WBC Histogram Interpretations of 3-Part Differentiation

Sysmex Xtra Online | July 2011

Basis for automated cell counts is the concentration analysis of corpuscular peripheral blood components per defined volume. The performance spectrum in the class of automated compact systems (for example, Sysmex KX-21 or Sysmex K-4500) also includes a pre-differentiation of white blood cells, in addition to the eight parameters of the CBC. Based on this trimodal pre-differentiation, a lot of information is provided for the interpretation of results and indications of abnormalities. Deviations from normal ranges are detected such as, for example, an increased or reduced white blood cell count. Such abnormal values require more precise clarifications, and, respectively, a microscopic differentiation to detect and identify haematologic diseases. Initial information about the reason for an abnormal white blood cell count is provided by a classification of white blood cells into three separate subpopulations which is called a pre-differentiation and which immediately provides important information to the examiner. These are results on the differential blood count, especially regarding the percentage of lymphatic and myeloid white blood cell in the total white blood cells count. With a careful interpretation of all parameters, pre-differentiation allows the detection of normal blood samples and the results can be transmitted without delay to the treating physician. Samples are considered normal when their analysis results are within the reference ranges and if the ordering physician did not request further haematologic examination. Accordingly, pre-differentiation will bring about a reduction of the number of differential blood counts so that pathological and suspicious blood counts can be concentrated on. Without a specific problem at issue, it is entirely sufficient to separate white blood cells into lymphocytes and neutrophils. Any suspicion of a viral or inflammatory disease can thus also be confirmed or disproved.

Reagent effect on cells Various white blood cells forms are fixed to a defined size by means of a special lyse reagent. The lyse reagent effect on white blood cells causes the cells to shrink to a defined volume according to their cell type to be thus presented as a separate population in the histogram. After this special lysis treatment, cells will be shown in a histogram according to their size (Fig. 1).

Sysmex Xtra Online | July 2011 | 5 pages WBC Histogram Interpretations of 3-Part Differentiation

Before adding lyse reagent

Neutrophils Basophils Eosinophils Monocytes Lymphocytes m

Cell diameter in m Neutrophils 10 15 Basophils 9 14 Eosinophils 11 16 Monocytes 12 20 Lymphocytes 7 12

After adding lyse reagent

Lymphocytes Monocytes Basophils Neutrophils Eosinophils Cell volume in fl Lymphocytes 30 80 Basophils 60 120 Eosinophils 70 130 Monocytes 80 140 Neutrophils 120 250 fl Fig. 1 Normal haematologic result

Monocytes present the largest population in the peripheral blood with a diameter of approx. 20 m and are shrunk the most. The effect on eosinophils and basophils is also relatively major, so that these three populations find themselves in one mixed population. The lyse reagents influence is most minor for lymphocytes and it is also minor for neutrophils. The x-axis of the WBC histogram indicates cell size in femtolitres. This cell size in the WBC histogram does not show the actual volume of the white blood cells presented since the measuring signals following lysis treatment are no longer in relation to the volume of native white blood cells.

Histogram interpretation All measured pulses are shown in the volume distribution curve. Red blood cells are lysed in the separate WBC channel so as not to interfere with the white blood cells. Platelets are separated from the white blood cells by means of the lower discriminator (threshold value LD = lower discriminator) so that only white blood cells are actually shown. The volume distribution curve should be located within the two discriminators (LD + UD) and not only begin at the base line but also end there. If a deviation from the base line occurs, the analyser provides a corresponding warning message to indicate required follow-up actions. The various deviations are explained in the following text.

