J Clin Pathol 2000;53:105–109 105 Artefactual serum hyperkalaemia and hypercalcaemia in essential thrombocythaemia J Clin Pathol: first published as 10.1136/jcp.53.2.105 on 1 February 2000. Downloaded from

M R Howard, S Ashwell, L R Bond, I Holbrook

Abstract the release of ions from platelets Aim—To investigate possible abnormali- which are entrapped within the clot in a serum ties of serum potassium and sample. This phenomenon generally occurs levels in patients with essential thrombo- with platelet counts in excess of 600 × 109/litre, cythaemia and significant thrombocyto- with a roughly predictable increment in serum sis. potassium for every further increase in platelet Methods—24 cases of essential thrombo- count. The potassium level is normalised if the cythaemia with significant thrombocyto- estimation is made using plasma rather than sis (platelet count > 700 × 109/litre) had serum.45 serum potassium and calcium estimations Abnormalities of other ions have not been performed at the time of maximum well described in essential thrombocythaemia. thrombocytosis before treatment, and at Despite the presence of calcium in platelet- the time of low platelet count after dense granules and its secretion from platelets treatment with cytoreductive drugs. Se- during activation, there has been no systematic lected patients were further investigated study of serum calcium levels in patients with with plasma sampling and estimation of essential thrombocythaemia and significantly ionised calcium and parathyroid hor- increased platelet counts. There has been a mone. single case report of serum hypercalcaemia Results—At the time of maximum throm- associated with essential thrombocythaemia. In bocytosis six patients had serum hyperka- this case the hypercalcaemia rapidly resolved laemia (> 5.5 mmol/litre) and five had following reduction of the platelet count.6 We serum hypercalcaemia (> 2.6 mmol/litre). have investigated a group of patients with Following treatment and reduction of the essential thrombocythaemia and significant platelet count, hyperkalaemia resolved in thrombocytosis to further characterise the all cases and hypercalcaemia in four of the nature of pseudohyperkalaemia and also to five cases. Mean serum potassium and establish whether there are similar alterations calcium concentrations were raised in serum calcium levels. (p < 0.0001) at maximum thrombocytosis compared with the values when the plate- let count was low. Serum potassium and Methods http://jcp.bmj.com/ calcium values were significantly corre- PATIENTS lated at all stages. Measurements on Thirty three patients with essential thrombo- plasma consistently corrected the hyper- cythaemia were identified from clinical records kalaemia but not the hypercalcaemia. and 24 were eligible for the study. To be eligi- Serum hypercalcaemia was associated ble, patients had to have a presentation platelet with raised ionised calcium and normal count in excess of 700 × 109/litre and measure-

concentrations. ments of serum calcium and serum potassium on October 2, 2021 by guest. Protected copyright. Conclusions—Essential thrombocythae- made at the time of maximum platelet count mia with significant thrombocytosis is before treatment and at the time of a low plate- associated with serum hyperkalaemia and let count after appropriate treatment of throm- hypercalcaemia. The probable mech- bocytosis. Where relevant biochemical tests anism of hypercalcaemia is the secretion were available at more than one normal platelet of calcium in vitro from an excessive count after treatment, the lowest platelet count number of abnormally activated platelets. was used. Department of It is thus likely that the hypercalcaemia is The mean age of the patients was 66 years Haematology, York an artefact, as is the hyperkalaemia. (range 17–84 years). The male:female ratio was District Hospital, (J Clin Pathol 2000;53:105–109) 11:13. The mean pretreatment platelet count Wigginton Road, York × 9 YO31 8HE, UK was 1144 10 /litre (range 736–2291). The Keywords: thrombocythaemia; hypercalcaemia; patients were treated with either oral hydroxy- MRHoward hyperkalaemia S Ashwell urea (n = 20) or busulphan, anagrelide, or L R Bond interferon alfa (n = 4). Essential thrombocythaemia is a chronic my- Following cytoreductive treatment the Department of eloproliferative disorder characterised by a platelet count fell to a mean level of 288 × 109/ Biochemistry, York 1 District Hospital persistently raised platelet count. It has litre (range 61–526). Two patients with poor I Holbrook previously been reported that the marked compliance with treatment and fluctuating thrombocytosis that is often associated with levels of severe thrombocytosis (cases 1 and 2) Correspondence to: essential thrombocythaemia may lead to associated with serum hyperkalaemia and Dr Howard. spurious laboratory abnormalities including hypercalcaemia had serial monitoring of 2 3 Accepted for publication pseudohyperkalaemia and pseudohypoxaemia. serum potassium and serum calcium during 8 July 1999 Serum hyperkalaemia appears to result from their clinical course, and also the following 106 Howard, Ashwell, Bond, et al

