Pneumatic transport is critical for leukaemic patients with major leukocytosis: what precautions to measure lactate dehydrogenase,potassium and aspartate aminotransferase?

Abstract

False elevations of plasma lactate dehydrogenase (LDH), potassium and aspartate aminotransferase (AST) have been described, in relation to haemolysis, occurring most often by mechanical release during phlebotomy or specimen processing. We present the cases of two leukaemic patients with severe hyperleukocytosis for whom LDH, potassium and AST were dramatically but falsely elevated. This false elevation was not caused by haemolysis but could be related to white cells lysis during transport through a pneumatic transportation system, enhanced by a specific fragility of leukaemic cells. Interestingly, this interference almost completely disappeared when serum rather than plasma was used, or when leukocytosis came back to normal. This work is meant to alert clinicians to the risks of errors in LDH, potassium and AST in leukaemic patients and suggest what precautions to take.

Introduction

Reports of pseudohyperkalaemia have been made in leukaemic patients, in the serum or even in the plasma. The causes have been variably attributed to diffusion of potassium from the buffy coat layer, heparin-mediated cell membrane damage, ¹ mechanical disruption of white blood cells by pneumatic tube transport. ² We describe here two cases of falsely elevated plasma potassium, lactate dehydrogenase (LDH) and aspartate aminotransferase (AST) in leukaemic patients with major leukocytosis after pneumatic transportation. This interference disappeared when pedestrian transport was used instead of pneumatic transport, or when serum was used instead of plasma.

Case report

Case 1 was a 25-year-old patient admitted for chemotherapy after diagnosis of acute T-cell lymphoblastic leukaemia. White cell count (WBC) was 309 × 10⁹ cells/L (90% blasts) and 86 × 10⁹ platelets/L (reference range for WBC: 4–10 × 10⁹ cells/L and for platelets: 150–400 × 10⁹/L). The plasma potassium concentration obtained from a non-haemolyzed lithium-heparin tube transported to the laboratory via a pneumatic system was 9 mmol/L on Advia 2400 (Siemens Medical Solutions Diagnostics, Tarrytown, NY, USA). The patient presented no symptoms associated with hyperkalaemia. The possibility of pseudohyperkalaemia was investigated. Venous blood specimens were collected from a catheter both on lithium-heparin Vacutainer tubes and on red top Vacutainer tubes without anticoagulant. Tubes used were all 5 mL plastic tubes (diameter: 13 mm, length: 75 mm), they did not contain separator gel and were filled to capacity. Samples were sent to the laboratory about 15 min after phlebotomy, either via a pneumatic system or via pedestrian transport (<5 min both ways). In the pneumatic system used (Swisslog, Switzerland), tubes are inserted in a first disposable plastic bag which is placed in a second bag with parachutes propelled in a one-way air-flow system with speed limited to 6 m/s and soft landing capabilities. Upon receipt at the laboratory, tubes were promptly centrifuged (10 min at 2465 g ) and analysed within 30 min. Routine chemistry analysis was performed on Advia 2400 and verified on Modular DP (Roche Diagnostics, Indianapolis, IN, USA). Electrolytes were also analysed immediately after phlebotomy and before transportation to the laboratory on whole-blood lithium-heparin specimens with a point of care i-STAT one analyser (Abbott, Point-of-Care, East Windsor, NJ, USA) using E3+ cartridges. The overall process was repeated on the following days until normalization of the WBC. Results showed a dramatic increase of potassium (up to 9.5 mmol/L) and LDH, and a moderate increase of AST on plasma lithium-heparin when transported via the pneumatic system versus pedestrian transport (Table 1). Point of care analysis of whole-blood heparinized specimens performed before pneumatic transportation confirmed the absence of hyperkalaemia in the patient. Values matched those of plasma obtained from specimens transferred via pedestrian transport. Interestingly, results obtained on plasma were increased compared with serum, whereas pneumatic transportation had no effect when serum was used. Potassium, AST and LDH concentrations did not show anymore significant differences on day 9 when WBC dropped to 24.5 × 10⁹ cells/L, between the different types of transport and tubes used (Table 1). Variation in results was considered as clinically significant when exceeding total allowable error (7% for potassium, 16% for AST and 18% for LDH).

Table 1

Measured K, AST and LDH concentrations, obtained in different specimens, using different transport routes

Date	Day 1			Day 3			Day 6			Day 9
Analyte	K	AST	LDH	K	AST	LDH	K	AST	LDH	K	AST	LDH
Pneumatic transport
Lithium-heparin tube	9.5	48	4732	6	39	2992	4.5	22	1487	3.4	16	830
Tube without anticoagulant	4.3	22	2233	4.1	25	1809	4	18	1159	3.4	18	774
Pedestrian transport
Lithium-heparin tube	5.4	38	3435	3.5	20	1666	3.6	19	1168	3.2	15	782
Tube without anticoagulant	4.3	25	2302	3.9	19	1523	4	17	1148	3.4	17	744
Point of care analysis
Lithium-heparin tube	nr	nr	Nr	3.5	nr	nr	3.7	nr	nr	3.1	nr	nr
Haemolysis index	0			0			0			0
WBC, 109 cells/L	297			198			85			24.5

