HAEMATOLOGY CELL COUNTERS

HAEMATOLOGY CELL

COUNTERS

Jayne Cohen

SSWPS – Liverpool Hospital

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Wallace Coulter (1913-1998)

Wallace Coulter, an American electronics engineer, developed the Coulter Principle of the counting and sizing of microscopic particles (including blood cells) during the late 1940’s into the early 1950’s. He successfully secured a US patent for the first Coulter counter in 1953

About the same time in Kobe, Japan, scientists were developing using radio frequency to count cells.

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Technologies Used in Automated Cell Counters

Impedance TechnologyAbsorbance SpectrophotometryOptical TechnologyFluorescent TechnologyConductivity (radio frequency)Cytochemical stainingMonoclonal Antibodies are starting to be

utilised.

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Impedance Technology

Earliest form of automated particle (cell) counting.Based on cells being suspended in an electrically

conductive fluid.Cells pass through an aperture of known size.

Hydrodynamic focussing is applied to keep diluted specimen in the middle of the stream.

Current is applied across the aperture.

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Impedance Technology cont’d

Cells impede current as they pass through the aperture.

Each time a cell “impedes”, a pulse is created.Number of pulses equal the number of cells

passing through the aperture.Size of the pulse (resistance) is proportional to

the size of the cell.

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Impedance Transducer

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Pitfalls of Impedance Technology

Co-incidence Counting.2 or more cells passing through the aperture at the same

time.

Non-axial Passage.Cells “swirling” back into the sensing zone

Deformability of cells and different cell shapes.Vacuum applied, pulling cells through the aperture distorts

the shape of the cell and hence the size of the cell.

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Elliptocytes Passing through an Impedance Transducer

Aperture

Elliptocytes passing

through the aperture impedes

the current differently depending

on the orientation of the cell

as it passes

through the aperture.

Aperture

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Aperture

However if we make the cells

uniform, we see that we remove

the issue of shape and orientation

having an effect on the amount of current impeded.

Aperture

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Absorbance Spectrophotometry Used to measure the quantity of Haemoglobin

in blood.Complete lysis of the red blood cells is required

to release Haemoglobin into solution.Haemoglobin is measured at 540nm ( an LED

is the light source).Haemoglobin is calculated by measuring the

absorbance readings at 540nm of both the lysed specimen and the blank Haemoglobin reagent. A Photodetector measures the amount of light that passes through the lysed specimen and the blank reagent.

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Absorbance Spectrophotometry cont’d

The transmitted light signal is lower when the Haemoglobin flow cell is filled with lysed specimen than when filled with the blank reagent. This difference is due to absorption by the Haemoglobin complex and is used to calculate the concentration of Haemoglobin in the blood specimen.

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Specimen- Related Interference with Haemoglobin Calculations

High White Blood Cell counts (refer to the manufacturer’s guidelines).

High level of plasma lipids.High levels of bilirubin (obstructive jaundice).Haemolysed specimens.

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Optical TechnologyCells are counted and classified using flow

cytometry technology.Lasers are used as the light beam source

and light scatter patterns at various angles are measured and collated to classify cell types.

Fluorescence technology also incorporated to enumerate RNA (reticulocytes)and DNA (nucleated red blood cells and white cell abnormalities)

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Optical Technology cont’d

Hydrodynamic focusing of cells is achieved by sheath reagent being injected into the flow cell at a greater rate than the diluted specimen. This causes laminar flow which positions and spaces the cells into the centre of the flow cell allowing the cells to pass into the sensing zone.

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Optical Technology Cont’dThe laser beam is perpendicular to the

path of the cells.Light hits the cell causing light to scatter

at various angles. Hence the size of the cell, it’s internal complexity and granulation are measured.

If fluorescence technology is incorporated, PMT’s will measure the amount of fluorescence emitted.

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Benefits of Optical Technology

Laser light can be focused on individual cells.More than 2 measurements can be made.