8 Serum and plasma potassium estimation

Potassium was measured on a Hitachi 917 J Clin Pathol: first published as 10.1136/jcp.53.2.105 on 1 February 2000. Downloaded from analyser (Roche Diagnostics, Lewes, E Sussex, 6.5 UK) using an indirect ion selective electrode method. The was 3.5–5.5 6 mmol/litre.

ULk Serum and plasma calcium estimation 5.5 Calcium was measured on a Hitachi 917 analyser using an o-cresolphthalein complex- 5 one method. The reference range was 2.10– 2.60 mmol/litre.

4.5 Serum estimation Serum potassium (mmol/l) Phosphate was measured on a Hitachi 917 4 analyser using an ammonium molybdate method, the reaction being monitored at an ULp LLp ultraviolet wavelength. The reference range 3.5 was 0.8–1.4 mmol/litre. 1400 1200 1000 800 600 400 200 0 1500 1300 1100 900 700 500 300 100 × 9 Ionised calcium estimation Platelet count ( 10 /l) Serum ionised calcium was measured on a Figure 1 Platelet count and serum potassium Ciba Corning 634 Ca2+/pH analyser (Beckman concentrations in 24 patients with essential Instruments, High Wycombe, Buckingham- thrombocythaemia. Serum potassium values are shown at the highest (C) and low (x) platelet counts. Broken lines shire, UK) using a calcium ion selective and indicate the upper limit (UL) and lower limit (LL) of pH electrodes. The reference range was normal reference ranges for potassium (k) and platelet 1.18–1.38 mmol/lire. count (p). Parathyroid hormone (PTH) estimation investigations: plasma sampling in addition to Intact PTH was analysed on a Nichols Advan- serum sampling for potassium and calcium tage analyser (Nichols Institute Diagnostics, concentrations, and measurement of serum Newport, Gwent, UK) using a chemilumines- phosphate, ionised calcium, and parathyroid cent immunoassay method. The reference hormone. range was 10–60 ng/litre.

LABORATORY METHODS STATISTICAL METHODS Blood count Possible diVerences in the mean serum calcium All blood counts were performed using a and serum potassium levels at the time of

Sysmex automated cell counter. The normal maximum thrombocytosis and low platelet http://jcp.bmj.com/ platelet reference range was defined as 150– count were analysed using the paired t test. 400 × 109/litre. Blood films were routinely Possible correlations between serum calcium inspected to confirm the degree of thrombocy- and serum potassium levels were detected tosis. using the Pearson correlation coeYcient test.

Case 1 Case 2

7 7 on October 2, 2021 by guest. Protected copyright. 6 6 5 5 4 4

(mmol/l) 3 3 Serum potassium

3.0 3.0 2.8 2.8 2.6 2.6 2.4 2.4

(mmol/l) 2.2 2.2

2000 2000 /l) Serum calcium 9 1000 1000 700 700 × 10 500 500 300 300 200 200 100 100 Platelets ( 321 45 910876 14 15131211 19 20181716 21 321 45 9876 Time (months) Time (months) Figure 2 Platelet count, serum potassium concentration, and serum calcium concentration in two patients with poorly controlled essential thrombocythaemia (cases 1 and 2). Broken lines indicate the upper limits of the normal range. Hyperkalaemia and hypercalcaemia in thrombocythaemia 107

