Pre analytical variables affecting laboratory results

PRE-ANALYTICAL
VARIABLES AFFECTING
LABORATORY RESULTS
DR. OFONMBUK UMOH1

OUTLINE
INTRODUCTION
CLASSIFICATION OF PRE-ANALYTICAL VARIABLES
OVERVIEW OF VARIABLES AND THEIR EFFECTS
CONCLUSION
REFERENCES
2

INTRODUCTION
Laboratory services play a pivotal role of clinical decision
making process. Effective laboratory service is the
amalgamation of precision, accuracy and promptness.
Recently, Johns Hopkins University School of Medicine in
Baltimore made headlines when they estimated that medical
error is the third leading cause of death in the United States.
While patient safety remains a struggle in many areas of
healthcare, laboratory medicine has been a leader in
reducing error, with an estimated total error rate of 0.33%,
the lowest in diagnostic medicine.
3

…introduction
The classic paradigm of Total Testing Process
beginning with Ordering of test to Reporting of
test results, encompasses the Pre-analytical,
Analytical and Post-analytical phases of testing.
Of the laboratory-associated errors, pre-analytical
errors currently accounts for up to 75% of all
mistakes.
While the likelihood of variation in any of these three phases is
not negligible, the vast majority of laboratory variation
emerges from the many factors affecting laboratory specimens
prior to testing.
4

…introduction
Sadly, since most activities in the pre-analytical
phase are neither performed entirely in the
clinical laboratory nor under the control of
laboratory personnel, they are harder to monitor
and improve.
Besides, most laboratories often leave pre-
analytical activities to healthcare personnel who
have little to no formal training in laboratory
medical practice.
5

CLASSIFICATION OF PRE-ANALYTICAL
VARIABLES
Pre-analytical processes includes all the steps
that occur from test ordering until right
before sample analysis.
Pre-analytical variables is classified into;
Controllable factors and
Non-controllable factors
6

CONTROLLABLE FACTORS
REQUEST & TEST SAMPLE FACTORS
PHYSIOLOGICAL FACTORS
POSTURE/RECUMBENCY
CIRCADIAN VARIATIONS
LIFESTYLE FACTORS
FOOD & FASTING STATE
HERBAL PREPARATIONS
RECREATIONAL SUBSTANCES
PHYSCAL FITNESS, EXERCSE
AND TRAINING
TRAVELS
DIET
STIMULANTS
DRUGS
7

NON-CONTROLLABLE FACTORS
AGE
SEX
RACE
ENVIRONMENTAL
MEDICAL CONDITIONS / COMORBIDITY
8

REQUEST & TEST SAMPLE FACTORS include:
Test ordering,
Patient identification
Patient preparation
Site Selection & Preparation
Tourniquet Application & Time
Venipuncture Technique…
Use of appropriate sample bottles
Order of draw
9

Correct Specimen Volume
Proper sample Handling and Specimen Processing
Serum or Plasma Samples ?
Centrifugation
Proper handling of blood samples
Stability and storage for Whole
Blood, Serum and Plasma
10

Test ordering
Test requisition forms should contain a minimum of
patient’s name, Age, Sex, Hospital Number,
Location of patient, Clinical diagnosis and brief
clinical information, if necessary.
Ordering of the wrong test. This could be due to:
Confusion over tests with similar names (such as 25-
hydroxyvitamin D versus 1,25-dihydroxyvitamin D),
Transcription errors and misinterpreted verbal orders,
which occur when physicians do not place test orders
themselves.
11

Patient identification
Identification bracelet for in-patients,
Positive Vocal identification (spell their name
correctly) from the conscious & alert,
or identification by staff/family member; for the
unconscious.
Label sample collection tubes with a minimum of
Patients full name, proposed test, Date/time of
collection, and the Laboratory processing serial
number.
12

Patient preparation
Patient should be informed, and given a brief
education on the test to be performed.
If pertinent for the test, it should be verified that the
patient is fasting.
Ideally, a patient should’ve remained in the same
position for at least 30mins before a specimen is
collected, and should be in the same likely position
for the next similar specimen, e.g. supine if an
inpatient, or sitting if outpatient.
13

Site selection
Ante cubital and median cubital veins are the most
suitable for use in a conscious patient.
Basilic, cephalic or dorsal dorsum veins of the hands
should be secondary sites for consideration – as they are
prone to frequent trials and errors, thereby increasing
stress and tourniquet time.
Swab with alcohol – based antiseptic in cotton wool or
gauze and allow to air-dry.
Using the same / similar vascular site for serial sample is
advised. Minor difference occurs from use of different
vascular location.
14

