Preventing factitious hyperkalaemia in general practice by using blood collection tubes with a new rapid clot activating solution (BD Vacutainer®Rapid serum tubes) Tine Huyghe, KUL Promotor: Prof. Dr. Frank Buntinx, KUL Co-promotor: Prof. Norbert Blanckaert, KUL Master of Family Medicine Masterproef Huisartsgeneeskunde December 2011

ABSTRACT Objective: To evaluate the performance and efficacy of the BD Vacutainer® Rapid Serum Tube (RST) compared to the BD Vacutainer® SST™ II Advance Blood Collection Tube (SST), the effect of correct mixing, place of sample collection, degree of filling, travel time and difficulty of vein punction for haemolysis reduction. Setting: Routine general practice Participants: 31 Flemish General Practitioners (GPs).353 patients that visited their general practitioner for a blood test. Two regional laboratories. Intervention: A pair of SST tubes and a pair of RST tubes were drawn in a randomised order in replacement of the regularly used serum tube. Of each pair, one was inverted 5 times according to manufacturer’s instructions, the other was not inverted. Main Outcome measure: Potassium, LDH and Haemolytic Index. Results: The use of the RST tube and correct mixing have no benefit over the SST tube and no mixing in preventing hyperkalaemia or increased LDH levels. Tubes that were incompletely filled show higher mean serum potassium values and higher LDH levels. Too few abnormal haemolytic indices were observed to make a proper analysis possible. Conclusion: For determination of serum potassium levels, RST and SST Blood Collection Tubes are equally valid serum transporters to be used in general practice. Inverting of the tubes seems unnecessary with regards to serum potassium and LDH values. A new factor effecting serum potassium and LDH levels seems to be revealed: correct filling of the serum tubes. This can easily be taken into account while taking a blood sample.

SAMENVATTING Doelstelling: Evalueren van het gebruik van BD Vacutainer® Rapid Serum Tube (RST) in vergelijking met BD Vacutainer® SST™ II Advance Blood Collection Tube (SST) in de huisartsenpraktijk, het effect van omdraaien van de tubes volgens aanwijzingen van de producent, plaats van bloedname, vullingsgraad, reistijd van het staal en moeilijkheid van de staalname voor hemolysereductie. Setting: Huisartsenpraktijk Deelnemers: 31 Vlaamse huisartsen.353 patiënten die hun huisarts raadpleegden voor een bloedname. Twee regionale labo’s. Opzet: Een paar SST tubes en een paar RST tubes werden in gerandomiseerde volgorde afgenomen in plaats van de normaal gebruikte serumtube. Van elk paar tubes werd een vijf keer omgedraaid volgens aanwijzingen van de producent, de ander werd niet omgedraaid. Voornaamste uitkomstmaten: Kalium, LDH en Hemolytische Index. Resultaten: Gebruik van de RST tube en correct omdraaien van de tubes hebben geen voordeel tegenover gebruik van de SST tube en niet omdraaien van de tubes voor preventie van hyperkaliëmie of gestegen LDH. Tubes die onvolledig gevuld waren hebben hogere gemiddelde kalium en LDH waardes. Er werden te weinig afwijkende hemolytische indexen geobserveerd om hier zinvolle analyse op uit te voeren. Conclusie: Voor het bepalen van kaliumwaardes in serum zijn de RST en SST collectietubes evenwaardig in de huisartsenpraktijk. Het omdraaiden van de serum tubes lijkt onnodig wat betreft de kalium- en LDH waarden. Met de invloed van het volledig vullen van de serumtubes lijkt een nieuwe invloed op kalium –en LDH waarden aan het licht gekomen. Hier kan gemakkelijk rekening mee gehouden worden tijdens bloedname.

