Automated Blood Cell Analysis PDF

15 Automated Blood Cell Analysis Sharral Longanbach and Martha K. Miers OUTLINE OBJECTIVES General Principles of Au- After completion of this chapter, the reader will be able to: tomated Blood Cell Anal- ysis...

15 Automated Blood Cell Analysis Sharral Longanbach and Martha K. Miers OUTLINE OBJECTIVES General Principles of Au- After completion of this chapter, the reader will be able to: tomated Blood Cell Anal- ysis 1. Explain the different principles of automated blood cell 6. Interpret and compare patient data, including white Electronic Impedance counting and analysis. blood cell, red blood cell, and platelet histograms or Radiofrequency 2. Describe how the general principles are implemented cytograms or both, obtained from the four major Optical Scatter on different instruments. hematology instruments. Principal Instruments 3. Identify the parameters directly measured on the four 7. Explain the general principles of automated reticulo- Overview analyzers discussed. cyte counting. Beckman Coulter Instru- 4. Explain the derivation of calculated or indirectly 8. Identify sources of error in automated cell counting mentation measured parameters for the same four analyzers. and determine appropriate corrective action. Sysmex Instrumentation 5. Explain the derivation of the white blood cell differen- Abbott Instrumentation tial count on the different instruments discussed. Siemens Healthcare Diag- nostics Instrumentation Automated Reticulocyte Counting Limitations and Interfer- ences Calibration Instrument Limitations Specimen Limitations Clinical Utility of Auto- mated Blood Cell Analy- sis S ince the 1980s, automated blood cell analysis has virtu- and complexity, most rely on only two basic principles of ally replaced manual hemoglobin, hematocrit, and cell operation: electronic impedance (resistance) and optical counting, due to its greater accuracy and precision, with scatter. Electronic impedance, or low-voltage direct current (DC) the possible exception of phase platelet counting in certain resistance, was developed by Coulter in the 1950s1,2 and is the circumstances. Hematology analyzers are marketed by multi- most common methodology used. Radiofrequency (RF), or ple instrument manufacturers. These analyzers typically pro- alternating current resistance, is a modification sometimes used vide the eight standard hematology parameters (complete in conjunction with DC electronic impedance. Technicon Instru- blood count [CBC]), plus a three-part, five-part, or six-part ments Corporation (Tarrytown, NY) introduced darkfield differential leukocyte count in less than 1 minute on 200 !L optical scanning in the 1960s, and Ortho Clinical Diagnostics, or less of whole blood. Automation allows more efficient Inc. (Raritan, NJ), followed with a laser-based optical instrument workload management and more timely diagnosis and treat- in the 1970s.3 Optical scatter, using both laser and nonlaser light, ment of disease. is frequently employed in today’s hematology instrumentation. Electronic Impedance GENERAL PRINCIPLES OF AUTOMATED The impedance principle of cell counting is based on the detec- BLOOD CELL ANALYSIS tion and measurement of changes in electrical resistance pro- Despite the number of hematology analyzers available from dif- duced by cells as they traverse a small aperture. Cells suspended ferent manufacturers and their varying levels of sophistication in an electrically conductive diluent such as saline are pulled 208  CHAPTER 15 Automated Blood Cell Analysis 209 through an aperture (orifice) in a glass tube. In the counting Oscilloscope chamber, or transducer assembly, low-frequency electrical cur- rent is applied between an external electrode (suspended in the cell dilution) and an internal electrode (housed inside the 100 fL aperture tube). Electrical resistance between the two electrodes, 98 fL or impedance in the current, occurs as the cells pass through 96 fL 94 fL the sensing aperture, causing voltage pulses that are measur- 92 fL able (Figure 15-1).4,5 Oscilloscope screens on some instru- 90 fL 88 fL ments display the pulses that are generated by the cells as they 86 fL interrupt the current. The number of pulses is proportional to 84 fL 82 fL the number of cells counted. The height of the voltage pulse is 80 fL 78 fL directly proportional to the volume of the cell, which allows discrimination and counting of cells of specific volumes through the use of threshold circuits. Pulses are collected and sorted (channelized) according to their amplitude by pulse height analyzers. The data are plotted on a frequency distribu- Histogram tion graph, or volume distribution histogram, with relative num- ber on the y-axis and volume (channel number equivalent to a specific volume) on the x-axis. The histogram produced depicts the volume distribution of the cells counted. Figure 15-2 illus- trates the construction of a frequency distribution graph. Volume thresholds separate the cell populations on the histo- gram, and the count is the cells enumerated between the lower and upper set thresholds for each population. Volume distribution histograms may be used for the evaluation of Relative number one cell population or subgroups within a population.5 The use of proprietary lytic reagents to control shrinkage and lysis of specific cell types, as in the older Coulter S-Plus IV, STKR, and Sysmex E-5000 models, allows separation and quantitation of white blood cells (WBCs) into three popula- 78 80 82 84 86 88 90 92 94 96 98 100 102 tions (lymphocytes, mononuclear cells, and granulocytes) Femtoliters for the three-part differential on one volume distribution Figure 15-2 Oscilloscope display and histogram showing the construc- histogram.6-8 tion of a frequency distribution graph. (Modified from Coulter Electronics: Significant advances in hematology: hematology education series, PN 4206115A, Hialeah, FL, 1983, Coulter Electronics.) Vacuum (6" Hg) Several factors may affect volume measurements in imped- ance or volume displacement instruments. Aperture diameter Aperture current is crucial, and the red blood cell (RBC)/platelet aperture is smaller than the WBC aperture to increase platelet counting Internal sensitivity. On earlier systems, protein buildup occurred, de- electrode + creasing the diameter of the orifice, slowing the flow of cells, and increasing their relative electrical resistance. Protein External electrode buildup results in lower cell counts, which result in falsely el- Blood cell – suspension evated cell volumes. Impedance instruments once required frequent manual aperture cleaning, but current instruments incorporate burn circuits or other internal cleaning systems to prevent or slow protein buildup.6-9 Carryover of cells from one sample to the next also is minimized by these internal cleaning systems. Coincident passage of more than one cell at a time through the orifice causes artificially large pulses, which results in falsely increased cell volumes and falsely decreased cell Aperture Aperture bath Aperture tube counts. This count reduction, or coincident passage loss, is statis- Figure 15-1 Coulter principle of cell counting. (From Coulter Electronics: tically predictable (and mathematically correctable) because of Coulter STKR product reference manual, PN 4235547E, Hialeah, FL, 1988, its direct relationship to cell concentration and the effective Coulter Electronics.) volume of the aperture.7-9 Coincidence correction typically is 210 PART III Laboratory Evaluation of Blood Cells completed by the analyzer computer before final printout of Two different cell properties, such as low-voltage DC imped- cell counts from the instrument. Other factors affecting pulse ance and RF resistance, can be plotted against each other height include orientation of the cell in the center of the aper- to create a two-dimensional distribution cytogram or scatterplot ture and deformability of the RBC, which may be altered by (Figure 15-4). Such plots display the cell populations as clus- decreased hemoglobin content.10,11 Recirculation of cells back ters, with the number of dots in each cluster representing the into the sensing zone creates erroneous pulses and falsely concentration of that cell type. Computer cluster analysis can elevates cell counts. A backwash or sweep-flow mechanism determine absolute counts for specific cell populations. The use prevents recirculation of cells back into the sensing zone, and of multiple methods by a given instrument for the determina- anomalously shaped pulses are edited out electronically.6,7,9 tion of at least two cell properties allows the separation of The use of hydrodynamic focusing avoids many of the WBCs into a five-part differential (neutrophils, lymphocytes, potential