Quality Lectures - Pre-Analytic Variables - Dr. Tamer Soliman
Quality Lectures - Pre-Analytic Variables - Dr. Tamer Soliman
Quality Lectures - Pre-Analytic Variables - Dr. Tamer Soliman
Documents
and Occurrence
management Assessment
Records
Facilities
Process Customer and
Improvement Service Safety
Total Quality Management 5. Process Control
Process Control
1. Pre-analytic Sample Management
Test Influencers and Interferences
2. Analytic Quality Control
Method Validation
3. Post-analytic Reporting
Interpretation
Total Quality Management 5. Process Control
Laboratory Errors
Statistics
• Up to 96% of patients perform in vitro diagnostic tests 1
• Up to 80% of clinical decisions involve consideration of laboratory results 1
• 40 – 94% of all objective health record data are laboratory results 2 – 4
• Diagnostic errors:
̶ accounted for 26 - 78% of identified medical errors 5
̶ nearly 60% of malpractice claims 6
̶ were involved in 17% of adverse effects due to medical errors in one large study 7
1. Rohr UP, Binder C, Dieterle T, Giusti F, Messina CG, Toerien E, et al. The value of in vitro diagnostic testing in medical practice: a status report. PLoS One 2016;11(3):e0149856.
2. Forsman RW. The value of the laboratory professional in the continuum of care. Clin Leadersh Manag Rev 2002;16(6):370e3.
3. Forsman RW. Why is the laboratory an afterthought for managed care organizations? Clin Chem 1996;42(5):813e6.
4. Hallworth MJ. The ‘70% claim’: what is the evidence base? Ann Clin Biochem 2011;48(Pt 6):487e8.
5. Sandars J, Esmail A. The frequency and nature of medical error in primary care: understanding the diversity across studies. Fam Pract 2003;20(3):231e6.
6. Gandhi TK, Kachalia A, Thomas EJ, Puopolo AL, Yoon C, Brennan TA, et al. Missed and delayed diagnoses in the ambulatory setting: a study of closed malpractice claims. Ann
Intern Med 2006;145(7):488e96.
7. Leape LL, Brennan TA, Laird N, Lawthers AG, Localio AR, Barnes BA, et al. The nature of adverse events in hospitalized patients. Results of the Harvard Medical Practice Study II.
N Engl J Med 1991;324(6):377e84.
Total Quality Management 5. Process Control
Laboratory Errors
Factors that constitute an accurate laboratory result involve more than analytical accuracy
and can be summarized as follows:
1. The right test,
• with the right costs
• and right method
• was ordered for the right patient
• at the right time
• for the right reason
At least 20% of all test orders are inappropriate, 8
Up to 68% of tests ordered do not contribute to improve patient management 9
Conversely, tests were not ordered when needed in nearly 50% of patients. 8
8. Zhi M, Ding EL, Theisen-Toupal J, Whelan J, Arnaout R. The landscape of inappropriate laboratory testing: a 15-year meta-analysis. PLoS One 2013;8(11):e78962.
9. Miyakis S, Karamanof G, Liontos M, Mountokalakis TD. Factors contributing to inappropriate ordering of tests in an academic medical department and the effect of an educational
feedback strategy. Postgrad Med J 2006;82(974):823-9.
Total Quality Management 5. Process Control
Laboratory Errors
Factors that constitute an accurate laboratory result involve more than analytical accuracy
and can be summarized as follows:
2. The right sample was collected on the right patient, at the correct time, with appropriate
patient preparation.
3. The right technique was used collecting the sample to avoid contamination with intravenous
fluids, tissue damage, prolonged venous stasis, or hemolysis.
4. The sample was properly transported to the laboratory, stored at the right temperature,
processed for analysis, and analyzed in a manner that avoids artifactual changes in the
measured analyte levels.
Total Quality Management 5. Process Control
Laboratory Errors
Factors that constitute an accurate laboratory result involve more than analytical accuracy
and can be summarized as follows:
5. The analytical assay measured the concentration of the analyte corresponding to its “true”
level (compared to a “gold standard” measurement) within a clinically acceptable margin of
error (the total acceptable analytical error (TAAE)).
6. The report reaching the clinician contained the right result, together with interpretative
information, such as a reference range and other comments, aiding clinicians in the
decision-making process.
Total Quality Management 5. Process Control
Laboratory Errors
Test ordering
• Duplicate Order
• Ordering provider not identified
• Ordered test not performed
• Order misinterpreted (test ordered <> intended test)
• Inappropriate/outmoded test ordered
• Order not drawn by specimen collector
Total Quality Management 5. Process Control
Laboratory Errors
Laboratory Errors
Laboratory Errors
Laboratory Errors
Laboratory Errors
Laboratory Errors
Laboratory Errors
Laboratory Errors
Laboratory Errors
Other Errors
ﻓﺸﻞ ﰲ اﺧﺘﺒﺎر اﻟﻜﻔﺎءة • Proficiency test failure
ﱂ ﻳﺘﻢ ﺗﺴﻠﻴﻢ اﻟﺘﻘﺎرﻳﺮ ﰲ اﻟﻮﻗﺖ اﳌﻨﺎﺳﺐ • Reports not delivered timely
ﺳﺤﺐ اﻟﺘﻘﺮﻳﺮ • Report recall
إﺻﺎﺑﺔ اﳌﻮﻇﻒ • Employee injury
ﻓﺸﻞ اﻟﺴﻼﻣﺔ • Safety failure
ﻓﺸﻞ ﺑﻴﺌﻲ • Environmental failure
ﺗﻠﻒ اﳌﻌﺪات • Damage to equipment
Total Quality Management 5. Process Control
Laboratory Errors
When classifying sources of error, it is important to distinguish between:
اﻷﺧﻄﺎء اﳌﻌﺮﻓﻴﺔ
1. Cognitive errors, or mistakes, which are due to poor knowledge or judgment.
This type can be prevented by:
• Increased training,
• Competency evaluation,
• Job aids such as checklists or “cheat sheets” summarizing important steps in a procedure.
زﻻت ﻫﻔﻮات
2. Non-cognitive errors, commonly known as slips and lapses, due to interruptions in a process
that is routine or relatively automatic.
This types is best addressed by:
• Process improvement
• Environment re-engineering to minimize distractions and fatigue.
Total Quality Management 5. Process Control 1. Pre-analytic phase Sample Management
Factors that modify certain analyte quantity concentration Factors that modify laboratory test result
in a method-independent way through a mechanism that interfere with the analytic method
A
Pre-analytic
Influencing Factors
Total Quality Management 5. Process Control 1. Pre-analytic phase
Race CK higher in black than in white males. Provide race-specific reference intervals
Granulocytes higher in white than in black males.
Creatinine higher in black than in white males.
Pregnancy TGs ↑, homocysteine ↓ during pregnancy Document months of pregnancy with lab results
Altitude CRP ↑, hemoglobin ↑, transferrin ↓ Consider weeks of adaptation, when coming from
or going to high altitude
Total Quality Management 5. Process Control 1. Pre-analytic phase
• Aging is a complex physiological change that is not fully understood and significantly
affect clinical laboratory test results as an individual transitions through different phases:
prenatal, infancy, childhood/puberty, adulthood and elderhood.
• The recognition of age-related changes allows clinicians to distinguish clinically significant
changes in laboratory test results that are attributed to disease from changes that are
associated with healthy aging and so can increase diagnostic accuracy
• Reference ranges for a majority of analytes measured have been established for the healthy
adult population. However, standardized age-specific reference ranges for the newborn,
childhood to puberty, and elderly adult populations are not complete.
In newborn subjects, the body fluids reflect the trauma of birth and early postnatal
events related to the adaptation of the baby to new extrauterine life.
― Immediately after birth, infants usually experience a mild metabolic acidosis of
transient nature, due to the accumulation of lactates.
― This acid-base disturbance is usually normalized within the first day after birth.
Young DS. Preanalytical variables and biological variation In: Tietz textbook of clinical chemistry and molecular diagnostics. 5th edition.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Colantonio DA, Kyriakopoulou L, Chan MK, et al. Closing the gaps in pediatric laboratory reference intervals: a CALIPER database
of 40 biochemical markers in a healthy and multiethnic population
Total Quality Management 5. Process Control 1. Pre-analytic phase
Levels of other markers are very low in after birth and gradually ↑ within the first 2 weeks
of extrauterine life. Examples: amylase, transferrin, cholesterol, IgA and IgM
Colantonio DA, Kyriakopoulou L, Chan MK, et al. Closing the gaps in pediatric laboratory reference intervals: a CALIPER database
of 40 biochemical markers in a healthy and multiethnic population
Total Quality Management 5. Process Control 1. Pre-analytic phase
This upward trend in analyte concentrations continues over time from birth to 18 years.
Most of the biochemistry parameters (albumin, ALP, AST, total bilirubin, creatinine, IgM,
iron, lipase, transferrin, HDL cholesterol, and uric acid) exert differences between genders
during the early childhood years.
However, these changes are most significant during puberty (age 14–18 years), due to
the strong influence of sexual development and growth.
Colantonio DA, Kyriakopoulou L, Chan MK, et al. Closing the gaps in pediatric laboratory reference intervals: a CALIPER database
of 40 biochemical markers in a healthy and multiethnic population
Total Quality Management 5. Process Control 1. Pre-analytic phase
HB, HCT, and the other RBC indices follow a similar pattern, showing the gradual ↑ during
the first 10 years of life.
Colantonio DA, Kyriakopoulou L, Chan MK, et al. Closing the gaps in pediatric laboratory reference intervals: a CALIPER database
of 40 biochemical markers in a healthy and multiethnic population
Total Quality Management 5. Process Control 1. Pre-analytic phase
100 FSH
Monthly Surges
(mU/mL)
LH
70
10
100 FSH
Monthly Surges
(mU/mL)
LH
70
10
Menopause
Puberty
Female E2
Male E2
Female Testosterone
10 20 30 40 50 60 70 80 90 Age
(Years)
Total Quality Management 5. Process Control 1. Pre-analytic phase
Skeletal growth and muscle mass development accounts for the ↑ ALP, GGT, creatinine,
and GH concentrations seen in the childhood to puberty developmental period :
• Alkaline phosphatase :
̶ ALP ↓ after the age of 12 in girls and after the age of 14 in boys.
̶ Notably, appreciable ALP concentrations are present during growth spurts but can also be
associated with bone diseases (osteoblastic bone cancers, osteomalacia, Paget’s disease
and rickets).
̶ ALP concentrations are approximately 3-fold higher in adolescents compared to adults.
• Creatinine: ↑ with age from 12 – 19 years whereas cystatin C concentration ↓ during the
same age range, particularly in females.
• Uric acid: The high uric acid level seen in a newborn declines for the first 10 years of life,
then ↑, especially in boys, until the age of 16.
Total Quality Management 5. Process Control 1. Pre-analytic phase
In both sexes,
• Total cholesterol ↑ with advancing age (men age 60 and women age 55).
• Uric acid levels peak in men in their 20s but do not peak in women until 50s.
In post-menopausal women,
• Total cholesterol ↑, which are thought to be due to ↓ estrogen levels.
• HDL cholesterol also ↓ up to 30%.
More than 14.0 Indicative of pregnancy unless the clinical setting dictates
mIU/mL otherwise.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Liver enzymes
̶ AST: Small ↑ in serum AST are noted between 60 – 90 years of age
̶ ALT: ALT peaks in the 50s and by the 60s gradually ↓ to levels below those of young adults
̶ ALP: ↑ by 20% between the 3rd – 8th decade),
̶ GGT: ↑ during aging.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Immunoglobulin
̶ IgA concentration ↑ slightly in elderly men
̶ but overall IgG and IgM concentrations gradually ↓.
Blood gases
̶ Age significantly impacts lung elastic architecture, alveoli function and diaphragm
strength and significantly alters respiratory function.
̶ Consequently, the arterial pO2 is ↓ while pCO2 and HCO3– concentration are ↑.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Kidney Function
̶ A 30–40% ↓ in kidney function and ↓ GFR is associated with ↓ creatinine clearance (CrCl).
