MEDICAL MYSTERIES

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MEDICAL MYSTERIES

• Using Hematology Instrument Data to Troubleshoot

DR PETER JOHN LOGA, PhD MS MSc BSc SDMLT
DMLT FIBMS FZIMLS
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OBJECTIVES

• Review instrument technology compare contrast
  normal vs abnormal
• Apply this technology / knowledge to a variety of
  cases
• SOLVE the medical mystery

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PROVEN BECKMAN COULTERTECHNOLOGIES
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The Coulter Principle
Vacuum
Detail of Aperture
Aperture Current Pathway
Internal Electrode
External Electrode
Suspension of Cells
External Housing (Aperture Bath)
Aperture
Aperture Housing
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Aperture Impedance SystemSystem with Sweep Flow

• Eliminates recirculation of cells
• Cells pushed away by diluent
• More accurate counts

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Coincidence Correction

• Electronic pulse-editing coincidence
  correction
• Provides accurate histograms and cell sizing for
  reliable RBC and PLT indices

Pulse to be edited
Diluent stream
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Aperture Impedance System

• Triplicate Counting
• Ensures Precision
• Reduces Repeats

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The Coulter Principle
A red cell passes through RBC aperture
Red Blood Cell
Sensing Zone
Oscilloscope
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RBC HISTOGRAM
NORMAL
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RBC HISTOGRAM
MACROCYTIC, TARGET CELLS, DI RBC
COLD AGGLUTININ
DI RBCs
Post Transfusion
RBC FRAGMENTS, MICROCYTIC RBCs, Giant PLT
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PLT HISTOGRAMS
NORMAL
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PLT Curve Fitting

• The Curve Fitting Process Allows More Accurate
  Counts When Platelets of Larger Than 20 fL Are
  Present

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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

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PLT HISTOGRAMS

• Giant Platelets

• Small Platelets

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The Coulter Principle
A white cell passes through WBC aperture
Neutrophil
Sensing Zone
Oscilloscope
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Coulter WBC Histogram
Monos 90 -160 fL
Neuts 160 - 450 fL
Lymphs 50 90 fL
Eos
Baso
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WBC HISTOGRAMS
ImmNE1 ImmNE2
Lymphocytosis
Variant Lymph
ImmNE2
Eosinophilia
Blasts
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WBC Interference

• Percentage of interference analyzed for
  statistical significance
• Flagging based on all three histograms instead
  of one
• Histogram positional parameters used for further
  definition

Cellular Interference
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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

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VCS TECHNOLOGY

• Automated Differential Analysis

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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

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Flow Cytometry

• Technique for counting, examining and sorting
  microscopic particles suspended in a stream of
  fluid. It allows simultaneous multiparametric
  analysis of the physical and/or chemical
  characteristics of single cells flowing through a
  detection apparatus.

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BioPhysical Flow Cytometry

• Cells are hydrodynamically focused
• An electro-optical flow cytometer provides
  concurrent electronic and optical measurements

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The Triple Transducer Module
RF Detector Pre-Amp
Laser
Lens Block
Flow Cell
LS Sensor
Light Scatter Pre-Amp

• A major advance in technology
• An electro-optical flow cytometer
• Provides concurrent electronic and optical
  measurements

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VCS Technology
Volume
Total Cell Volume
Total Cell Volume
Nuclear Volume Nuc/Cyto Ratio
Cell Surface Characteristics
Light Scatter
Conductivity
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The 3-D VCS Scatterplot
NUCLEAR SHAPE AND COMPOSITION
GRANULES
CYTOPLASM
CELL SIZE
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COULTER VCS TECHNOLOGY

• VOLUME SIZE
• CONDUCTIVITY INTERNAL COMPOSITION
• LIGHT SCATTER CELL SHAPE / SURFACE

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VOLUME

• DC Measures Total Cell Volume Using the Reference
  Method of Direct Current Impedance
• Unaffected by cell orientation

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CONDUCTIVITY

• RF Measures Internal Cell Structure Using
  Radiographic Imaging Similar to Ultrasound
• Conductivity Is a Proprietary Technology

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LASER LIGHT SCATTER

• Light Scatter Measures Cell Surface Granularity
  Using a Broad Range of Angles. Over 60 angles of
  light scatter are analyzed.

