Haemglobin Estimation by Automated Cell Counters

In the past, blood counts were performed by slow and labour-intensive manual techniques using counting chambers, microscopes, glass tubes, colorimeters, centrifuges and a few simple reagents.

The only tests done with any frequency were estimations of Hb concentration, PCV and WBC.

The latest fully automated blood cell counters aspirate and dilute a blood sample and determine 8–46 variables relating to red cells, white cells and platelets.

Many counters are also capable of identifying a blood, mixing it, transporting it to the sampling tube and checking it for adequacy of volume and absence of clots.

Some are also linked to an automated film spreader.

To avoid any unnecessary handling of blood specimens by instrument operators, sampling is usually done by piercing a cap.

Apart from the measurement of Hb, all variables depend on counting and sizing of particles, whether red cells, white cells or platelets.

Automated instruments have at least two channels.

In one channel a diluent is added and red cells and platelets are counted and sized.

In another channel a lytic agent is added, together with diluent, to reduce red cells to stroma, leaving the white cells intact for counting and also producing a solution in which Hb can be measured.

Further channels are required for a differential WBC, which is often dependent on study of cells by a number of modalities, e.g. impedance technology with current of various frequencies, light scattering and light absorbance.

Various Automated Cell Counters

Beckman–Coulter instruments - Coulter STKS, MAXM, HmX, Gen S and LH750

Sysmex instruments - SE9000, XE 2100, SE9500

Siemens instruments – H.1 series (H.1, H.2, H.3) Advia 120, Advia 2120

Abbott (Cell-Dyn) instruments – Cell Dyn 3500, Cell Dyn 4000, Cell Dyn Sapphire

Horiba ABX instruments - ABX Pentra 60 and ABX Pentra DX 120

Basic Principle

Electronic Impedance Radiofrequency Optical Scatter

Electrical impedance

Blood cells are extremely poor conductors of electricity. When a stream of cells in a conducting medium flows through a small aperture across which an electric current is applied there is a measurable increase in the electrical impedance across the aperture as each cell passes through, this increase being proportional to the volume of conducting material displaced.

The change in impedance is therefore proportional to the cell volume. Cells can thus be both counted and sized from the electrical impulses that they generate.

This is the principle of impedance counting, which was devised and developed by Wallace Coulter in the late 1940s and 1950s and which ushered in the modern era of automated blood cell counting.

Radiofrequency

Impedance - size cells Conductivity (RF) – proportional to cell interior density

(granules and nucleus)

Five-part WBC differential Scatterplot (RF X DC) Computer cluster analysis provides absolute counts

Optical Scatter

Flow cytometry (measurement in a ‘flow’) Deflects light beam (‘mirrors’ bouncing light)

Forward angle light scatter correlates to cell volume/size Side angle (orthogonal) light scatter correlates to degree of

internal complexity (granules and nucleus)

Laser

Beckman–Coulter instruments

Principles of Measurement

Direct Measurement: RBC – Impedance WBC - Impedance, hydrodynamic focusing Platelets – Impedance (2-20 fl) Hgb – mod. Cyanmethemoglobin (525 nm) MCV – mean RBC volume (histogram)

Indirect Measurement: Hct, MCHC, and MCH (calculations)

RDW and MPV

WBC differential VCS – volume, conductivity, scatter employs differential shrinkage

Reticulocyte Supravital stain (new methylene blue) VCS

Flags

Cell shape is relevant, as well as cell volume, so that cells of increased deformability, which can elongate in response to shear forces as they pass through the aperture, appear smaller than their actual size and rigid cells appear larger.

Abberant impulses

Cells that recirculate through the edge of an electrical field produce an aberrant impulse, which is smaller than that produced by a similar cell passing through the aperture; a recirculating red cell can produce an impulse similar to that of a platelet passing through the aperture.

Furthermore, cells that pass through the aperture off centre produce aberrant impulses and appear larger than their actual size.

