IMMUNOHEMATOLOGY
them.
 One of the specialized branches of
medical science involved in blood
banking and blood transfusions.
FUNCTIONS OF MODERN BB:
1. Recruiting blood donors.
2. Collecting and storing WB or
components from volunteer; paid or
autologous donors.
3. Typing, screening and preparing
patient and donor blood for
transfusion.
4. Detecting and identifying unexpected
Abs in potential blood recipients or
pregnant women.
5. Establishing a database of HIV + or
questionable donors.
6. Processing and dispensing blood
components.
7. Performing paternity testing.
8. Conducting tissue typing prior to
organ transplantation and
participating in the processing of
human materials as in bone banking.
SAFETY IN THE BB:
 OSHA
 CDC – “Universal Precautions” (1987)
 Occupational transmission of HV and
HIV
 Protective techniques for infection
control
 Safety practices (handwashing, needle
precautions)
 Hazardous waste management and
control
RISK CLASSIFICATION IN LAB:
CATEGORY I. Tasls that involves exposure
to blood, body fluids or tissues. All
procedures or job-related tasks that involve
inherent potential for mucous membrane or
skin contact with blood, body fluids or
tissues or a potential for spills or splashes of
CATEGORY II. Tasks that involve NO
exposure to blood, body fluids or tissues but
employment may require performing
unplanned Category I tasks.
CATERGORY III. Tasks that involve NO
exposure to blood, body fluids or tissues and
Category I tasks are not a condition of
employment.
BIOSAFETY LEVELS OF RISK (PHS)
LEVEL I. Work that involves agents of no
known or minimal potential hazard to
laboratory personnel and the environments.
LEVEL 2. Work that involves agents of
moderate potential hazard to personnel and
the environment. Exceptions may be
appropriate if no open specimens will be
encountered.
BASIC SEROLOGICAL AND BLOOD BANKING
PROCEDURES:
SPECIMEN COLLECTION & PREPARATION
1. SERUM – supernatant portion of whole
blood that has been allowed to clot;
most frequently assayed specimen in
serology.
2. PLASMA
3. CSF
4. URINE
SEPARATION OF SERUM: blood for all
serotests except cold agglutinins can be
allowed to clot at RT. If > quantitites of
serum are required & delay is not significant,
the clot maybe allowed to retract more
completely overnight in a refrigerator (4-
10celcius).
CENTRIFUGATION: depending on the
amount of serum required for testing,
centrifugation for as little as 5 minutes at
1,500 rpm may be adequate.
SEPARATION: Never store serum with the
clot.
PRESERVATION OF SERUM: Ideally,
serotests should be performed ASAP after
the serum is collected.
A. PHYSICAL PRESERVATION
I. Refrigeration – the best short
term preservation method for
serum & the least likely to
interfere with the test. (4-
10celcius)
II. FREEZING – serum can be kept
nearly indefinitely if frozen (-20
to -70 celcius) in tightly
stoppered containers.
III. LYOPHILIZATION – removing
water from serum by freeze
drying the serum under high
vacuum is an effective way to
preserve. Lyophilized serum can
be stored in tightly stoppered
containers for long periods of
time, especially if refrigerated.
Lyophilized serum can be
shipped w/o refrigeration.
B. CHEMICAL PRESERVATION – chemical
preservatives should be added to
serum that is to be used for serotests.
Preservation is only by bacteriostasis
& does not prevent denaturation of
Abs by heat or other means.
ex. merthiolate (used in serum & CSF)
PRINCIPLES OF ANTIGENS & ANTOBODIES
ANTIGEN:
 foreign substance that is able to elicit
an antibody response
1. A person’s immunologic ability both
recognize an antigen as foreign or
no self and to respond to this
foreigness.
2. The physical and chemical nature
of the antigen.
ANTIGEN CHARACTERISTICS:
 foreign substances such as
erythrocytes can be immunogenic or
antigenic (capable of provoking an
immune response) if their membrane
contains a number of areas recognized
as foreign (ANTIGENIC
DETERMINANTS or EPITOPES)
 each EPITOPE is capable of eliciting an
Ab response & each can be the target
of these Abs.
CELLULAR ANTIGENS:
1. Blood group antigens
2. Histocompatability antigens (HLA)
3. Autoantigens
I.BLOOD GROUP ANTIGENS
 blood group substances that are
widely distributed throughout the
tissues, blood cells and body fluids.
2.HLA
 Human Leukocyte Antigen (HLA)
system a.k.a. Major Histocompatibility
Complex (MHC) or a supergene
 glycoprotein in nature & are found on
the surface membranes of nucleated
body cells comprising both solid
tissues & most circulating blood cells.
 plays an important role in the immune
response & diseases found in the short
arm of Chromosome 6.
3.AUTOANTIGENS:
 in some situations (not always
abnormal), Abs may be produced in
response to normal self-antigens. This
failure to recognize self-antigens can
result in autoAbs directed at hormones
(thyroglobulin & cell membrane Ag).
CHEMICAL NATURE OF Ags:
  large organic molecules that are either
proteins or polysaccharides & rarely,
lipids.
 can be composed of combinations of
the biochemical classes (ex.
glycoproteins or glycolipids)
PHYSICAL NATURE OF Ags:
1. FOREIGNESS: the more foreign the
antigenic determinant or the > the
degree to which it is recognized as
non-self by an individuals immune
system, the more antigenic it is.