The decline in muscle mass results in ↓ creatinine production;
thus serum creatinine levels remain within normal limits despite the ↓ CrCl capacity.
̶ Mean CrCl ↓ by ~ 10 mL/min/1.73 m2 per decade and is significantly different between
the adult and geriatric populations.
• The mean CrCl for a 30-year old is 140 mL/min/1.73 m2 of body surface area (BSA),
• whereas in an 80-year old the CrCl concentration is 97 mL/min/1.73 m2 of BSA.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Others
̶ Serum 2hr PP glucose (after age 40) ↑ 30 – 40 mg/dL per decade,
̶ Serum cholesterol (by age 60 ↑ by 30 – 40 mg/dL),
̶ ESR (values as high as 40 can be non-pathogenic),
̶ Mg2+ (↓ by 15%),
̶ FT4, T3, ACTH, and cortisol concentrations are ↓.
̶ In men, free testosterone ↓ with age, without significant changes in total testosterone.
̶ In men, PSA levels ↑ up to 6.5 ng/mL in men > 70 without evidence of prostate cancer.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Sample Mis-identification
Total Quality Management 5. Process Control 1. Pre-analytic phase
Pre-collection
Variables
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
• Different analytes have different rhythms, ranging from a few hours to monthly changes.
• Awareness of such changes can be relevant to proper interpretation of laboratory results.
• These changes can be divided into circadian, ultradian, and infradian rhythms according
to the time interval of their completion.
Circadian rhythm
Recurring naturally on cycles every 24 hours
Infradian rhythm
Recurring naturally on cycles more than 24 hours
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Circadian rhythm
• Recurring naturally on cycles every 24 hours
• Analytes fluctuate between a maximum and a minimum value during the day
• Can be influenced by individual rhythms concerning meals, exercise and sleep.
GH ↑ at start of sleep and ↓ during the day Cortisol ↑ during the day and ↓ at night
40 12 am 300
250 Cortisol
30 1 am (Somatostatin)
2 am 200
2 pm
20 150
11 pm
3 am
10 pm 100
10
50
0 Time 0 Time
12 am 2 pm 8 am 8 pm
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Circadian rhythm
Examples
Cortisol Peaks 4-6 am; lowest 8 pm –12 am; 50% lower at 8 pm than at 8 am
Plasma renin activity Lower at night
Aldosterone Lower at night
Insulin Lower at night
Growth hormone Higher in afternoon and evening
Acid phosphatase Higher in afternoon and evening
TSH Higher levels at 2 – 4 am and minimum levels at 6 – 10 pm
Prolactin Higher levels at 4 – 8 am and at 8 – 10 pm
Iron Peaks early to late morning; decreases up to 30% during the day
Potassium Higher in the morning than in the afternoon
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Ultradian rhythm
• Recurring naturally on cycles less than 24 hours.
• Analytes that are secreted in a pulsatile manner throughout the day.
• Example Testosterone, which usually peaks between 10:00 am and 5:00 pm.
Infradian rhythm
• Recurring naturally on cycles more than 24 hours
• The example most commonly cited is the monthly menstrual cycle.
Constituents such as pituitary gonadotropin (FSH, LH), ovarian hormones (progesterone,
estrogen), and prostaglandins are significantly affected by this cycle.
• T3 is 20 % lower in summer than in winter
• Vitamin D3 exhibits higher serum concentrations in summer
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Estrogen Progesterone
FSH
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Acute Effects of Diet and Other Influencing Factors
• Prior to blood sampling, the confounding influences of foods and fluid intake should be excluded.
• Diet and fluid intake are major factors influencing a number of analytes.
• The effect of food is dependent on:
― The composition of diet
― The elapsed time between food intake and sampling
• Changes of 5% or less may be neglected (below 1.05 in) for being clinically insignificant.
Therefore samples for these analytes do not require strict fasting.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Acute Effects of Diet and Other Influencing Factors
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Acute Effects of Diet and Other Influencing Factors
• The activity of some enzymes (e.g. ALP, AST, ALT) ↑ up to 20% following a meal.
• TGs and glucose concentrations in serum ↑ during the absorptive phase.
• The turbidity of the plasma/serum sample, caused by chylomicrons following absorption of lipids,
can also interfere with various measurement procedures.
• The serum cholesterol and TGs are influenced by various factors, such as food composition,
physical activity, smoking, and consumption of alcohol and coffee.
• In response to a meal, HCl secretion in the parietal cells of the stomach is associated with chloride
extraction and release of HCO3– into the plasma. Thus venous blood leaving the stomach is
enriched with HCO3–, and this phenomenon is responsible for a mild postprandial metabolic
alkalosis with concomitant ↑ of pCO2 and a subsequent ↓ of iCa2+ by 0.2 mg/dL (0.05 mmol/L).
To avoid any misinterpretation,
It is recommended that blood sampling be done after 12 hours of fasting and ↓ activity (bed rest).
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Acute Effects of Diet and Other Influencing Factors
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Long-term Effects of Diet
Proteins
― The changes in protein intake that occur over a couple of days may affect the composition of
nitrogenous components of plasma and the excretion of end products of protein metabolism.
― Creatinine is an important example of the effect of diet on the composition of plasma.
It has been shown that ↑ up to 20% of plasma creatinine concentration (measured by kinetic
Jaffe method) is observed after ingesting a cooked meat.
― Serum urea and urate concentrations is also affected by protein-rich foods
Fat
― A diet rich in fat leads to ↑ serum TGs, ↓ serum urate, a depletion of the body’s nitrogen pool.
The nitrogen pool is affected because NH3 excretion is required to maintain acid-base balance.
― The relative ratio in which various dietary fats are consumed closely relates to serum lipid
concentrations. A diet rich in monounsaturated and polyunsaturated fats causes a ↓ of LDL-C
and HDL-C concentrations, although in some situations HDL-C may be ↑.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Long-term Effects of Diet
Carbohydrates
― A diet rich in carbohydrates ↓ serum protein and lipid (TGs, and total and LDL-C).
• It should be emphasized that not only the proportion but also the source of nutrients in the diet
affect the composition of serum. For example, some early studies have shown that serum ALP and
LD activities are ↑, whereas AST and ALT activities are ↓ in individuals who consume carbohydrates
rich in sucrose or starch rather than other sugar types.
• Moreover, total LDL-C and HDL-C levels tend to be much ↓ in those who consume the same amount
of food in many small meals throughout the day than in individuals who eat 3 meals per day.
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Special diet-related changes
Long-time vegetarian diet LDLs, VLDLs,, phospholipids, cholesterol, and TGs
Vitamin B12 deficiency (unless supplements are taken)
High protein diet ↑ plasma NH3, uric acid. and urea compared to vegetarian diet
The ketogenic diet ↑ Blood urea, ketosis and ketonuria within several days
(low CHO, moderate-protein, high-fat diet)
Diuresis within 2 weeks.
↓ in serum TGs and an ↑ in HDL-C occur over several weeks.
The hCG diet Positive blood and urinary pregnancy test results.
(hCG sublingual drops or injections
paired with a low 500-calorie diet)
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Fasting/starvation-related changes
• Fasting: decreased caloric intake
• Starvation: No caloric intake
Within 3 days of fasting,
̶ Glucose concentrations ↓ by as much as 18 mg/dL
̶ Subsequently, insulin rapidly ↓ while glucagon secretion ↑ to restore blood glucose to
pre-fasting concentrations.
̶ The fasting individual undergoes:
a. Lipolysis and hepatic ketogenesis (↑ fatty acids, ↑ blood and urine ketones)
b. Metabolic acidosis state (↓ pH, pCO2 and HCO3).
Burtis CAAE, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 4 ed. St. Louis, MO: Elsevier Saunders; 2006.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Fasting/starvation-related changes
T3 Significant ↓ up to 50% in both TT3 and FT3
Growth hormone Early in fasting: Sharp ↑ up to 15 times the pre-fast plasma in GH levels.
Within 3 days of completing a fast: the plasma GH returns to pre-fast levels.
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Caffeine (e.g. tea, coffee, cola and energy drinks)
Caffeine stimulates the adrenal cortex and medulla,
― leading to the subsequent ↑ of the concentration of catecholamines and their metabolites, as
well as free cortisol, 11-hydroxycorticoids, and 5-HIAA (5-OH indole-acetic acid) in serum.
― These hormonal changes are followed by the ↑ in plasma glucose concentration.
Plasma renin activity may also be ↑ following caffeine ingestion which results in:
― Induction of diuresis (H2O loss) within 2 hours following caffeine ingestion
― Inhibition of electrolytes reabsorption (Ca2+, Mg2+, Na+, Cl–, K+), leading to ↑ in their excretion.
Caffeine also has a marked effect on lipid metabolism.
Ingestion of coffee ↑ the rate of lipid catabolism, thus leading to the ↑ of plasma lipids, free fatty
acids, glycerol, and lipoproteins.
Finally, caffeine is a strong stimulant of gastrin release and gastric acid secretion and also
induces the secretion of pepsin.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Alcohol
Alcohol consumption, depending on its duration and extent, may affect a number of analytes.
Acute alcohol ingestion
― ↓ plasma glucose and ↑ lactate are occur within 2 – 4 hours of ethanol consumption.
― Ethanol is metabolized to acetaldehyde and then to acetate. This ↑ hepatic formation of uric
acid and inhibits renal urea excretion, thus causing an ↑ of uric acid in plasma.
― Acetate and lactate ↓ plasma HCO3, resulting in mild to severe metabolic acidosis, depending
on the amount of ingested alcohol.
― Also, acute alcohol ingestion ↑ the activity of serum GGT.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Alcohol
Alcohol consumption, depending on its duration and extent, may affect a number of analytes.
Chronic ethanol ingestion
― ↑ in serum TGs concentration due to ↓ plasma TGs breakdown
― ↑ in the serum activity of many enzymes (GGT, AST, ALT).
― ↑ MCV is related to the direct toxic effect of alcohol on erythropoiesis or a folate deficiency.
― Chronic alcohol consumption affects pituitary and adrenal function.
Pituitary: ↓ formation of vasopressin with ↑ diuresis.
Adrenal: ↑ secretion of renin and aldosterone as a result of diuresis.
To assess the effect of alcoholic drinks on test results and to avoid misinterpretations of laboratory
results, it is recommended that the history of alcohol intake (i.e. the ingested amount and
frequency/time of ingestion) be documented in clinical records.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Smoking Tobacco
The extent of smoking-related changes also depends on:
• The amount, kind (cigarettes, cigars, pipes) and technique of smoking (with or without inhalation).
• Age and gender.
Smoking tobacco leads to a number of acute and chronic changes in analyte concentrations.
Acute changes (within 1 hour of smoking a cigarette)
― ↑ serum fatty acids, epinephrine, free glycerol, aldosterone, and cortisol.
― ↑ urinary excretion of catecholamines and their metabolites.
― ↑ in serum TGs, LDL, and total cholesterol concentrations.
― ↑ blood Glucose level, within10 minutes of smoking a single cigarette, glucose level ↑ by up to
10 mg/dL. This ↑ may persist for 1 hour.
― Similar to caffeine, nicotine is also a very potent stimulant of the secretion of gastric juice
and an inhibitor of duodenal HCO3 secretion.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Smoking Tobacco
Chronic changes
Chronic changes induced by nicotine and its metabolites and reflect responses to their toxic effects
― ↑ carboxyhemoglobin concentration.
― ↓ pO2, which is lower in tobacco smokers than in nonsmoking individuals by about 5 mmHg.
― ↑ RBCs (↑HB, HCT) as a compensation for the impaired O2 transport capacity in heavy smokers.
― ↑ WBCs may be ↑ (up to 30%) with a proportional ↑ in the lymphocyte count.
― ↑ Total cholesterol and LDL-C, Heavy metals (Copper, lead, cadmium) and CEA.
― ↓ HDL-C, prolactin and ACE (due to destruction of lung tissue)
― CEA, show higher levels which is caused by ↑ synthesis and secretion of CEA in the colon.