Aberrant impulses can be edited out electronically.

Coincidence correction

Cells that pass through the aperture simultaneously, or almost so, are counted and sized as a single cell; the inaccuracy introduced requires correction, known as coincidence correction.

Sheathed flow or hydrodynamic focusing can direct cells to the centre of the aperture to reduce the problems caused both by coincidence and by aberrant impulses. Both sheathed flow and sweep flow behind an aperture can prevent recirculation of cells.

Coulter instruments count and size red cells, white cells and platelets by impedance technology.

Platelets and red cells are counted and sized in the same channel. There is often some overlap in size between small red cells and large platelets.

Depending on the model of instrument, platelets and red cells may be separated from each other by a fixed threshold, e.g. at 20 fl, or by a moving threshold, or the data from counts between two thresholds, e.g. 2 and 20 fl, may be used to fit a curve, which is extrapolated so that platelets falling beyond these thresholds, e.g. between 0 and 70 fl, are also included in the count.

The measurement of MCV and RBC allow the Hct to be derived, and the measurement of mean platelet volume (MPV) and the platelet count allow the derivation of an equivalent platelet variable, the plateletcrit (Pct).

MCH is derived from the Hb and the RBC. MCHC is derived from the Hb and Hct

Hct = RBC x MCVMCH = Hb/RBCMCHC = Hb/ Hct

The variation in size of red cells is indicated by the red cell distribution width (RDW), which is the SD of individual measurements of red cell volume. The equivalent platelet variable is the platelet distribution width (PDW).

Histograms of volume distribution of white cells, red cells and platelets are provided.

The fully automated Coulter instruments - Coulter STKS, MAXM, HmX, Gen S and LH750- produce a five-part differential WBC, which is based on various physical characteristics of WBCs, following partial stripping of cytoplasm.

Three simultaneous measurements are made on each cell: i. Impedance measurements with low-frequency electromagnetic current,

dependent mainly on cell volume.ii. Conductivity measurements with high-frequency (radiofrequency)

electromagnetic current, which alters the bipolar lipid layer of the cell membrane allowing the current to penetrate the cells and is therefore dependent mainly on the internal structure of the cell, including nucleocytoplasmic ratio, nuclear density and granularity.

iii. Forward light scattering at 10–70° when cells pass through a laser beam, determined by the structure, shape and reflectivity of the cell.

In recent instruments (e.g. the Gen S and LH750), the software permits further analysis of this data:

Conductivity measurements are corrected for the effect of cell volume so that they more accurately reflect internal cell structure and nucleocytoplasmic ratio – designated as ‘opacity’.

Light-scatter measurements are corrected for the effect of cell volume so that separation of different cell types is improved - designated as ‘rotated light scatter’.

The abbreviation VCS (volume, conductivity, scatter) is used.

Latest Beckman–Coulter instrument

Able to count NRBC and corrects the WBC for NRBC interference.

Reticulocytes can be counted in a separate mode.

A new red cell variable, the mean sphered cell volume (MSCV), represents the average volume of sphered red cells, measured in the reticulocyte channel.Normally the MSCV is greater than the MCV (of non-sphered cells) and an inversion of this relationship suggests the presence of spherocytosis or a similar shape.

The Coulter Gen S and LH750 VCS differential white cell data can be used as an ‘alarm’ for samples that are likely to contain malaria parasites.

AcT 5diff counter - current instrument marketed by Beckman–Coulter instrument, performs a five-part differential count by means of measurements in two channels.

The WBC and the basophil count are determined by impedance measurements following differential lysis, basophils being more resistant to stripping of cytoplasm in acid conditions.

Other cell types are determined in a second channel using a combination of measurements of volume (by impedance technology) and absorbance cytochemistry (after interaction with chlorazole black).

Chlorazol black binds to the granules of eosinophils (most strongly), neutrophils (intermediate) and monocytes (least strongly); lymphocytes are unstained.