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

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.

Fig. 1: Antibody-producing immunocytes

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

1/6

Leucocyte differentiation with the XT-series Mai 2005

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 cytoplasm 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 according 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 differenFig. 2: Differentiation by the DIFF channel

tiation since neither impedance nor forward scatter signals, both of which are related to cell volume or size, are recorded.

2/6

Leucocyte differentiation with the XT-series Mai 2005

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

stained slide. For promyelocytes, 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

3/6

Leucocyte differentiation with the XT-series Mai 2005

Imm. Gran? This suspect message shows pathologic left shifts, i.e. when promyelocytes, myelocytes or metamyelocytes 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 automatic 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.

4/6

Leucocyte differentiation with the XT-series Mai 2005

Blasts? This suspect message is generated if blasts are detected in the corresponding flagging area (lymphoblasts 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 promyelocyte. 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 Pfeiffers 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 immunoglobulins 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.

5/6

Leucocyte differentiation with the XT-series Mai 2005

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 orthochromatic 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 lymphocytes and the corresponding flagging range is below that of the lymphocyte population (Fig. 4). In the xt-series, the
Fig. 4: Presentation of the flagging areas in the DIFF scattergram of the xt-series and the cell types detectable in these areas

lymphocyte count and the total leucocyte count must be microscopically 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 9:117124 (2003)

SYSMEX EUROPE GMBH

6/6

Bornbarch 1, 22848 Norderstedt, Germany Tel.: +49 (40) 527 26-0 Fax: +49 (40) 527 26-100 info@sysmex-europe.com www.sysmex-europe.com