― ↓ Immunoglobulin (Ig)A, IgG, and IgM but IgE may be ↑
― ↓ male fertility: ↓ Sperm counts and motility and abnormal morphology have been reported
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Smoking Tobacco
Chronic changes
The effect of chronic smoking may persist even after smoking cessation.
― It usually takes 5 years, or even longer, for most parameters to normalize (e.g. CRP and
fibrinogen concentrations, HCT).
― Some parameters (e.g. WBCs), may take up to 20 years to return to baseline value.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Smoking Tobacco
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Exercise (Muscular Activity)
Physical activity of varying duration and intensity may lead to changes in the plasma composition,
and the extent of this change depends on several factors, such as:
― Training status,
― Intake of fluid, electrolytes and carbohydrates
― The surrounding temperature.
For example,
even a mild physical effort, like clenching the fist during venous blood sampling, can ↑ the
concentration of K+ and should therefore be avoided. This occurs due to the release of K+ from
skeletal muscles and even without a tourniquet.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Exercise (Muscular Activity)
Intensive exercise
• Associated with transient changes related to metabolic activities for energy, such as:
― ↑ cardiac and muscle damage biomarkers (CK, CKMB, AST, LDH and sometimes cTnI)
― ↑ Lactate (as much as 300%)
― Leukocytosis, enhanced platelet aggregation, ↑ tPA, activation of the fibrinolytic system
― Hormones: e.g. catecholamines, ACTH, cortisol, T4, TSH, prolactin, GH, hCG, vasopressin,
gastrin, aldosterone, testosterone, insulin, glucagon, and β-endorphin.
• These transient changes usually return to pre-exercise levels soon after exercise cessation
(within 12 hours before blood sampling).
Long-term effects (professional sportsmen)
A large proportion of laboratory results may fall outside the usual reference intervals
― ↑ CK, aldolase, AST, and LDH values
― In long-distance athletes: ↓ serum FSH, LH, E2, progesterone and testosterone while ↑ PRL.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Mental stress
May induce:
• ACTH ↑
• Cortisol ↑
• Catecholamines ↑
• Total Cholesterol ↑
• HDL ↓ as much as 15%
• Prolactin may ↑
Total Quality Management 5. Process Control 1. Pre-analytic phase
Biological Rhythm | Diet | Fluid Intake | Smoking | Exercise | Stress | Body Position
Body Posture
Body posture influences the blood concentrations of certain analytes.
• Ideally, reference intervals of these analytes should obtained with regard to body posture.
• Capillary filtration is ↑ in the lower extremities when changing from the supine (lying) to the
upright (sitting) position which ↑ the concentration of all constituents that usually do not pass the
capillary filtration barrier, including LMW molecules bound to proteins.
― Calcium: free Ca2+ does not change, whereas total Ca2+ ↑ by 5 – 10%, when changing
from the supine to upright position. This is observed in healthy and diseased individuals, the
change is usually greater in some disease states—e.g. cardiac insufficiency. Sampling
should be performed after at least 15 minutes of rest in a supine or sitting position.
― Aldosterone and renin show different serum levels between supine and upright position.
Sampling should not be done before a period of 2 hour-rest in a supine or sitting position.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Patient Preparation
Preparing for Blood Sampling
• Because food, fasting time, circadian rhythm, muscular activity, smoking, drugs, and ethanol
consumption can affect the concentration of numerous analytes, standardization of all those
controllable variables is highly recommended.
• Proper standardization of controllable variables leads to significant ↓ of preanalytical variability.
Blood samples for routine testing should not be taken if a patient has not been appropriately prepared.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Patient Preparation
Specific Blood sampling requirements
Glucose Fasting level requires a diet restriction for 6- 8 hours with permission of water intake
Refrain from caffeinated products in the morning before sampling is recommended
Glucagon Fasting for at least 12 hour before specimen collection (for baseline values)
Gastrin Fasting for at least 12 hour before specimen collection (for baseline values)
Lactic acid Fasting for 12 hours before the test
Refrain from alcohol consumption or 12 hours before the test
No muscular exercise 12 hours before the test
Lipid Triglycerides Overnight fasting 12 – 14 hours before sampling
Ideally, the patient should be on a stable diet for 3 weeks
Refrain from alcohol consumption for 3 days before sampling
Cholesterol It was reported that there is no fasting required before sampling
Lipoproteins Overnight fasting 12 – 14 hours before sampling
Avoid excessive exercise for at least 12 hours before sampling
Refrain from alcohol consumption for 24 hours before sampling
Total Quality Management 5. Process Control 1. Pre-analytic phase
Patient Preparation
Specific Blood sampling requirements
Lipase Ensure specimen collection takes place prior to ERCP
(endoscopic retrograde cholangiopancreatography)
Aldosterone Supine or upright posture must be maintained for 2 hours before sampling
Patient Preparation
Specific Blood sampling requirements
Therapeutic drug monitoring May require timed-blood collection (trough and peak levels)
(A trough level is drawn before the next dose of the drug is administered)
Documentations of renal and hepatic function is recommended
Protein C and S Warfarin therapy should be discontinued for 2 weeks before collection
Collection should not be performed < 10 days following a thrombotic event
Female fertility hormones May require sampling at a specific time during the menstrual cycle
Specific Semen requirements No sexual activity for 3 days before specimen collection
Total Quality Management 5. Process Control 1. Pre-analytic phase
Patient Preparation
Specific Urine sampling requirements
Morning urine Urine sediment, test strip
Second morning urine Proteins, urinalysis
Timed urine (24 h) Urine Chemistry (proteins, electrolytes, …. ,etc.)
Timed urine (6, 12 h) Hormones, drugs,
First void urine Chlamydia
Midstream urine Urinalysis, microbiological examination
Metanephrines Avoid excessive exercise and stress during the 24-hours collection of urine
Specific Stool sampling requirements اﻟﻠﻔﺖ اﻟﺒﻨﺠﺮ اﻟﻘﺮﻧﺒﻴﻂ اﳉﺰر اﻷﺑﻴﺾ اﻟﻔﺠﻞ
Occult blood test Foods to avoid: beets, turnips, cauliflower, broccoli, parsnips, bananas, horseradish and
(Guaiac method) cantaloupe and meat
Instruction for not to using laxatives, enemas, or suppositories for 3 days before the test.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Collection Variables
Total Quality Management 5. Process Control 1. Pre-analytic phase
Red None (plan) N/A Serum / Chemistry & serology Not required
Tests that cannot be collected into SST tubes
Clot activator Silica clot activator Serum / Chemistry & serology 5 times
Tests that cannot be collected with gel;
e.g. some therapeutic drugs (antidepressants)
Na Citrate
Light • Completely fill tube to the line indicated Chelates calcium Plasma / coagulation 3 – 4 times
Blue • Over- or under-filling not allowed
• 1 part Na citrate + 9 parts Blood
0.2 ml + 1.8 ml
0.5 ml + 4.5 ml
Plasma / Chemistry
Green Na heparin or Inhibits thrombin Amino acids, blood gases 8 – 10
Li heparin formation Whole Blood / FCM
/ Cytogenetic
/ HLA Typing
/ G6PD
= 1 inversion
Total Quality Management 5. Process Control 1. Pre-analytic phase
Order of Draw
A standardized order of draw minimizes carryover contamination of additives between tubes.
The general order of draw is as follows:
1. Microbiological blood culture tubes (filled first to avoid bacterial contamination)
2. Trace element tubes (non-additive) (These tubes are trace- and heavy metals-free that
may be present in the next tubes)
3. Citrated coagulation tubes (clot activator in the next tube(s) may cause interference with
coagulation clotting factors).
4. Non-anticoagulant tubes for serum (clot activator, gel or no gel) (drown before other
anticoagulant/additive tubes to avoid contamination with Na heparin, K EDTA, and others
5. Heparin tubes (with or without gel)
6. EDTA tubes
7. Acid citrate dextrose tubes
8. Glycolytic inhibitor tubes
Total Quality Management 5. Process Control 1. Pre-analytic phase
Order of Draw
1 2 3 4 5 6 7 8 9 10 11
Blood Clot Activator Na Citrate Na Citrate clot activator Gel Separator Li Na K3EDTA Na Heparin ACD Na Fluoride
Culture ± Gel Clot Activator Heparin Heparin + K3EDTA
Plastic or glass serum tubes containing a clot activator may cause interference
in coagulation testing.
Plan tubes e.g. Glass nonadditive serum tubes or plastic serum tubes without a clot activator
may be drawn before the coagulation tube.
Plan
Total Quality Management 5. Process Control 1. Pre-analytic phase
ABG
4 Grey NaFl/Na2 EDTA 10x
EDTA
5 Yellow (Gold)
Clot Activator 5x Other Additives
and Gel for serum separation
Serum tubes
6 Red No additive 0x
Total Quality Management 5. Process Control 1. Pre-analytic phase
Tourniquet
• A similar mechanism occurs when a tourniquet is applied to facilitate finding appropriate
veins for venipuncture.
• The higher pressure obtained in veins leads to the loss of water and LMW substances,
Resulting in hemoconcentration and ↑ the proteins, cells, and analytes bound to them.
• This becomes clinically significant after 1 – 2 minutes.
• Therefore the tourniquet should be released 1 minute after it has been applied.
• Examples:
― Total protein and Albumin
― Potassium and Calcium
― Glucose and Lactic acid
― Cholesterol
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Problems
1. Blood gas testing is commonly requested in patients with a critical, life-threatening
condition or who are experiencing some unexpected deterioration. Such patients may
have a serious metabolic (acute complications of DM, drug intoxication) or respiratory disorder
(respiratory failure, sepsis, or multi-organ failure) and need immediate medical intervention.
2. Arterial blood sampling is an invasive procedure associated with a risk of complications such
as bruising, bleeding, infections, and arterial thrombosis.
3. Arterial blood samples have very limited stability. Due to the low biological variability of
many blood gas parameters, allowable total error is quite low, and even small differences in
serial measurements can be clinically meaningful.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Patient Condition
• To ensure that test results reflect the actual condition of the patient,
Blood sampling should be done when a patient is in a stable, resting state.
• Any deviation from the steady state should be:
― Noted as a comment
― Accompany the test result in order to allow proper interpretation of the results and patient
management.
• The exact collection time should always be:
― Recorded
― Reported with a test result.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Patient Condition
Relevant patient condition determinants (at the time of blood collection) include:
― Patient activity status (resting, exercising, crying (children), anxious)
― Ventilatory setting (spontaneous breathing or assisted mechanical ventilation)
― Mode of O2 delivery (fraction of inspired O2 (FiO2) through nasal cannula or Ventouri mask)
― Respiratory rate (hyperventilation, hypoventilation)
― Body temperature
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Patient Condition
* Activity Status
• If the patient’s condition is changing,
a sufficient time should be allowed for the patient to stabilize.
• For example,
― Crying leads to a rapid ↓ of O2 saturation.
― It has been shown that even a short walk or mild exercise may lead to a significant ↓ in
O2 saturation in patients who are suffering from COPD.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Patient Condition
* Body Temperature
• ↑ body temperature is associated with ↑ iCa2+ and pCO2 and ↓ pH.
• Thus, if patient temperature deviates from normal body temperature,
Information about that should accompany the report to allow proper interpretation of results.
• Although blood gas instruments offer temperature corrected values,
Their use is not recommended because currently data are not available to quantify the
balance between O2 delivery and O2 demand at temperatures other than 37°C.
• If the temperature-adjusted results are reported anyway,
It is absolutely mandatory that the report be clearly labeled as such and that the
uncorrected values are also made available on the test report.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Patient Condition
* Ventilatory setting
• Hypoventilation is associated with ↑ iCa2+ and pCO2 and ↓ pH.
• In the ventilatory setting or mode of O2 delivery,
The patient should be left in a resting state to stabilize.
― For patients without lung disease, a period of 3 – 5 min is usually enough to stabilize.
― However, in patients with lung disease, this period is significantly longer.
