Blutbild 2012: Automatisiert oder manuell? Georg Stüssi Servizio di Ematologia Istituto Oncologico della Svizzera Italiana

IS THERE STILL A ROLE FOR THE MANUAL BLOOD COUNT?

Indication for examination of a blood smear Bain BJ. Diagnosis from the blood smear. N Engl J Med. 2005;353(5):498 507.

The blood counters

Principles of cell counters n Light absorption n Light scatter n Impedance n Fluorescence n Electrical conductivity

Hemoglobin photometry Probe Cellpack PhotoSensor Diode Lens Filter Cuvette Photosensor 17

WBC Count: Light scattering

ADVIA: RBC/PLT channel

The RBC method

The Coulter principle

Coulter principle

Impedance Widerstand Gleichspannungsquelle (ca. 100 V) konst. Gleichstrom Vakuum externe Elektrode interne Elektrode Kapillaröffnung Blutzellen Blutsuspension 23

Impedance Opening of the capillary external electrode internal electrode Vacuum

Impedance Opening of the capillary external Electrode internal Electrode U = R x I U = Voltage I = Current R = Resistance

Impedance Opening of the capillary external Electrode internal Electrode U=R x I U = Voltage I = Current R = Resistance

Volumen [fl] Impedance Mixing chamber Vorlauf Impulse/Volumen Zeit [sec] Zählzeit Nachlauf (definiertes Volumen) RBC Sheath Injektionspumpe (Schrittmotor betrieben) Hauptventil Hüllstrom Abfallkammer verdünnte Blutprobe 27

Technologies employed for the differential counts Instrument and manufacturer Coulter Sysmex ADVIA Cobas Technology employed for differential count Impedance with low- and high-frequency electromagnetic current Laser light scattering Impedance with low-frequency direct current Impedance with radiofrequency current Light scattering Absorbance (Peroxydase) Electrical impedance Light absorbance

Advanced systems ACAS = Adaptive cluster analysis system VCS = Volume/Conductivity/Scatter

COMPARABILITY OF THE CELL COUNTERS

Difference Plot 60% Identity WBC Difference (advia2120i WBC (10E09/L) - advia120.wbc (10E9/L)) / Mean of All 40% 20% 0% -20% -40% -60% Bias (1.0%) 95% Limits of agreement (-10.9% to 13.0%) ADVIA120 vs. ADVIA2120-80% 0 20 40 60 80 100 120 140 Mean of All Difference Plot 60% Identity Bias (-0.1%) Difference (beckman DxH 800.WBC (10E9/L) - advia120.wbc (10E9/L)) / Mean of All 40% 20% 0% -20% -40% -60% 95% Limits of agreement (-14.6% to 14.4%) ADVIA120 vs. DxH800-80% 0 20 40 60 80 100 120 140 Mean of All Difference Plot 60% Identity Bias (1.8%) Difference (sysmex XT4000i.WBC (10E9/L) - advia120.wbc (10E9/L)) / Mean of All 40% 20% 0% -20% -40% -60% 95% Limits of agreement (-9.1% to 12.7%) ADVIA120 vs. XE4000i -80% 0 20 40 60 80 100 120 140 Mean of All

Difference Plot 200% Identity RBC Difference (advia 2120i.RBC (10E12/L) - advia120.rbc (10E12/L)) / Mean of All 150% 100% 50% 0% Bias (-0.7%) 95% Limits of agreement (-20.3% to 18.9%) ADVIA120 vs. ADVIA2120-50% 0 1 2 3 4 5 6 7 Mean of All Difference Plot 200% Identity Difference (beckman DxH 800.RBC (10E12/L) - advia120.rbc (10E12/L)) / Mean of All 150% 100% 50% 0% Bias (-1.9%) 95% Limits of agreement (-21.8% to 18.0%) ADVIA120 vs. DxH800-50% 0 1 2 3 4 5 6 7 Mean of Difference All Plot 200% Identity Bias (-0.5%) Difference (sysmex XT4000i.RBC (10E12/L) - advia120.rbc (10E12/L)) / Mean of All 150% 100% 50% 0% 95% Limits of agreement (-20.0% to 19.1%) ADVIA120 vs. XE4000i -50% 0 1 2 3 4 5 6 7 Mean of All