problems inherent in a rigid aperture system. The monocytes, eosinophils, and basophils). DC and RF detection sample stream is surrounded by a sheath fluid as it passes are two methods used by the Sysmex analyzers to perform WBC through the central axis of the aperture. Laminar flow allows differentials.15,16 the central sample stream to narrow sufficiently to separate and align the cells into single file for passage through the sensing Optical Scatter zone.12-14 The outer sheath fluid minimizes protein buildup Optical scatter may be used as the primary methodology or in and plugs, eliminates recirculation of cells back into the sens- combination with other methods. In optical scatter systems ing zone with generation of spurious pulses, and reduces pulse (flow cytometers), a hydrodynamically focused sample stream height irregularity because off-center cell passage is prevented is directed through a quartz flow cell past a focused light and better resolution of the blood cells is obtained. Coincident source (Figure 32-3). The light source is generally a tungsten- passage loss also is reduced because blood cells line up one halogen lamp or a helium-neon laser (light amplification by after another in the direction of the flow.15 Laminar flow and stimulated emission of radiation). Laser light, termed mono- hydrodynamic focusing are discussed further in Chapter 32. chromatic light because it is emitted at a single wavelength, differs from brightfield light in its intensity, its coherence Radiofrequency (i.e., it travels in phase), and its low divergence or spread. Low-voltage DC impedance, as described previously, may be These characteristics allow for the detection of interference in used in conjunction with RF resistance, or resistance to a high- the laser beam and enable enumeration and differentiation of voltage electromagnetic current flowing between both elec- cell types.12,17 Optical scatter may be used to study RBCs, trodes simultaneously. Although the total volume of the cell is WBCs, and platelets. proportional to the change in DC, the cell interior density is As the cells pass through the sensing zone and interrupt the proportional to pulse height or change in the RF signal. Con- beam, light is scattered in all directions. Light scatter results ductivity, as measured by this high-frequency electromagnetic from the interaction between the processes of absorption, probe, is attenuated by nucleus-to-cytoplasm ratio, nuclear diffraction (bending around corners or the surface of a cell), density, and cytoplasmic granulation. DC and RF voltage changes may be detected simultaneously and separated by two RF D I F F different pulse processing circuits.15,16 Figure 15-3 illustrates the simultaneous use of DC and RF current. Radiofrequency current Direct current RF signal DC DC supply Detection chamber Radiofrequency External supply electrode (+) Resistance Electrolyte Condenser solution DC signal Figure 15-4 Illustration of cell volume measurement with direct current Aperture Internal electrode (–) (DC) voltage change versus measurement of cell nuclear volume/complexity Figure 15-3 Radiofrequency/direct current (RF/DC) detection method, show- with change in the radiofrequency (RF) signal. The two measurements can be ing simultaneous use of DC and RF in one measurement system on the Sysmex plotted against each other to form a two-dimensional distribution scatterplot. SE-9500. (From TOA Medical Electronics Company: Sysmex SE-9500 operator’s (From TOA Medical Electronics Company: Sysmex SE-9500 operator’s manual [CN 461-2464-2], Kobe, Japan, 1997, TOA Medical Electronics Co.) manual [CN 461-2464-2], Kobe, Japan, 1997, TOA Medical Electronics Co.)  CHAPTER 15 Automated Blood Cell Analysis 211 refraction (bending because of a change in speed), and reflec- nucleated red blood cell, and WBC differential count deter- tion (backward scatter of rays caused by an obstruction).18 The mination on four major hematology instruments. detection of scattered rays and their conversion into electrical Hematology analyzers have some common basic compo- signals is accomplished by photodetectors (photodiodes and nents, including hydraulics, pneumatics, and electrical systems. photomultiplier tubes) at specific angles. Lenses fitted with The hydraulics system includes an aspirating unit, dispensers, blocker bars to prevent nonscattered light from entering the diluters, mixing chambers, aperture baths or flow cells or both, detector are used to collect the scattered light. A series of filters and a hemoglobinometer. The pneumatics system generates and mirrors separate the varying wavelengths and present them the vacuums and pressures required for operating the valves to the photodetectors. Photodiodes convert light photons to and moving the sample through the hydraulics system. The electronic signals proportional in magnitude to the amount of electrical system controls operational sequences of the total light collected. Photomultiplier tubes are used to collect the system and includes electronic analyzers and computing cir- weaker signals produced at a 90-degree angle and multiply the cuitry for processing the data generated. Some older-model photoelectrons into stronger, useful signals. Analogue-to-digital instruments have oscilloscope screens that display the electri- converters change the electronic pulses to digital signals for cal pulses in real time as the cells are counted. A data display computer analysis.12,17 unit receives information from the analyzer and prints results, Forward-angle light scatter (0 degrees) correlates with cell histograms, or cytograms. volume, primarily because of diffraction of light. Orthogonal Specimen handling varies from instrument to instrument light scatter (90 degrees), or side scatter, results from refraction based on degree of automation, and systems range from dis- and reflection of light from larger structures inside the cell and crete analyzers to walkaway systems with front-end load capabil- correlates with degree of internal complexity. Forward low- ity. Computer functions also vary, with the larger instruments angle scatter (2 to 3 degrees) and forward high-angle scatter having extensive microprocessor and data management capa- (5 to 15 degrees) also correlate with cell volume and refractive bilities. Computer software capabilities include automatic index or with internal complexity.17,19 Differential scatter is the start-up and shutdown, with internal diagnostic self-checks combination of this low-angle and high-angle forward light and some maintenance; quality control, with automatic review scatter and is primarily used on Siemens systems for cellular of quality control data, calculations, graphs, moving averages, analysis. The angles of light scatter measured by the different and storage of quality control files; patient data storage and flow cytometers are manufacturer and method specific. retrieval, with " checks (Chapter 5), critical value flagging, and Scatter properties at different angles may be plotted automatic verification of patient results based on user-defined against each other to generate two-dimensional cytograms or algorithms; host query with the laboratory or hospital infor- scatterplots, as on the Abbott CELL-DYN instruments.20,21 mation system to allow random access discrete testing capabil- Optical scatter may also be plotted against absorption, as ity; analysis of animal specimens; and even analysis of body on the Siemens systems,22,23 or against volume, as on the fluids. larger Beckman Coulter systems.9 Computer cluster analysis of the cytograms may yield quantitative and qualitative Beckman Coulter Instrumentation information. Beckman Coulter, Inc., manufactures an extensive line of he- matology analyzers, including the smaller Ac-T series that provide complete RBC, platelet, and WBC analysis with a five- PRINCIPAL INSTRUMENTS part differential. The LH 780 system, part of the LH 700 series, Overview provides a fully automated online reticulocyte analysis.27 The Hematology blood cell analyzers are produced by multiple LH series also has the capability to perform CD4 and CD8 manufacturers, including, but not limited to, Abbott Labo- counts.29 Coulter instruments typically have two measurement ratories (Abbott Park, IL);24 HORIBA Medical (Irvine, CA);25 channels in the hydraulics system for determining the hemo- Siemens Healthcare Diagnostics, Inc. (Deerfield, IL);26 Beck- gram data. The RBC and WBC counts and hemoglobin are man Coulter, Inc. (Brea, CA);27 and Sysmex Corporation considered to be measured directly. The aspirated whole-blood (Kobe, Japan).28 The following discussion is limited to sample is divided into two aliquots, and each is mixed with an instrumentation produced by four of these