Are shortened aPTT values always to be attributed only to preanalytical problems?

OBJECTIVES: It has been recognized that shortened activated partial thromboplastin time (aPTT) may be caused by various preanalytical conditions. As coagulation Factor VIII is included in the in vitro intrinsic coagulation cascade measured by aPTT, we hypothesized that the shortened aPTT could be a result of elevated FVIII activity. We aimed to inspect the connection of elevated FVIII with shortened aPTT, and the possible effect inflammation has on routine laboratory parameters. METHODS: 40 patients from various hospital departments with aPTT measurement below the lower limit of the reference interval (<23.0 s) were included in the study. To compare the obtained results with aPTT measurements in the non-inflammatory state, samples from 25 volunteers (laboratory personnel) were collected. White blood cell count, C-reactive protein, aPTT, and FVIII values were measured in the control group. RESULTS: Only two samples among 40 patients with shortened aPTT (5 %) were clotted. Out of the remaining 38, 26 had FVIII activity above 150 % (upper limit of a reference interval), median value of 194 % (IQR: 143-243 %). Seven samples in the control group had shortened aPTT results (36 %). However, all coagulation samples were clot and hemolysis-free. Multiple regression identified only FVIII activity as an independent variable in predicting aPTT values (p=0.001). CONCLUSIONS: Our results support the thesis that shortened aPTT is rarely a consequence of preanalytical problems. Elevated FVIII activity causes shortened aPTT, not only in the inflammatory state but also in individuals with concentration of inflammatory markers within reference intervals.

Reporting categories in urine test strip analysis: Croatian survey and call for action.

INTRODUCTION: In line with the national recommendations, Croatian medical laboratories report urine test strip qualitative analysis results using a categorized scale with defined number of categories. Since concentration ranges for measured analytes have not been provided by national professional authority, it is up to the laboratories to define their own categories. The aim of study was to assess the comparability of concentrations assigned to different categories used in reporting the results of dipstick urinalysis in Croatian laboratories. MATERIAL AND METHODS: A questionnaire was e-mailed to all Croatian medical laboratories (N = 195). They were asked to provide the number of categories and respective concentrations for each parameter. Data were described as numbers and percentages. Values above the upper reference range limit, which were assigned as normal and/or trace category, were considered as false negative. RESULTS: Response rate was 71% (139/195). Seventy percent (98/139) of laboratories report their results with either higher (77/98; 79%) or lower (2/98; 2%) number of categories, relative to the national recommendation, whereas 19/98 (19%) report their results as concentrations. Great heterogeneity of reporting categories was observed. Multiple categories were assigned to same concentrations and there was a large overlap of concentrations for most categories. Considerable proportion of laboratories reported false negative results for ketones (42%), leukocytes (30%) and glucose (21%). CONCLUSIONS: The concentrations assigned to categories used to report the results of dipstick urinalysis are not comparable among Croatian medical laboratories. There is an urgent need for harmonization and standardization of reporting the results of urine dipstick analysis in Croatia.

Haemolysis and lipemia interfere with resistin and myeloperoxidase BioVendor ELISA assays.

INTRODUCTION: The aim of our study was to investigate the influence of haemolysis and lipemia on resistin (RES) and myeloperoxidase (MPO) measurement by BioVendor enzyme-linked immunosorbent assays (ELISA). MATERIALS AND METHODS: Blood was taken from healthy volunteers into lithium heparin tubes. Plasma samples were spiked with Lipofundin® emulsion (B. Braun Melsungen AG, Germany) for lipemia interference testing. Haemolysed samples were obtained by drawing aliquots of heparinized blood through a 26 gauge needle. Index of haemolysis (H), lipemia (L) and triglyceride concentration were measured on Abbott Architect c8000. Haemoglobin concentration was measured on Sysmex XN-1000. Concentrations of RES and MPO in all samples were determined with RES and MPO ELISA kits (BioVendor, Czech Republic). All measurements were performed in triplicate. Biases from the native samples were calculated for both analytes and compared with an arbitrary value (e.g. ± 10%). RESULTS: Triglyceride concentration in the investigated samples ranged from 0.57 to 38.23 mmol/L, which corresponds to L index from - 0.01 to 13.77. Haemoglobin concentration in all samples ranged from 0 to 8 g/L which correspond to H index from 0.05 to 8.77. Both MPO and RES showed significant biases at 1 g/L haemoglobin (58.7% and 66.7%, respectively). Also, both MPO and RES showed significant biases at 4.66 mmol/L triglycerides (33.8% and - 12.2%, respectively). CONCLUSIONS: Resistin BioVendor assays are affected by haemolysis and lipemia already at low degree of interferent. Haemolysis was found to interfere at 1 g/L haemoglobin for both assays, while lipemia interferes at 4.66 mmol/L of triglycerides.