Tourniquet time
Tourniquet – while used to dam venous pool to facilitate
easy location of a vein for venipuncture, application for
longer than 1min begins to induce hemoconcentration. It
mimics the effect of change from lying to standing
position.
Venipuncture technique
Appropriately sized needles should be used, to lessen the
possibility of hemolysis. Aim at successful venipuncture
and blood draw with at most 2 attempts.
No puncturing of skin before the alcohol used for skin
asepsis has evaporated.
15

Correct sample bottles & sample volume
Use of appropriate sample bottle as indicated for the
test to be carried out.
Collect adequate sample quantity as labelled on tube,
unless in peculiar situations, e.g. neonates.
Order of Draw
Blood Culture
Serum tubes
Heparin tubes
EDTA tubes
Fluoride oxalate tubes
16

Proper sample handling and specimen processing
No vigorous shaking with blood sample, to avoid massive
hemolysis. Gentle rocking of the sample tube to mix, for
anticoagulant tubes. (…)
Sample collected into one type of tube should never be
transferred into another tube.
If blood collection site is distant from the laboratory,
specimen should be collected into evacuated tubes
containing thixotropic polymer gel and should be
centrifuged on site. The gel forms an effective barrier
between the separated serum / plasma and cells, so that
no leakage of cellular constituents into the supernatant
above the gel.
17

Serum or Plasma ?
Generally, plasma allows for more rapid processing of
specimen for clinical chemistry tests. But anticoagulants
may interfere with some analytical methods. E.g. K+ and
phosphate.
Also, note that the type of urine specimen needed for
different tests can be quite different. For e.g. the first
morning urine specimen is usually the most
concentrated, and appropriate for microscopic
examination, while a collection of 24hr urine is
appropriate for quantitative measurement.
18

Storage
Within the laboratory, if specimen cannot be
tested timely, it should be stored under
appropriate conditions until testing can take
place.
For example, whole blood sample collected
into Fluoride – Oxalate bottle and separated
as soon as possible can last as long as 3 days
at room temperature, or up to 21 days when
frozen.
19

Prolonged bed rest:
Prolonged bed rest can dramatically affects body constituents.
Plasma volume and ECF volume decrease within few days
Hematocrit increase by up to 10%. Creatine kinase also increases
from muscle release.
There is reduction in plasma protein concentration. Protein-
bound constituents decreases.
Serum potassium, reduce by 0.5mmol/L. Hydrogen ion excretion
decreases – (!)Skeletal muscle metabolism.
PHYSIOLOGICAL FACTORS
20

Calcium: Hypercalcemia, hypercalciuria, from bone
resorption.
The amplitude of circadian variation of cortisol is
reduced with prolonged immobilization.
As a result, some hospitalized patients should delay
certain tests until after they leave the hospital and
resume normal activity.
When an individual becomes active after a period of
bedrest, longer than 3 weeks is required before calcium
balance is achieved.
21
…Prolonged bed rest:

10% reduction in blood volume, equivalent to
approximately 600-700mls.
Resultant increase in plasma proteins by approx. 8-10%.
5 – 15% increase in concentration of protein-bound
molecules
Increase secretion of catecholamines, aldosterone,
angiotensin II, Renin and ADH (Vasopressin).
Increase in Heart Rate, Systolic & Diastolic blood
pressures
Posture / Recumbency
In an adult, a change from lying to an upright position, results in:
10mins to happen &
30mins to revert back, on
switching posture.
22

Decrease in GFR…, Decrease in urinary pH
Reduced excretion of bicarbonate as Hydrogen
ions are exchanged for Sodium.
Increase in K+: 0.2 – 0.3 mmol/L
Increases in: Calcium 3%, Cholesterol 7%, TG 6%,
IgG 7%, IgA 7%, IgM 5%, Amylase 6% ALP 7%,
ALT 7%, AST 5%, Albumin 9%, Thyroxine 11%
23

Exercise:
Blood pressure, increase, Arterial pH is reduced
Blood glucose, increased by up to 2folds (M), but
hypoglycemia in prolonged strenuous exercise
Lactate, urates, increase approx. 2folds,
Strenuous exercise: increases plasma renin by 400%
Cortisol secretion is stimulated, the normal diurnal variation
may be abolished. Urinary free cortisol excretion and plasma
concentration of cortisol, aldosterone, growth hormone and
prolactin are increased.
24

