THE THEORY OF

IMMUNOLOGIC AND
SEROLOGIC
PROCEDURES
Giane Trixia Mae Buño │ Ronabel Ilagan

1
 CHAPTER 6 :
Safety in the
Immunology-
Serology Laboratory
 ◦ U.S. Department of Labor, Occupational
Safety and Health Administration (OSHA)
◦ Clinical and Laboratory Standards Institute
SAFETY (CLSI), a nonprofit educational organization
STANDARDS that provides a forum for the development,
AND promotion, and use of national and
AGENCIES international standards
◦ Centers for Disease Control and Prevention
(CDC), part of the U.S. Department of Health
and Human Services Public Health Service
◦ College of American Pathologists (CAP)
◦ In 1991, OSHA mandated that all
clinical laboratories must implement a
chemical hygiene plan and an
exposure control plan.
◦ As part of the chemical hygiene plan,
a copy of the material safety data
sheet (MSDS) must be on file and
readily accessible and available to all
employees at all times.

4
◦ The MSDS describes hazards, safe
handling, storage, and disposal of
hazardous chemicals.
◦ Each MSDS contains basic information
about the specific chemical or product,
including its trade name, chemical name
and synonyms, chemical family,
manufacturer’s name and address,
emergency telephone number for further
information about the chemical, hazardous
ingredients, physical data, fire and
explosion data, and health hazard and
protection information.

5
◦ In 2006, the CDC introduced
the National Healthcare Safety
Network (NHSN). This
voluntary system integrates a
number of surveillance
systems and provides data on
devices, patients, and staff.

6
 ◦ Staff must wear laboratory coats and be
additionally protected from contamination by
infectious agents.

◦ Food and drinks should not be consumed in work

ADHERENCE TO areas or stored in the same area as specimens.
GENERAL SAFETY
Containers, refrigerators, or freezers used for
PRACTICES WILL
REDUCE THE RISK specimens should be marked as containing a
OF INADVERTENT biohazard.
CONTAMINATION
◦ Specimens needing centrifugation are capped
WITH BLOOD OR
BODY FLUIDS, AS and placed into a centrifuge with a sealed dome.
FOLLOWS:
◦ A gauze square is used when opening rubber-
stoppered test tubes to minimize aerosol
production.

◦ Autodilutors or safety bulbs are used for

pipetting. 7
 ◦ According to the CDC concept of Standard
Precautions, all human blood and other
body fluids are treated as potentially

PREVENTION infectious for human immunodeficiency

OF virus (HIV), hepatitis B virus (HBV), and
TRANSMISSION other blood-borne microorganisms that can
OF INFECTIOUS
DISEASES cause disease in human beings.

◦ Compliance with the OSHA Bloodborne

Pathogens Standard and the Occupational
Exposure Standard is required to provide a
safe work environment.

8
 ◦ Educate and train all health
OSHA care workers in Standard
MANDATES Precautions and in preventing
THAT THE bloodborne infections.
EMPLOYER
DO THE ◦ Provide proper equipment and
FOLLOWING: supplies (e.g., gloves).
◦ Monitor compliance with
protective biosafety policies.

9
◦ Blood is the most important source of
HIV, HBV, and other bloodborne
pathogens in the occupational setting.
◦ HIV is usually found in lower
concentrations. HBV may be stable in
dried blood and blood product at 25° C
for up to 7 days. HIV retains infectivity
for more than 3 days in dried specimens
at room temperature and for more than
1 week in an aqueous environment at
room temperature.

