This document discusses factors that can affect biochemical test results, including pre-analytical, analytical, and post-analytical errors. It describes a case where inappropriate collection of a blood sample into a potassium fluoride tube caused erroneous results for electrolyte and glucose tests. Using the wrong collection tube can lead to pre-analytical errors from chemical interference. The document also covers reference ranges, diagnostic accuracy, and how various lifestyle and medical conditions can influence biochemical markers.

1. CLINICAL BIOCHEMISTRY
By: Neli Syahida Ni'ma

2. BIOLOGIC SAMPLE
Whole
blood
Serum
Plasm
a Urine
Saliva
Feces
Spinal Synovial

3. Collecting Specimens
• Most biochemical analyses are made on serum or plasma but
occasionally whole blood is required (e.g. for ‘blood gases’).
• Haemolysis must be avoided when blood is drawn & sample is
processed → affect result (e.g. increases in plasma potassium and
phosphate concentrations and aspartate aminotransferase activity,
owing to leakage from red cells)
• Collecting in an appropriate container !!! → Collecting a blood
specimen into the wrong container can lead to erroneous results

4. CONTAINER

5. TYPES OF ANTICOAGULANT
Heparin
• most widely used
• unacceptable for polymerase chain reaction
(PCR) because inhibition of the polymerase
enzyme by this large molecule
EDTA
• Chelating agent of divalent cations (Ca2+ and
Mg2+)
• Preserves the cellular components of blood →
particularly useful for hematologic
examinations, isolation genomic DNA,
qualitative and quantitative virus
determinations by molecular techniques
Na-Citrate
• Citrate tube is rarely acceptable or
chemistry testing but may be
acceptable, usually used for genomic
DNA isolation
Sodium/Potassium Fluoride
• Weak anticoagulant that is often
added as preservative for blood
glucose analyses.
• Often used together with another
anticoagulant (such as potassium
oxalate)

6. Case
• The laboratory staff were concerned when a serum specimen from an
outpatient due to attend the diabetic clinic was analysed and the following
results were found:
• Investigation was conducted and disclosed that phlebotomist who had
taken the blood had collected the original specimen into a tube containing
(potassium) fluoride and oxalate, the correct container for an accurate
blood glucose measurement, but had then concealed his error by
transferring the sample to a plain tube.
• Explain this case, why inappropriate tube can cause erroneous result!
Potassium 12.2 mmol/L
Sodium 140 mmol/L
Creatinine 84 μmol/L
Calcium 0.34 mmol/L
Phosphate 1.22 mmol/L

7. ANALYTICAL METHOD
Must be accurate, precise, specific, sensitive
The imprecision of the analysis can be expressed as the CV,
where CV =
𝑆𝐷
𝑚𝑒𝑎𝑛
𝑥 100

8. ERRORS
Pre-analytical: wrong specimen, mislabelling,
incorrect preservation
Analytical: human or instrumental error
Post-analytical: transcription error

9. Factors affecting
biochemical result

10. Factors affecting biochemical result

11. Environment
• Altitude → blood hemoglobin and hematocrit increased because of
reduced atmospheric PO2
• People living in areas with hard water → statistically significant
increase in serum concentrations of cholesterol, TG, and Mg.
• Individuals who primarily work indoors → lower concentrations of
25-hydroxy vitamin D, → higher serum calcium concentrations and
greater urinary excretion o calcium.

12. Underlying Medical Condition
• Anxiety → stimulates increased secretion aldosterone, angiotensin,
catecholamines, cortisol, prolactin, renin, somatotropin, TSH, and
vasopressin. Plasma concentrations of albumin, cholesterol, fibrinogen,
glucose, insulin, and lactate also increase
• Shock and Trauma → corticotropin secretion is stimulated to produce 3-
5x increase in the serum cortisol concentration
• Pregnancy →
• Blood volume increase from about 2600 mL early in pregnancy to
3500 mL at about 35 weeks
• Deficiency of iron and ferritin.
• Blindness → In some blind individuals, the normal diurnal variation of
cortisol may or may not persist

13. Lifestyle
• Recreational drug →
• Morphine increases the activity of amylase, lipase, ALT, AST, ALP,
bilirubin, gastrin, TSH, and prolactin.
• Morphine decreases concentrations of insulin, norepinephrine,
pancreatic polypeptide, and neurotensin.
• Nicotine →
• Increases concentration of epinephrine plasma and the urinary
excretion of catecholamines and their metabolites.
• Increase glucose concentration by 10 mg/dL (0.56 mmol/L) within 10
minutes of smoking a cigarette.
• Affects the body’s immune response (serum IgA, IgG, and IgM levels
are lower in smokers, whereas the IgE concentration is higher)
• Sperm count reduced, abnormal forms of sperm is greater, and
sperm motility is less → fertility decreased

14. Lifestyle
• Alcohol →
• Ingestion of enough alcohol may increase the blood glucose
concentration by 20% to 50%.
• Chronic alcohol ingestion increased activity o γ-glutamyltransferase
(GGT) → often used as a marker o persistent drinking.
• Chronic alcoholism: abnormal pituitary, adrenocortical, medullary
function.
• Chronic alcoholism increased mean cell volume (MCV) : caused by
decreased folic acid or direct toxicity alcohol on RBC precursor
• Diet →
• Fasting: decreases the variability in the concentrations of many
analytes → to standardize blood collections, particularly lipids
• Vegetarian: concentrations of cholesterol and triglycerides may be
only 2/3 of people with mixed diet
• Malnutrition → total serum protein, albumin, β-globulin concentrations
are reduced

15. Interpretation

16. REFERENCE VALUE OR NORMAL VALUE?
What is
normal
anyway?
• The range of the mean ± 2 SD = 95.5% → considered ‘normal’
• The range of the mean ± 3 SD = 99.7% of the total number
Many biological variation follow normal distribution curve
Non normal distribution?
Healthy
Not healthy
Reference value,
not normal value

17. CHOLESTEROL PROFILE
Mabuchi et al., 2005, Cutoff Point Separating
Affected and Unaffected Familial
Hypercholesterolemic Patients

18. SIGNIFICANT CHANGES
Between measurement there will always be variation: analytical
variation & biological variation. So, how to differ whether different
result is due to variation or significant changes?
SD = 𝑆𝐷𝐴
2
+ 𝑆𝐷𝐵
2 In which
SDA = standard deviation of analytical
SDB = standard deviation of biological
If the difference between two test results > 2.8 X SD, the difference
can be regarded as of potential clinical significance: the
probability of this difference being a result of analytical and
biological variation is <0.05

19. CLINICAL SENSITIVITY & SPECIFITY
TEST RESULT
Positive Negative
REALITY
Positive
True Positive (TP) False Negative (FN)
Negative
False Positive (FP) True Negative (TN)
Ideal diagnostic test would be 100% sensitive and 100% specific. But practically, it’s impossible!! Factors that increase
specificity tend to decrease the sensitivity, and vice versa
Upper limit range plasma free
thyroxine = 26 pmol/L
To diagnose hyperthyroidism
If set > 32 pmol/L →
specificity 100%, sensitivity
low (many false negative)
If set > 20 pmol/L →
sensitivity 100%, specificity
low (many false positive)

20. Win-win solution:
𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦 =
𝑇𝑃 + 𝑇𝑁
𝑇𝑜𝑡𝑎𝑙 𝑡𝑒𝑠𝑡
𝑥 100%

21. PREDICTIVE VALUES
• To answer: given a positive result, what is the likelihood that a patient actually has the disease ?
• Predictive value combines disease prevalence with the test’s sensitivity and specificity.

22. Thank you