Julia Niez

Titer (endpoint) measurement of the amount or

concentration of a substance in a solution
RBCS SAMPLE PROBLEM
IMPORTANCE OF PREPARING DILUTIONS
1. A third year BMLS student was asked to prepare a
Preparing standards
5.00% red cell suspension (RCS), using 0.25 mL from a
Preparing quality control materials
60.00% RCS. How much normal saline solution is
Preparing patient sample to be read/ analyzed
needed? What would be the total volume?
by machine
Given:
Test result cannot be read/ analyzed by machine
%RBCS= 5.00% (tube 1)
Less specimen available to test
%RCBCS= 60.00% (tube 2)
Looking for least amount of Ab that will react to
PCV= 0.25% mL
Ag
Asked:
Aliquot- a portion of a larger whole, especially a sample
NSS=?
taken for chemical analysis or other treatment
TV=?
TYPES OF SERIAL DILUTION
Computation:
1. Random

%RCBS= 60.00% 1:2 1:8 1:20

* not 100% because dili siya gkan sa fresh washing,

instead from a prepared RCS na daan which is 60.00%* 2. 2 fold ratio = 1:1
(1 part solute/ 1 part dilution)
5. % 0.25 60%
=
100 100

NSS 0.20 mL 0.20 mL 0.20 mL

TV= 0.25 mL x 0.06
Serum 0.20 mL 0.20 mL 0.20 mL
TV=3 mL
TV- 3mL Dilution 1:2 1:4 1:8

NSS=TV-PCV 0.20 0.20 0.20

= 1: 2 1: 2 1: 4
NSS=3 mL- 0.25 mL 0.40 0.40 0.40

NSS = 2.75 mL 3. 3 fold ratio= 1:2

(1 part solute/ 2 parts diluent)
WHAT IS DILUTION?
Dilution- process to reduce the strength of a
concentrated solution
NSS 0.40 mL 0.40 mL 0.40 mL
Two parts:
Serum 0.20 mL 0.20 mL 0.20 mL
Solute- material being diluted
Dilution 1:3 1:9 1:27
Solvent( diluents) makes up the rest of the
4. 4 fold ratio= 1:3
solution
(1 part solute/ 3 parts diluent)
Types of dilution
Singly- If it is a single-step process
NSS 0.60 mL 0.60 mL 0.60 mL
Series/ serial- when the steps are repeated
Serum 0.20 mL 0.20 mL 0.20 mL
and when solutions need to
Dilution 1:4 1:16 1:64
be diluted several orders of magnitude
Julia Niez

=
COMPUTATION

= 1 0.1
=
5 0.1 +

= X= 5 (0.1 mL) 0.1 mL

X= 0.4 mL of diluent

5 0.1 +
=
1 0.1

5(0.1 mL0 0.1 mL= x

SAMPLE PROBLEMS
X= 0.4 mL of diluents
1. Two (2) mL of a 1:20 dilution is needed to run a
3. Using 0.20 mL from a 1:6 prediluted serum, a medical
specific test. How much diluents and serum are needed
technologist was tasked to prepare a fourfold serial
to make this dilution?
1 dilution in 3 test tubes using 0.20 mL serum in each
=
tube. How much NSS is needed in each test tube? 5
What would be the resulting total volume in each tube?
1 Indicate the dilution in each tube.
=
20 2

2 20 =
=
20 20
X= 0.1 mL of serum
1 0.20
=
= 4

TV= 0.20 mL (4)

TV= solute + solvent
TV= 0.80 mL
2.0 mL= 0.1 + x
2.0 mL- 0.1 =x
NSS= TV- solute (serum)

X= 1.9 mL of diluent NSS= 0.80 mL- 0.20 mL

NSS= 0.60 mL
2. A 1:5 dilution of patient serum is necessary to run a
serologic test. There is ).1 mL of serum that can be
Serum NSS TV ID FD
used. What amount of diluents is necessary to make
Prediluted 1:6
this dilution using all of the serum?
TT1 0.20 ml 0.60 ml 0.80 ml 1:4 1:24
TT2 0.20 ml 0.60 ml 0.80 ml 1:4 1:96
Julia Niez
TT3 0.20 ml 0.60 ml 0.80 ml 1:4 1:384
LYMPHOCYTE ONTOGENY AND ANTIGEN
PRESENTATION
(DN4) Double Negative 4 will become Double Positive
(DP)
Cortical epithelial cell(CEC) will present DP to Self-MHC
class II recognition and will become CD4-committed DP
if negative reaction it will undergo apoptosis by the
Medullary epithelial cell (MEC)
if positive it becomes CD4+ SP (Cluster Of
Differentiation 4 Single Positive)
CEC will present DP to Self-MHC class I recognition
and will become CD4-committed DP
if negative reaction it will undergo apoptosis by the
Medullary epithelial cell
if positive it becomes CD8+ SP (cluster of
differentiation 4 single positive)
RECOGNITION AND AFFINITY TO SELF-MHC
ANTIGENS
(+)Check if T cell is able to recognize self-MHC
antigens
(-) Check the T cells affinity of binding or
recognizing self-MHC antigen *wag masyadong
clingy; masasaktan ka rin*
B CELL ONTOGENY
(Bone Marrow)
Pro Bcell
Pre Bcell having heavy chain and CD19
Immature Bcell having IgM, CD19 and CD20
(2 lymphoid organ) proliferate into Plasma
becomes Mature Bcell having IgM, IgD, CD19 and
CD20
becomes activated Plasma cell (short lived as it
act upon an infxn) after infxn
becomes Memory cell having IgA, IgD, IgG, IgE,
IgM, CD19, CD20 and CD27
*but the antibody that was used in the infxn will be
the only one being activated*
by the next infxn, higher production of Plasma Cell
(PC) and becomes long lived
Julia Niez