Circadian variation:
Cortisol: highest around 0600 and 0800 hours
Renin & Aldosterone: maximum renin activity occurs in
the morning during sleep. Minimum in the afternoon.
Plasma aldosterone concentration shows a similar
pattern.
Growth Hormone: secretion is increased threefold to
fivefold from its minimum around afternoon & night, to
its maximum between midnight and waking.
Thyroid Stimulating Hormone: TSH is maximum
between 0200 and 0400, and minimum between 1800
and 2200…varying between 50 – 200%
26

LIFESTYLE
 Plasma urea conc. doubles, within 4days of change from a normal
diet to a high protein diet, along with increase in urinary excretion.
Serum cholesterol, phosphate, urates and NH3 conc also increase
concomitantly.
 A high-fat diet, increases serum TG concentration, but depletes the
nitrogen pool because of Nitrogen requirement for excretion of
ammonium ions to maintain acid-base homeostasis.
 A high-carbohydrate diet decreases the serum concentrations of LDL-
cholesterol, triglycerides, cholesterol, and protein
 The effects of a meal may be long lasting. For e.g. ingestion of a
protein-rich meal in the evening may cause increases in concentration
of serum urea nitrogen, phosphorus, and urate that are still apparent
12 hours later.
Diet:27

Food & Fasting state:
Food ingestion is a significant source of pre-analytical
variability. This effect varies based on the analyte and the time
between meal ingestion and blood collection. For e.g, glucose
and TGs significantly increase after meals with high
carbohydrates and fat, respectively. An overnight fasting
period of 10 to 14 hours prior to blood collection is optimal for
minimizing variations.
Glucagon and insulin secretions are stimulated by a protein
meal, and insulin is also stimulated by carbohydrate meals. In
response to a meal, the stomach secretes hydrochloric acid,
causing a reduction in the plasma chloride concentration.
28

Venous blood from the stomach contains an increased
amount of bicarbonate. This condition reflects a mild
metabolic alkalosis (“alkaline tide”) and increased PCO2.
The metabolic alkalosis is sufficient to reduce serum-free
ionized calcium by 0.2 mg/dL (0.05 mmol/L).
Caffeine ingestion stimulates adrenal medulla:
increases plasma epinephrine by 2-3folds,
increases plasma cortisol: free cortisol,, so much so great that
the normal diurnal variation of plasma cortisol may be
suppressed.
Ingestion of two cup of coffee may increase the plasma free
fatty acid concentration, glycerol, total lipids and lipoproteins by
as much as 30%.
29
…Food & Fasting state

Malnutrition:
 Plasma protein – reduced. Complement C3, retinol binding globulin, prealbumin
and transferrin decrease more rapidly.
 Plasma cortisol – increased, from free cortisol moiety, and decreased metabolic
clearance.
 T3, T4 – reduced because of reduced TBG and Prealbumin.
Vegetarianism
 Total lipids and phospolipids concentration – reduced to 2/3rd of normal diet.
 In strict vegetarians, the LDL-cholesterol concentration may be 37% less and the
HDL-cholesterol concentration 12% less than in nonvegetarians. The cholesterol :
HDL-cholesterol ratio is decreased.
 Urinary pH is usually higher in vegetarians than in meat-eaters
 Vitamin B12 & folate – reduced.
30

Travel
Travel across several time zones affects the normal circadian rhythm.
Five days is required to establish a new stable diurnal rhythm after
travel across 10 time zones.
Changes in laboratory test results are attributable to altered pituitary
and adrenal function.
Urinary excretion of catecholamines – increased for 2days.
Serum cortisol – reduced.
During a 20-hour flight, serum glucose and TG concentration
increases, while glucocorticoids is stimulated.
During a prolonged flight, fluid and sodium retention occurs, but
urinary excretion return to normal after 2days.
31

Alcohol:
Glucose metabolism: inebriation level induces transient
hyperglycemia (20-50%) that inhibits gluconeogenesis,
then results in hypoglycemia and ketonemia, as alcohol
gets metabolized to acetaldehyde and acetate
High lipids, as Liver TG formation is increased (up to
0.23mmol/L), and there is impaired removal of VLDL
& Chylomicrons from circulation.
AST and ALT activities – may be increased by 250% and
60% respectively.
GGT, Cortisol, Lactate & Urate – all increased.
33

