BRIEF COMMUNICATION Spurious hyperglycaemia impairs automated leucocyte counting. A pilot study with two different haematological analysers Ruggero Buonocore, Alessandra Picanza, Daniela Gennari, Silvia Pipitone, Giuseppe Lippi Laboratory of Clinical Chemistry and Haematology, Academic Hospital of Parma, Parma, Italy
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Pre-analytical errors are a frequent problem that can compromise the quality of the total testing process and, along with analytical mistakes, can have a negative impact on the reliability of test results and patients' safety1. There is now consolidated evidence that the vast majority of pre-analytical problems emerge from mishandling or scarcely standardised procedures during collection, management and preparation of biological specimens2. In particular, sample contamination by exogenous fluids (e.g., saline) or therapeutics (e.g., antibiotics, thrombolytic agents, glucose or potassium solutions) is a frequent source of problems in diagnostic testing, wherein test values may be variably biased by dilution or direct interference from the specific contaminating substance3. A paradigmatic example is contamination of biological samples with glucosecontaining solutions. The administration of intravenous standard glucose solutions, typically 5% (i.e., 278 mmol/L) dextrose in water, is commonplace in clinical practice not only for the treatment of hypoglycaemia, but also for maintaining tissue hydration after acute disease or surgery, and as a means of parenteral nutrition4. Although it has been recommended that blood samples should be drawn from the opposite arm from which glucose solutions and other intravenous fluids are administered5, blood samples are frequently collected from infusion routes, in order to avoid a second venipuncture and save precious time in short stay or critical wards such as emergency departments and intensive care units. Even the best practice of the Clinical and Laboratory Standards Institute for collecting blood from intravenous lines, that is clearance of fluid before the sample is collected by discarding an adequate amount of fluid (i.e., typically 5 mL or 6 times the dead space volume)6, is frequently overlooked. Therefore, blood sample contamination is a relatively common occurrence and may lead to diagnostic errors and adverse consequences for patients' health, including inappropriate therapeutic correction of spurious abnormalities (e.g., hypoglycaemia or hyperkalaemia), as well as unjustified transfusion of blood components in the case of spurious dilution of blood7,8. It has been previously shown that blood sample contamination by glucose solutions may generate a significant bias in clinical chemistry9 and coagulation
testing10. Nevertheless, to the best of our knowledge, no information exists on the effect of spurious hyperglycaemia on leucocyte counts and differential. This is of paramount importance, since biased results may confound diagnostic reasoning and lead to inappropriate therapeutic decisions. The aim of this study was, therefore, to evaluate whether contamination of whole blood samples with standard glucose solutions (as may occur during normal venipuncture contaminated by exogenous glucose) may affect leucocyte counts measured by two widely used haematological analysers (ADVIA 2120 [Siemens Diagnostic Solutions, Milan, Italy] and XE-2100 [Dasit SpA, Cornaredo, Italy]), characterised by different analytical technologies.
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Introduction
Materials and methods
©
SI
M
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The study population consisted of 12 ostensibly healthy subjects recruited from the laboratory staff, for whom routine laboratory testing was requested. A total of 9 mL of blood was collected into three evacuated blood tubes containing K2EDTA (13×75 mm, 3.0 mL BD Vacutainer® Plus plastic whole blood tube containing 5.4 mg spray dried K2EDTA [Becton Dickinson Italia SpA, Milan, Italy]). The autologous blood was pooled and then divided in four aliquots of 2.0 mL each. The first specimen was left untreated (i.e. without contamination), whereas scalar concentrations of a standard 5% glucose-containing solution (25 g of glucose monohydrate in 500 mL of water, 278 mOsm/L; Baxter SpA, Rome, Italy) were added to the remaining three aliquots, to achieve final glucose contaminations of 5%, 10% and 20%. The aliquots were left capped in upright position for exactly 1 hour at room temperature, and then analysed on an ADVIA 2120 and XE-210011,12. Both instruments were used properly, according to manufactures' instructions. After the haematological analyses had been completed, all aliquots were centrifuged and plasma glucose was assayed by the reference hexokinase method, using the clinical chemistry analyser Beckman DxC (Beckman Coulter Inc., Brea, CA, USA). The normality of the distribution of values was preliminarily verified by the Kolmogorov-Smirnov test and values were then reported as mean and standard error of the mean (SEM). Difference between the uncontaminated samples and the contaminated aliquots Blood Transfus 2015; 13: 656-61 DOI 10.2450/2015.0104-15 © SIMTI Servizi Srl
656 All rights reserved - For personal use only No other uses without permission
Spurious hyperglycaemia and leucocyte counting
er v
The main results of this study are shown in Table I. As intended, the concentration of plasma glucose increased progressively from the uncontaminated samples to the glucose-contaminated aliquots. As regards white blood cells (WBC), the total WBC, neutrophil and lymphocyte counts appeared to be significantly decreased starting from 5% glucose contamination with both analysers, the monocyte count from 10% glucose contamination with both analysers, and the basophil count from 20% contamination with both analysers. The eosinophil count was significantly decreased starting from 10% glucose contamination with ADVIA 2120 and from 20% glucose contamination with XE-2100. The large and unstained cell count was significantly decreased after 20% contamination.
