Journal of Automatic Chemistry, Vol. 13, No. 5 (September-October 1991), pp. 235-239

International Federation of Clinical Chemistry, Scientific Division

IFCC recommendation on sampling,transport and storage for the determinationof the concentration of ionized calcium inwhole blood, plasma and serum

A. B. T. J. Boink (NL), B. M. Buckley (GB), T. F.Christiansen (DK), A. K. Covington (GB), A. H. J.Maas (NL), O. Miiller-Plathe (DE), Ch. Sachs (FR)and O. Siggaard-Andersen (DK)

The substance concentration of ionized calcium (CCa2+) in blood,plasma or serum preanalytically may be affected bypH changes ofthe sample, calcium binding by heparin, and dilution by theanticoagulant solution.

pH changes in whole blood can be minimized by anaerobicsampling to avoid loss of COe, by measuring as soon as possible, orby storing the sample in iced water to avoid lactic acidformation.CCa2+ and pH should be determined simultaneously.

Plasma or serum: If centrifuged in a closed tube, and measuredimmediately, the pH of the sample will be close to the originalvalue. If a delay has occurred between centrifugation and themeasurement, causing substantial loss of COe, equilibration ofthesample with a gas mixture corresponding to Pco2 5"3 kPa priorto the measurement is recommended. Conversion of the measuredvalues to CCa2+ (7.4) is only valid ifthepH is in the range 7"2-7"6.

Ca2+ binding by heparin can be minimized by using either of thefollowing:

(1) A final concentration of sodium or lithium heparinate of 15IU/ml blood or less

(2) Calcium titrated heparin with afinal concentration ofless than50 IU/ml blood.

Dilution effect can be avoided by use ofdry heparin in capillaries or

syringes. When heparin solutions are used, errors due to dilution orcalcium binding can be reduced by using syringes with a heparinsolution containing free calcium ions corresponding to the meanconcentration of ionized calcium in normal plasma.

Conditions for blood collection, storage, and transport to avoidpreanalytical errors are described in this paper.

Introduction

The calcium fractions in plasma are in equilibrium andinclude ionized calcium (free calcium) and boundcalcium (complex-bound calcium and protein-bound

calcium). After sampling, the substance concentration orionized calcium is mainly influenced by pH and suchligands as bicarbonate, lactate, phosphate andanticoagulants.

For clinical interpretation ionized calcium concentrationand pH should be measured simultaneously [1].

The substance concentration of ionized calcium can bemeasured in heparinized blood, heparinized plasma orserum. The reported value should refer to the actual pHof anaerobically sampled blood (cc2+(act)) because thismost closely reflects the in vivo ionized calcium activity inplasma.

Besides the actual cc2+, the ionized calcium concent-ration adjusted to a pH of 7"4 can be calculated(cc2+(7"4)). This eliminates the influence of Pcoinduced alterations of the pH on the concentration ofionized calcium in vivo and in vitro. It should beemphasized that cc2+(7"4) is calculated on the basis ofthe relationship between CCa+ and pH obtained when

Pco varies in vitro. The relationship is almost the same foracute Pco2 changes in vivo. Little is known about theoptimal values of CCa+ or CCa+(7"4) in patients withchronic acidemia or alkalaemia.

The calculation of cc+(7"4) can compensate for theinfluence of preanalytical pH alterations on ionizedcalcium results. For separated plasma or serum,CCa+ (7"4) should be used, because plasma or serum, evenwhen handled anaerobically, tend to give erroneouslyhigh pH values unless separated at 37 C [2].

The determination of CCa2+ is affected by specificpreanalytical factors which may induce changes in thefinal result of the same order of magnitude as physiologi-cal or pathological variations. The main factors are:

(1) Influences of pH on the ionized calciumconcentration.

(2) Calcium binding by the anticoagulant.(3) Sample dilution by the anticoagulant solution.

Correspondence should be addressed to Professor Dr A. H. J. Maas,Zwaardemakerlaan 45, 3571 ZB Utrecht, The Netherlands.