- more information can be gathered giving better separation of cell populations.

Cells are passed in single file through the flow cell.- no issue with backflow into sensing zone

More realistic results. E.g. Mean Cell Volume.

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Optical Technology – Flow Cytometry

Laser Beam Focussed on individual cells.

Scattered laser light, which is picked up by a series of detectors, allowing us to classify cells on their scatter characteristics.

Sheath Fluid Surrounding sample stream

Cells pass through flow cell in single file due to HYDRODYNAMIC FOCUSSING

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Fluorescent Technology

Fluorescence is obtained when light is emitted at a wavelength that is higher than that of the light source

RNA fluorescence measured ReticulocytesDNA fluorescence measures white cell viability,

nucleated red blood cells and fragile white blood cells (eg. Smudge cells)

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Analyser Running to Manufacturer’s Specifications

Analyser background limits essentially zero.No carryover following high cell counts.Good precision.Commercial Quality Controls running within

limits.Patient Moving Averages within acceptable

limits

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Cell Dyn SapphireProcesses 106 specimens per hour in CBC mode, and

only requires 117ul of blood (both open and closed modes). If there are a lot of cytopenic patients processed, there will be fewer processed per hour

Processes 76 specimens per hour in CBC/Reticulocyte mode, and still only requires 117ul of blood (for both modes).

WBC linearity is up to 250 x 109/L.Platelet linearity is up to 2,000 x 109/L.Utilises impedance, absorbance spectrophotometry,

optical and fluorescence technologies (3 colour fluorescence).

Nucleated red cells counted as part of an FBC analysis (no reflex testing required) utilising DNA fluorescence

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Cell Dyn Sapphire Cont’dHas an Argon ion solid phase (blue) laser with a

wavelength of 488nm. Ability to run specimens in an extended lyse mode if

lyse resistant red cells present.Red cells are “sphered” to enable consistent MCV

measurement.Fully automated and intergrated Monoclonal Antibody

testing for CD61 platelet counts and CD3,4 and 8 for T-cell lymphocyte typing. Kits are available for CD64 Neutrophil sepsis marker and Hb F for foetal cell enumeration. CD34 stem cell counting is at the developmental stage.

White cell viability measured using DNA fluorescence.

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Multi Angle Polarised Scatter Separation

Hydrodynamic focusing

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Red Blood Cell and Platelet Counting

Red cell count and platelet counts are obtained by both impedance and optical methods.

Optical platelets are enumerated using 2 dimensional light scatter at 7° and 90º using the complexity of the platelets to effectively separate the platelets from small or fragmented red blood cells

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Benefits Of Reporting the Optical Platelet Count

Interfering substances NOT included in the optical platelet count. These include: circulating blast fragments, lipids, some protein complexes and cryoglobulins.

Already stated improved separation between platelets and fragmented and/or microcytic red cells.

The lower the platelet count, the longer the count period to give a more accurate count.

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Impedance Red Cell and Platelet plots vs. Optical Platelet plots

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New Software Upgrades

Reticulated platelet counts processed when a specimen is processed in CBC/Reticulocyte mode

Reportable extended White cell DifferentialExtended red cell parameters including red cell

cytogramsExtended Reticulocyte parameters

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Normal Patient Reports from the Cell Dyn Sapphire

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Sysmex XE-5000Haemoglobin is measured by absorbance

spectrophotometry (cyanide free)using the SLS method (reagent is sodium-lauryl-sulphate).

Red cells are measured by impedance technologyPlatelets are measured by impedance and optical

light scatter technology.White cell count and differential using 3

technologies.Nucleated red blood cells and Reticulocytes are

measured using Fluorescence technology.The laser is a semiconductor type.

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White Cell Count and Differential on the Sysmex XE-5000

Uses Impedance Technology, direct current (DC).

Radio frequency technology (RF).Optical and fluorescent light scatter.

