Mckenzie Ch39 Lecture

Clinical Laboratory Hematology
THIRD EDITION
CHAPTER 39
Automation in
Hematology
Copyright © 2015, © 2010, © 2004 by Pearson Education, Inc.

All Rights Reserved
Learning Objectives—Level I
At the end of this unit of study, the student

should be able to:
1. Cite the electrical impedance principle of
cell counting, and identify the instruments
that use this technology.
2. Describe the use of radio frequency in cell
counting, and identify the instruments
that use this technology.
continued on next slide

Clinical Laboratory Hematology, Third Edition
Shirlyn B. McKenzie | J. Lynne Williams

should be able to:
3. State the principles of light scatter used
in cell counting, and identify the
instruments that use this technology.
4. List the reported parameters for each
blood cell–counting instrument.
5. Categorize cell parameters as directly
measured and derived from a histogram,
scattergram or cytogram, or calculation.

should be able to:
6. Describe the principle of reticulocyte
count enumeration by automated blood
cell–counting instruments.

Learning Objectives—Level II

should be able to:
1. Compare and contrast the methods of
analysis for the described histograms,
scatterplots, scattergrams, and
cytograms, and interpret the results.
2. Describe and interpret the automated
reticulocyte parameters.

Automated Blood
Cell–Counting Instruments

Automated Blood
• Primary testing vehicle in hematology
lab
• Automated instruments can perform
– Complete blood cell count (CBC)
including
 Platelet count
 Five-part leukocyte differential
 Absolute reticulocyte count

Automated Blood
• Two principles of blood counting
currently used
– Impedance
 Based on increased resistance that
occurs when a blood cell with poor
conductivity passes through an electrical
field

Automated Blood
• Two principles of blood counting
currently used
– Optical light scattering
 Based on light scattering measurements
obtained as a single blood cell passes
through a beam of light (optical or laser)

Automated Blood
• Automated reticulocyte counts
– Enumerates reticulocyte
– Reticulocyte hemoglobin concentration
(CHr or Ret He)
– Reticulocyte mean cell volume (MCVr)

Automated Blood
• Automated reticulocyte counts
– Immature reticulocyte fraction (IRF)
– Useful in
 Measuring BM engraftment following
transplant
 Determining BM response to iron or EPO
therapy
 Diagnosing iron deficient states in infants
and adolescents

Automated Blood
• Body fluid analysis
– Newer feature available on some
instruments
– Includes
 Total nucleated cell count (TNC)
 Leukocyte counts
 Erythrocyte count
 Partial leukocyte differential counts

Automated Blood
– Determined by same technologies as
blood counts
– Improves TAT, increases precision, less
variability

Automated Blood
• Impedance instruments
– Coulter® LH Series
– Beckman-Coulter® Unicel DxH 800
– Sysmex XE-Series™
– Sysmex XN-Series™
– Abbott CELL-DYN Sapphire®

Automated Blood
• Light-scattering instruments
– Siemens Healthcare ADVIA 120
– Siemens Healthcare ADVIA 2120

Coulter® LH Series
• LH 750 and LH 780—single blood

aspiration
– Provides
 CBC
 Five-part leukocyte differential
 Reticulocyte count
– Same basic principles as earlier Coulter
models

Coulter® LH Series
• LH 750 and LH 780—single blood

aspiration
– Provide additional parameters
 NRBC%, Corrected WBC, RDW-SD

Coulter® LH Series
• EDTA blood sample is aspirated

– Closed tube system
– Divided into four aliquots which are
delivered to
 RBC/PLT chamber
 WBC/Hb chamber
 Orbital mixing chamber—differential
 Reticulocyte dilution chamber

Coulter® LH Series
• Coulter principle
– Blood cells diluted in an electrically
conductive dilutent
– Within dilution chamber is an external
electrode and three apertures with
internal electrodes

Coulter® LH Series
– Individual blood cells pass through an
aperture
 Increase in resistance (pulse height)
between internal and external electrode
that is proportional to cell volume
 Steady stream of diluent behind aperture
prevents cells from reentering the
aperture

Coulter® LH Series
– Threshold limits established for cells
based on cell volumes