suppliers. isotonic diluent. The first dilution is delivered to the RBC aper- Emphasis is not placed on sample size or handling, speed, ture chamber, and the second is delivered to the WBC aperture level of automation, or comparison of instruments or man- chamber. In the RBC chamber, RBCs and platelets are counted ufacturers. Likewise, technology continues to improve, and and discriminated by electrical impedance as the cells are the newest (or most recent) models produced by a manufac- pulled through each of three sensing apertures (50 !m in turer may not be mentioned. Instead, a detailed description diameter, 60 !m in length). Particles 2 to 20 fL are counted as of primary methods used by these manufacturers is given to platelets, and particles greater than 36 fL are counted as RBCs. show the application of, and clarify further, the principles In the WBC chamber, a reagent to lyse RBCs and release hemo- presented earlier and to enable the medical laboratory globin is added before WBCs are counted simultaneously by scientist or technician to interpret patient data, including impedance in each of three sensing apertures (100 !m in di- instrument-generated histograms and cytograms. Table 15-1 ameter, 75 !m in length). Alternatively, some models employ summarizes methods used for the hemogram, reticulocyte, consecutive counts in the same RBC or WBC aperture. After 212 PART III Laboratory Evaluation of Blood Cells TABLE 15-1 Methods for Hemogram, Reticulocyte, Nucleated RBC, and WBC Differential Counts on Four Major Hematology Instruments Beckman Coulter UniCel Siemens ADVIA Parameter DxH 800 Sysmex XN Series Abbott CELL-DYN Sapphire 2120i WBC Impedance volume and con- Fluorescent staining; forward Light scatter (primary count), Light scatter and ductivity and five-angle light light scatter and side fluores- impedance (secondary count) absorption scatter measurement cent light detection RBC Impedance Impedance Impedance Low-angle and high- angle laser light scatter HGB Modified cyanmethemoglobin Sodium lauryl sulfate-HGB Modified cyanmethemoglobin Modified cyanmethemo- (525 nm) (555 nm) (540 nm) globin (546 nm) HCT (RBC # MCV)/10 Cumulative RBC pulse height (RBC # MCV)/10 (RBC # MCV)/10 detection MCV Mean of RBC volume (HCT/RBC) # 10 Mean of RBC volume distribution Mean of RBC volume distribution histogram histogram distribution histogram MCH (HGB/RBC) # 10 (HGB/RBC) # 10 (HGB/RBC) # 10 (HGB/RBC) # 10 MCHC (HGB/HCT) # 100 (HGB/HCT) # 100 (HGB/HCT) # 100 (HGB/HCT) # 100 Platelet count Impedance volume and con- Impedance; light scatter; fluores- Dual angle light scatter analysis; Low-angle and high- ductivity and five-angle light cent staining, forward light impedance count for verification; angle light scatter; scatter measurement scatter, and side fluorescent optional CD61 monoclonal refractive index light detection antibody count RDW RDW as CV (%) of RBC histo- RDW-SD (fL) or RDW-CV (%) Relative value, equivalent to CV CV (%) of RBC histogram gram or RDW-SD (fL) Reticulocyte Supravital staining; Fluorescent staining; forward light Fluorescent staining; low-angle Supravital staining count impedance volume and scatter and side fluorescent scatter, and fluorescent light (oxazine 750); low- conductivity and light scatter light detection detection angle and high-angle measurement light scatter and absorbance NRBC* Impedance volume and con- Fluorescent staining; forward light Red fluorescent dye staining; Multi-angle light scatter ductivity and five-angle light scatter and side fluorescent forward light scatter and fluores- measurements in the scatter measurement light detection cent light detection two WBC differential channels WBC differential Impedance volume and con- Fluorescent staining; forward and Multi-angle polarized scatter sepa- Peroxidase staining, light ductivity and five-angle light side light scatter, and side ration (MAPSS) and three-color scatter and absorp- scatter measurement fluorescent light detection fluorescence detection tion; for basos, differ- ential lysis, low-angle and high-angle laser light scatter CV, Coefficient of variation; DC, direct current; HCT, hematocrit; HGB, hemoglobin; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume; NRBC, nucleated red blood cell count; RBC, red blood cell (or count); RDW, RBC distribution width; SD, standard deviation; VCS, volume, conductivity, scatter; WBC, white blood cell (or count). *Instruments auto-correct the WBC count for the presence of nucleated RBCs. counting cycles are completed, the WBC dilution is passed 64 channels are used for platelet analysis. Volume-distribution to the hemoglobinometer for determination of hemoglobin histograms of WBC, RBC, and platelet populations are gener- concentration (light transmittance read at a wavelength of ated. The RBC mean cell volume (MCV) is the average volume 525 nm). Electrical pulses generated in the counting cycles are of the RBCs taken from the volume distribution data. The he- sent to the analyzer for editing, coincidence correction, and matocrit (HCT), mean cell hemoglobin (MCH), and mean cell digital conversion. Two of the three counts obtained in the hemoglobin concentration (MCHC) are calculated from mea- RBC and the WBC baths must match within specified limits for sured and derived values. The RBC distribution width (RDW) the counts to be accepted by the instrument.5,9 This multiple is calculated directly from the histogram as the coefficient of counting procedure prevents data errors resulting from aper- variation (CV) of the RBC volume distribution, with a refer- ture obstructions or statistical outliers and allows for excellent ence interval of 11.5% to 14.5%.5 The RDW is an index of an- reproducibility on the Beckman Coulter instruments. isocytosis, but it may be falsely skewed because it reflects the Pulse height is measured and categorized by pulse height ratio of the standard deviation (SD) to MCV. That is, an RBC analyzers; 256 channels are used for WBC and RBC analysis, and distribution histogram with normal divergence but a decreased  CHAPTER 15 Automated Blood Cell Analysis 213 MCV may imply a high RDW, falsely indicating increased immature granulocytes, and reactive lymphocytes); and parti- anisocytosis. MCV and RDW are used by the instrument to flag cles between 160 and 450 fL are considered granulocytes. This possible anisocytosis, microcytosis, and macrocytosis.9 allows the calculation of relative and absolute numbers for Platelets are counted within the range of 2 to 20 fL, and a these three populations (Figure 15-5).6 Proprietary computer- volume-distribution histogram is constructed. If the platelet ized algorithms further allow flagging for increased eosino- volume distribution meets specified criteria, a statistical least- phils or basophils or both and interpretation of the histogram squares method is applied to the raw data to fit the data to a differential, including flagging for abnormal cells, such as reac- log-normal curve. The curve is extrapolated from 0 to 70 fL, tive lymphocytes and blasts.7 When cell populations overlap or and the final count is derived from this extended curve. This a distinct separation of populations does not exist, a region fitting procedure eliminates interference from particles in the alarm (R flag) may be triggered that indicates the area of inter- noise region, such as debris, and in the larger region, such as ference on the volume-distribution histogram. An R1 flag rep- small RBCs. The mean platelet volume (MPV), analogous to resents excess signals at the lower threshold region of the WBC the RBC MCV, also is derived from the platelet histogram. The histogram and a questionable WBC count. This interference is reference interval for the MPV is about 6.8 to 10.2 fL. The MPV visualized as a high takeoff of the curve and may indicate the increases slightly with storage of the specimen in ethylenedi- presence of nucleated RBCs, clumped platelets, unlysed RBCs, aminetetraacetic acid (EDTA).5 or electronic noise.6,7 Many older-model Beckman Coulter instruments, such as More recent Beckman Coulter instruments, the LH 700 the STKR, and the newer, smaller models, such as the Ac-T se- Series and UniCel DxH series, generate hemogram data (in- ries, provide three-part leukocyte subpopulation analysis, cluding the WBC count) as before but use Coulter’s proprietary which differentiates WBCs into lymphocytes, mononuclear VCS (volume, conductivity, scatter) technology in a separate chan- cells, and granulocytes. In the WBC channel, a special lysing nel to evaluate WBCs for the determination of a five-part dif- reagent causes differential shrinkage of the leukocytes, which al- ferential. The VCS technology includes the volumetric sizing of lows the different cells to be counted and