According to the CLSI C46-A2 standard for blood gas and pH analysis, adequate time for
most patients to reach a stable state following ventilatory changes is 20 – 30 minutes.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Type
The composition of arterial blood is constant throughout the body,
whereas the composition of venous blood largely variable, depending on:
― The time of blood sampling,
― Local and global circulatory conditions,
― Metabolic activity of the organ or tissue from which it carries blood to the heart.
• The major difference between arterial and venous blood is in their O2 content.
• However, other parameters (pCO2, pH) may also vary.
• The differences are more pronounced in conditions associated with compromised local or global
circulation.
Arterial blood collected under anaerobic conditions is therefore the only acceptable sample
type for an accurate evaluation of the gas exchange function of the lungs (pO2 and pCO2).
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Type
Capillary blood
• If arterial blood is not available (e.g. neonates, small children, patients with burns) and during
medical transport and prehospital critical care, a capillary sample is an acceptable alternative.
• Capillary blood is obtained by puncturing the dermis layer of the skin and collecting it from the
capillary beds running through the subcutaneous layer of the skin.
• Capillary blood is a mixture of unknown proportions of the blood from the smallest veins
(venules) and arteries (arterioles), the capillaries, and surrounding interstitial and intracellular
fluids.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Type
Capillary blood
• Due to large differences in O2 content between arterial and capillary blood, the results obtained
from a capillary sample should be interpreted with extra caution.
• Whereas capillary blood, if sampled properly,
― May accurately reflect arterial pCO2 and pH over a wide range of values,
― But never serve as an adequate substitute for arterial accurate pO2 measurement.
• Capillary blood sampling is not recommended in patients with:
― Hypotension, circulatory shock (↑ arterial pO2, which mean greater difference)
― With poorly perfused (cyanotic)
― Infected, inflamed, swollen, or edematous tissues
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Type
Capillary blood
• Capillary blood should be collected using an arterialization technique by warming the skin to
40 to 45°C with a warm towel or by using a vasodilating cream containing.
• Arterialization ↑ the blood flow through the capillary beds and thus the proportion of arterial
blood relative to venous blood in the capillary sample.
• An earlobe is better sampling site than a fingertip because the blood sampled from an
arterialized earlobe better reflects arterial blood values.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Anticoagulants
The recommended anticoagulant for ABG and iCa2+ testing is lyophilized balanced Li-heparin.
• According to the CLSI C46-A2 standard on blood gas and pH analysis and related measurements,
the final heparin concentration in the sample should be 20 IU/mL blood.
• Because the heparin pH of is 7.0 and its pO2 and pCO2 values are near room air values,
the excess of heparin in the sample can alter sample pH, pO2, and pCO2.
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Anticoagulants
The recommended anticoagulant for ABG and iCa2+ testing is lyophilized balanced Li-heparin.
Why Use Lyophilized Heparin?
― Syringes for arterial blood sampling either
1. Commercially dedicated syringes for ABG sampling contain spray-dried balanced heparin
2. Syringes with liquid heparin are also available.
― Whereas liquid heparin enables better sample mixing,
it may introduce sample dilution in cases of incomplete draw.
― Using ordinary syringes (without heparin) and flushing them before use are strongly discouraged.
Flushing the syringe with liquid heparin causes sample contamination with heparin and sample
dilution, resulting in significant differences among blood gas parameters.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Contamination
Sample contamination may substantially affect blood sample quality and cause significant bias.
ABG samples are most commonly contaminated with :
― Liquid heparin (discussed previously),
― Venous blood
― Air bubbles
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Contamination
Contamination with venous blood
• Occurs if a vein is accidentally punctured.
• Contamination of arterial blood by venous blood may show falsely ↓ pO2, ↓ sO2 and ↑ pCO2.
• This may happen if the needle is not correctly positioned during arterial blood sampling.
• To decrease this contamination, it is recommended to:
1. When making an arterial puncture, the needle should be inserted at a 30 – 45 degree angle.
2. Using short-beveled needles because they are much easier to position inside the artery.
3. Dedicated self-filling syringes are also highly recommended.
These syringes fill more quickly & much easier when a needle is puncturing an artery instead
of a vein as a result of the difference in blood pressure between the vein and the artery.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Contamination
Contamination with Air
• The aspiration of air during ABG sampling or bubble formation can result in significant changes in
the concentration of some blood gas parameters (↑pH, ↑pO2,↑sO2, ↓pCO2).
• The exchange between the air bubble and the arterial blood sample is rapid.
• It starts immediately and becomes significant after only 1 to 2 minutes.
The exchange rate does not depend on the size of the bubble.
― The longer the delay between blood sampling and sample analysis, the greater the effect of the
contamination with atmospheric air and deviation from the true patient values.
― It should be noted that even a bubble as small as 1% of the total sample volume may cause
significant changes in the O2 content of the specimen.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Contamination
Contamination with Air
Prevention
1. By visual inspection of the specimen immediately after sampling.
― If air bubbles are present in the sample, they should be expelled as soon as possible and
certainly prior to the sample mixing.
― If there is a visible froth in the sample, such samples should not be analyzed because froth
may contain a significant amount of atmospheric air.
― The degree of contamination also depends on the agitation/turbulences during transport.
2. The use of blood gas syringes with a vented mechanism. Once such a syringe has been filled
up to the dedicated volume, the vent allows the air to be pushed out from the syringe. After the air
has been pushed out, the vent is closed, preventing the subsequent air contamination.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Hemolysis
• Although arterial blood sample hemolysis is difficult (or almost impossible), it has been found that
a significant proportion (up to 4%) of arterial blood samples are hemolyzed.
• Hemolysis leads to a significant ↓ in pO2 and an ↑ in pCO2.
• Electrolyte concentrations (K+, Ca2+) are also dramatically affected by hemolysis.
• The most common cause of hemolysis:
1. Vigorous mixing. Sample mixing should be done gently.
2. Any source of sample turbulence
3. High force during sample aspiration
4. Cooling the sample directly on ice cubes. An ice slurry should be used instead.
5. If capillary blood is collected, excessive pressure (“milking”) should be avoided.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Hemolysis
Sample milking
― Sample milking leads to significant hemolysis and contamination with surrounding tissue fluid.
― If milking is applied, many parameters in the sample will deviate from the true values.
― Possible difficulties during capillary blood sampling should always be recorded and reported
with the test results to enable proper interpretation of test results by the clinician.
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Mixing
• Must be properly mixed to:
― Prevent clot formation (A clotted sample cause analyzer malfunction and false ↑ K+ levels)
― Promote heparin dissolution,
― Ensure that blood cells are uniformly suspended in the sample.
• Should be mixed immediately after the sampling
• Only after expelling visible air bubbles.
• Should be done gently to avoid hemolysis.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Mixing
Samples can be mixed manually and automatically.
• Manual sample mixing
― is done by gently inverting the syringe several times and rolling it between the palms.
― If manual mixing is not performed properly, the sample is unsuitable for analysis.
• The automatic arterial sample mixing
― is done with the use of small metal ball located in the syringe barrel.
― The ball in the sample is moved through the sample by the force of the external magnet.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Mixing
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Mixing
If the analysis is not done immediately and the sample needs to be transported to another location,
the sample must be mixed again immediately prior to analysis.
This is necessary to obtain a homogeneous sample and to ensure accurate test results.
Mixing time depends on the time span between the sample collection and analysis.
― The shorter the time span (less than 2 minutes), a shorter mixing time (< 1 minute) is acceptable.
― The longer the time span (20 – 30 minutes), the longer the mixing time required.
In samples that have been left to stand for, the homogeneity of the samples can be achieved by
continuous mixing for at least 2 minutes.
― If longer delays occur between sampling and analysis, longer mixing intervals are required.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Transport
― Arterial blood samples should be transported by hand and at room temperature.
― Avoid vigorous movement during sample transport.
― Avoid delays and to analyze the sample as soon as possible.
SPECIFIC CONSIDERATIONS
Preanalytical Aspects of Arterial Blood Gas (ABG) Testing
Blood Gas Sample Transport
According to the CLSI C46-A2 standard,
1. Samples should be transported by hand in a plastic syringe at room temperature and analyzed
within 30 min of collection.
2. In cases when expected delivery time is longer than 30 minutes, glass syringes should be used
and the sample should be transported on ice to reduce the rate of metabolism and exchange of
gases between the sample and the ambient air.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hemostasis Testing
Some specific considerations related to hemostasis testing are associated with:
• The type of anticoagulant
• Sampling technique (fasting state, length of the venous stasis)
• Order of draw, sampling from a catheter)
• Sample handling (centrifugation)
• Transport
• Storage prior to analysis
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hemostasis Testing
The type of anticoagulant
• Laboratories is commonly use 3.2% Na+ citrate, (3.8% may also be acceptable).
• Clotting times may be longer in 3.8% than in 3.2% Na+ citrate (3.8% binds more Ca2+ ions)
• It has been reported that:
― PT and APTT may be overestimated in 3.8% Na+ citrate,
― Fibrinogen is underestimated in 3.8% compared to values obtained in 3.2% citrated samples.
• Other anticoagulants (e.g. oxalate, heparin, or EDTA) are unacceptable
SPECIFIC CONSIDERATIONS
Hemostasis Testing
Mixing
Mixing of samples is extremely important for adequate sample coagulation.
― Samples must be promptly mixed to avoid in vitro clot formation
― Tubes should be mixed by gentle tubes inversion (at 180 degrees) several times (3 – 5 inversions)
― For proper mixing, instructions from the tube manufacturer should be followed.
― Vigorous mixing and shaking the tubes are discouraged
because that may lead to sample hemolysis; the activation of
platelets and coagulation factors, resulting in ↓ shortening of
clotting times; and possibly a false ↑ in clotting factor activity.
1 inversion =
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hemostasis Testing
Sample Clotting
Samples for hemostasis testing that contain visible clots must be rejected. To prevent clot formation,
― some precautionary measures should be taken during blood sampling, handling, and transport,
― and the following errors should be avoided:
1. Blood flow (during blood sampling) too slow
2. Collection of the sample into a syringe and then transfer into a citrated tube
3. Tubes underfilled
4. Prolonged use of a tourniquet
5. Considerable manipulation of the vein by the needle
6. Incomplete mixing
Clot formation induces platelets and clotting factors activation, which may cause false assay results.
Even small clots that are invisible to the human eye may significantly impact coagulation assays.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hemostasis Testing
Effect of Tourniquet
Longer venous stasis should be avoided because it results in:
― Hemoconcentration
― Activation of fibrinolysis
― ↑ in fibrinogen and factors VII, VIII, and XII
Order of Flow
• A standardized order of draw has been recommended, with the coagulation tube as the first tube
and a tube drawn and discarded before the citrate tube. However, more recent evidence
suggests that a “discard” tube may not be necessary.
• If intravenous catheter systems are used for blood sampling, 5 – 6 times the dead space volume
of the catheter should be discarded prior to blood sampling.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hemostasis Testing
Effect of Temperature
Following collection, citrated samples should ideally be transported to the laboratory immediately
and at room temperature (RT)but no later than within 1 hour of blood draw.
Blood samples for coagulation testing must be kept at RT (20 – 25°C) until analysis.
Storage at lower temperatures or on ice is discouraged, because this may result in:
― Platelets activation
― FVII activation
― Significant time-dependent of FVIII and VWF loss.
PT and aPTT should not be performed in samples that were stored frozen.
Freezing leads to a marked ↓ in FVIII activity, and an apparent ↑ in fibrinogen levels.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hemostasis Testing
Effect of Centrifugation
Whole blood coagulation assays should be performed within 4 hours after blood sampling.
• For PT and aPTT
― Should be performed using fresh plasma within 4 hours after sampling and stored at RT.
― If the centrifuged plasma is left to sit on the blood cells, this may result in shortening of PT
and prolongation of aPTT.
• For platelet function assays,
― Samples should rest at RT for 30 minutes before analysis.
― To obtain PPP, centrifugation is performed at 1500 g at RT for 10 – 15 min.
― Higher speeds with shorter centrifugation times are not recommended because this may
induce hemolysis and activation of platelets.
