Quality Lectures - Pre-Analytic Variables - Dr. Tamer Soliman

Quality in Clinical Laboratories
Dr. Tamer Soliman

Total Quality Management 5. Process Control
Organization Personnel Equipment
Purchasing Process Information

and Control Management
Inventory
Documents
and Occurrence
management Assessment
Records
Facilities
Process Customer and
Improvement Service Safety
Process Control
1. Pre-analytic Sample Management
Test Influencers and Interferences
2. Analytic Quality Control
Method Validation
3. Post-analytic Reporting
Interpretation
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.
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.
Laboratory Errors
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.
Laboratory Errors
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.
Laboratory Errors
Pre-analytic Errors Analytic Errors Post-analytic Errors

~65% ~15% ~20%
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
Laboratory Errors

~65% ~15% ~20%
Test ordering Specimen

Collection
• Unsuccessful phlebotomy
• Traumatic phlebotomy
• Patient complaint about phlebotomy
• Check-in not performed (in the LIS)
• Wrong patient preparation (e.g., non-fasting)
• Therapeutic drug monitoring test timing error
Laboratory Errors

~65% ~15% ~20%
Test ordering Specimen Specimen

Collection Transport
• Inappropriate sample
• transport conditions
• Specimen leaked in transit
• Specimen damaged during transport
• Specimen damaged during centrifugation/analysis
Laboratory Errors

~65% ~15% ~20%
Test ordering Specimen Specimen Specimen

Collection Transport Identification
• Unlabeled sample or label is illegible
• Mislabeled sample: No or incomplete Name or ID on tube
• Wrong name or ID on tube
• Wrong blood type (serum, plasma, whole blood) or sample
• Missing Date/time or Collector’s initials
• Two contradictory labels or overlapping labels
• Mismatch requisition/label
• Specimen information misread by automated reader
Laboratory Errors

~65% ~15% ~20%
Test ordering Specimen Specimen Specimen High

Collection Transport Identification pre-analytical
TAT
• Delay in receiving specimen in lab
• Delay in performing test
• STAT not processed urgently
Laboratory Errors

~65% ~15% ~20%
Test ordering Specimen Specimen Specimen High Specimen

Collection Transport Identification pre-analytical Quality
TAT
• Specimen contaminated with infusion fluid

• Specimen contaminated with microbes
• Specimen too old for analysis
• Hemolyzed
• Clotted or platelet clumps
Laboratory Errors

~65% ~15% ~20%
Test ordering Specimen Specimen Specimen High Specimen Specimen

Collection Transport Identification pre-analytical Quality Container
TAT
• No specimens received/Missing tube • Insufficient specimen quantity for analysis

• Specimen lost in laboratory • Tube filling error (too much anticoagulant)
• Wrong specimen type • Tube filing error (too little anticoagulant)
• Inappropriate container/tube type • Empty tube
• Wrong tube collection instructions
• Wrong preservative/anticoagulant
Laboratory Errors

~65% ~15% ~20%
• High analytical turnaround time

• Instrument caused random error
• Instrument malfunction
• QC failure
• QC not completed
• Test perform by unauthorized personnel
• Results discrepant with other clinical or laboratory data
• Testing not completed
• Wrong test performed (different from test ordered)
Laboratory Errors

~65% ~15% ~20%
‫اﻟﺘﻘﺮﻳﺮ ﱂ ﻳﻜﺘﻤﻞ‬ • Report not completed

‫اﻟﺘﺄﺧﲑ ﰲ اﻟﺘﻘﺎرﻳﺮ ﻋﻦ اﻟﻨﺘﺎﺋﺞ‬ • Delay in reporting results
‫ﻋﺪم أو اﻟﺘﺄﺧﲑ ﰲ اﻹﺑﻼغ ﻋﻦ اﻟﻨﺘﺎﺋﺞ اﳊﺮﺟﺔ‬ • Critical results not called or delayed
‫ﻛﺘﺎﺑﺔ اﻟﻨﺘﺎﺋﺞ ﺑﺸﻜﻞ ﺧﺎﻃﻲء‬ • Results reported incorrectly
‫أﺑﻠﻐﺖ اﻟﻨﺘﺎﺋﺞ إﱃ ﻃﺒﻴﺐ ﻣﻌﺎﰿ ﺧﺎﻃﺊ‬ • Results reported to wrong provider
‫ﻛﺘﺎﺑﺔ اﻟﻨﺘﺎﺋﺞ اﳌﺸﻜﻮك ﻓﻴﻬﺎ‬ • Reported questionable results
‫ﻋﺪم إﳊﺎق اﻟﺘﻌﻠﻴﻖ اﳌﻨﺎﺳﺐ‬ • Failure to append proper comment
‫ﱂ ﻳﺘﻢ ﻣﺮﺟﻌﺔ اﻟﺘﻘﺮﻳﺮ ﻣﺮة أﺧﺮى‬ • Read back not done
‫ﺗﻔﺴﲑ اﻟﻨﺘﺎﺋﺞ ﺑﺸﻜﻞ ﺧﺎﻃﺊ‬ • Results misinterpreted
‫ﻋﺪم اﻟﺘﺼﺮف ﺑﻨﺎء ﻋﻠﻰ ﻧﺘﺎﺋﺞ اﻻﺧﺘﺒﺎرات‬ • Failure to act on results of tests
Laboratory Errors

~65% ~15% ~20%
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
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
Influencing Pre-analytic Interfering

Factors Variables Factors
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
• Affect analytes concentration (↓ or ↑) • Not Affect analytes concentration

• Not affect the analytic method • Affect a specific analytic method
• Can not be eliminated or reduced • Could be eliminated or at least reduced
Classified into Classified into

1. Controllable Factors 1. In Vivo Interferents of the sample
caused by all tasks that occur before the sample (e.g. acetoacetate interferes with creatinine Jaffe method)
arrives in the core laboratory for analysis
2. In Vitro Interferents present in the sample
2. Uncontrollable Factors (e.g. drug interferences fall into this category)
i.e. physiological factors such as gender and age
Total Quality Management 5. Process Control 1. Pre-analytic phase
Pre-analytic variables ► A. Pre-analytic Influencing Factors
A
Pre-analytic
Influencing Factors
A. Pre-analytic Influencing Factors
1. Uncontrollable variables 2. Controllable variables

Physiologic variables caused by all tasks that occur before the sample
1. Gender
2. Age
3. Ethnicity/race
4. Pregnancy
5. Altitude
Pre-collection Collection Post-collection
Variables Variables Variables
Patient preparation Sample collection Sample processing
Sample storage
1. Time of sampling Sample transport
2. Diet
3. Fluid intake
4. Tobacco Smoking
5. Mental Stress

1. Gender
2. Age
3. Ethnicity/race
4. Pregnancy
5. Altitude
Patient preparation Sample collection Sample processing
Sample storage
1. Time of sampling Sample transport
2. Diet
3. Fluid intake
4. Tobacco Smoking
5. Mental Stress
Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

| Age | Gender | Ethnicity/Race | Pregnancy | Altitude |

Unavoidable Influences on Laboratory Results
Influence Examples of Analyte Concentrations Changed Remarks
Age ALP, LDL-cholesterol, hormones, creatinine. Provide age-dependent reference intervals
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.
Gender ALT, γ-GT, creatinine Provide gender-specific reference intervals
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

• 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.
The Canadian CALIPER study is an excellent source of reference intervals in childhood

http://www.sickkids.ca/Caliperproject/intervals/index.html

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly
Intra-uterine Infancy Childhood Adult-life Elderly

Life
Birth Puberty
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.


Life
Birth Puberty
In the early hours of extrauterine life,

― The concentration of some biochemical markers is ↑, thus reflecting the maternal levels,
but it then ↓ within the first 2 weeks of life.
― Examples: (AST, direct and total bilirubin, creatinine, uric acid, CRP, GGT, IgG, LDH,
Mg2+, and phosphate)
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


Life
Birth Puberty
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


Life
Birth Puberty
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.


Life
Birth Puberty
HB, HCT, and the other RBC indices follow a similar pattern, showing the gradual ↑ during
the first 10 years of life.


• FSH and LH
― In boys and girls,
* LH levels ↑ 2 weeks following birth but ↓ to pre-pubertal levels by their 1st year.
* FSH levels follow the same trend as LH levels after birth but ↓ to pre-pubertal levels in
boys by the 1st year of life and in girls by the 2nd year of life.
Fetus Infancy Childhood Puberty Reproductive Years Menopause

Plasma Gonadotropins
100 FSH
Monthly Surges
(mU/mL)
LH
70
10
Birth 6 months 10 – 14 years 50 years



• FSH and LH
― Reduced LH and FSH levels in the early to mid-teen years are not sensitive enough to
distinguish between pubertal delay and hypogonadotropic hypogonadism.
Fetus Infancy Childhood Puberty Reproductive Years Menopause

Plasma Gonadotropins
100 FSH
Monthly Surges
(mU/mL)
LH
70
10
Birth 6 months 10 – 14 years 50 years



• Estrogen
― E2 pre-pubertal concentrations is ~ 0.5 - 5.0 ng/dL for girls and 1.0 - 3.2 ng/dL for boys.
― E2 levels is markedly ↑ at birth then rapidly ↓ to reach the pre-pubertal levels by the
6th month in boys and by 1st year in girls.
Sex Hormones
Menopause
Puberty
Female E2
Male E2
Female Testosterone
10 20 30 40 50 60 70 80 90 Age
(Years)

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.

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%.
Menopausal pre- and post- period (age between 41 – 55 years )

• The transition from the peri-to post-menopausal stage is associated with endocrine changes.
• A strong correlation between age and hCG is observed.
• Notably, the pituitary gland produces hCG in addition to LH, FSH and TSH, which are all
structurally similar glycoprotein.

Menopausal pre- and post- period (age between 41 – 55 years )

• The transition from the peri-to post-menopausal stage is associated with endocrine changes.
• A strong correlation between age and hCG is observed.
• Notably, the pituitary gland produces hCG in addition to LH, FSH and TSH, which are all
structurally similar glycoprotein.
• Clinical confusion surrounds how to appropriately interpret hCG laboratory results since
hCG synthesis is appreciable during healthy pregnancy, cancer or trophoblastic disease.
• Routine measurements of serum hCG (reference limit hCG < 0.5 mIU/mL) in women are
performed to either identify or to rule out pregnancy prior to performing invasive medical
procedures or administering medications that may be harmful to a developing fetus.

less than 5.0 No problem

≤ 45.0 IU/L means
mIU/mL
hCG is mostly of placental origin
Serum hCG 5.0 – 14.0 Measure FSH

> 45.0 IU/L means
(41 – 55 years) mIU/mL (FSH Reflex) hCG is not of placental origin
More than 14.0 Indicative of pregnancy unless the clinical setting dictates
mIU/mL otherwise.


Aging-related diseases are diagnosed, treated and monitored using various in vitro diagnostic testing.
However, interpretation of lab findings in the elderly adult population is challenging due to multiple
confounding factors:
1. Physiologic changes that naturally occur with healthy aging
2. Acute and chronic conditions (kidney disease, diabetes and CVS disease)
3. Diets
4. Lifestyles
5. Medication regimens
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.


Serum proteins
̶ Age-related changes in serum proteins are evident in elderly adults including albumin,
an indicator of malnutrition and disease, which is more significantly affected.
̶ After the age of 60, albumin concentrations ↓ each decade, with significant ↓ noted in
individuals > 90 years old
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 ↑.


Iron
̶ During aging ↓ iron storage, serum iron, and TIBC are noticed .
̶ Depletion of iron stores may be followed by ↑ serum ferritin and ↓ in serum transferrin.
̶ Dysregulated liver synthesis during aging may account for the low transferrin levels.
̶ It is hypothesized that the anemia in this age group may be explained by the age-related ↓
in stomach HCl, a key acid responsible for iron absorption in the intestines.
Vitamin B12
̶ Elderly adults may exhibit age-related ↓ in serum vitamin B12 concentrations.
̶ The underlying cause may be ↓ HCI concentrations or chronic atrophic gastritis, which
subsequently accounts for insufficient intrinsic factor and vitamin B12 absorption.


Electrolytes
̶ Serum electrolytes, such as K+ and Ca2+, ↑ during aging.
̶ Blood Ca2+ ↑ in individuals aged 60–90 provided that serum albumin (Ca2+ protein carrier)
is normal. However, after the age of 90, Ca2+ gradually ↓. Hypercalcemia may be due to a
simultaneous ↓ in serum pH and ↑ in PTH
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.


Glucose and Insulin
̶ A steady ↑ in serum glucose concentrations and a ↓ in glucose tolerance are prevalent.
̶ Lower glucose concentrations may be due to poor diet and reduced body mass.
̶ Higher serum insulin levels are prevalent and may be associated with insulin resistance.
which may be explained by the ↓ in response capacity of insulin receptors in elderly.
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.

