MEDICAL MYSTERIESMEDICAL MYSTERIESUsing Hematology Instrument Data to

Troubleshoot

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

OBJECTIVESOBJECTIVES

Review instrument technology; compare & contrast normal vs abnormal

Apply this technology / knowledge to a variety of cases

“SOLVE” the medical mystery

PROVENPROVEN BECKMAN COULTERBECKMAN COULTER

TECHNOLOGIESTECHNOLOGIES

The Coulter PrincipleThe Coulter Principle

External Electrode

Vacuum

External Housing(Aperture Bath)

Detail of Aperture

Internal Electrode

Suspension of Cells

Aperture Current Pathway

Aperture

Aperture Housing

RBC HISTOGRAMRBC HISTOGRAM

NORMALNORMAL

RBC HISTOGRAMRBC HISTOGRAM

RBC FRAGMENTS, MICROCYTIC RBCs, Giant PLTRBC FRAGMENTS, MICROCYTIC RBCs, Giant PLT DI RBCsDI RBCs

MACROCYTIC, TARGET CELLS, DI RBCMACROCYTIC, TARGET CELLS, DI RBCCOLD AGGLUTININCOLD AGGLUTININ

Post TransfusionPost Transfusion

PLT HISTOGRAMSPLT HISTOGRAMS

NORMALNORMAL

PLT Curve FittingPLT Curve FittingThe Curve Fitting Process Allows

More Accurate Counts When Platelets of Larger Than 20 fL Are Present

PLT Counting & SizingPLT 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 HISTOGRAMSPLT HISTOGRAMS

Giant Platelets

Small Platelets

Sensing Zone

Neutrophil

Oscilloscope

The Coulter PrincipleThe Coulter Principle

A white cell passes through WBC aperture

Eos

Baso

Coulter WBC HistogramCoulter WBC Histogram

Lymphs

50 – 90 fL

Monos

90 -160 fL

Neuts

160 - 450 fL

WBC HISTOGRAMSWBC HISTOGRAMS

ImmNE2 Eosinophilia

ImmNE1 & ImmNE2

Blasts

Lymphocytosis Variant Lymph

WBC InterferenceWBC 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

AccuCount TechnologyAccuCount 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 TECHNOLOGYVCS TECHNOLOGYAutomated Differential

Analysis

Near Native WBC AnalysisNear 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 CytometryBioPhysical Flow Cytometry

SHEATH-FLUID IN

SHEATH-FLUID IN

SAMPLE IN

FLOW CELL

SAMPLE DILUTION

SHEATH FLUIDSENSING

AREA

Cells are hydrodynamically focused An electro-optical flow cytometer provides concurrent

electronic and optical measurements

NUCLEAR SHAPE

AND COMPOSITION

CELL SIZECYTOPLASM

GRANULES

The 3-D VCS ScatterplotThe 3-D VCS Scatterplot

COULTER COULTER VCSVCS TECHNOLOGYTECHNOLOGY

VOLUME = SIZE

CONDUCTIVITY = INTERNAL COMPOSITION

LIGHT SCATTER = CELL SHAPE / SURFACE

VOLUMEVOLUME

DC Measures Total Cell Volume Using the Reference Method of Direct Current Impedance

Unaffected by cell orientation

CONDUCTIVITYCONDUCTIVITY

RF Measures Internal Cell Structure Using Radiographic Imaging Similar to Ultrasound

Conductivity Is a Proprietary Technology

LASER LIGHT SCATTERLASER LIGHT SCATTER

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

Lymphs

Monos

Basos

Neuts

Eos

3-D Cellular Analysis - VCS3-D Cellular Analysis - VCS

VOLUME (Y) CONDUCTIVITY (Z)

LIGHT SCATTER (X)

The 3 probes (DC, RF and Scatter) interrogateeach of the 8192 cells simultaneously.

Every cell is treated in the same manner and each cell is given an X, Y, and Z coordinate onthe dataplot; with 16 million points in the matrix.

ALL cell populations are DIRECTLYmeasured

Better Better Abnormal Cell DetectionAbnormal 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 Platelets10 RBC Parasites (Malaria,

etc)

1

2

34

56

7

8 9 10

7a

DIFF TECHNOLOGYDIFF TECHNOLOGY

NORMALNORMAL

NORMALNORMAL

NORMAL DATAPLOTNORMAL DATAPLOT

MONOCYTESNEUTROPHILS

EOSINOPHILS

LYMPHOCYTES

BASOPHILS

NRBC, PLT CLUMPS, GIANT PLT, MALARIAL PARASITES, DEBRIS, ETC

C O N

D U

C T

I V I

T Y

VOLUME

S C A T T E R

CONDUCTIVITY

SCATTER

VO

LU

ME

CONDUCTIV

ITY

CUBE ROTATIONCUBE ROTATION

RED = VOLUME = SIZEGREEN = SCATTER = SURFACEBLUE = CONDUCTIVITY = INTERNAL

RED = VOLUME = SIZEGREEN = SCATTER = SURFACEBLUE = CONDUCTIVITY = INTERNAL

SCATTER

VO

LU

ME

LH 700 SeriesLH 700 SeriesThe “6-Part Diff”The “6-Part Diff”

Fully automated

– No reflex or repeat testing required

– No additional reagent packs required

WBC count

automatically corrected

NRBC enumeration automatic with differential

Decision RulesDecision Rules

4 Rule Types

Message-Action To Be Taken

And/Or Joins

Automatically Make Slide

UNLIMITEDRULES!