10
 ◦ Concentration of HBV or HIV; viral
concentration is higher for HBV than
THE LIKELIHOOD
OF INFECTION IN HIV
HEALTH CARE
WORKERS AFTER ◦ Duration of the contact
EXPOSURE TO
BLOOD INFECTED ◦ Presence or skin lesions or
WITH HBV or HIV
DEPENDS ON THE abrasions on the hands or exposed
FOLLOWING
FACTORS: skin of the health care worker

◦ Immune status of the health care

worker for HBV

11
 ◦ The components of this
regulation include the following:
SAFE
WORK ◦ A workplace hazard
assessment, with a written
PRACTICES
hazard certification
FOR
◦ Proper equipment selection
INFECTION
◦ Employee information and
CONTROL training, with written
competency certification
◦ Regular reassessment of work
hazards
12
 ◦ Selection and Use of
PROTECTIVE Gloves
TECHNIQUES ◦ Facial Barrier Protection
FOR
INFECTION and Occlusive Bandages
CONTROL ◦ Laboratory Coats or
Gowns as Barrier
Protection

13
 ◦ Frequent handwashing is an
important safety precaution. It should
be performed after contact with
patients and laboratory specimens.

◦ Gloves should be used as an adjunct

HANDWASHING
to, not a substitute for, handwashing.

◦ The efficacy of handwashing in

reducing the transmission of
microbial organisms has been
demonstrated.
14
◦ After completing laboratory work and before
leaving the laboratory
◦ After removing gloves. The Association for
Professionals in Infection Control and
Epidemiology has reported that extreme
variability exists in the quality of gloves, with
leakage in 4% to 63% of vinyl gloves and in 3%
to 52% of latex gloves.

◦ Before eating, drinking, applying makeup, and

changing contact lenses, and before and after
using the bathroom

◦ Before all activities that involve hand contact

with mucous membranes or breaks in the skin
15
◦ Immediately after accidental skin contact with
blood, body fluids, or tissues

a. If the contact occurs through breaks in

gloves, the gloves should be removed immediately
and the hands thoroughly washed.

b. If accidental contamination occurs to an

exposed area of the skin or because of a break in
gloves, wash first with a liquid soap, rinse well with
water, and then apply a 1:10 dilution of bleach or
50% isopropyl or ethyl alcohol. The bleach or
alcohol is left on skin for at least 1 minute before
final washing with liquid soap and water.

16
17
18
 VOLUME VOLUME RATIO SODIUM
PREPARATION BLEACH H2O HYPOCHL
ORITE
OF DILUTED
HOUSEHOLD 1 mL 9 mL 1:10 0.5%

BLEACH

19
 ◦ Decontaminate nondisposable
equipment by soaking
overnight in a dilute (1:10)
DECONTAMINATION bleach solution and rinsing
OF WORK
SURFACES,EQUIPM with methyl alcohol and water
ENT, AND SPILLS before reuse. Disposable
glassware or supplies that
have come into contact with
blood should be autoclaved or
incinerated.

20
 ◦ Wear gloves and a laboratory coat.
◦ Absorb the blood with disposable towels.
Bleach solutions are less effective in the
THE
presence of high concentrations of protein.
FOLLOWING
Remove as much liquid blood or serum as
PROTOCOL IS
possible before decontamination.
RECOMMENDE
D FOR ◦ Using a diluted bleach (1:10) solution, clean
MANAGING the spill site of all visible blood.
SPILLS IN A
◦ Wipe down the spill site with paper towels
CLINICAL
soaked with diluted bleach.
LABORATORY:
◦ Place all disposable materials used for
decontamination into a biohazard container.

21
 ◦ OSHA has defined infectious waste as blood
and blood products, contaminated sharps,
pathologic wastes, and microbiological wastes

◦ Conspicuously marked “Biohazard” with the

DISPOSAL OF universal biohazard symbol
INFECTIOUS ◦ Universal color—orange, orange and black, or
LABORATORY
red
WASTE
◦ Rigid, leakproof, and puncture resistant.
(Cardboard boxes lined with leakproof plastic
bags are available.)

◦ Used for blood and other potentially infectious

body fluids, as well as disposable

22
 ◦ Body fluid specimens, including
blood, must be placed in well
constructed biohazard
BIOHAZARD
containers with secure lids to
CONTAINERS
prevent leakage during
transport and for future
disposal.

