IMMUNOSERO AND BLOOD BANKING

THE CLASSICAL PATHWAY

 It was the first complement pathway being
NATURAL/INNATE IMMUNITY studied, hence, named as classical
 -Present at birth  It is initiated by Ag-Ab complex/binding (1
 Standardized response for all Antigen molecule of IgM, and 2 molecules of IgG for
 Lacks memory activation)
 Responsible for the first and second line of
 Recognition Units: C1q, C1r, C1s - which
defense in the body
 Pathogen recognized by receptors encoded in the require the presence of calcium to maintain
germline structure
 Receptors have broad specificity
 Immediate response C1q C1q has a molecular weight of
410,000 and is composed of six
strands that form six globular
ADAPTIVE/ACQUIRED IMUNITY heads with a collagen-like tail
 Not present at birth portion. C1q “recognizes” the
 Diverse response for each antigen fragment crystallizable (Fc) region
 Capable of recalling previous antigen thus with of two adjacent antibody
memory molecules, but at least two of the
globular heads of C1q must be
 Secondary immune response is greater than
bound to initiate the classical
primary immune response pathway.
 Responsible for the Third line of defense in the  Activation Units: C4, C2, C3
body  Membrane attack complex(MAC) :
 Pathogen recognized by receptors generated C5,C6,C7,C8 ,and C9
randomly  STUDY THE CLASSICAL PATHWAY
 Receptors have very narrow specificity; i.e., (LAST PAGE) FROM INITIATION TO
recognize a specific epitope MAC (ag-ab binding to MAC)
 Slow (3-5 days) respons
THE PROPERDIN/ALTERNATIVE PATHWAY
COMPLEMENT SYSTEM  It was the first complement pathway
discovered
CLASSICAL PATHWAY  Originally thought to be initiated/activated by
C1q Binds to Fc of IgM and IgG Properdin or Factor P
C1r Activates C1s  Factor P acts as a stabilizer for C3
C1s Cleaves C4 and C2 convertase and C5 convertase in the
C4 Part of C3 convertase (C4b) presence of Magnesium
C2 Binds to C4b – form C3 convertase
 It is Initiated or activated by cobra venom
C3 KEY INTERMEDIATE IN ALL PATHWAY ,
factor, Lipopolysaccharide, bacterial cell wall,
most concentrated complement component
C5 Initiates membrane attack complex yeast, virally infected cells, tumor cell lines,
C6 Binds to C5b in MAC some parasites (e.g., trypanosomes) , and
C7 Binds to C5bC6 in MAC aggregation of IgA
C8 Starts pore formation on membrane  It bypasses C1q,C1r, and C1s
C9 Polymerizes to cause cell lysis  Activation starts with native C3 (iC3) through
ALTERNATIVE PATHWAY water hydrolysis, or from classical and lectin
Factor B Binds C3b to form C3 convertase pathways.
Factor D Cleaves factor B
Properdin Stabilizes C3 convertase and C5 convertase THE LECTIN/MBL PATHWAY
in the presence of magnesium  The most recently described complement
MBL PATHWAY pathway
MBL Binds to mannose
 The lectin pathway represents another
MASP-1 Helps cleave C4 and C2
means of activating complement without
MASP-2 Cleaves C4 and C2
antibody being present. Lectins ,which are
 TYPE I TYPE II TYPE III TYPE IV
Anapahylactic Cytotoxic Immune Complex Delayed or Cell mediated
Immune mediator IgE IgM / IgG IgM/ IgG T- Cells
Effector cells Basophil, Mast RBC, WBC, PLATELETS Host tissue cells Macrophages, T cells
cell
Immune Release of Cytolysis due to Deposits of antigen Antigen-sensitized Th1 cells
mechanism mediators from antibody and antibody complexes that release cytokines that recruit
mast cells and complement, activates complement. macrophages
basophils opsonization, or ADCC Neutrophil are recruited and induce inflammation or
and release lysosomal activate cytotoxic T cells to cause
enzymes that cause direct cell damage
tissue damage
Antigen Heterologous Cellsurface; autologous Soluble: autologous or Autologous or heterologous
or heterologous heterologous
Complement NO YES YES NO
involvement
Cytokines invol. Yes No Yes Yes
EXAMPLES 1.Anaphylaxis 1.Transfusion rxn 1. Serum sickness 1. Contact dermatitis (poison
2. Hay fever 2. AIHA 2.Arthus reaction ivy,nickel,mercury, copper rubber,
3.Food allergies 3. HDN 3. SLE formaldehyde, hair dyes,
4.Asthma 4.GOODPASTURE’S 4.Rheumatoid arthritis sunscreen agents, disinfectants,
5.Drugs syndrome 5.Post streptococcal perfumes, and pesticides )
(e.g Penicillin) 5.Myasthenia gravis glomerulonephtritis 2. Tuberculin/ PPD test
6.Rhinitis (most 6.Graves disease associated in SLE 3. Pneumonitis
common) 7.ITP 6. Other autoimmune 4.Mantoux
7.latex allergy disorders 5.anergy skin test
7.Neoplastic diseases 6.hypersensitivity pneumonitis
7.Type 1 DM
8. GVHD
calcium dependent, are proteins that bind to  MBL - binds to mannose or related sugars in
carbohydrates a calcium-dependent manner to initiate this
 The lectin pathway molecules are structurally pathway
similar to those of the classical
PLEASE WATCH VIDEO FOR THE PATHWAY
MBL (Mannan Binding Homolgous to
Lectin) C1q HYPERSENSITIVITY REACTIONS
 Defined as Heightened state of immune
MASP-1 (Mannose- Homologous to responsiveness.
binding-lectin- C1r  Typically, it is an exaggerated response to a
associated serine harmless antigen that results in injury to the
protease) tissue, disease, or even death