INTRODUCTION Detecting abnormal potassium blood levels in general practice may be important. Hyperkalaemia may result from treatment with ACE inhibitors, potassium-sparing diuretics or other drugs and can result in sudden death. (1,2) In Intego, a general practice-based morbidity network database with more than 1.8 million patient-years, all results of laboratory tests are filed. The proportion of increased potassium results is 4.6% in patients on potassium-sparing diuretics. (3) The Accident & Emergency department of the Leuven University Hospital on average receives two patients with hyperkalaemia each day (personal communication). However, factitious elevations in potassium levels often occur. It can be the result of patient-related factors, specimen acquisition, processing, handling and transport conditions. (4, 5, 6) It is on the in vitro haemolysis due to the sample handling and transportation prior to analysis that we would like to focus in this article. For general practitioners (GPs) in Belgium, and probably elsewhere, proper monitoring of serum levels of potassium is almost impossible. A typical blood sample is taken by the GP in his office during morning hours, kept in the office for one or more hours, then collected by a transport service, transported by a frequently stopping and accelerating car for some hours and received and dealt with in the laboratory somewhere in the afternoon. GPs and transporters can be instructed about storage procedures including temperature control, etc. in order to minimize haemolysis. No method has been found, however, preventing the sample from haemolysis during transport. Centrifugation prior to transport is not a realistic option because small practices do not have the time or resources to do this. In a small-scale pilot study (n=6 patients) on multiple samples taken from each patient at the same time we found no systematic difference between serum samples transported using a gel separator tube, or either pipetted or poured before transport of the plasma. In another pilot study dual samples were taken from 50 subsequent patients. From each patient, one sample was transported without previous manipulation, the other sample was centrifuged within five minutes and the serum was poured and separately transported. The results were largely similar: In a regression analysis, results of centrifuged serum explained 70% of the variation of the results of the other samples. Bland and Altman analysis showed that for samples that were not centrifuged in patients with broadly normal serum values, there was a lower frequency of elevated serum results compared to the centrifuged serum samples. An opposite trend was observed if the mean result was above 4.5meq/L. A systematic literature review showed no additional methods that may solve the problem.

BD has recently released a new transport tube with a thrombin based rapid clot activated solution (BD Vacutainer® Rapid Serum Tubes - RST). It was assumed that the rapid clotting promoted by the tube leads to a more compact and robust clot which protects the red cells and leads to a reduction in haemolysis. Previous evaluation has shown slightly lower mean values for LDH and potassium compared to the standard BD SST II tube (SST) in a hospital environment. (7) Therefore, it has been proposed to evaluate the impact of this tube in preventing factitious hyperkalaemia in general practice.

METHODS Laboratories and GPs were recruited via letter, email or personal connections of the authors. The GPs were provided with all material necessary for the blood draw, an overview card with the exclusion criteria and the tube handling protocol, and a poster for the waiting room to point out to the patients that their GP was participating in a trial and that the patients could voluntary participate too. Patients above the age of 20 that visited their GP to purchase a blood test willing to cooperate, were included in the trial after signing an informed consent form. Exclusion criteria were age, samples taken at night or in weekends, samples taken in the laboratories, and expected interference of participation with any current or future medical treatment. Devices and procedures The study tubes are listed in Table 1. All tubes are 13x100mm evacuated BD Vacutainer® blood collection tubes with sterile interior, a draw volume of 5 ml and a BD Hemogard™ closure. All tubes are serum blood collection tubes and contain a clot activator to promote clotting of blood. Both tubes also contain a gel polymer, which moves up the tube during centrifugation to form a barrier between the serum and clot. BD currently markets both tubes in Europe. BD provided 500 sets of the four study tubes in sealable bags. Each bag carried a label with the subject number and a randomised draw order of the four tubes. All tubes were labelled with the subject number and tube type, tubes that needed no mixing carried a red label indicating so. Each bag provided by BD was completed with an informed consent form and a data report form (DRF), used to gather some additional information. GPs marked date, time of vein puncturing, use of ACE-inhibitors or diuretics by the patient, site and difficulty of vein puncturing and confirmation of correct mixing of the tubes on the DRF. Laboratories added travel time and correct filling on the DRF. Appendix Analysis Prior to starting the study, inter-laboratory testing was performed, to verify harmonisation of test results, including haemolytic index and centrifugation practices. All research forms, except the informed consent forms, were anonymous. For each patient, four serum tubes were taken, two RST and two SST Tubes. The drawing order of these four serum tubes was randomised, and for each patient the drawing order of the four tubes was indicated on the study bag. One tube of each type was inverted 5 times, according to manufacturer’s guidance; the other tubes were not inverted at all. Serum potassium (mmol/L), LDH (%) and haemolytic index were tested on all samples according to normal laboratory procedures. As the process of determination is automated, there was no need for test blinding. Normal levels of serum potassium range between 3,5 and 5,1 mmol/L. LDH levels above 248U/L are considered to be raised for the Practimed Tessenderlo laboratory, levels above 480 U/L for the MCH Leuven laboratory. Haemolytic index was displayed in six categories from “normal” until “+++++”. Those categories represent serum haemoglobin levels expressed in mg/dL Haemoglobin: “normal”=500.