Gender differences in laboratory analytes can be explained by:

a. Differential endocrine organ related functions
b. Skeletal muscle mass.
Men have:
― higher ALP, ALT, AST, CK, and aldolase levels than women (larger muscle mass in men).
― higher 24-h urinary excretions of catecholamines, cortisol, and creatinine excretion.
Women have:
― lower levels of serum Mg2+, Ca2+, albumin, bilirubin, Hb, iron (menstrual blood loss), and ferritin.
― higher serum GGT and copper, and reticulocyte count (↑ RBCs turnover) compared to male.
Lipids (the lipid profile difference is partially due to the effect of estrogen in females)
― Below 20 years, females have higher total cholesterol compared to males.
― Between the ages of 20 – 45, males commonly have higher total cholesterol than females
― Males experience a lipid peak between the ages of 40 – 60 whereas females between 60 – 80.
― Between the ages of 30 – 80, mean HDL ↓ by 30% in females but ↑ by 30% in males.
In contrast, LDL-C is higher in men.

• When interpreting laboratory results during pregnancy,

it is necessary to take into account the gestational week at which each sample was taken.
• During a healthy pregnancy,
― Physiological ↑ in mean plasma volume ~ 2.6 – 3.9 Liters especially in the 2nd & 3rd trimester.
― Physiological ↑ in urine volume by up to 25% in the 3rd trimester.
― Physiological ↑ in GFR by 50% in the 3rd trimester.
• Examples of pregnancy-related changes:
― Changes in hormone production e.g. fertility and thyroid hormones
― Changes in metabolites levels (amino acids ↑, urea ↓)
― Changes in electrolytes (Ca2+ ↓, Mg2+ ↓, iron ↓, zinc ↓, copper ↑)
― Changes in proteins (especially acute phase proteins ↑)
― Changes in lipids (TGs ↑, cholesterol ↑)
― Changes in enzymes (alkaline phosphatase ↑, cholinesterase ↑)
― Changes in plasma levels of coagulation and fibrinolytic factors.
― The ESR is ↑ 5-fold during pregnancy

When interpreting laboratory results during pregnancy,

it is necessary to take into account the gestational week at which each sample was taken.
During a healthy pregnancy,
― Physiological ↑ in mean plasma volume ~ 2.6 – 3.9 Liters especially in the 2nd & 3rd trimester.
― Physiological ↑ in urine volume by up to 25% in the 3rd trimester.
― Physiological ↑ in GFR by 50% in the 3rd trimester.
Examples of pregnancy-related changes:

Hormones Fertility and thyroid hormones
Metabolites levels Amino acids ↑ Urea ↓
Electrolytes Ca2+ ↓, Mg2+ ↓, iron ↓, zinc ↓, copper ↑
Proteins ↑ acute phase proteins, changes in coagulation and fibrinolytic factors levels
Lipids ↑ TGs, ↑ cholesterol
Enzymes ↑ ALP, ↑ cholinesterase
The ESR ↑ Up to 5-fold

1. Gender
2. Age
3. Ethnicity/race
4. Pregnancy
5. Altitude
Patient preparation Sample collection Sample transport
Sample processing
1. Biological Rhythm
Sample storage
2. Diet
3. Fluid intake
4. Tobacco Smoking
5. Muscular Exercise
6. Mental Stress
7. Body Position
Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 2. Controllable variables

| Pre-collection Variables | Collection Variables | Post-collection Variables |
Patient preparation Blood sample Sample transport

1. Biological Rhythm • Anticoagulant/Preservatives • Time
2. Diet • Plastic vs Glass tubes • Temperature
3. Fluid intake • Venous vs Arterial samples • Handling
4. Tobacco Smoking • Tourniquet effect
Sample processing
5. Muscular Exercise Other Considerations • Centrifugation
6. Mental Stress • Urine • Cross-contamination
7. Body Position • ABG
• Hemostasis Sample storage
• Hematology
Sample Mis-identification

Pre-collection
Variables

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
Biological rhythm Ultradian rhythm

Recurring naturally on cycles less than 24 hours
Infradian rhythm
Recurring naturally on cycles more than 24 hours

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
Cortisol Blood level (μg/dl)

Growth hormone
GH Blood level (mg/ml)
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

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

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

Reproductive Hormone Level Ovulation

LH
Estrogen Progesterone
FSH
Day 1 Follicular Phase Day 14 Luteal Phase Day 28


• Diet substantially affects the composition of plasma.

• Differences in serum composition may occur respective to:
― The source of nutrients
― Number of meals
― Proportion of nutrients in a diet
• Moreover, malnutrition or obesity, prolonged fasting, starvation, and vegetarianism may also
influence plasma composition.
• The effects from diet can be divided into long-term and acute effects.

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.

Change in serum concentrations

after Two hours of a standard meal
― The percentage of change in different analyte levels
concentrations as a function of food intake.
― Changes of 5% or less (< 1.05) may be neglected
for being clinically insignificant.

• 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).

Carbohydrate meals ↑ glucose and insulin, ↓ phosphorus

Protein meals ↑ cholesterol and GH within 1 hour of food
consumption and ↑ glucagon and insulin levels
Intake of omega-3 oils May ↓ TGs and VLDL concentrations
Serotonin-rich foods ↑ Urinary excretion of 5-HIAA.
(bananas, pineapples, kiwis, and avocados)
Food such as meat, fish, and horseradish May cause a false-positive stool occult blood reaction
(have substances that mimic heme structure) if the Guaiac method is used.
(with no effect with the immunochemical method)

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 ↑.

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.
Vegetarians tend to have:

― Lower plasma concentrations of cholesterol, TGs, and creatinine,
― ↓ urinary creatinine with higher urinary pH as a result of ↓ intake of acid metabolites precursors.

Special diet-related changes
Long-time vegetarian diet  LDLs, VLDLs,, phospholipids, cholesterol, and TGs
Vitamin B12 deficiency (unless supplements are taken)
Lacto-vegetarians Have higher LDL-C and HDL-C compared to vegetarians

(vegetarians that consume dairy products)
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)

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.

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.
Proteins Albumin, prealbumin and C3 ↓ during an extended fast

these analytes are rapidly restored following protein supplementation
Lactate and pyruvate Begin to ↑ after short term 14 hours fast.
Starvation for 4 weeks Significantly ↑ AST with ↓ GGT,

↑ creatinine, ↑ uric acid (20 – 40%) with ↓ urea (20 – 50%)
↓ TGs,

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.

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.

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.

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.

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

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.

Smoking Tobacco
Chronic effects of smoking. Deviation (%) of blood

analyte concentrations between current smokers
and nonsmokers. (Reproduced from Guder WG, Narayanan
S, Wisser H, Zawta B. Diagnostic Samples: From the Patient
to the Laboratory. 4th updated ed. Weinheim: Wiley-Blackwell;
2009)

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.

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.

Mental stress
May induce:
• ACTH ↑
• Cortisol ↑
• Catecholamines ↑
• Total Cholesterol ↑
• HDL ↓ as much as 15%
• Prolactin may ↑

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.
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.
General recommendations should be applied to all blood tests:

1. Blood should be drawn preferably in the morning between 7 a.m. and 9 a.m.
2. Fasting should last for 12 hours, during which only water consumption is permitted.
3. Alcohol should be avoided for 24 hours before blood sampling.
4. In the morning before blood sampling, patients should cease cigarette smoking and caffeine-
containing drinks (tea, coffee, etc.).
Blood samples for routine testing should not be taken if a patient has not been appropriately prepared.
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
Patient Preparation
Lipase Ensure specimen collection takes place prior to ERCP
(endoscopic retrograde cholangiopancreatography)
Creatinine High protein diet restriction a day before sampling

Instruct the patient to refrain from excessive exercise for 8 hours before the test.
Renin There are no fluid restrictions unless by medical direction

Normal sodium diet (1–2 g/day) for 2 to 4 weeks before sampling
Supine or upright posture must be maintained for 2 hours before sampling
Aldosterone Supine or upright posture must be maintained for 2 hours before sampling
Cortisol Timing sampling at 8 am and 8 pm (8 am specimens are preferred)

(The 8 a.m. cortisol can be drawn anywhere between 6 am and 10:30 am in the morning).
Resting at least 15 minutes before sample collection
GH Avoid strenuous exercise for 12 hours before specimen collection.

Patient Preparation
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
Lupus anticoagulant Heparin therapy should be discontinued 2 days before collection

Warfarin therapy should be discontinued 2 weeks before collection
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
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
5-HIAA Serotonin-rich foods (bananas, pineapples, kiwis, eggplant and avocados)

should be avoided for 3 days prior to and during urine collection;
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.

Collection Variables

Color Anticoagulant/Additive Mechanism of Action Specimen types/use Inversions
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)
Gold SST Thrombin Clot activator Serum / Chemistry 5 times

(Orange) Gel Separator Gel Separator
Clot Activator
(Allow to clot 30 min.
prior to centrifugation)
Light Li Heparin and Gel Heparin Inhibits Plasma / Chemistry 8 – 10

Green thrombin formation • Acute care chemistry (STAT)
• Ammonia
Gel barrier separates
Plasma from RBCs

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
Na Citrate Chelates calcium Whole blood / ESR 3 – 4 times

• Completely fill tube to the line indicated
Black • Over- or under-filling not allowed

Plasma / Chemistry
Green Na heparin or Inhibits thrombin Amino acids, blood gases 8 – 10
Li heparin formation Whole Blood / FCM
/ Cytogenetic
/ HLA Typing
/ G6PD
Purple K2EDTA or Chelates calcium Whole blood / Hematology 8 – 10

K3EDTA / Blood Bank
(Spray-dried or liquid) / Parathormone
/ ACTH
Pink K2EDTA Chelates calcium Whole blood / Blood bank 8 – 10

(Spray-dried) / Hematology
/ molecular testing

Royal Clot activator Clot activation Serum / Toxicology 8 – 10

Blue Transporting and handling Trace elements e.g. times
in upright position Copper
Zinc
Aluminum
Chromium
Nickel
Royal Na Heparin Heparin inhibits Plasma / Toxicology 8 – 10

Blue + K2EDTA or Na2EDTA thrombin formation Trace elements e.g. times
Transporting and handling Lead
in upright position EDTA binds calcium Arsenic
Cadmium
Cobalt
Heavy Metal-free Manganese
tube Mercury
Molybdenum
Thallium

Grey Na Fluoride + K oxalate Inhibit glycolysis Plasma / Glucose 8 – 10

Preserves glucose up to / Lactate
5 days Not suitable for urea
(inhibit urease enzyme)
Pale ACD WBC preservative Plasma / Blood bank 8 – 10

Yellow Acid Citrate Dextrose / FCM
/ HLA typing
(paternity testing)

• Tubes with additives must be thoroughly mixed by

gentle inversion as per manufacturer recommended
protocols.
• Erroneous test results may be obtained when the
blood is not thoroughly mixed with the additive.
= 1 inversion

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

Order of Draw
1 2 3 4 5 6 7 8 9 10 11
Royal Light Gold Light Royal

Blue Blue Black Red SST Green Green Purple Pink Blue Yellow Grey
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

Order of Draw for capillary (micro) collection
1 Lavender K2 EDTA 10x The microcollection order of draw

is designed to prevent microclot
2 Green Lithium Heparin 10x formation and platelet clumping
3 Light Green Lithium Heparin 10x If ABG is ordered,

and Gel for plasma separation it must be collected firstly
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

Anticoagulant/Additive Citrate Oxalate

Effect on Blood Tests ALT and AST
Alkaline phosphatase
Inhibit
Inhibits
Acid phosphatase
Alkaline phosphatase
Inhibits
Inhibits
Acid phosphatase Stimulates Amylase Inhibits
Amylase Decreases LD Inhibits
Calcium Decreases Calcium Decreases
Sodium and potassium Increase Sodium and potassium Increase
Labile coagulation factors Preserve Cell morphology Distorts
Heparin EDTA Fluorides

Triiodothyronine Increases Alkaline phosphatase Inhibits Acid phosphatase Decreases
Thyroxine Increases Creatine kinase Inhibits Alkaline phosphatase Decreases
PT and PTT Increase Calcium and iron Decrease Amylase Decreases
Wright’s stain blue background PT and PTT Increase Creatine kinase Decreases
Lithium (LiHep tubes only) Increases Sodium and potassium Increase ALT and AST Decrease
Sodium (NaHep tubes only) Increases Platelet aggregation Prevents Cell morphology Distorts

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

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.

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.

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
Natural air includes 21% oxygen, which is equivalent to FiO2 of 0.21.

Oxygen-enriched air has a higher FiO2 than 0.21; up to 1.00 which means 100% oxygen.
FiO2 is typically maintained below 0.5 even with mechanical ventilation, to avoid oxygen toxicity.

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.

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.

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.

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).

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.

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

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.

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.
Why Use Balanced Heparin?