volumetrically sized cells by impedance, conductivity measurements of cells, and based on their impedance. A WBC histogram is constructed laser light scatter, all performed simultaneously for each cell. from the channelized data. Particles between approximately After RBCs are lysed and WBCs are treated with a stabilizing 35 and 90 fL are considered lymphocytes; particles between reagent to maintain them in a near-native state, a hydrody- 90 and 160 fL are considered mononuclears (monocytes, blasts, namically focused sample stream is directed through the flow REL No. LYMPHOCYTOSIS WBC 4.3 WBC 9.0 NORMAL DISTRIBUTION SUSPECT: EOS IMM GRANS LY 36.7 LY 51.3 R2 ATYP LYMPHS BLASTS MO 9.2 MO 6.6 R2 GR 54.1 GR 42.1 RM EO# 0.7 EO# BA# 0.2 BA# 0.2 WBC 50 100 200 300 400 50 100 200 300 400 NORMAL DISTRIBUTION RBC 3.98 ANISO RBC 3.84 HGB 12.9 HGB 10.6 HCT 37.0 HCT 31.0 MCV 93.0 MCV 80.6 MCH 32.4 MCH 27.6 MCHC 34.9 MCHC 34.2 H RDW 15.0 RDW 19.9 RBC 50 100 200 300 50 100 200 300 NORMAL DISTRIBUTION SUSPECT: ABNORMAL DISTRIBUTION PLT 418. PLT 26. R L MPV 7.0 MPV 6.9 R A PLT 2 10 20 FEMTOLITERS B 2 10 20 FEMTOLITERS Figure 15-5 Printouts from the Coulter STKR showing the interpretive differential. A, Note the three distinct white blood cell (WBC) populations, Gaussian or normal distribution of red blood cells (RBCs), and right-skewed or log-normal distribution of platelets. B, Note the left shift in the WBC histogram with pos- sible interference at the lower threshold region. R2 flag indicates interference and loss of valley owing to overlap or insufficient separation between the lym- phocyte and mononuclear populations at the 90-fL region. RM flag indicates interference at more than one region. Eosinophil data have been suppressed. Also note the abnormal platelet volume distribution with a low platelet count. Manual 200-cell differential counts on the same samples: A, 52.5% neutrophils (47% segmented neutrophils, 5.5% bands), 41.5% lymphocytes, 4.0% monocytes, 1% basophils, 0.5% metamyelocytes, 0.5% reactive lymphocytes; B, 51% neutrophils (23% segmented neutrophils, 28% bands), 12% lymphocytes, 9.5% monocytes, 1% metamyelocytes, 1.5% myelocytes, 25% reactive lymphocytes, and 17 nucleated RBCs/100 WBCs. 214 PART III Laboratory Evaluation of Blood Cells cell past the sensing zone. Low-frequency DC measures cell resolution. On the LH 700 series, Coulter utilizes an IntelliKinetics volume, whereas a high-frequency electromagnetic probe mea- application. This application is used to ensure consistency sures conductivity, an indicator of cellular internal content. with the kinetic reactions. It provides the instrument the best The conductivity signal is corrected for cellular volume, which signals for analysis independent of laboratory environment yields a unique measurement called opacity. Each cell also is variations. Compared with earlier models Coulter IntelliKinetics scanned with monochromatic laser light that reveals informa- provides better separation of cell populations for WBCs tion about the cell surface, such as structure, shape, and reflec- and reticulocytes, which enables better analysis by the system tivity. Beckman Coulter’s unique rotated light scatter detection algorithms.33,34 method, which covers a 10-degree to 70-degree range, allows The UniCel DxH 800 also includes the number of nucleated for separation of cells with similar volume but different scatter RBCs as part of the standard CBC report. They are identified, characteristics.27 Beckman Coulter’s newest analyzer, the Uni- counted, and subtracted from the white blood cell count using Cel DxH 800, uses volume and conductivity as well as five volume, conductivity, and the same five light scatter measure- additional parameters: axial light loss (AL2), low-angle light ment described above. The AL2 measurement (which reflects scatter (LALS), median-angle light scatter (MALS), lower me- the amount of light absorbed as it passes through the flow cell) dian–angle light scatter (LMALS), and upper median–angle initially separates the nucleated RBCs from the WBCs. Algo- light scatter (UMALS).30,31 Using the data collected by the rithms are applied using the scatter from the other angles to parameters listed above, the instrument applies data transfor- electronically separate and count the nucleated RBCs. Two scat- mation, the process by which populations of cells are separated, terplots display the nucleated RBC data by plotting axial light allowing the determination of major populations as well as the loss (AL2) on the x-axis against low-angle light scatter (RLALS) enhancement of subpopulations of cells. Once those popula- and upper-median angle light scatter (RUMALS) on y-axis. tions are established, a technique called the watershed concept Figure 15-6 represents a standard patient printout from the searches for those populations and aids in determining counts Beckman Coulter UniCel DxH 800. as well as flagging based on all the populations found for that sample.30,31 Sysmex Instrumentation This combination of technologies provides a three- Sysmex Corporation, formerly TOA Medical Electronics Com- dimensional plot or cytograph of the WBC populations, which pany, Ltd., manufactures a full line of hematology analyzers are separated by computer cluster analysis. Two-dimensional that provide complete RBC, platelet, and WBC analysis with scatterplots of the measurements represent different views of three-part differential; the larger XT-1800i (SF-3000 and SE- the cytograph. The scatterplot of volume (y-axis) versus light 9000) that performs a CBC with five-part differential; and the scatter (x-axis) shows clear separation of lymphocytes, mono- XE series and the newest XN series that also provide a fully cytes, neutrophils, and eosinophils. Basophils are hidden be- automated reticulocyte count.28,33 The newest XN series is hind the lymphocytes but are separated by conductivity owing modular. The series is scalable, and multiple modules can be to their cytoplasmic granulation. Single-parameter histograms combined onto one platform. Each module contains the of volume, conductivity, and light scatter also are available.9 XN-CBC and XN-DIFF with other options available, including Two types of WBC flags (alarms or indicators of abnormal- XN-BF, the body fluid application. Included standard on the ity) are generated on all hematology analyzers that provide a CBC and DIFF modules are NRBCs, RET-He (reticulocyte he- WBC differential count: (1) user defined, primarily set for dis- mogloblin), and IRF (immature reticulocyte fraction). The tributional abnormalities, such as eosinophilia or lymphocyto- platelet analysis on the XN also utilizes a fluorescent count, in penia (based on absolute eosinophil or lymphocyte counts); addition to the impedance count and optical count, called the and (2) instrument specific, primarily suspect flags for mor- PLT-F, performed by optical measurement.35-38 The PLT-F can phologic abnormalities. For distributional flags, the user estab- be performed on each sample or set up as a reflex based on the lishes reference intervals and programs the instrument to flag laboratory’s PLT criteria. The method uses a fluorocell fluores- each parameter as high or low. Suspect flags indicating the pos- cent dye (oxazine) combined with an extended PLT counting sible presence of abnormal cells are triggered when cell popu- volume and time. The PLTs can be differentiated from other lations fall outside expected regions or when specific statistical cells based on differences in intensity of the fluorescence limitations are exceeded. Instrument-specific suspect flags on combined with forward scattered light.35 The WBC, RBC, the Coulter UniCel DxH 800 system and LH 700 series include platelet counts, hemoglobin, and hematocrit are considered immature granulocytes/bands, blasts, variant lymphocytes, to be measured directly. Three hydraulic subsystems are used nucleated RBCs, and platelet clumps. The UniCel DxH 800 for determining the hemogram: the WBC channel, the RBC/ also utilizes the International Society for Laboratory Hematol- platelet channel, and a separate hemoglobin channel. In the ogy (ISLH) consensus rules in addition to the user defined and WBC and RBC transducer chambers, diluted WBC and RBC system defined flags for complete data analysis.32,33 In addition samples are aspirated through the different apertures and to the flags listed above, inadequate separation of cell popula- counted using the impedance (DC detection) method for tions may disallow reporting of differential results by the in- counting and volumetrically sizing cells. Two unique features strument and may elicit a review slide message.9,33,34 enhance the impedance technology: in the RBC/platelet chan- The UniCel DxH 800 