Neutrophils, Lymphocytes and Monocytes initially sorted by size and light scatter patterns following lysis of red cells and platelets. Eosinophils are classified by a different light scatter due to their granulation. Basophils are counted using low angle forward scatter and side scatter at 90º after further lysis of all other white cells except Basophils.

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Sysmex XE-5000

Capable of processing 150 specimens per hour on CBC, CBC+NRBC, CBC+ NRBC+WBC5DIFF modes.

113 specimens per hour in CBC+NRBC+WBC5DIFF+RET and CBC+RET modes.

Approximately 38 body fluid analysis per hour.

Requires 200uL of blood in the closed mode.Requires 130uL of blood in the open mode

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Radio Frequency and Direct Current Methods

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White Cell Count and Differential - Optical

Values for white cell differential derived from the scatterplots.

Scatterplots are a graphical representation of electronic data generated by the laser /cell interaction.

Each coloured area represents a reportable parameter (the dotted regions indicate flagging areas).

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Special Features of the XE- 5000Ability to quantitate the RNA content of platelets

when processing specimens in the reticulocyte mode.

Measurement of stem cells (Human Progenitor Cells) utilising the DC/RF white cell counts and differentials.

Body Fluid analysisHigh fluorescent Lymphocytes – reactive B

LymphocytesAdditional red cell parameters ie % microcytes

and % macrocytes

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Technicon Advia 2120i

CBC 120 Samples/hrCBC/Diff 120 Samples/hrCBC/Diff/Retic 120 Samples/hrCBC/Retic 74 Samples/hrRetic 74 Samples/hr

Reagents used specific to request

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Advia 2120i - Improved Efficiency

Enhanced LinearityWBC 0.02 - 400 x 10^9/LRBC 0-7.5 x 10^12/LHb 0 - 225 g/LPlt 5 - 3500 x 10^9/LRetic 0 - 24.5%

175uL of blood required for processing in both the open and closed modes.

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White Blood Cell TechnologyDual WBC counts and Differentials.Optical Light Scatter.Peroxidase Staining.

Cel

l S

ize

Peroxidase ActivityNuclear Complexity

Cel

l S

ize

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Red Blood Cell TechnologyCellular Haemoglobin and Volume of rbc’s.Dual Haemoglobin.Accurate Morphology - rbc differential.

Micro

Hypo Hyper

Macro

120fL

60fL28g/dL 41g/dL

Hgb Concentration (g/dL)

RB

C V

olu

me

(fL

)

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Red Blood Cell Cytogram: Visual Analysis

Normal -Thalassemia Trait

Iron Deficiency Anemia Sickle Cell Anemia

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2 Dimensional Platelet Analysis

Linearity - 5 to 3500 x109 PLT/LSize range - 1 to 60 fLRefractive Index - 1.35 to 1.40Large PLTs included RBC fragments excluded

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Reticulocytes and Nucleated Red Blood Cells

Nucleated red blood cells are automatically counted when specimens are run in the CBC/DIFF mode (based on size and intensity of the nucleus).

Reticulocytes are measured in a specific reticulocyte channel, and additional reticulocyte parameters are derived such as the cellular haemoglobin reticulocyte.

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Special Features of the Advia 2120i

CSF Assay - FDA approved cell count and differential

Cellular Haemoglobin cross checked with the colorimetric Haemoglobin method.

Red cell differential - %hypo, %hyper,

% micro and % macro.Cross checked WCC - Peroxidase and Baso

channel.

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Beckman Coulter UniCel DxH 800

Utilises Impedance technology for White Cell Counts, platelet counts and red cell counts.

Utilises Volume, Conductivity and Scatter (VCS) principles for white cell differential and NRBC(5 angles of scatter)

Random access reticulocyte testing using new methylene blue staining of reticulocytes and analysis using VCS technology

Monoclonal capabilities

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UniCel DxH 800Processes 100 specimens per hour in CBC and

CBC/Diff modes (can take longer if there are a lot of cytopenic patients).