― Heparin is negatively-charged and binds cations (e.g. Ca2+, Na+, K+) in a dose-dependent manner.
― This may cause underestimation of electrolyte concentration.
― To prevent such direct binding, balanced heparin was introduced in which the binding sites of
balanced heparin are pre-saturated with Ca2+.

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.

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

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.

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.

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.

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.

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.
Example of the effect of excessive repetitive pressure “milking” on

Without milking Milking applied sample hemolysis and sample contamination with tissue fluid.
These two samples were obtained from the same patient in the
Na+ 140.1 Na+ 137.1
resting state within 2 minutes.
K+ 3.76 K+ 4.12
Ca2+ 0.99 Ca2+ 0.71 Sample without milking was obtained after an arterialization with a
Cl− 104 Cl− 101 warm towel, while the other sample was obtained by the use of
excessive finger pressure.

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.

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.

Small metal ball

located in the syringe barrel

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.

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.
Plastic vs Glass Syringes

• As a general rule, samples drawn in plastic syringes should be analyzed immediately.
• If analysis is delayed, the samples should be kept in glass syringes.
• Plastic syringes should not be cooled because plastic molecules contract when cooled to 0 – 4°C.
Contraction of plastic molecules creates pores in the syringe wall through which O2 easily diffuses.
Because CO2 is a much larger molecule than O2 , it cannot diffuse through the syringe wall.

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.

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

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
• The required blood to anticoagulant ratio is 9 : 1

It is very important that tubes be filled to the mark noted on the tube. Maximum
• Acceptable deviation is maximum ±10% of the total volume. Minimum

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 =

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.

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.

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.

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.

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.

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.
ICSH : International Council for Standardization in Haematology


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

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.

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).

Hematology Testing
Antibodies may affect the cell count of RBCs, WBCs, and platelets.
The following antibodies are known to interfere with hematological analytes:

― Cold agglutinins (RBCs specific)
― Cryoglobulins
― EDTA-dependent antibodies with platelet and leukocyte specificity

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.
Rarely, cold agglutinins target platelets, causing pseudothrombocytopenia independent of EDTA.

Cold agglutinins, if undetected, may cause diagnostic confusion and subsequent extensive diagnostic
workup as well as incorrect and unnecessary therapy, risking patient safety and increasing costs. It is
therefore very important to recognize cold agglutinins promptly.

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.
For adequate analysis of samples in which cold agglutinins are suspected,

― Warm up the EDTA blood sample at 37°C and analyze immediately afterward.
― With Re-cooling at 4°C , the previous findings will appear again.

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.

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.

Specimen labelling
Two Steps for vacutainer labeling
1. Line up notch with arrow & align edge of label with

color-coded sidebar, leaving the sidebar exposed.
2. Smooth label around the tube like a graceful turn

Specimen labelling
For Perfect Labeling 1

1. Label directly under cap 2
2. Color-coded notch clearly
3. Sidebar clearly visible 3
4. Easy-to-read patient info 6
4
5. Barcode straight
6. Open window to see sample 5

Specimen labelling
Examples of wrong labeling
Backward Belt Wrinkled Scarfy Twisted Turtle neck Upside Down The Group

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.
How to Prevent Specimen Misidentification?
Implementing rules The use of Specimen rejection Auditing

and the instructions Delta checks procedures And monitoring
Preanalytical Analytical Post-analytical


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.

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.

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.

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.

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.

• 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.

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.

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.

Post-collection
Variables

Sample Transport | Sample processing | Sample storage
Sample transport
• Time
• Temperature
• Handling
Sample processing
• Sample Mis-identification
• Centrifugation
• Cross-contamination
Sample storage

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

Sample Transport ► 1. Transportation time

Many obstacles prevent timely transportation from collection site to laboratory.
• Specimens can get lost or misplaced for hours.
• Tubes can get stuck or delayed in pneumatic tubes systems.
• Couriers may get stuck in traffic while samples sit in the car.
• A nurse or phlebotomist may slip a tube into his or her pocket.
No matter the reason,

sample processing is often delayed, and this can be a source of pre-analytical error.
Every reasonable effort should be made to ↓ transport time between drawing and processing
the sample.


A. Analytes concertation affected by in vitro metabolic activity of blood cells
especially when maintained at room temperature (RT) or higher, and uncentrifuged
Glucose
― Glucose levels fall at 5-7% per hour due to on-going glycolysis by the blood cells.
― Even in samples that have been centrifuged, but not separated, glucose in serum or plasma
will continue to ↓, falling outside a clinically significant change range in < 4 hrs.
Lactate
― Lactate ↑ with the glucose ↓, as pyruvate is reduced to lactate during glycolysis.
• Samples with bacterial growth or leukocytosis undergo more glycolysis,
• Care should be taken to avoid prolonged time to processing, especially in patients with
bacteremia and/or leukocytosis, as hypoglycemia may otherwise be misinterpreted and
prompt an unnecessary workup for hypoglycemia (e.g. low CSF glucose may misinterpreted
as bacterial meningitis).


B. Elevations in intracellular RBCs components can occur via trans-membrane diffusion of
cellular constituents.
Phosphorus
― Phosphorus is ~ 7-times more concentrated in red cells than plasma, and leaks from the
RBCs cell into plasma with extended storage time.
Potassium
― The same is true for K+, though the intracellular gradient may be maintained if the cells are
kept at RT and can consume glucose to generate the ATP required to feed the Na+/k+
ATPase.
Chloride and CO2
― Cl‒ and CO2 concentrations ↓ over time, likely due to the Cl‒ -HCO3‒ shift into RBCs


C. Hemolysis infrequently occurs during handling and transportation which can significantly ↑
the concentration of intracellular RBCs components, such as K+, AST, and LDH.
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,
E. Finally, the effect of hemoconcentration, where significant ↑ may be observed in several

analytes, such as Na+ as water moves into the cells as the samples stand.


Specimen integrity must be maintained from draw to analysis.
CLSI guidelines recommend separating plasma/serum from the cells within 2 hrs of collection as
most analytes are stable for > 2 hrs, so rejection of all specimens received 2 hrs or more after
draw is unnecessary.
Tactics to optimize specimen integrity during transportation include:

― Implementation of strategies to reduce transport time
― Use of appropriate tubes (Gel tubes for most analytes, Grey-top tubes for glucose and lactate)
― Development of strict guidelines for specimen storage conditions during transport
― Reducing transit time which preserves sample stability and shortens TAT.


Examples of Measures to Reduce the effect of time on the measurement of analytes
Automated delivery systems
• For inpatient specimens, robotic couriers and pneumatic tube systems can cut-down on
staffing and ↓ transport time to the laboratory. These solutions may provide an alternative to
opening a satellite lab in far sites from a medical center.
• One study estimated that robotic couriers could ↓ turnaround time by 34% in a 591 bed
hospital. Rossetti MD, Felder RA, Kumar A. Simulation of robotic courier deliveries in hospital distribution services.
Health Care Manag Sci 2000;3(3):201-13.
• 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.






Gel separator tubes
― Provide a single, closed system to draw, process, test, and store samples.
― These tubes have the added advantage of helping to prevent labeling errors as there is less
need to aliquot and re-label, reducing labeling and misidentification errors.
― Use of serum and plasma separator tubes extends the stability of most chemistry analytes.
However, many drugs, such as phenytoin, phenobarbital, carbamazepine, lidocaine, and
tricyclic antidepressants absorb into the gel, so should these tubes not be used for therapeutic
drug monitoring.


Gray-top tubes (Na+ fluoride/K+ oxalate)
― Fluoride and oxalate inhibit glycolysis, so it is helpful when highly accurate glucose results are
necessary, such as for glucose tolerance tests (glucose is stable for 72 hours) .
― Prevention of glycolysis ensures that lactate concentrations also remain stable, making the
gray-top tube the preferred tube type for lactate samples.
― However,
• It takes up to 2 hours to completely inhibit glycolysis, so some glucose loss can still occur.
• Require immediate separation of plasma or serum from the cells remains the best means
for stabilizing glucose.
• Not suitable for electrolyte determinations as they contain Na+ and K+ salts;
• Not suitable for certain enzymatic assays as they interfere with certain enzymatic assays,
such as the urease method for blood urea determination.

Sample Transport ► 2. Transportation Temperature

• Manipulation of transport temperature is essential to ↑ analyte stability.
• Extreme heat denatures proteins and can ↓ enzyme activity.
• Lower temperatures generally enhance analyte stability.
• Most analytes are stable at 4°C than RT, However, there are several notable exceptions.
― Cold inhibits glycolysis, which provides the ATP required for cell surface Na+/K+ pumps.
― Without ATP, intracellular K+ accumulates and begins to leak out of cells over time.
So, extracellular K+ may be artificially ↑ in specimens stored in the cold for > 6 hours.
― Thus, to maintain appropriate K+ concentration, false ↓ in glucose may be resulted.
― The problem is solved by separating the plasma from the cells before transporting.
• When transporting samples outside, special considerations should be made for the weather.
― On very hot days, samples should be sealed in a plastic bag, and then immersed in
ice slurry, to provide better cooling than ice packs alone.
― In very cold climates, extra insulation may needed to keep the sample from freezing.


Chilling
• Some analytes must be chilled because they rapidly degrade at RT
• Examples: NH3, lactate, pyruvate, PTH-related protein, catecholamines and gastrin.
• These, specimens of such analytes should be chilled immediately after collection, and this
temperature should be maintained throughout the pre-analytical phase.
• Chilling inhibits the blood cells metabolism and stabilizes thermolabile constituents.
• Chilling whole blood > 2 hours is contraindicated for a specimen intended for K+.
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.


Freezing of specimens
• Testing is often intentionally delayed, for example for batch analysis or to send specimens
to reference labs and specimens may be frozen to preserve sample integrity until they are
tested.
• However, some enzymes are sensitive to freezing temperatures:
― AST, ALT, ALP, GGT, and LDH remain stable
― amylase ↑
― CK activity ↓ (CK activity ↓ significantly when frozen to –20°C for even short periods).
Cryoglobulins
Certain analytes, such as cryoglobulins, must be maintained at body temperature, and require
transit in a warm water bath, kept around 37°C.


Humidity
• Humidity affects:
― Dried blood spots: Humidity is also a consideration, particularly for samples that will be
exposed to outside air, such as dried blood spots.
High humidity speeds the degradation of analytes in dried blood spots, e.g. biotinidase,
galactose-1-phosphate uridyltransferase, G6PD and T4.
― Self-monitoring blood glucose meters: High humidity may cause inaccurate results.
• Controlling and reduction of humidity: ‫اﺠﻤﻟﻔﻔﺔ‬

― Can be prevented by transporting samples in plastic bags with desiccant packets,
― Can be monitored with humidity indicator cards.
― Care must be exercised in the selection of a device, reagent system, and collection system
that will minimize errors at high temperature and high humidity.

Sample Transport ► 3. Transportation Handling and Turbulence

Outreach specimens are often brought to the main laboratory by courier
• Although not perfect, courier services are the preferred for transporting from remote locations.
• Compared to mailing specimens, couriers are faster and can provide better control of the
sample environment, both temperature and jostling. ‫ﺗﺰاﺣﻢ‬
Inpatient samples, however, are frequently delivered via pneumatic tubes or robots.
• Robotic couriers replace full time employees, and transport samples efficiently and safely.
• Pneumatic tube systems are widely used because they transport samples.
― The Speed of pneumatic tube systems may reach up to 7.5 m/s, and often go through rapid
accelerations, sharp corners, and abrupt decelerations.
― The mechanical forces experienced by the samples in pneumatic tubes can cause
container breakage or leakage [particularly when they are glass and/or difficult to close
(e.g., urine collection cups)] and blood cell damage and subsequent hemolysis.
― Smartphones equipped with accelerometers have been suggested for use in monitoring
pneumatic tube system performance.


Hemolysis
Blood cells damage and subsequent hemolysis may occur because of bad handling of specimen
(caused by turbulence and agitations) (‫اﻻﺿﻄﺮاب واﻹﻫﺘﺰاز )اﻟﺮﺟﺮﺟﺔ‬
• This is true of specimens from patients with hematologic disorders or on chemotherapy and
other conditions associated with ↑ RBCs and WBCs fragility.
• LDH is a good marker for evaluating exposure to turbulence.
Full tubes are less subject to damage by agitation compared to partially filled tubes.
Effect of Hemolysis on Laboratory Test (CLSI C44-A4)

― Tests Seriously Affected : LDH (↑), AST (↑), free HB( ↑), K+, Troponin I and T (↓)
― Tests Noticeably Affected : ALT (↑), Iron (↑), T4 (↓)
― Tests Slightly Affected : Albumin (↑,↓ or N), ALP (N or ↓) Ca2+ (↑), Mg+2 (↑), P–(↑),
Haptoglobin (↓), Total Bilirubin (N or ↓) , Total Protein (↑)


Tube Orientation
It is recommended that blood tubes be kept in a vertical, closure-up position.
This positioning promotes complete clot formation and ↓ agitation of the tube contents,
which, in turn, ↓ the potential for hemolysis.
Non-anticoagulated tubes that contain gel:

• Should always be stored in an upright position as soon as the mixing is completed.
This prevents fibrin from attaching to the tube closure.
• If this attachment is allowed to occur, these fibrin strands, usually trapping RBC, will be
pushed above the gel plug following centrifugation (particularly in fixed angle-head
centrifuges) and will then contaminate the resultant serum with both RBC and fibrin
that can cause obstructions in instruments.


