4/5/2014
XN Hematology Case Studies
Every Picture Tells a Story
Barbara J Connell MS, MT(ASCP)SH
Senior Product Manager
Technical Marketing & Communications
Sysmex America, Inc
Objectives
• Describe the advantages of the new
technologies available on the XN-Series
• Utilize case studies to demonstrate how
fluorescent flow cytometry technology used on
the XN can improve efficiency and productivity.
• Understand and be able to explain the
advantages of utilizing non-traditional
Hematology parameters to aid the clinician in
the diagnosis and treatment of anemia and
thrombocytopenia)
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4/5/2014
… to core lab
Complete Hematology
Testing Solutions
XN-9000
Consistent operation, reagents, controls and workflow…
Providing comparable results across your health network
XN-2000
XN-3000
XN-1000
XT-Series
from satellite lab …
ISP
XS-1000
WAM
XP-300
DM96
pocH-100i
DM1200
EasyCell
Sysmex XN Technology
RBC,
PLT,
Hgb
WNR
WDF
NEW
NEW
RET
PLT-F
NEW
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FSC
WNR Channel
Scattergram - Normal Pattern
BASO
NRBC
WBC
Debris
SFL
NRBC - XN WNR
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Case Study - Clinical History
• 23 year old male diagnosed with juvenile CML at age 11.
• Multiple admissions for blast crisis with white blood cell
count >400K
• Transferred to Hershey Medical Center on December 13,
2013 with shortness of breath on exertion, productive
cough, and nasal symptoms.
• Treated with the tyrosine kinase inhibitor Nilotinib and
allopurinol which decreased his white blood cell count.
Laboratory Values
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Peripheral Smear
FLORID LEUKOCYTOSIS WITH
LEFT SHIFT INCLUDING BLASTS
AND BASOPHILIA
BONE MARROW BIOPSY
• Cell counts within the bone marrow show blasts
within the normal range
• High percentage of the immature myeloid cells
• High basophil and eosinophil percentages.
• Erythroid precursors are low, likely the primary
reason for his anemia.
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Analyzer results
XE-5000
XN-1000
Molecular testing
• He was known to have a p210
BCR/ABL molecular fusion
transcript
• ABL Gene Mutation
• Resistance to certain tyrosine
kinase inhibitor drugs
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.
Interesting CML Facts
• Up to 5% will not have this 9;22 Philadelphia chromosome
translocation – BCR/ABL negative CML
• These patients have:
– a poorer prognosis
– Are generally older
– Lower platelet and basophil counts
– Lower rate of blast transformation, but shorter mean survial
Martiat P, Michaux J, et al. “Philadelphia-negative chronic myeloid leukemia: comparison with Ph+ CML
and chronic myelomonocytic leukemia”. 1991; 78: 205-211.
FSC
Advantage of Fluorocell WNR
SFL
SFL
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WNR Channel
• Fluorescent Flow Cytometry Technology
• Maximized Efficiency
• NRBC the first time – all the time
– No additional steps
– Accurate WBC Counts in the presence of NRBCs
– No additional reagent needed
• Virtually eliminates interference from:
• Lyse resistant RBCs
• Lipids
WDF Channel
Scattergram - Normal Pattern
MONO
IG
LYMPH
NEUT+BASO
Debris
EO
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Immature Granulocytes
Autoverify
Immature Granulocytes
Review
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Enhanced Flagging Algorithms
SAFLAS method
(Sysmex Adaptive Flagging Algorithm based on Shape-recognition)
Detects abnormal cells - (with high sensitivity)
LDA (Linear Discriminant Analysis)
WDF SAFLAS Method
Normal
Abnormal (Blasts?)
Abnormal (Abn Lympho?)
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Case Study - Clinical History
• 75 year old female diagnosed with
monoclonal gammopathy of uncertain
significance (MGUS) February 2001
• Progressed to plasma cell myeloma
September 2005.
• She showed relapse in 2012 (rapid
increase in IgG-lambda restricted).