Analytical verification of 12 most commonly used urine dipsticks in Croatia: comparability, repeatability and accuracy.

INTRODUCTION: Variability among manufacturers of urine dipsticks, respective to their accuracy and measurement range, may lead to diagnostic errors and thus create a serious risk for the patient. Our aims were to determine the level of agreement between 12 most commonly used urine dipsticks in Croatia, examine their accuracy for glucose and total protein and to test their repeatability. MATERIALS AND METHODS: A total of 75 urine samples were used to examine comparability and accuracy of 12 dipstick brands (Combur 10 TestM, ChoiceLine 10, Combur 10 TestUX, ComboStik 10M, ComboStik 11M, CombiScreen 11SYS, CombiScreen 10SL, Combina 13, Combina 11S, Combina 10M, UriGnost 11, Multistix 10SG). Agreement between each dipstick and the reference (Combur 10 TestM) was expressed as kappa coefficient (acceptable κ ≥ 0.80). Accuracy for glucose and total protein was tested by comparison with quantitative measurements on analysers: AU400 (Beckman Coulter, USA), Cobas 6000 c501 (Roche Diagnostics, Germany) and Architect plus c4000 (Abbott, USA). Repeatability was assessed on 20 replicates (acceptable > 90%). RESULTS: Best agreement was achieved for glucose, total protein and nitrite (11/11, k > 0.80) and the lowest for bilirubin (5/5, k < 0.60). Sensitivities for total protein were 41-75% (AU400) and 56-92% (Cobas and Architect); while specificities were 41-75% (AU400, Cobas, Architect). Dipsticks' sensitivity and specificity for glucose were 68-98%. Most of the dipsticks showed unacceptable repeatability (6/12, < 90%) for one parameter, most prominently for pH (3/12, < 90%). CONCLUSIONS: Most commonly used dipsticks in Croatia showed low level of agreement between each other. Moreover, their repeatability varies among manufacturers and their accuracy for glucose and proteins is poor.

Various glycolysis inhibitor-containing tubes for glucose measurement cannot be used interchangeably due to clinically unacceptable biases between them.

BACKGROUND: The aim of our study was to determine the difference between glucose concentration measured 30 min after venipuncture in ice-chilled heparin plasma sample and all currently available citrate buffer-containing tubes (Greiner Glucomedics, Greiner FC Mix and Sarstedt GlucoEXACT) and still widely used sodium fluoride/potassium oxalate (NaF/Kox) tubes from Greiner. METHODS: Blood was collected from 20 healthy volunteers and 20 patients with diabetes into LiH, NaF/KOx, Glucomedics, FC mix and GlucoEXACT tubes. Glucose was measured within 30 min from blood sampling in duplicate on the Architect c8000 analyzer. Mean biases between all tube types were calculated and compared to the recommended criteria (1.95%). Additionally, glucose concentrations measured in all five tube types were compared using the Friedman test. RESULTS: In the entire studied population, glucose concentrations measured in Glucomedics, FC mix and GlucoEXACT were higher (7.3%, 3.2% and 2.0%, respectively) than in the ice-chilled LiH tubes. When all glycolysis inhibitor-containing tubes were compared, Glucomedics tubes significantly differed from GlucoEXACT and FC mix tubes (biases -4.9% and 4.0%, respectively). In addition, there was a significant difference between the NaF/KOx tube and Glucomedics, as well as FC mix tubes (biases 7.1% and 3.0%, respectively). CONCLUSIONS: Glucose concentrations measured in recommended ice-chilled lithium heparin- and citrate buffer-containing tubes are not comparable. Significant biases exist between various glycolysis inhibitor-containing tubes; therefore, they cannot be used interchangeably.

The evidence for clinically significant bias in plasma glucose between liquid and lyophilized citrate buffer additive.

