MEDICAL MYSTERIES

Using Hematology
Instrument Data to
Troubleshoot

DR PETER JOHN LOGA, PhD; MS;

MSc; BSc; SDMLT; DMLT; FIBMS;
FZIMLS
 OBJECTIVES
 Review instrument technology;
compare & contrast normal vs abnormal

 Apply this technology / knowledge to a

variety of cases

 “SOLVE” the medical mystery

 PROVEN
BECKMAN COULTER
TECHNOLOGIES
 The Coulter Principle
Vacuum

Aperture Current Detail of Aperture

Pathway
Internal Electrode

External Electrode Suspension of Cells

External Housing
(Aperture Bath)

Aperture

Aperture Housing
RBC HISTOGRAM

NORMAL
NORMAL
 RBC HISTOGRAM

COLD MACROCYTIC,
MACROCYTIC,TARGET
TARGETCELLS,
CELLS,DI
DIRBC
COLDAGGLUTININ
AGGLUTININ RBC

RBC DI
DIRBCs
RBCs Post
PostTransfusion
Transfusion
RBCFRAGMENTS,
FRAGMENTS,MICROCYTIC
MICROCYTICRBCs,
RBCs,Giant
GiantPLT
PLT
PLT HISTOGRAMS

NORMAL
NORMAL
 PLT Curve Fitting
The Curve Fitting Process Allows
More Accurate Counts When Platelets
of Larger Than 20 fL Are Present
 PLT Counting & Sizing
Coulter impedance counting has a
PATENTED CURVE FITTING process
that is used in conjunction with WBC
histogram review for platelet clump and giant
platelet flags
 PLT HISTOGRAMS

 Giant Platelets

 Small Platelets
 The Coulter Principle

A white cell passes

Neutrophil
through WBC
aperture

Sensing Zone

Oscilloscope
 Coulter WBC Histogram
Monos
Lymphs Neuts
90 -160 fL Eos
50 – 90 fL 160 - 450 fL

Baso
 WBC HISTOGRAMS

ImmNE1 & ImmNE2 Lymphocytosis Variant Lymph

ImmNE2 Eosinophilia Blasts

 WBC Interference

 Percentage of
interference analyzed for
statistical significance
Cellular Interference
 Flagging based on all
three histograms instead
of one

 Histogram positional
parameters used for
further definition
AccuCount Technology
– LH700 Series
 WBC 0 – 400,000
 RBC 0 – 8,000,000
 HGB 0 - 25
 PLT 0 – 3,000,000

AccuCount WBC and

AccuCount Plt Counts have
been “validated” by
Reference Flow
Cytometry
VCS TECHNOLOGY
Automated Differential
Analysis
Near Native WBC Analysis
 Red Cells Removed From
Sample Dilution Using a
Lytic Process

 Second Agent Prevents

Alteration of the White
Cells

 Hydrodynamically
Focused Flowcell
– Laminar Flow Ensures
Single File Cell Passage
– Coincidence Effects Are
Minimized
 BioPhysical Flow Cytometry
FLOW CELL

SHEATH-FLUID IN
SAMPLE DILUTION

SENSING
AREA SHEATH FLUID

SHEATH-FLUID IN

SAMPLE IN

 Cells are hydrodynamically focused

 An electro-optical flow cytometer provides concurrent
electronic and optical measurements
 The 3-D VCS Scatterplot
APE N GR
R SH TIO AN
E A OS I UL
C L M P ES
NU O
D C
AN

S M
CEL LA
L SI O P
ZE T
CY
 COULTER VCS
TECHNOLOGY

 VOLUME = SIZE

 CONDUCTIVITY = INTERNAL COMPOSITION

 LIGHT SCATTER = CELL SHAPE / SURFACE

VOLUME
 DC Measures Total
Cell Volume Using
the Reference
Method of Direct
Current Impedance
 Unaffected by cell
orientation
CONDUCTIVITY
 RF Measures
Internal Cell
Structure Using
Radiographic
Imaging Similar to
Ultrasound
 Conductivity Is a
Proprietary
Technology
LASER LIGHT SCATTER
 Light Scatter
Measures Cell
Surface
Granularity Using a
Broad Range of
Angles. Over 60
angles of light
scatter are
analyzed.
3-D Cellular Analysis - VCS
VOLUME (Y) CONDUCTIVITY (Z)