system utilizes VCS as well as digital nel, a sheathed stream with hydrodynamic focusing is used signal processing from five light scatter angles for clear cellular to direct cells through the aperture, which reduces coincident  CHAPTER 15 Automated Blood Cell Analysis 215 Beckman Coulter UniCel DxH 800 Test Result Flags Units D WBC Suspect A WBC 9.143 10^3/uL F UWBC 9.143 10^3/uL RBC 3.931 10^6/uL HGB 11.17 g/dL HCT 32.96 % 35 100 200 300 400 fL MCV 83.85 fL RBC MCH 28.41 pg MCHC 33.88 g/dL RDW 14.32 % RDW-SD 40.69 fL PLT 223.0 10^3/uL 36 100 200 300 fL MPV 9.46 fL PLT B NE 67.14 % LY 21.77 % MO 9.57 % EO 0.78 % BA 0.74 % 2 10 20 30 fL NE# 6.140 10^3/uL 1.990 E 5PD1 NRBC1 RETIC1 LY# 10^3/uL V R V MO# 0.875 10^3/uL L A L EO# 0.071 10^3/uL S BA# 0.067 10^3/uL NRBC 0.10 /100WBC NRBC# 0.009 10^3/uL C RET 1.858 % RLSn AL2 LLSn RET# 0.07305 10^6/uL 5PD2 NRBC2 RETIC2 MRV 100.72 fL V R U V M IRF 0.387 A L S OP AL2 OP Figure 15-6 Coulter UniCel DxH 800. The DxH 800 printout displays the CBC, DIFF and reticulocyte data for the same patient in Figures 15-7, 15-9, and 15-12. A, CBC data; B, Differential with the nucleated red blood cells (NRBCs); C, Reticulocyte data, including the IRF (immature reticulocyte fraction); D, Impedance histograms for the WBC, RBC, and PLT; E, Advanced two-dimensional optical scatterplots for WBCs, NRBCs, and reticulocytes; F, Suspect area in which any sample or system flags will display. passage, particle volume distortion, and recirculation of blood distribution. Likewise, the RBC lower and upper thresholds cells around the aperture; and in the WBC and RBC/platelet may be set in the 25- to 75-fL and 200- to 250-fL volume channels, floating thresholds are used to discriminate each cell ranges. This floating threshold circuitry allows for discrimina- population.8,15,16 tion of cell populations on a sample-by-sample basis. Cell As cells pass through the apertures, signals are transmitted counts are based on pulses between the lower and upper auto- in sequence to the analogue circuit and particle volume distri- discriminator levels, with dilution ratio, volume counted, and bution analysis circuits for conversion to cumulative cell vol- coincident passage error accounted for in the final computer- ume distribution data. Particle volume distribution curves are generated numbers. In the RBC channel, the floating discrimi- constructed, and optimal position of the autodiscrimination nator is particularly useful in separating platelets from small level (i.e., threshold) is set by the microprocessor for each cell RBCs. The hematocrit also is determined from the RBC/platelet population. The lower platelet threshold is automatically ad- channel, based on the principle that the pulse height generated justed in the 2- to 6-fL volume range, and the upper threshold by the RBC is proportional to cell volume. The hematocrit is adjusted in the 12- to 30-fL range, based on particle volume is the RBC cumulative pulse height and is considered a true 216 PART III Laboratory Evaluation of Blood Cells relative percentage volume of erythrocytes.8,15 In the hemoglo- In the XN-1000, fluorescent flow cytometry is used for the bin flow cell, hemoglobin is oxidized and binds to sodium WBC count, WBC differential, and enumeration of nucleated lauryl sulfate (SLS) forming a stable SLS–hemoglobin com- RBCs. In the WDF channel, RBCs are lysed, WBC membranes plex, which is measured photometrically at 555 nm.16 are perforated, and the DNA and RNA in the WBCs are stained The following indices are calculated in the microprocessor with a fluorescent dye. Plotting side scatter on the x-axis using directly measured or derived parameters: MCV, MCH, and side fluorescent light on the y-axis enables separation and MCHC, RDW-SD, RDW-CV, MPV, and plateletcrit. RDW-SD is enumeration of neutrophils, eosinophils, lymphocytes, mono- the RBC arithmetic distribution width measured at 20% of the cytes, and immature granulocytes. In the WNR channel, the height of the RBC curve, reported in femtoliters, with a refer- RBCs are lysed including nucleated RBCs, and WBC mem- ence interval of 37 to 54 fL. RDW-CV is the RDW reported as a branes are perforated. A fluorescent polymethine dye stains the CV. Plateletcrit is the platelet volume ratio, analogous to the nucleus and organelles of the WBCs with high fluorescence hematocrit. MPV is calculated from the plateletcrit and platelet intensity and stains the released nuclei of the nucleated RBCs count just as erythrocyte MCV is calculated from the hemato- with low intensity. Plotting side fluorescent light on the x-axis crit and RBC count. The proportion of platelets greater than and forward scatter on the y-axis enables separation and enu- 12 fL in the total platelet count may be an indicator of possible meration of the total WBC count, basophils, and nucleated platelet clumping, giant platelets, or cell fragments.8,15,16 RBCs. The WBC count is automatically corrected when nucle- The XE series has the capability to run the platelet counting in ated RBCs are present in the sample. A WPC channel detects the optical mode, which eliminates common interferences blasts and abnormal lymphocytes in a similar manner using a found with impedance counting. In the optical mode, the, im- lysing agent and fluorescent dye and plotting side scatter on mature platelet fraction or IPF, can be measured to provide the x-axis and side fluorescent light on the y-axis. Figure 15-7 additional information concerning platelet kinetics in cases of shows a patient report from the Sysmex XN-1000 analyzing the thromobocytopenia.39 same patient specimen for which data are given in Figure 15-6. The SE-9000/9500 uses four detection chambers to analyze WBCs and obtain a five-part differential: the DIFF, IMI (imma- Abbott Instrumentation ture myeloid information), EO, and BASO chambers. The high- Instruments offered by Abbott Laboratories include the smaller end instrumentation such as the XE-series and the XN series CELL-DYN Emerald, which provides complete RBC, platelet, has a six-part differential: neutrophils, lymphocytes, mono- and WBC analysis with three-part differential, and the larger cytes, eosinophils, basophils, and immature granulocytes. Every CELL-DYN Sapphire and the midrange CELL-DYN Ruby, both differential performed generates a percentage and absolute of which provide a CBC with five-part differential and random number for immature granulocytes, thus providing valuable fully automated reticulocyte analysis.33 The CELL-DYN 4000 information about the complete differential.28 In the DIFF de- system has three independent measurement channels for de- tection chamber, RBCs are hemolyzed and WBCs are analyzed termining the hemogram and differential: an optical channel simultaneously by low-frequency DC and high-frequency cur- for WBC count and differential data, an impedance channel for rent (DC/RF detection method). A scattergram of RF detection RBC and platelet data, and a hemoglobin channel for hemo- signals (y-axis) versus DC detection signals (x-axis) allows globin determination.20,21 The WBC, RBC, hemoglobin, and separation of the WBCs into lymphocytes, monocytes, and platelet parameters are considered to be measured directly. A granulocytes. Floating discriminators determine the optimal 60- to 70-!m aperture is used in the RBC/platelet transducer separation between these populations. Granulocytes are ana- assembly for counting and volumetrically sizing of RBCs and lyzed further in the IMI detection chamber to determine im- platelets by the electronic impedance method. mature myeloid information. RBCs are lysed, and WBCs other A unique von Behrens plate is located in the RBC/platelet than immature granulocytes are selectively shrunk by tempera- counting chamber to minimize the effect of recirculating cells. ture and chemically controlled reactions. Analysis of the treated Pulses are collected and sorted in 256 channels according to sample using the DC/RF detection method allows separation their amplitudes: particles between 1 and 35 fL are included in of immature cells on the IMI scattergram. A similar differential the initial platelet data, and particles greater than 35 fL are shrinkage and lysis method is also used in the EO and BASO counted as RBCs. Floating thresholds are used to determine the chambers. That is, eosinophils and basophils are counted by best separation of the platelet population and to eliminate in- impedance (DC detection) in separate chambers in which the terference, such as noise, debris, or small RBCs, from the count. RBCs are lysed, and WBCs other than eosinophils or basophils Coincident passage loss is corrected for in the final RBC and are selectively shrunk by temperature and chemically con- platelet counts. RBC pulse editing