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3-D Cellular Analysis - VCS
VOLUME (Y)
CONDUCTIVITY (Z)
LIGHT SCATTER (X)
The 3 probes (DC, RF and Scatter)
interrogate each of the 8192 cells simultaneously.
Every cell is treated in the same manner and
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
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AccuGate Software Technology

• Population Boundaries Curve Around Clusters
• Overlapping Clusters Are Separated
• Each Population Is Independently Categorized
• Rare Event Clusters Are Easily Identified
• Older samples more accurately evaluated

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Better Abnormal Cell Detection

• 1 Mono-Blasts
• 2 Myelo-Blasts
• 3 Immature Granulocytes
• 4 Band Neutrophils
• 5 Lympho-Blasts
• 6 Variant Lymphocytes
• 7 Low Volume Lymphocytes
• 7a NRBCs
• 8 PLT Clumps
• 9 Giant Platelets
• 10 RBC Parasites (Malaria, etc)

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DIFF TECHNOLOGY
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CONFUSED????
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NORMAL
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NORMAL
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NORMAL DATAPLOT
MONOCYTES
NEUTROPHILS
EOSINOPHILS
V O L U M E
BASOPHILS
LYMPHOCYTES
NRBC, PLT CLUMPS, GIANT PLT, MALARIAL PARASITES,
DEBRIS, ETC
C O N D U C T I V I T Y
S C A T T E R
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CUBE ROTATION
VOLUME
CONDUCTIVITY
SCATTER
VOLUME
RED VOLUME SIZE GREEN SCATTER
SURFACE BLUE CONDUCTIVITY INTERNAL
SCATTER
CONDUCTIVITY
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LH 700 SeriesThe 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

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Decision Rules
UNLIMITED RULES!
4 Rule Types
And/Or Joins
Automatically Make Slide
Message-Action To Be Taken
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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

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Research Population Data
Mean and SD are typically consistent from one
normal population to the next
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Research Population Data
NE1
The increasing SD corresponds to a more immature
population of cells
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Research Population Data (RPD)

• WBC Research Population Data has been studied in
  the following clinical cases
• CLL
• Left Shift
• Malaria
• Lymphoproliferative Disorders
• Myelodysplasia
• Sepsis

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CLINICAL APPLICATION
Steve Marionneaux Laboratory Manager The Saint
Vincents Comprehensive Cancer Center New York,
New York
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MYSTERY 1
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CBC Results8 Year Old Female
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DataPlot Results
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MANUAL DIFF RESULTS
MANUAL DIFF Seg 20 Band 2 Lymph 51 Blast
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Diff Cube Rotation
VOLUME
VOLUME
SCATTER
CONDUCTIVITY
SCATTER
CONDUCTIVITY
PRE-B CELL Acute Lymphoblastic Leukemia
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PRECURSOR B-CELL ALL

• Low WBC, neutropenia
• Anemic
• Mononuclear population with smooth chromatin
• CD34, TdT population

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MYSTERY 2
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WBC PLTHISTOGRAMSAUTODIFF RESULTS
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RBC HISTOGRAM
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CBC / RBC RESULTS
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MYSTERY 3
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46 Year Old Female
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46 / Female
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Acute Promyelocytic Leukemia (Microgranular)
VOLUME
CONDUCTIVITY
SCATTER
MANUAL DIFF Lymph 2 Mono 1 Blast 97
CONDUCTIVITY
SCATTER
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MYSTERY 4
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Medical Mystery 4
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Chronic Lymphocytic Leukemia
MANUAL DIFF Seg 6 Lymph 92 Mono 2
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CLL WITH SMUDGE CELLS
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CHRONIC LYMPHOCYTIC LEUKEMIA

• Typically gt60 years of age
• Initially asymptomatic
• Increased WBC
• Increased of small, normal lymphs (as disease
  progresses, more immature lymphs appear
• Smudge cells

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MYSTERY 5
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12 Month Old Male
HGB 7.0
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12 Month Old Male
RBC Morphology 2 Poik 3 Aniso 4 Hypo 4
Micro 1 Target 2 Ellipto 1 Teardrop 1
Poly
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MANUAL DIFF Seg 42 Lymph 46 Mono 5 Eo
5 Baso 2 NRBC 1
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?????
Iron Deficiency Thalassemia
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RETIC RESEARCH POPULATIONS
Sickle Thalassemia Low Volume Lymphs CLL

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MYSTERY 6
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Case Study History

• 74 year old female
• 20lb unexplained weight loss
• Fever
• Malaise
• Sore throat
• Muscle aches

2 weeks duration
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Blasts or large lymphs
HISTOGRAM DATA
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Auer rods are defined as a coalescence of the
azurophilic granules and are only seen in
non-lymphocytic leukemias
???????
AUER ROD
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Manual Differential

