Dynamic reference intervals for coagulation parameters from infancy to adolescence.

INTRODUCTION: Practical and ethical challenges as well as time and costs have restricted the generation of pediatric reference intervals. Therefore, pediatric reference intervals on coagulation parameters based on solid evidence are still scarce. Furthermore, reference intervals by age-group cannot reflect the dynamics of age and sex specific coagulation values during childhood. This study is the first to close this gap and provide continuous age and sex dependent reference intervals during childhood in hemostasis. METHODS: We used an innovative indirect method for providing continuous reference intervals for five common coagulation parameters: Activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin clotting time (TT), fibrinogen (FIB) and antithrombin (AT). Calculations were performed using retrospective laboratory data from pediatric patients between 2005 and 2015 of two major Austrian hospitals, resulting in a total of 195.360 measurements (aPTT: 55,100; PT: 35,492; TT: 35,295; FIB: 49,789; AT: 19,684). RESULTS: This multicenter study provides calculations of continuous reference intervals for five common coagulation parameters in a large pediatric cohort, accounting for age and gender. CONCLUSION: To the best of our knowledge, this is the first multicenter study, determining continuous pediatric coagulation reference intervals based on a large retrospective dataset.

Are laboratory tests always needed? Frequency and causes of laboratory overuse in a hospital setting.

BACKGROUND: Inappropriate utilization of laboratory resources is an increasing concern especially in high-throughput facilities. Until now, no reliable information has been published addressing to which extent laboratory results are actually used for clinical decision-making. Therefore, we aimed to close this gap using a novel retrospective approach including a survey of clinicians and nurses. METHODS: We retrospectively evaluated the number of re-orders for potassium (K), lactate dehydrogenase (LD), aspartate-aminotransferase (AST), activated partial thromboplastin-time (APTT) and prothrombin-time/INR (PT/INR), after the initial order had to be cancelled due to preanalytical non-conformities. We analyzed subgroups regarding time to re-order, ward and sample priority (urgent vs. routine). Subsequently, we surveyed clinicians and nurses, asking for their estimate of the amount of failed re-orders as well as for possible reasons. RESULTS: From initially cancelled tests, only ~20% of K, LD, AST and ~30% of APTT and PT/INR tests were re-ordered within 24 h. 70% of the investigated clinical chemistry and 60% of coagulation tests were re-ordered one week after cancellation or not at all. Survey participants quite accurately estimated these numbers. Routine laboratory panels, short stay of out-patients, obsolete test results and avoiding additional phlebotomies were the main reasons for not re-ordering cancelled tests. CONCLUSIONS: Overall, 60-70% of test results in the investigated assays ordered in a high throughput laboratory are potentially inappropriate or of doubtful clinically importance. Although clinicians and nurses are aware of this situation, it is the duty of laboratory specialists to overcome overutilization in close collaboration with all involved healthcare workers.

Influence of centrifugation conditions on the results of 77 routine clinical chemistry analytes using standard vacuum blood collection tubes and the new BD-Barricor tubes.

INTRODUCTION: Although centrifugation is performed in almost every blood sample, recommendations on duration and g-force are heterogeneous and mostly based on expert opinions. In order to unify this step in a fully automated laboratory, we aimed to evaluate different centrifugation settings and their influence on the results of routine clinical chemistry analytes. MATERIALS AND METHODS: We collected blood from 41 healthy volunteers into BD Vacutainer PST II-heparin-gel- (LiHepGel), BD Vacutainer SST II-serum-, and BD Vacutainer Barricor heparin-tubes with a mechanical separator (LiHepBar). Tubes were centrifuged at 2000xg for 10 minutes and 3000xg for 7 and 5 minutes, respectively. Subsequently 60 and 21 clinical chemistry analytes were measured in plasma and serum samples, respectively, using a Roche COBAS instrument. RESULTS: High sensitive Troponin T, pregnancy-associated plasma protein A, ß human chorionic gonadotropin and rheumatoid factor had to be excluded from statistical evaluation as many of the respective results were below the measuring range. Except of free haemoglobin (fHb) measurements, no analyte result was altered by the use of shorter centrifugation times at higher g-forces. Comparing LiHepBar to LiHepGel tubes at different centrifugation setting, we found higher lactate-dehydrogenase (LD) (P = 0.003 to < 0.001) and lower bicarbonate values (P = 0.049 to 0.008) in the latter. CONCLUSIONS: Serum and heparin samples may be centrifuged at higher speed (3000xg) for a shorter amount of time (5 minutes) without alteration of the analytes tested in this study. When using LiHepBar tubes for blood collection, a separate LD reference value might be needed.