MASP-2 Homologous to COMPARISON OF HYPERSENSITIVITY

REACTIONS
C1s
 III. OTHER AUTOIMMUNE DISORDERS
DISORDER PATHOLOGY ANTIBODIES
Type 1 diabetes Selective destruction of the insulin producing B cells of a. Anti- glutamic acid
the islets of Langerhans in the pancreas decarboxylase (GAD)
b. Anti-insulin antibodies
Grave’s Disease Unregulated secretion of T3 and T4 due to stimulation a. Anti-TSH receptor antibodies
of TSH receptor by antibody b. Anti-thyroid peroxidase
c. Anti- thyroglobulin antibodies
Hashimoto’s Thyroiditis Destruction of the thyroid gland a. Anti-thyroid peroxidase
b. Anti- thyroglobulin antibodies
c. Anti- microsomal antibodies
Pernicious Anemia Destruction of the parietal cells of the stomach a. Anti- parietal cell
mucosa leading to intrinsic factor deficiency b. Anti- intrinsic factor
Multiple Sclerosis an immune-mediated inflammatory disease that a. Anti- myelin sheath
attacks myelinated axons in the central nervous
system, destroying the myelin and the axon in variable
degrees and producing significant physical disability. It
is characterized by the formation of lesions called
plaques in the white matter of the brain and spinal
cord, resulting in the progressive destruction of the
myelin sheath of axons.
Good pasture’s syndrome Combination of glomerulonephritis with alveolar a. Anti- glomerular basement
hemorrhage and anti-GBM antibodies. membrane
Primary Billiary cirrhosis An autoimmune cholestatic liver disease characterised a. Anti- mitochondrial
by a breakdown of immune tolerance to mitochondrial
and nuclear antigens, causing injury to the biliary
epithelial cells (BEC) lining the small intrahepatic bile
ducts

Chronic active hepatitis Liver disease that is characterized by diffuse a. Anti-smooth muscle antibody
parenchymal inflammation and hepatic cell necrosis
Myasthania Gravis Disease of the neuromuscular junction (NMJ) caused a. Anti-acetycholinesterase
by antibodies that attack components of the antibodies
postsynaptic membrane, impair neuromuscular
transmission, and lead to weakness and fatigue of
skeletal muscle.
Wegener’s Granuloma formation, "pauci-immune" vasculitis and a. Anti- neutrophilic cytoplasmic
Granulomatosis glomerulonephritis (= renal vasculitis) are the antibodies (ANCA)
histologic hallmarks
Sjogren’s syndrome Keratoconjunctivitis sicca(dryness of eyes) and a. Anti SSA , Anti SSB
xerostomia(dry mouth) due to lymphocytic infiltrates
of lachrymal and salivary glands
Scleroderma Involves the hardening and tightening of the skin and a. Anti-centriole , anti-Scl
connective tissues

DIFFERENT TUMOR MARKERS

AFP Hepatic , testicular cancers, Germ cell cancers
ALP Lung cancer
Amylase Pancreatic cancer
 Bence jones protein Multiple myeloma
BRCA-1 Breast or ovarian caner
CA- 125 Ovarian cancer ( treatment and recurrence)
CA-15.3 Breast cancer ( treatment and recurrence)
CA-19.9 Gastric, pancreatic and colorectal cancers
CA-50 Gastric and pancreatic cancers ( treatment and recurrence)
CA-27.29 Breast cancer ( treatment and recurrence)
CEA Colorectal, stomach, breast, lung cancer ( treatment and recurrence)
Estrogen receptor Breast cancer
GGT Hepatoma
HER-2/neu Breast cancer (efficiency of trastuzumab or herceptin therapy)
PSA Prostate cancer
CYFRA 21-1 Lung cancer / Breast cancer (Henry’s)