K (mmol/L), AST (UI/L), LDH (UI/L) concentrations and haemolysis index (semi-quantitative results) were determined on an Advia 2400 analyser; point of care analysis used i-STAT analysers; WBC was measured on a Sysmex XE-2100 analyser (Sysmex Corporation, Kobe, Japan); nr: no result Reference range for K: 3.5–5 mmol/L, AST: 15–40 UI/L, LDH: 220–480 UI/L and WBC: 4–10 × 10⁹ cells/L, K, potassium; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; WBC, white blood cell

Case 2 was a 15-year-old girl with acute T-cell lymphoblastic leukaemia (WBC: 358 × 10⁹ cells/L, 95% blasts, 4 × 10⁹ platelets/L), with no clinical signs of hyperkalaemia. Results were similar as in case 1 (data not shown) with high erroneous potassium, LDH and AST plasma values for specimens transported via the pneumatic system, and normal values when pedestrian transport point of care analysis, or serum was used. No sign of haemolysis was present on either specimen.

Additionally, haemolysis index, potassium, LDH and AST were evaluated in 59 non-leukaemic patients (WBC: 1.24–33.23 × 10⁹ cells/L), after sampling in pairs on 5-mL lithium-heparin tubes and transportation either through the pneumatic system or by human porter. Measurements performed on both Advia 2400 and Modular DP did not show any of the abnormalities reported in the two described cases. In particular, potassium was not affected by the different transport processes, and nor was AST. However, when sent through the pneumatic system, mild haemolysis was more frequent (12% of cases versus 3.5% when delivered by porter), and so was mild elevation of LDH (data not shown), which will be investigated further.

Discussion

Pseudohyperkalaemia, in a reverse pattern (absent in serum), has been recently described in a leukaemic patient on a site using pneumatic transport, ¹ but imputability to the pneumatic transport was not examined. Our results are in favour of the responsibility of the pneumatic transport, acting via mechanical disruption of heparin-weakened white cells, ¹ from leukaemic patients with extreme leukocytosis, especially as leukaemic cells may inherently show increased fragility to lysis and leaky membranes. ³ This phenomenon may also cause errors in other analytes measurement such as LDH and AST, which are largely released with cell lysis. Interestingly, such interference is not observed when serum is used. We hypothesize that clotting process may protect the cells therein imprisoned from mechanical damage notably during pneumatic transport. Pseudohyperkalaemia is observed in association with clotted specimen, but gives comparatively minor rises. ⁴ Other hypothesis have been evoked such as heparin-mediated cell membrane damage. ¹ Although no clear evidence, as far as we are aware, has been brought that heparin contained in tubes could have damaging effects on normal blood cells, we cannot rule out that it could exert some damaging effects on fragilized membranes. Centrifugation conditions and delays have been suggested as causes of false elevations of potassium. ⁵ In our case, centrifugation, performed according to the manufacturer's recommendation, does not appear to be a major cause of hyperkalaemia as delays never exceeded 20 min prior to centrifugation, and, following transport, both pedestrian and pneumatic transported tubes underwent the same process of centrifugation, showing normal potassium values in one case and false elevated values in the other. Premature centrifugation could also be cause of serum haemolysis by disruption of the clot, which was not observed here. Type of tubes (with or without separator gel) used to collect samples subjected to pneumatic transport, and tube size to a lesser extent, may play a role in susceptibility to haemolysis. ⁶ Reduced leakage of plasma potassium ¹ in the presence of gel separator and a protective effect of separator gel on haemolysis in serum-gel tubes ⁶ were reported after pneumatic transportation. However, unexpectedly high and erroneous plasma potassium values have also been attributed to the presence of gel in the tube in another case of leukaemic patient. ⁷ Moreover, when sent through a pneumatic system, plain serum samples are more prone to haemolysis ⁶ than heparin ones. Curiously, false elevation of potassium was not observed in our case when plain serum samples were used, which matches what had been observed by Abraham et al. ¹ Moreover, as we did not use serum-gel tubes, we cannot evoke a gel protecting effect in our case. Although the pneumatic system we use (Swisslog, Switzerland) is modern, properly maintained and used according to the manufacturer's recommendations, its weakness could well lie in the weak protection of the tubes during the run. ⁸ Tubes are not protected in solid carriers with foam padding or bubble wraps, they are just inserted in disposable plastic bags with parachutes propelled by compressed air. Moreover, the system configuration including multiple turns may cause sufficient trauma to lyse blood cells. Given the increased incidence of haemolysis in control subjects with normal blood count that pass through this system, the setting of this pneumatic transportation system will require further investigations. In those leukaemic patients with high white cell count, more reliable results require point of care analysis, pedestrian transport or analysis on serum. Possible effect of the use or not of separator gel should also be considered. Awareness of this phenomenon could avoid inappropriate diagnostic or therapeutic measures.

DECLARATIONS

Competing interests: All authors have no conflicts of interest for this work.

Funding: This work has been kindly funded by the CHRU of Lille.

Ethical approval: NA.

Guarantor: IV.

Contributorship: Each author contributed to the design, acquisition of data or analysis, and interpretation of data, drafting or revising the manuscript for intellectual content, and final approval.

Acknowledgment: We thank Aurelie Chuin for technical assistance.