• Seg 4
• Band 1
• Lymph 17
• Mono 3
• BLAST 75 w/ occ Auer rod

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ACUTE MYELOCYTIC LEUKEMIA

• Sudden onset
• Anemic
• Variable WBC
• Decreased PLT count
• gt10 Blasts in peripheral blood
• Special Stains Flow markers for myelogenous
  cell lines

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FLOW CYTOMETRY DATA PLOTS
CD45 is a generic marker for all cell lines.
CD117 is considered a myelocytic marker. If a
patient is positive for this marker, they are
considered a good candidate for a newer
chemotherapeutic drug called GLEVEC.
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IMMUNOPHENOTYPIC RESULTS

• 60 population of myeloid blasts
• CD34 CD11b (partial)
• CD64, CD33, CD15, CD56
• CD117, MPO
• Negative for HLA-DR, CD7, CD19, CD20, CD22, CD3,
  CD8, and TDT

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MYSTERY 7
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Case StudyHistory

• 83 year old male
• Unexplained weight loss
• Malaise
• Night sweats
• Slight hepatosplenomegaly

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LAB RESULTS

• Manual Diff
• Seg 33
• Band 15
• Lymph 19
• Mono 6
• Meta 11
• Myelo 10
• Blast 6

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???
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VCS 3-D Data Plot

• Neutrophil Series
• Neutrophils
• Bands
• Metas
• Myelos
• Pros
• Ne Blasts

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VCS 3-D Data Plot

• Monocytes
• Monoblasts

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FLOW CYTOMETRY DATA PLOT
CD14 CELLS
CD14 Monocytic Cells
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FLOW CYTOMETRYPATHOLOGIST 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.

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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.

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SUMMARY

• Look at ALL the information provided by the
  instrument
• CBC parameters
• WBC Histograms
• RBC Histograms
• PLT Histograms
• Dataplots
• Suspect Flags
• Research Parameter

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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
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Questions ???????
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ANY QUESTIONS
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Case Study History

• 14 year old female
• Hgb SS
• Asthmatic
• Admitted in crisis

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Lab Results

• CBC Results
• WBC 11.5 corrected for NRBCs
• RBC 2.10
• HGB 6.6 corrected for icterus
• PLT 349
• RDW 25.4

• Morphology
• 3 Aniso
• 3 Poik
• 2 Poly
• 3 Sickle
• 3 Pappenheimer Bodies
• 1 Target Cells

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Lab Results

• Chemistry
• Glucose 104
• Sodium 142
• Potassium 3.9
• BUN 3 L
• Creatinine 0.5 L
• CO2 28
• Chloride 108
• Calcium 8.3

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Sickle Cell Pappenheimer bodies
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Cellular interference with corrected and
uncorrected WBC
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NRBCs Giant platelets Platelet clumps RBC
fragments Lyse resistant RBCs Malaria very small
lymphs
Manual Differential 55 Seg 1 Band 36 Lymph 7
Mono 1 Eo 6 NRBC
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NRBC Enumeration Cells must be present in BOTH
the signature position of the scatterplot as well
as a population of events consistent with NRBCs
at 35fl on the WBC threshold.
Threshold Interference
Signature Position
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Derivation of NRBCs

• WBC Histogram
• Presence of high take-off
• Standard deviation and shape of lymphocyte
  population
• Lymphocyte mean channel

THE WBC IS ONLY CORRECTED FOR NRBCs gt35fl

• VCS Dataplot
• Volume and light scatter mean channels
  differentiate suspected NRBCs from lyse resistant
  RBCs
• Conductivity channel differentiates NRBCs from
  PLT clumps and giant platelets

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RETICULOCYTE COUNT
WBCs
Retics
Mature RBCs
Platelets/Debris
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Sickle Cell Disease

• Leukocytosis
• Howell-Jolly Bodies
• Increased NRBCs
• Increased bilirubin
• Numerous Target cells

• Anemia
• Numerous sickle cells
• Pappenheimer bodies
• Retic 10-40
• Hgb Electrophoresis
• Hgb S (gt50)
• Hgb F (variable)

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medical
technologists
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Research Population Data
NE2
The increasing SD corresponds to a more immature
population of cells
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Research Population Data
NE BLAST
The increasing SD corresponds to a more immature
population of cells
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Bonus - RBC Grading

• Accurately measure MCV
• Accurately measure RDW
• Detect dimorphic populations

• Graded RBC morphology
• Anisocytosis , ,
• Microcytosis , ,
• Macrocytosis , ,
• Hypochromia , ,
• Dimorphic RBC Population
• Micro RBCs/RBC Fragments
• RBC Agglutination