3 patients developed clinical symptoms of hyper-

2.9 calcaemia. The mean serum calcium was J Clin Pathol: first published as 10.1136/jcp.53.2.105 on 1 February 2000. Downloaded from significantly raised at the time of maximum 2.8 thrombocytosis compared with the value when 2.7 the platelet count was low (2.52 2.33 mmol/ UL v 2.6 c litre; p < 0.0001). 2.5 2.4 Plasma calcium In two patients with coexisting serum hyperka- 2.3 laemia and hypercalcaemia (cases 1 and 2), the 2.2 LL degree of hypercalcaemia was not significantly 2.1 c altered by the collection and analysis of a 2 plasma sample in case 1, but there appeared to

Serum calcium (mmol/l) 1.9 be a small correction in the plasma sample in case 2 (case 1: serum calcium 2.79 mmol/litre, 1.8 plasma calcium 2.72 mmol/litre; case 2: serum 1.7 calcium 2.64 mmol/litre, plasma calcium 2.51 1.6 ULp LLp mmol/litre). 1.5 1400 1200 1000 800 600 400 200 0 1500 1300 1100 900 700 500 300 100 Correlation of serum calcium with the platelet count in two patients with poorly controlled 9 Platelet count (×10 /l) thrombocythaemia Figure 3 Platelet count and serum calcium concentration In these patients (cases 1 and 2) there was a in 24 patients with essential thrombocythaemia. Serum close correlation between the degree of serum calcium is shown at the time of highest (C) and low (x) hypercalcaemia and the degree of thrombocy- platelet counts. Broken lines indicate the upper limit (UL) and lower limit (LL) of normal reference ranges for calcium tosis (fig 2). (c) and platelet count (p). CORRELATION BETWEEN SERUM CALCIUM AND Results SERUM POTASSIUM SERUM AND PLASMA POTASSIUM ESTIMATIONS There was a significant correlation between Serum potassium serum potassium and serum calcium, both at Serum hyperkalaemia (> 5.5 mmol/litre) was the time of maximum thrombocytosis observed in six patients at the time of (p = 0.02) and at the time of low platelet count maximum thrombocytosis and in no patients at (p = 0.02) (fig 4). the time of low platelet count (fig 1). The mean serum potassium level was significantly raised FURTHER INVESTIGATIONS IN TWO PATIENTS at the time of maximum thrombocytosis com- WITH SERUM HYPERCALCAEMIA pared with the value when the platelet count In the two patients with most marked serum was low (5.29 v 4.57 mmol/litre; p < 0.0001). hypercalcaemia (cases 1 and 2) there was an associated increase in ionised calcium (case 1, http://jcp.bmj.com/ Plasma potassium 1.43 mmol/litre; case 2, 1.42 mmol/litre) but In two cases of combined serum hyperkalaemia normal parathyroid hormone concentrations and hypercalcaemia (cases 1 and 2), there was (case 1, 41.3 ng/litre; case 2, 20.2 ng/litre). The normalisation of the potassium level on a simultaneously measured plasma sample (case 3 1; serum potassium 6.5 mmol/litre, plasma 2.9

potassium 4.5 mmol/litre; case 2; serum potas- on October 2, 2021 by guest. Protected copyright. sium 6.3 mmol/litre, plasma potassium 4.2 2.8 mmol/litre). 2.7

ULc Correlation of serum potassium with the platelet 2.6 count in two patients with poorly controlled thrombocythaemia 2.5 In these patients (cases 1 and 2) there was a close correlation between the degree of serum 2.4 hyperkalaemia and the fluctuating degree of 2.3 thrombocytosis (fig 2). 2.2

