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8 Special Issues: Blood Gas and Ionized Calcium Analysis

8 Special Issues: Blood Gas and Ionized Calcium Analysis

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P r e -A na ly t ica l V ar i ab le s

Therefore, specimens sent to the lab for ionized calcium determinations

should be handled with the same caution as other blood gas samples since

pre-analytical errors in pH will impact ionized calcium results [7].


Errors in the clinical laboratory can occur in pre-analytical, analytical, or postanalytical steps. Most errors (almost two-thirds of all errors) occur in pre-analytical


During specimen collection, a patient must be identified by matching at least two

criteria. Blood should be collected in the correct tube following the correct order

of draw.

Correct order of drawing blood: (1) microbiological blood culture tubes (yellow

top), (2) royal blue tube (no additive) if trace metal analysis is desired, (3) citrate

tube (light blue), (4) serum tube (red top) or tube with gel separator/clot activator

(gold top or tiger top), (5) heparin tube (green top), (6) EDTA tube (purple/lavender

top), and (7) oxalate-fluoride tube (gray top).

Proper centrifugation (in the case of analyzing serum or plasma specimens) and

proper transportation of the specimen to the laboratory are required, as well as

maintaining proper storage of the specimen prior to analysis in order to avoid

artifactual changes in the analyte.

EDTA (purple top tube) is the anticoagulant of choice for the complete blood

count (CBC). The EDTA tube is also used for blood bank pre-transfusion testing,

flow cytometry, hemoglobin A1C, and most common immunosuppressive drugs

such as cyclosporine, tacrolimus, sirolimus, and everolimus; another

immunosuppressant, mycophenolic acid, is measured in serum or plasma instead

of whole blood.

Heparin (green top tube) is the only anticoagulant recommended for the

determination of pH blood gases, electrolytes, and ionized calcium. Lithium

heparin is commonly used instead of sodium heparin for general chemistry tests.

Heparin is not recommended for protein electrophoresis and cryoglobulin testing

because of the presence of fibrinogen, which co-migrates with beta-2 monoclonal


For coagulation testing, citrate (light blue top) is the appropriate anticoagulant.

Potassium oxalate is used in combination with sodium fluoride and sodium

iodoacetate to inhibit enzymes involved in the glycolytic pathway. Therefore the

oxalate/fluoride (gray top) tube should be used for collecting specimens for

measuring glucose level.

Ideally, all blood gas specimens should be measured immediately and never

stored. A plastic syringe, transported at room temperature, is recommended if

analysis will occur within 30 minutes of collection. Otherwise, a specimen must be

stored in ice. Glass syringes are recommended for delayed analysis because glass


does not allow the diffusion of oxygen or carbon dioxide. Bubbles must be

completely expelled from the specimen prior to transport, as the pO2 will be

significantly increased and pCO2 decreased within 2 minutes.


[1] Carraro P, Plebani M. Errors in STAT laboratory; types and frequency 10 years later. Clin

Chem 2007;53:1338À42.

[2] Murphy JE, Ward ES. Elevated phenytoin concentration caused by sampling through the

drug-administered line. Pharmacotherapy 1991;11:348À350.

[3] Dunn EJ, Morga PJ. Patient misidentification in laboratory medicine: a qualitative analysis of

227 root cause analysis reports in the Veteran Administration. Arch Pathol Lab Med


[4] Aleccia J. Patients still stuck with bill for medical errors. 2008 2/29/2008 8:26:51 AM ET

[cited 2012 06/28/2012]; Available from: ,http://www.msnbc.msn.com/id/23341360/ns/


[5] Lee DC, Klachko MN. Falsely elevated lithium levels in plasma samples obtained in lithium

containing tubes. J Toxicol Clin Toxicol 1996;34:467À9.

[6] Knowles TP, Mullin RA, Hunter JA, Douce FH. Effects of syringe material, sample storage

time, and temperature on blood gases and oxygen saturation in arterialized human blood

samples. Respir Care 2006;51:732À6.