In addition, some special aspects of collection, storage,and choice of sampling technique with regard to theclinical situation have to be considered.

( IFCC 1991235

A. B. T. J. Boink et al. IFCC recommendation on sampling, transport and storage of blood, plasma and serum

Avoidance of pH changes

The main causes ofpH changes in vitro are loss ofCO2 bynon-anaerobic handling and formation of lactic acid byglycolysis in red cells and leukocytes.

Whole blood samples in syringes or capillaries

Loss of CO2 must be minimized by applying the rules ofanaerobic sampling as used in blood gas analysis. Smallamounts ofCO are absorbed by some plastic materials,leading to a gradual increase of pH. Formation of lacticacid can be avoided by minimizing the delay beforemeasurement (<15min). If storage is unavoidable,syringes and capillaries should be kept in iced water. Ofcourse, the ionized calcium concentration is valid only ifpH alterations have been avoided.

Plasma or serum samples in tubOs

The tube should be completely filled with blood andshould remain closed during centrifugation and through-out processing until the measurement is performed. Aftercentrifugation, serum or plasma should be sampled byaspiration from a layer just above the cells [3]. Pouringthe liquid from one tube to another is not suitable [4].

To minimize the effect of lactic acid formation, serum orplasma should be separated from erythrocytes within h:An increase in pH above 7"9 may lead to a decrease ofCca+ by precipitation of calcium phosphate, especially ifelevated phosphate levels are present, as in haemodia-lyzed patients [5], or by formation of calcium carbonate.

or lactic acid [1], or when the albumin concentration isabnormal [9].

Because of the abundance of influences causing differentslopes, and because the relation between lg CCa+ and pHis approximately linear only for a limited pH intervalaround 7"4, these conversions have to be limited to the pHinterval 7"2 to 7"6 of samples with normal albumin/totalprotein levels.

Avoidance of Ca2+-binding by the anticoagulant

At present, the only adequate anticoagulant for ionizedcalcium determinations is heparin. Heparin is known tobind small but significant amounts ofcalcium to an extentwhich depends on the quantity and type ofheparin beingused [7].

Calcium titrated heparinate is the best available means tominimize calcium binding. The heparinate should betitrated to an ionized calcium concentration correspond-ing to the midpoint of the reference range in plasma. Theoriginal CCa+ of the sample will then be modifiedproportionally to the difference between its value and thetitration value. These changes, however, are negligible ifthe final concentration of calcium titrated heparin isbelow 50 IU/ml blood [4].

If heparin preparation with calcium titration are used the finalheparin concentration should not be higher than 15 IU/ml blood. In this case the extent of calcium binding issmall and will not exceed 2%.

If anaerobic handling is impossible, serum or plasmamust be equilibrated immediately prior to measurementwith a gas mixture containing 5"7% CO2 correspondingto Pcoo 5" 3 kPa (these figures are valid for equilibrationat 37 C; equilibration at 25 C requires a gas mixturecontaining: 3"2% CO). The pH induced by thisprocedure should be in the range 7"2-7"6, otherwise theconversion of the measured ionized calcium concen-tration to pH 7’4 is invalid [6].

pH dependent transformation of ionized calcium results

The measured concentration of ionized calcium may beconverted to pH 7"40 by equation (1) [6]:

lg CCa+(7"4) lg Cca2+(x)--S (7.40-pH(x)) (1)

where S is a slope factor and x indicates the measuredvalues. Consequently, the concentration of ionized cal-cium at the actual pH may be estimated from theconverted value by equation (2) for example if the actualpH has been measured independently.

lg Cca+(act) lg CCa2+(7"4) 4- S (7"40-pH(act))(2)

S was originally determined as 0"24 for serum and 0"22 forwhole blood ]. Values of S between 0" 16 and 0"20 havebeen reported with different instruments [7,8]. Thevalues were determined by equilibration with differentCO mixtures. The slope differs if pH is changed by HC1

To ensure proper anticoagulation, careful mixing imme-diately after sampling is necessary, especially when theanticoagulant is in a dry state. Mixing is achieved byrolling syringes between the hand, by moving a mixingwire along the full length of a capillary tube with amagnet, or by inverting tubes repeatedly.