is applied before MCV deri- trolled reactions. Eosinophils and basophils are subtracted vation to compensate for aberrant pulses produced by nonax- from the granulocyte count derived from the DIFF scattergram ial passage of RBCs through the aperture. The MCV is the aver- analysis to determine the neutrophil count. User-defined dis- age volume of the RBCs derived from RBC volume distribution tributional flags may be set, and instrument-specific suspect data. Hemoglobin is measured directly using a modified hemi- flags, similar to those described for the Beckman Coulter LH globincyanide method that measures absorbance at 540 nm. 700 series, are triggered for the possible presence of morpho- Hematocrit, MCH, and MCHC are calculated from the directly logic abnormalities.15,16 A POSITIVE or NEGATIVE interpretive measured or derived parameters. The RDW, equivalent to CV, message is displayed. is a relative value, derived from the RBC histogram by using the  CHAPTER 15 Automated Blood Cell Analysis 217 Sysmex XN-1000 Sample comments: D Negative WDF WNR SFL FSC A WBC 9.04 [10^3/uL] RBC 3.84 [10^6/uL] HGB 11.0 [g/dL] HCT 32.3 [%] MCV 84.1 [fL] MCH 28.6 [pg] SSC SFL MCHC 34.1 [g/dL] PLT &F 240 [10^3/uL] RDW-SD 45.1 [fL] RDW-CV 14.8 [%] MPV 11.1 [fL] B NRBC 0.00 [10^3/uL] 0.0 [%] NEUT 6.01 [10^3/uL] 66.4 [%] LYMPH 2.06 [10^3/uL] 22.8 [%] MONO 0.83 + [10^3/uL] 9.2 [%] E EO 0.07 [10^3/uL] 0.8 [%] RET PLT-F BASO 0.05 [10^3/uL] 0.6 [%] FSC FSC IG 0.02 [10^3/uL] 0.2 [%] RET 2.08 [%] 0.0799 [10^6/uL] IRF 10.6 [%] C RET-He 33.1 [pg] IPF 5.0 [%] SFL SFL WBC-BF [/uL] RBC PLT RBC-BF [10^3/uL] MN [/uL] [%] PMN [/uL] [%] TC-BF# [/uL] 250 fL 40 fL F WBC IP Message RBC IP Message PLT IP Message Figure 15-7 Sysmex XN-1000. The XN-1000 printout displays the CBC, DIFF, and reticulocyte data for the same patient in Figures 15-6, 15-9, and 15-12. A, CBC data; note the “&F” to indicate the fluorescent platelet count result; B, Nucleated red blood cells (NRBCs), six-part differential, including the IG (immature granulocyte), reticulocyte count (RET), and immature reticulocyte fraction (IRF); C, Reticulocyte hemoglobin (RET-He), immature platelet fraction (IPF), and body fluid counts, if done; D, Two scatterplots, WDF (lymphocytes, monocytes, neutrophils, eosinophils, and immature granulocytes) and WNR (WBC count, basophils, and nucleated RBCs); E, Reticulocyte (RET) and platelet (PLT-F) scatterplots, and RBC and PLT impedance histograms; F, Sample-related flags are listed at the bottom of the printout, if any are generated. 20th and 80th percentiles. The platelet analysis is based on scatter separation (MAPSS) technology with three-color fluo- a two-dimensional optical platelet count using fluorescent rescent technology. A hydrodynamically focused sample technology, the same technology used for direct nucleated stream is directed through a quartz flow cell past a focused RBC counting by adding a red fluorescence to the sample light source, an argon ion laser. Scattered light is measured at to stain nucleated red cells.40 Further analysis of platelets multiple angles: 0-degree forward light scatter measurement is and platelet aggregates can be performed by using an auto- used for determination of cell volume, 90-degree orthogonal mated CD61 monoclonal antibody to generate an immuno- light scatter measurement is used for determination of cellular platelet count.41-43 Other indices available include MPV and lobularity, 7-degree narrow-angle scatter measurement is used plateletcrit.20,21 to correlate with cellular complexity, and 90-degree depolar- The WBC count and differential are derived from the optical ized light scatter measurement is used for evaluation of channel using CELL-DYN’s patented multiangle polarized cellular granularity. Orthogonal light scatter is split, with one 218 PART III Laboratory Evaluation of Blood Cells portion directed to a 90-degree photomultiplier tube and the each angle of light measured, and graphically presented as other portion directed through a polarizer to the 90-degree scatterplots. Scatter information from the different angles is depolarized photomultiplier tube. Light that has changed plotted in various combinations: 90 degrees/7 degrees, or polarization (depolarized) is the only light that can be de- lobularity versus complexity; 0 degrees/7 degrees, volume tected by the 90-degree depolarized photomultiplier tube. versus complexity; and 90 degrees depolarized/90 degrees, Various combinations of these four measurements are used to granularity versus lobularity. Lobularity or 90-degree scatter differentiate and quantify the five major WBC subpopula- (y-axis) plotted against complexity or 7-degree scatter (x-axis) tions: neutrophils, lymphocytes, monocytes, eosinophils, and yields separation of mononuclear and segmented (polymor- basophils.20,40,44 Figure 15-8 illustrates CELL-DYN’s MAPSS phonuclear neutrophil) subpopulations. Basophils cluster technology. with the mononuclears in this analysis, because the basophil The light scatter signals are converted into electrical sig- granules dissolve in the sheath reagent, and the degranulated nals, sorted into 256 channels on the basis of amplitude for basophil is a less complex cell. Each cell in the two clusters Various angles Focused of scattered light laser beam 90º Scatter 0º Scatter 90ºD Scatter 10º Scatter A B C D Figure 15-8 A, Multiangle polarized scatter separation (MAPSS) technology. Cells are measured and characterized by plotting light scatter from four differ- ent angles. B, Mononuclear and polymorphonuclear scatter with MAPPS technology. It plots 10 degree scatter (complexity) on the x-axis and 90 degree scatter (lobularity) on the y-axis. The system uses algorithms to further separate the two populations, displaying mononuclear on the lower left and polymorphonuclear on the upper right. C, Separation and plotting of the polymorphonuclear cells into neutrophils and eosinophils based on MAPPS technology. It plots 90 degree scatter (lobularity) on the x-axis and 90 degree depolarized (90 D) scatter on the y-axis. The system uses algorithms to further separate the two populations of cells. D, Scatter of all WBC populations by MAPPS technology plotting 10 degree scatter (complexity) on the x-axis and 0 degree scatter (size or volume) on the y-axis. On the newer instruments, a 7-degree angle for complexity is now used instead of the 10-degree angle. The change reflects use of the midrange of the angle instead of the end range; however, it still provides the same information. (From Abbott Laboratories: CELL-DYN 3700 system operator’s manual [914032C], Abbott Park, IL, 2000.)  CHAPTER 15 Automated Blood Cell Analysis 219 is identified as a mononuclear or segmented neutrophil for as cells, in a sheath-stream, pass through a flow cell by a laser further evaluation. optical assembly (laser diode light source). RBCs and platelets The mononuclear subpopulation is plotted on a 0-degree/ are isovolumetrically sphered before entering the flow cell to 7-degree scatterplot, with volume on the y-axis and complexity eliminate optical orientation noise. Laser light scattered at on the x-axis. Three populations (lymphocytes, monocytes, two different angular intervals—low angle (2 to 3 degrees), and basophils) are seen clearly on this display. Nucleated correlating with cell volume, and high angle (5 to 15 degrees), RBCs, unlysed RBCs, giant platelets, and platelet clumps fall correlating with internal complexity (i.e., refractive index or below the lymphocyte cluster on this scatterplot and are ex- hemoglobin concentration)—is measured simultaneously cluded from the WBC count and differential. Information from (Figure 15-10). This differential scatter technique, in combina- the WBC impedance channel also is used in discriminating tion with isovolumetric sphering, eliminates the adverse effect these particles.21 of variation in cellular hemoglobin concentration on the deter- The segmented neutrophil subpopulation is plotted on a mination of RBC volume (as seen by differences in cellular 90-degree depolarized/90-degree scatterplot, with granularity deformability affecting the pulse height generated on imped- or 90-degree depolarized scatter on the y-axis and lobularity or ance instruments).10,48 The Mie theory of light scatter of dielec- 90-degree scatter on the x-axis. Because of the unique nature of tric spheres18 is applied to plot scatter-intensity signals from eosinophil granules, eosinophils scatter more 90-degree depo- the two angles against each other for a cell-by-cell RBC volume larized light, which allows clear separation of eosinophils and (y-axis) versus hemoglobin concentration (x-axis) cytogram or neutrophils on this display. Dynamic thresholds are used for RBC map (Figure 15-11).19 