Receptor Editing - Group of functionality defined cells capable of taking

Heavy chain- more amino acids
up antigens and presenting them to lymphocytes in a
form that they can recognize
-pathways of antigen processing for APC and target cell:
Endogenous pathway (CD8 Tc cell + MHC I)
Intracellular antigens
Exogenous pathway (CD4 Th cell + MHC II)
Soluble proteins are taken up from the
extracellular environment, then processed in a series of
intracellular acidic vesicles called endosomes.
Antigen Presentation (MHC II) to CD8 Tc cell :
ENDOGENOUS PATHWAY

Other B cell Surface Markers

Electron microscope
- hairy B cells
- Surface immunoglobulin
Processes proteins that have benn internalized,
Mitogen generates Mitosis separated into fragments, an re-expressed at the cell
surface membrane in association with MHC molecules
Lymphocytes+ Mitogen=
1. B cells: Pokewed seed
Lipopolysaccharide
Dextran
2. T cells:Pokeweed seed
Concanavalin A
Phytohemagglutinin
Nylon Test- B cells will be trapped due to surface Igs
Soluble proteins are taken up from the extracellular
ANTIGEN PRESENTATION MECHANISMS
encironment, then processed in a series of intracellular
1. Antigen- presenting cells (APCPs) acidic vesicles called endosomes.
Julia Niez

MHC CLASS I VS MHC CLASS II PATHWAYS Dendritic cell most potent phagocytic cell in the tissue
-covered with long membranous extensions
FACTOR MHC CLASS I MHC CLASS II
resembling nerve cell dendrites
Only in Fxn: APC to the Th cells
All nucleated macrphage, B Two subtypes:
Present in
cells except RBC cells, and Interstitial dendritic cells- major organs
dendritic cells Interdigitating dendritic cells-present in T lymphocyte
Obligate areas of secondary lymphoid organs and thymus
intracellular
Extracellular/
tpes of Ag bacteria/
exogenous Ag
engdogenous
Ag(virus)
CD8/ T cytotoxic CD4/ T helper
Recognized by
cell cell
Cell lysis (Th) Ab
End goal Cell lysis
production (Th2)
Comparison

THIRD POPULATION/ NATURAL KILLER CELLS

2.Autoimmune regulatory gene/ Autoimmune regulator
(AIRE)
-transcription factor exressed in the medulla of the
thymus and controls the mechanism that prevents the
immune system from attacking the body itself.
-lack concentional antigen receptors of T or B cells
-previously classified as null cells
-phenotypic (surface) markers:
CD2+, CD16+, CD56+, CD57+, occasionaly CD8+
-essential mediators of virus immunity
-can also bind and lyse antibody-coated nucleated cells;
mediates antibody-dependent, cell-mediated
cytotoxicity(ADCC)
-target cells include: tumor cells, microbial agents,some
cells of the embryo, norma; bone marrow and thymus
cells
-destroy target cell through extracellular non-
phagocytic mechanism: cytotoxic reaction, MHC-
unrestricted cytolysis
MECHANISM OF NATURAL KILLER CELL
Julia Niez

CLUSTERS OF DIFFERENTIATION

Cluster of designation or classification determinant

Markers can be classified as follows:
1.Specific fro cells of a particular lineage or
maturational pathway (e.g CD19 for Bcells) Immune Response Curve
2. Depending on the state of activation or
differentiation of the same cells (e.g CD4 and CD8 to
classify Tcells)
Functions of CD antigens
1. Promotion of cell to cell interactions and adhesion
2. Transduction of signals that lead to lymphocyte
activation
IMPORTANT CD MARKERS
1. CD2 - present in all Tcells (pan-T cell marker)
-rosette formation in combination with SRBCs

SRBC+ serumlymphocytes agglutination

(+) CD2 = Tcells in
serum sample
No agglutination
(-) CD2 =B cells present in serum sample