Syringes

Filling a 5 ml syringe with blood up to the nominalvolume and assuming a dead space of 0"2 ml containingcalcium-titrated heparin (1000 IU/ml), a final concen-tration of 40 IU/ml can be expected. (Of course syringesshould always be filled to the specified volume, see section4.)

Capillary tubes

To ensure proper anticoagulation in capillary tubes,higher concentrations of dry heparinate are required.However, the heparinate concentration should not exceed50 IU/ml. With such high concentrations it is necessaryto use calcium titrated heparinate.

Tubes

It should be noted that the final concentration ofheparinshould not exceed 15 IU/ml of blood.

Constituents like fluid separators or clotting activatorsmay only be used if they do not influence the concen-tration or the measurement of ionized calcium 10,11 ].

236

A. B. T.J. Boink et al. IFCC recommendation on sampling, transport and storage of blood, plasma and serum

Avoidance of dilution effects caused by anticoagu-lant solutions

Choice of sampling technique with regard to theclinical situation

Sample dilution by anticoagulant solutions may occurparticularly when syringes are used for sampling, or, lesscommonly, when heparinate solution is added to collec-tion tubes. Glass and plastic syringes are often used foranaerobic blood sampling. The dead space of the syringeand the needle is filled with a heparin solution containingfree calacium ions to an extent corresponding to themidpoint of the reference range for ionized calcium inplasma. Alterations in the original concentration ofionized calcium can thus be minimized.

The dead space ofa 10 ml or 5 ml syringe is 2 to 6% whenthe syringe is filled to their nominal volume. With 2 ml orml syringes, the dilutions may be even greater, and the

effect is enhanced if syringes are filled with less then theirnominal volume [4,12,13]. Disposable syringes contain-ing dry calcium titrated heparinate are preferable.

Site of blood collection

tea2+ can be measured in arterial capillary and venousblood. Arterial and capillary sampling requires the sameprecautions as for pH and blood gas analysis. For venoussampling, it is important to avoid factors modifying thepH value of the specimen. Venous sampling shouldpreferably be done without a tourniquet. If the use of atourniquet is unavoidable, sampling should be completedwith 2 min. Stasis and muscular action like ’pumping’cause changes of the ionized calcium concentration[5,14].

Storage and transport

Storage of whole blood samples

Cca2+(act) and pH(act) should be measured within15 min in samples kept at room temperature, and within 4h in samples stored at 4 C. CCa2+ (7"4) can be determinedwithin 6 h in samples kept at room temperature, andwithin 24 h in samples stored at 4C [5].

Storage of serum or plasma samples

In separated plasma or serum, Cca2+(7’4) should bedetermined (see section 1). Plasma or serum can be storedin plain glass tubes at 4C for week and at -20C for 6weeks without significant changes in Cca2+(7’4), whereasa significant fall (-2’4%) is observed after 4 months [5].

Routine

CCa2+ determinations can usually be performed either inanaerobically handled whole blood samples, or in venousplasma or serum samples handled as described in thepreceding sections. If only CCa+(7"4) could be deter-mined, and if the patient’s pH has been measuredindependently, Cca+(act) can be estimated by calcula-tion according to Section 2.3.

In non-emergency situations, preanalytical factors likevenous stasis, position, nutritive state and diurnalvariation have to be respected. If possible, the bloodcollection should be done between 8 and 10 a.m. on thefasting subject in a supine position using the samplingtechnique as described in section 5.

Urgent situations

In urgent situations (for example requests from intensivecare units, haemodialysis, cardiovascular surgery) theresults have to be reported as quickly as possible. This isensured by working with strictly anaerobically handledwhole blood samples collected in syringes or capillarieswhich require no further sample preparation. In thesecases, both the actual ionized calcium concentration andpH should be reported.

