Which Parameters are truly useful in XbarM analysis and which are a waste of time!

Reviewing the XbarM analysis parameters used in our laboratory XbarM control program and looking at whether the current parameters being monitored are providing truly useful information on anlayser and reagent performance. I read this paper https://www.sysmex.co.uk/education/library/documents/detail/the-xbarm-control-program-use-it-wisely.html, which is really helpful. The section I found particularly interesting is where parameters are divided into three groups depending on their significance in analyser monitoring. These groups should be observed and dealt with differently as some have very little biological variation and are not effected by the patient population whereas others depend on a multitude of variables. These are named ‘major’, ‘supporting’ and ‘other’ parameters.

Major parameters- These are useful! 

The paper states that the major parameters are mostly independent of factors such as gender and age and patient population and therefore are very useful in XBarM monitoring. These include the MCHC and Sensitivity parameters. They also are involved in two technical units of the XN analysers, the FCM detector and RBC/platelet detector and HGB detector.

MCHC

The MCHC only varies naturally within a tight range as it is calculated from the HGB and HCT and abnormal results for the MCHC are usually related to sample quality issues or analyser failure, rather than clinical reasons. The clinical reasons I can think of are slightly raised MCHC can be seen when spherocytes are present in haemolysis and low value can been seen when there is a microcytic, hypochromic anaemia. On the XN series, the MCHC is two different technical detectors, the RBC/PLT channel and haemoglobin channel are used and can therefore be monitored at the same time.

 

Sensitivity Parameters 

The sensitivity parameters again are useful in XbarM analysis as biologically will not vary very much and will monitor the technical units used for flow cytometry . They relate to the scattergrams that are produced in different measurement channels and the position of the cell populations. For example below the WDF-X and WDF-Y will mark the position of the neutrophil population on the WDF scattergram and this shouldn’t vary significantly biologically.

Supporting Parameters- Less useful This group is not so important in XbarM analysis as they do have more biological variation and do depend on the patient population . IQC is a better way of monitoring these. These include MCH and MCV, WBC, RBC, PLTS, HGB and HCT. 

Other parameters- Waste of time!!! These again are highly variable parameters that depend on patient population, type of ward, season etc. These are therefore not useful in XbarM control program. They mainly include the WBC differential and distribution parameters such as RDW for example. 

Those parameters that have a very narrow biological range i.e MCHC and sensitivity parameters are very useful in monitoring analyser performance including reagents used, stability of analyser performance and sensitivity of the electronics. For the supporting parameter and other parameters, it would be far better to monitor these by IQC.

Unstable Haemoglobin Causing Inaccurate White Cell Differential on Sysmex XN

 

Look at these white cell differentials analysed on the Sysmex XN. The first run to the right labelled  run 1, gives a neutrophil count of 1.21 x 10⁹/L and Eosinophils of 8.5 x 10⁹/l. The white cell differential on second run is completely reversed.

This can also be clearly seen on the WDF plots. On run 1, the large orange population of white cells were counted as eosinophils.  On the second run, they were counted as neutrophils represented by the blue population.

Run 1

Run 2

It is also clear that the white cell populations are ‘squashed’ and restricted to lower portion of the plot. Usually all populations would be higher up the plot, with the monocytes being the highest . This is because side flouorescent light (SFL) corresponds to the amount of DNA and RNA in a cell and monocytes have more than other normal white cells.

The inaccuracy of the white cell differential here, was caused by an unstable haemoglobin. These are haemoglobinopathies where the solubility of haemoglobin is altered, leading to precipitates of haemoglobin and haemolytic anaemia due to reduced red cell lifespan.

The reason the unstable haemoglobin leads to inaccuracies in the white cell differential, is because it does not act in the same way as it’s stable counterparts when the red cells are lysed with Lysercell WDF on the Sysmex XNs.. This is an important step in the measurement of white blood cells. Reading up on this White_Paper_RBC_Diseases.pdf (sysmex-europe.com), it is suggested that the unstable haemoglobin interferes with the fluorescence marker used to differentiate white blood cell populations, leading to decreased flouorescence and inaccurate differentials. This is why the entire plot is shifted down and appears that all cells have little DNA or RNA content.

Therefore if you encounter an abnormal plot where the fluorescence is reduced, it is firstly important to perform a manual differential. Secondly there should be the suspicion of an unstable haemoglobin particularly if haemolysis is present and HPLC and or/genetic sequencing would be indicated.