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Copyright© Sysmex America, Inc. – Confidential, Not for Distribution

The Future of Hematology – XN-Series Technology

Pending FDA Clearance. For Investigational Use OnlyNot Available for Sale in the United States

Performance Characteristics have not been Established.

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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XN-Series

It all begins here….

XN-Series:Addresses Needs Of All Segments

Single Analytical Module

Multiple Options

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Copyright© Sysmex America, Inc. – Confidential, Not for Distribution

Compact Automation Solutions

XN-2000

XN-1000

XN-3000

Copyright© Sysmex America, Inc. – Confidential, Not for Distribution

Scalable Automation Solutions

XN-9000

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XN-Series Technology

Sysmex XE vs. XN Technology

RBC, PLT, Hgb

WNR WDF RET PLT-F

NEW NEW NEW

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Fluorescent Flow Cytometry

Side Fluorescent LightDNA/RNA information

Side Scattered LightCell inside structure information

Forward Scattered LightCell size information

Dichroic Mirror

Laser Beam wavelength=633nm

WNR Channel

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WNR ChannelReagent Reaction

BASO LYMPH MONO Granulocyte

NRBC RBC

WNR Channel Scattergram - Normal Pattern

NRBC

WBC

BASO

Debris SFL

FS

C

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NRBC - XE-5000

NRBC - XN WNR

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Acute Erythroid Leukemia (AML-M6)

Acute Erythroid Leukemia (AML-M6)

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FS

C

SFLSFL

Advantage of Fluorocell WNR

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

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WDF Channel

WDF Channel Reagent Reaction

LYMPH MONO NEUT EO

Atypical LYMPH Immature WBC

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WDF Channel Scattergram - Normal Pattern

LYMPH NEUT+BASO

EO

MONO

Debris

IG

Immature Granulocytes

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IG – XN DIFF Abstract

So what’s different about the WDF?

XN flagging system improves work flow by reducing manual reviews

Enhanced Flagging

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Enhanced Flagging Algorithms

(Sysmex Adaptive Flagging Algorithm based on Shape-recognition)

LDA (Linear Discriminant Analysis)

Detects abnormal cells - (with high sensitivity)

SAFLAS method

Normal Abnormal (Blasts?) Abnormal (Abn Lympho?)

WDF SAFLAS Method

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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.

Low WBC Analysis Mode

WB (Whole Blood) mode LW (Low WBC) mode

Dilution Rate :Analysis Volume :Whole Counts *2 :

Dilution Rate :Analysis Volume :Whole Counts *2 :

x 61.158.2µL9,530

x 61.1174.6µL28,600

Analysis Time extended to 3 times the Whole Blood WBC count time

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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

PLT-F Channel

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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: falsely high

– Microcytic RBCs counted as platelets: falsely high

– Large platelets counted as RBC: falsely low

PLT-F Channel

Reagent Reaction

RBC PLT IPF

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PLT-F Channel

Reportable Parameters

PLT-FIPF

.

Fluorocell PLT Staining

PLT WBCRBC

Flu

oroc

ell P

LTR

ET

SE

AR

CH

II

Low RBC background

(because of using only one staining dye, oxazine) WBCs were stained by both dyes.

Dye binds diffusely spread throughout.

Dye binds some structures more strongly.

High RBC background WBCs were stained by both dyes.

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Binding sites of Fluorocell PLT

open tubuleα granule

←glycogen granule

microtubule

deep dyeing granule

rough-surfaced endoplasmic reticulum

Fluorocell PLT stains nucleic acid rich organelle

• Rough-surfaced endoplasmic reticulum (ribosomal RNA)

• Mitochondria (MtDNA)

mitochondria

RBCRBC

PLT-F separates platelets from RBC fragment by the differences in staining

• CD41/CD61 positive ＝ Platelets

• Staining by PLT-F dedicated reagent

– Platelets - Strong, especially within the cell

– RBC fragments - Weak, only cell membrane

RBC Fragment

Platelet

Transmitted light

CD61/PLT-F

Transmitted light

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Interference with Routine Impedance Count

ß-Thalassemia Major with numerous fragmented red cells

XN: PLT-FXE: PLT-O

PLT-I

XE: PLT-I

XEPLT-I = 477*109/L PLT-0 = 111*109/LPLT-CD61 =152.2*109/LIPF% = 13.9%

XNPLT-I = 514*109/L PLT-F = 140.8*109/LPLT-CD61 = 152.2*109/LIPF%= 12.9%

Microcytic RBC

Microcytic RBCMicrocyticRBC

Improved Performance of PLT-FAcute Prolymphocytic Leukemia / chemo: white blood cell fragments

XNPLT-I = 28*109/L PLT-F = 24.2*109/LPLT-CD61 = 24.2*109/L IPF% = 1.1%IPF# = 0.3*109/L

XEPLT-I = 25*109/L PLT-0 = 181*109/LPLT-CD61 = 24.2*109/LIPF% = 41.2%IPF# = 74.6*109/L

XN: PLT-FXE: PLT- O

WBC cytoplasm fragments

IPF

PLT-F

WBC cytoplasm fragments

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PLT-F ChannelScattergram - Normal Pattern

RBC

PLT-F

IPF

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

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Reticulocyte Channel

RET Channel

Reagent Reaction

RET RBCWBC PLT

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RET Channel Scattergram on Normal Pattern

PLT

RET

HFRRBC

MFRLFR

IRF

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

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Reticulocyte Hemoglobin(Ret-He/CHr)

• Measured at cellular level

• Monitor acute changes in hemoglobin incorporation into the erythron

• Real-time estimate of iron availability in bone marrow

• Shown as a more sensitive tool for early detection of iron deficiency

• Changes rapidly, more sensitive screen than Hb

• Less variation than acute phase reactants

• Provides additional information for managing iron requirements for ESA therapy

• Limitation in specificity addressed by interpreting Ret-He results in conjunction with other tests and clinical picture

K-DOQI Guidelines Evaluation of Anemia

Initial anemia evaluation– Cellular Assessment

CBC• Hgb < 12 g/dL

• RBC indices

• Absolute Retic

• WBC & Diff

• Platelet

– Iron Assessment• Serum ferritin

• Serum TSAT or Hb content of reticulocytes

Iron Assessment– HD-CKD Target

• Hemoglobin > 11 g/dl

• Tsat > 20%

• Ferritin > 200 ng/ml or Reticulocyte Hgb > 29 pg/cell

NKF/K-DOQI May 2006

Analysis of Iron Status in Clinical Laboratories, M. Schaefer et al, SJI Vol 11 No 2 (2001)

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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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Body Fluid Analysis

Body Fluid Analysis

Cerebrospinal fluid (CSF)

Body fluid mode (target species)

Pleural fluid

Peritoneal fluid

Synovial fluid

HF-BF

MN

PMN

MO-BF

LY-BF

EO-BFNE-BF

WDF Scattergram

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Body Fluid Analysis

• No pre-analysis preparation

• No additional reagents

• Automated background counts

• Rapid analysis

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

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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

XN Technology

Hematology Technology of the Future Today