022 Leucocyte Differentiation With the XT-Series

Irrespective of how one proceeds when microscoping a slide, three characteristics will always remain

determinative for leucocyte differentiation which, in their entirety, are unique for each cell type:

■ cell size

■ structure, position and colour of the nucleus

■ amount, staining and granulation of the cytoplasm

Even inexperienced eyes will fast be able to differentiate mature

leucocytes, but in-depth experience is required to clearly classify

all immature or reactively changed cell forms. Certain immature

cell forms are relatively easy to recognize: Promyelocytes, for

example, are conspicuous for their dark-blue, reddish granulated

cytoplasm staining and their size; plasma cells can be recognized

because of their marginal nuclei. Yet, even after many years of

practice, some cell forms can only be classified correctly upon

direct comparison with other cells, e.g. reactive lymphocytes

such as they are found in a specific immune response. Such

immunocytes are mostly detectable only if the blood film as a

whole suggests a reactive immune response.

Now, there are parallels between these morphological observations in the smear and the results

offered by the automated differentiation systems of the sysmex xt- and the xe-series. With the

fluorescence flow cytometry which they are using, structures are examined – as in the microscopic

picture – which, in their development, are characterised by the metabolic activity of cells.

‘Only by looking into the inside of the cells will exact differentiation be possible.’

This statement applies for the manual as well as the automatic differentiation and is certainly the

decisive aspect these two methods have in common. A prerequisite for this will be, however, to keep

the cells in approximately native condition, stain them and differentiate them, as with a supravital

Leucocyte differentiation with the XT-series: Suspect messages by the system, compared with the microscopic picture

Fig. 1: Antibody-producing immunocytes

Leucocyte differentiation with the XT-seriesMai 20051/6

staining. This is the only way that cells will keep their characteristic properties and can be clearly

identified by viewing nucleus and cytoplasm together. It is nearly impossible to allocate isolated

nuclei to the cell types; this holds true for both the manual as well as the automated differentiation.

Another parallel to be emphasised between the different methods is the differentiation of mature

and immature or, respectively, reactive cells on the basis of their cytoplasmic RNA part and their

DNA structure. In the slide, immature and reactive cells can be recognised because of their dark-blue

stained cytoplasm, as well as their loose nuclear structure or existing nucleoli. These temporarily

developed nuclear structures, consisting of rRNA, tRNA and mRNA, as well as basic proteins, suggest

a highly increased metabolic activity of the cell. The xt-series and the xe-series are using special

fluorescence dyes which specifically stain nucleic acids in the nucleus or in the cytoplasm. After

excitation with laser light, such as it is used in flow cytometry, those cells with their dark-blue cyto-

plasm and their loose nuclear structure will emit a significantly increased fluorescence signal. The

higher this signal, the larger the nucleus and the darker appears the blue stain of the cytoplasm in

the smear. That means, metabolically active cells – such as blasts (proliferation) or plasma cells

(antibody production), because of their activity, will always show a loose, mostly nucleoli-containing

nuclear structure with an increased RNA part, as well as an increased RNA part in the cytoplasm.

This is visible in the smear and, respectively, measurable in the DIFF channel of the xt-series.

The xe- and xt-systems perform

leucocyte differentiation in the

DIFF channel (Fig. 2). In this

channel, the individual cell

populations are distinguished

according to their nucleic acid

percentage (corresponds to the

fluorescence signal) and accord-

ing to their interior structure,

thus e.g. the complexity of their

granulation (corresponds to the

side scatter light). In this channel,

the size of the cells does not

make any impact on the differen-

tiation since neither impedance

nor forward scatter signals, both

of which are related to cell

volume or size, are recorded.

Fig. 2: Differentiation by the DIFF channel


Cells are found in a defined position in the scattergram

Metabolically active cells with a deep-blue cytoplasm edge and a high fluorescence signal are found

on the Y-axis in the DIFF scattergram distinctly above the mature cell populations (Fig. 3, cell type

shown under 1).