Figure 39-1 Coulter principle. Increased electrical resistance, or impedance, occurs when the poorly conduc-
tive blood cell passes through the aperture.
Pierre R. Seminar and Case Studies: The Automated Differential. Hialeah, FL: Coulter Electronics, Inc.; 1985.
Reprinted with permission

Coulter® LH Series
• Red blood cells

– Particles > 36fL counted as RBCs
– Three RBC counts obtained
– Reported RBC count is average of three
counts
• RBC histogram
– Size distribution curve
– Based on cell volume and relative cell
number
Figure 39-2 Histogram, or size distribution curve, allows the visualization of the blood cell population based on
the relative cell number and its volume (in fL).
Significant Advances in Hematology. Hialeah, FL: Coulter Electronics, Inc.; 1983. Reprinted with permission.

Figure 39-3 Red blood cell (RBC) histogram and count. The shaded area represents those cells used in the red
cell distribution width (RDW) (i.e., RDW–coefficient of variation) calculation. The excluded cells can represent
large platelets, platelet clumps, or electrical interference on the left and RBC doublets, RBC triplets, RBC
agglutinates, or aperture artifacts on the right.

Coulter® LH Series
• RBC/platelet dilution
– RBC count—direct measure
– MCV and RDW—derived from RBC
histogram
– HCT—calculated from MCV and RBC
count
– MCH—calculated from RBC count and
Hgb
– MCHC—calculated from Hgb and Hct

Coulter® LH Series
• RDW-CV
– Based on SD of RBC population and MCV
 RDW-CV = 1SD/MCV × 100%

Coulter® LH Series
• RDW-CV
– Can be false in certain situations
 IF both SD and MCV elevated
– RDW-CV can be normal when anisocytosis
is present
 If SD is normal and MCV is decreased
– RDW-CV can be increased when no
anisocytosis is present

Coulter® LH Series
• RDW-SD
– Red cell distribution width—standard
deviation
 Determined from RBC histogram by
– Calculating width in fL of RBC population
at the 20% height level on RBC histogram

Coulter® LH Series
• RDW-SD
– Red cell distribution width—standard
deviation
 Reflects degree of anisocytosis
 Direct measure
 Not affected by MCV
– Better indicator of anisocytosis

Figure 39-4 Calculation of red cell distribution width (RDW)–standard deviation (RDW-SD). The RDW-SD
represents the width of the erythrocyte population at the 20% height level on the red blood cell histogram and is
reported in fL. For example, if ChannelL is 70 fL and ChannelR is 111 fL, the RDW-SD is 41 fL (i.e., 111–70 = 41).
RDW-SD = red cell distribution width–standard deviation
Red Cell Distribution Parameters—(1) RDW-SD (2) RDW (CV), Technical Bulletin 9617. Brea, CA: Beckman-
Coulter®; 2007. Reprinted with permission

Coulter® LH Series
• Platelet count
– Particles between 2–20 fL are counted
– Raw platelet histogram is obtained
– Evaluated to determine whether it is a
log normal curve

Coulter® LH Series
• Platelet count
– Raw histogram is electronically
smoothed and extrapolated over 0–
70 fL
– Platelet count derived from
extrapolated histogram

Coulter® LH Series
• Additional parameters
– MPV—mean platelet volume
 Analogous to RBC MCV

Figure 39-5 Normal platelet histogram. The jagged line represents the raw data collected from 2–20 fL. The
smooth line represents the extrapolated histogram from 0–70 fL.

Coulter® LH Series
• WBC/hemoglobin chamber
– Hemoglobin determination
 Lytic agent added to dilution
– Lyses RBCs
– Coverts released Hgb to
cynamethemoglobin
– Shrinks WBC membrane and cytoplasm

Coulter® LH Series
• WBC/hemoglobin chamber
– WBC count
 Measure of cell volume not native cell
size
 Particles > 35 fL counted as WBCs by
impedence
 Three WBC counts obtained from three
apertures
 Reported WBC count is average of three
counts
Coulter® LH Series
• WBC count histogram

– Size distribution curve for WBC data
– Visualization of subpopulations of cells
based on WBC relative sizes
– WBC histogram—three-part differential
 Lymphocytes 35–90 fL
 Mononuclear cells 90–160 fL
 Granulocytes 160–450 fL

Coulter® LH Series
• WBC count histogram

– Interferences within cell populations
 generate region flags

Figure 39-6 White blood cell (WBC) histogram and count. In a normal patient, the lymphocyte region
represents lymphocytes, the mononuclear region represents monocytes, and the granulocyte region represents
neutrophils, eosinophils, and basophils.