Sysmex Xtra Online | July 2011 | 5 pages WBC Histogram Interpretations of 3-Part Differentiation

Deviation of the histogram on the lower discriminator In case of a deviation of the base line on the lower discriminator, the analyser generates a WL warning (WL = WBC lower discriminator Fig. 2). There are various causes for this: n Platelets aggregates (clotted sample, EDTA incompatibility) n Lyse-resistant red blood cells n Erythroblasts n Cryoagglutinates n Giant Platelets

Fig. 2 WL warning due to interference on lower discriminator

Fig. 3 erythroblasts and one lymphocyte

In case of a WL warning, the WBC count value should be checked since it might have a false increase due to the effect of one of the above-mentioned interference factors. The most frequent cause for such an interference are platelets aggregates. These aggregates affect the WBC result because the analyser cannot differentiate between aggregates and white blood cells. In this case, however, the platelets histogram also suggests the presence of aggregates which can also be seen in the manual differential blood count. To obtain a correct result, it is necessary to take another blood sample. The presence of erythroblasts can also lead to a WL warning. The nucleated erythroblasts (Fig. 3) are also counted as white blood cells. Accordingly, the WBC value of these samples must be corrected using the formula you can find to the left.

Correct WBC value =

measured WBC value x 100 100 + number of erythroblasts*

* Number of erythroblasts counted to 100 WBC

Sysmex Xtra Online | July 2011 | 5 pages WBC Histogram Interpretations of 3-Part Differentiation

White blood cells values may be distorted due to lyse-resistant red blood cells in WBC WL* 49.4x10 /L LYM % WL -. premature infants and in patients with MXD % WL -. higher osmotic resistance. The lyse reaNEUT % WL -. gents effect on the red blood cells membrane which is very firm in this case will not be sufficient so that the white blood cells value is interfered with. The typical slide curve in the white blood Fig. 4 slide curve cells histogram is characteristic for this (Fig. 4). In this case, the sample must be diluted to increase the effect of the lyse reagent on the red blood cells. A falsified white blood cells count due to giant platelets is extremely rare but should be taken into account for the sake of completeness.
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Deviation of the histogram on the upper discriminator With a deviation on the upper discriminator (UD) (Fig. 5), the system generates the warning WU (WU = WBC upper discriminator). In this case, the linearity limit of the measurement is mostly exceeded. Due to the greatly increased cell count (WBC > 100 x 10 L), not all cells can be shown in the histogram; the WBC count is perhaps higher yet. The sample should be diluted at a ratio of 1:5 to obtain a reliable count.

Fig. 5 WU warning on upper discriminator

Fig. 6 T2 alarm. T1 was found, but not T2

T1 and T2 flags Aside from the two delimiting discriminators, so-called valley discriminators are additionally used. These discriminators seperate the white blood cells into the three populations. The two valley discriminators T1 or T2 are flexible in certain areas; i. e. they can largely adjust to the sample. With certain abnormalities of the sample, a reliable separation of the populations through valley discriminators will not be possible. In this case, the equipment generates flag T1 or T2 (Fig. 6). When a T1 or T2 flag is set, the pre-differentiation values should not be used because the populations have not been precisely separated from each other. Here, clarification is recommended through manual differential blood count. However, the total WBC count is correct if no WL or WU warning has been generated.

Sysmex Xtra Online | July 2011 | 5 pages WBC Histogram Interpretations of 3-Part Differentiation

F1, F2 and F3 flags Contrary to the T1 and T2 flag, the corresponding valley was found in this case; however, the valleys are conspicuously far away from the base line. It is accordingly possible that the fractions are mixed; i. e. fraction 1 and fraction 2, or fraction 2 and 3 merge into each other over large areas. Even in such a case, the total number of white blood cells is counted correctly, provided that no additional WL or WU warning is given; however, the pre-differentiation should be verified by manual differential count.

F1

F2

F3

Fig. 7 F1, F2, F3 abnormal differential

Summary The above described warning messages enable the user to detect positive samples and to react with follow-up actions because of the warnings. Morphological changes mostly preliminary stages or abnormalities of the myeloid and lymphatic cell series require manual differentiation. Pre-differentiation information will reduce the rate of microscopic differentiation to clinically relevant cases. Working time can thus be saved without loss of quality.

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