― Moreover, centrifuge breaks should also be avoided to prevent remixing of samples.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hemostasis Testing
Effect of Centrifugation
• The preparation of PRP for platelet function assays requires special care, and centrifugation
speeds and duration need to be carefully optimized to ensure optimal results.
― Generally, centrifugation is done at 200 – 250 g for 10 min without application of a rotor brake.
― If possible, all coagulation analyses should be performed using fresh material; freezing of
samples should be an exception.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hematology Testing
EDTA (ethylenediaminetetraacetic acid) is the anticoagulant of choice for hematology testing.
― Of supreme efficacy for preserving cellular morphology
― Mechanism: Ca2+ chelation.
― EDTA is a free non water-soluble acid in nature. It comes as Na2EDTA, K2EDTA, and K3EDTA salt.
Solubility (depend on nature of ions) K2EDTA, and K3EDTA > Na2EDTA
pH (depend on the number of ions) K3EDTA (pH 7.5) > Na2EDTA and K2EDTA > EDTA acid (pH 2.5)
Cell swelling Na2EDTA and K2EDTA, cell swelling is counteracted by cell shrinkage
Cell Shrinkage K3EDTA > K2EDTA
MCV (the mini-hematocrit values) Na2EDTA and K2EDTA is more acceptable than K3EDTA
For these reasons, due to its higher solubility, lower osmotic effect, and best overall performance,
the ICSH recommends K2EDTA salt as the anticoagulant of choice for hematology testing.
SPECIFIC CONSIDERATIONS
Hematology Testing
• Tubes need to be mixed immediately after the blood is drawn to allow proper additive mixing with
blood and to prevent clotting.
• Adequate mixing is achieved by gentle inversion at 180 degrees and back to the upright position.
• The number of turns (~ 8 – 10 inversions) depends on the tube type, and for optimal results,
manufacturer instructions should be followed.
• Blood tubes should be filled to ±10% of the stated draw volume.
― In underfilled EDTA tubes, cell count and HCT might be
falsely ↓ due to the excess EDTA.
― In overfilled tubes, clot formation and platelet clumping are
likely to occur due to the difficulty of appropriate mixing.
= 1 inversion
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hematology Testing
EDTA – induced pseudothrombocytopenia
In some individuals, EDTA may be a cause of pseudothrombocytopenia—either platelet clumping or
platelet satellitism (platelets adhering to neutrophils) and subsequently inaccurate platelet results.
• Because most cell counters are not able to identify this preanalytical problem, platelets are thus
counted as WBCs, resulting in spurious leukocytosis and false thrombocytopenia.
• EDTA-induced pseudothrombocytopenia has so far been observed in healthy and diseased
individuals and is not related to gender and age.
• The hypothesized mechanism in pseudothrombocytopenia involves IgM autoantibodies directed
against platelet IIb/IIIa fibrinogen receptors. This is further supported by the fact that platelets
from patients with Glanzmann thrombasthenia (in which platelets have either defective or low
levels of gp IIb/IIIa) do not react to autoantibodies from pseudothrombocytopenic patients.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hematology Testing
Stability during transport and storage has been studied for a number of analytes.
• Some parameters are very stable (HB, RBCs), while others are not (reticulocytes, MCV, HCT).
• The analyte stability may differ depending on the parameters being measured, instrument type,
and transport and storage conditions.
As a general rule, the EDTA blood should be stored at RT and analyzed within 3 hours of collection.
The stability of hematological parameters is improved if samples are kept at 4°C.
Here are the ICSH data on the stability of some hematology parameters:
― Hb concentration and RBCs count are stable up to 72 hours if blood is kept at 4°C.
― Platelet and reticulocyte counts are stable for 24 – 72 hours if blood is stored at 4°C.
― WBC count with automated differential count is stable for at least 24 hours if blood is kept at 4°C
and up to 6 hours at room temperature.
― PB smear should be made from blood stored not >3 hours at room temperature (18 – 25°C).
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hematology Testing
Antibodies may affect the cell count of RBCs, WBCs, and platelets.
SPECIFIC CONSIDERATIONS
Hematology Testing
Cold Agglutinins
Cold agglutinins are antibodies that are specific for RBCs surface carbohydrate antigens, which bind
to the RBC surface at temperatures of 0 – 4°C. Binding of agglutinins causes RBCs agglutination,
induces complement activation and hemolysis, and impairs peripheral circulation.
Cold agglutinins may be monoclonal or polyclonal.
― Monoclonal agglutinins are found in cold agglutinin disease or lymphoproliferative disorders,
― Polyclonal antibodies are often found in patients recovering from some infectious diseases.
SPECIFIC CONSIDERATIONS
Hematology Testing
Cold Agglutinins
Cold agglutinins should be suspected if the following criteria are observed:
1. RBCs count too low even at normal Hb concentration
2. Grossly elevated MCV values
3. Low values of calculated HCT with too high MCH and MCHC values without any clear explanation.
4. Falsely elevated WBCs count and platelet count, depending on their size, are counted in either the
leucocyte or platelet channel.
5. The blood smear may also show agglutination of RBCs.
SPECIFIC CONSIDERATIONS
Hematology Testing
Cryoglobulins
Cryoglobulins are Igs with temperature-dependent solubility that precipitate at temperatures < 37°C.
• Often associated with infections, autoimmune disorders, and malignancies,
• Cause organ damage via immune-mediated process and vascular damage by ↑ blood viscosity.
Cryoglobulins precipitation depends on the Ig class and the pH (ppt is absent at pH < 5.0 or > 8.0)
In samples kept at room temperature, cryoglobulins tend to form globular or cylindric precipitates
that are then counted as cells, leading to false leucocytosis and/or thrombocytosis depending on:
― The time of exposure
― The temperature
― Cryoglobulin concentration
― The interaction of cryoglobulins with other plasma proteins.
Total Quality Management 5. Process Control 1. Pre-analytic phase
SPECIFIC CONSIDERATIONS
Hematology Testing
Cryoglobulins
The following indices may point to the presence of cryoglobulins:
1. Very different cell counts in different investigations
2. Blue sediments in differential count samples
3. In a sample warmed up to 37°C, significantly lower cell counts
As in the case of cold agglutinins, for adequate analysis of samples in which cryoglobulins are
suspected, blood samples should be kept warm at 37°C and analyzed immediately afterward.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen labelling
Specimen labelling
Specimen labelling
Examples of wrong labeling
Backward Belt Wrinkled Scarfy Twisted Turtle neck Upside Down The Group
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
Accurate patient and specimen identification are required for quality patient care.
Patient and specimen misidentification occurs in numerous phases of the testing process.
Specimen Misidentification
How to Prevent Specimen Misidentification?
1. Following the rules and the instructions
I. During the preanalytical phase
A. when the patient presents to the hospital, doctor’s office, or phlebotomist.
― Accurate identification requires collection of at least two unique identifiers from the
patient and ensuring that those match the patient’s prior records. If a patient is unable
to provide identifiers (i.e. neonate or patient in a coma) a family member or nurse
should verify the identity of the patient.
― Information on laboratory requisitions or electronic orders must also match patient
information in their chart or electronic medical record.
― Specimens should not be collected unless all identification discrepancies have been
resolved.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
How to Prevent Specimen Misidentification?
1. Following the rules and the instructions
I. During the preanalytical phase
B. At specimen collection,
― Ensuring that the collection area is cleared of other patients identification information.
― The sample(s) should be collected and labeled in front of the patient.
― The specimen should be sent to the laboratory with the test request.
C. Upon acceptance into the laboratory,
― The identifiers on the specimen should match the requisition and/or electronic order.
― For non-barcoded specimens, specimens should be accessioned, labeled with a
barcode (or re-labeled,), processed, and sent for analysis.
― Identification of the specimen should be carefully maintained during centrifugation,
aliquoting and analysis.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
How to Prevent Specimen Misidentification?
1. Following the rules and the instructions
II. During the analytical phase
― Automated analyzers use bar codes to identify specimens during analysis and result
reporting.
― For manual assays, carefully match identifiers on specimens with work lists.
― Laboratories should carefully monitor repeats, dilutions, add-ons and reflex testing,
particularly if these are manual processes.
― Ensuring that barcodes are accurately printed, as poorly or misprinted barcodes
may be read incorrectly by laboratory instruments. Such procedures may include
regular cleaning and services of label printers.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
How to Prevent Specimen Misidentification?
1. Following the rules and the instructions
III. During the post-analytical phase
― With automated analyzers, results are electronically transferred to middleware or the
laboratory information system (LIS), where rules may dictate whether results are auto
verified or require attention from a technologist or pathologist.
― With manual assays, results are manually entered and technologists must match
patient identifiers on specimens, worklists, or result print-outs with information in the LIS.
― Most LISs are interfaced with hospital information systems to report results in
individual patient’s charts. In the absence of electronic medical record, laboratory
representatives must print laboratory results and fax or mail them to treating physicians.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
How to Prevent Specimen Misidentification?
2. The Use of Delta Checks
• Delta checks are a simple way to detect mislabels.
• A delta check is “a process of comparing a patient’s result to his/her previous result for any
one analyte over a specified period of time”.
• The difference or “delta”, if outside pre-established rules, may indicate a specimen mislabel
or other preanalytical error.
• Laboratories should determine:
― The difference in concentration (or relative change) as well as
― The time interval that is most appropriate for each analyte’s delta check.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
How to Prevent Specimen Misidentification?
2. The Use of Delta Checks
• With the increasing use of automation, delta checks have become a common practice for
core laboratories.
• Delta checks are most often set up for assays with little intra-individual variation that are
tightly regulated within patients including:
― MCV, HCT
― Creatinine, BUN
― Bilirubin
― Total protein
• Simulation studies demonstrated that delta checks for MCV, HCT, BUN and creatinine are
the most sensitive for detecting mislabeled specimens.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
How to Prevent Specimen Misidentification?
2. The Use of Delta Checks
Delta checks limitations:
• Further, medical centers should establish their own delta checks based on their individual
patient population. For example,
― Creatinine and BUN delta checks for may not be appropriate for a large dialysis clinic,
― HCT delta checks may be ineffective in a large hematology/oncology practice.
• Delta checks are not appropriate for all analytes because of high intra-individual
variabilities. For example, growth hormone from the case above shows diurnal variation;
concentrations at night are significantly higher than in the morning. Thus, laboratories should
employ measures to prevent mislabels at their source.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Specimen Misidentification
How to Prevent Specimen Misidentification?
3. Specimen rejection procedures
• Laboratories should adopt a strict specimen rejection policy to reduce entry of questionable
specimens into the analytical process.
• For example, laboratories may decide to reject all specimens that arrive unlabeled or that
show disagreement between the requisition and label on the specimen unless they are
irreplaceable (i.e., CSF specimens, surgical specimens, etc.).
4. Auditing
• Laboratories should conduct periodic audits of patient records from requisition to result into
the patient chart to look for errors.
• Intermittent and continual audits of patient ID bands are also helpful in ↓ misidentification.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Post-collection
Variables
Total Quality Management 5. Process Control 1. Pre-analytic phase
Sample transport
• Time
• Temperature
• Handling
Sample processing
• Sample Mis-identification
• Centrifugation
• Cross-contamination
Sample storage
Total Quality Management 5. Process Control 1. Pre-analytic phase
Sample Transport
• All specimens must travel from the collection site to processing and/or testing sites.
• This ranges from short trips down the hall, to long cross-country trips to reference labs.
• No matter the nature of transportation, laboratory staff must be aware of time, temperature,
and turbulence, which can all influence specimen integrity.
CLSI GP44-A4
Specimens must be transported in the appropriate biohazard bags or containers to the
laboratory in as short a time as possible. Unless chilling of the specimen is required, all
specimens should be transported at room temperature. Prompt removal of specimens from the
collection area is especially important if the area temperature is above 22°C, which may cause
some measurands to deteriorate.
