochemistry Some current hematology analyzers still rely on Electrical impedance is a cell counting and sizing impedance technology, though with additional technique based on the measurement of changes technologies to improve the white cell differential. in electrical impedance (resistance) produced by a The utilization of a high frequency signal, defined particle (i.e. a blood cell). It was the first automated as radio frequency (RF) conductivity, allows technology to count cells and measure the size of the discrimination between different WBC WBCs, Red Blood Cells (RBCs) and Platelets (PLTs). subpopulations.8 RF signals pass through the cell, Blood cells are suspended in a conductive fluid producing a response that is related to nuclear and pass through an aperture of known size with composition, cytoplasmic density and other electrodes on either side. As cells pass through the differences in internal structures. This technology orifice, they cause a change in electrical conductance aims at mitigating the inherent limitations of which is proportional to the size of the particle impedance technology. Cytochemical staining (Coulter Principle,8 Figure 2a). Early challenges is another technology that is used to distinguish to this method were managing coincidence events, between cells containing myeloperoxidase (a where two cells pass through the aperture too lysosomal enzyme located in the azurophilic granules close together and cells being recounted due to the of the neutrophils and its precursors, eosinophils and recirculation of cells around the detection area of monocytes) and peroxidase-negative cells, which the aperture. Impedance technology can deliver a include lymphocytes and basophils.9 three-part WBC differential, where cells are grouped To reduce the occurrence of coincidence and re- into three sizes: lymphocytes, mid-range cells and circulation, a technique called hydrodynamic granulocytes (Figure 2b). It does not allow for the focusing was developed. Hydrodynamic focusing differentiation of the granulocyte subtypes. The enables cells to follow a path of least resistance, limitation of this technology is that it is based purely surrounded by sheath fluid, and to proceed in single on measuring cell size. Therefore, abnormal cells, such file based on the principal of laminar flow. This as nucleated red blood cells (NRBCs), PLT clumps, technique can be used in both electrical impedance giant PLTs or unlysed red cells cannot be separated as well as with optical cell counting methods. and may interfere with normal cell populations. + Electrode - Electrode ID: WBC PARTICLE B PARTICLE A PARTICLE C PARTICLE VOLUME PARTICLE A PARTICLE B PARTICLE C APERTURE Figure 2a. Representation of Coulter Principle. LYM MID GRAN 50 100 150 200 250 300 350 Figure 2b. Three-part WBC differential by impedance. 3 DIAGNOSTICS Optical Technologies and depending on the sophistication of the instrument, some current analyzers provide six-part Optical flow cytometry provides several advantages WBC differentials, including immature granulocyte over traditional impedance methods. During optical (IG) counts.10 light scatter measurement, a beam of laser light is passed through a diluted blood specimen stream Adding the detection capability of a fluorescent signal that is projected into the flow cell by hydrodynamic further enhances the potential of optical technology. focusing. As each cell passes through, the focused Fluorescent flow cytometry captures the light light is scattered in various directions and detected emitted from internal cellular components that are by photodetectors which convert the signal into an stained with a fluorescent dye as cells pass through electric pulse. The electronic signals are transmitted the flow cell in front of the laser beam. Fluorescence to a computer for storage and analysis. The signals is frequently integrated with multiple-angle optical provide information about cellular characteristics light scatter methods to further improve the WBC such as size, internal complexity, nuclear lobularity/ differential subtype classification. segmentation and cytoplasmic granularity, which are used to identify the cells. Cells with similar light MAPSS™ Technology scatter properties form a cluster in the scattergram, The Multi-Angle Polarized Scatter Separation and can be separated from other cell clusters using (MAPSSTM) technology from Abbott uses four light advanced software algorithms. Some analyzers only scatter detectors to determine various cellular use two angles of light, whereas others use multi- features. The application of a depolarized light angle optical scatter analysis (Figure 3). detector is a unique characteristic of this method and allows for specific identification of eosinophil VARIOUS ANGLES granulocytes. The four detectors generate the DSS PSS OF SCATTERED following signals: LIGHT • 0° or Axial Light Loss (ALL): related to size FOCUSED LASER BEAM IAS • 0° to 10° Intermediate Angle Scatter (IAS): ALL related to cellular complexity • 90° Polarized Side Scatter (PSS): related to nuclear lobularity/segmentation SAMPLE STREAM • 90° Depolarized Side Scatter (DSS): related to eosinophil granules These signals correlate with morphological characteristics that can be determined visually under the microscope from a stained slide. Various combinations of these four measurements are used to classify the WBC subpopulations and provide morphological flagging. Fluorescent flow cytometry is used to detect NRBCs based on DNA staining when additional reagent and a detector for fluorescence signal is added. Multi- vs. Single-Channel Analysis