On the DRF, all movements of the serum tubes were marked. Statistical analysis Serum potassium and serum LDH are continuous and were analysed by the following model: y=β0 + β1Subject + β2Tube + β3Inversion + β4Tube : Inversion + error, where y is either the response or the natural log of the response, depending on the distribution of the data. The model reported differences for each possible combination of the different tube types (e.g. RST 5 inversions versus RST 0 inversions). Subject is accounted for as a grouping variable for the continuous responses. Haemolytic index, hyperkalaemia and elevated LDH were evaluated using logistic regression analysis. Additional characteristics, such as transport time, place of sample collection, and correct filling, were taken into account as independent variables. Differences in the proportions of increased results were calculated and tested with a fisher exact test for each of the transport systems. The Fisher test was done for the entire matrix, if significance was found, additional testing was done to isolate the source. Finally Bland & Altman analysis for serum potassium and serum LDH levels was performed. RESULTS 1. Description of the population Two regional Laboratories cooperated: MCH Leuven and Practimed Tessenderlo. 405 sets of tubes were distributed among 31 GPs of which five worked with MCH Leuven and 26 with Practimed Tessenderlo. Blood samples were taken between the 26th of April and the 20th of June, 2011. 353 sets were finally processed by the two laboratories. After exclusion of all incomplete data sets and an additional nine datasets in which mixing of the tubes was not executed according to study protocol, a total of 296 full data sets remained. Table 2 Six percent of all included samples were taken at the patients’ homes; all other samples were taken at the GP’s office. In 14 (5%) vein punctures, there were some difficulties to collect the blood samples. Travel time – from pick-up in the GP’s office until arrival in the laboratory – ranged from 10 to 265 minutes. Out of the 296 included subjects, 22% was on diuretics, 6% on potassium sparing diuretics and 26% on ACE-inhibitors. 1128 (95%) of all 1184 tubes were completely filled. Eight percent of them were hyperkalaemic. Ten percent showed elevated LDH levels and two percent had an abnormal haemolytic index (of which 80% was only slightly haemolytic in category “+”). 2. Performance of the different tubes The RST samples had little or no difference in serum potassium and slightly lower serum LDH levels compared to the SST. There is no significant difference between RST and SST for hyperkalaemia and elevated serum LDH. Table 3. Bland & Altman plots showed no influence on the mean potassium values in our results. 3. Performance of correct mixing

Inverting the tubes resulted in little or no difference for serum potassium or serum LDH compared to non-inverting. Table 3 it also did not result in a significant difference in hyperkalaemia or elevated serum LDH. Table 4. Bland & Altman plots showed no influence on the mean potassium values in our results. 4. Effect of other variables Table 5 For all tube types together, mean serum potassium values are significant higher for tubes that were incompletely filled (0,5mmol higher on average, p