Paediatrics

Because of the limited sample volume, sampling shouldbe done anaerobically in capillary tubes or ml syringescontaining calcium titrated heparinate in a dry state (seesections 2.1 and 3.2). The syringe should be filled to itsnominal volume. The sample has to be mixed carefully,avoiding haemolysis. The measurement should be per-formed on whole blood. This procedure minimizes theeffects ofsample dilution, calcium binding, and CO loss.

Establishing reference intervals

From the preceding paragraphs, it is clear that, to definereference intervals, samples must be collected andhandled under strictly standardized preanalytical con-ditions. Selection and partition factors, kind of specimen(blood, plasma, serum) and site of collection have to betaken into account.

Transport conditions

Only separated serum or plasma kept in closed tubesshould be used for transport exceeding 4 hours. Thesamples may be transported at ambient temperature, butthey should be stored at 4C. The samples should beprocessed within 7 days with measurement of Cca+ andpH after preceding CO2-equilibration (see section 2.2)and subsequent calculation of CCa2+(7"4). Valid resultsfor the actual pH and the actual ionized calciumconcentration ofthe patient cannot be expected ifsampleshave been mailed.

Reports of results

The laboratory report on CCa2+ should contain thefollowing additional information:

(1) Type of sample: blood, plasma or serum.(2) Site of collection: arterial, capillary or venous.(3) Kind of quantity: actual substance concentration of

ionized calcium, substance concentration of ionizedcalcium converted to a pH of 7.4 and actual pH.

237

A. B. T. j. Boink et al. IFCC recommendation on sampling, transport and storage of blood, plasma and serum

Table 1. Types ofspecimen for determination of cca2+.

Ca2+-binding by Dilution bySpecimen anticoagulant anticoagulant Handling Limitations

Whole blood, liquidsodium or lithiumheparinate.Whole blood liquidCa-titrated heparin.

Below 2% as long as Depending upon theheparin conc. is below volume of heparin15 IU/ml blood, solution in a tube or theNegligible for specimens relative dead space of awith normal cca2+, syringe, respectively. In

many cases the deadspace is about 5% of thenominal volume.

Whole blood, dry so- Approx. 5% with a he-dium or lithium hepar- parin conc. of 50 IU/mlinate, blood, which is regarded

Whole blood, dry Ca-titrated heparin.Serum

None

as necessary for safeanti-coagulation with adry preparation.Negligible for specimens Nonewith normal CCa2+.None None

1. Pretreated samplingcontainers necessary,normally syringes orcapillaries.

2. Anaerobic samplingnecessary, arterial,venous or by skinprocedures.

3. To be stored at 4C

If the specimen is sam-pled and handled anaer-obically and stored ap-propriately, and albu-min and total proteinare normal, Cca+(act)and pH(act) can bemeasured accuratelyand CCa:+(7’4) can be

if not measured with- calculated (limited pHin 15 min. range).

1. Sampling and sample Preferably cca+(7"4)handling routineavoiding CO lossesas far as possible.

2. Pre-equilibration re-commended if COloss may haveoccurred.

should be reported. Thevalidity of this quantityis limited in presence ofacid-base disorders and/or in situations wherethe serum proteins,especially albumin aresignificantly abnormal.

References

1. THODE, J., FOGH-ANDERSEN, N., WIMBERLEY, P. D.,MOLLER-SORENSEN, A. and SIGGAARD-ANDERSEN, O., Rela-tion between pH and ionized calcium in vitro and in vivo inman. Scandinavian Journal of Clinical Laboratory Investigation,43, Suppl. 165 (1983), 79-82.

2. SIGGAARD-ANDERSEN, O., The Acid-Base Status of the Blood,4th revised edition. Munksgaard, Copenhagen (1974),149-154.

3. GRAHAM, G. A. and JOHNSON, L. A., The stability of serumionized calcium during sampling handling and analysis. In:Methodology and Clinical Applications of Ion-Selective Electrodes.Vol. 10, Mass, A. H.J., Buckley, B, Manzoni, A., Moran,R. F., Siggaard-Andersen, O. and Sprokholt, R. (Eds).Elinkwijk, Utrecht, The Netherlands (1979), 55.