A second criterion of diffe-

rentiation is the cells’ internal

structure, the side scatter light,

plotted on the X-axis of the DIFF

scattergram. This is primarily

synonymous with the degree

of granulation, i. e. the density

of granulocytes. In contrast to

myeloblasts, promyelocytes

have already formed some

granulae which are visible as

a reddish granulation in the

stained slide. For promyelocy-

tes, this granulation results in

a higher optical density versus the myeloblasts. The higher this density, the higher the signal of the

side scatter light of these cells (see cell type shown in Fig. 3 under 3). Accordingly, with existing

granulae, mature or immature neutrophilic granulocytes will have a stronger side scatter light than

mononuclear cells, such as e.g. lymphocytes.

Leucocyte suspect messages

According to the diagram shown in Fig. 3, the corresponding cell types can be assigned to the suspect

messages. A comparison with their morphological characteristics explains the position of the various

cell types in the scattergram.

The following 6 leucocyte suspect messages are to be described in more detail:

■ ‘Imm. Gran?’ = immature granulocytes

■ ‘Left Shift?’ = left shift due to the increased occurrence of band cells

■ ‘Blasts?’ = lymphoblasts or myeloblasts

■ ‘Abn lymph/blasts?’ = abnormal, mostly malignant lymphocytes

■ ‘Atyp. lymph?’ = activated lymphocytes, but also certain forms of malignant lymphocytes

■ ‘NRBC?’ = erythroblasts

Fig. 3: Diagram of differentiation in the DIFF channel of the xt-series


‘Imm. Gran?’

This suspect message shows pathologic left shifts, i.e. when promyelocytes, myelocytes or meta-

myelocytes are contained in peripheral blood [1,2,3]. In contrast, band cells are detected in a separate

flagging area.

The position of the three cell types which are measurable in the ‘IG’ flagging area is in accordance

with the sequence indicated in Fig. 4 (see page 6): The most immature cells with the largest nucleus

and the highest RNA percentage also show the highest fluorescence signal (promyelocytes). In the

course of continuing maturation processes, the RNA percentage becomes ever smaller, the cytoplasm

appears paler in the smear and the fluorescence signal decreases (myelocytes). The most mature

cells of this group are found in the transition phase to band cells and come to lie in the scattergram

on the bottom edge of the ‘IG’ area (metamyelocytes).

Promyelocytes are only detected in this flagging area if they have already formed some granulae.

As a rule, the percentage of myelocytes and metamyelocytes is then at the same time very high so

that the warning message ‘IG’ will primarily be triggered by the last-named cells.

In addition to the flagging information, the number of detected immature granulocytes will be

indicated as an additional ‘research’ parameter (IG# and IG%).

‘Left Shift?’

This message refers to band cells in peripheral blood. In the xt-series equipment, the still slightly

increased residual RNA content of the cell is decisive in comparison with segmented granulocytes.

However, what the equipment does not define is the special nuclear form of a band cell such as it

is observed in the smear, using the filament rule or the third rule in the slide. The system determines

only those band cells which are clearly more immature than the segmented.

There is great diversity of opinions regarding the clinical relevance of the band cells, and hardly any

other warning message is handled so differently in the various laboratories than the physiological

left shift for which there is no general standardisation. Thus, it is possible that manual differentiation

which is primarily oriented on nuclear morphology will result in other counting values than the auto-

matic differentiation which determines the degree of immaturity of the cells. To comply with the various

manual criteria of evaluation, the sensitivity of the individual flags and thus the sensitivity of the

suspect message ‘Left Shift?’ can be adjusted to individual requests by means of the q-flag system.


‘Blasts?’

This suspect message is generated if blasts are detected in the corresponding flagging area (lympho-

blasts or myeloblasts) and requires morphological evaluation. Blasts are characterised primarily

by a significantly increased RNA percentage in cytoplasm – for example, in comparison with latent

lymphocytes, or a lack of granulation.

The appearance of blasts can be highly variable – depending on cell type and degree of maturity.

Usually, blasts are significantly smaller than their successor cells, and a stem cell in the slide can

almost look just like a lymphocyte – except for its darker staining. Thus, the fluorescence signal

of blasts is lower than that of a plasma cell. At the same time, its side scatter light signal is lower

than with granular cells which ensures a clear separation of all granulocytes starting with the pro-

myelocyte.