Research Population Data (RPD)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

The increasing SD correspondsto a more immature populationof cells

NE1

Research Population Data

Research Population Data Research Population Data (RPD)(RPD)

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

Steve MarionneauxLaboratory ManagerThe Saint Vincent’s Comprehensive Cancer CenterNew York, New York

CLINICALCLINICAL APPLICATION APPLICATION

MYSTERY #1MYSTERY #1

CBC ResultsCBC Results8 Year Old Female8 Year Old Female

DataPlot DataPlot ResultsResults

MANUAL DIFF RESULTSMANUAL DIFF RESULTS

MANUAL DIFFSeg = 20Band = 2Lymph = 51Blast = 27

Diff Cube RotationDiff Cube Rotation

CONDUCTIVITY

SCA

TT

ER

VO

LU

ME

SCATTER

VO

LU

ME

CONDUCTIV

ITY

PRE-B CELL Acute Lymphoblastic Leukemia

PRE-B CELL Acute Lymphoblastic Leukemia

PRECURSORPRECURSOR B-CELL ALL B-CELL ALL

Low WBC, neutropenia Anemic Mononuclear population with smooth

chromatin CD34+, TdT+ population

MYSTERY #2MYSTERY #2

WBC &PLTWBC &PLTHISTOGRAMSHISTOGRAMS

AUTODIFF RESULTSAUTODIFF RESULTS

RBC HISTOGRAMRBC HISTOGRAM

CBC / RBC RESULTSCBC / RBC RESULTS

MYSTERY #3MYSTERY #3

46 Year Old Female46 Year Old Female

46 / Female46 / Female

Acute Promyelocytic Leukemia (Microgranular)

CONDUCTIVITY SCATTER

MANUAL DIFFLymph = 2Mono = 1Blast = 97

SCATTERCONDUCTIVIT

Y

VO

LU

ME

MYSTERY #4MYSTERY #4

Medical Mystery #4Medical Mystery #4

Chronic Chronic LymphocyticLymphocytic

LeukemiaLeukemia

MANUAL DIFFSeg = 6Lymph = 92Mono = 2

CLL

WITH

SMUDGE

CELLS

CHRONIC CHRONIC LYMPHOCYTIC LYMPHOCYTIC

LEUKEMIALEUKEMIA Typically >60 years of age Initially asymptomatic Increased WBC Increased % of small, normal

lymphs (as disease progresses, more ‘immature’ lymphs appear

Smudge cells

MYSTERY #5MYSTERY #5

12 Month Old Male12 Month Old Male

HGB = 7.0

12 Month Old Male12 Month Old Male

RBC Morphology2+ Poik 3+ Aniso 4+ Hypo4+ Micro 1+ Target 2+ Ellipto1+ Teardrop 1+ Poly

MANUAL DIFFSeg = 42Lymph = 46Mono = 5Eo = 5Baso = 2NRBC = 1

??????????

Iron Deficiency

Thalassemia

Iron Deficiency

Thalassemia

RETIC RETIC RESEARCH POPULATIONSRESEARCH POPULATIONS

SickleThalassemiaLow Volume Lymphs =CLL

MYSTERY #6MYSTERY #6

Case Study Case Study HistoryHistory

74 year old female20lb unexplained weight lossFeverMalaiseSore throatMuscle aches

2 weeks duration

Blasts or large Blasts or large lymphslymphs

HISTOGRAMDATA

???????

AUER ROD

Auer rods aredefined as acoalescence ofthe azurophilicgranules and areonly seen in non-lymphocyticleukemias

Manual DifferentialManual Differential

Seg = 4 Band = 1 Lymph = 17 Mono = 3 BLAST = 75 w/ occ

Auer rod

ACUTE MYELOCYTIC ACUTE MYELOCYTIC LEUKEMIALEUKEMIA

Sudden onset Anemic Variable WBC Decreased PLT count >10% Blasts in peripheral blood Special Stains & Flow markers

+ for myelogenous cell lines

MYSTERY #7MYSTERY #7

Case StudyCase StudyHistoryHistory

83 year old maleUnexplained weight lossMalaiseNight sweatsSlight

hepatosplenomegaly

LAB RESULTSLAB RESULTS

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

???

Neutrophil Series

•Neutrophils

•Bands

•Metas

•Myelos

•Pros

•Ne Blasts

VCS3-D Data Plot

MonocytesMonoblasts

VCS3-D Data Plot

FLOW CYTOMETRYFLOW CYTOMETRYPATHOLOGIST PATHOLOGIST

INTERPRETATIONINTERPRETATIONThe 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 MYELOPROLIFERATIVE DISORDERSDISORDERS

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.

SUMMARYSUMMARY

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

SUMMARYSUMMARY

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 QUESTIONSANY QUESTIONS