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Results
When these changes were compared to the expected variations attributable to effect of dilution (Figures 1A and 1B), a different pattern was observed with the two haematological analysers. More specifically, the percentage decreases of WBC (r=1.00 and p=0.005), neutrophil (r=1.00 and p=0.002), lymphocyte (r=1.00 and p=0.002) and monocyte (r=0.99 and p=0.004) counts on ADVIA 2120 followed the expected trend precisely, whereas the decreases of eosinophil (r=0.84; p=0.070), basophil (r=0.80; p=0.103) and large and unstained cell (r=0.79; p=0.109) counts were non-linear and virtually unpredictable. On the XE-2100 analyser the percentage decreases of all WBC populations appeared to be linear up to 10% glucose contamination, whereas a substantial deviation from linearity was observed starting from aliquots with 20% glucose contamination (WBC: r=0.97 and p=0.057; neutrophils: r=0.97 and p=0.066; lymphocytes: r=0.95 and p=0.236; monocytes: r=0.97 and p=0.108; eosinophils: r=0.96 and p=0.156; basophils: r=0.98 and p=0.086). More specifically, all the leucocyte counts appeared to be overestimated by 3% to 5% compared to their theoretical values corrected for the dilution coefficient.
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were assessed with Student's paired t-test. Linearity was evaluated with linear regression analysis. Statistical analyses were performed using Analyse-it (Analyse-it Software Ltd., Leeds, UK). The subjects provided written consent to participation in the study, which was carried out in accordance with the Declaration of Helsinki and under the terms of all relevant local legislation.
No contamination
TI S
Table I - Effect of spurious contamination of diagnostic blood samples on total white blood cell (WBC), neutrophil (NEU), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS), basophil (BASO) and large and unstained cell (LUC) counts (mean ± standard error of the mean). Glucose solution contamination
5%
Value WBC×10 /L 9
6.58±0.37
- XE-2100
6.56±0.40
- ADVIA 2120
20%
Value
p
Value
p
6.24±0.39
0.022
5.98±0.36
Sr l
er v
Pre-analytical errors are a frequent problem that can compromise the quality of the total testing process and, along with analytical mistakes, can have a negative impact on the reliability of test results and patients' safety1. There is now consolidated evidence that the vast majority of pre-analytical problems emerge from mishandling or scarcely standardised procedures during collection, management and preparation of biological specimens2. In particular, sample contamination by exogenous fluids (e.g., saline) or therapeutics (e.g., antibiotics, thrombolytic agents, glucose or potassium solutions) is a frequent source of problems in diagnostic testing, wherein test values may be variably biased by dilution or direct interference from the specific contaminating substance3. A paradigmatic example is contamination of biological samples with glucosecontaining solutions. The administration of intravenous standard glucose solutions, typically 5% (i.e., 278 mmol/L) dextrose in water, is commonplace in clinical practice not only for the treatment of hypoglycaemia, but also for maintaining tissue hydration after acute disease or surgery, and as a means of parenteral nutrition4. Although it has been recommended that blood samples should be drawn from the opposite arm from which glucose solutions and other intravenous fluids are administered5, blood samples are frequently collected from infusion routes, in order to avoid a second venipuncture and save precious time in short stay or critical wards such as emergency departments and intensive care units. Even the best practice of the Clinical and Laboratory Standards Institute for collecting blood from intravenous lines, that is clearance of fluid before the sample is collected by discarding an adequate amount of fluid (i.e., typically 5 mL or 6 times the dead space volume)6, is frequently overlooked. Therefore, blood sample contamination is a relatively common occurrence and may lead to diagnostic errors and adverse consequences for patients' health, including inappropriate therapeutic correction of spurious abnormalities (e.g., hypoglycaemia or hyperkalaemia), as well as unjustified transfusion of blood components in the case of spurious dilution of blood7,8. It has been previously shown that blood sample contamination by glucose solutions may generate a significant bias in clinical chemistry9 and coagulation
testing10. Nevertheless, to the best of our knowledge, no information exists on the effect of spurious hyperglycaemia on leucocyte counts and differential. This is of paramount importance, since biased results may confound diagnostic reasoning and lead to inappropriate therapeutic decisions. The aim of this study was, therefore, to evaluate whether contamination of whole blood samples with standard glucose solutions (as may occur during normal venipuncture contaminated by exogenous glucose) may affect leucocyte counts measured by two widely used haematological analysers (ADVIA 2120 [Siemens Diagnostic Solutions, Milan, Italy] and XE-2100 [Dasit SpA, Cornaredo, Italy]), characterised by different analytical technologies.