Stability of common chemistry and immunochemistry analytes with varying time, temperature, and tube types
Analyte Serum Heparin plasma EDTA plasma Urine < 24 h 24 h 48 – 72 h >14 days
ACTH × 4°, RT
Alpha-fetoprotein × 4°, RT
Albumin × × × 4°, RT
Aldosterone × 4°, RT
Alkaline Phosphatase × × 4°, RT
ALT × × × 4°, RT
Amylase × 4°, RT
AST × × 4°, RT
Bilirubin, total × 4°, RT
BUN × × RT 4°, RT
Calcium × × 4°, RT
Catecholamines × 4°, – 20°
Cholesterol, total × × × RT
hCG × 4°, RT
Creatine Kinase × × 4°, RT
Carbon Dioxide × × RT
Creatinine × × × × RT 4°, RT 4°, – 20°
Cortisol × × RT RT
C-Reactive Protein × 4°, RT
Estradiol × 4°, RT
Stability of common chemistry and immunochemistry analytes with varying time, temperature, and tube types
Analyte Serum Heparin plasma EDTA plasma Urine < 24 h 24 h 48 – 72 h >14 days
Ferritin × × RT
GGT × × 4°, RT
Growth Hormone 4°, RT
Glucose × × 4°, RT 4°
Hemoglobin A1C × RT
HDL × × RT
Homocysteine × 4°
Lactate × × RT
LDH × × RT 4°
Microalbumin × 4°, – 20°
Metanephrines × RT
Phosphorus × × RT 4°
Potassium × × 4° RT RT
Protein, Total × × × 4°, RT
Sodium × × 4° RT
Triglycerides × × RT
TSH × × RT
Free T4 × × RT
4°, RT
Uric Acid × ×
Vitamin B12 × × RT
Total Quality Management 5. Process Control 1. Pre-analytic phase
Every reasonable effort should be made to ↓ transport time between drawing and processing
the sample.
Total Quality Management 5. Process Control 1. Pre-analytic phase
D. Many analytes are simply unstable in vivo and in vitro, and remain intact for a relatively
short time after specimen collection.
ACTH and brain natriuretic peptide (BNP), which are rapidly degraded.
insulin and parathyroid hormone, are subject to a slower degradation,
• Pneumatic tube systems can send carriers containing laboratory specimens, paperwork,
pharmaceuticals, and more throughout a hospital at high speeds. Thus, pneumatic tube
systems are in wide use in medical centers around the world.
Total Quality Management 5. Process Control 1. Pre-analytic phase
To chill a specimen,
― Place it immediately in a mixture of ice and water.
― Provide a good contact between the cooling medium and the specimen.
Large cubes of ice instead of ice water are not acceptable because of inadequate contact
between the coolant and the specimen.
― Avoid direct contact between specimen and ice, this may cause hemolysis.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Cryoglobulins
Certain analytes, such as cryoglobulins, must be maintained at body temperature, and require
transit in a warm water bath, kept around 37°C.
Total Quality Management 5. Process Control 1. Pre-analytic phase
These specimens should be protected with an aluminum foil wrap, an amber specimen
container, or the equivalent. ﻛﻬﺮﻣﺎن
Total Quality Management 5. Process Control 1. Pre-analytic phase
• Refrigerated specimens should be sent with sufficient frozen packs to keep the interior of the
container between 0 – 10°C.
― Frozen specimens should be sent with dry ice.
― One solid piece of ice (1″x3″x4″) should be enough to keep a sample frozen for 48 hours.
― Staff must be properly trained for the shipping of biological specimens and dry ice.
• Biological specimens should be treated as infectious agents and therefore are subject to
specific laws and regulations; dry ice is considered a hazardous material to ship and thus
requires special considerations. Overnight or next-day shipping ↓ transit time and preserves
specimen integrity.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Ideally, all blood gas specimens should be measured immediately and never stored.
― If analysis will occur within 30 min, a plastic syringe, transported at RT, is recommended.
― If testing is delayed for more than 30 min, specimens should be collected in a glass syringe
and immediately immersed and kept in a mixture of water and crushed ice to chill the
specimen. Plastic contracts with cooling, making pores large enough for atmospheric O2 to
cross into the tube, but not CO2.while glass does not allow the diffusion of O2 or CO2.
Total Quality Management 5. Process Control 1. Pre-analytic phase
pH patient’s adjusted pH
pH = pHm – [ 0.0147 + 0.0065 × ( [ pHm – 7.40 ) ] ( T – 37° ) T
pHm
patient’s temperature
measured pH
iCa2+ CO2 loss causes ↑ pH, which lower the iCa2+ due to ↑ Ca2+ ions binding to albumin
On average, iCa2+ ↓ by about 0.036 mmol/L for each 0.1 ↑ in pH
iMg2+ pH alterations also affect iMg2+ concentrations, the effect is about 1/3 that of iCa2+
On average, each 0.1 change in pH changes iMg2+ by about 0.012 mmol/L
Total Quality Management 5. Process Control 1. Pre-analytic phase
Sample Processing
• Due to instability of certain analytes in unprocessed serum or plasma,
CLSI recommend that plasma or serum be separated from cells as soon as possible,
but definitely within 2 hours of collection.
• Residual fibrin occur secondary to improper specimen handling (during or after collection)
― Fibrin may be present as a visible clot or as invisible microfibers or as strands.
― These invisible fibrin strands may directly affect some assays, like troponin.
― Fibrin interference usually is not reproducible and disappears with time as the fibrin
settles out of the sample.
― Fibrin strands can be eliminated if the recommended times for blood clotting and
subsequent centrifugation are employed.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Manufacturers provide recommendations for appropriate RCF and spin times for individual tube types
Total Quality Management 5. Process Control 1. Pre-analytic phase
Swinging bucket rotors allow for a more reliable barrier seal and will not cause hemolysis at
appropriate speed and temperatures and are recommend by most tube manufacturers.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Due to the conflicting data on the effect of re-centrifugation on the different analytes, to avoid
erroneous results, re-centrifugation should be avoided.
Stability of common chemical and immunochemical analytes in serum or heparin plasma separator tubes after re-centrifugation
Potassium × RT
× 4°C
Sodium × RT, 4°C
Chloride × RT, 4°C
CO2 × RT, 4°C
BUN × RT, 4°C
Creatinine × RT 4°C
Glucose × RT 4°C
Calcium × RT, 4°C
Total protein × RT, 4°C
Albumin × 4°C RT
Total Bilirubin × RT, 4°C
ALP × RT, 4°C
AST × RT, 4°C
ALT × RT 4°C
HDL-C × RT, 4°C
Cholesterol × RT, 4°C
Triglycerides × RT, 4°C
LDL-C × RT, 4°C
TSH × RT, 4°C
Free T3 × 4°C RT
Ferritin × RT, 4°C
Vitamin B12 × RT, 4°C
Shafi H, Sadrzadeh H. The effect of recentifugation on serum separator tubes on concentration of serum analytes. Ann Clin Lab Sci 2012;42:318-9.
Total Quality Management 5. Process Control 1. Pre-analytic phase
The manufacturer’s guidelines should always be followed unless there is proven documentation
that deviations do not affect test results.
Different testing methods may have different stability requirements for the same analyte.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Sample Storage
CLSI guidelines recommend the following as “general” guidelines for in-lab specimen storage.
1. Serum/plasma should be separated from cellular part immediately after centrifugation, either
by transferring to a new tube, or by use of physical separators, such as gel.
2. Separated specimens can be stored, tightly capped to avoid evaporation and concentration,
up to 8 h at RT (preferably 20 – 25°C ), up to 48 h at 4°C, and after 48 h specimens should
be frozen at –20°C.
3. Samples should be sudden frozen on dry ice or liquid nitrogen to avoid gradient formation.
4. Prior to analysis,
― Thawing specimens at room temperature because heating may denature analytes.
― Gentle inversion can remove gradients formed with freezing.
― Centrifugation will sediment cellular material and/or fibrin strands that form upon freezing.
5. Although repeat freeze/thaw cycles are not recommended by CLSI, very few analytes are
affected by this process.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Sample Storage
There are many analytes that cannot be stored according to the CLSI “general” recommendations.
Whole blood specimens should not be centrifuged. whole blood Storage depends on the analyte.
Blood freezing induces hemolysis and is not recommended for hematological or ABG testing.
Sample Storage
Because of the number of exceptions to the general in-lab specimen handling procedures,
laboratories should:
• Consult manufacturers’ package inserts for appropriate storage conditions prior to analysis.
• Conduct in-house stability studies prior to changing approved in-lab storage conditions.
Some studies suggest that utilization of gel separator tubes eliminates the need to physically
separate plasma/serum from cells for short term in lab storage. However,
― Gel separator tubes should not be used for certain analytes as gel polymers may cause
significant negative interference (e.g. drugs and estradiol).
― Further, plasma separator tubes may not be appropriate for storage of common chemistry
analytes at 4°C for greater than 48 hours.
• After long-term storage, barrier seals should be inspected on all separator tubes prior to analysis.
• Laboratories should consult manufacturers’ instructions for a list of analytes that are stable in
these tubes for long-term storage.
Total Quality Management 5. Process Control 1. Pre-analytic phase
B
Pre-analytic
Interfering Factors
Total Quality Management 5. Process Control 1. Pre-analytic phase
Interferents
Any substance whose presence:
― interferes with an analytical procedure
― generates incorrect results.
Either:
1. In Vivo interferents originate from the substances present in the patient sample,
2. In Vitro interferents relate to the effect of various substances added to the patient
sample, such as separator gels, anticoagulants, surfactants, and so on.
Low Result
Low 25-OH vit. D result despite replacement therapy Incorrect diagnosis of hypovitaminosis D
Negative hCG level Missed diagnosis of choriocarcinoma
False low digoxin results Wrong treatment (digoxin overdosing, digoxin toxicity)
Low insulin level Missed diagnosis of insulinoma
False negative troponin results Missed diagnosis of myocardial infarction
Total Quality Management 5. Process Control 1. Pre-analytic phase
1. In Vivo Interferents
Total Quality Management 5. Process Control 1. Pre-analytic phase
I
Interference caused by
Endogenous Blood Components
Triglycerides
OxyHemoglobin
Bilirubin Absorption curves of:
• Oxyhemoglobin in serum with characteristic peaks at 415, 540,
and 570 nm (red);
• Triglycerides absorption curve cover wide range of wavelengths,
Absorbance
Wavelength (nm)
300 350 400 450 500 550 600 650 700 750
380
780
UV Visible Light IR
Total Quality Management 5. Process Control 1. Pre-analytic phase
1
Hemolysis
Interference
Total Quality Management 5. Process Control 1. Pre-analytic phase
Hemolysis is defined as a process of membrane disruption of RBCs and other blood cells, accompanied
by the subsequent release of cell components into the plasma and red coloration of the serum (or
plasma) to various degrees after centrifugation.
Hemolysis is the most common preanalytical error and the most common cause of sample rejection.
Types of Hemolysis
Published Recommended criteria for differentiation between in vivo and in vitro hemolysis
1. Collect both serum and plasma sample.
2. Because anticoagulation of blood helps minimize in vitro hemolysis, perform all tests on
plasma whenever in vivo hemolysis is suspected.
3. Measure Hb and K+ concentrations and LDH activity in the serum and plasma specimens.
4. Specimens with ↑ LDH activity and Hb concentrations, but normal K+ concentrations suggest
in vivo hemolysis.
5. Measure haptoglobin, hemopexin and the reticulocyte count to confirm in vivo hemolysis
Blank DW, Kroll MH, Ruddel ME, Elin RJ. Hemoglobin interference from in vivo hemolysis. Clin Chem 1995;31:1566-9.