OBJECTIVES: Citrate buffer additive has been suggested to be of supreme performance in inhibiting glycolysis. However, there is little evidence in the literature regarding the comparability of glucose concentrations in liquid and lyophilized citrate buffer containing tubes. The aim of this study was to compare glucose concentrations in tubes containing liquid (Glucomedics) and lyophilized citrate buffer (Terumo VENOSAFE™ Glycemia) additive, measured immediately after centrifugation. DESIGN AND METHODS: Blood was collected from forty volunteers into both Glucomedics and Venosafe Glycemia tubes. Blood was centrifuged within 15min from venipuncture and glucose concentration was measured immediately after centrifugation, on the Abbott Architect analyzer. Differences between glucose concentrations in Glucomedics and Terumo tubes were tested using the paired t-test. Mean bias was calculated and compared to recommended quality specification for glucose (i.e. 2.2%). RESULTS: Glucose concentration in Terumo tubes was 3.4% lower than in Glucomedics tubes (P<0.001). The mean bias was clinically significant. CONCLUSIONS: There is a clinically significant difference between glucose concentrations in liquid and lyophilized citrate buffer additive tubes (Glucomedics vs. Terumo tubes) measured immediately after centrifugation. This difference may affect the patient outcome due to the misclassification of diabetes.

Glucose is stable during prolonged storage in un-centrifuged Greiner tubes with liquid citrate buffer, but not in serum and NaF/KOx tubes.

BACKGROUND: Delayed sample processing can affect accurate glucose measurement. Our aim was to investigate the stability of glucose in samples collected in serum, sodium fluoride/potassium oxalate (NaF/KOx) and Glucomedics tubes processed according to different controlled pre-centrifugation delays (up to 180 min after venipuncture) in order to simulate prolonged sample transport between venipuncture and centrifugation. METHODS: Samples were collected from healthy volunteers (n=80) into either serum or NaF/KOx and Glucomedics tubes. Glucose concentration was measured in samples centrifuged immediately after venipuncture and compared with tubes processed with a delay of 60, 120 and 180 min prior to centrifugation. Differences between baseline and respective delayed centrifugation glucose value for each tube type were tested using the paired t-test. Mean bias calculated for each tube type and delay protocol was compared to recommended quality specifications for glucose (2.2%). RESULTS: Glucose concentrations measured in all three delayed tube types were lower in comparison to respective baseline glucose concentrations measured in immediately processed tube (p<0.001). The highest decrease in glucose was observed in serum tubes in all specified time points (p<0.001), while glucose was most stable in Glucomedics tubes (p<0.001). The decrease in glucose observed for serum and NaF/KOx tubes was clinically significant at all specified time points while the bias for Glucomedics tubes did not exceed the criteria even with a centrifugation delay of 180 min. CONCLUSIONS: Glucose stability in un-centrifuged Glucomedics tubes is much superior to serum and NaF/KOx tubes. Glucomedics tubes can be left un-centrifuged for up to 3 h without affecting glucose concentration.

Liquid citrate acidification introduces significant glucose bias and leads to misclassification of patients with diabetes.

BACKGROUND: Glucose measurements are crucial in diabetes diagnosis. We aimed to assess the effectiveness of liquid citrate acidification in preventing glycolysis and investigate glucose stability in serum, sodium fluoride (NaF/KOx) and Glucomedics tubes. METHODS: Samples from 40 participants were collected in serum, lithium-heparin (LiH), sodium fluoride/potassium oxalate (NaF/KOx) and Glucomedics tubes. Glucose was measured within 60 min (baseline), 120 and 180 min from venipuncture. Serum, NaF/KOx and Glucomedics values at baseline were compared to LiH glucose concentration. Additionally, glucose values measured at 120 and 180 min from each tube were compared with the baseline value. Mean absolute bias for each tube and time point was calculated and compared to recommended criteria. The regression equation obtained comparing citrate to NaF/KOx tubes was used to recalculate glucose results retrieved from the laboratory information system. RESULTS: Glucose measured in Glucomedics was higher (9.9%; p<0.001), while glucose in NaF/KOx and serum was lower compared to LiH (2.4%; p<0.001 and 3.2%; p<0.001, respectively). Biases for all tubes were clinically significant. Glucose remained unchanged at room temperature in all tubes for up to 180 min after venipuncture. Observed bias caused by Glucomedics leads to a 10.6% increase in diabetes prevalence (p<0.001). CONCLUSIONS: Inhibition of glycolysis is most effectively achieved using liquid citrate acidification, compared to LiH, NaF/KOx or serum. Due to clinically significant bias relative to reference glucose, the interchangeable use of different tube types for serial glucose measurements is not recommended. The replacement of NaF/KOx with Glucomedics tubes substantially impacts glucose results, giving marked rise in diabetes prevalence.

How compliant are technicians with universal safety measures in medical laboratories in Croatia?--A pilot study.