Difference Plot Hemoblobin Difference (advia 2120i.HGB (g/dl) - advia120.hgb (g/dl)) / Mean of All 20% 15% 10% 5% 0% -5% -10% -15% -20% -25% -30% 4 6 8 10 12 14 16 18 Mean of All Difference Plot 20% 15% Identity Bias (0.8%) 95% Limits of agreement (-4.4% to 5.9%) ADVIA120 vs. ADVIA2120 Identity Bias (-1.0%) Difference (beckman DxH 800.HGB (g/dl) - advia120.hgb (g/dl)) / Mean of All 10% 5% 0% -5% -10% -15% -20% -25% -30% 4 6 8 10 12 14 16 18 Mean of All 95% Limits of agreement (-4.8% to 2.9%) ADVIA120 vs. DxH800 Difference (sysmex XT4000i.HGB (g/dl) - advia120.hgb (g/dl)) / Mean of All Difference Plot 20% 15% 10% 5% 0% -5% -10% -15% -20% -25% Identity Bias (0.4%) 95% Limits of agreement (-3.4% to 4.2%) ADVIA120 vs. XE4000i -30% 4 6 8 10 12 14 16 18 Mean of All

Difference Plot 80% Identity Platelets Difference (advia 2120i PLT (10E09/L) - advia120.plt (10E9/L)) / Mean of All 60% 40% 20% 0% -20% -40% -60% -80% Bias (0.4%) 95% Limits of agreement (-15.7% to 16.6%) ADVIA120 vs. ADVIA2120-100% 0 200 400 600 800 1000 1200 1400 1600 Difference Plot Mean of All 80% Identity Difference (beckman DxH 800.PLT (10E9/L) - advia120.plt (10E9/L)) / Mean of All 60% 40% 20% 0% -20% -40% -60% -80% Bias (-12.2%) 95% Limits of agreement (-29.4% to 5.1%) ADVIA120 vs. DxH800-100% 0 200 400 600 800 1000 1200 1400 1600 Mean of All Difference Plot 80% Identity 60% Bias (-2.1%) Difference (sysmex XT4000i.PLT (10E9/L) - advia120.plt (10E9/L)) / Mean of All 40% 20% 0% -20% -40% -60% -80% 95% Limits of agreement (-23.1% to 18.8%) ADVIA120 vs. XE4000i -100% 0 200 400 600 800 1000 1200 1400 1600 Mean of All

250% Difference Plot Identity Monocytes Difference (advia 2120i MO(ass/10E3uL) - advia120.mo (ass/10e3ul)) / Mean of All 200% 150% 100% 50% 0% -50% -100% -150% -200% Bias (24.4%) 95% Limits of agreement (-30.0% to 78.9%) ADVIA120 vs. ADVIA2120-250% -5 5 15 25 35 45 Mean of All 250% 200% Difference Plot Identity Difference (beckman DxH 800.MO (ass/10e3ul) - advia120.mo (ass/10e3ul)) / Mean of All 150% 100% 50% 0% -50% -100% -150% Bias (46.5%) 95% Limits of agreement (-41.9% to 134.9%) ADVIA120 vs. DxH800-200% -250% -5 5 15 25 35 45 Mean of All 250% 200% Difference Plot Identity Difference (sysmex XT4000i.MO (ass/10e3ul) - advia120.mo (ass/10e3ul)) / Mean of All 150% 100% 50% 0% -50% -100% -150% Bias (64.6%) 95% Limits of agreement (-4.3% to 133.6%) ADVIA120 vs. XE4000i -200% -250% -5 5 15 25 35 45 Mean of All

KANG SH, KIM HK, HAM CK, LEE DS, CHO HI. Comparison of four hematology analyzers, CELL-DYN Sapphire, ADVIA 120, Coulter LH 750, and Sysmex XE-2100, in terms of clinical usefulness. Int J Lab Hematol. 2008;30(6):480 486.