Eos
Monos
LIGHT SCATTER (X)

The 3 probes (DC, RF and Scatter) interrogate

each of the 8192 cells simultaneously. Neuts
Every cell is treated in the same manner and Lymphs Basos
each cell is given an X, Y, and Z coordinate on
the dataplot; with 16 million points in the matrix.
ALL cell populations are DIRECTLY
measured
 Better
Abnormal Cell Detection
1 Mono-Blasts
2 Myelo-Blasts
2 3 Immature Granulocytes
1 3
4
4 Band Neutrophils
5 Lympho-Blasts
6 Variant Lymphocytes
5
6 7 Low Volume Lymphocytes
7a NRBCs
8 PLT Clumps
7 7a
9 Giant Platelets
8 9 10
10 RBC Parasites (Malaria,
etc)
DIFF TECHNOLOGY
NORMAL
NORMAL
NORMAL DATAPLOT
MONOCYTES
NEUTROPHILS

EOSINOPHILS

V
O
L
U
M
E

BASOPHILS

LYMPHOCYTES

Y
IT
IV
T
U
C NRBC, PLT CLUMPS, GIANT PLT,
N
D MALARIAL PARASITES, DEBRIS, ETC
O
C SCATTER
 CUBE ROTATION
VOLUME

Y
IT
IV
CT

SCATTER
U
ND
CO

VOLUME
RED==VOLUME
RED VOLUME==SIZE
SIZE
GREEN==SCATTER
GREEN SCATTER==SURFACE
SURFACE
SC
BLUE==CONDUCTIVITY
BLUE CONDUCTIVITY==INTERNAL
INTERNAL AT
T ER
CONDUCTIVITY
 LH 700 Series
The “6-Part Diff”
NRBC enumeration automatic with differential

 Fully automated
– No reflex or repeat testing
required
– No additional reagent
packs required
 WBC count
automatically corrected
 Decision Rules
UNLIMITED
RULES!

4 Rule Types

And/Or
Joins

Message-
Action To Automatically
Be Taken Make Slide
 Research Population Data (RPD)

When VCS 3D Dataplot is

optimized;
 There is a change in the
WBC Research Population
Data

 This appears to correlate

with the presence of
abnormal cells in previously
undiagnosed patients
 Research Population Data

NE1

The increasing SD corresponds

to a more immature population
of cells
Research Population Data (RPD)

 WBC Research Population Data has

been studied in the following clinical
cases:
– CLL
– Left Shift
– Malaria
– Lymphoproliferative Disorders
– Myelodysplasia
– Sepsis
 CLINICAL
APPLICATION

Steve Marionneaux
Laboratory Manager
The Saint Vincent’s Comprehensive Cancer Center
New York, New York
MYSTERY #1
CBC Results
8 Year Old Female
DataPlot
Results
MANUAL DIFF RESULTS

MANUAL DIFF
Seg = 20
Band = 2
Lymph = 51
Blast = 27
 Diff Cube Rotation

VOLUME
VOLUME

TY

SCA
VI
TI

SCATTER
UC
ND

T
CO

TER
CONDUCTIV
ITY

PRE-BCELL
PRE-B CELLAcute
Acute
LymphoblasticLeukemia
Lymphoblastic Leukemia
 PRECURSOR
B-CELL ALL

 Low WBC, neutropenia

 Anemic

 Mononuclear population with smooth

chromatin
 CD34+, TdT+ population
MYSTERY #2
 WBC &PLT
HISTOGRAMS
AUTODIFF RESULTS
RBC HISTOGRAM
CBC / RBC RESULTS
MYSTERY #3
46 Year Old Female
46 / Female
 Acute Promyelocytic
Leukemia (Microgranular)
VOLUME