2. CD3 recognizes antigen during presentation

-important in T cell activation of APC
-New pan-T cell marker

3. CD4- present only in T helper cells

-recognizes MHC clas II
Primary response
4.CD8- present only in T cytotoxic cells - the first contact with an antigen, due to natural
-recognize MHC Class I exposure or vaccination, leads to the activation of naive
BL.
5. CD19 and CD20- identification of B cells (via -which differentiate into antibody-producing
monoclonal Abs in lab) plasmocytes and memory cells, resulting in the
production of specific antibodies against the inducing
6. CD21 serves as receptor for Epstein Barr virus in B antigen.
lymphocytes -after the start of the response, there is a phase with an
-seen in infectious Monocleosis exponential increase in antibody levels, followed by a
phase called plateau, in which levels do not change.
7. CD16, CD56, CD57 seen in Natural Killer cells (NK cells) -that is followed by the last phase of the primary
response, the decline phase, in which a progressive
decrease in the number of circulating specific
antibodies occurs.
Julia Niez

Secondary response
-differe from the primary one in the ff aspects:
the dose of Ag necessary to induce the response is
smaller
the latency phase is shorter
the exponential phase is more marked
the production of Ab is faster and higher levels are
attained
the plateau phase is reached faster and last longer
the decline phase is slower and persistent

LINES OF DEFENSE

Roles of keratin, prostaglandin, histamine

First Line of defense -functions to prevent entry of

microorganism and toxins
1. Anatomical / Physical skin, mucus membranes, cilia Leukocyte extravasion- refers to the movement of
2. Chemicals sweat, sebum, cerumen, lactic acid, tears WBCs from the capillaries to the tissues surrounding
and saliva, HCL them (via chemotaxis)
3. Mechanical- urination, defecation, sneezing,
vomiting, coughing

Second line of defense- fights off microorganism that

evaded the first line of defense
1. Natural killer cells- provides immunity to virus or
tumor cells
-stress molecules: MICA/MICB

2. Beta lysine- Is an amino acid produceedmby platelets

during coagulation and Directly antibacterila to GP
bacteria
-Can damage bacterial cell wall
3. Inflammation- primary objective of inflammation is to
localize and eradicate the irritant and repair the
surrounding tissue
4. Phagocytosis- from ancient greek phagien, meaning
to devour (kytos), meaning cell and osis, meaning
process.
- is the process by which a cell engulfs a solid
particle to form an internal vesicle known as a
phagosome
Julia Niez

Divided into seven stages: TYPES OF OPSONINS:

1. Chemotaxis -IgG: receptors for Fc/ tail portion
2. Adherance -C3b: is the largest of the two elements formed by the
3.Engulfment cleavage of complement component 3
4. Phagosome formation -C-reactive protein (CRP)- acute phase reactant
5. Fusion
6.Digestion
7.Destruction

Chemotactic response- change in direction of

movement of a motile cell in response to a
concentration gradient of a specific chemical
(chemotaxin = can be positive or negative)

Phagocyte killing mechanisms

O2-NADPHsuperoxide dismutase H202 -

myeloperoxidaseCLO

Pathogen Associated Molecular Patterns (PAMPs)

-molecules associated with groups of pathogens that
are recognized by cells of the innate immune system
-found in plants and animals, not in humans
Examples: cell wall, capsule, flagella

Pattern recognition receptors ( PRRs)- receptors of the

innate system that recognize these PAMPs
Example: Toll-like receptors

5.Opsonins- from the greek opsonein meaning to Respiratory burst (oxidative burst) rapid release of
prepare for eating is any molecule that enhances reactive oxygen
phagocytosis by marking an antigen for an immune a. Superoxide radial
response b. hydrogen peroxide

6. interferons- group of signaling proteins made and

released by host cells in response to the presence of
pathogens
Named so fof their ability to interfere with
viral replication by protecting cells from virus infections
Julia Niez

Types of interferons:
Type I mediate the early innate response to viral infxn
IFN alpha (IFN-a): produced by leukocytes/ WBCs
IFN beta (IFN-) : produced by fibroblasts
Type II- decrease viral replication in cells
IFN gamma (IFN-y) :produced by T cells (Tdh or Th1)
AKA immune interferon
Increases phagocytic activity of macrophage
Stimulates expression of MHC class I and II
Acts on B cells to promote switching to certain
different IgG subclasses

7. Acute Phase Reactants- AKA acute phase proteins

-increased synthesis of these proteins takes
place shortly after a trauma or early stage of infection
where Ab are still absent

Examples : Ceruloplasmin: copper-binding proteins

Haptoglobulin: prevents loss of iron by urinary
excretion
Fibrinogen: forms clot critical in healing process
Alpha-1 antitrypsin: neutralize elastase
Lactoferrin: iron-binding protein
CRP (as opsonin): during phagocytosis

8. Fever
Pyrogens- chemical messages that WBCs send out to
recruit cells towards site of infxn increase heat:
1. increaded mobility of white blood cells
2. enhanced white blood cell phagocytosis

PYROGENS:

IL-1: acts on hypothalamus to increase body

temperature
IL-6: acts on liver to produce acute phase
reactants.
Tumor necrosis factor- (TNF):
Inducing death in tumor cells and contributes in
inflammatory signal

THIRD LINE OF DEFENSE

Recall:
B CELLS
T CELLS
Adaptive / acquired immunity