xtra_online_wbc_histogram_Â-_interpretations_of_3-part_differentiation

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WBC HistogramInterpretations of 3-Part Differentiation

Sysmex Xtra Online | July 2011

Basis or automated cell counts is the concentration analysis o corpuscular peripheral

blood components per deined volume. The perormance spectrum in the class o

automated compact systems (or example, Sysmex KX-21 or Sysmex K-4500) also

includes a pre-dierentiation o white blood cells, in addition to the eight parameterso the CBC. Based on this trimodal pre-dierentiation, a lot o inormation is provided

or the interpretation o results and indications o abnormalities. Deviations rom

normal ranges are detected such as, or example, an increased or reduced white

blood cell count.

Such abnormal values require more precise clariications, and, respectively, a micro-

scopic dierentiation to detect and identiy haematologic diseases. Initial inormation

about the reason or an abnormal white blood cell count is provided by a classiication

o white blood cells into three separate subpopulations which is called a pre-dierenti-

ation and which immediately provides important inormation to the examiner. Theseare results on the dierential blood count, especially regarding the percentage o

lymphatic and myeloid white blood cell in the total white blood cells count. With

a careul interpretation o all parameters, pre-dierentiation allows the detection o

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

reerence ranges and i the ordering physician did not request urther haematologic

examination. Accordingly, pre-dierentiation will bring about a reduction o the number

o dierential blood counts so that pathological and suspicious blood counts can be

concentrated on. Without a speciic problem at issue, it is entirely suicient to separate

white blood cells into lymphocytes and neutrophils. Any suspicion o a viral or inlam-matory disease can thus also be conirmed or disproved.

Reagent effect on cells

Various white blood cells orms are ixed to a deined size by means o a special lyse

reagent. The lyse reagent eect on white blood cells causes the cells to shrink to a

deined volume according to their cell type to be thus presented as a separate

population in the histogram. Ater this special lysis treatment, cells will be shown

in a histogram according to their size (Fig. ).


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2Sysmex Xtra Online | July 011 | pagesWBC Histogram Interpretations o 3-Part Diferentiation

Monocytes present the largest population in the peripheral blood with a diameter o

approx. m and are shrunk the most. The eect on eosinophils and basophils is also

relatively major, so that these three populations ind themselves in one mixed popula-

tion. The lyse reagents inluence is most minor or lymphocytes and it is also minor

or neutrophils. The x-axis o the WBC histogram indicates cell size in emtolitres.This cell size in the WBC histogram does not show the actual volume o the white

blood cells presented since the measuring signals ollowing lysis treatment are no

longer in relation to the volume o 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 interere with the white blood cells.

Platelets are separatedrom the white blood cells by means o 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. I a deviation rom the

base line occurs, the analyser provides a corresponding warning message to indicate

required ollow-up actions. The various deviations are explained in the ollowing text.

Lymphocytes 30 80

Basophils 60 120

Eosinophils 70 130

Monocytes 80 140

Neutrophils 120 250

Neutrophils 10 15

Basophils 9 14

Eosinophils 11 16

Monocytes 12 20

Lymphocytes 7 12

Cell diameter in m

Cell volume in f

Fig. 1 Normal haematologic result

NeutrophilsBasophilsEosinophilsMonocytesLymphocytes

m

Before adding lyse reagent

After adding lyse reagentLymphocytes

Neutrophils

l

MonocytesBasophils

Eosinophils


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In case o a WL warning, the WBC count value should be checked since it might havea alse increase due to the eect o one o the above-mentioned intererence actors.

The most requent cause or such an intererence are platelets aggregates. These

aggregates aect the WBC result because the analyser cannot dierentiate between

aggregates and white blood cells. In this case, however, the platelets histogram also

suggests the presence o aggregates which can also be seen in the manual dierential

blood count. To obtain a correct result, it is necessary to take another blood sample.

The presence o erythroblasts can also

lead to a WL warning. The nucleated

erythroblasts (Fig. ) are also countedas white blood cells. Accordingly, the

WBC value o these samples must be

corrected using the ormula you can

ind to the let.

Correct WBC value =

measured WBC

value x + number oerythroblasts*

* Number o erythroblasts counted to 100 WBC

Fig. 3 erythroblasts and one lymphocyteFig. 2 WL warning due to intererence on

lower discriminator

Deviation o the histogram on the lower discriminator

In case o a deviation o the base line on the lower discriminator, the analyser

generates a WL warning (WL = WBC lower discriminator Fig. ). There are variouscauses or this:

n Platelets aggregates (clotted sample,

EDTA incompatibility)

nLyse-resistant red blood cells

n Erythroblasts

n Cryoagglutinates

n Giant Platelets


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T1 and T2 lagsAside rom the two delimiting discriminators, so-called valley discriminators are addi-

tionally used. These discriminators seperate the white blood cells into the three popu-

lations. The two valley discriminators T or T are lexible in certain areas; i.e. they

can largely adjust to the sample. With certain abnormalities o the sample, a reliable

separation o the populations through valley discriminators will not be possible. In

this case, the equipment generates lag T or T (Fig. ). When a T or T lag is set,

the pre-dierentiation values should not be used because the populations have not

been precisely separated rom each other. Here, clariication is recommended through

manual dierential blood count. However, the total WBC count is correct i no WL

or WU warning has been generated.

Deviation o the histogram on the upper discriminator

With a deviation on the upper discriminator (UD) (Fig. ), the system generates the

warning WU (WU = WBC upper discriminator). In this case, the linearity limit o 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 o : to obtain a reliable count.

Fig. 6 T2 alarm. T1 was ound, but not T2Fig. 5 WU warning on upper discriminator

White blood cells values may be distorted

due to lyse-resistant red blood cells in

premature inants and in patients withhigher osmotic resistance. The lyse rea-

gents eect on the red blood cells mem-

brane which is very irm in this case

will not be suicient so that the white

blood cells value is interered with. The

typical slide curve in the white blood

cells histogram is characteristic or this

(Fig. ). In this case, the sample must be diluted to increase the eect o the lyse rea-

gent on the red blood cells. A alsiied white blood cells count due to giant platelets

is extremely rare but should be taken into account or the sake o completeness.

Fig. 4 slide curve

WBC WL* 49.4x10/LLYM% WL -.MXD% WL -.NEUT% WL -.


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Please fnd your local Sysmex representative address under www.sysmex-europe.com

Fig. 7F1, F2, F3 abnormal dierential

F1 F2 F3

F1, F2 and F3 lags

Contrary to the T and T lag, the corresponding valley was ound in this case;

however, the valleys are conspicuously ar away rom the base line. It is accordinglypossible that the ractions are mixed; i.e. raction and raction , or raction and

merge into each other over large areas. Even in such a case, the total number o white

blood cells is counted correctly, provided that no additional WL or WU warning is

given; however, the pre-dierentiation should be veriied by manual dierential

count.

Summary

The above described warning messages enable the user to detect positive samples

and to react with ollow-up actions because o the warnings. Morphological changes mostly preliminary stages or abnormalities o the myeloid and lymphatic cell series

require manual dierentiation. Pre-dierentiation inormation will reduce the rate o

microscopic dierentiation to clinically relevant cases. Working time can thus be saved

without loss o quality.

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