INTRODUCTION: This pilot study aimed to investigate the use of personal protective equipment (PPE) and compliance to the code of conduct (rules defined in institutional, governmental and professional guidelines) among laboratory technicians in Croatian medical laboratories. In addition, we explored the differences in compliance between participants of different age groups, laboratory ownership and accreditation status. MATERIALS AND METHODS: An anonymous and voluntary survey with 15 questions was conducted among Croatian medical laboratory technicians (N=217). The questions were divided into two groups: demographic characteristics and the use of PPE. The questions of the second part were graded according to the Likert scale (1-4) and an overall score, shown as median and range (min-max), was calculated for each participant. Differences between the overall scores were tested for each group of participants. RESULTS: The majority of participants always wear protective clothes at work, 38.7% of them always wear gloves in daily routine, more than 30.0% consume food and almost half of them drink beverages at workplace. A significantly lower overall score was found for participants working in public compared to private laboratories (36 (16-40) vs. 40 (31-40), P<0.001). There were no statistically significant differences in overall scores for participants of different age groups (P=0.456) and laboratory accreditation status (P=0.081). CONCLUSION: A considerable percentage of laboratory technicians in Croatian medical laboratories do not comply with safety measures. Lack of compliance is observed in all personnel regardless laboratory accreditation and participants' age. However, those working in private laboratories adhere more to the code of conduct.

The evaluation and comparison of consecutive high speed centrifugation and LipoClear® reagent for lipemia removal.

BACKGROUND: The aim of this study was to evaluate and compare the efficiency of high speed centrifugation and LipoClear® reagent for lipemia removal in plasma samples spiked with Intralipid®, for 26 biochemistry analytes. MATERIALS AND METHODS: A plasma pool was collected. Aliquots of the pool were spiked with Intralipid® (final concentrations of 300mg/dL and 500mg/dL Intralipid®). The lipemia was removed from the aliquots by high speed centrifugation or LipoClear® reagent. 26 analytes were determined in native, lipemic plasma and in samples after lipemia removal. The bias from the concentration in the native sample was calculated for each parameter for Intralipid® concentrations, 300 and 500mg/dL of Intralipid®, respectively. Also, the recovery for each parameter after processing the samples using high speed centrifugation and LipoClear® was calculated. The biases and test recoveries were compared with the desirable specification for imprecision (DSI) according to Ricos available at the Wesgard's website. The bias and recovery for procalcitonin were compared with DSI according to Barassi and colleagues. RESULTS: The bias of the spiked samples exceeded the DSI at 300mg/L Intralipid® for creatinine, glucose, total protein, iron and albumin; and for all previously mentioned parameters including CK-MB, sodium, potassium, chlorides, magnesium and ALP at concentration of 500mg/L Intralipid®. For the test recovery the DSI criteria were not met for calcium, total protein, sodium and chlorides after high speed centrifugation and for glucose, calcium, phosphates, magnesium, sodium, potassium, chlorides, ALP, GGT, CK-MB, total protein, albumin and troponin T after using LipoClear®. CONCLUSIONS: LipoClear® is not suitable for lipemia removal from samples designated for glucose, sodium, potassium, chlorides, phosphates, magnesium, CK-MB, ALP, GGT, total protein, albumin, CRP and troponin T measurements. High speed centrifugation should be used for lipemia removal instead for glucose, potassium, phosphates, magnesium, CK-MB, ALP, GGT, albumin, CRP and TnT measurements.

The stability of ethanol in unstoppered tubes.

OBJECTIVES: The exact time frame within which ethanol can be reliably measured in unstoppered tubes is not known. The aim of this study was to investigate the stability of alcohol concentration in unstoppered tubes. DESIGN AND METHODS: 44 samples with alcohol concentration >2.7 mmol/L were included in the study. Measurements were done on Vitros 250 analyzer with original Vitros reagents. After the initial alcohol measurement, each sample was aliquoted into two separate clean tubes (1 mL). One of the aliquoted tubes was stoppered immediately after aliquoting and remained stoppered during the experiment; while the other two tubes (original sample tube and the second aliquoted tube) remained open. During the experiment all three tubes were kept at room temperature. Alcohol concentration was measured at 30 minutes, 1, 2 and 3 hours after the initial measurement in all 3 tubes. The differences between the time intervals for each test tubes were examined using repeated measures Anova or Friedman test. The deviation from the initial concentration was calculated for all three test tubes for each time interval. The calculated deviations were compared with desirable imprecision specifications (DI) according to the RiliBÄK (DI<9%). RESULTS: We found a statistically significant difference between the initial concentration and the concentration in unstoppered tubes for all the investigated time intervals; however, the DI was exceeded only in the original tube and in the tube B, 3 hours after the initial measurement (-9.2% and -12.6%, respectively). CONCLUSIONS: Alcohol concentration can be accurately measured in the unstoppered samples within two hours upon decapping the tube, when stored at room temperature. Longer storage time (>2 hours) in the unstoppered samples introduces significant bias in alcohol concentration.