best separation of the different populations in the various scat- Independent histograms of RBC volume and hemoglobin terplots. Each cell type is identified with a distinct color, so that concentration also are plotted. On the ADVIA 2120 and 120 after all classifications are made and volume (0-degree scatter) platelets are counted and volumetrically sized using a two- is plotted on the y-axis against complexity (7-degree scatter) on dimensional (low-angle and high-angle) platelet analysis, the x-axis, each cell population can be visualized easily by which allows better discrimination of platelets from interfering the operator on the data terminal screen. Other scatterplots particles, such as RBC fragments and small RBCs.22 Larger (90 degrees/0 degrees, 90 degrees depolarized/0 degrees, platelets can be included in the platelet count.23,47 90 degrees depolarized/7 degrees) are available and may be Several parameters and indices are derived from the mea- displayed at operator request. On earlier instruments, the 7-de- surements described in the previous paragraph. MCV and MPV gree angle measurement for complexity was referred to as the are the mean of the RBC volume histogram and the platelet 10-degree angle. The change reflects use of the midrange of the volume histogram. Hematocrit, MCH, and MCHC are mathe- angle instead of the end range; however, it still provides matically computed using RBC, hemoglobin, and MCV values. the same information.45-46 As on the previously described RDW is calculated as the CV of the RBC volume histogram, instruments, user-defined distributional flags may be set, and whereas hemoglobin distribution width (HDW), an analogous instrument-specific suspect flags may alert the operator to index, is calculated as the SD of the RBC hemoglobin concen- the presence of abnormal cells.20,45 Figure 15-9 represents a tration histogram. The reference interval for HDW is 2.2 to patient printout from the CELL-DYN Sapphire analyzing the 3.2 g/dL. Cell hemoglobin concentration mean (CHCM), same patient specimen for which data are given in Figures 15-6 analogous to MCHC, is derived from cell-by-cell direct mea- and 15-7. sures of hemoglobin concentration. Interferences with the hemoglobin colorimetric method, such as lipemia or icterus, Siemens Healthcare Diagnostics affect the calculated MCHC but do not alter measured Instrumentation CHCM. CHCM generally is not reported as a patient result Siemens Healthcare Diagnostics Inc. manufactures the ADVIA but is used by the instrument as an internal check for the 2120 and 2120i, the next generation of the ADVIA 120.26,33,47 MCHC and is available to the operator for calculating the Siemens has simplified the hydraulics and operations of the cellular hemoglobin if interferences are present. Unique RBC analyzer by replacing multiple complex hydraulic systems with flags derived from CHCM include hemoglobin concentration a unified fluids circuit assembly, or Unifluidics technology. The variance (HC VAR), hypochromia (HYPO), and hyperchromia ADVIA 2120, 2120i, and 120 provide a complete hemogram (HYPER).22,23 and WBC differential, while also providing a fully automated Siemens hematology analyzers determine WBC count and a reticulocyte count.22,23 six-part WBC differential (neutrophils, lymphocytes, mono- Four independent measurement channels are used in deter- cytes, eosinophils, basophils, and large unstained cells [LUCs]) mining the hemogram and differential: RBC/platelet channel, by cytochemistry and optical flow cytometry, using the PEROX hemoglobin channel, and peroxidase (PEROX) and basophil- and BASO channels. LUCs include reactive or variant lympho- lobularity (BASO) channels for WBC and differential data. cytes and blasts. WBC, RBC, hemoglobin, and platelets are measured directly. Hemoglobin is determined using a modified cyanmethemo- Peroxidase (PEROX) Channel globin method that measures absorbance in a colorimeter flow In the PEROX channel, RBCs are lysed, and WBCs are stained cuvette at approximately 546 nm. The RBC/platelet method for their peroxidase activity. The following reaction is catalyzed uses flow cytometric light scattering measurements determined by cellular peroxidase, which converts the substrate to a dark 220 PART III Laboratory Evaluation of Blood Cells Abbott CELL-DYN Sapphire D WBC Differential NEU - EOS 90DGRNLRTY WBC 9.09 10e3/uL NEU 6.14 %N 67.5 B LYM 2.11 %L 23.2 0 SIZE MONO.677 %M 7.44 EOS.074 %E.810 BASO.092 %B 1.01 7º - COMPLEXITY 90º - LOBULARITY Low-Hi FL RETC A RBC 3.95 10e3/uL HGB 11.1 g/dL HCT 32.1 % MCV 81.1 fL MCH 28.2 pg FL1 RNA MCHC 34.7 g/dL 0 SIZE RDW 13.4 % C RETC 77.7 10e3/uL %R 1.97 IRF.249 FL3 - DNA 7º NRBC 0.00 10e3/uL NR/W 0.00 Impedance PLT Optical PLT PLT 267. E 10e3/uL MPV 8.26 fL 90 Volume (fL) 7º F Interpretive Report WBC RBC/RETC PLT Figure 15-9 CELL-DYN Sapphire. The Sapphire printout displays the CBC, DIFF, and reticulocyte data for the same patient in Figures 15-6, 15-7, and 15-12. A, CBC data; B, Differential count data. Note that the WBC is listed with the differential instead of the CBC data; C, Reticulocyte and nucleated RBC data dis- played under the CBC data but before the PLT data; D, Two scattergrams for the differential; both 7-degree scatter (complexity) vs 0-degree scatter (size or volume) and 90-degree scatter (lobularity) vs 90-degree depolarized (90D) scatter (granularity) are plotted for the WBCs; two histograms are also plotted for the nucleated RBC and reticulocyte data; E, Impedance histogram and optical platelet scatterplot side by side; F, At the bottom where the interpretative report flags display if there are any for the sample. precipitate in peroxidase-containing cells (neutrophils, mono- signals in this channel and is used as an internal check of cytes, and eosinophils): the primary WBC count obtained in the basophil-lobularity channel (WBC-BASO). If significant interference occurs in the H2O2 $ 4-chloro-1-naphthol ucellular peroxid daser uuuuuuuuuuuuuuuuu dark precipitate WBC-BASO count, the instrument substitutes the WBC-PEROX value.23 A portion of the cell suspension is fed to a sheath-stream Computerized cluster analysis allows classification of the flow cell where a tungsten-halogen darkfield optics system is different cell populations, including abnormal clusters such as used to measure absorbance (proportional to the amount of nucleated RBCs and platelet clumps. Nucleated RBCs are ana- peroxidase in each cell) and forward scatter (proportional to lyzed for every sample using four counting algorithms, which the volume of each cell). Absorbance is plotted on the x-axis permits the system to choose the most accurate count based on of the cytogram, and scatter is plotted on the y-axis.22,23 A total internal rules and conditions. Neutrophils and eosinophils WBC count (WBC-PEROX) is obtained from the optical contain the most peroxidase and cluster to the right on  CHAPTER 15 Automated Blood Cell Analysis 221 High-angle detector Cell Laser 0 angle Low-angle detector A Figure 15-10 Differential scatter detection as used in the ADVIA 120. Forward high-angle scatter (5 to 15 degrees) and forward low-angle scatter (2 to 3 degrees) are detected for analysis of red and white blood cells. (From Miles: Technicon H systems training guide, Tarrytown, NY, 1993, Miles.) the cytogram. Monocytes stain weakly and cluster in the midregion of the cytogram. Lymphocytes, basophils, and LUCs (including variant or reactive lymphs and blasts) contain no peroxidase and appear on the left of the cytogram, with LUCs appearing above the lymphocyte area. Basophils cluster with B the small lymphocytes and require further analysis for classifi- Figure 15-11 Cytograms or red blood cell (RBC) maps derived using the cation.22,23,49 Mie theory of light scatter of dielectric spheres. A, Transformation between scatter angles (2 to 3 degrees and 5 to 15 degrees) and RBC volume (V) and Basophil-Lobularity (BASO) Channel hemoglobin concentration (HC). B, RBC map for a patient sample. (From In the BASO channel, cells are treated with a reagent contain- Groner W: New developments in flow cytochemistry technology. In Simson E, ing a nonionic surfactant in an acidic solution. Basophils are editor: Proceedings of Technicon H-1 hematology symposium, October 11, 1985, Tarrytown, NY, 1986, Technicon Instruments Corp, p. 5.) particularly resistant to lysis in this temperature-controlled reaction, whereas RBCs and platelets lyse and other leuko- cytes (nonbasophils) are stripped of their cytoplasm. Laser optics, using the same two-angle (2 to 3 degrees and 5 to 15 degrees) forward scattering system of the RBC/platelet or suspected left shift. As indicated earlier, this channel pro- channel, is used to analyze the treated cells. High-angle scat- vides the