Exposure to Light
It is important to avoid exposing blood specimens for photosensitive analytes to artificial light or
sunlight (UV) for any length of time.
• For bilirubin, this is critically important when an icteric newborn is monitored for the
possibility of an exchange transfusion.
• Other examples include vitamins A and B6, beta-carotene, and porphyrins.
These specimens should be protected with an aluminum foil wrap, an amber specimen
container, or the equivalent. ‫ﻛﻬﺮﻣﺎن‬


Shipping to reference laboratory
Systems should be in place for sending specimens to distant reference labs.
As with transporting samples from nearby locations, time, temperature, and handling must be
considered when shipping samples over greater distances.
• In most cases, plasma or serum samples should be separated from cells and aliquot into a
separate tube, rather than shipping the primary tube.
• Certain tests may have special specimen requirements. Always consult the reference
laboratory’s test directory for appropriate specimen type and storage conditions prior to
collection of send out samples.
• Specimens may also be placed into a secondary container or packing to ↓ turbulence.


Shipping to reference laboratory
• Generally, samples are sent either frozen or refrigerated in a Styrofoam
container, with walls at least 1 inch thick, to ensure adequate insulation.
• 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.


Special case: blood gases and ionized calcium
Specimens collected for blood gas determinations require special care, as the analytes are very
sensitive to time, temperature, and handling.
In standing whole blood samples,
Rate of change Rate of change Rate of change

Analyte Direction at 37°C at 22°C at 4 °C
pH ↓ 0.04 – 0.08 /h 0.02 – 0.03 /h < 0.01 /h
pCO2 ↑ 5.0 mmHg/h 1.0 mmHg/h 0.5 mmHg/h
pO2 ↓ 5 – 10 mmHg/h 2 – 10 mmHg/h ---
The drop in pH is concordant with ↓ glucose and ↑ lactate.


Metabolic activity is affected also by the number of metabolically active cells present.
― Specimens with leukocytosis and/or thrombocytosis may demonstrate a spurious hypoxemia,
as leukocytes and platelets consume O2 in vitro.
― pO2 loss is best prevented by immediate analysis, as even rapid cooling may not sufficiently
reduce metabolism.
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.


Blood gas analyzers re-heat samples to 37°C for analysis to restore physiological temperature.
• However, for patients with abnormal body temperature, either hyperthermia due to fever,
or induced hypothermia in patients undergoing cardiopulmonary bypass, a temperature
correction should be made to determine accurate pH, pO2, and pCO2 results.
• This adjustment is particularly important in hypothermia, in which the temperature change
has marked impact on pH, pO2, and pCO2 .


Equations are recommended by CLSI for temperature corrections:
pH patient’s adjusted pH
pH = pHm – [ 0.0147 + 0.0065 × ( [ pHm – 7.40 ) ] ( T – 37° ) T
pHm
patient’s temperature
measured pH
pCO2 patient’s adjusted pCO2

pCO2 = pCO2m × 10 0.019 ( T – 37° ) T patient’s temperature
pCO2 measured pH
(5.49 × 10 –11 pO23.88) + 0.071 pO2 patient’s adjusted pO2

( T – 37° )
(9.72 × 10 –9 pO23.88) + 2.30 T patient’s temperature
pO2 = pO2m × 10 pO2 measured pO2


Air Exposure
― Blood gas specimen exposure to air should be minimized, as gases in the sample will rapidly
equilibrate with atmospheric air.
― Anaerobic technique should be used to draw all blood gas samples.
― However, even with the most careful collection, air bubbles can arise from the syringe hub dead
space. Bubbles must be completely expelled from the specimen prior to transport, as the pO2
will be significantly ↑ and pCO2 ↓ within 2 min


• iCa2+ is often measured in by ion sensitive electrodes in blood gas analyzers.
• iCa2+ is inversely related to pH: ↓ pH = ↓ albumin-binding to calcium, thereby ↑ free iCa2+ ,
• Therefore, specimens sent to the lab for iCa2+ determinations should be handled with the same
caution as other blood gas samples, since pre-analytical errors in pH will impact iCa2+ results.
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


Special case: Receipt of Unprocessed Specimens in the Laboratory
1. Time
On receipt, specimens should be sorted and prepared for centrifugation.
― Allow sufficient time for clotting to occur. Blood collected using a tube containing a
clotting activator) can be processed as early as 5 – 30 minutes after the blood is drawn.
― Anticoagulated specimens can be centrifuged immediately. Also refer to the specific
recommendations of the manufacturer’s.
― Some tests require an unseparated, anticoagulated whole blood specimen (e.g.
blood lead, cyclosporin, and A1c). Do not centrifuge these specimens.
If a specimen is accidentally centrifuged, do not discard it; send the centrifuged tube to
the testing location for evaluation and determination if the specimen can be remixed and
assayed.


Special case: Receipt of Unprocessed Specimens in the Laboratory
2. Temperature
― Chilled specimens (2 – 8°C) are kept at this temperature until they are ready for centrifuging.
― Temperature-controlled centrifuges are recommended .
3. Tube orientation
― It is recommended that tubes of blood be placed in a vertical, closure-up position on.
4. Tube Closure
― Tubes of blood are kept closed at all times.
― Certain test results can be inaccurate with improper tube closure, because of an ↑ in pH
resulting from the loss of CO2 (e.g. pH [↓], iCa2+ [↓], and acid phosphatase [↓]).
― Keeping the tube in a closed position eliminates possible exogenous contamination of the
specimen and prevents evaporation and the possibility of spills and aerosols.

Criteria for Rejected Sample

Under the following conditions, blood specimens may not be acceptable for testing purposes.
Professional judgment at the laboratory supervisor level must be exercised in applying these criteria:
1. Inappropriate Specimen Containers

― Specimens container should be rigid, robust, leakproof with a tight-fitting lid.
― Specimen containers should be labeled and transported in accord with institutional policy.
2. Inadequate or Incorrect Specimen Identification

Examples:
― A tube of blood is not labeled or it is inadequately labeled.
― The identification on the specimen does not match the identification on the requisition.


3. Inappropriate Volume of Blood
― The amount of additive placed into a tube is intended for a certain volume of blood.
― If less blood than required is drawn, the excess amount of additive has the potential to
adversely affect the accuracy of test results.
― If more blood is drawn than is required, the amount of additive may be insufficient for its
intended purpose and may similarly adversely affect the accuracy of test results.
― Examples
Sodium citrate: prothrombin time.
EDTA: packed cell volume, cell count, cell morphology, lipids.
Heparin: CK, aminoglycosides (gentamicin, tobramycin).


4. Using the Wrong Collection Tube
Wrong additive can interfere with the analyte to be determined. Examples:
― Sodium fluoride interferes in the urease BUN method.
― The wrong order of draw during multiple blood specimen collection can invalidate results
because of contamination by the additive.
5. Hemolysis
― Hemolysis can occur in vivo or in vitro.
― Repeated blood collection and continued receipt of hemolyzed specimens often indicates IV
hemolysis, and the physician should be notified.
― This items is described in details in “Hemolysis as pre-analytic interferant”
6. Improper Storage/Transportation
Examples
― A specimen that should have been chilled (2 – 8°C) is received in the laboratory unchilled
― A serum or plasma sample that should have been frozen is received thawed.

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.
• Centrifugation is an integral part of specimen processing.

• Improper centrifugation techniques may lead to erroneous results for several laboratory tests.

Sample Processing ► 1. Preparing Specimens for Centrifugation

Mixing
• Immediately after blood collection, plasma specimens must be mixed gently according to
manufacturer’s instruction to ensure efficient release of additive/anticoagulant.
• Insufficient mixing leads to:
― Inefficient clotting in serum tubes
― Platelet clumping and/or clotting in plasma tubes,
• Thus, inadequate separation of plasma/serum from cellular material during the centrifugation.
Time for clotting
Serum specimens must be allowed enough time to clot prior to centrifugation.
― Tubes with clot activators require sufficient mixing and at least 30 min of clotting time
― Serum tubes may require up to 60 min.
― Patients taking anticoagulants may require longer clotting times.
Specimens should not be opened prior to or during centrifugation.

Sample Processing ► 1. Preparing Specimens for Centrifugation

Clot Release (Rimming the Tube)
• The use of a wooden applicator stick or similar device for the release of a clot attached to the
tube closure or the sides of the tube is not recommended. Rimming the tube is a potential
source for laboratory induced hemolysis.
• Clot/cell hang-up has been virtually eliminated by technical improvements in tube/closure
design and manufacture.
• 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.

Sample Processing ► 2. Centrifugation

Labs should select appropriate centrifugation speed, time, and temperature for each analyte
Inadequate centrifugation
= Specimens spun at too slow speeds or not enough time
= No adequate separation of serum from the clot or plasma from cellular components.
Erroneous results may occur for chemistry/immunochemical assays because of such materials:
― Invisible microfibers or other particulate matter in incompletely centrifuged serum or
plasma interferes with assays such as Troponin.
― Platelet contamination of the plasma in inadequately separated specimens leads to
inaccurate results in coagulation studies.
― Further, centrifugation at slow speeds leads to ↑ “trapped plasma” in the RBC fraction,
leading to abnormal results for analytes found in RBCs (i.e., K+ and G6PD).
Specimens spun too fast are subject to in vitro hemolysis and/or cell lysis and release of
intracellular constituents like K+.


Relative Centrifugal force (speed)
In order to avoid abnormal results due to improper centrifugation, all laboratories should establish
appropriate centrifugation time and speed for tube type, centrifuge, and rotor.
Calculate relative centrifugal force (RCF), not revolutions per minute (RPM),
for each centrifuge model, rotor, head and the rotor radius in order to determine appropriate speed.
The equation for RCF, expressed as multiples of gravitational force (g).
RCF = 1.118 × 10-5 × r × n2

r = rotating radius (cm)
n = speed of rotation (RPM)
Manufacturers provide recommendations for appropriate RCF and spin times for individual tube types


Type of Centrifuge Rotor
Rotating Tip Radius. The distance measured from the rotor axis to the tip of the liquid inside the tubes
at the greatest horizontal distance from the rotor axis..
Swinging Bucket Rotors
Fixed Angle Rotors



Type of Centrifuge Rotor
• Laboratories should revise tube manufacturer’s guidelines when deciding which centrifuge is
appropriate for their specimen.
• No matter the type of centrifuge, they should always be balanced prior to processing.
• Improper balancing can lead to tube cracking, breakage and/or inadequate separation of
serum or plasma from cells, as well as centrifuge damage.
The use of fixed angle rotors, particularly with separator tubes will cause the gel to form at an
angle. Angled gel formation may indicate an inadequate barrier seal, leading to mixing of serum
or plasma with cells; numerous abnormalities are associated with storage on the clot or cells.
Further, centrifugation in fixed angled rotors for prolonged times may induce hemolysis.
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.


Centrifuge Temperature
Recommendations
1. Laboratories should have temperature-controlled centrifuges for temperature-sensitive analytes.
2. Specific thermolabile measurands should be separated at 4°C (e.g. ACTH) as centrifuges can
generate internal heat that may be inappropriate for analyte stability.
3. Check to make sure that the centrifuge is at its intended setting.
4. Unless a specific temperature for a certain analyte is needed, a centrifuge setting of 20 – 22°C
is recommended.
5. Any heat-sensitive specimens arriving to the lab chilled should be spun in the cold temperature.


Centrifuge Temperature
Tight control of internal temperature is critical during the centrifugation phase.
• The internal temperature of a centrifuge may become hot with activity, leading to:
― degradation of temperature sensitive analytes like ACTH.
― Induction of hemolysis.
• Centrifugation at refrigerated temperatures is not appropriate for all specimens.
― Serum K+ should not tested on specimens stored and/or centrifuged in the cold (<15°C) for
more than 2 hours, as K+ leaks from blood cells with prolonged cold exposure.
― Gel in separator tubes are often temperature dependent, thus centrifugation of these tubes
in a chilled centrifuge may result in inefficient barrier formation. Laboratories should follow
manufacturer’s guidelines for spin temperatures for barrier tubes.
― Serum pH and iCa2+ are affected by centrifugation temperatures (iCa2+ change by 0.006
mmol/L per 1°C). Centrifuge temperature should not deviate > ± 2.5°C for iCa2+ or pH.