Serum calcium (mmol/l) LLc SERUM AND PLASMA CALCIUM ESTIMATIONS 2.1 Serum calcium There was serum hypercalcaemia (> 2.6 mmol/ 2 litre) in five patients at time of thrombocytosis 1.9 and in one patient at the time of low platelet LLk ULk count (fig 3). In the single patient with persist- 1.8 43.53 4.5 5 5.56 6.5 7 ing hypercalcaemia at low platelet count, the low platelet count was still above the normal Serum potassium (mmol/l) range, at 413 × 109/litre. In the remaining four Figure 4 Correlation of serum potassium and serum patients, serum hypercalcaemia entirely re- calcium at time of highest (C) and low (x) platelet counts in 24 patients with essential thrombocythaemia. Broken solved following normalisation of the platelet lines indicate the upper limit (UL) and lower limit (LL) of count with oral cytoreductive treatment. No normal reference ranges for potassium (k) and calcium (c). 108 Howard, Ashwell, Bond, et al

serum phosphate was normal in case 1 (1.11 lets acquire abnormalities within the circula-

mmol/litre) but raised in case 2 (1.80 mmol/ tion or in vitro. Certainly there is evidence to J Clin Pathol: first published as 10.1136/jcp.53.2.105 on 1 February 2000. Downloaded from litre). In both of these cases bone marrow tre- suggest that these platelets are abnormally phine samples had been collected at diagnosis activated and may become “exhausted” within and there was no morphological evidence of the circulation or in vitro, with release of their bone resorption in these samples. granule contents.910 Specific findings to sup- port this are decreased levels of á granule â Discussion thromboglobulin associated with raised plasma An increase in serum potassium, commonly concentrations of this platelet specific termed “pseudohyperkalaemia,” has been well protein,11 12 and the finding of a low platelet described in patients with essential thrombo- calcium content in myeloproliferative disorders cythaemia, correlating with the raised platelet in comparison with normal controls.13 We sug- count. There is a similarly documented but gest that the most likely cause of hypercalcae- lesser eVect in reactive thrombocytosis. The mia in essential thrombocythaemia is the finding of a normal potassium concentration in release of calcium ions in vitro from increased a simultaneously collected plasma sample numbers of abnormally activated platelets. It is suggests that the high serum potassium is an of interest that, although serum hyperkalaemia artefact. It is presumed that potassium leaks was not reproducible in a plasma sample, into the serum sample from the numerous significant hypercalcaemia occurred in both platelets which are trapped within the clot. serum and plasma samples in one case, and in Although this is considered to be an artefact, it a second case there was only a slightly lower is nevertheless of clinical significance as in our calcium concentration in the plasma sample. experience patients may be wrongly investi- This is presumably because potassium is only gated or even treated for hyperkalaemia owing released slowly from degranulating platelets to ignorance of the association with thrombo- within a clot, whereas the release of calcium cytosis. Our study has confirmed the relatively ions will occur promptly into the sample as an high frequency of serum hyperkalaemia in this early stage of the platelet secretory response. patient group, with the presence of frank This mechanism of in vitro hypercalcaemia, as hyperkalaemia in six of the 24 patients. It is opposed to a more long standing in vivo abnor- noteworthy that, in contrast to some other mality in calcium haemostasis, is further studies, there was not a very close correlation supported by the lack of any clinical symptoms between the magnitude of the increase in the of hypercalcaemia and by the very close corre- platelet count and the degree of hyperkalaemia, lation between calcium and platelet levels over and also that significant hyperkalaemia (> 6 time in two of our patients with poorly control- mmol/litre) occurred in patients with a platelet led thrombocythaemia owing to lack of compli- count of less than 1000 × 109/litre. ance with treatment. The high incidence of serum hypercalcaemia Our observations in these patients confirm observed in this series of patients with essential that serum hyperkalaemia is a common com- thrombocythaemia has not been described plication of essential thrombocythaemia with before. As with hyperkalaemia, the increased significant thrombocytosis. In addition we have http://jcp.bmj.com/ serum calcium appears to be related to throm- documented for the first time that there is a bocytosis as the hypercalcaemia quickly re- significant incidence of serum hypercalcaemia solved following the normalisation of the plate- in this disorder. It is likely that these biochemi- let count with cytoreductive chemotherapy. cal abnormalities are artefactual, but it is The cause of hypercalcaemia in these patients important that clinicians understand these is conjectural. Hypercalcaemia has been de- associations to avoid unnecessary investigation