What´s floating on my plasma?

We report on a preanalytical issue we encountered during routine clinical chemistry analyses, potentially leading to deviated analysis results and believe that it might help other laboratories to overcome similar problems. In a heparin-gel tube we measured an implausible glucose value of 0.06 mmol/L. Re-measurement of the same sample resulted in a glucose value of 5.4 mmol/L. After excluding an analytical error, we inspected the sample closer and found a white material as well as fatty droplets floating on the surface of the plasma tube. Evaluation of these structures revealed that the white particulate matter (WPM) consisted of fibrinogen, platelets and leukocytes and the fatty droplets most probably originated from the separator gel. We concluded that these structures formed a temporary clot in the instruments pipetting needle thereby altering the sampling volume and subsequently the measured glucose value. The formation of WPM might be attributable to high speed centrifugation, high cholesterol levels, the gel formulation or a combination of several issues such as temperature, heparin concentration, pH and patient-specific factors. The gel droplets were most probably caused by an aberrant gel formulation in combination with an improper storage of the empty tubes on the wards prior to phlebotomy. After adding an additional instrument cleansing cycle and changing to another batch of heparin tubes the problems could be significantly reduced.

The relationship between vacuum and hemolysis during catheter blood collection: a retrospective analysis of six large cohorts.

BACKGROUND: Blood collection through intravenous (IV) catheters is a common practice at emergency departments (EDs). This technique is associated with higher in vitro hemolysis rates and may even be amplified by the use of vacuum collection tubes. Our aim was to investigate the association of five different vacuum tubes with hemolysis rates in comparison to an aspiration system under real-life conditions and to propose an equation to estimate the amount of hemolysis, depending on the vacuum collection tube type. METHODS: We retrospectively evaluated hemolysis data of plasma samples from our ED, where blood is drawn through IV catheters. Over the past 5 years, we compared 19,001 hemolysis index values amongst each other and against the respective vacuum pressure (Pv) of the collection tubes, which were used within the six observational periods. RESULTS: The highest hemolysis rates were associated with full-draw evacuated tubes. Significantly reduced hemolysis was observed for two kinds of partial-draw tubes. The hemolysis rate of one partial-draw blood collection tube was comparable to those of the aspiration system. Regression analysis of Pv and mean free hemoglobin (fHb) values yielded the formula fHb (g/L)=0.0082*Pv2-0.1143*Pv+ 0.5314 with an R2 of 0.99. CONCLUSIONS: If IV catheters are used for blood collection, hemolysis rates directly correlate with the vacuum within the tubes and can be estimated by the proposed formula. By the use of partial-draw vacuum blood collection tubes, hemolysis rates in IV catheter collections can be reduced to levels comparable with collections performed by aspiration systems.

Hemolysis rates in blood samples: differences between blood collected by clinicians and nurses and the effect of phlebotomy training.

BACKGROUND: Hemolytic samples are one of the most challenging preanalytical issues in laboratory medicine. Even causes leading to hemolytic specimen are various, including phlebotomy practices. Respective educational interventions as well as the reduction of the number of people involved in blood collections are claimed to influence the sample quality for the better. In our hospital 70 junior doctors were in charge of routine phlebotomy until 2012, when this task was shifted to 874 nurses, including a preceding training in phlebotomy and preanalytics. Our aim was to evaluate the impact of this training effect and the increase of people involved on sample quality. METHODS: The hemolysis index (HI) of 43,875 samples was measured before (n=21,512) and after (n=22,363) the switch of blood collection responsibilities. Differences in overall hemolysis rates and the amount of plasma samples with a concentration of free hemoglobin (fHb) above 0.5 g/L and 1 g/L were calculated. RESULTS: Overall HI as well as the percentage of samples with an fHb concentration >0.5 g/L decreased after the responsibility for phlebotomy changed. The rate of samples with an fHb concentration >1 g/L remained unchanged. CONCLUSIONS: Hemolysis rates were reduced upon passing phlebotomy tasks from untrained physicians on to a trained nursing staff. We therefore conclude that the number of people performing phlebotomy seems to play a minor role, compared to the effect of a standardized training. However, whether a reduction in the number of people involved in blood collection could lead to further improvement of sample quality, remains to be investigated in future studies.