Re-centrifugation
Re-centrifugation of tubes is not recommended by CLSI and tube manufacturers as several
studies demonstrated falsely ↑ K+ after re-centrifugation.
(If necessary to further separate serum or plasma from cells, transfer the serum or plasma to
another tube and centrifuge)
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
Specimen Time before re-centrifugation

Analyte Serum Heparin plasma < 4 hours < 12 hours Up to 24 hours
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.

Sample Processing ► 3. Post-centrifugation

It is recommended that serum/plasma be separated from contact with cells as soon as possible,
unless conclusive evidence indicates that longer contact times do not contribute to error of the
results.
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.

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.

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.
In unseparated plasma specimens,

― K+ is falsely ↑ in prolonged storage at 4°C ( K+ leakage from the blood cells).
― Catecholamines are released from lysed RBCs and reuptake is slowed, falsely ↑ results with
prolonged storage at 4°C.
― Cryo-activation of pro-renin with long-term colds storage falsely ↑ plasma renin activity assays.
― Specimens collected for LDH isoenzyme testing should be stored at room temperature prior
to analysis because freezing and long-term 4°C storage results in loss of LDH 5 activity.
― Prothrombin time is significantly ↑ with prolonged frozen storage and these specimens should
only be stored refrigerated or at room temperature.

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.
Pre-analytic variables ► B. Pre-analytic Interfering Factors
B
Pre-analytic
Interfering Factors
B. Pre-analytic Interfering Factors
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.
Characteristics of pre-analytic interferents:

― Not Affect analytes concentration
― Affect a specific analytic method
― Could be eliminated or at least reduce
1. In Vivo interferents 2. In Vitro interferents

Substances already present in the patient sample Substances added to the patient sample
• Sample Contaminants
 Lubricants
 Surfactants
I. Endogenous blood component II. Exogenous component  Separator gels
introduced to the blood  Additives
• Hemolysis  Anticoagulants
• Lipemia • Prescribed medication
Supportive medical therapy
 Clot activators
• Icterus •
• Paraproteins • Natural preparations • Sample carryover
• Heterophilic antibodies • Accidental exposure and poisoning • Fibrin clot
• Nutritional Supplements (Biotin)
Maximum allowable deviation

• Interfering factors are considered clinically relevant when the bias caused by their
interference is greater than the maximum allowable deviation of a measurement
procedure.
• How this “maximum allowable deviation” should be established is still debated.
• The CLSI EP7-A2 guideline, for example, sets this criterion at ±10% as a general rule.

Effects of Laboratory Errors on Patient Outcome
Wrong Result Consequence

Elevated Result
Raised hCG) indicating gonadal tumor Unnecessary surgery, chemotherapy
Raised calcitonin indicating medullary thyroid cancer Unnecessary fine-needle aspiration
Raised prolactin level Misdiagnosis of prolactinoma
Increase in urine free cortisol Unnecessary diagnostic follow-up
Elevated testosterone in women Unnecessary diagnostic follow-up
Elevated LH and FSH levels Unnecessary diagnostic follow-up
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
1. In Vivo Interferents
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. Endogenous interfering factors
I
Interference caused by
Endogenous Blood Components
Hemolysis Lipemia Icterus Paraproteins Heterophilic Abs


 Lubricants
 Surfactants
I. Endogenous blood component II. Exogenous component  Separator gels
• Lipemia • Prescribed medication
Supportive medical therapy
 Clot activators
• Icterus •
• Paraproteins • Natural preparations • Sample carryover
• Heterophilic antibodies • Accidental exposure and poisoning • Fibrin clot
• Nutritional Supplements (Biotin)
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents ► I. Endogenous 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
with a maximum in the lower part of the spectrum (Grey);

• Bilirubin has one distinct peak at 460 nm (Yellow).
Wavelength (nm)
300 350 400 450 500 550 600 650 700 750
380
780
UV Visible Light IR
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 1. In Vivo Interferents
1
Hemolysis
Interference
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.
Though hemoglobin is the most abundant protein in RBCs,

hemolysis is not necessarily always associated with the release of hemoglobin into the surrounding ECF.
• For example, if the blood sample is stored at a low temperature, LMW intracellular components like
electrolytes diffuse from the cells, but hemoglobin will not.
• Furthermore, efflux of cell components due to cell lysis affects platelets and WBCs) and not only RBCs.
• So, it is important to remember that red coloration of the serum or plasma can never accurately
predict the concentration of blood cell components.
Hemolysis Grading Chart
Mild Moderate Gross

+ + ++ +++ ++++ ++++
0.00 0.02 0.05 0.10 0.25 0.50 1.00 g/dl

Types of Hemolysis Hemolysis
In vivo hemolysis In vitro hemolysis

(~ 3%) (~ 97%)
• Improper blood sampling
• Improper sample handling
Intravascular Extravascular
• Improper sample delivery to the laboratory
within the vasculature within the RES
• Improper sample storage
• Transfusion Reactions • Enzyme deficiencies
• Microangiopathic (TTP, HUS) • RBCs membrane defects
• Prosthetic heart valves • Hemoglobinopathies
• Infections (e.g. sepsis, malaria) • Infection (e.g., Bartonella, Babesia, malaria)
• PCH • Autoimmune hemolytic anemia
• PNH • Others (e.g. Hypersplenism and liver diseases)
Types of Hemolysis
Laboratories should have a procedure in place

for distinguishing in vivo and in vitro 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.
Causes of In Vitro Hemolysis

1 2 3 4
Collection Transport Processing Storage
• 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
Mechanisms of Interference by Hemolysis

1. Spectrophotometric Interference
• Due to the ability of hemoglobin to absorb light at 415-, 540-, and 570-nm wavelengths.
Leading to either falsely ↑ or ↓ concentrations of the measured parameters.
• The direction and degree of the interference largely depend on the analyte and the method.

2. Release of the Cell Components Into the Sample
• Some components are present in higher levels in blood cells as compared to plasma or
serum.
• 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.

2. Release of the Cell Components Into the Sample
Intracellular Concentration
Analyte (Compared to Extracellular)
LDH ↑ 160 ×
Phosphorus ↑ 100 ×
Potassium ↑ 40 ×
AST ↑ 40 ×
Folic acid ↑ 30 ×
ALT ↑7×
Magnesium ↑3×

3. Sample dilution
• Some analytes are present in much higher concentrations in plasma than in blood cells
like albumin, bilirubin, glucose, sodium, and a few others.
• This dilution effect causes clinically significant bias only at higher degree of hemolysis.
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.
2
Lipid
Interference
• Lipemia is defined as a turbidity of the sample visible to the naked eye.

• Sample turbidity is caused by the light scattering due to the presence of large lipoprotein particles.
• The ↑ in concentration of lipoproteins in blood most commonly occurs due to:
1. postprandial TGs ↑,
2. parenteral lipid infusions,
3. Lipid disorders.
• Not all lipoproteins have equal contribution to the sample turbidity.
• The effect of lipoprotein particles on the sample turbidity depends on the size of the particles.
Chylomicrons and VLDL lipoproteins, the largest lipoprotein particles in the circulation, have the
greatest contribution to the sample turbidity.
• To avoid postprandial lipemia, patients are therefore requested to fast for 12 hours before the
blood sampling.
S. Turbid Turbid Milky

Clear + + ++ ++ +++ ++++

Mechanism of Interference
a. Interference by Light Absorption
• The lipemic sample absorbs light causing a ↓ in
the light intensity passing through the sample.
• The lipemic interference affects a wide range of
wavelengths (300–700 nm).
• Sample absorbance ↑ with the ↓ wavelength and
is maximal in the UV range.
• That is why many enzymatic methods in which
the end product is measured at 340 nm (NAD[P]
or NADP[H]) are strongly affected by lipemia.

b. Interference by Light Scattering
• Lipemic samples also cause light scattering.
• Light scattering occurs in all directions, and its intensity depends on:
― The number and size of lipoprotein particles
― The wavelength of measurement.
• For this reason, light scattering of lipoprotein particles causes significant interference with
turbidimetry and nephelometry.
― In methods where the transmittance of light is inversely proportional to the
concentration of the analyte, in the absence of the sample blank, sample turbidity
causes positive bias.
― However, in some competitive assays where the transmittance of light is directly
proportional to the analyte concentration, sample turbidity will cause negative bias.

Healthy Lipemic
2. Interference Caused by The Volume Depletion Effect Individual Individual
• The ↑ in the lipoprotein particles leads to an ↑ in the plasma volume
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
Total Plasma Volume

concentration in all methods in which the concentration of respective
analyte is measured in the total plasma volume.
• One example of this interference is pseudo-hyponatremia. This type of
interference affects electrolytes only if measured by flame photometry
and ion-selective electrodes but not in direct potentiometry.
• However, it must be noted that the volume displacement effect of the
lipemic sample will affect the electrolyte measurement only in grossly
lipemic samples (TGs >1500 mg/dL).

3. Interference Caused by Partitioning of The Sample
Upon centrifugation of a lipemic sample,
• Lipoproteins are not homogeneously distributed in the serum/plasma Top layer of
Lipid-soluble
due to the lipid gradient. analytes
― 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

Albumin
4. Interference Caused by Physicochemical Mechanisms
a. An excess of lipoproteins in the blood may interfere in
electrophoretic and chromatographic methods by
causing abnormal peaks.
α1 α2
Elevated levels of TGs and lipoprotein particles may Pre-albumin
disturb the electrophoretic pattern and morphology,
as well as falsely ↑ the relative % of the prealbumin, Protein Electrophoretic pattern
albumin, and α1- and α2-globulin regions.
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.

One additional complication of excessive lipemia is the ↑ sample susceptibility to hemolysis leading
to the specific turbid and reddish appearance of the sample (the so-called “strawberry milk”
appearance).
This effect is most probably caused by the ↑ fragility of the RBC membranes due to the alterations
in the content of the phospholipid membrane layer and is more pronounced with the ↑ in lipid
(particularly TGs) concentrations.

Removal of Lipids From the lipemic Samples
― In the hospital environment, lipemic samples are not infrequent. They most often originate from
emergency departments, ICUs, endocrinology and GIT clinics from patients suffering from
conditions e.g. acute pancreatitis, acute or chronic kidney failure, thyroid disorders, and DM.
― Unlike hemolysis, the interference caused by lipemia can be fully eliminated, or at least
reduced, by removing the excess of lipids from the sample (delipidation).
― Still, even if lipids have been successfully removed from the sample, any visible turbidity
of a sample should be documented and reported with the test results because it offers
clinically useful information about the patient.
― Moreover, lipid testing and testing for lipid-soluble drugs (e.g., benzodiazepines) and
hormones (e.g., thyroid hormones) should always be done before delipidation.

Methods for lipid removal

a.Ultracentrifugation
b.Highspeed centrifugation
c. Lipid-clearing agents

a. Lipid Removal by Ultracentrifugation
• Ultracentrifugation is considered a gold standard for lipid removal from lipemic samples,
However it is not widely available in many laboratories.
• Ultracentrifuges use the centrifugation force of almost 200,000 g and are very effective in
clearing lipemic sera by separating lipids, especially chylomicrons (top layer) from the
aqueous part (lower layer) of the sample. After centrifugation, the infranatant (lower part of
the sample) can be transferred into the clean tube and analyzed.
• It should be kept in mind that by removing the lipid layer, one also removes lipid-soluble
analytes like drugs and hormones.
• Results reported from ultracentrifuged samples or samples from which lipids have been
removed in any other way should be appropriately annotated to ensure clinicians are
aware that the sample has been manipulated to obtain the reported results.

a. Lipid Removal by Ultracentrifugation
RCF = (RPM)2 × 1.118 × 10-5 × r
RCF (G-force) Relative Centrifugal Force

rpm Revolutions per minute (rpm)
r Radius of rotor (mm)
• 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.

b. Lipid Removal by High-Speed centrifugation
• High-speed centrifugation using the microcentrifuge with a maximum centrifugation
speed of up to 20,000 g may therefore serve as an acceptable alternative and is the
method of choice for most laboratories.
• The effectiveness of high-speed centrifugation depends on the concentration of lipids
in the lipemic sample.
• However, it must be emphasized that ultracentrifugation is superior to high-speed
centrifugation for grossly lipemic samples.
― By using the ultracentrifuge, TGs concentration may be reduced 7-fold.
― whereas the high-speed centrifuge may achieve only 3.4-fold reduction.

c. Lipid Removal by Lipid-Clearing Agents
• Lipid-clearing agents are widely used in many labs due to their low cost,
convenience, and ease of use.
• Lipid-clearing agents
(cyclodextrin, polyethylene glycol, dextran sulphate, hexane, and others):
― may vary in their ability to extract lipids from a lipemic sample
― may also lead to reduction of a significant amount of protein from
the sample.
• It is therefore extremely important for laboratories to verify the performance of such
reagents before their routine use because they may not be appropriate for a wide range of
analytes due to their low recovery.