Table 39-1 Coulter® WBC Histogram Region Flags

Coulter® LH Series
• Hemoglobin concentration
– Measure absorbance of
cyanmethemoglobin in the WBC/Hgb
chamber at 525 nm
– Beer's law
 Absorbance reading is proportional to the
concentration of Hgb
 Reagent blank at beginning of each
cycle

Coulter® LH Series
• Five-part differential
– Aliquot sent to orbital mixing chamber
– Blood is mixed by gentle agitation with
heated lysing agent
 Remove RBCs, leaves WBCs in near
native state
– Second stablizing agent is added
 Stops lytic activity
 Preserves integrity of WBCs
Coulter® LH Series
• Five-part differential
– Sent to VCS flow cell for five-part WBC
differential
 Volume, Conductivity, Scatter
 Cells pass through flow cell singly by
hydrodynamic focusing

Coulter® LH Series
• VCS flow cell

– Three separate measurements taken
simultaneously
 Cell volume
– Determined by impedance
 Cell conductivity
– High-frequency electromagnetic probe
– Evaluates internal physical and chemical
constituents of cell

Coulter® LH Series
• VCS flow cell

– Three separate measurements taken
simultaneously
 Cell light scatter
– Helium-neon laser
– Determines cell surface, shape, reflectivity

Coulter® LH Series
• VCS flow cell

– Different WBCs are identified and
classified by contour gating
 Individualized for each samples
 Enables differentiation of overlapping cell
populations

Coulter® LH Series
• NRBCs
– First suspected on WBC scatterplot
– WBC histogram examined
 Computer system looks for presence of
cells to left of lymphocyte population
– If both plots indicate NRBCs
 Giant platelets and small lymphocytes
are ruled out

Coulter® LH Series
• NRBCs
– %NRBC (number of NRBCs/100 WBCs)
is derived from VCS data
– NRBC# = NRBC% × WBC count
– WBC is corrected

Figure 39-7 White blood cell (WBC) scatterplot.WBC scatterplot graphs volume versus light scatter and reveals
the locations of four leukocyte populations in two-dimensional space. The basophil population is located behind
the lymphocytes but in three-dimensional space is clearly delineated.

Coulter® LH Series
• Reticulocytes
– Sample mixed with new methylene blue
reagent in heated reticulocyte dilution
chamber
– Residual RNA precipitated within
reticulocytes

Coulter® LH Series
• Reticulocytes
– Stained sample is mixed with acidic,
hypotonic solution
 Elutes Hgb from RBCs
– Precipitated RNA remains
 Spheres the RBCs
– Eliminates error of flow cytometric
examination of irregularly shaped
erythrocytes

Coulter® LH Series
• Reticulocytes
– Sample sent to VCS flow cell
 Contour gating
– classifies reticulocytes vs. mature RBCs
– Identifies immature reticulocyte fraction

Coulter® LH Series
• Reticulocytes
– Parameters
 Absolute reticulocyte count
 Reticulocyte %
 Immature reticulocyte fraction
 MCVr

Coulter® LH Series
• Computer system
– Compiles data
– Corrects for coincidence
 Two or more cells passing through the
aperture at the same time
– Records "vote-out”
 Two of three counts do not agree

Coulter® LH Series
• Computer system
– Reports average of counts when there is
agreement
– Evaluates data to detect abnormalities
– Results, histograms/scatterplots
displayed on screen and printed out or
transferred to LIS
– Suspect flags (software-generated flags)
– User-defined flags (definitive flags)

Figure 39-8 Coulter® LH 750 white blood cell (WBC) scatterplot and WBC histogram depiction of the presence of
nucleated erythrocytes. On the WBC scatterplot, detection of cells between the lymphocyte population and red
blood cell ghosts suggests nucleated erythrocytes, which are suspected if there is a population of cells to the left
of the lymphocyte population on the WBC histogram. The nucleated red blood cell percentage is determined if
both observations are made and other potential interferences are ruled out (e.g., giant platelets).