Drugs:
Opiates – increased pancreatic enzymes
Oral contraceptives – reduces HDL & increases LDL
Thiazides - Hypokalemia, hypercalcemia, Hyperglycemia,
hyperuricemia, prerenal azotemia, increased LDL-Cholesterol, total
cholesterol and TGs
Phenytoin – Reduces calcium and phosphate concentration (with
risk of osteomalacia), increases ALP activity and induces synthesis of
Bilirubin conjugating enzymes in the liver. Also cause hyperglycemia
and glycosuria.
Barbiturates induce the hepatic cytochrome P450 enzyme system,
which could interfere with the metabolism and subsequent plasma
level of co-administered drugs.
34

NON-CONTROLLABLE FACTORS
 After puberty, the serum activities of ALP, ALT, AST, CK, and
aldolase are greater in men than in women.
 Serum iron is low during a woman’s fertile years, and her plasma
ferritin may be only one third of the concentration in men.
 Total cholesterol and LDL-cholesterol concentrations are typically
higher in men than in women.
 Creatinine clearance is greater in men than in women.
Sex:
35

Age:
Newborn and jaundice: In infants, even in the absence
of disease, the concentration of bilirubin rises after
birth because of enhanced erythrocyte destruction.
Newborn and low Blood glucose:
RBC count in childhood lower than in adults.
Childhood and Plasma Proteins.
36

Race:
Total Proteins & Albumin: total serum protein
concentration is known to be higher in blacks than in
whites. This is largely attributable to a much higher γ-
globulin. However, Serum albumin is typically less in
blacks than in whites.
In black men, serum IgG is often 40% higher and serum
IgA may be as much as 20% higher than in white men
Hemoglobin concentration is lower in blacks
The activity of CK and LD is usually much higher in both
black men and women than in whites.
37

Environmental:
Altitude and Hemoglobin/blood gases:
In individuals living at a high altitude, blood hemoglobin
and hematocrit are greatly increased because of reduced
atmospheric PO2. Erythrocyte 2,3-diphosphoglycerate is also
increased, and the oxygen dissociation curve is shifted to the
right….with increased erythrocyte production
Plasma and urine concentrations of catecholamine are
increased with increased altitude, up to twofold, largely
caused by increase in norepinephrine secretion. Urinary
Creatinine concentration and clearance are decreased as a
result.
38

Medical conditions:
Obesity: Serum concentrations of cholesterol,
triglycerides, and β-lipoproteins are positively correlated
with obesity. HDL-C reduces, LDL-C increases, Cortisol
increases. Increased plasma concentration of acute
phase reactants. Serum iron and transferrin
concentrations are low.
Pregnancy: Blood volume increases by about 2600 –
3500ml. Total protein reduces, mostly Albumin.
Cholesterol and TG increases. Urine volume increases by
up to 25%. Increased fibrinogen concentration.
39

 Stress: Anxiety stimulates increased secretion of
aldosterone, angiotensin, catecholamines, cortisol,
prolactin, renin, growth hormone, TSH, and antidiuretic
hormone. Plasma cholesterol, fibrinogen, glucose, insulin
and lactate are increased by a large degree Plasma
concentrations of albumin are decreased by up to 5%.
Fever provokes lipolysis and glycogenolysis with
hyperglycemia, which stimulates insulin secretion. Fever is
often associated with a respiratory alkalosis caused by
hyperventilation.
40

CONCLUSION
Major advancements in methodologies, automation
and analytical instrumentation have helped increase
accuracy over the last decade.
To help reduce pre-analytical errors, vis-a-vis
laboratory-associated errors, laboratory medicine
professionals should also expand their focus to what is
happening outside of the laboratory that can affect the
outcome of test results.
42

REFERENCES
 Tietz Clinical Chemistry and Molecular Diagnostics, 5th Ed
 Bishops Clinical Chemistry – Principles, Techniques, Correlations, 7th Ed.
 Makary MA, Daniel M. Medical error-the third leading cause of death in the us. BMJ
2016;353:i2139.
 Carraro P, Plebani M. Errors in a stat laboratory: Types and frequencies 10 years later.
Clin Chem 2007;53:1338-42.
 Bonini P, Plebani M, Ceriotti F, Rubboli F. Errors in laboratory medicine. Clin Chem
2002;48:691-8.
 Standardization IOf. Iso 15189:2012: Medical laboratories: Particular requirements for
quality and competence. Vol. Geneva, Switzerland, 2012.
 Young DS. Effects of preanalytical variables on clinical laboratory tests. 3rd ed.
Washington, DC: AACCPress, 2007.
44

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