Vcs Technology Case Studies

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 Pathway

Detail of Aperture Internal Electrode

External Electrode

Suspension of Cells

External Housing (Aperture Bath)

Aperture

Aperture Housing

Aperture Impedance System System with Sweep Flow 

Eliminates recirculation of cells



Cells pushed away by diluent



More accurate counts

Coincidence Correction



Electronic pulse-editing & coincidence correction:

Pulse to be edited

– Provides

accurate histograms and cell sizing for reliable RBC and PLT indices

Diluent stream

Aperture Impedance System

Triplicate Counting – Ensures Precision – Reduces Repeats

The Coulter Principle Red Blood Cell

A red cell passes through RBC aperture

Sensing Zone Oscilloscope

RBC HISTOGRAM

NORMAL

RBC HISTOGRAM

COLD AGGLUTININ

MACROCYTIC, TARGET CELLS, DI RBC

RBC FRAGMENTS, MICROCYTIC RBCs, Giant PLT

DI RBCs

Post Transfusion

PLT HISTOGRAMS

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





Small Platelets

Giant Platelets

The Coulter Principle Neutroph il

A white cell passes through WBC aperture

Sensing Zone Oscilloscope

Coulter WBC Histogram Monos Lymphs 50 – 90 fL

90 -160 fL Baso

Neuts Eos

160 - 450 fL

WBC HISTOGRAMS

ImmNE1 & ImmNE2

ImmNE2

Lymphocytosis

Variant Lymph

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

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.

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 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

VCS Technology Volume Total Cell Volume

Nuclear Volume Nuc/Cyto Ratio

Conductivity

Total Cell Volume

Cell Surface Characteristics

Light Scatter

The 3-D VCS Scatterplot PE N A SH TIO R I A S E O L P C M U O N C D AN

CEL

GR

AN

UL ES

L SI

M S A

ZE

T Y C

L P O

COULTER VCS TECHNOLOGY  VOLUME

= SIZE

 CONDUCTIVITY

 LIGHT

= INTERNAL COMPOSITION

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  ScatterNew.wmv

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)

Allinone.avi ALLINONE.AVI Allinone.avi

Allinone.avi

CONDUCTIVITY (Z)

Monos

Eos

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

Neuts Lymphs Basos

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

Better Abnormal Cell Detection 2 1

3 4

5

6

7

7a 8

9

10

1 2 3 4 5 6 7 7a 8 9 10

Mono-Blasts Myelo-Blasts Immature Granulocytes Band Neutrophils Lympho-Blasts Variant Lymphocytes Low Volume Lymphocytes NRBCs PLT Clumps Giant Platelets RBC Parasites (Malaria, etc)

DIFF TECHNOLOGY

CONFUSED????

NORMAL

NORMAL

NORMAL DATAPLOT MONOCYTES

NEUTROPHILS EOSINOPHILS

V O L U M E

BASOPHILS LYMPHOCYTES

C

O

N

D

U

C

T

IV

IT

Y

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

SCATTER VOLUME

CO

ND U

CT

IV

IT Y

VOLUME

CUBE ROTATION

RED = VOLUME = SIZE GREEN = SCATTER = SURFACE BLUE = CONDUCTIVITY = INTERNAL

SC 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

MessageAction To Be Taken

Automatically 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

Mean and SD are typically consistent from one normal population to the next

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

TI VI

SCATTER CONDUCTIV ITY

PRE­B CELL Acute  Lymphoblastic Leukemia

TER

CO

ND UC

T SCA

TY

VOLUME

VOLUME

Diff Cube Rotation

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

VOLUME

Acute Promyelocytic Leukemia (Microgranular)

IT

V TI C U ND

CO

Y

SCATTER

MANUAL DIFF Lymph = 2 Mono = 1 Blast = 97

CO ND UC

TIV

I TY

R

TE T A SC

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 Deficiency 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 DATA

Blasts or large 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 

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.

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

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 DATA PLOT

CD14 = Monocytic Cells

CD14+ CELLS

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 ???????

ANY QUESTIONS

Case Study History  14

year old female  Hgb SS  Asthmatic  Admitted in crisis

Lab Results CBC Results  WBC = 11.5 corrected for NRBC’s  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

Lab Results Chemistry  Glucose = 104  Sodium = 142  Potassium = 3.9  BUN = 3 L  Creatinine = 0.5 L  CO = 28 2 Chloride = 108  Calcium = 8.3 

Sickle Cell Pappenheimer bodies

Cellular interference with corrected and uncorrected WBC

NRBC’s 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

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

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

THE WBC IS ONLY CORRECTED FOR NRBCs >35fl

RETICULOCYTE COUNT Retics

Mature RBC’s

Platelets/Debris

WBC’s

Sickle Cell Disease   



 

Anemia Numerous sickle cells Pappenheimer bodies Retic = 10-40% Hgb Electrophoresis: Hgb S (>50%) Hgb F (variable)

   

Leukocytosis Howell-Jolly Bodies Increased NRBC’s Increased bilirubin Numerous Target cells

medical technologists

Research Population Data NE2

The increasing SD corresponds to a more immature population of cells

Research Population Data NE BLAST

The increasing SD corresponds to a more immature population of cells

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