Case Study - Clinical History
• The patient was considered for an
autologous stem cell transplant
• Her peripheral plasma cell count was 30%,
which indicated secondary plasma cell
leukemia.
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Laboratory Values
XE5000
XN1000
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Peripheral Smear
• Numerous plasmacytoid
lymphocytes
• Plasma cells, consistent with
plasma cell leukemia
Bone Marrow Biopsy
Bone marrow biopsy showed 48% plasma cells
FISH showed t(14;16)
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Plasma Cell Leukemia
• The peripheral blood shows clonal plasma
cells in excess of 2x109/L or 20%.
• They can be present at diagnosis (primary
PCL 2-5% of cases) or evolve as a late
feature in the course of plasma cell
myeloma (secondary PCL).
• This is an aggressive disease with short
survival.
WDF Channel
• Enhanced Flagging
• Better separation between lymphs and
monos (SAFLAS)
• Better identification of platelet clumps
(multiple channel detection)
• Auto-correction of lymphs when NRBCs are
present
• 6-part differential (including IG)
Copyright © 2012 by Sysmex America, Inc. All rights reserved.
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Low WBC Analysis Mode
WB (Whole Blood) mode
LW (Low WBC) mode
Dilution Rate : x 61.1
Analysis Volume : 58.2µL
Whole Counts *2 : 9,530
Dilution Rate : x 61.1
Analysis Volume : 174.6µL
Whole Counts *2 : 28,600
Analysis Time extended to 3 times the Whole Blood WBC count time
Low WBC Mode Example
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Low WBC Mode Example
Low WBC Analysis Mode
• Analysis time extended to 3 times WB WBC
count time
• Better accuracy and precision on
counts < 0.50 x 103
• No Vote Outs – Differential results on all counts
• Increased reportable differentials
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PLT-F Channel
PLT performance
• Impedance platelet analysis (size) has limitations in the identification
and discrimination of platelets from interfering particles with the
same size.
• Possible interferences
– RBC fragments counted as platelets:
– Microcytic RBCs counted as platelets:
– Large platelets counted as RBC:
falsely high
falsely high
falsely low
PLT-F Channel
Reportable Parameters
PLT-F
IPF
.
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4/5/2014
Binding sites of Fluorocell PLT
Fluorocell PLT stains nucleic acid rich
organelle
• Rough-surfaced endoplasmic
reticulum (ribosomal RNA)
• Mitochondria (MtDNA)
open tubule
mitochondria
RBC
RBC
roughsurfaced
endoplasmic
reticulum
α granule
microtubule
deep
dyeing
granule
←glycogen
granule
Am J Clin Pathol 2013;140:495-499
“Conclusions: The new PLT-F method demonstrated excellent results for
reproducibility in samples with platelet counts less than 50 × 109/L. PLT-F could be
helpful in making better decisions for platelet transfusions.”
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Interference with Routine Impedance Count
ß-Thalassemia Major with numerous fragmented red cells
XE: PLT-I
XE: PLT-O
XN: PLT-F
Microcytic RBC
PLT-I
Microcytic
RBC
Microcytic RBC
XE
PLT-I = 477*109/L
PLT-0 = 111*109/L
PLT-CD61 =152.2*109/L
IPF% = 13.9%
XN
PLT-I = 514*109/L
PLT-F = 140.8*109/L
PLT-CD61 = 152.2*109/L
IPF%= 12.9%
Improved Performance of PLT-F
Acute Promyelocytic Leukemia / chemo: white blood cell fragments
XE: PLT- O
XN: PLT-F
WBC cytoplasm
fragments
WBC cytoplasm
fragments
IPF
XE
PLT-I = 25*109/L
PLT-0 = 181*109/L
PLT-CD61 = 24.2*109/L
IPF% = 41.2%
IPF# = 74.6*109/L
PLT-F
XN
PLT-I = 28*109/L
PLT-F = 24.2*109/L
PLT-CD61 = 24.2*109/L
IPF% = 1.1%
IPF# = 0.3*109/L
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4/5/2014
Severe Burn Patient
The feature:
Huge micro-spherical FRCs appeared because of the heat shock.