Figure 39-9 Coulter® LH 750 report from a normal individual.

Beckman-Coulter Unicel® DxH 800
• Determines CBC using same methods

as Coulter LH series
• Improvements to
– WBC differential
– NRBC determination
– Reticulocyte enumeration

• VCSn module
– Next generation of VCS technology
– Diff, NRBC and reticulocyte
measurements are made here

• Light scatter detection by flow cell

– Multitransducer module (MTM)
 Multiple sensors around flow cell
 Collect three measures of light scatter—
lower, upper, and low-angle (LMALS),
(UMALS), (LALS)

• Light scatter detection by flow cell

– Multitransducer module (MTM)
 Additional sensor measures axial light
loss (AL2)
 Fifth light scatter measure
– Median angle light scatter (MALS)
– Sum of LMALS and UMALS

• Multitransducer module (MTM)

– LMALS, UMALS and UMAL—detect
granularity and membrane topography
– AL2—evaluates cellular transparency
– LALS—evaluates cellular complexity
index

• System manager
– Evaluates raw data from flow cell
– Applies algorithm
– Calculates cell frequency (percent and
absolute count)
– Data-plots
 Two-dimensional and three-dimensional

Figure 39-10 Light scatter measurements from the Unicel® DxH 800 flow cell. The upper median angle light
scatter (UMALS) and lower median angle light scatter (LMALS) detect the cell’s granularity and membrane
topography. The axial light loss (AL2) evaluates cellular transparency and the low-angle light scatter (LALS) is
used as a cellular complexity index.
Advancements in Technology: WBC Differential Methodology, Technical Bulletin 9403. Brea, CA: Beckman-
Coulter®; 2009, Reprinted with permission

Figure 39-11 Unicel® DxH 800 white blood cell differential dataplots. Diff 1 dataplot graphs volume versus
rotated median angle light scatter (RMALS) and Diff 2 dataplot graphs volume versus opacity (OP). These
dataplots reveal five leukocyte populations in two- dimensional space.
Advancements in Technology: WBC Differential Methodology, Technical Bulletin 9403. Brea, CA: Beckman-
Coulter®; 2009, Reprinted with permission

• NRBC%
– RBCs enumerated in VCSn module

• NRBC%
– NRBC mix chamber
 Sample is diluted
 Lyse reagent is added
– Lyses red cells but keeps NRBCs, WBCs,
Platelets, cellular debris intact
 Flow cell analysis for VCS
– AL2 and LALS—used to determine NRBCs
– Two 2-dimensional plots created
– NRBC% determined from collected data

Figure 39-12 Unicel® DxH 800 nucleated red blood cell (NRBC) dataplots. NRBC 1 dataplot graphs rotated low-
angle light scatter (RLALS) versus axial light loss (AL2) and NRBC 2 dataplot graphs rotated upper median angle
light scatter (RUMALS) versus AL2. These dataplots clearly delineate the nucleated erythrocyte population from
the leukocyte population.
Advancements in Technology: NRBC Enumeration, Technical Bulletin 9404. Brea, CA: Beckman-Coulter®; 2009,
Reprinted with permission

• Reticulocyte mix chamber

– Blood is mixed with NMB and RNA is
precipitated
– Clearing reagent is added
 Stabilizes dye–reticulum complex
 Spheres RBCs and reticulocytes (similar
to Coulter LH)

• MTM evaluates each cell for:

– Volume, conductivity, and five light
scatter characteristics
– System manager applies algorithm
 Discriminates reticulocytes from mature
RBCs using
– RETIC1 AND RETIC2 dataplots (vol vs LLS
and vol vs OP)

• MTM evaluates each cell for:

– Volume, conductivity, and five light
scatter characteristics
– System manager applies algorithm
 Determines RETIC%, absolute count
(RETIC% xRBC count), IRF, and mean
reticulocyte volume (MRV)

Figure 39-13 Unicel® DxH 800 Reticulocyte dataplots. RETIC 1 dataplot graphs volume versus linear light
scatter (LLS) and RETIC 2 dataplot graphs volume versus opacity (OP). These dataplots provide delineation of
reticulocytes from mature erythrocytes and platelets.
Advancements in Technology: Reticulocyte Methodology, Technical Bulletin 9405. Brea, CA: Beckman- Coulter®;
2009, Reprinted with permission

• System manager
– Evaluates all data
– Determines if an interference is present
– Corrects CBC for clearly identified
interferences
 WBC for NRBCs
 Hemoglobin for WBC > 11 × 103/mcL
 RBC for WBC > 140 × 103/mcL

• System manager
– Abnormalities generate suspect, system,
and definitive flags to alert operator


– RBC and TNC on body fluids
– Uses CBC module data
 CSF, serous, synovial fluids—all
acceptable
 Pre-treat synovial fluids with
hyluronidase to reduce viscosity
– Each lab should establish acceptable
background counts

Sysmex XE-Series™
• Uses four different technologies

– Impedance (i.e., direct current)
– Radio frequency
– Absorption spectrophotometry
– Flow cytometry—fluorescent dyes

Sysmex XE-Series™
• Parameters reported
– CBC
– Five-part leukocyte differential
– Nucleated erythrocytes
– Reticulocyte count

Sysmex XE-Series™
• Hemoglobin
– Measured using the sodium lauryl
sulfate (SLS) method (cynanide free)
– RBCs are lysed—release Hgb
– SLS converts ferrous iron to ferric iron—
methemoglobin

Sysmex XE-Series™
• Hemoglobin
– Methemoglobin combines with SLS
 SLS-hemichrome molecule
 Measured at 555 nm
 Beer's law

Sysmex XE-Series™
• RBCs and platelets

– Counting using impedance with
hydrodynamic focusing

Sysmex XE-Series™

– Resistance detected between the
internal and external electrodes
 RBCs counted between a lower
discriminator (25–75 fL) and a upper
discriminator (200–250 fL)

Sysmex XE-Series™

– Resistance detected between the
internal and external electrodes
 Platelets counted between a lower
discriminator (2–6 fL) and a upper
discriminator (12–30 fL)
– Fixed discriminator at 12 fL detects
potential interferences (e.g., RBC
fragments)

Sysmex XE-Series™

– RBC and platelet histograms are
generated
– HCT calculated from pulse heights of
individual RBCs
 Pulse height is proportional to RBC
volume
– Other derived or calculated parameters
 MCV, MCH, MCHC, RDW-SD, RDW-CV,
MPV
Sysmex XE-Series™
• WBC count and differential generated

from:
– Two channels
 DIFF
 WBC/BASO

Sysmex XE-Series™
• Diff channel
– Sample mixed with lyse reagent, RBCs
lysed
– Creates "holes" in cytoplasmic
membranes of nucleated cells
– Fluorescent dye enters cells, binds to
DNA and RNA
– Sent to optical detection block
 Cells pass singly through laser
Sysmex XE-Series™
• Diff channel
– Each cell generates
 Forward scatter (size), side scatter (cell
complexity), fluorescent intensity
 Scattergram—side scatter vs. fluorescent
intensity

Sysmex XE-Series™
• Diff channel
– Adaptive cluster analysis system
 Clear separation of cell populations
– RBC ghosts, lymphocytes, monocytes,
eosinophils, and neutrophils + basophils

Sysmex XE-Series™
• Diff channel
– IG master software
 Immature granulocyte population
counted
– Metamyelocytes, myelocytes,
promyelocytes

Sysmex XE-Series™
• WBC/BASO channel
– Sample mixed with lyse reagent, RBCs
lysed
– Shears all WBCs except basophils

Sysmex XE-Series™
• WBC/BASO channel
– Analyzed by optical detection block
 Captures forward and side scatter as
cells pass through laser beam
 Adaptive cluster analysis
– Total WBC count and basophil count
(percentage and absolute)

Figure 39-14 DIFF scattergram, Sysmex XE-2100™. RBC ghosts, lymphocytes, monocytes, eosinophils, and
nutrophils + basophils are identified. Basophils are specifically identified in the WBC/BASO scattergram. Courtesy
of Sysmex Corporation, Kobe, Japan.