c. Lipid Removal by Lipid-Clearing Agents True High-speed
Concentration Lipoclear centrifugation
140
For example, 120

LipoClear spin columns
(Iris International Inc., Westwood, Mass.) 100
may lead to :
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

Lipid removal takes time and may cause delays in reporting the results.
• 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.
3
Icteric
Interference

Introduction
For human plasma/serum:
• The normal bilirubin concentration : up to 1.2 mg/dl (20 μmol/L)
• Color change becomes detectable : when bilirubin > 1.2 mg/dl (34 μmol/L)
• Icterus is clinically considered : when bilirubin > 6.0 mg/dl (100 μmol/L)
• Icteric plasma is commonly seen in patients from ICUs, GIT centers, and pediatric clinics.
Bilirubin interferes with numerous chemistry tests such as:

― Enzymes (ALT, ALP, CK, lipase)
― Electrolytes
― Metabolites (urea, creatinine, glucose)
― Lipids (cholesterol, TGs)
― Proteins (albumin, TP, IgG)
― Hormones (estradiol, beta-HCG, free T3)
― Some drugs (gentamicin, phenobarbital, theophylline, tobramycin)

Introduction
Bilirubin is present in the blood in several distinct forms:
― Unconjugated bilirubin is not water-soluble and therefore transported in blood bound to albumin.
― Conjugated bilirubin are soluble in water.
― Additionally, bilirubin photoisomers may be found in the blood of neonates.
All these different molecular forms of bilirubin have:
― Different physical properties
― Different chemical properties
― Behave differently in different chemistry assays.
The total amount of measured bilirubin in the patient is a mixture of these different forms.
― The different forms of bilirubin cause interference to various degrees with different methods,
― The same forms of bilirubin can act differently with the same assays on different instruments.
Most interference studies performed by manufacturers or others were done on commercially available
forms of unconjugated bilirubin that may not correspond to what is found in the blood.
This is why data obtained by interference studies does not mimic real scenarios in human blood.

Example
Interference caused by bilirubin differs among instruments and assays.
For example, In serum creatinine measurement:
Bilirubin exerts interference of different magnitudes (strong, moderate, or negligible) and directions
(positive & negative interferences), both with Jaffe & enzymatic methods from different manufacturers.
While some methods are not affected by bilirubin at all, others may exhibit strong interference by
bilirubin, causing clinically significant bias for creatinine measurement and compromising the
adequate management of patients with kidney disease.
• It has been demonstrated that even if two methods have identical reagents, differences in their
susceptibility to interference by bilirubin may still occur. These differences may be due to the
different incubation times and temperatures and other parameters related to the assay setup.
• Interestingly, although enzymatic methods are often considered the method of choice due to
being less susceptible to various interferences, bilirubin has been reported to cause greater
interference in some enzymatic creatinine assays than in Jaffe methods.

Minimizing the icteric effect
• Unfortunately, laboratories cannot do much about removing or minimizing the effect of
icteric interference.
― Bilirubin oxidase and blanking procedures have been recommended.
― Possible options are dilution of the sample (possible only for analytes present at high
enough concentrations in the blood)
― Testing the requested analytes with a different method or on a different instrument for
which icterus does not cause clinically significant interference.
• 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.

Mechanisms of Interference
Mechanisms of Interference Caused by Icterus
Icterus interferes through 2 mechanisms that may occur simultaneously in one sample:
Bilirubin causes spectrophotometric interference due
to its ability to absorb light in the wide range of
wavelengths between 400 and 540 nm.
2. Chemical Interference
Bilirubin produces negative bias on assays that
involve H2O2 as an intermediate reaction
(e.g. cholesterol, glucose, uric acid, TGs).
Detection of Hemolytic, Icteric, and Lipemic Samples


1. Visual Detection of Serum HIL Indices
Such an approach is historically-used, highly unreliable and not recommended because the
degree of hemolysis, lipemia and icterus is either over- or underestimated.
― Lack of accuracy
Lab personnel are not able to accurately assess the degree of hemolysis, icterus, or lipemia
in serum, even if well trained and when using a color standard for comparison.
― Inter-individual differences in visual sensitivity to different colors
Poor reproducibility in estimating the serum indices degree between different lab members.
― Combined presence of hemolysis and icterus (e.g. neonatal samples),
This combination makes the evaluation more difficult.
― Presence of other serum substances, such as medical contrast media (e.g. Patent Blue
dye), may also influence the human ability to detect not only hemolysis but also icterus and
lipemia.

1. Visual Detection of Serum HIL Indices
Hemolysis becomes visible at the concentration

of 0.03 - 0.05 g/dL of free Hb
Lipemia causes sample turbidity, which

approximately corresponds to the serum TGs
concentration.
↑ concentrations of serum indirect bilirubin lead

to yellow to orange coloration of the serum,
Color standard scales provided by Clinical Institute of Chemistry, University Hospital Center “Sestre milosrdnice,” Zagreb, Croatia.

2. Automated Serum HIL Indices
• Today, most mainstream chemistry analyzers can detect serum indices by the use of:
― Semi-quantitatively
― Spectrophotometric measurement
― Grading the interfering substances into categories
• The serum index is automatically reported for every sample and can be used to determine
the degree of interference and its effect on the requested parameters.
• Advantages of automated serum index detection:
― High precision
― Provide an objective and standardized way to screen for common interferences and
manage specimen rejection via built-in rules.
― Improves laboratory turnaround time, leads to an ↑ in laboratory efficiency.
Management of Hemolytic, Icteric, and Lipemic Samples


Management of Hemolytic, Icteric, and Lipemic Samples
Unfortunately, there is a large discrepancy among the ways results are reported from samples
with interferences, among different countries, institutions, and even individuals, for example:
― Analyze and report all components or
― Reject the sample and not analyze anything or
― Analyze only selected components that are not affected by the interferent.
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).
4
Paraproteins
Interference

Overview
• Paraproteins are monoclonal heavy chains, light chains, or intact immunoglobulins present
in the blood or urine produced by a clone of plasma cells or B cells.
• Paraprotein interferences are not uncommon.
• The paraprotein interference frequency has been estimated to be as high as 3–4% in hospitals.
• Laboratory staff should carefully review every case in which a measured concentration of an
analyte does not correlate with the clinical condition of the patient after all potential sources of
errors have been investigated.
• Paraprotein interferences have been observed on different analytical instruments, and they
appear to be methodology and concentration dependent.
― They affect not only turbidimetry and nephelometry but also some common chemistry
assays with spectrophotometric detection.
― The likelihood of paraprotein-caused interferences ↑ with an ↑ paraprotein concentration.

Paraprotein Interference With Different Assays
Group of Analytes Molecule
Enzymes ALP, GGT, LDH

Electrolytes, minerals Ca2+,P ̶ ,Iron
Metabolites Bilirubin, Cholesterol, Creatinine, Glucose, Urea, Uric acid
Proteins CRP, IgA, IgG
Hormones TSH, hCG
Drugs Gentamicin, Vancomycin, Valproic acid, Phenytoin
Cardiac markers Troponin I
Tumor markers AFP, CA-125
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

a. Precipitation of the Paraprotein
Precipitation depends on:
• The assay parameters, such as:
― Reaction components
― Presence of assay (reagent) additives such as preservatives and surfactants
― Ionic strength
― pH (protein precipitation can occur at both very low and very high pH)
• The paraprotein physicochemical characteristics.
• The specimen type (choice of anticoagulant in specimen tubes).
For example, IgM interfered with a glucose hexokinase method for using reagents on Hitachi
Modular systems (Roche Diagnostics GmbH). Glucose in lithium heparin plasma was
extremely low in that patient, but the interference was not present in serum tubes. Moreover,
the interference with glucose was absent when using dry-chemistry on a Vitros 950 analyzer
(Ortho Clinical Diagnostics).

b. Binding of Paraprotein to Assay Components
• Paraproteins may bind to the analyte or any other component of the reaction mixture.
• The effect of such interference depends on which component the paraprotein binds.
• For example:
― IgM paraprotein may bind to latex particles resulted in high CRP, ASO, IgG, IgA or IgM
values resulting in false ↑ of these results.
― When measured with another method, the concentration of these tests were within the
reference interval.
• This kind of interference does not cause sample turbidity.
Reaction kinetics for the unaffected sample and a sample with an interfering paraprotein are
very similar, and therefore this type of interference cannot be detected by reviewing the
reaction curve on the instrument.

c. Paraprotein Interference Due to Volume Displacement
• Paraproteins affect chemistry assays by the volume displacement effect (the same as lipemia).
• This paraprotein effect causes pronounced changes in electrolytes testing, especially Na+ .
― Using indirect ISE technology causes false hyponatremia
― while direct ISE technology can accurately determination of serum Na+ concentration
• As a general rule, in samples with TP more than 10.0 g/dL or less than 0.4 g/dL, electrolytes
(especially Na+) should be measured by direct ISE technology.

Complexed
Direct vs Indirect ISE technology
Electrostatically bound
Measured by
Results from sample with Indirect ISE
Measured by
abnormal proteins, lipids, and electrolytes Active Direct ISE
will not match.
Direct ISE results are most physiologically correct
Non-aqueous Solution
(Lipids, Proteins)
Indirect ISE cannot correct for Measured by

volume displaced by lipids and proteins Indirect ISE Aqueous Solution Measured by
(Serum) Direct ISE

d. Paraprotein Interference Due to Change in Sample Viscosity
• Viscosity is much higher in samples with very high paraprotein levels or in refrigerated
samples in which a gel has been formed (e.g. in the case of cryoglobulinemia).
• Sample viscosity affects the volume of the sample pipetted in the reaction mixture.
• Many instruments are able to detect such changes in sample viscosity and trigger an alarm
if viscosity is not within predefined limits.
― In such cases, a rerun in a dilution mode is the recommended corrective action,
provided the analyte can be accurately measured in the diluted specimen.
― In instruments where this feature is not available, ↑ sample viscosity may lead to
incorrect sample volume and cause falsely ↑ or ↓ results, depending on the assay
format.

Elimination of Paraprotein Interference
Laboratories may also apply various approaches to eliminate paraprotein interference:
1. Change the analytic method: samples can be analyzed on an alternative instrument with
a different method.
2. Paraproteins Removal: Proteins in the sample can be precipitated by a blocking agent,
ethanol, ammonium sulphate, or polyethylene glycol, while the analyte of interest
remains in the supernatant.
3. Sample serial dilutions may be performed.
4. Sample filtration to remove the proteins.
5
Heterophilic Antibody
Interference
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).
3. Auto-antibodies endogenous human antibodies interacting to an analyte.

4. Therapeutic antibodies where the antibody given therapeutically interferes with an assay
5. Macro-analytes which are oligomeric or polymeric conjugates of an analyte,
or macro-enzymes either conjugated with the endogenous antibody or not.
6. Rheumatoid factors IgM directed nonspecifically against Fc portion of IgG
• HAs may arise in a patient in response to:

― Exposure to certain animals
― Exposure to animal products
― Infection by bacterial or viral agents
― Arise nonspecifically
• 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.
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.
A patient history with respect to:

― exposure to animals or
― exposure to animal products, or
― autoimmune diseases
alerts the laboratorian towards the possibility of encountering heterophilic antibody interference.

How problematic is heterophilic antibody interference?
• Heterophilic antibodies (HAs):
― Are polyreactive antibodies,
― Have no clear immunogen and are of more non-specific type
― Recognize IgG from different species.
― Bind mostly to the Fc region of assay antibodies. However, binding to other parts of the
assay antibody (e.g., idiotope or the ‘hinge’) have been reported.
• HAs interference is:
― More pronounced if the assay antibody-pair is from the same (animal) source.
― Less frequent if monoclonal antibodies (mostly of murine type) are used in the assay,
but the magnitude of interference is higher.
• HAs are found more in sick and hospitalized patients with reported prevalence of 0.2 – 15%.
• During the last decade most commercial assays have started to incorporate blocking reagents
against the HAs in their assay reagent formulation, reducing this percentage.
• However, No blocking-reagent can guarantee 100% protection against such interference.

Thus, it is important to note that while frequency of HAs interference may have gone down and
The cases that are reported now, have a large interference magnitude, affecting the clinical
interpretation of assay results much more.
D-Dimer immunoassays seem more susceptible to HA interference;

Wu et al. reports that 66.7% of incorrect D-Dimer results were caused by HAs.
Use of HA blocker reagents minimized the interference.
Wu Y, Xiao YX, Huang TY, et al. What makes D-dimer assays suspicious e heterophilic antibodies? J Clin Lab Anal October 15, 2018:e22687.
Thyroid function immunoassays are also frequently impacted by HA interference;

― the HA interference falsely ↑ the TSH (commonly, a sandwich assay)
― while ↓ free thyroid results (a competition assay)
Favresses J, Paridaens H, Pirson N, et al. Massive interference in free T4 and free T3 assays misleading clinical judgement. Clin Chem Lab Med 2017;55:e84-6.
Monchamp T, Chopra IJ, Wah DT, et al. Falsely elevated thyroid hormone due to anti-sheep antibody interference in an automated chemiluminescent immunoassay. Thyroid 2007;17:271-5.
PSA was falsely elevated in a patient after radical prostatectomy;

they resolved the clinical versus PSA result discrepancy by repeating the result in several immunoassays.
Kiyo H False elevation of prostate-specific antigen caused by heterophilic antibody interference after radical prostatectomy: a case report. Acta Urol Jpn 2017;63:435-7.