TY
I VI
T
D UC
N
CO SCATTER

MANUAL DIFF
Lymph = 2 CO
ND
Mono = 1 UC
TIV ER
T T
Blast = 97 ITY SCA
MYSTERY #4
Medical Mystery #4
 Chronic
Lymphocytic
Leukemia
MANUAL DIFF
Seg = 6
Lymph = 92
Mono = 2
CLL

WITH

SMUDGE

CELLS
 CHRONIC
LYMPHOCYTIC
LEUKEMIA
 Typically >60 years of age
 Initially asymptomatic

 Increased WBC

 Increased % of small, normal

lymphs (as disease progresses,
more ‘immature’ lymphs appear
 Smudge cells
MYSTERY #5
 12 Month Old Male

HGB = 7.0
12 Month Old Male

RBC Morphology
2+ Poik 3+ Aniso 4+ Hypo
4+ Micro 1+ Target 2+ Ellipto
1+ Teardrop 1+ Poly
MANUAL DIFF
Seg = 42
Lymph = 46
Mono = 5
Eo = 5
Baso = 2
NRBC = 1
Iron
Iron Deficiency
Deficiency

Thalassemia
Thalassemia

?????
 RETIC
RESEARCH POPULATIONS
Sickle
Thalassemia
Low Volume Lymphs =CLL
MYSTERY #6
 Case Study
History

74 year old female

20lb unexplained weight loss

Fever

Malaise

Sore throat 2 weeks duration

Muscle aches
HISTOGRAM
Blasts or large
DATA lymphs
Auer rods are
defined as a
coalescence of
the azurophilic
granules and are
only seen in
non-lymphocytic
leukemias

???????

AUER ROD
Manual Differential
 Seg = 4
 Band = 1
 Lymph = 17
 Mono = 3
 BLAST = 75 w/ occ
Auer rod
ACUTE MYELOCYTIC
LEUKEMIA
 Sudden onset
 Anemic

 Variable WBC

 Decreased PLT count

 >10% Blasts in peripheral blood

 Special Stains & Flow markers

+ for myelogenous cell lines
MYSTERY #7
 Case Study
History
 83 year old male
 Unexplained weight loss

 Malaise

 Night sweats

 Slight
hepatosplenomegaly
LAB RESULTS
Manual Diff:
Seg = 33
Band = 15
Lymph = 19
Mono = 6
Meta = 11
Myelo = 10
Blast = 6
???
 VCS
3-D Data Plot

Neutrophil Series
•Neutrophils
•Bands
•Metas
•Myelos
•Pros
•Ne Blasts
 VCS
3-D Data Plot

Monocytes
Monoblasts
 FLOW CYTOMETRY
PATHOLOGIST
INTERPRETATION
The immunophenotypic findings reveal increased
monocytes (26%) and 52% granulocytes with a shift
toward immaturity and diminished side scatter.
There is no evidence of increased blasts, a
monoclonal B cell or aberrant T cell process.

The immunophenotypic findings are suggestive of a

myeloproliferative process. Acute monocytic
leukemia cannot be entirely excluded. Clinical
pathologic correlation is required for final diagnosis.
 MYELOPROLIFERATIVE
DISORDERS
Defined as a hypercellular bone marrow
with increased quantities of one or more
of the cells lines: erythrocytes, leukocytes
or platelets in the peripheral blood.

It is thought to be a neoplastic, clonal

proliferation of a single multipotential
stem cell w/ one cell line predominating
and often transforming into another.
 SUMMARY
 Look at ALL the information provided by
the instrument:
– CBC parameters
– WBC Histograms
– RBC Histograms
– PLT Histograms
– Dataplots
– Suspect Flags
– Research Parameter
 SUMMARY
 Combine this information with what you
see at the microscope
 Ask for a “second opinion” from a peer
 Create an “abnormal file”

SAVED LIST
FOLDER
Questions
Questions
???????
???????
ANY QUESTIONS