Automated Blood Cell Count

Automated blood cell count

Veronique Stove Clinical biologist Core lab

20 oktober 2013

Automation in Hematology J. LEHNER, B. GREVE, U. CASSENS Transfus Med Hemother 2007; 34:328-339

The CBC at the Turn of the Millenium: An Overview P. WARD Clin Chem 2000; 46:1215-1220

Blood cell counts (red cells, white cells, platelets): appropriately diluted blood samples and a ruled counting chamber (hemocytometer).

Hemoglobin concentration: colorimetrically by the cyanomethemoglobin method.

The hematocrit (packed cell volume): high speed centrifugation of a column of blood, either in a specially designed tube (the Wintrobe tube), or in sealed microcapillary tubes (ie, the "spun" hematocrit, often obtained by fingerstick blood collection).

Microscopic reticulocyte counting: based on supravital staining of cytoplasmic ribosomal RNA

The white blood cell differential: by examining and enumerating by class (eg, granulocytes, lymphocytes, monocytes) 100 to 200 individual white blood cells on a suitably stained blood smear. © 2008 Universitair Ziekenhuis Gent 3 3 First half of the 20th century Calculated indices

HCT (%) x 10 MCV (fL) = ______Maxwell Wintrobe RBC (millions/µL) (1932)

MCH (pg/RBC) = ______HGB (g/dL) x 10 RBC (millions/µL)

MCHC (g/dL) = ______HGB (g/dL) x 100 HCT (%)

Laborious

Imprecise (hemocytometer)

Errors in leukocyte differential count: Distributional error Error due to inter-observer variability Statistical error

Total number of cells counted Differential 100 200 500 1000 10 000 3 0-9 1-7 1-5 2-5 2.7-3.3 6 2-13 3-11 4-9 4-8 5.5-6.5 15 8-24 10-21 12-19 12-18 14.6-15.4

40 30-51 33-48 35-45 36-44 39.5-40.5

Anticoagulated whole blood samples were aspirated into the apparatus automatically aliquoted and diluted into RBC and WBC counting chambers (baths) for cell counting and sizing with impedance apertures

into a spectrophotometric cuvette for hemoglobin determination using the cyanmethemoglobin method.

calculations of red cell indices were automatically performed.

Towards high throughput, low cost and fast TAT with the ability to produce immediate (STAT) results.

© 2008 Universitair Ziekenhuis Gent 8 8 © 2008 Universitair Ziekenhuis Gent 9 9 1970s: Automated platelet counting Coincidence -> Hydrodynamic focussing improvement of the cell counting apertures  More accurate cell sizing  Reliable and accurate platelet counts.

Diluted blood sample

Hydrodynamic focussing (Sysmex) Pulse amplitude ~ Cell volume

Flow cell

PREPARATION Selective lysis of red cells and counting of white cells Special stains

DETECTION Impedance technology Direct current (DC): pulse height ~ cell volume Radiofrequency current (RF): pulse height ~ nuclear size and density Optical system Scattered laser light Fluorescence light

WBC WBC

Impedance PLT Immuno PLT

Optical PLT

Excellent analytical performance Closed-tube analysis No inter-observer variability No slide distribution error Eliminate statistical variations Potential of reflex testing Availability of extra parameters e.g. MCV, RDW, Ret-He, … More efficient (> 100 analyses/hour) and cost effective than manual method

Cell Dyn® (Abbott)

Coulter® LH 780 and other (Beckman Coulter)

ADVIA® 2120 and other(Siemens Diagnostics)

X-Class Hematology Systems (Sysmex)

ESR analyser Tube sorter Smear-stainer Cell counters

Microscope Smear-stainer Analysers

Every hematology analyser is Specific condition of patient affected ! Operators must be aware of the Sampling characteristics of their analyser + be able to recognize and conditions circumvent anomalous results Total testing process All suspected spurious cell counts / morphologies should be validated with microscopic examination of slides Technical problems

© 2008 Universitair Ziekenhuis Gent 23 23 Spurious counts and spurious results on haematology analysers: a review. Part I: platelets M. ZANDECKI, F. GENEVIEVE, J. GERARD, A. GODON Volume 29, Issue 1, pages 4–20, February 2007