scribed in other myeloproliferative disorders and treatment. on October 2, 2021 by guest. Protected copyright. and has been attributed to several possible mechanisms, including bone destruction medi- 1 Jandl JH, ed. Essential thrombocythaemia. In: Textbook of ated by leukaemic cells, ectopic production of , 2nd ed. Boston: Little, Brown, 1996:940–5. parathyroid hormone, and tumour derived 2 Hartmann RC, Auditore JV, Jackson DP. Studies on throm- bocytosis. I. due to release of potassium transforming growth factors stimulating from platelets during coagulation. J Clin Invest 1959;37: osteoclastic bone resorption.78In our series the 699–707. 3 Hess CE, Nichols AB, Hunt WB, et al. Pseudohypoxaemia two patients with most marked hypercalcaemia secondary to leukaemia and thrombocytosis. Medical Intel- had normal parathyroid hormone levels, mak- ligence 1979;301:361–3. ing ectopic production of this hormone an 4 Wolkan RW, Michiels JJ. Pseudohyperkalaemia in thrombo- cythaemia. J Clin Chem Clin Biochem 1990;28:489–91. unlikely cause. Bone marrow trephine samples 5 Makela K, Kairisto V, Peltola O, et al.EVect of platelet count in these patients showed no morphological evi- on serum and plasma potassium; evaluation using database information from two hospitals. Scand J Clin Lab Invest dence of leukaemic transformation or bone Suppl 1995;222:95–100. resorption. 6 Varkel V, Braester A, Mutlak D, et al. Hypercalcemia complicating essential thrombocythemia. Eur J Haematol The close correlation between calcium and 1990:4:204. potassium concentrations in these patients 7 Waller RM, Greenburg BR. Hypercalcaemia in the acceler- ated phase of chronic myelogenous leukaemia. suggests that the mechanism of hypercalcaemia 1980;46:1174–8. may be similar to that of hyperkalaemia. 8 Libnoch JA, Ajlorini K, Millman WL, et al. Acute myelofibrosis and malignant hypercalcaemia. Am J Med Various qualitative defects have been described 1977;62:432–8. in the platelets in essential thrombocythaemia. 9 Mitus AJ, Schafer AI. Thrombocytosis and thrombo- cythemia. Hematol Oncol Clin North Am 1990;4:157–78. It is often diYcult to establish whether these 10 Cortelazzo S, Viero P, Barbu T. Platelet activation in myelo- abnormalities arise because of the production proliferative disorders. Thromb Haemost 1981;45:211–18. 11 Luzzatto G, Fabris F, Mazzucato M, et al.PF4 versus âTG of intrinsically abnormal platelets from neo- as evidence for platelet activation in myeloproliferative dis- plastic megakaryocytes, or whether the plate- orders. Scand J Haematol 1985;35:299–304. Hyperkalaemia and hypercalcaemia in thrombocythaemia 109

12 Belluci S, Ignatova E, Jaillet N, et al. Platelet hyperactivation 13 Minder K, Ostermann G, Hochaus A, et al. Blood platelet in patients with essential thrombocythemia is not associ- calcium content and aggregation behaviour in myelopro-

ated with vascular endothelial cell damage as judged by the liferative disorders and secondary thrombocytosis. Folia J Clin Pathol: first published as 10.1136/jcp.53.2.105 on 1 February 2000. Downloaded from level of plasma thrombomodulin, protein S, PAI-1, t-PA Haematol Int Mag Klin Morphol Blutforsch 1988;115: and VWF. Thromb Haemost 1993;70:736–42. 461–4.

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