23
 ◦ These biohazard bags should
be used for all blood, body
fluids, tissues, and other
BIOHAZARD
BAGS disposable

◦ materials contaminated with

infectious agents and should be
handled with gloves

24
 The ACIP-HICPAC recommendations determine
which vaccines to include based on documented
nosocomial transmission and significant risk for
acquiring or transmitting the following vaccine-
preventable diseases:
◦ Hepatitis B

◦ Influenza
IMMUNIZATION
◦ Measles

◦ Mumps

◦ Rubella

◦ Varicella

Optional immunizations include hepatitis A, diphtheria,

pneumococcal disease, and tetanus.

25
 ◦ The standard recommended tuberculin
Purified test is the Mantoux test, which is
Protein administered by injecting a 0.1 mL of
Derivative liquid containing 5 TU
Tuberculin (tuberculin units) PPD (purified protein
Skin Test derivative) into the top layers of skin of
the forearm. Doctors should read skin
tests 48-72 hours after the injection.

26
 ◦ QuantiFERON-TB Gold (QFT)
is a blood test used to detect
QuantiFERON- infection with TB bacteria. The
TB Gold QFT measures the response to
TB proteins when they are
mixed with a small amount of
blood.

27
 ◦ All phlebotomists & laboratory
staff need to demonstrate
immunity to rubella. If antibodies
RUBELLA are not demonstrable,
vaccination is necessary.

28
 ◦ All phlebotomists and
HEPATITIS laboratory staff need to
B SURFACE demonstrate immunity to
ANTIGEN hepatitis B. If a positive test is
not demonstrable, vaccination
is necessary.

29
 ◦ “prophylaxis” means to
POSTEXPOSURE prevent or control the
PROPHYLAXIS
spread of an infection or
disease.

30
 ◦ After occupational exposure to
HEPATITIS Hepatitis B virus (HBV), appropriate
B VIRUS and timely prophylaxis can prevent
EXPOSURE HBV infection and subsequent
development of chronic infection or
liver disease.

31
 CHAPTER 7 :
Quality Assurance
and Quality
Control
 ◦ The Clinical Laboratory
Improvement Amendments of
1988 (CLIA ’88) established a
CLINICAL minimum threshold for all aspects
LABORATORY of clinical laboratory testing.
REGULATORY
& ACCREDITING ◦ The International Organization for
ORGANIZATIONS Standardization (ISO) is the
world’s largest nongovernmental
developer and publisher of
international standards.

33
 ◦ The competence of personnel is an
important determinant of the quality
of the laboratory result.
NONANALYTICAL ◦ A complete laboratory procedure
FACTORS
RELATED
manual for all analytical
TO TESTING procedures performed in the
ACCURACY laboratory must be provided.
◦ The manual must be reviewed
regularly or annually, by the
supervisory staff.

34
WRITTEN PROCEDURAL
PROTOCOL
• Procedure name
• Name of the test method
• Principle and purpose of the test
• Specimen collection and storage
• Quality control
• Reagents, supplies, and equipment
• Procedural protocol
• Expected or normal (reference) values
• Procedural notes:
• Sources of error
• Limitations
• Clinical applications
35
◦ The information on the accompanying
specimen container must exactly
match the patient identification on the
test request.
◦ Microscopes, centrifuges, and other
pieces of equipment need to be
cleaned and checked for accuracy,
Failure to monitor equipment regularly
can produce inaccurate test results
and lead to expensive repairs.

36
 ◦ Systematic errors can be
eliminated by a program that
monitors equipment, reagents, and
Inaccuracies in
other supplies.
testing can be
◦ Sporadic or isolated errors in
systematic or
technique can produce false-positive
sporadic
and false-negative results,
depending on the technique used for
testing

37
◦ The Institute for Quality Laboratory
Medicine has developed measures
to evaluate quality in the laboratory
based on the phase of testing:
preanalytical, analytical, and
postanalytical.
◦ Currently, most laboratory errors
are related to the preanalytical and
postanalytical phases of testing.