Sysmex XE-Series™
• IMI channel
– Reagents act selectively on lipid
membranes of WBCs
– Mature WBCs are lysed, leave bare
nuclei
– Immature myeloid cells remain intact

Sysmex XE-Series™
• IMI channel
– Cells are analyzed by direct current and
radio frequency (cells internal structure
and density)
– Determine degree of immaturity
 Immature granulocytes and blasts
 Hematopoietic progenitor cell (HPC)
count
– Correlates with CD34+ count by flow
cytometry
Sysmex XE-Series™
• XE-5000
– Multichannel messaging (eMM) software
 Improved detection of
– Blasts
– Abnormal lymphocytes/lymphoblasts
– Atypical lymphocytes

Sysmex XE-Series™
• NRBC channel
– Counts NRBCs, corrects WBC and
lymphocyte count
– Lyse RBCs, removes NRBC cytoplasmic
membrane, shrinks nucleus, fluorescent
dye binds to nuclei

Sysmex XE-Series™
• NRBC channel
– WBC membrane perforated, dye enters
and binds to organelles and nucleus
 Fluorescence is stronger in WBCs than
NRBCs

Sysmex XE-Series™
• NRBC channel
– Pass through laser beam and generate
FS, SS, and fluorescence
 Used to create NRBC scattergram
 Sensitive to NRBCs as low as
0.1/100WBCs
 #NRBC used to correct WBC count and
lymphocyte count

Figure 39-15 Nucleated red blood cell (NRBC) scattergram, Sysmex XE-2100™. Nucleated erythrocytes are
clearly delineated from leukocytes based on their lower fluorescence intensity and smaller size (i.e., less forward
scatter) as seen on this schematic pattern.WBC, white blood cell; PLT, platelet
Rowan RM, Linssen J. A picture is worth a thousand words. Sysmex Journal International. 2005;15(1):27–38.
Reprinted with permission.

Sysmex XE-Series™
• RET channel
– Fluorescent dye binds to residual RNA
within reticulocytes and another dye
binds to RNA and DNA in nucleated cells

Sysmex XE-Series™
• RET channel
– Each cell evaluated by diode laser for FS
and fluorescence
 RET scattergram is generated
 Mature erythrocytes, reticulocytes and
platelets
 Reticulocytes further classified for
maturity
– Immature platelet fraction (IPF)—
reticulated platelets
Sysmex XE-Series™
• RET channel
– Absolute reticulocyte count, %
reticulocyte, low, middle, high
fluorescence ratio (LFR, MFR, HFR),
immature reticulocyte fraction (IRF),
reticulocyte hemoglobin content (ReT
He), and platelet count

Sysmex XE-Series™
• RET channel
– IRF
 Represents MFR and HFR combined
 Reported as % of total reticulocyte count

Figure 39-16 Reticulocyte (RET) scattergram, Sysmex XE-2100™. Mature erythrocytes, RETs, and platelets
(PLTs) are identified.
RBC = red blood cell
Rowan RM, Linssen J. A picture is worth a thousand words. Sysmex Journal International. 2005;15(1): 27–38.
Reprinted with permission.

Sysmex XE-Series™
• XN-10 instrument—latest Sysmex

(2012)
– Modular component capable of
determining
 CBC, six-part diff (neutrophils,
lymphocytes, monocytes, eosinophils,
basophils, and IGs, NRBCs, and
reticulocyte count)
 SP-10 slide maker/stainer creates
complete workstation
Sysmex XE-Series™
• XN-10 instrument
– Uses same methods for RBC count, Hgb,
RBC parameters, platelet count,
reticulocyte count and reticulocyte
parameters

Sysmex XE-Series™
– New methods
 Adaptive Flagging Algorithm based on
Shape recognition (SAF-LAS)
– Allows better discrimination of WBCs,
abnormal WBCs, and NRBCs
 Low WBC mode
– Accurate diffs on low WBC counts (< 0.5 ×
103/mcL)

Sysmex XE-Series™
– New methods
 White cell nucleated (WNR) channel
– Clear delineation between NRBC and WBC
– Total WBC count does not require
correction

Figure 39-17 Sysmex XE-Series™ report from a normal individual.