Total Quality Management 5. Process Control 1. Pre-analytic phase
• Via partially obstructed catheter • Transport via pneumatic tube • Freezing thawing cycles
• Collection of capillary blood • Significant time delay • Improper storage temperature
• Excessive aspiration force • Inappropriate temperature • Length of storage time
• Needle gauge
• Length of time that tourniquet is used
• Fist clenching by patient • Significant time delay between receipt of specimen
• Tube under filled and centrifugation
• Vigorous sample mixing after collection • Centrifuge temperature extremes
• Lack of mixing following collection • Speed of centrifugation
• Transfer from syringe into vacutainer • Re-centrifugation of previous centrifuged specimen
• Poor barrier separation if gel barrier tubes are used
Total Quality Management 5. Process Control 1. Pre-analytic phase
• The most pronounced effect of hemolysis is seen for LDH as it may be increased by:
― over 20% at 0.027 g/dL free Hb concentration (mildly hemolyzed samples)
― over 60% at 0.075 g/dL free Hb concentration
― over 350% at 0.334 g/dL free Hb concentration (grossly hemolyzed samples)
• K+ may also escape from platelets during clotting, so is a marked difference in the
concentration between serum and plasma,
Therefore, plasma is more the recommended for accurate K+ measurement.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Intracellular Concentration
Analyte (Compared to Extracellular)
LDH ↑ 160 ×
Phosphorus ↑ 100 ×
Potassium ↑ 40 ×
AST ↑ 40 ×
Folic acid ↑ 30 ×
ALT ↑7×
Magnesium ↑3×
Total Quality Management 5. Process Control 1. Pre-analytic phase
4. Chemical interference
• Free hemoglobin pseudo-peroxidase activity interferes in measurement of bilirubin
through the inhibition of the formation of diazonium salt.
• Proteolytic enzymes released from RBCs may mask or enhance epitope recognition in
various immunoassays.
̶ The degree and direction of bias are analyte- and method-dependent.
̶ Examples of –ve interference: cTnT, insulin, cortisol, testosterone and vit. B12
̶ Examples of false-positive increases: PSA and cTnI.
Total Quality Management 5. Process Control 1. Pre-analytic phase
2
Lipid
Interference
Total Quality Management 5. Process Control 1. Pre-analytic phase
Lipid
occupied by lipids. Particles that are not lipid soluble are displaced by the
lipids to the water part of the plasma.
Lipid
• Therefore, lipemia leads to a false decrease in the measured analyte
― Water-soluble analytes are more concentrated in the lower layer, Lower layer of
― whereas lipids and lipid-soluble analytes, such as drugs and lipid- water-soluble
analytes
soluble hormones, are more concentrated in the top lipid-rich layer.
• This is especially important in automated chemistry analyzers with
fixed path lengths of the sample probe. Test results may differ for
those analytes that are not evenly distributed between the lipid and
water portion of the sample, depending on the part of the sample from
which the sample probe is taking the sample for analysis.
Lipid gradient
in centrifuged sample
Total Quality Management 5. Process Control 1. Pre-analytic phase
b. Moreover, lipemia may even affect some immunochemistry assays by masking the binding
sites on antigens and antibodies and thus physically interfering with antigen–antibody
binding.
Total Quality Management 5. Process Control 1. Pre-analytic phase
• RPM (revolutions per minute) is the way in which we describe how fast a centrifuge is going.
• This is the rate at which the rotor is revolving regardless of its size.
• G-Force or RCF (relative centrifugal force) is the force being exerted on the rotor contents.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Recovery %
80
― underestimation of CRP (−92%),
― underestimation of CK-MB (−25%), 60 cTn-T
― underestimation of and GGT (−30%) P
Alb
― overestimation of Troponin T (+20%) 40 Ca
― overestimation of phosphates (+7%) TP
CK-MB
20
GGT
CRP
0
Total Quality Management 5. Process Control 1. Pre-analytic phase
• It is up to each individual laboratory to establish its own procedure for managing lipemic
samples, bearing in mind to ensure the highest possible accuracy of results and speed.
• To minimize the prolongation of the turnaround time and subsequent delays in reporting the
results for grossly lipemic samples, laboratories may consider analyzing electrolytes using
the blood gas analyzers (direct ISE methodology) while manipulating the rest of the
sample to remove the lipids.
Total Quality Management 5. Process Control 1. Pre-analytic phase
3
Icteric
Interference
Total Quality Management 5. Process Control 1. Pre-analytic phase
• For maximal patient benefit, labs may consider having special protocols (dilutions or different
methods) for some critical analytes in icteric samples to avoid unnecessary sample
rejections.
Total Quality Management 5. Process Control 1. Pre-analytic phase
2. Chemical Interference
Bilirubin produces negative bias on assays that
involve H2O2 as an intermediate reaction
(e.g. cholesterol, glucose, uric acid, TGs).
Total Quality Management 5. Process Control 1. Pre-analytic phase
Color standard scales provided by Clinical Institute of Chemistry, University Hospital Center “Sestre milosrdnice,” Zagreb, Croatia.
Total Quality Management 5. Process Control 1. Pre-analytic phase
When interferences are causing unacceptable bias and results are clinically inaccurate,
1. Such results should not be reported and sample redraw should be requested.
2. Such a test report should always be accompanied with comments informing the clinical staff
about the reasons for not reporting the originally requested test results.
3. It is also important that the lab notify the staff when sample appearance (color, turbidity)
deviates from a normal state by including a comment on a test report (e.g. sample hemolyzed,
icteric, lipemic, or turbid), even if the tests are not affected by this change in appearance (Such
comments provide useful information to the clinicians).
4. Comments should also indicate if the sample has been treated in any way to minimize the
effect of interfering substances (e.g., delipidation).
Total Quality Management 5. Process Control 1. Pre-analytic phase
4
Paraproteins
Interference
Total Quality Management 5. Process Control 1. Pre-analytic phase
Mechanisms of Interference
Paraprotein may affect chemistry assays through several mechanisms:
a. Precipitation of the Paraprotein
b. Binding of Paraprotein to Assay Components
c. Volume displacement
d. Change of sample viscosity
Total Quality Management 5. Process Control 1. Pre-analytic phase
Non-aqueous Solution
(Lipids, Proteins)
5
Heterophilic Antibody
Interference
Total Quality Management 5. Process Control 1. Pre-analytic phase
Heterophilic antibodies (HAs) are polyclonal heterogeneous human antibodies in a specimen that
interact with assay antibodies (of animal source) to give false positive or negative results.
Types
1. Nonspecific Antibodies Interact poorly and nonspecifically with the assay antibodies.
2. Anti-animal antibodies Which interact strongly and specifically
(with respect to the animal species in which the assay antibodies have been raised).
• Amongst all HAs, Only those with sufficient titer and affinity towards the reagent antibody used in
an assay will cause interference.
• Among the anti-animal antibodies, the most common occurrence is of human anti-mouse
antibodies (HAMA), because of wide use of murine monoclonal antibody products in therapy
or imaging.
• Heterophilic antibody interference may cause critical impact and clinical misjudgment, resulting
in unnecessary follow-up testing and unneeded but potentially dangerous therapy, leading to
significant patient morbidity and even to mortality.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Since heterophilic antibodies are found mainly in serum, plasma or whole blood, but not in urine,
such interference is not found in urine specimens.
― This gives an excellent way to detect the interference for analytes that may be present in both
matrices (e.g. serum and urine).
― For example, many case studies with false +ve hCG in serum/plasma, if hCG was measured
in parallel urine samples, the false results could have been easily avoided.
• However, when the sample was assayed in another digoxin immunoassay that required protein
precipitation before the assay, they found no digoxin in the sample.
• Similar results were found in 2 other digoxin assays, using different assay antibodies, as well.
• These results demonstrated that the original false +ve results were caused by HA interference.
Liendo C, Ghali JK, Graves SW. A new interference in some digoxin assays: anti-murine heterophilic antibodies. Clin Pharmacol Ther 1996;60:593-8.
Total Quality Management 5. Process Control 1. Pre-analytic phase
RF interference follows the same mechanism as interference from other types of HAs.
― In two-antibody immunometric assays, RF bridges the capture and label antibodies
without involving the antigen and generates false positive signal and results.
― In single-antibody competition immunoassays, RF binds to assay antibody, preventing its
reaction to the label reagent, thus reducing signal and generating false positive results.
If RF is suspected, the patient history needs to be examined for diseases that may ↑ serum RF.
― RF concentration in the sample may be measured by many of the commercial assays.
― RF can be removed from the sample by the many separation steps.
Total Quality Management 5. Process Control 1. Pre-analytic phase
A case study of false positive thyroglobulin in a RA woman with confirmed RF interference by:
a. Non-linear dilution
b. Alternate thyroglobulin immunoassay employing different antibodies
c. Precipitating out interfering RF and retesting
Astarita G, Gutie´rrez S, Kogovsek N, et al. False positive in the measurement of thyroglobulin induced by rheumatoid factors. Clin Chim Acta 2015;447:43-6.
A case of 60-year old male where RF was suspected to interfere in multiple assays (LH, FSH, SHBG,
PRL, hCG, TSH) resulting in false positive results; PEG precipitation brought all results to normal.
Mongolu S, Armston AE, Mozley E, Nasruddin A. Hetrophilic antibody interference affecting multiple hormone assays: is it due to rheumatoid factor? Scand J Clin Lab
Invest 2016;76:240-2.
Total Quality Management 5. Process Control 1. Pre-analytic phase
• The HAAA produced against animal Igs, can have anti-idiotype or anti-isotype specificity.
― Anti-idiotype antibodies are directed against the hypervariable region of the Ig molecule,
― Anti-isotype antibodies (more common) are directed against the constant regions. The anti-
idiotype antibodies may again generate endogenous anti-anti-idiotype antibodies.
Idiotypic Isotypic
difference difference
Total Quality Management 5. Process Control 1. Pre-analytic phase
• The prevalence estimates of HAAA, especially HAMA, have been studied by many authors; they
vary between less than 1% to 80% among different hospitalized or outpatient hospitals.
― Several commercial assay kits for HAMA estimation are available.
― However, because HAMA are so heterogeneous, a negative HAMA assay result does not
confirm absence of all types of HAMA in a sample.
Total Quality Management 5. Process Control 1. Pre-analytic phase
As expected, the HAMA can be of varied prevalence, specificity, titer, and binding capacity.
• Most common HAMA concentration is less than 10 mg/mL, however HAMA concentrations as
high as 1000 mg/mL have been reported.
• Since HAMA arise from exposure of patients to mouse antibodies, cancer patients who may
have used such antibodies as part of imaging or therapeutic agents, have higher prevalence
of HAMA occurrences (40-70%).
Total Quality Management 5. Process Control 1. Pre-analytic phase
1 2
Total Quality Management 5. Process Control 1. Pre-analytic phase
Signal Signal
Capture Antibody
Signal
Capture Antibody
Signal
The interfering antibody
interact with the
assay antibody
Labelled antibody
Capture Antibody
Labelled antibody
Capture Antibody
2. The assay insert should be examined for the types of antibodies and if heterophilic
antibody blockers used in the assay.
3. A dilution linearity study with the specimen after successive dilutions with the assay diluent,
the interfering antibody is diluted enough as to not cause any assay interference.
4. Blocking of the interfering antibody. There are various commercial blockers for heterophilic
antibody or HAAA (preferably from the same species used to raise the reagent antibodies). The
blocker can be non-immune animal serum, polyclonal antibody, polymerized IgG, nonimmune
(irrelevant) mouse monoclonalor a mixture of monoclonal antibodies or fragments of IgG.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 2. In Vivo Interferents ► II. Exogenous components
II
Exogenous component
introduced to the blood
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Mechanism of interference
A. Biological influences are the result of an in vivo action of a drug or metabolite.
Drugs can alter levels of a large number of tests by several mechanisms:
― Induction of hepatic microsomal enzymes (phenytoin raises levels of GGT)
― Enzyme inhibition (finasteride and dutasteride cause a ↓ in PSA by inhibition of 5α-reductase)
― Displacement of the drug from the protein-binding site (tizoxanide alters free warfarin
fraction by its displacement from the protein-binding site; this effect can be monitored by
alterations in coagulation parameters)
All of these drug actions occur in vivo, and changes in parameters reflect a true state in the human
body. Therefore, alteration in concentration of the measured parameter is not an analytical error.
Total Quality Management 5. Process Control 1. Pre-analytic phase
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Mechanism of interference
B. Analytical (chemical) interference is caused when the presence of the drug directly or
indirectly leads to falsely ↑ or ↓ concentration/result of a measured analyte.
