International Journal of Laboratory Hematology The Official journal of the International Society for Laboratory Hematology
LETTER TO THE EDITOR
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Letter to the Editor
Screening of hemolysis in whole-blood specimens Sir, The recent article by Lippi et al. [1] is a valuable contribution to the literature on pre-analytical factors in hematology; a subject that has attracted much less attention than in other areas of laboratory medicine. As already explained by these authors, the ease of detecting hemolysis in clinical chemistry and coagulation specimens is obviously a determining factor for this difference. Lippi et al. are to be complimented with the simplicity of their formulas for detecting hemolysis, because simple methods are likely to be adopted on a wider scale. Yet, some items in their study need a comment or further elucidation. The authors mention that their formula Hct/Hb measured on the Advia 2120 analyzer has an area under the ROC curve (AUC) of 1.00, meaning perfect discrimination between native and hemolyzed samples. On the other hand, the AUC of the MCHC is reported as 0.95 only, which is surprising as MCHC (Hb/Hct) is actually the inverse of the Hct/Hb formula, and one would expect identical discriminative power. The reasons for this difference are unfortunately not explained in the study. Apart from the MCHC, which is calculated from Hb and Hct, Advia hematology analyzers report the CHCM, which is also the mean cellular Hb concentration, but directly measured in intact RBC’s using the Mie light scattering theory. As traditional Hb measurements do not distinguish between intra- and extracellular Hb, and Hct obviously only refers to intact RBC, the calculated MCHC may be spuriously increased in samples with hemolysis. This is not the case for CHCM, and consequently, a discrepancy between MCHC and CHCM can be used as an alert for possibly compromised sample integrity. Therefore, it would have been very interesting to know how the MCHC–CHCM discrepancy correlated with the measured plasma Hb concentration in the experiment performed by Lippi et al. The explanation provided by the authors for the lack of increase in RBC ghosts is somewhat puzzling. The median increase in apparent PLT count found in the
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, e87–e88
Sysmex XE-2100 represented about 190 109/L (their Table 1), or roughly 0.2 1012/L. If the Advia 2120 would have classified all these particles as RBC ghosts or RBC fragments, this value would have accounted for an approximate 10-fold (and not 0.05%) increase over the baseline. Moreover, the median decrease in RBC count after purposefully causing hemolysis amounted to 0.5–0.8 1012/L (or 500–800 109/L), which is several fold higher than the observed increase in PLT count. This can only mean that not every single hemolyzed RBC produced one or more vesicles that interfered in the XE-2100 PLT count. Further, it remains to be elucidated why Advia 2120 did not classify (part of) these vesicles as RBC ghosts or RBC fragments as theoretically might have been expected. In a previous study, the same authors suggested that RBC ghosts in Advia 2120 and immature platelet fraction (IPF) in Sysmex XE-2100 can be reliably used for distinguishing hemolyzed and nonhemolyzed blood samples. They based this conclusion on demonstrating spurious increase of RBC ghosts and IPF in blood samples after mechanical hemolysis [2], using the same model as in their current study [1]. Although this model certainly can be used for generating artificial hemolysis in vitro and for studying the effect of hemolysis on hematology parameters, these findings cannot be extrapolated to unselected patient samples, as the effect of hemolysis was exclusively documented in paired samples (without and with artificial hemolysis) and a control sample is obviously never available when analyzing patient samples in a routine laboratory setting. Thus, before the Hct/ Hb ratio or any other parameter associated with hemolysis can be used in daily laboratory practice, it is necessary to confirm the findings by Lippi et al. using ‘real-world’ hemolytic samples, preferably also including samples from patients with active in vivo hemolysis. Finally, we like to mention that neither plasma Hb nor H-index nor whichever formula can distinguish between spurious in vitro hemolysis and genuine in vivo hemolysis as can occasionally be observed in patients with severe intravascular hemolytic disorders. Therefore, the authors might wish reconsidering the word ‘spurious’ in the title of their article.
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e88
LETTER TO THE EDITOR
†
C o n fl i c t s o f I n t e r e s t S t a t e m e n t
Medical & Scientific Affairs, Abbott Hematology, Santa Clara, CA, USA
The authors are scientific employees of Abbott Diagnostics.
E-mail: [email protected]
J. J. M. L. Hoffmann*, T. Yu†
doi: 10.1111/ijlh.12324
*Medical & Scientific Affairs, Abbott Diagnostics, Wiesbaden-Delkenheim, Germany
References 1. Lippi G, Pavesi F, Avanzini P, Chetta F, Aloe R, Pipitone S. Development of simple equations for effective screening of spurious
hemolysis in whole-blood specimens. Int J Lab Hematol 2014. doi:10.1111/ijlh.12277. 2. Lippi G, Pipitone S, Gennari D, Franchini M. Identification of spurious hemolysis in anticoagulated blood with Sysmex XE-2100
and Siemens Advia 2120. Clin Lab 2012;58: 801–4.