Sysmex XE-Series™
• Fluorescent platelet channel (PLT-F)

– Determines IPF and platelet count
– Platelets are clearly delineated based on
size and fluorescent intensity
 Depicted in PLT-F scattergram
 Immature platelets have high
fluorescence intensities

Sysmex XE-Series™
• Fluorescent platelet channel (PLT-F)

– Provides accurate platlet count when
 Numbers are low
 Red cell or WBC fragments are present

Sysmex XE-Series™
• XN-Series
– Have body fluid module
– No pretreatment or off-line prep is
required
– RBC-BF
 Determined by impedance

Sysmex XE-Series™
• XN-Series
– WBC-BF, TC-BF, and two-part diff
(PMNS and mononuclears)
 Determined by fluorescent FC in WDF
channel
– HF-BF cells (e.g., macrophages,
mesothelial cells) can also be
distinguished on the BF scattergram

Figure 39-18 WDF scattergram, Sysmex XN-Series™. Lymphocytes (LYMPH), monocytes (MONO), eosinophils
(EO), neutrophils (NEUT)+basophils (BASO), and immature granulocytes (IG) are identified. Basophils are
specifically identified in the WNR scattergram.
SSC = side scatter; SFL = fluorescence intensity
Courtesy of Sysmex Corporation, Kobe, Japan.

Abbott CELL-DYN Sapphire®
• Technologies used
– Flow cytometry, fluorescence staining,
impedance
– Enumerate
 WBCs, RBCs, platelets, reticulocytes,
five-part differential

• Hemoglobin dilution cup

– RBCs lysed, free Hb complexes with
imidazole
– Hgb concentration determined
spectrophotometrically at 540 nm
 Cyanide free method

• WBC dilution cup

– Lyses RBCs, strips cytoplasmic
membrane from NRBCs and fragile
WBCs
– Stains DNA of nuclei with PI—goes to
flow cell


– MAPPS technology
 Light scatter and fluorescence
characteristics determined
– 0° (forward) = cell size
– 7° = cell complexity
– 90° (side) = nuclear lobularity
– 90° depolarized light scatter = cytoplasmic
granularity

Figure 39-20 Four light scatter measurements from the CELL-DYN Sapphire ®. The 0° scatter reflects cell size;
7° scatter reflects cell complexity; 90° scatter reflects nuclear lobularity; and 90° depolarized scatter reflects
cytoplasmic granularity.


– 0° vs 7° = neutrophils, monocytes,
lymphocytes
– 90° D vs 90° = eosinophils
differentiated from neutrophils
– Lymphocytes separated from basophils
based on size and complexity


– Total WBC, five-part diff, NRBCs,
nonviable WBCs
 NRBCs clearly distinguished from WBCs
– Total WBC and five-part differential not
affected by their presence

Figure 39-21 0° (Size) vs 7° (complexity) scatterplot, CELL-DYN Sapphire ®. Neutrophils, monocytes, and
lymphocytes are identified. Lymphocytes are located lower left in the scatterplot; the middle cell population is
composed of monocytes; and neutrophils are located upper center in the scatterplot.

• RBC/Plt cup
– One dilution—spheres RBCs, counts
using impedance
 RBC count and histogram, platelet count
and histogram
 Part of dilution sent to optical flow cell
– RBCs and platelets enumerated by light
scatter

• RBC/Plt cup
– Second dilution—reticulocytes
 Nucleic acid stained with fluorescent dye
 Reticulocytes determined based on
fluorescence and light scatter
– Reticulocyte count and immature
reticulocyte fraction (IRF)

Figure 39-22 Reticulocyte histogram, CELL-DYN Sapphire ®. Reticulocytes are found between the two gates.
The peak to the left represents mature erythrocytes.
x-axis = fluorescence; y-axis = relative number of cells

Figure 39-23 CELL-DYN Sapphire® report depicting WBC differential.

