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MEDICAL MYSTERIES Using Hematology Instrument Data to Troubleshoot DR PETER JOHN LOGA, PhD; MS; MSc; BSc; SDMLT; DMLT; FIBMS; FZIMLS.

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Presentation on theme: "MEDICAL MYSTERIES Using Hematology Instrument Data to Troubleshoot DR PETER JOHN LOGA, PhD; MS; MSc; BSc; SDMLT; DMLT; FIBMS; FZIMLS."— Presentation transcript:

1 MEDICAL MYSTERIES Using Hematology Instrument Data to Troubleshoot DR PETER JOHN LOGA, PhD; MS; MSc; BSc; SDMLT; DMLT; FIBMS; FZIMLS

2 OBJECTIVES Review instrument technology; compare & contrast normal vs abnormal Apply this technology / knowledge to a variety of cases SOLVE the medical mystery

3

4 PROVEN BECKMAN COULTER TECHNOLOGIES

5 The Coulter Principle External Electrode Vacuum External Housing (Aperture Bath) Detail of Aperture Internal Electrode Suspension of Cells Aperture Current Pathway Aperture Aperture Housing

6 Aperture Impedance System System with Sweep Flow Eliminates recirculation of cells Cells pushed away by diluent More accurate counts

7 Pulse to be edited Diluent stream Coincidence Correction Electronic pulse-editing & coincidence correction: – Provides accurate histograms and cell sizing for reliable RBC and PLT indices

8 Aperture Impedance System Triplicate Counting – Ensures Precision – Reduces Repeats

9 Sensing Zone Red Blood Cell The Coulter Principle A red cell passes through RBC aperture Oscilloscope

10 RBC HISTOGRAM NORMAL

11 RBC HISTOGRAM RBC FRAGMENTS, MICROCYTIC RBCs, Giant PLT DI RBCs MACROCYTIC, TARGET CELLS, DI RBC COLD AGGLUTININ Post Transfusion

12 PLT HISTOGRAMS NORMAL

13 PLT Curve Fitting The Curve Fitting Process Allows More Accurate Counts When Platelets of Larger Than 20 fL Are Present

14 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

15 PLT HISTOGRAMS Giant Platelets Small Platelets

16 Sensing Zone Neutrophil Oscilloscope The Coulter Principle A white cell passes through WBC aperture

17 Eos Baso Coulter WBC Histogram Lymphs 50 – 90 fL Monos fL Neuts fL

18 WBC HISTOGRAMS ImmNE2 Eosinophilia ImmNE1 & ImmNE2 Blasts LymphocytosisVariant Lymph

19 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

20 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

21 VCS TECHNOLOGY Automated Differential Analysis

22 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

23 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.

24 BioPhysical Flow Cytometry Cells are hydrodynamically focused An electro-optical flow cytometer provides concurrent electronic and optical measurements

25 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

26 VCS Technology Volume Conductivity Light Scatter Total Cell Volume Cell Surface Characteristics Nuclear Volume Nuc/Cyto Ratio

27 NUCLEAR SHAPE AND COMPOSITION CELL SIZE CYTOPLASM GRANULES The 3-D VCS Scatterplot

28 COULTER VCS TECHNOLOGY VOLUME = SIZE CONDUCTIVITY = INTERNAL COMPOSITION LIGHT SCATTER = CELL SHAPE / SURFACE

29 VOLUME DC Measures Total Cell Volume Using the Reference Method of Direct Current Impedance Unaffected by cell orientation

30 CONDUCTIVITY RF Measures Internal Cell Structure Using Radiographic Imaging Similar to Ultrasound Conductivity Is a Proprietary Technology

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

32 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

33 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

34 Better Abnormal 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 Platelets 10 RBC Parasites (Malaria, etc) a

35 DIFF TECHNOLOGY

36 CONFUSED????

37 NORMAL

38 NORMAL

39 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

40 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

41 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

42 Decision Rules 4 Rule Types Message- Action To Be Taken And/Or Joins Automatically Make Slide UNLIMITED RULES!

43 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

44 Research Population Data Mean and SD are typically consistent from one normal population to the next

45 The increasing SD corresponds to a more immature population of cells NE1 Research Population Data

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

47 Steve Marionneaux Laboratory Manager The Saint Vincents Comprehensive Cancer Center New York, New York CLINICAL APPLICATION