Burn injury
PLT measurement over time in the sever burn injury patient.
Day 1
PLT
(x10^3/uL)
PLT-I
1118
PLT-O
515
PLT-F
228
CD61
204
PLT-F
PLT-O
PLT-I
Day 2
PLT
(x10^3/uL)
PLT-I
795
PLT-O
359
PLT-F
161
PLT-F
PLT-O
PLT-I
CD61
150
XN Conference
2012 Kobe Japan
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Burn injury
PLT measurement over time in the sever burn injury patient.
Day 3
PLT
(x10^3/uL)
PLT-I
632
PLT-O
211
PLT-F
116
CD61
115
PLT-F
PLT-O
PLT-I
Day 4
PLT
(x10^3/uL)
PLT-I
PLT-F
PLT-O
PLT-I
201
PLT-O
59
PLT-F
54
CD61
51
Specimen RBC morph
2101
Comments
Amended
2+ giant plts ESTIMATE 200
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DXH Platelet 114
Manual plt estimate 200
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OB Patient with Giant Platelets
Microscopic Images of Slide
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OB Patient with Giant Platelets
OB Patient with Giant Platelets
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Megakaryocytes
Megakaryocytes
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PLT-F Channel
Scattergram - Normal Pattern
RBC
IPF
PLT-F
Differentiate Physiological Mechanisms
Low PLT + Low IPF
Consistent with disorder
of production
Normal
Low PLT+ High IPF
Destruction consistent
with autoimmune or other
destruction mechanism
(ITP, TTP, DIC)
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Immature Platelet Fraction to Assess
Bone Marrow Recovery
How well can IPF predict platelet recovery
following peripheral blood HPC
transplantation?
Zucker, M. Laboratory Hematology, 2006 12:125 - 130
Potential IPF Applications
•
•
•
•
•
•
•
•
HIT
Drugs
Corticosteroids
Estrogens
Thrombopoietic growth factors
Other chemokines (eg. IL-11)
Platelet substitutes
Factor VIIa
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PLT-F Channel
• Second method of platelet
• Fluorescent Dye specific for platelet organelle
• Extended count time (6 times) for accurate platelet
enumeration, especially in low platelet counts
• Good comparison with CD41/CD61
• Minimizes interference from RBC fragments, microcytic
RBC’s and WBC fragments
• Automated Action message and reflex with on board
rules
Reticulocyte Channel
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RET Channel
Reagent Reaction
WBC
RET
RBC
RET Channel
PLT
Scattergram on Normal Pattern
RET
RBC
LFR
MFR
HFR
IRF
PLT
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Reticulocyte Parameters
• Reticulocytes
– # and % of immature RBC’s
• Immature Reticulocyte
Fraction
– Newly released from the
marrow, a direct cellular
measurement of
erythropoiesis
• Reticulocyte Hemoglobin
– Direct cellular measure of iron
availability
Reticulocyte Channel
• Operational Efficiency
– Reduced Interference From:
•
•
•
•
WBCs
Howell Jolley Bodies
Parasites
Sickle cells
– Quick and automatic
• Monitor RBC development at the cellular level
– IRF for Retic production
– RET-He for iron incorporation in hemoglobin of the erythron
• Clinically relevant information for the management of
anemia in conjunction with other available clinical
information.
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XN Technology Summary
• WNR Channel
– Maximized efficiency
– NRBC the first time – all the time
– Accurate WBC Counts in the presence of
NRBCs
• WDF, Low WBC
– Enhanced Flagging
– 6-part differential (including IG)
– Improved Sensitivity and Specificity
XN Technology Summary
• Low WBC
– Better accuracy and precision on counts < 0.50 x 103
– No Vote Outs – Differential results on all counts
– Increased Reportable differentials
• PLT-F
– Fluorescent Dye specific for platelet organelle
– Extended count time (6 times) for accurate low platelet
enumeration
– Good comparison with CD41/CD61
– Automated Action message and reflex with on board rules
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XN Technology
Hematology Technology of the Future Today
Questions ?
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