38
 ◦ Consists of procedures used to
detect errors that result from test
system failure, adverse
environmental conditions, and
differences between technologists,
QUALITY
as well as the monitoring of the
CONTROL accuracy and precision of test
performance over time.
◦ Monitors the accuracy and
reproducibility of results through the
use of control specimens.

39
◦ Accuracy describes how close
a test result is to the true
value.
◦ Precision describes how close
the test results are to one
another when repeated
analyses of the same
specimen are performed.

40
 ◦ Used to compare the standard
deviations of two samples.
◦ Used to compare a day’s work with
COEFFICIENT that of a similar day or to compare
OF VARIATION test results from one laboratory with
the same type of test results from
another laboratory.
𝑆𝐷
◦ CV(%) = × 100
𝑀𝑒𝑎𝑛

41
◦ True positives are subjects who have a
positive test result and who also have the
disease in question.
◦ True negatives represent those who
have a negative test result but do not
have the disease.
◦ False positives are those who have a
positive test result but do not have the
disease.
◦ False negatives are those who have a
negative test result but do have the
disease.

42
 ◦ Defined as the proportion of
subjects with the specific
disease or condition who have
a positive test result.
◦ Sensitivity represents how
SENSITIVITY
much of a given substance is
measured.
◦ 𝑆𝑒𝑛𝑠𝑖𝑡𝑖𝑣𝑖𝑡𝑦(%) =
𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠
× 100
𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 +𝐹𝑎𝑙𝑠𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠

43
 ◦ Defined as the proportion of
subjects without the specific
disease or condition who have
a negative test result.
SPECIFICITY ◦ Specificity represents what is
being measured.
◦ 𝑆𝑝𝑒𝑐𝑖𝑓𝑖𝑐𝑖𝑡𝑦(%) =
𝑇𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠
× 100
𝐹𝑎𝑙𝑠𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠 +𝑇𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠

44
 ◦ Assessing the predictive value (PV)
requires knowledge of the
sensitivity, specificity, and disease
prevalence.
Predictive ◦ Prevalence is the proportion of a
Values population who has the disease.
◦ Incidence is the number of subjects
who have the disease within a
defined period per 100,000
population.

45
◦ A positive predictive value for
a test indicates the number of
patients with an abnormal test
result who have the disease
compared with all patients with
an abnormal result:
◦ 𝑃𝑜𝑠𝑖𝑡𝑖𝑣𝑒 𝑃𝑉 =
𝑇𝑟𝑢𝑒 𝑝𝑜𝑠𝑖𝑡𝑖𝑣𝑒𝑠
𝑇𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠 +𝐹𝑎𝑙𝑠𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠

46
◦ A negative predictive value for
a test indicates the number of
patients with a normal test result
who do not have the disease
compared with all patients with a
normal (negative) result:
◦ 𝑁𝑒𝑔𝑎𝑡𝑖𝑣𝑒 𝑃𝑉 =
𝑇𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠
𝑇𝑟𝑢𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠 +𝐹𝑎𝑙𝑠𝑒 𝑛𝑒𝑔𝑎𝑡𝑖𝑣𝑒𝑠

47
 ◦ Proficiency testing (PT) is
incorporated into the CLIA
requirements.
PROFICIENCY ◦ In addition to internal QC
TESTING programs, each laboratory
should participate in an
external PT program to verify
laboratory accuracy.

48
 ◦ Is the range of values that
includes 95% of the test
results for a healthy reference
Reference
population.
Range
◦ Formerly referred to as
normal values or normal
range.

49
◦ Mean is the mathematical
average of the values.
◦ Median is the middle value.
◦ Mode is the most frequently
occurring value.
◦ Standard deviation (SD) is
the square root of the
variance of the values.