[7] Toffaletti J, Blosser N, Kirvan K. Effects of storage temperature and time before centrifugation

on ionized calcium in blood collected in plain vacutainer tubes and silicone-separator (SST)

tubes. Clin Chem 1984;30(4):553À6.



Laboratory Statistics and Quality Control




In an ideal situation, when measuring a value of the analyte in a specimen,

the same value should be produced over and over again. However, in reality,

the same value is not produced by the instrument, but a similar value is

observed. Therefore, the most basic statistical operation is to calculate the

mean and standard deviation, and then to determine the coefficient of variation (CV). Mean value is defined as Equation 4.1:

Mean ðXÞ 5

X 1 1 X 2 1 X 3 1 ?? 1 Xn



Here, X1, X2, X3, etc., are individual values and “n” is the number of values.

After calculation of the mean value, standard deviation (SD) of the sample

can be easily determined using the following formula (Equation 4.2):



SD 5

ðx1 2xÞ2



Here, X1 is the individual value from the sample and n is again the number

of observations.

Standard deviation represents the average deviation of an individual value

from the mean value. The smaller the standard deviation, the better the precision of the measurement. Standard deviation is the square root of variance.

Variance indicates deviation of a sample observation from the mean of all

values and is expressed as sigma. Therefore (Equation 4.3):

σ 5 OSD

A. Dasgupta and A. Wahed: Clinical Chemistry, Immunology and Laboratory Quality Control

DOI: http://dx.doi.org/10.1016/B978-0-12-407821-5.00004-8

© 2014 Elsevier Inc. All rights reserved.


4.1 Mean, Standard

Deviation, and

Coefficient of

Variation................ 47

4.2 Precision and

Accuracy ............... 48

4.3 Gaussian

Distribution and

Reference Range .. 48

4.4 Sensitivity,

Specificity, and


Value...................... 50

4.5 Random and

Systematic Errors in

Measurements...... 51

4.6 Laboratory

Quality Control:

Internal and

External ................. 52

4.7 LeveyÀJennings

Chart and Westgard

Rules ...................... 54

4.8 Delta Checks.. 56

4.9 Method


Evaluation of a

New Method ......... 58

4.10 How to

Interpret the


Equation? .............. 59




Laboratory Statistics and Quality Control

4.11 BlandÀAltman

Plot ......................... 60

4.12 ReceiverÀ

Operator Curve..... 60

4.13 What is Six

Sigma? ................... 61

4.14 Errors

Associated with


Range .................... 62

4.15 Basic Statistical

Analysis: Student

t-Test and Related

Tests ...................... 63

Coefficient of variation is also a very important parameter because CV can be

easily expressed as a percent value; the lower the CV, the better the precision

for the measurement. The advantage of CV is that one number can be used

to express precision instead of stating both mean value and standard deviation. CV can be easily calculated with Equation 4.4:

CV 5 SD=Mean 3 100


Sometimes standard error of mean is also calculated (Equation 4.5).

Standard error of mean 5 SD=On


Here, n is the number of data points in the set.

Key Points ............. 63

References ............ 66


Precision is a measure of how reproducible values are in a series of measurements, while accuracy indicates how close a determined value is to

the target values. Accuracy can be determined for a particular test by analysis of an assayed control where the target value is known. This is typically provided by the manufacturer or made in-house by accurately

measuring a predetermined amount of analyte and then dissolving it in a

predetermined amount of a solvent matrix where the matrix is similar to

plasma. An ideal assay has both excellent precision and accuracy, but

good precision of an assay may not always guarantee good accuracy.



Gaussian distribution (also known as normal distribution) is a bellshaped curve, and it is assumed that during any measurement values will

follow a normal distribution with an equal number of measurements

above and below the mean value. In order to understand normal distribution, it is important to know the definitions of “mean,” “median,” and

“mode.” The “mean” is the calculated average of all values, the “median”

is the value at the center point (mid-point) of the distribution, while the

“mode” is the value that was observed most frequently during the measurement. If a distribution is normal, then the values of the mean,

median, and mode are the same. However, the value of the mean,

median, and mode may be different if the distribution is skewed (not

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8 Special Issues: Blood Gas and Ionized Calcium Analysis

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