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
AccuCount Technology – LH700 Series WBC 0 – 400,000 RBC 0 – 8,000,000 HGB 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 Cells are hydrodynamically focused An electro-optical flow cytometer provides concurrent electronic and optical measurements
The Triple Transducer Module A major advance in technology An electro-optical flow cytometer Provides concurrent electronic and optical measurements RF Detector Pre-Amp Lens Block Light Scatter Pre-Amp LS Sensor Laser Flow Cell
VCS Technology Volume Conductivity Light Scatter Total Cell Volume Cell Surface Characteristics Nuclear Volume Nuc/Cyto Ratio
NUCLEAR SHAPE AND COMPOSITION CELL SIZE CYTOPLASM GRANULES The 3-D VCS Scatterplot
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.
Lymphs Monos Basos Neuts Eos 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
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 AccuGate Software Technology
NORMAL DATAPLOT MONOCYTES NEUTROPHILS EOSINOPHILS LYMPHOCYTES BASOPHILS NRBC, PLT CLUMPS, GIANT PLT, MALARIAL PARASITES, DEBRIS, ETC C O N D U C T I V I T Y VOLUMEVOLUME S C A T T E R
CONDUCTIVITY SCATTER VOLUME CONDUCTIVITY CUBE ROTATION RED = VOLUME = SIZE GREEN = SCATTER = SURFACE BLUE = CONDUCTIVITY = INTERNAL RED = VOLUME = SIZE GREEN = SCATTER = SURFACE BLUE = CONDUCTIVITY = INTERNAL SCATTER VOLUME
LH 700 Series 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 Rules 4 Rule Types Message- Action To Be Taken And/Or Joins Automatically Make Slide UNLIMITED RULES!
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
The increasing SD corresponds to a more immature population of cells NE1 Research Population Data
Research Population Data (RPD) WBC Research Population Data has been studied in the following clinical cases: – CLL – Left Shift – Malaria – Lymphoproliferative Disorders – Myelodysplasia – Sepsis
Steve Marionneaux Laboratory Manager The Saint Vincents Comprehensive Cancer Center New York, New York CLINICAL APPLICATION
CBC Results 8 Year Old Female
MANUAL DIFF RESULTS MANUAL DIFF Seg = 20 Band = 2 Lymph = 51 Blast = 27
MANUAL DIFF Seg = 42 Lymph = 46 Mono = 5 Eo = 5 Baso = 2 NRBC = 1
????? Iron Deficiency Thalassemia Iron Deficiency Thalassemia
RETIC RESEARCH POPULATIONS Sickle Thalassemia Low Volume Lymphs =CLL
Case Study History 74 year old female 20lb unexplained weight loss Fever Malaise Sore throat Muscle aches 2 weeks duration
Blasts or large lymphs HISTOGRAM DATA
??????? AUER ROD Auer rods are defined as a coalescence of the azurophilic granules and are only seen in non-lymphocytic leukemias
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
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
Neutrophil Series Neutrophils Bands Metas Myelos Pros Ne Blasts VCS 3-D Data Plot
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
Case Study History 14 year old female Hgb SS Asthmatic Admitted in crisis
Lab Results Chemistry Glucose = 104 Sodium = 142 Potassium = 3.9 BUN = 3 L Creatinine = 0.5 L CO 2 = 28 Chloride = 108 Calcium = 8.3
Sickle Cell Pappenheimer bodies
Cellular interference with corrected and uncorrected WBC
Manual Differential: 55% Seg 1% Band 36% Lymph 7% Mono 1% Eo 6 NRBC NRBCs Giant platelets Platelet clumps RBC fragments Lyse resistant RBCs Malaria very small lymphs
Signature Position Threshold Interference 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.
Derivation of NRBCs 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 WBC Histogram Presence of high take-off Standard deviation and shape of lymphocyte population Lymphocyte mean channel THE WBC IS ONLY CORRECTED FOR NRBCs >35fl