50
◦ Reference range for a
particular measurement is
usually a normal bell-shaped
curve.
◦ Biometrics attempts to
describe statistical variations
in biological observation.

51
◦ The QC program also determines
how new procedures are validated
before being included as one of the
methods routinely used by the
laboratory.
◦ The QC program includes
calculation of the mean (or average
value) and standard deviation and
the preparation of control charts for
each procedure.

52
 CHAPTER 8 :
Basic Serologic
Laboratory
Techniques
◦ Serologic testing has long been
an important part of diagnostic tests
in the clinical laboratory for viral and
bacterial diseases.
◦ Immunologic testing is done in
many areas of the clinical
laboratory, including: microbiology,
chemistry, toxicology, immunology,
hematology, surgical pathology,
cytopathology, immunohematology
(blood banking).

54
◦ Most immunology tests are done on
serum; Lipemia, hemolysis, and
bacterial contamination can make
the specimen unacceptable.
◦ Icteric or turbid serum may give
valid results or may interfere.
◦ Blood specimens should be
collected before a meal to avoid
chyle.
◦ Contamination with alkali or acid
must be avoided.

55
 ◦ Are used in the immunology
serology laboratory for the
quantitative transfer of
PIPETTES
reagents and the preparations
of serial dilutions of
specimens.

56
Graduated Pipette
◦ Is a straight piece of glass
tubing with a tapered end
and graduation marks on
the stem separating into
parts.
◦ delivers the liquid between
two calibration marks.
◦ Used when great accuracy
is not required.

57
Serologic Pipettes
◦ resembles the graduated
pipette, but has a frosted
ring and enlarged tip
opening.
◦ The rate of fall of liquid is
much too fast for great
accuracy or precision.

58
Automatic pipette
◦ allow fast repetitive
measurement and
delivery of solutions
of equal volumes.

59
 ◦ Dilution is an indication of
relative concentration.
DILUTION ◦ All dilutions are a form of
ratio.

60
 ◦ expressed as concentration per some
convenient standard volume. In a
certain procedure, 0.5mL of blood is
diluted to a total of 10 mL with
DILUTING various reagents, and 1 mL of this
SPECIMEN dilution is then analyzed for a
particular chemical constituent.

◦ The final result is to be expressed in

terms of the concentration of that
substance per 100 mL of blood.
61
 ◦ A dilution factor is used to
correct for having used a
DILUTION
diluted sample in a
FACTOR
determination rather than
the undiluted sample.

62
 ◦ These single dilutions are usually expressed
as a ratio, such as 1:2, 1:5, or 1:10, or as a
fraction, ., 1⁄5, or 1⁄10. These ratios or
fractions refer to 1 unit of the original
specimen diluted to a final volume of 2, 5, or
SINGLE 10 units, respectively.
DILUTIONS ◦ A dilution refers to the volume or number of
parts of the substance to be diluted in the
total volume, or parts, of the final solution.

◦ A dilution is an expression of concentration,

not volume; it indicates the relative amount
of substance.
63
64
 ◦ If serum is being tested for antibody
levels with a specific infectious
organism, generally the blood
should be drawn during the acute
ANTIBODY
phase of the illness—when the
TESTING
disease is first discovered or
suspected— and another sample
drawn during the convalescent
phase, usually about 2 weeks later.

65
 ◦ The antibody titer is defined as
the reciprocal of the highest
dilution of the patient’s serum in
which the antibody is still
ANTIBODY
detectable.
TITER
◦ That is, the titer is read at the
highest dilution of serum that
gives a positive reaction with
the antigen.
66
Determination of the concentration
of antibody (titer) for a specific
antigen involves the following two
steps:
◦ Preparing a serial dilution of the
antibody-containing solution (e.g.,
serum).
◦ Adding an equal volume of antigen
suspension to each dilution.

A high titer indicates that a considerable

amount of antibody is present in the
serum.
67
THANK YOU

68