48 MYSTERY #1

49 CBC Results 8 Year Old Female

50 DataPlot Results

51 MANUAL DIFF RESULTS MANUAL DIFF Seg = 20 Band = 2 Lymph = 51 Blast = 27

52 Diff Cube Rotation CONDUCTIVITY SCATTER VOLUME SCATTER VOLUME CONDUCTIVITY PRE-B CELL Acute Lymphoblastic Leukemia

53 PRECURSOR B-CELL ALL Low WBC, neutropenia Anemic Mononuclear population with smooth chromatin CD34+, TdT+ population

54 MYSTERY #2

55 WBC &PLT HISTOGRAMS AUTODIFF RESULTS

56 RBC HISTOGRAM

57 CBC / RBC RESULTS

58 MYSTERY #3

59 46 Year Old Female

60 46 / Female

61 Acute Promyelocytic Leukemia (Microgranular) CONDUCTIVITY SCATTER MANUAL DIFF Lymph = 2 Mono = 1 Blast = 97 SCATTER CONDUCTIVITY VOLUME

62 MYSTERY #4

63 Medical Mystery #4

64 Chronic Lymphocytic Leukemia MANUAL DIFF Seg = 6 Lymph = 92 Mono = 2

65 CLL WITH SMUDGE CELLS

66 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

67 MYSTERY #5

68 12 Month Old Male HGB = 7.0

69 12 Month Old Male RBC Morphology 2+ Poik 3+ Aniso 4+ Hypo 4+ Micro 1+ Target 2+ Ellipto 1+ Teardrop 1+ Poly

70 MANUAL DIFF Seg = 42 Lymph = 46 Mono = 5 Eo = 5 Baso = 2 NRBC = 1

71 ????? Iron Deficiency Thalassemia Iron Deficiency Thalassemia

72 RETIC RESEARCH POPULATIONS Sickle Thalassemia Low Volume Lymphs =CLL

73 MYSTERY #6

74 Case Study History 74 year old female 20lb unexplained weight loss Fever Malaise Sore throat Muscle aches 2 weeks duration

75 Blasts or large lymphs HISTOGRAM DATA

76 ??????? AUER ROD Auer rods are defined as a coalescence of the azurophilic granules and are only seen in non-lymphocytic leukemias

77 Manual Differential Seg = 4 Band = 1 Lymph = 17 Mono = 3 BLAST = 75 w/ occ Auer rod

78 ACUTE MYELOCYTIC LEUKEMIA Sudden onset Anemic Variable WBC Decreased PLT count >10% Blasts in peripheral blood Special Stains & Flow markers + for myelogenous cell lines

79 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.

80 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

81 MYSTERY #7

82 Case Study History 83 year old male Unexplained weight loss Malaise Night sweats Slight hepatosplenomegaly

83

84

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

86 ???

87 Neutrophil Series Neutrophils Bands Metas Myelos Pros Ne Blasts VCS 3-D Data Plot

88 Monocytes Monoblasts VCS 3-D Data Plot

89 FLOW CYTOMETRY DATA PLOT CD14+ CELLS CD14 = Monocytic Cells

90 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.

91 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.

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

93 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

94 Questions ???????

95 ANY QUESTIONS

96 Case Study History 14 year old female Hgb SS Asthmatic Admitted in crisis

97 Lab Results CBC Results WBC = 11.5 corrected for NRBCs 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

98 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

99 Sickle Cell Pappenheimer bodies

100 Cellular interference with corrected and uncorrected WBC

101 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

102 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.

103 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

104 Mature RBCs Retics WBCs Platelets/Debris RETICULOCYTE COUNT

105 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 NRBCs Increased bilirubin Numerous Target cells

106 technologists medical

107 The increasing SD corresponds to a more immature population of cells NE2 Research Population Data

108 The increasing SD corresponds to a more immature population of cells NE BLAST Research Population Data

109 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


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