Processes 45 specimens per hour in CBC/Reticulocyte mode.

White cells linear from 0 – 400 x109/LPlatelet counts linear from 0-3,000x109/LSample path the same in open and closed

modes and requires 165uL of bloodPre-dilute mode available - 50uL of blood to

200uL of diluent

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VCS Technology - Volume

V is for Volume and is obtained when the impedance white cell count is performed and the cells are grouped by size.

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VCS Technology - ConductivityConductivity is a measurement of cellular

internal content using a high-frequency electromagnetic probe. The information gained by this method looks at the cell size, the nucleus to cytoplasm ratio as well as cell granularity and chemical composition.

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VCS Technology - ScatterScatter is the measurement of the light

scattering characteristics of a cell , which gives valuable information on the granularity and the nuclear structure of a cell. The laser is a Helium-Neon Laser.

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DxH 800 – Multi-transducer module –29 data measurements for each cell

Direct CurrentRadio Frequency :

OpacityStretched Opacity

5 angles of Light Scatter :Axial Light lossLow angle light scatterMedian angle light scatterLower Median angle light scatterUpper Median angle light scatter

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The VCS Cube

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VCS Technology - cont’d

Eosinophils separated from other populations.

Monocytes identified as a separate population and then classified.

Neutrophils are separated from Lymphocytes and Basophils.

Finally Lymphocytes and Basophils are classified.

2-Dimensional separation completed.Actual 3-Dimensional display.

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Example of a Normal Scatterplot

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UniCel DxH 800

Body Fluid analysis for CSF, serous and synovial fluids.

White cell differentials by monoclonal flow cytometry technology (for white cells flagged as being abnormal).

Red cell counts automatically corrected in the presence of high White cell counts and other interfering substances.

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Normal Neonate on the CD Sapphire

Neonatal specimens are run in an extended lyse mode.Note high MCV and Hb and low number of NRBC’s

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Patient with Red Cell Agglutination - RTRCC 2.16x1012/LHb 111g/LMCV 81.0 FlMCH 51.4pgMCHC 634g/LRDW 16.3%CVWCC 8.9x109/LPlatelets 392x109/L

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Patient with Red Cell agglutination – warmed to 37°C

RCC 4.21x1012/LHb 107g/LMCV 82.9fLMCH 25.5pgMCHC 308g/LRDW 15.0%CVWCC 10.5x109/LPlatelets 361x109/L

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Patient with Platelet Clumping

Patient with knownPlatelet clumping with EDTA collectionPlatelet count of 57 x 109/L

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Patient with lyse resistant red cells – in CBC mode

WBC 31.4 x 109/L

Absolute Lymphocyte 22.0 x 109/L

Analyser gave a “resistant red cell” flag

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Patient with lyse resistant Red Cells – ran in extended lyse mode

WBC is now 15.2 x 109/L

Lymphocyte Absolute count is 6.0 x 109/l

Resistant red cell flag has now disappeared

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Acquired Education Spend as much time in the laboratory as

possible.Familiarise yourself completely with analyser

capabilities (and limitations) and information given by the analyser.

Analyser flagging needs to be as accurate as possible – incorrect flagging (either false positive or negative flags) have a drastic effect on blood film review rates.

Talk to the staff responsible for running and maintaining the analysers.

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Where to Next?

Integration of multiple analysers including automated slide makers and stainers with a “tracking” system especially in institutions with an extremley heavy workload. Already in use in the largest Private Laboratories

Further development of use of monoclonal antibodies – reducing the workload for Flow Cytometry Depts.

?????????? And that’s an exciting aspect.

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Acknowledgements

Stephen Lang, PHS, SWAPS, LiverpoolDr David Rosenfeld, Director of Haematology, SWAPS,

Liverpool

The Haematology Application Specialists from: Abbott Diagnostics, Australia Roche Diagnostics Siemens DiagnosticsBeckman Coulter

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The true stars of this presentation