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