A Look Into the Determination of Cell Morphology in Hematology in the 21 st Century Ramon Simon-Lopez, MD Global Scientific Director Beckman Coulter
Is cell morphology important?
AML M7 CLL CD5 CD19 NHL Mantle cell CD5 CD19 AML t(15,17) Plasma cell Leukemia NHL Villous S L M Zone AML t(8;21)(q22;q22) AML M2
Normal human blood cells
Normal Cells
Normal cells as seen in peripheral blood Erythrocyte Neutrophil Monocyte Eosinophil Basophil Platelets B Cell T Cell T Cell CD1C + NK Cell
Normal cells as seen in peripheral blood VCS Technology
Newly described/classified blood cells
Newly described normal cells: Subtypes of dendritic cells Human blood DCs DCs plasmacytoid CD303+ =CD303, =CD1c, =CD141 myeloid CD1c+ myeloid CD141+ CD16+ Dendritic Cell Myeloid Related Dendritic Cell Lymphoid Related Dendritic Cell
Immature and abnormal cells
Neutrophil maturation
Abnormal neutrophils M1 M2 M0
Abnormal monocytes A, B Monoblasts C, D Promoncytes Mono-Histocyte E, F Abnormal Monocytes M5b M5a
Abnormal Lymphocytes
How can we detect abnormal or immature cells in an automated complete blood count (CBC)?
Parameters and Reference Values Reference Range Studies A Normal Range study was con ducted to assess the Reference Ranges for the DxH 800. Whole-blood samples were collected from approximated 240 donors (males and females). The selection of donors was consistent with the guidelines stated in CLSI C28-A2. These ranges are used as the System manager default normal range flags. Your patient population ranges may be different. Parameter Units Overall Mean 95% Confidence Low Limit 95% Confidence High Limit WBC x10 3 /µl 6.3 3.6 11.2 RBC x10 6 /µl 4.52 3.73 5.50 HGB g/dl 13.4 11.4 15.9 HCT % 39.0 33.3 45.7 MCV fl 86.4 73.7 95.5 MCH pg 29.6 24.3 33.2 MCHC g/dl 34.2 32.5 35.8 From the DxH 800 operators manual
Parameters and Reference Values cont. Parameter Units Overall Mean 95% Confidence Low Limit RDW % 13.8 12.3 17.0 RDW- SD fl 41.4 37.1 47.8 PLT x10 3 /µl 257 159 386 MPV fl 9.2 7.5 11.2 NE % 58.5 43.3 76.6 LY % 29.6 16.0 43.5 MO % 8.3 4.5 12.5 EO % 2.8 0.6 7.9 BA % 0.7 0.2 1.4 NE# x10 3 /µl 3.7 1.8 7.8 LY# x10 3 /µl 1.8 1.0 3.0 MO# x10 3 /µl 0.5 0.3 1.0 EO# x10 3 /µl 0.2 0.0 0.5 BA# x10 3 /µl 0.0 0.0 0.1 NRBC /100 WBC 0.1 0.0 0.4 NRBC# x10 3 /µl 0.01 0.00 0.02 From the DxH 800 operators manual 95% Confidence High Limit
Parameters and Reference Values cont. Parameter Units Overall Mean 95% Confidence Low Limit RET % 1.10 0.50 2.17 RET# x10 6 /µl 0.0498 0.0221 0.0963 MRV fl 108.8 97.4 120.2 IRF 0.40 0.29 0.53 95% Confidence High Limit TNC Total Nucleated Cell (Body Fluids) From the DxH 800 operators manual
With Suspect Flags
Suspect Flags
Case with Blasts (Acute Lymphoblastic Leukemia) Lymphoblast
Are suspect flags and reference values enough?
Intended Use, Clinical Sensitivity of the DxH 800/600 Intended Use The UniCel DxH 800 Analyzer is a quantitative multi-parameter, automated hematology analyzer for in vitro diagnostic use in screening patient populations found in clinical laboratories. The UniCel& DxH 800 Analyzer identifies and enumerates the parameters indicated below on the following sample types: Whole Blood (Venous and Capillary) WBC, RBC, HGB, UCT, MCV, MCII, MCHC, RDW, RDW-SD,PLT, MPV, NE%, NE#, LY%, LY#, MO%, MOlt, EO %, EOD#,%, BA#l, NRBC%, NRBC#l, RET0/, RET#, MRV, IRF Pre-Diluted Whole Blood (Venous and Capillary) WvBC, RBC, HGB, HCT, MCV, MCH, MCI-C, RDW, RDW-SD, PLT, MPV Body Fluids (cerebrospinal, serous and synovial) TNC and RBC Clinical Sensitivity and Specificity Performance Characteristics Clinical sensitivity and specificity of WBC differential flagging performance can be influenced by a number of factors relating to instrument technology, cellular frequency, uncertainty in the reference determination of a positive and the sample population evaluated. The DxH 800 provides the ability to set the levels and sensitivities of a variety of Flags and Messages to meet individual laboratory requirements. Beckman Coulter, Inc. recommends completion of sensitivity and specificity studies using your sample population to establish these settings.
TABLE 2. Truth Table Summary Number % True positive 1483 11.20 False positive 2476 18.60 True negative 8953 67.30 False negative 386 2.90 Total number of samples 13298 Is 2.9% false negatives acceptable? 2.9% of 300 samples x day Everyday 8 or 9 patients with a disease that will be reported as normal. Is this acceptable? Barnes PW, McFadden SL, Machin SJ, Simson E. The interna^onal consensus for hematology review: suggested criteria for ac^on following automated CBC and WBC differen^al analysis. Laboratory Hematology. 2005.
Think about the faces of these people
How we can tailor or adapt the instrument sensitivity to our needs?
Decision Rules allow for user-defined flags, system messages and actions to be written
DxH 800/600 an automated hematological consult for every sample If a pathologist or a technologist identifies certain morphologic findings in a sample, he will take in consideration information about the patient to determine its significance. The DxH 800 does just that, automatically, for every single CBC-diff run in a lab. Users just have to have the decision rule of their interest. The end result: Optimal patient care with review of significant morphologic abnormalities. Minimized unnecessary manual reviews (false positives)
Lymphoid Myeloid Reactive, HIV Monocytes Lymphoproliferative Disorders Normal low B12 or Folate
Conclusion Beckman Coulter s user-defined decision rules allow each lab the ability to activate, modify and adjust them according to individual lab requirements.