Angiotensin-converting enzyme insertion/deletion polymorphism genotyping error: the cause and a possible solution to the problem.

Rigat and colleagues were the first ones to develop a rapid PCR-based assay for identifying the angiotensin converting enzyme insertion/deletion (I/D) polymorphism. Due to a big difference between the length of the wild-type and mute alleles the PCR method is prone to mistyping because of preferential amplification of the D allele causing depicting I/D heterozygotes as D/D homozygotes. The aim of this study was to investigate whether this preferential amplification can be repressed by amplifying a longer DNA fragment in a so called Long PCR protocol. We also aimed to compare the results of genotyping using five different PCR protocols and to estimate the mistyping rate. The study included 200 samples which were genotyped using standard method used in our laboratory, a stepdown PCR, PCR protocol with the inclusion of 4 % DMSO, PCR with the use of insertion specific primers and new Long PCR method. The results of this study have shown that accurate ACE I/D polymorphism genotyping can be accomplished with the standard and the Long PCR method. Also, as of our results, accurate ACE I/D polymorphism genotyping can be accomplished regardless of the method used. Therefore, if the standard method is optimized more cautiously, accurate results can be obtained by this simple, inexpensive and rapid PCR protocol.

ABCC8 polymorphisms are associated with triglyceride concentration in type 2 diabetics on sulfonylurea therapy.

BACKGROUND: The failure of therapy with oral hypoglycemic drugs leads to not only poorly regulated glycemic status, but also dyslipidemia and increased body weight and body mass index (BMI). Sulfonylureas act as insulin secretagogues by binding to the sulfonylurea receptor (SUR-1) encoded by the gene ABCC8. The aim of this study was to explore whether there is an association of ABCC8 polymorphisms SUR1 exon 16 (-3C/T), SUR-1 exon 31 (Arg1273Arg), and SUR-1 exon 33 (S1369A) with lipid concentration and BMI in type 2 diabetics on sulfonylurea therapy. MATERIALS AND METHODS: This study included 251 unrelated type 2 diabetics on sulfonylurea therapy. Height and weight were measured for BMI calculation. All polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism methods. Lipid concentrations and BMI were measured at inclusion into the study and after 6 months of follow-up. RESULTS: Wild-type allele carriers for the SUR-1 exon 31 polymorphism (Arg1273Arg) had a significantly higher triglyceride (TG) concentration when compared with the carriers of two variant alleles (p=0.023). Polymorphic allele carriers of the SUR-1 exon 16 (-3C/T) polymorphism were more frequent in the subgroup of patients with the TG concentration increase after 6 months (p for genotype and allelic differences: 0.024 and 0.015, respectively). CONCLUSION: ABCC8 polymorphisms in exon 16 and 31 are associated with the TG concentration in type 2 diabetics on sulfonylurea therapy.

Insulin receptor H1085H C>T and insulin receptor substrate 1 G972R polymorphisms and prostate cancer risk: a pilot study.

BACKGROUND: In recent years, numerous studies have focused their attention on genes that are part of the insulin/insulin-like growth factor 1 signaling pathway, such as the insulin receptor (INSR) and the insulin receptor substrate 1 (IRS1) genes. AIM: We aimed to examine the association of INSR H1085H C>T and IRS1 G972R polymorphisms with prostate cancer (PC). We also aimed to examine possible association with cancer severity assessed by Gleason score. MATERIALS AND METHODS: We have studied 180 consecutive patients referred for PC screening. The genotyping of two polymorphisms (INSR H1085H C>T and IRS1 G972R) was performed by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: There was no difference in genotype (p=0.794) or allelic (p=0.621) frequency of the IRS1 G972R polymorphism between PC (n=119) and control (n=61) groups. However, a significant difference was found in INSR H1085H C>T polymorphism genotype and allelic distribution. Carriers of the polymorphic allele (C/T+T/T) were more frequent in control group patients than in the PC group (54.10% vs. 37.82%; p=0.040; odds ratio [95% confidence interval]=0.52 [0.28-0.96]). The IRS1 and INSR polymorphism distribution did not differ in subgroups according to Gleason score. CONCLUSION: INSR H1085H C>T polymorphism seems to be associated with PC risk, whereas IRS1 G972R is not. However, because of the limited power of this study, there is a possibility that some modest effects of the IRS1 G972R polymorphism on PC risk went undetected. Neither polymorphism is associated with the degree of PC malignancy.