2. DEGRADABILITY: for an antigen to be
recognized as foreign by an
individual’s immune system, a
sufficient amount of Ag must be
present to stimulate an immune
response.
3. MOLECULAR WEIGHT: the higher the
molecular weight, the better the
molecule functions as antigen.
*HAPTENS; very small molecules can
bind to a larger carrier molecule & behave
as Ags.
4. STRUCTURAL STABILITY: if a molecule
is to function as an effective Ag,
structural stability is an essential
characteristic.
*Inferior Ags: lipids, inert molecules
5. COMPLEXITY: the more complex an
antigen is, the more effective it will be.
Complex proteins are better antigens
than large repeating polymers like lipids,
carbohydrates & nucleic acids (poor Ags)
ANTOBODIES:
 chemically classified as globulins
 specific glycoproteins are referred
to as immunoglobulins (Ig)
Ab RESPONSE TO Ag:
1. Primary Ab Response: following a
foreign Ag challenge, the I IgM Ab
producing response proceeds in 4
phases.
a. Lag phase, when NO Ab is
detectable.
b. Log phase, the Ab titer rises
logarithmically.
c. Plateau phase, which the Ab titer
stabilizes.
d. Decline phase, the Ab is
catabolised.
FUNCTIONS OF Abs:
 binding to antigen
 secondary effector function
Complement fixation
Placental transfer
IN VITRO DETECTION OF Ag-Ab REACTIONS
1. AGGLUTINATIN, the clumping of
particles with Ags on their surface like
RBCs by Ab molecules that form
bridges between the antigenic
determinants.
*STAGES:
I.I SENSITIZATION – 1st phase,
represents the physical attachment of
Ab molecules to Ags on the
erythrocytic membrane.
FACTORS influencing Ag-Ab association:
1. The Ag-Ab ratio or the number of Ab
molecules in relation to the number of
Ag sites per cell.
2. Physical conditions susch as pH,
temperature and length of time of
incubation, ionic strength and steric
hindrance.
Ag-Ab RATIO:
 Ab excess. a surplus of molecular Ag-
combining sites which are not bound
to antigenic determinants exists
 (PROZONE PHENOMENON) and can
cause false (-) results.
pH:
 a pH of 7.0 is used for routine
laboratory testing
 some Abs such as anti-M and anti-D
react best at a lower pH (6.5-7.0)
TEMPERATURE & LENGTH OF INCUBATION:
 The optimum temperature for
reaching equilibrium in an Ab-Ag
reaction differs for different Abs.
IgM (19S) are cold-reacting
(thermal range 4-22celcius)
IgG (7S) are warm reacting
(37celcius)
 The length of time of incubation
required to achieve maximum results
depends upon the rate of association
& dissociation of each specific Ab.
o 15-60minutes or 30mins
IONIC STRENGTH:
 the concentration of salt in the
reaction medium has an effect on Ab
uptake by the membrane-bound
erythrocytes Ags (ex. Na & Cl ions in a
solution have a shielding effect)
 LISS – low ionic strength saline or
polybrene enhances Ab uptake – these
are reducing medium which lowers the
ionic strength of a reaction.
STERIC HINDRANCE:
 an important physiochemical effect
that influences Ab uptake by cell
surface Ags. Their If dissimilar Abs
with approximately the same binding
constant are directed against antigenic
determinants located in close
proximity to each other, they will
compete for space in reaching their
specific receptor sites. The effect of
this competition can be MUTUAL
BLOCKING or STERIC HINDRANCE, &
neither Ab type will be bound to its
respective antigenic determinant.
LATTICE FORMATION – the 2nd phase, the
establishment of crosslinks between
sensitized particles such as RBCs & Abs
resulting in aggregation (clumping), is a
much slower process than the sensitization
phase.
FACTORS affecting the crosslinking:
a. ZETA POTENTIAL, an electrostatic
potential dependent on the actual net
surface charge density at the surface
of the cell membrane and the
dielectric constant of the surrounding
medium.
b. Ab TYPE, Abs differ in their ability to
agglutinate. IgM Abs (complete Abs)
are more efficient than IgG or IgA Abs
in exhibiting in vitro agglutination
when the Ag-bearing erythricytes are
suspended in NSS.
c. EFFECT OF Ag DOSAGE , a greater
proportion of erythrocytes is
agglutinated to some blood group Ags
possessing a double dose (such as
“c”).
d. METHODS on ENHANCING
AGGLUTINATION, reffered to as the
techniques that are commonly used in
routine blood banking.
- centrifugation
- enzyme treatment; alteration of
the zeta potential to enhance the
chances of demonstrable
agglutination. The risk of enzyme
treatment is that it destroys some
blood group Ags such M, N & Duffy.
- colloidal media: colloids can
enhance agglutination (ex. 22%
Bovine Albumin Solution @pH 7.2
& added preservative 0.1% Na
azide).
- use of other enhancement media
like LISS, polybrene & polyethylene
glycol (PEG) to detect low titered
Abs in routine alloAb screening or
compatibility testing.
- use of chemically modified
antisera: involves the conversion of
some “incomplete” IgG Abs which
are not capable of causing direct
agglutination of saline-suspendend
erythrocytes, into saline
agglutinins.