• New software package—2010

– Extends RBC and reticulocyte
parameters
 Calculated from data collected

• New software package—2010

– Determines microcytic RBC% (%MIC),
macrocytic RBC% (%MAC),
hypochromic RBC% (%HPO),
hyperchromic RBC% (%HPR), MCVr,
MCHr, CHCr
 Shown to correlate well with similar
parameters from ADVIA

• Two additional assays

– Fluorescent immunophenotyping
methods
 Immuno T-cell assay
– CD4 and CD8 populations
 ImmunoPlt assay
– CD61

Light-Scattering Instruments
• Siemens Healthcare ADVIA 120

• Siemens Medical ADVIA 2120

Siemens HealthCare ADVIA 120
• Methodology
– Continous flow analysis, peroxidase
staining, flow cytometry
– CBC, five-part WBC differential,
reticulocyte count

• RBC/Plt channel
– Isovolumetric sphering of RBCs and Plts
– Diluted cells pass singly through a laser
beam

• RBC/Plt channel
– RBCs counted and sized by high-angle
and low-angle light scatter
 MCV, CHCM
– Generate RBC cytogram, RBC histogram,
Hgb histogram
• RDW, HDW are derived from these
histograms

• RBC/Plt channel
– Platelets measured with high-angle and
low-angle light scatter
– Platelet cytogram and RBC cytogram
• Hgb channel
– RBCs lysed, free Hgb converted to
cyanmethemoglobin
– Measured at 546 nm

• WBC count and five-part differential

– RBCs lysed
– Peroxidase channel
 Neutrophils, monocytes, eosinophils—
degree of peroxidase positivity
 Lymphocytes and large unstained cells
(LUCs) remain unstained
 Pass through flow cell
 Peroxidase cytogram

• WBC count and five-part differential

– Basophil/lobularity channel
 Basophil diluent lyses RBCs, platelets,
strips all WBCs except basophils of
cytoplasm
 Flow cell

• Reticulocyte channel
– Cellular RNA stained
– Flow cell
– Absolute and relative reticulocyte count
– CHr™, MCVr, reticulocyte cellular Hgb
concentration mean

Figure 39-24 ADVIA 120 report from a normal individual.

Siemens Medical ADVIA 2120
• Basic principles same as ADVIA 120

• Hgb measurement
– Cyanide-free methodology
• Neither ADVIA 120 and ADVIA 2120
can enumerate NRBCs

• First to produce extended RBC

parameters
– %HYPO, %HYPER, %MICRO, %MACRO
 Combined use of %HYPO and CHR can be
useful in early detection of iron deficiency
in chronic renal disease dialysis patients

• DNI index
– Delta neutrophil index
 DNI = (neutrophils + eosinophils from
peroxidase channel)—
(polymorphonuclear cells from
basophil/lobularity channel)
– Reflects number of immature granulocytes
– May be useful as prognostic indicator for
sepsis

• CSF analysis
– Use dedicated CSF mode

• CSF analysis
– Sample is mixed with CSF assay reagent
 Spheres and fixes cells
 Incubated for 4 minutes
 Determines cell counts and two-part diff
by light scatter and absorbance
 CSF WBC count, CSF RBC count, and
two-part differential
– PMNs and MNs reported in absolute and
relative values


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Clinical Laboratory Hematology

Copyright © 2015, © 2010, © 2004 by Pearson Education, Inc.

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At the end of this unit of study, the student

At the end of this unit of study, the student

Clinical Laboratory Hematology, Third Edition

At the end of this unit of study, the student

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Clinical Laboratory Hematology, Third Edition

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• LH 750 and LH 780—single blood

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• LH 750 and LH 780—single blood

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• EDTA blood sample is aspirated

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• Red blood cells

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• WBC count histogram

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• WBC count histogram

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• VCS flow cell

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• VCS flow cell

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• VCS flow cell

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Clinical Laboratory Hematology, Third Edition