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) 1
Complete Hematology Testing Solutions to core lab XN-9000 Consistent operation, reagents, controls and workflow Providing comparable results across your health network XN-2000 XN-3000 XN-1000 from satellite lab XP-300 XS-1000 XT-Series WAM ISP DM96 poch-100i EasyCell DM1200 Sysmex XN Technology RBC, PLT, Hgb WNR WDF RET PLT-F NEW NEW NEW 2
WNR Channel Scattergram - Normal Pattern FSC BASO NRBC WBC Debris SFL NRBC - XN WNR 3
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 4
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. 5
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 6
. 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. Advantage of Fluorocell WNR FSC SFL SFL 7
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 8
Immature Granulocytes Autoverify Immature Granulocytes Review 9
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?) 10
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. 11
Laboratory Values XE5000 XN1000 12
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) 13
Plasma Cell Leukemia The peripheral blood shows clonal plasma cells in excess of 2x10 9 /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. 14
Low WBC Analysis Mode WB (Whole Blood) mode LW (Low WBC) mode Dilution Rate : Analysis Volume : Whole Counts *2 : x 61.1 58.2µL 9,530 Dilution Rate : Analysis Volume : Whole Counts *2 : x 61.1 174.6µL 28,600 Analysis Time extended to 3 times the Whole Blood WBC count time Low WBC Mode Example 15
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 10 3 No Vote Outs Differential results on all counts Increased reportable differentials 16
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 Reportable Parameters PLT-F IPF. 17
Binding sites of Fluorocell PLT Fluorocell PLT stains nucleic acid rich organelle Rough-surfaced endoplasmic reticulum (ribosomal RNA) Mitochondria (MtDNA) open tubule mitochondria α granule microtubule RBC RBC roughsurfaced endoplasmic reticulum 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 10 9 /L. PLT-F could be helpful in making better decisions for platelet transfusions. 18
Interference with Routine Impedance Count ß-Thalassemia Major with numerous fragmented red cells XE: PLT-I XE: PLT-O XN: PLT-F PLT-I Microcytic RBC Microcytic RBC Microcytic RBC XE PLT-I = 477*10 9 /L PLT-0 = 111*10 9 /L PLT-CD61 =152.2*10 9 /L IPF% = 13.9% XN PLT-I = 514*10 9 /L PLT-F = 140.8*10 9 /L PLT-CD61 = 152.2*10 9 /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*10 9 /L PLT-0 = 181*10 9 /L PLT-CD61 = 24.2*10 9 /L IPF% = 41.2% IPF# = 74.6*10 9 /L PLT-F XN PLT-I = 28*10 9 /L PLT-F = 24.2*10 9 /L PLT-CD61 = 24.2*10 9 /L IPF% = 1.1% IPF# = 0.3*10 9 /L 19
Severe Burn Patient The feature: Huge micro-spherical FRCs appeared because of the heat shock. Day 1 Burn injury PLT measurement over time in the sever burn injury patient. PLT (x10^3/ul) PLT-F PLT-O PLT-I 1118 PLT-I PLT-O 515 PLT-F 228 CD61 204 Day 2 PLT (x10^3/ul) PLT-I 795 PLT-F PLT-O PLT-I PLT-O 359 PLT-F 161 CD61 150 XN Conference 2012 Kobe Japan 20
Day 3 Burn injury PLT measurement over time in the sever burn injury patient. PLT (x10^3/ul) PLT-F PLT-O PLT-I 632 PLT-I PLT-O 211 PLT-F 116 CD61 115 Day 4 PLT (x10^3/ul) PLT-I 201 PLT-F PLT-O PLT-I PLT-O 59 PLT-F 54 CD61 51 Specimen RBC morph Comments Amended 2101 2+ giant plts ESTIMATE 200 21
DXH Platelet 114 Manual plt estimate 200 22
OB Patient with Giant Platelets Microscopic Images of Slide 23
OB Patient with Giant Platelets OB Patient with Giant Platelets 24
Megakaryocytes Megakaryocytes 25
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) 26
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 27
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 28
RET Channel Reagent Reaction WBC RET RBC PLT RET Channel Scattergram on Normal Pattern RET RBC LFR MFR HFR IRF PLT 29
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. 30
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 10 3 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 31
XN Technology Hematology Technology of the Future Today Questions? 32