iz i
Introduction
Materials and methods
©
SI
M
TI S
The study population consisted of 12 ostensibly healthy subjects recruited from the laboratory staff, for whom routine laboratory testing was requested. A total of 9 mL of blood was collected into three evacuated blood tubes containing K2EDTA (13×75 mm, 3.0 mL BD Vacutainer® Plus plastic whole blood tube containing 5.4 mg spray dried K2EDTA [Becton Dickinson Italia SpA, Milan, Italy]). The autologous blood was pooled and then divided in four aliquots of 2.0 mL each. The first specimen was left untreated (i.e. without contamination), whereas scalar concentrations of a standard 5% glucose-containing solution (25 g of glucose monohydrate in 500 mL of water, 278 mOsm/L; Baxter SpA, Rome, Italy) were added to the remaining three aliquots, to achieve final glucose contaminations of 5%, 10% and 20%. The aliquots were left capped in upright position for exactly 1 hour at room temperature, and then analysed on an ADVIA 2120 and XE-210011,12. Both instruments were used properly, according to manufactures' instructions. After the haematological analyses had been completed, all aliquots were centrifuged and plasma glucose was assayed by the reference hexokinase method, using the clinical chemistry analyser Beckman DxC (Beckman Coulter Inc., Brea, CA, USA). The normality of the distribution of values was preliminarily verified by the Kolmogorov-Smirnov test and values were then reported as mean and standard error of the mean (SEM). Difference between the uncontaminated samples and the contaminated aliquots Blood Transfus 2015; 13: 656-61 DOI 10.2450/2015.0104-15 © SIMTI Servizi Srl
656 All rights reserved - For personal use only No other uses without permission
Spurious hyperglycaemia and leucocyte counting
er v
The main results of this study are shown in Table I. As intended, the concentration of plasma glucose increased progressively from the uncontaminated samples to the glucose-contaminated aliquots. As regards white blood cells (WBC), the total WBC, neutrophil and lymphocyte counts appeared to be significantly decreased starting from 5% glucose contamination with both analysers, the monocyte count from 10% glucose contamination with both analysers, and the basophil count from 20% contamination with both analysers. The eosinophil count was significantly decreased starting from 10% glucose contamination with ADVIA 2120 and from 20% glucose contamination with XE-2100. The large and unstained cell count was significantly decreased after 20% contamination.
Sr l
Results
When these changes were compared to the expected variations attributable to effect of dilution (Figures 1A and 1B), a different pattern was observed with the two haematological analysers. More specifically, the percentage decreases of WBC (r=1.00 and p=0.005), neutrophil (r=1.00 and p=0.002), lymphocyte (r=1.00 and p=0.002) and monocyte (r=0.99 and p=0.004) counts on ADVIA 2120 followed the expected trend precisely, whereas the decreases of eosinophil (r=0.84; p=0.070), basophil (r=0.80; p=0.103) and large and unstained cell (r=0.79; p=0.109) counts were non-linear and virtually unpredictable. On the XE-2100 analyser the percentage decreases of all WBC populations appeared to be linear up to 10% glucose contamination, whereas a substantial deviation from linearity was observed starting from aliquots with 20% glucose contamination (WBC: r=0.97 and p=0.057; neutrophils: r=0.97 and p=0.066; lymphocytes: r=0.95 and p=0.236; monocytes: r=0.97 and p=0.108; eosinophils: r=0.96 and p=0.156; basophils: r=0.98 and p=0.086). More specifically, all the leucocyte counts appeared to be overestimated by 3% to 5% compared to their theoretical values corrected for the dilution coefficient.
iz i
were assessed with Student's paired t-test. Linearity was evaluated with linear regression analysis. Statistical analyses were performed using Analyse-it (Analyse-it Software Ltd., Leeds, UK). The subjects provided written consent to participation in the study, which was carried out in accordance with the Declaration of Helsinki and under the terms of all relevant local legislation.
No contamination
TI S
Table I - Effect of spurious contamination of diagnostic blood samples on total white blood cell (WBC), neutrophil (NEU), lymphocyte (LYM), monocyte (MONO), eosinophil (EOS), basophil (BASO) and large and unstained cell (LUC) counts (mean ± standard error of the mean). Glucose solution contamination
5%
Value WBC×10 /L 9
6.58±0.37
- XE-2100
6.56±0.40
- ADVIA 2120
20%
Value
p
Value
p
6.24±0.39
0.022
5.98±0.36