Aqueous humor ferritin in hereditary hyperferritinemia cataract syndrome.

PURPOSE: Hereditary hyperferritinemia cataract syndrome (HHCS) is a rare autosomal dominant hereditary disease, characterized by hyperferritinemia but with absence of body iron excess and early onset of bilateral cataracts. Although 5- to 20-fold increased serum ferritin concentrations have been reported in HHCS patients, data of ferritin levels in aqueous humor have not been obtained. We therefore aimed to investigate the ferritin levels in aqueous humor and serum and further present histological and ultrastructural data of the lens. METHODS: During cataract extraction and intraocular lens implantation, aqueous humor and lens aspirate of a 37-year-old HHCS patient were obtained from both eyes. Ferritin levels in serum and aqueous humor were quantitatively analyzed via immunoassays in the HHCS patient and healthy control subjects (n = 6). Lens aspirate in HHCS was analyzed histologically and at the ultrastructural level. Further, genetic mutation screening by polymerase chain reaction and DNA sequencing in blood was performed. RESULTS: Serum ferritin levels in the control group were 142.2 ± 38.7 µg/L, whereas in the HHCS patient, this parameter was excessively increased (1086 µg/L). Analysis of ferritin in aqueous humor revealed 6.4 ± 3.8 µg/L in normal control subjects and 146.3 µg/L (OD) and 160.4 µg/L (OS) in the HHCS patient. DNA analysis detected a C>A mutation on position +18, a T>G mutation on position +22, a T>C mutation on position +24, and a T>G polymorphism on position +26 in the iron-responsive element of the light-chain ferritin (L-ferritin) gene. CONCLUSIONS: In the HHCS patient, a 23-fold (OD) to 25-fold (OS) increased aqueous humor ferritin level was detected. Therefore, the formation of bilateral cataract in HHCS is most likely a result of elevated aqueous humor ferritin. In addition, a novel mutation in this rare disease in the iron-responsive element of L-ferritin gene is reported.

Relevance of EDTA carryover during blood collection.

BACKGROUND: The order of draw is regarded as a preanalytical issue to prevent carryover of additives during blood collection. Our objective was to prove the theory of ethylenediaminetetraacetic acid (EDTA) carryover for a closed vacuum system and the influence of EDTA on concentrations of selected biomarkers. METHODS: To test the carryover of EDTA, a blood collection with tripotassium EDTA (K3EDTA) and subsequent non-additive tubes was simulated using distilled water as substitute for blood. EDTA concentrations were measured by tandem mass spectrometry. Then we added increasing concentrations of EDTA to heparinized blood and measured routine biomarkers, thereby simulating a carryover of EDTA whole blood and pure EDTA, respectively. Additionally, we tested for EDTA contamination and biomarker alteration in samples collected from 10 healthy volunteers by a syringe with subsequent transfer into sample tubes. RESULTS: No EDTA contamination was detected in samples collected subsequent to a K3EDTA tube when adhering to guidelines of blood sampling. Magnesium, calcium, and potassium levels were altered by artificial K3EDTA whole-blood contamination as well as when adding 1 µL pure K3EDTA. Iron values were altered at EDTA concentrations of 4.4 mmol/L. All other parameters remained unaffected. A slight EDTA carryover was observed in syringe collection and subsequent transfer into EDTA and heparin tubes, however, without any biomarker alteration. CONCLUSIONS: An EDTA carryover during blood collection using a closed vacuum system is highly unlikely. Even if carryover of EDTA whole blood occurs, an absolute volume larger than 10 µL would be necessary to alter test results. However, contamination of samples with preloaded pure K3EDTA solution by severe neglect of current recommendations in blood collection may significantly alter testing results.