NEW PARAMETERS IN BLOOD CELL COUNTING

Newer parameters DxH800 XE4000i ADVIA 2120 Nucleated RBC (NRBC) Immature granulocytes (IG) ( ) Reticulocyte hemoglobin ( ) ( ) Reticulocyte volume (MRV) Immature reticulocytes (IR) Hypochromic RBC ( ) Fragmented red cells (FRC) Immature platelet fraction (IPF) Reticulocyte hemoglobin DxH800: RSF, XE4000i Ret-Hb, ADVIA2120 CHr Hypochromic RBC: DxH800: LHD, ADVIA2120 Hypochromic RBC

Nucleated red blood cells n Clinical significance q Disease detection q Automatic correction of WBC and lymphocyte counts n Different techniques q Signature position on the impedance size WBC histogram (DxH800) q Basophil channel and the Peroxidase channel (ADVIA) q Flow cytometry (XE-2100)

NRBC ADVIA

NRBC Beckman Coulter

Immature reticulocyte fraction (IRF) n Most immature reticulocytes n Clinical significance q Earliest increase of the reticulocyte fraction q Monitoring BM regeneration q Hemolysis q Therapy of iron-deficiency

Immature reticulocyte fraction RBC IRF LFR MFR HFR WBC/ NRBC Forwardscatter Fluorescence

Parameters of functional iron deficiency n CHr, hypochromic RBC (ADVIA) q Measurement: Two-angle scatter n Ret-He (Sysmex) q Reticulocyte fluorescence and FSC n MRV (mean reticulocyte volume), LHD (Low hemoglobin density), MAF (microcytic anemia factor, RSf (Red blood cell size factor), (Beckman Coulter) n Clinical utility q Predicting of iron deficiency q Manage iron supplementation therapy

Immature granulocytes

Immature granulocytes n Sysmex only n Promyelocytes, myelocytes, metamyelocytes n Clinical application?? q Detection of inflammation

Platelet measurement Briggs C, Harrison P, Machin SJ. Continuing developments with the automated platelet count. Int J Lab Hematol. 2007;29(2):77 91. Segal HC, Briggs C, Kunka S, et al. Accuracy of platelet counting haematology analysers in severe thrombocytopenia and potential impact on platelet transfusion. Br J Haematol. 2005;128(4):520 525.

Immunological platelet counting

Immature platelet fraction Briggs C, Kunka S, Hart D, Oguni S, Machin SJ. Assessment of an immature platelet fraction (IPF) in peripheral thrombocytopenia. Br J Haematol. 2004;126(1):93 99.

Conclusions n Cell counters are increasingly sophisticated n The basic values are comparable n Increasing amount of new parameters q Often no internal or external QC n The complex analysis matrices of the new cell counters offer interesting options n Their clinical applications have to be evaluated

Automated digital imaging systems Yu H, Ok CY, Hesse A, et al. Evaluation of an automated digital imaging system, nextslide digital review network, for examination of peripheral blood smears. Arch Pathol Lab Med. 2012;136(6):660 667.

Is there still a role for manual blood count? Yes, there is, but qualitative, not quantitative

Manual vs automated: RBC Automated Manual Hemoglobin +++ - Micro-/Macrocytosis +++ - Hypo-/Hyperchromia +++ - Anisocytosis +++ + Fragmentocytes + +++ Poikilocytosis - +++ RBC inclusions - +++ Reticulocytes +++ + (Supravital stainings) Dysplasia + +++

Manual vs automated: WBC Automated Manual Neutrophils ++ ++ Basophils +++ (if neg), - (if pos) +++ Lymphocytes ++ ++ Eosinophils +++ ++ Monocytes + ++ Myeloid precursors + +++ Blasts +++ +++

Gerber, Blood, 2011

Case report n A 34-year-old patient n Coombs negative hemolytic anemia with numerous spiculated RBC n Spur cell anemia q end-stage alcoholic liver disease, renal failure q accumulation of excess membrane cholesterol q impaired deformability and reduced RBC survival q Poor prognosis (median survival of a few months) n Orthotopic liver transplantation q Primary graft failure n Urgent retransplantation n 3 months follow-up: q Alcohol abstinence q Sustained improvement of the anemia and transfusion independence.

Gerber, Blood, 2011

Automated vs manual Automated analyzers Pro n Superior accuracy and precision n Fast n Online possibilities Manual evaluation Pro n Overview n Dysplatic changes n Long experience Con n Complexity and variation of cells remains challenging for analyzers n Acquisition costs Con n Labor-intensive n Expensive n Requires highly-trained stuff n Delayed results