Spurious counts and spurious results on haematology analysers: a review. Part II: white blood cells, red blood cells, haemoglobin, red cell indices and reticulocytes M. ZANDECKI, F. GENEVIEVE, J. GERARD, A. GODON Volume 29, Issue 1, pages 21–41, February 2007

Flags to signal poor reliability of quantitative results (e.g. turbidity/Hb interference, WBC abnormal scattergram, PLT clumps, RBC agglutination, …)

Morfology flags (blasts, IG, atypical lymph, RBC fragments, …)

Limit check

Delta check

DM96 (CellaVision)

MCHC = HGB x 100 / HCT (%) normal range: 31.9 - 34.9 g/dL

Parameter Run 1 Run 2 Run 3 30MCV min = Plasma 37HCT°C / RBCexchange x 10

WBC 5.02 5.25 6.70 10 3/uL MCH = RBC 0.71 HGB0.90 / RBC x 3.4410 10 6/uL HGB 11.9 11.9 11.6 g/dL HCT 8.6 10.9 36.9 % MCV 121.1 121.1 107.3 fL MCH 167.6 132.2 33.7 pg MCHC 138.4 109.2 31.4 g/dL PLT 195 196 205 10 3/uL

MCHC = HGB x 100 / HCT (%)

Parameter Run 1 Run 2 Plasma exchange WBC 12.07 13.28 10 3/uL RBC 2.47 2.62 10 6/uL HGB 15.9 8.8 g/dL HCT 23.4 24.9 % MCV 94,7 95.0 fL MCH 64.4 33.6 pg MCHC 67.9 35.3 g/dL PLT 519 192 10 3/uL

Hypertriglyceridemia (>2000 mg/dL)

HEMATOLOGY Screening Red blood cells 4.78 10E6/µL 3.79 - 5.23 Hemoglobin 14.7 g/dL 11.7 - 15.7 Hematocrit 43.7 % 34.9 - 46.9 MCV 91.4 fL 80.5 - 99.7 MCH 30.8 pg/cel 26.6 - 33.8 MCHC 33.6 g/dL RBC 33 - 36 Red cell distribution width 12.6 % 11.1 - 14.2

Platelets - 54 10E3/µL 170 - 394

Thrombocytes - citrate 198 10E3/µL Screening thrombocytes Thrombocyte aggregates

EDTA-dependent pseudothrombocytopenia

Normal Lipids Lysis resistant RBC Nucleated red (Parental nutrition) (HbC, HbS) blood cells (Neonates, Path. circumstances)

© 2008 Universitair Ziekenhuis Gent 33 Parameter TAT MCV Delta check (p50 percentile) Chemistry 44 min | Current – Previous result | Coagulation 39 min Average > 5% Hematology 15 min Change in MCV indicates Transfusion Sample mishandling Sample mix-up

Example (93 – 87) / 93 = 7%

Extended differential count Immature granulocytes (infections, neonatal sepsis, cancer, …) Nucleated red blood cells (neonates / numerous conditions in adults) Hematopoietic progenitor cells (time to harvest)

RBC and Reticulocyte parameters Immature reticulocyte fraction (IRF) - early index of erythropoeisis Reticulocyte Hemoglobin content (Ret-He) – iron status Hypochromic RBC Microcytic RBC

Platelet parameters Immature platelet fraction (IPF) – early index of thrombopoiesis

Thalassemia

Index Formula TT Hereditary spherocytosis England and Fraser index (1973) MCV – RBC – 5 x Hb – K (3,4) < 0 Iron status – Iron therapy Mentzer index (1973) MCV / RBC < 13

Srivastava index (1973) MCH / RBC < 3,8 Shine and Lal index (1977) MCV ² x MCH x 0.01 < 1530 RDW index (1987) MCV x RDW / RBC < 220 Ricerca index (1987) RDW / RBC < 3,3 Green and King index (1988) MCV ² x RDW / (100 x Hb) <72 Ehsani index (2005) MCV – 10 x RBC < 15 Sirdah et al.’s index (2007) MCV – RBC – 3 x Hb < 27

Thalassemia

Hereditary spherocytosis Mullier et al., Annals of hematology (2011); 90:759-768. Persijn et al., Annals of hematology (2011)

Essential role of automation in the modern hematology laboratory

Microscopic control of pathologic samples remains indispensable

Of major importance for correct interpretation of results: Knowledge of the limits of your specific analyser