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, e87–e88
LETTER TO THE EDITOR
INTERNAT IONAL JOURNAL OF LABORATO RY HEMATO LOGY
Letter to the Editor
Screening of hemolysis in whole-blood specimens Sir, The recent article by Lippi et al. [1] is a valuable contribution to the literature on pre-analytical factors in hematology; a subject that has attracted much less attention than in other areas of laboratory medicine. As already explained by these authors, the ease of detecting hemolysis in clinical chemistry and coagulation specimens is obviously a determining factor for this difference. Lippi et al. are to be complimented with the simplicity of their formulas for detecting hemolysis, because simple methods are likely to be adopted on a wider scale. Yet, some items in their study need a comment or further elucidation. The authors mention that their formula Hct/Hb measured on the Advia 2120 analyzer has an area under the ROC curve (AUC) of 1.00, meaning perfect discrimination between native and hemolyzed samples. On the other hand, the AUC of the MCHC is reported as 0.95 only, which is surprising as MCHC (Hb/Hct) is actually the inverse of the Hct/Hb formula, and one would expect identical discriminative power. The reasons for this difference are unfortunately not explained in the study. Apart from the MCHC, which is calculated from Hb and Hct, Advia hematology analyzers report the CHCM, which is also the mean cellular Hb concentration, but directly measured in intact RBC’s using the Mie light scattering theory. As traditional Hb measurements do not distinguish between intra- and extracellular Hb, and Hct obviously only refers to intact RBC, the calculated MCHC may be spuriously increased in samples with hemolysis. This is not the case for CHCM, and consequently, a discrepancy between MCHC and CHCM can be used as an alert for possibly compromised sample integrity. Therefore, it would have been very interesting to know how the MCHC–CHCM discrepancy correlated with the measured plasma Hb concentration in the experiment performed by Lippi et al. The explanation provided by the authors for the lack of increase in RBC ghosts is somewhat puzzling. The median increase in apparent PLT count found in the
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, e87–e88
Sysmex XE-2100 represented about 190 109/L (their Table 1), or roughly 0.2 1012/L. If the Advia 2120 would have classified all these particles as RBC ghosts or RBC fragments, this value would have accounted for an approximate 10-fold (and not 0.05%) increase over the baseline. Moreover, the median decrease in RBC count after purposefully causing hemolysis amounted to 0.5–0.8 1012/L (or 500–800 109/L), which is several fold higher than the observed increase in PLT count. This can only mean that not every single hemolyzed RBC produced one or more vesicles that interfered in the XE-2100 PLT count. Further, it remains to be elucidated why Advia 2120 did not classify (part of) these vesicles as RBC ghosts or RBC fragments as theoretically might have been expected. In a previous study, the same authors suggested that RBC ghosts in Advia 2120 and immature platelet fraction (IPF) in Sysmex XE-2100 can be reliably used for distinguishing hemolyzed and nonhemolyzed blood samples. They based this conclusion on demonstrating spurious increase of RBC ghosts and IPF in blood samples after mechanical hemolysis [2], using the same model as in their current study [1]. Although this model certainly can be used for generating artificial hemolysis in vitro and for studying the effect of hemolysis on hematology parameters, these findings cannot be extrapolated to unselected patient samples, as the effect of hemolysis was exclusively documented in paired samples (without and with artificial hemolysis) and a control sample is obviously never available when analyzing patient samples in a routine laboratory setting. Thus, before the Hct/ Hb ratio or any other parameter associated with hemolysis can be used in daily laboratory practice, it is necessary to confirm the findings by Lippi et al. using ‘real-world’ hemolytic samples, preferably also including samples from patients with active in vivo hemolysis. Finally, we like to mention that neither plasma Hb nor H-index nor whichever formula can distinguish between spurious in vitro hemolysis and genuine in vivo hemolysis as can occasionally be observed in patients with severe intravascular hemolytic disorders. Therefore, the authors might wish reconsidering the word ‘spurious’ in the title of their article.
e87
e88
LETTER TO THE EDITOR
†
C o n fl i c t s o f I n t e r e s t S t a t e m e n t
Medical & Scientific Affairs, Abbott Hematology, Santa Clara, CA, USA
The authors are scientific employees of Abbott Diagnostics.
E-mail: [email protected]
J. J. M. L. Hoffmann*, T. Yu†
doi: 10.1111/ijlh.12324
*Medical & Scientific Affairs, Abbott Diagnostics, Wiesbaden-Delkenheim, Germany
References 1. Lippi G, Pavesi F, Avanzini P, Chetta F, Aloe R, Pipitone S. Development of simple equations for effective screening of spurious
hemolysis in whole-blood specimens. Int J Lab Hematol 2014. doi:10.1111/ijlh.12277. 2. Lippi G, Pipitone S, Gennari D, Franchini M. Identification of spurious hemolysis in anticoagulated blood with Sysmex XE-2100
and Siemens Advia 2120. Clin Lab 2012;58: 801–4.
© 2014 John Wiley & Sons Ltd, Int. Jnl. Lab. Hem. 2015, 37, e87–e88
