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

Internal Electrode

Detail of Aperture

External Electrode

Suspension of Cells

External Housing (Aperture Bath)

Aperture

Aperture Housing

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

Giant Platelets

Small Platelets

The Coulter Principle

Neutrophil
A white cell passes through WBC aperture

Sensing Zone
Oscilloscope

Coulter WBC Histogram

Monos Lymphs 50 90 fL
Baso

90 -160 fL

Neuts
Eos

160 - 450 fL

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 IN SAMPLE IN

SHEATH FLUID

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

The 3-D VCS Scatterplot

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. 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
Lymphs Basos

Neuts

Better Abnormal Cell Detection

2

3 4

5 6

7 8

7a
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

NORMAL

NORMAL

NORMAL DATAPLOT
MONOCYTES

NEUTROPHILS
EOSINOPHILS

V O L U M E

BASOPHILS LYMPHOCYTES

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

SCATTER

CUBE ROTATION
VOLUME

SCATTER
VOLUME CONDUCTIVITY

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

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

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 Vincents 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

SCATTER

PRE-B CELL Acute Lymphoblastic Leukemia

VOLUME

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

SCATTER

MANUAL DIFF Lymph = 2 Mono = 1 Blast = 97

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 2 weeks duration Sore throat 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

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 ???????

ANY QUESTIONS