― Structural similarity ― The parent drug or its metabolite can have structural similarity to the
tested analyte and therefore interfere in immunochemical or photometric methods.
― Reaction interference ― can occur when the compound or its metabolites catalyze or inhibit
some steps of the chemical or immunochemical reaction.
― Alteration of Sample Integrity ― Some drugs can interfere with the integrity of the sample
by changing sample density (viscosity) and cause obstruction problems on analytical systems
(e.g., iodine-based contrast media).
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Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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There is evidence that ethamsylate also causes significant false ↓ in the concentrations of
cholesterol (9.2%), TGs (15.6%), and uric acid (15.4%).
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Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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Metamizole Acetaminophen
Ethamsylate
Ethamsylate cefpirome
Acetylsalicylic acid
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Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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Leflunomide product
in Egyptian market
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Medical contrast media are used during medical imaging procedures to enhance the contrast of
organs and fluids.
* Iodine-based compounds
― e.g. iohexol, iodixanol, and ioversol
― Mostly used for the x-ray methods.
* Other contrast media may interfere with some clinical chemistry assays
― Such as ACE, TIBC, zinc, magnesium, and creatinine.
― All of these interferences are assay-specific, and contrast media agent-specific
Total Quality Management 5. Process Control 1. Pre-analytic phase
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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Physical interference:
• Any measurement can be obstructed because of the interference with the sample integrity.
• Iodine molecules have high density and can prevent proper formation of the barrier in the
serum gel separator tubes.
Chemical interference:
• Iopromide is used as a contrast media agent in coronary angiography.
• Sometimes, if the sample is taken immediately after coronary angiography,
A false ↓ in cTnI concentration is detected when measure by reagents of certain manufacturers
For example: Opus Magnum reagent (Opus cTnI immunoassay; Behring Diagnostics, Siemens)
Total Quality Management 5. Process Control 1. Pre-analytic phase
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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* Problems
a. Patients consume herbal and other dietary products, but they fail to report the
usage to their doctors or to laboratory staff when they come in for blood sampling.
b. The exact content of these herbal preparations is not always known.
c. Labeling of herbal products is inaccurate.
d. The influence of these products on laboratory tests is not fully known.
Total Quality Management 5. Process Control 1. Pre-analytic phase
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* Cross-reactivity
Herbal medicines can cause direct interference with immunoassays due to cross-reactivity.
Due to their structural similarity to the tested analyte, active compounds that are present in herbal
products can react with the antibody in the assay result in both falsely ↑ and ↓ analyte levels.
• Preparations used in Chinese medicine, like Chan Su, can contain bufadienolides
(which is used for the treatment of tonsillitis, sore throat, furuncle, and heart palpitations).
― The structural similarity of bufadienolides and digoxin is responsible for both cardiotoxicity
and interference in the immunochemistry method.
― Both false ↑ and ↓ of the digoxin measurement can occur, depending on the assay format.
• Herbal supplements that are used widely throughout the world, like ginseng,
― Can also interfere with digoxin measurement,
― However, some more recently introduced chemiluminescent microparticle assays seem to
be free of such interference.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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In the case of accidental poisonings with herbs, household cleaning products, or any other
exogenous compounds, interferences on test results can prolong the diagnostic procedures in
acute patient care and cause harm to the patient.
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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• Potential drug interferences are numerous, and not all of them can be recognized or predicted.
• The largest available online source for analytical interferences is the Effects on Clinical
Laboratory Tests series, edited by Young and colleagues.*
― This database has compiled the large body of evidence from the published literature and
is the most extensive source of analytical interferences.
― For example, the database lists 307 results of potential drug interferences for creatinine.
• Laboratory professionals should be alerted by any unexpected result and discuss it
with the clinical staff.
• If the source of suspected interference cannot be determined, laboratories should try to
involve manufacturers of the reagents to identify the potential interfering substance and
quantify its effect.
* Young DS. Effects on clinical laboratory tests: drugs, disease, herbs and natural products.
Available at: <http://eu.wiley.com/WileyCDA/WileyTitle/productCd-1118477979.html>.
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Background
• Biotin (vitamin B7) is a coenzyme involved in multiple metabolic processes, including:
― CHO metabolism
― Fatty acid synthesis
― Amino acid catabolism
― Gluconeogenesis
• Humans and other mammals cannot synthesize biotin and it must be derived exogenously.
• Recommended daily biotin intake is 30 µg. Although the inclusion of biotin in over-the-counter
multivitamins, biotin supplementation is not usually necessary because biotin is found in
numerous foods (e.g., meat, fish, nuts, grains, eggs, and dairy products).
• High doses of biotin in supplements (up to 3333 times the recommended daily dose, i.e., 100 000
µg [100 mg]) have been used for a variety of medical conditions, including diabetes, lipid
disorders and peripheral neuropathy, as well as for hair, nail, and skin health.
Total Quality Management 5. Process Control 1. Pre-analytic phase
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Background
• Biotin is also an essential reagent in biochemical applications.
• Its binding with avidin (e.g., streptavidin), one of the strongest noncovalent irreversible
bonds, has been exploited in biochemical studies since the 1970s.
• Many laboratory tests that have been approved or cleared by the US FDA are immunoassays that
utilize biotin and streptavidin binding in the assay design (i.e., biotinylated immunoassays).
• Their strong binding is particularly useful in:
― Increases the ability of the assay to detect lower quantities of the analyte
― Decreases the number of steps required for analyte measurement
― Allows for more rapid measurement of biomolecules of interest
Total Quality Management 5. Process Control 1. Pre-analytic phase
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Background
• Normal circulating concentrations of biotin derived from the diet are too low to interfere with
biotinylated immunoassays.
• Biotin in multivitamins (doses < 1 mg) has not been reported to cause interference.
• However, ingestion of high-dose biotin supplements (e.g., ≥ 5 mg) results in significantly ↑
their blood concentrations that can interfere with commonly used competitive biotinylated
immunoassays.
• Evidence of interference has been described in case reports, in vivo studies (with study
participants), and in vitro studies (biotin addition to mimic high blood concentrations).
• In November 2017, the FDA released a Safety Communication warning the public that biotin
supplementation may interfere with laboratory tests.
Total Quality Management 5. Process Control 1. Pre-analytic phase
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Mechanism of interference
In competitive (sandwich) immunoassay model Signal
B
• The remaining signal will be inversely proportional Streptavidin
to the concentration of analyte.
Streptavidin-coated
microparticle B Biotin in sample
or solid phase
Total Quality Management 5. Process Control 1. Pre-analytic phase
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Mechanism of interference
In competitive (sandwich) immunoassay model
B. However, if there is excess biotin in the specimen
• The biotin will bind to the streptavidin sites, blocking
washed off
the biotinylated antibody and hence the analyte of
interest.
• The biotinylated antibody will bind to the analyte of B
interest.
B
• But without being tethered to the solid phase, it will
B B
be washed off, resulting in a signal that is falsely ↓,
which will lead to a false ↑ result.
Streptavidin-coated
microparticle B
or solid phase
Total Quality Management 5. Process Control 1. Pre-analytic phase
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Mechanism of interference
• Assays in which biotinylated antibodies are already bound to streptavidin coated microparticles
are generally not affected by biotin interference.
• The biotin: streptavidin interaction is almost irreversible and pre-formed biotin: streptavidin is not
readily displaced by excess free biotin in the sample.
B
B
B
B
B
B B
Streptavidin-coated
microparticle B
or solid phase
Total Quality Management 5. Process Control 1. Pre-analytic phase
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Biotin Tolerance
• Many manufacturers report thresholds of biotin tolerance to indicate the concentration of biotin
expected to cause significant analytical bias, usually defined as 10%.
Roche Elecsys immunoassays
― All Roche Elecsys immunoassays are sensitive to biotin
― with tolerances ranging from 10 – 120 ng/mL
― and more than 90% of immunoassays at 30 ng/mL or above
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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Biotin Tolerance
Siemens Healthineers
― Many of the Centaur ® and Immulite ® immunoassays do not employ biotin: streptavidin, but for
those that do, the majority are resistant to biotin interference, using streptavidin-magnetic particles
pre-complexed with biotinylated reagents.
― About 15% of Centaur ® and Immulite ® assays are biotin-sensitive, including the Troponin I.
― The Dimension EXL ® and Dimension Vista LOCI ® include numerous biotin-sensitive assays, but
the tolerance for the majority is greater than 100 ng/mL.
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► II. Exogenous components
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2. Medication management
― Biotin interference can be viewed as a particular case of adverse drug event.
― The biotin supplementation should be given in a right way to the right person.
4. Surveillance
―Electronic surveillance is used to target high-risk samples for further integrity checks and/or
reflex testing. High-risk patients and results can be identified through clinical notes or
drug/script alerts, and suspicious analytical results.
Total Quality Management 5. Process Control 1. Pre-analytic phase
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2. In Vitro Interferents
Total Quality Management 5. Process Control 1. Pre-analytic phase
Lubricants
• Lubricants like silicone oils and glycerol facilitate insertion and removal of the stoppers.
― Glycerol should not be used as a stoppers lubricant in tubes that will be used for TGs testing
because their interference with most TGs assays.
― Silicone-based lubricants are less likely to interfere with TGs assays, although silicone can
falsely ↑ Mg2+ and T3 levels. Additional peaks in mass spectrometry in the presence of
silicone-based lubricants can interfere with interpretation of results.
Surfactants
• Silicone surfactants used to ↓ nonspecific adsorption of components on tube walls may interfere
with measurement of vitamin B12 and CA15-3.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Clot activators
• Plastic tubes require clot activators to ensure rapid clot formation.
• Some clot activators based on silica particles affect some analytes like lithium and
testosterone.
Pink Purple EDTA is a commonly used additive, especially in the fields of hematology and
endocrinology, because it offers ↑ stability of cells and analytes.
Most hormones (except ACTH) are stable for up to 5 days in EDTA plasma if they
are kept refrigerated at 4°C.
• The main action of EDTA is chelation of cations (e.g., Ca2+, Mg2+, and zinc).
― If EDTA is present in higher concentrations in the sample (when tubes are
underfilled), its chelating activity is enhanced.
― This may lead to interferences in some chemiluminescence immunoassays
that use conjugated ALP as a secondary enzyme in their reactions.
K3EDTA
• For example, underfilling the EDTA tubes by half or more causes clinically
significant bias (the reported concentration was < 75% of the true value) in the
measurement of intact PTH with the DPC IMMULITE assay.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Potassium oxalate
― K+ oxalate is often combined with anti-glycolytics (Na+ fluoride or Na+ iodoacetate)
― Acts as Ca2+ chelating anticoagulant
― As with EDTA, oxalate can also inhibit some enzymes (e.g. amylase, LDH, ALP)
by chelating bivalent cations that are necessary for their activity.
Total Quality Management 5. Process Control 1. Pre-analytic phase
Separator gels are used to ensure rapid and good separation of serum/plasma from clotted blood
and cells, respectively.
Separation of the sample is enabled due to:
― The specific gravity of the gel (1.030–1.060).
― Its ability to undergo a temporary change in viscosity during centrifugation,
― Its ability to lodge between the packed cells and the top serum/plasma layer.
Hydrophobic compounds may bind to the gel, which is why tubes containing separator gels are
not appropriate for some hydrophobic drugs and hormones such as the following:
1. Drugs: phenytoin, phenobarbitol, carbamazepine, tricyclic antidepressants, quinidine, lidocaine
2. Hormones: testosterone, E2, cortisol, free T4, total T3
Total Quality Management 5. Process Control 1. Pre-analytic phase
Analyte Concentration
the amount of
an analyte transferred
Carryover (%) = 100 × (B1− B3) (A2 − B3) from sample A2 to
sample B1
A1 A2 B1 B2 B3
Total Quality Management 5. Process Control 1. Pre-analytic phase
With new tube types containing clot activators (thrombin-based clotting agent),
serum clotting time is reduced (on average < 2.5 minutes) without compromising the sample quality
and stability for most chemistry analytes.
Some analyzers have the ability to detect clots and flag such samples for rerun,
This feature may not available on other analyzers.