4. SACHS, CH. and THODE, J., Preanalytical errors in ionizedcalcium determination in blood: A review for a subsequentrecommendation for blood sampling. In: Methodology andClinical Applications of Ion Selective Electrodes, Vol. 7, Maas,A. H.J., Boink, A. B. T. J., Saris, N.-E. L., Sprokholt, R.and Wimberley, P. D. (Eds). Radiometer, Copenhagen(1986), 259-270.

5. THODE, J., FOGs-ANDERSON, N., AAS, F. and SIGGAARD-ANDERSEN, O., Sampling and storage of blood determi-nation of ionized calcium. Scandinavian Journal of ClinicalLaboratory Investigation, 45 (1985), 131-138.

6. SIGGAARD-ANDERSEN, O., THODE, J. and WANDRUP, J., Theconcentration of free calcium ions in the blood plasma:’ionized calcium’. In: Blood pH, Carbon Dioxide, Oxygen andCalcium-ion. Siggaard-Andersen, O. (Ed.). Private Press,Copenhagen (1981), 163-190.

7. SACHS, CH., CHANEAC, M., RABOUINE, PH. and KINDER-MANS, C., Effect of calcium ligands on the pH standardiz-ation of ionized calcium measurements in blood: a theoreti-cal approach. In: Methodology and Clinical Applications ofIon-Selective Electrodes. Vol. 9, Burritt, M. F., Cormier, A. D.,Mass, A. H.J., Moran, R. F. and O’Connell, K. M. (Eds).Davies Printing Company, Rochester (1988), 228-233.

8. RAWSON, K. and VERGHESE, D. A. Personal com-munication.

9. BUCKLEY, B. M., RAWSON, K., RUSSEL, L. J. and SMITH,S. C. H., The influence of protein concentration on themathematical adjustment ofionized calcium measurementsfor pH. In: Methodology and Clinical Applications ofIon-SelectiveElectrodes. Vol. 9, Burritt, M. F., Cormier, A. D., Mass,A. H.J., Moran, R. F. and O’Connell, K. M. (Eds). DaviesPrinting Company, Rochester (1988), 121-130.

10. LARSSON L. and OHMAN ST., Effect of silicon-separatortubes and storage time on ionized calcium in serum. ClinicalChemistry, 31 (1985), 169-170.

11. TOFFALETTI, J., BLOSSER, N. and KIRVAN, K., Effects ofstorage temperature and time before centrifugation onionized calcium in blood collected in plain tubes andsilicon-separator (SST) Tubes. Clinical Chemistry, 30 (1984),553-556.

12. SACHS, CH., RABOUINE, PH., KINDERMANS, C. and SAGE, C.Preanalytical dilution errors in plasma and whole bloodionized calcium determinations on small sized samples. In:Methodology and Clinical Applications of Ion-Selective Electrodes,Vol. 8, Maas, A. H. J., Buckley, B., Marsoner, H., Saris,N.-E. L. and Sprokholt, R. (Eds). Radiometer, Copen-hagen (1987), 117-123.

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13. SACHS, CH., RABOUINE, PH., CHANEAC, M. and KINDER-MANS, C. Preanalytical dilution errors in ionized calcium onplasma and whole blood. Part II. Effect ofspecimen volumeto syringe nominal volume ratio. In: Methodology and ClinicalApplications of Ion-Selective Electrodes, Vol. 9, Burritt, M. F.,Cormier, A. D., Maas, A. H. J., Moran, R. F. and

O’Connell, K. M. (Eds). Davies Printing Company,Rochester (1988), 108-113.

14. RENOE, B. W., McDONALD, J. M. and LADENSON, J. H., The,effect of stasis with and without exercise on free calcium,various cations and related parameters. Clinical ChimicaActa, 103 (1980), 91-100.

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