‘Abn Lymph/Blasts?’

This warning message indicates all changed lymphocytes which differ from the latent lymphocytes

primarily by an increased side scatter light signal, i.e. a more complex interior cell structure.

For example, ‘natural killer’ (NK) cells are found in this area in case of an increased occurrence.

The same applies for lymphoma cells as far as they have no increased cytoplasmic RNA percentage.

Also, an increased number of abnormal lymphocytes can be expected in this flagging area in case of

Pfeiffer’s disease (mononucleosis). However, in such cases, the warning message ‘Abn Lymph/Blasts?’

is usually not the only warning because a heterogeneous morphological picture also always elicits

a combination of warning messages with the automatic differentiation.

Principally, blast-similar cells, such as e.g. centroblasts or immunoblasts can also be detected in

this area. However, as soon as they show dark-blue cytoplasm and a larger nucleus in the smear,

they will be detected instead in the blast area due to the significantly increased fluorescence signal.

‘Atyp. Lymph?’

In this flagging area, reactive B-lymphocytes are mostly found which massively synthesise immuno-

globulins and are characterised by an enlarged, often vacuolated dark-blue cytoplasm in the smear.

The development of B-cells towards reactive forms is flowing. Thus, the plasma cells which are at the

end of the development series of the B-lymphocytes are always detected in this area. However, it is

at present not yet ensured that this applies for all lymphoplasmocytoid cells (immunocytes). Their RNA

percentage in cytoplasm is lower than that of plasma cells and can vary greatly although they also

participate actively in the specific immune response. The ‘colourful’ microscopic picture of different

lymphocytes for a specific immune response is accordingly also reflected in the scattergram itself.


The number of detected reactive lymphocytes can be viewed in the additional ‘Research Display’

of the xt- and xe-systems (‘OTHER#’ and ‘OTHER%’).

‘NRBC?’

Any automatic leucocyte count or differentiation always requires as a condition the haemolysis of

the erythrocytes, thus also in the xt- and xe-systems. Erythrocyte fragments will remain which have

neither nucleic acids nor any detectable inner structure. In contrast, erythroblasts have a clearly

detectable fluorescence signal since their DNA in the cell nucleus is stained, as is also the case with

leucocytes. However, an ortho-chromatic erythroblast has no appreciable cytoplasmic RNA percentage

or nucleoli; thus, its fluorescence signal intensity is less compared with a latent lymphocyte. As a rule,

the flagging range for ‘NRBC’

comprises all stages from ortho-

chromatic via the poly-chromatic

to the basophilic erythroblasts.

They have all lost their ability to

divide. Their nucleic acid content

is lower than that of lympho-

cytes and the corresponding

flagging range is below that

of the lymphocyte population

(Fig. 4). In the xt-series, the

lymphocyte count and the total

leucocyte count must be micro-

scopically checked in case of

an NRBC warning message as soon as the manual NRBC count exceeds the internal laboratory limit

for this correction (usually >10%). With the xe-2100, the erythroblasts are sensitively detected and

counted in a specific NRBC channel with specific reagents. This enables automatic correction of the

lymphocyte and leucocyte result.

References:

1) Fujimoto et al.: Flow Cytometric Method for Enumeration and Classification of Reactive Immature Granulocyte Populations, Cytometry 42:371–378 (2000)

2) Weiland et al.: Evaluation of the Automated Immature Granulocyte Count (IG) on sysmex xe-2100 Automated Haematology Analyser vs. Visual Microscopy (NCCLS H20-A), sysmex Journal International, Vol.12 No.2 (2002)

3) Briggs et al.: Evaluation of Immature Granulocyte Counts by the xe-ig master: Upgraded Software for the xe-2100 Automated Haematology Analyzer, Laboratory Hematology 9:117–124 (2003)

Fig. 4: Presentation of the flagging areas in the DIFF scattergram

of the xt-series and the cell types detectable in these areas

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022 Leucocyte Differentiation With the XT-Series