primary WBC count, the WBC-BASO. Relative dif- ter (proportional to nuclear complexity) is plotted on the ferential results (in percent) are computed by dividing abso- x-axis, and low-angle scatter (proportional to cell volume) is lute numbers of the different cell classifications by the total plotted on the y-axis. Cluster analysis allows for identifica- WBC count.22,23 tion and quantification of the individual cellular popula- The nucleated RBC method is based on the physical charac- tions. The intact basophils are identifiable by their large teristics of volume and density of the nucleated RBC nuclei. low-angle scatter. The remaining nuclei are classified as These characteristics allow counting in both WBC channels on mononuclear, segmented, and blast cell nuclei based on the ADVIA 2120, and algorithms are applied to determine the their nuclear complexity (shape and cell density) and high- absolute number and percentage of nucleated RBCs. Informa- angle scatter.22,23 tion from the PEROX and BASO channels is used to generate Basophils fall above a horizontal threshold on the differential morphology flags indicating the possible presence cytogram. The stripped nuclei fall below the basophils, with of reactive lymphocytes, blasts, left shift, immature granulo- segmented cells to the right and mononuclear cells to the cytes, nucleated RBCs, or large platelets or platelet clumps.22,23,49 left along the x-axis. Blast cells uniquely cluster below the Figure 15-12 shows a patient printout from the ADVIA 2120i mononuclear cells. Lack of distinct separation between the seg- analyzing the same patient specimen for which data are given mented and mononuclear clusters indicates WBC immaturity in Figures 15-6, 15-7, and 15-9. 222 PART III Laboratory Evaluation of Blood Cells Siemens ADVIA 2120i C D Routine Retic Perox Baso % # Retic 2.13 87.0 x109 cells/L CHr H 31.3 pg CHm 28.5 pg RBC Volume A Routine CBC Additional Parameters Eval RBC HC WBC H 8.62 X103 cells/!L WBCB 8.62 X103 cells/!L RBC L 4.08 X106 cells/!L WBCP 9.12 X103 cells/!L HGB L 11.3 g/dL Cellular HGB 11.8 g/dL HCT L 34.5 % %Micro 1.6 MCV 84.7 fL %Macro 0.4 Retic CH RBC V/HC Retic Scatter Absorption E MCH 27.8 pg %Hypo 0.9 MCHC L 32.9 g/dL %Hyper 1.0 CHCM 34.1 g/dL MNx 14.0 CH 28.7 pg MNy 15.8 Platelet Vol RDW H 14.6 % Neut X 70.5 HDW 2.68 g/dL Neut Y 75.7 PLT 245 X103 cells/!L Large Plt 6 X103 cells/!L MPV H 9.4 fL HGB Trans B F Routine WBC Differential Morphology Flags % # WBC H 8.62 X103 cells/!L Neut 69.7 6.01 X103 cells/!L F Sample/System Flags Lymph 20.9 1.80 X103 cells/!L Mono 6.2 0.54 X103 cells/!L Eos L 0.9 0.08 X103 cells/!L Baso 0.5 0.04 X103 cells/!L LUC 1.8 0.15 X103 cells/!L NRBC 0.0 0.00 X109 cells/L LI 2.09 MPXI 9.7 WBCP 9.12 X103 cells/!L Figure 15-12 ADVIA 2120i. The ADVIA 2120i printout displays the CBC, DIFF, and reticulocyte data for the same patient in Figures 15-6, 15-7, and 15-9. A, CBC data; B, Six-part differential, including large unstained cells (LUCs); nucleated RBCs; C, Reticulocyte information includes the CHr (cellular hemoglobin reticulocyte). D, Cytograms for the differential, both the perox and baso channels, on the right; E, Scattergram for the reticulocyte and RBC counts; F, Morphology flags and Sample/System Flags where flags are displayed. Available automated reticulocyte analyzers include flow AUTOMATED RETICULOCYTE COUNTING cytometry systems such as the FACS system from Becton, Dick- Reticulocyte counting is the last of the manual cell-counting inson and Company (Franklin Lakes, NJ) or the Coulter EPICS procedures to be automated and has been a primary focus of system; the Sysmex R-3500, R-500, XE-2100, XE-5000, and XN- hematology analyzer advancement in recent years. The impre- series systems; the CELL-DYN 3500R, 3700, and 4000 systems; cision and inaccuracy in manual reticulocyte counting are the Coulter LH 750 systems and the UniCel DxH800; and the due to multiple factors, including stain variability, slide dis- Siemens ADVIA 2120, 2120i, and 120. All of these analyzers tribution error, statistical sampling error, and interobserver evaluate reticulocytes based on optical scatter or fluorescence error.50 All of these potential errors, with the possible excep- after the RBCs are treated with fluorescent dyes or nucleic acid tion of stain variability, are correctable with automated retic- stains to stain residual RNA in the reticulocytes. Because nei- ulocyte counting. Increasing the number of RBCs counted ther the FACS nor EPICS system is generally available in the produces increased precision.51 This was evidenced in the routine hematology laboratory, the discussion here is limited 1993 College of American Pathologists pilot reticulocyte pro- to the other analyzers. ficiency survey (Set RT-A, Sample RT-01) on which the CV for The Sysmex R-3000/3500 is a stand-alone reticulocyte ana- the reported manual results was 35% compared with 8.3% lyzer that uses auramine O, a supravital fluorescent dye, and for results obtained using flow cytometry.52 Precision of auto- measures forward scatter and side fluorescence as the cells, in a mated methods has continued to improve. The manual re- sheath-stream, pass through a flow cell by an argon laser. The ticulocyte results for one specimen in the 2000 Reticulocyte signals are plotted on a scattergram with forward scatter inten- Survey Set RT/RT2-A showed a CV of 28.7%, whereas the CV sity, which correlates with volume, plotted against fluorescence was 2.8% for results obtained using one of the automated intensity, which is proportional to RNA content. Automatic methods.53 Automated reticulocyte analyzers may count discrimination separates the populations into mature RBCs 32,000 RBCs compared with 1000 cells in the routine manual and reticulocytes. The reticulocytes fall into low-fluorescence, procedure.54 middle-fluorescence, or high-fluorescence regions, with the less  CHAPTER 15 Automated Blood Cell Analysis 223 mature reticulocytes showing higher fluorescence. The imma- as the cells pass through the flow cell. Three cytograms are ture reticulocyte fraction (IRF) is the sum of the middle- generated: high-angle scatter versus absorption, low-angle scat- fluorescence and high-fluorescence ratios and indicates the ter versus high-angle scatter (Mie cytogram or RBC map), and ratio of immature reticulocytes to total reticulocytes in a sam- volume versus hemoglobin concentration. The absorption cy- ple. The XE-5000, the XT-2000i and the XN series also deter- togram allows separation and quantitation of reticulocytes, mines the reticulocyte count and IRF by measuring forward with additional subdivision into low-absorbing, medium- scatter and side fluorescence. They also have a parameter called absorbing, and high-absorbing cells based on amount of stain- RET-He (reticulocyte hemoglobin equivalent) that measures ing. The sum of the medium-absorbing and high-absorbing the hemoglobin content of the reticulocytes.55 It uses a propri- cells reflects the IRF. Volume and hemoglobin concentration etary polymethine dye to fluorescently stain the reticulocyte for each cell are derived from the RBC map by applying Mie nucleic acids. This is similar to the reticulocyte hemoglobin scattering theory.26,57 Unique reticulocyte indices (MCVr, content (CHr) parameter on the ADVIA 2120i (discussed CHCMr, RDWr, HDWr, CHr, and CHDWr) are provided. below). Platelets, which also are counted, fall below a lower The CHr or reticulocyte hemoglobin content of each cell is discriminator line.56 The Sysmex SE-9500/9000$RAM-1 mod- calculated as the product of the cell volume and the cell ule uses the same flow cytometry methodology for reticulocyte hemoglobin concentration. A single-parameter histogram counting as the R-3500.16 Off-line sample preparation is not of CHr is constructed, with a corresponding distribution required. The smaller Sysmex R-500 uses flow cytometry with width (CHDWr) calculated.22,23 These reticulocyte indices are a semiconductor laser as the light source and polymethine not reported on the routine patient printout but are available supravital fluorescent dye to provide automated reticulocyte to the operator. Figure 15-13 is a reticulocyte printout from counts.28 an ADVIA 120, showing the cytograms and reticulocyte The CELL-DYN 3500R performs reticulocyte analysis by indices. measuring 10-degree and 90-degree scatter in the optical Automation of reticulocyte counting has allowed in- channel (MAPSS technology) after the cells have been iso- creased precision and accuracy and has greatly expanded the volumetrically sphered to eliminate optical orientation noise. analysis of immature RBCs, providing new parameters and The RBCs are stained with the thiazine dye new methylene indices that may be useful in the diagnosis and t