Digoxin with high serum levels (4.2 ng/mL) were observed in a patient 24 h after his last dose.
However, there were no clinical symptoms of digitalis toxicity.
• The affected immunoassay, a competition type, used a monoclonal assay antibody.
• 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.
• The specimen showed marked non-linearity in the original assay.

• Removal of HAs by both sample ultrafiltration and protein A adsorption
followed by digoxin re-assaying in the original assay, no digoxin was detected.
• 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.

Rheumatoid Factors (RF) are IgM type antibodies interact with the assay antibodies Fc portion.
― RF is present in serum from >70% patients with rheumatoid arthritis.
― RF is also may found in patients with other autoimmune diseases.
― RF concentration may ↑ in infection or inflammation.
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.

In an interesting study where RF interference gave false positive cardiac troponin I results, the
authors inactivated RF by sample incubation with anti-RF and the interference was removed.
Dasgupta A, Banerjee SK, Datta P. False positive Troponin I in the MEIA due to the presence of rheumatoid factors in serum. Am J Clin Pathol 1999;112:753-6.
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.

Macromolecules
Another source of HA-related interference is the conjugation of analytes to antibodies in certain
patients’ sera, leading to macromolecules of altered antigenicity (e.g. macroamylasemia and
macroprolactinemia).
Additionally antigens can polymerize in patient sera, resulting in altered antigenicity as well. This is
the case in so named ‘big prolactin’ or ‘big big prolactin’; these macroprolactin molecules often
produce reduced prolactin immunoassay results compared to the physiologically active hormone
concentrations.

Interference from autoantibodies and therapeutic antibodies
• Autoantibodies are endogenous patient’s antibodies that bind to either the analyte itself or
to one of the reagents used in the assay.
• Such antibodies are more frequently observed in autoimmune disease patients.
A. Autoantibodies to the analyte

The autoantibody may:
― bind to the analyte-label conjugate in a competition type immunoassay, reducing the signal,
and giving a false positive result.
― bind to the analyte in a sandwich assay or competition assay that uses a label containing an
analog of the analyte, giving false negative results.
An example has been described in a cardiac Troponin I assay where the autoantibody to
troponin caused negative interference.
Eriksson S, Halenius H, Pulkki K, et al. Negative interference in cardiac troponin I immunoassays by circulating troponin autoantibodies.
Clin Chem 2005;51:839-47.

B. Autoantibodies to a reagent component
• In one example, an endogenous anti-avidin antibody interfered in a theophylline assay that
used the avidin-biotin system.
― In this competition type immunoassay, the autoantibody interacted to avidin in the reagent,
interfering in complex formation, thus lowering signal giving false positive results.
― In the sandwich type, the reduced signal would have caused false negative results.
Banfi G, Pontillo M, Sidoli A, et al. Interference from antiavidin antibodies in thyroid testing in a woman with multiendocrine neoplasia
syndrome type 2B. J Clin Ligand Assay 1995;18:248-51
• There are many examples of anti-streptavidin antibodies mimicking heterophilic antibodies

in assays using streptavidin-biotin reaction.
Rulander NJ, Cardamone D, Senior M, et al. Interference from anti-streptavidin antibody. Arch Pathol Lab Med 2013;137:1141-6.

B. Autoantibodies to a reagent component
• Auto-antibodies to various label enzymes (e.g., ALP, peroxidase) have been reported; these
may interact with the label enzyme in an ELISA, ↓ the signal, and falsely ↑ the results in a
competition type immunoassay.
• Therapeutic antibodies are antibodies used to bind and inactivate toxic components from
circulation and treat toxicity of various substances.
― Thus, Digibind (Glaxo/Burroughs Wellcome), Fab-fragments from ovine anti-digoxin
antibodies and used to treat digoxin toxicity, interferes in most digoxin assays that do
not use sample pretreatment.
― This interference is removable by ultrafiltration or protein precipitation.
Dasgupta A, Wells A, Datta P. Effect of digoxin fab-antibody on the measurement of total and free digitoxin by fluorescence polarization
and a new chemiluminescent immunoassay. Ther Drug Monit 1999;21:251e5.

• Characteristics: HAAA are different from other heterophilic antibodies by:
― Their specificity to the antibodies from certain species
― Their stronger avidity.
• Source:
1. Exposure to antibodies of animal source (in the majority of the cases) in the form of
diagnostic (i.e. tumor-targeted imaging agent) or therapeutic monoclonal antibodies.
― Most antibody-targeted imaging agents and antibody-targeted drugs use murine antibodies.
― Digibind, used to treat digitalis toxicity, are Fab fragment of sheep anti-digoxin antibodies.
― Factor VIII used as drug is also from pigs.
― Many vaccines are generated in rabbits or chickens (eggs).
2. Exposure to animals:
― Animal contact (e.g., animal husbandry or keeping of animals as pets)
― The transfer of dietary antigens across the gut wall in conditions such as celiac disease

• The human anti-animal antibodies can belong to IgG, IgA, IgM, and rarely, IgE class and can be
directed to any part of the monoclonal antibody used in the assay (Fc, Fab, idiotope, etc.).
• 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

• Since these antibodies are mostly murine in source, the most prevalent examples of HAAA
interference are from human anti-mouse antibodies (HAMA).
• The magnitude and duration of HAAA vary wildly.

― Magnitude: serum concentrations of HAAA range from micrograms to grams per Liter.
― Duration: The HAAA may be transient lasting a few days to months and years.
The possibility of interfering antibodies being transient may make detection and solution of
discordant results from such types of interference difficult.
• 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.

As more and more HAMA monoclonal antibodies are used in commercial immunoassays
(because of the specificity of the antibody and the reliability of the antibody supply), the impact of
HAMA interference in terms of incorrect result and resulting inaccuracy in diagnosis and therapy
has become more serious.
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%).

Mechanism of heterophilic antibody interference
A. In the sandwich type immunoassays
1. Heterophilic antibodies can form the ‘sandwich complex’ even in absence of the target
antigen, generating mostly false positive results.
2. However, if the interfering antibody binds only one of the assay reagent antibodies
(capture or label), reducing reagent concentration, false negative results (though less
frequent than false positive results) are observed.
1 2

B. In the competition type immunoassays
1. If the assay uses a labeled analyte (or its analog), the interfering antibody (e.g. auto-antibody)
may interact with the label, reducing the assay signal, and generating a false positive result.
Without interference With interference
Signal Signal
The interfering antibody

interact with the label
Labeled analyte Target Analyte
Capture Antibody
Solid Phase Solid Phase


2. The same thing happens even if the assay using labeled antibody; the competing analyte
(or its analogs) on the ‘capture reagent’ will bind to the interfering antibody, again generating
a reduced signal, and false positive results.
Signal
Labelled antibody The interfering antibody

interact with the
Analyte analog Target Analyte Competing analyte
(Competing analyte)
Capture Antibody


3. Heterophilic antibody may interact with the assay antibody, ↓ its effective concentration (via
steric-hindrance), which then ↓ the assay signal, and generates a false positive result.
Signal
The interfering antibody
interact with the
assay antibody
Labelled antibody
Analyte analog Target Analyte

(Competing analyte)
Capture Antibody


4. If the HA interacts with the target analyte only (but not the competing analyte-reagent; this
happens when the assay uses not the analyte in the reagent to compete, but an analog), then
the analyte conc. in the assay reaction ↓, and false negative results are seen.
Signal The interfering antibody

interact with the target analyte
Labelled antibody
Analyte analog Target Analyte

(Competing analyte)
Capture Antibody


Approaches to deal with heterophilic antibody interferences
1. Examination of the patient history
― Exposure to animals
― Exposure to animal products
― History of hyperactive immune system (hypersensitivity or autoimmunity)
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.

Approaches to deal with heterophilic antibody interferences
5. Removal of the interfering antibody and re-assay
a. Sulfo-salicylic acid precipitation of proteins is routinely used by some commercial assays to
make protein-free specimens, which are then assayed in the analyzer.
b. Selective adsorption of human IgG by a solid phase containing protein A or protein G.
However, This does not work if the majority of the interfering antibodies are of IgM type.
c. Precipitation with polyethylene glycol (preferable PEG-6000): precipitates antibody fraction.
d. Protein precipitation (using TCA, sulfosalicylic acid, or ammonium sulfate)
More suitable for LMW analytes, if they are not highly protein bound, where they could be
extracted away from the interfering immunoglobulins by preparation of a protein-free filtrate.
e. The centrifugal ultrafiltration (suitable for LMW analytes)
A fast and easy method, using 10- or 30- kDa MW filter membrane.
6. Analyzing using a different method known to be free from such interferences.

Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 2. In Vivo Interferents ► II. Exogenous components
II
Exogenous component
introduced to the blood

• Sample carryover
• Fibrin clot
I. Biological blood component II. Exogenous component
• Hemolysis
introduced to the blood
• Lipemia 1. Prescribed medication
• Icterus 2. Supportive medical therapy (parenteral emulsions, contrast media agents)
• Paraproteins 3. Natural preparations
• Heterophilic antibodies 4. Accidental exposure and poisoning
5. Nutritional Supplements (Biotin)
Prescribed Supportive Natural Accidental Nutritional
Medications Medication Preparations Exposure & poisoning Supplementations
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.
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).
Interference with Creatinine measurements

False ↑ in colorimetric method (Jaffe Reaction)
1. Antibiotics: Cephalosporin antibiotics (cefpirome IV administration)
2. Analgesics: Certain Analgesics in subtherapeutic, therapeutic, and toxic concentrations (e.g.
acetaminophen, acetylsalicylic acid and metamizole)
False ↓ in enzymatic method

1. Toxic levels of metamizole (analgesic)
2. High concentrations of therapeutic catecholamines (e.g. epinephrine, dobutamine, dopamine)
3. Ethamsylate (hemostatic): significant ↓ in creatinine concentration (approximately by 50%)
There is evidence that ethamsylate also causes significant false ↓ in the concentrations of
cholesterol (9.2%), TGs (15.6%), and uric acid (15.4%).
Interference with Creatinine measurements
Metamizole Acetaminophen
Ethamsylate
Ethamsylate cefpirome
Acetylsalicylic acid
Interference with Ionized Calcium measurements
The active metabolite of leflunomide (teriflunomide), a synthetic drug with immunosuppressive

and antiviral properties, interference with ionized calcium (iCa2+).
In kidney transplant patients treated with leflunomide,
• Low iCa2+ concentrations are sometimes observed without any clinical signs of hypocalcemia
• After oral administration, leflunomide is rapidly converted into teriflunomide, which causes
falsely ↓ iCa2+ concentrations.
• The interference is dependent on the type of the blood gas analyzer:
―Affect the Rapidlab-1265 (Siemens Diagnostics) and i-Stat POC analyzer (Abbott),
―but not affect the ABL800-FLEX blood gas analyzer (Radiometer, Copenhagen, Denmark).
This drug is used also in patients with rheumatoid arthritis,
• Therefore, every hypocalcemia in laboratories using Rapidlab or i-Stat POC analyzers should
be interpreted with caution and compared with the patient’s clinical condition.
Interference with Ionized Calcium measurements
Leflunomide product
in Egyptian market
Interference with Sodium measurements
Thiopental (Pentothal) is a barbiturate used for the treatment of

↑ intracranial pressure.
• The central laboratory analyzer Dimension Vista (Siemens),
which uses the V-LYTE Integrated Multisensor Technology
system for electrolyte measurement, produces falsely ↑ Na+
concentrations in the presence of thiopental.
• However, when the Na+ is measured on a POC analyzer
using direct potentiometry (Rapidlab 1200, Siemens),
there is no evidence of thiopental interference.
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.
* Gadolinium contrast agents

― Either ionic, neutral, albumin-bound, or polymeric forms
― Typically used in magnetic resonance imaging.
* 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
* Iodine-based compounds Interference

Iodine-based compounds can interfere with chemical reactions in several ways.
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)
* Gadolinium contrast agents Interference

Interference with Calcium (Ca2+)
• Gadolinium contrast agents act as Ca2+ chelators,
― affect Ca2+ measurement using colorimetric assays
― ISE and inductively coupled plasma-atomic emission spectroscopy can still reliably determine
Ca2+ concentration.
Interference with Selenium (Se)
• Inductively coupled mass spectrometry (ICP-MS) is often used for trace elements measurement.
• There is many evidences that gadolinium interferes with ICP-MS measurement of selenium and
causes false ↑ of selenium concentrations in patients undergoing MRI.
* 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.
* 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.
* Inaccurate labeling of herbal products:

• Herbal products may not accurately reflect their content, and adverse events or interactions
attributed to a specific herb may be due to:
― Misidentification of plants.
― Contamination of plants with pharmaceuticals or heavy metals.
• For example,
― Some Chinese herbal remedies may contain steroids, with the potential to interfere with
some assays and to cause suppression of the hypothalamic-pituitary-adrenal axis.
― Some Ayurvedic herbal medicine products may be contaminated with lead, with the
potential to cause toxicity.
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.
* Example: Digoxin interference by cardiac glycoside

Numerous cases of poisonings by cardiac glycoside–containing
lily of the valley
plants like lily of the valley (Convallaria majalis) or oleander (Convallaria majalis )
(Nerium oleander) are reported Cardiac glycoside has a structural
similarity to the digoxin molecule, therefore can interfere with
the digoxin measurement.
Convallatoxin is a glycoside extracted from Convallaria majalis.
Due to the significant cross-reactivity between convallatoxin and Oleander
(Nerium oleander)
digoxin, the digoxin assay is proposed to be used as a screening
tool for detection of convallatoxin ingestion.
* Example: Creatinine interference by nitromethane

• Small racing cars run on nitromethane, which has been shown to interfere with
creatinine measurement, producing a falsely ↑ concentration.
• Extreme high creatinine level (may reach 90 mg/dl) without evident renal failure
has been observed in a suicide attempt in which Blue Thunder fuel
containing nitromethane was ingested.
• Nitromethane interferes with the Jaffe method, where reacts with alkaline
picrate and forms a red chromophore with absorbance similar to the creatinine
picrate chromophore. This reaction causes falsely ↑ creatinine concentration.
• When an enzymatic assay is used, creatinine can be accurately measured
in the presence of nitromethane.
• 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>.
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.
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
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.
In competitive (sandwich) immunoassay model Signal
A. The biotinylated antibody will bind to streptavidin, Labeled analyte
which is attached to the solid phase.

• The analyte of interest will compete with the labeled
Labeled antibody
analyte for binding to the biotinylated antibodies.
Analyte of interest
• Residual specimen and unbound analyte (labeled
analyte or the analyte present in the specimen) will Biotinylated antibody
be washed off. B
B
• The remaining signal will be inversely proportional Streptavidin
to the concentration of analyte.
Streptavidin-coated
microparticle B Biotin in sample
or solid phase
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
• 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
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
Immunodiagnostic Systems (IDS)

― The majority of immunoassays show a biotin tolerance of just > 70 ng/mL
― but the renin and aldosterone assays have thresholds under 10 ng/mL
Ortho Clinical Diagnostics Vitros assays

― More than 60% of these assays have biotin tolerances of 20 ng/mL or less,
― with several at 5 ng/mL and under, including Troponin ES.
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.
Beckman Coulter immunoassays are sensitive to biotin interference, e.g. thyroglobulin.
DiaSorin immunoassays are resistant to biotin interference

with the exception of a few serology assays that show sensitivities of 10 ng/mL or less.
Abbott Architect and Alinity assays are unaffected by biotin.

Solution of the problem of Biotin interference

1. Education and awareness
― At a General level, Biotin interference needs to be communicated to the wider scientific
community and relevant clinicians and healthcare professionals.
― At a local level, this be achieved through the existing channels of laboratory communication.
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.
3. Notification of biotin use at the time of blood collection
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.

5. Using the pharmacokinetic parameters of biotin
― An 8 h withdrawal period is required to ↓ biotin to below 30 ng/mL in individuals on 10 mg
daily dosing, which would suffice for many Roche assays, but would still exceed the tolerance
of all affected Vitros assays.
― For MS patients on 100-300 mg biotin, need longer withdrawal periods is awaited.
At least 48–72 h is recommended, and longer periods are necessary for very sensitive tests.
― Pharmacokinetic variability is likely, and examining such results is still important as an
additional safety check.
― Further cautions must be borne in mind:
a. In cases of renal impairment, biotin clearance will be delayed;
b. For some inborn errors of metabolism, withholding biotin may not be safe
c. Biotin washout is not an option in the acute setting.

6. Depletion of biotin
― Using streptavidin-coated agarose or magnetic beads
7. Assay re-design to improve biotin tolerance
8. Using Alternative methods

― Laboratories with access to multiple platforms may have scope to move certain tests to an
unaffected method if considered high-risk.
― Laboratories which do not have biotin-resistant platforms should implement a plan to deal
with such problems.
― For Emergency Departments served by biotin-sensitive troponin assays, access to a biotin-
unaffected cardiac biomarker would be important in the rare event of a patient on high dose
biotin therapy presenting with possible acute coronary syndrome.
2. In Vitro Interferents

 Lubricants
 Surfactants
I. Biological blood component II. Exogenous component  Separator gels
• Lipemia 1. Prescribed medication
2. Supportive medical therapy
 Clot activators
• Icterus
• Paraproteins 3. Natural preparations • Sample carryover
• Heterophilic antibodies 4. Accidental exposure and poisoning • Fibrin clot
5. Nutritional Supplements (Biotin)
Pre-analytic variables ► B. Pre-analytic Interfering Factors ► 2. In Vitro Interferents
Sample Contaminants Sample Fibrin

Lubricants | Surfactants | Clot Activators | Antibiotics | Additives | Gel Carryover Clot

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.

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.
Antibiotics (Gentamicin carryover)

• Antibiotics are commonly added into reagents and buffer solutions to prevent microbial growth.
• Carryover from reagents containing gentamicin may cause spuriously ↑ gentamicin results.
• Gentamicin is present in some diagnostic reagents (glucose, urate, direct bilirubin, CK, ALT,
AST) as an antibacterial additive and is known to cause spuriously high gentamicin results on the
Beckman Coulter AU480 analyzer due to reagent carryover.
• Therefore, gentamicin measurement should be processed in a separate batch
(i.e., before or after the measurement of any of the listed parameters) to prevent carryover.

Light Heparin interferes with numerous cardiac troponin (cTn) immunoassays

Green Green
by affecting the antigen – antibody binding and thus affecting the rate of reaction.
• A significant negative bias (up to 30%) in cTn measurement was documented.
― Affect earlier generation troponin assays by several major manufacturers.
― This bias did not correlate with cTn concentration.
• Mechanism:
― Heparin with – ve charge binds to troponin with +ve charge.
― This binding leads to the conformational change of troponin that affects the
immunoassay antibody-antigen interaction.
Li Na • Solution:
Heparin Heparin
This interference was neutralized in the 4th generation cTn assay by adding cationic
heparin blocking agent to the assay’s mixture, although in certain cases there is still
poor comparability between serum and plasma values.

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.

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.

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

Due to differences in the gel composition among different manufacturers,

it is quite possible that one manufacturer’s gel tube may be used for a particular analyte but another
manufacturer’s may not.
Moreover, if kept under improper storage conditions (time and temperature),

the gel may degrade and release small particles or globules into the supernatant.
These particles may:
• Interfere with the sample probe which affects instrument performance
• Coat the inner surface of the reaction cuvettes causing interference in immunoassays
It is therefore important to strictly follow recommendations provided by the tube manufacturers

on the appropriate storage and handling of gel tubes.

Carryover – is the contamination of a specimen by the previous one.

• Sample carryover in automated analyzers,
― Occurs due to the inefficient washing of probe and cuvette and subsequent inability of an
instrument to successfully remove any remnants of the sample or reagent.
― So, a certain amount of reagent or analyte can be transferred (carried) by the measuring
system from one assay reaction to a subsequent reaction causing erroneous results.
• As long as the instruments probes are not disposable, there is a susceptibly to carryover.
• Carryover is, of course, not unique to specific immunoassays and may occur in all assay types.
• The effect of carryover is more pronounced in highly sensitive immunoassays. (e.g. cTnI)
• To mitigate this problem,
― Implementing a reflex rule to reanalyze the affected analyte in 2 subsequent specimens.
― Also, additional probe and cuvette washing may be performed after the extremely elevated
results with an ↑ risk for carryover.

Sample Carryover Study
According to IUPAC (International Union of Pure and Applied Chemistry),

Sample carryover testing is performed by:
1. Running one sample (A) with extremely high level of an analyte at least 2 times,
2. Followed by at least 3 runs of a sample (B) with very low level of that analyte.
― If the instrument probe washing procedure is not done correctly, the results in the sample with low
analyte level will be higher, and subsequent results will show a gradually decreasing pattern.
― The performance of the instrument cuvette washing procedure is somewhat more difficult to
assess because it may require multiple runs of a sample with high and low analyte levels (the exact
number of runs depends on the number of cuvettes).

Sample Carryover Study

Carryover is expressed as the amount of analyte transferred from The red rectangle
sample A2 to sample B1, and it may be calculated as follows: represents
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

Under optimal clotting conditions,

serum is considered to be free of fibrin, fibrinogen, and cells,
and it is a preferred matrix for the most immunoassays.
To allow complete clot formation,

serum tubes should be allowed to clot for a minimum of 30 minutes.
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.
Blood from patients who are receiving heparin therapy

may require a longer time to completely clot,
and there is a greater likelihood for latent post-centrifugation clot formation.

Insoluble fibrin has been found in both serum and plasma.

Insoluble fibrin, fibrin strands, and microclots as a result of delayed and latent clotting may affect
instrument performance and cause interferences.
Some analyzers have the ability to detect clots and flag such samples for rerun,
This feature may not available on other analyzers.
If fibrin is aspirated for analysis and goes undetected,

Non-specific binding by the insoluble fibrin strands present in the sample may occur.
Resulting in a greater likelihood of getting false-positive results for that sample.
This is manifested by duplicate measurement errors
(i.e. unacceptable deviations in two measurements on the same sample).
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Quality Lectures - Pre-Analytic Variables - Dr. Tamer Soliman

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Quality in Clinical Laboratories

Dr. Tamer Soliman

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Pre-analytic Errors Analytic Errors Post-analytic Errors

Pre-analytic Errors Analytic Errors Post-analytic Errors

Test ordering Specimen

Pre-analytic Errors Analytic Errors Post-analytic Errors

Test ordering Specimen Specimen

Pre-analytic Errors Analytic Errors Post-analytic Errors

Test ordering Specimen Specimen Specimen

Pre-analytic Errors Analytic Errors Post-analytic Errors

Test ordering Specimen Specimen Specimen High

Pre-analytic Errors Analytic Errors Post-analytic Errors

Test ordering Specimen Specimen Specimen High Specimen

• Specimen contaminated with infusion fluid

Pre-analytic Errors Analytic Errors Post-analytic Errors

Test ordering Specimen Specimen Specimen High Specimen Specimen

• No specimens received/Missing tube • Insufficient specimen quantity for analysis

Pre-analytic Errors Analytic Errors Post-analytic Errors

• High analytical turnaround time

Pre-analytic Errors Analytic Errors Post-analytic Errors

‫اﻟﺘﻘﺮﻳﺮ ﱂ ﻳﻜﺘﻤﻞ‬ • Report not completed

Pre-analytic Errors Analytic Errors Post-analytic Errors

Influencing Pre-analytic Interfering

• Affect analytes concentration (↓ or ↑) • Not Affect analytes concentration

Classified into Classified into

Pre-analytic variables ► A. Pre-analytic Influencing Factors

Pre-analytic variables ► A. Pre-analytic Influencing Factors

A. Pre-analytic Influencing Factors

1. Uncontrollable variables 2. Controllable variables

Pre-analytic variables ► A. Pre-analytic Influencing Factors

A. Pre-analytic Influencing Factors

1. Uncontrollable variables 2. Controllable variables

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

Unavoidable Influences on Laboratory Results

Influence Examples of Analyte Concentrations Changed Remarks

Age ALP, LDL-cholesterol, hormones, creatinine. Provide age-dependent reference intervals

Gender ALT, γ-GT, creatinine Provide gender-specific reference intervals

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

The Canadian CALIPER study is an excellent source of reference intervals in childhood

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly

Intra-uterine Infancy Childhood Adult-life Elderly

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly

Intra-uterine Infancy Childhood Adult-life Elderly

In the early hours of extrauterine life,

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly

Intra-uterine Infancy Childhood Adult-life Elderly

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly

Intra-uterine Infancy Childhood Adult-life Elderly

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly

Intra-uterine Infancy Childhood Adult-life Elderly

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly

Fetus Infancy Childhood Puberty Reproductive Years Menopause

Birth 6 months 10 – 14 years 50 years

Pre-analytic variables ► A. Pre-analytic Influencing Factors ► 1. Uncontrollable (Physiological) variables

a. Newborn/infant population | b. Childhood to puberty | c. Adulthood | d. Elderly