AFFINITY VS. AVIDITY

Affinity Initial force of attraction that exists between a single Fab site on an antibody molecule and a single epitope or
determinant site of the corresponding antigen
1. Ionic bond
2. Hydrophobic bond
3. Hydrophilic bond
4. Van der waals force
Avidity Sum of all attractive forces between and antigen and an antibody. It represents the overall strength of antigen–
antibody binding

TURBIDIMETRY VS. NEPHELOMETRY

1. Turbidimetry
 Is a measure of the turbidity or cloudiness of a solution. A detection device is placed in direct
line with an incident light, collecting the light after it has passed through the solution. This
device measures the reduction in light intensity caused by reflection, absorption, or scatter. The
amount of scatter is proportional to the size, shape, and concentration of molecules present in
solution. It is recorded in absorbance units, a measure of the ratio of incident light to that of
transmitted light. The measurements are made using a spectrophotometer or an automated clinical
chemistry analyzer

2. Nephelometry
 Measures the light that is scattered at a particular angle from the incident beam as it passes
through a suspension. The amount of light scattered is an index of the solution’s concentration.
 Nephelometers typically measure light scatter at angles ranging from 10 degrees to about
90 degrees.
 Quantification of immunoglobulins such as IgG, IgA, IgM, and IgE, as well as kappa and lambda
light chains, is mainly done by rate nephelometry

STAGES OF SYPHILIS
STAGE Pathology Lab Diagnosis

Primary syphilis Hard Chancre (painless and firm) Dark field microscopy

Secondary Condylomatalata – a wart like lesions in moist area of the Darkfield microscopy
syphilis body Serologic test for syphilis

Latent syphilis Absence of clinical symptoms Positive for serologic test

Patients are noninfectious at this time, with the exception of pregnant
women, who can pass the disease on to the fetus even if they exhibit no
 symptoms.
Tertiary/late Gummas, Neurosyphilis Serologic test for syphilis
syphilis Gummas are localized areas of granulomatous inflammation that are most
often found on bones, skin, or subcutaneous tissue.

HEPATITIS
SEROLOGY FOR HEPATITIS
Hepatitis A  Member of the family Picornaviridae
 Known as Infectious Hepatitis
 Transmission by fecal oral route, close person to person contact , or ingestion of contaminated food/or
water
1.HAV Antigens – not clinically useful indicator of disease
2.HAV Antibodies - most commonly detected by automated, chemiluminescent microparticle IA
IgM Anti-HAV IgG Anti- HAV
marker of acute hepatitis A, detected by ELISA Indicate immunity to HAV, detected by competitive
inhibition ELISA

Hepatitis C  Member of the family Flaviviridae(Hepacivirus)

 Previously classified as “Non A-Non B”
 Transmission by sexual, parenteral, or perinatal
1.Surrogate test – Anti-HBc and ALT
2.Specific test – Anti-HCV , ELISA, RIBA
3.traditional test
-Traditional testing methods include a qualitative chemiluminescent immunoassay and qualitative EIA,
qualitative recombinant immunoblot assay, quantitative real-time PCR assay, qualitative PCR assay,
quantitative branched chain DNA test, polymerase chain reaction–nucleic acid sequencing. interleukin 28 B
(IL-28B)–associated variants test, and two single-nucleotide polymorphisms (SNPs) method—qualitative
PCR–qualitative fluorescence monitoring

HCV RNA- Viral load

It can be used to detect Current hepatitis C infection; viral load may be used to monitor effectiveness of
therapy; also used to determine HCV genotype

Hepatitis D  Unclassified, single stranded RNA virus

 Can occur in the presence of Hepatitis B (coinfection and superinfection)
 Antibodies can be detected by ELISA
 RNA or viral load can be measure using PCR
IgM ANTI-HDV IgG anti-HDV HDV RNA
Acute or chronic Hepatitis D Recovery or chronic hepatitis D Active HDV infection; may be
used to monitor effectiveness of
therapy
Hepatitis E  Member of caliciviridae reclassified to Hepeviridae
 Associated with a high rate of mortaliyty in pregnant women
 Transmission by fecal oral route
 IgM antibodies can be etected by ELISA, Western blot, and fluorescent antibody blocking assay
 HEV RNA Identified by PCR
IgM anti-HEV IgG anti-HEV HEV RNA
Current/new hepatitis E Current/formerhepatitis E Current hepatitis E infection
infection infection
HEPATITIS B
 Member of family Hepadnaviridae
 Known as serum hepatitis
 Complete HBV virion is called Dane particle
 Transmission by Sexual, Blood transfusion,Parenteral, and Perinatal/Vertical
 COMPLETE HEPATITIS PROFILE SEROLOGICAL TEST IS USED FOR THE STATUS OF THE PATIENT
 HBV DNA can be detected in the serum about 21 days before HBsAg and may be a useful adjunct in detecting early acute HBV
infection , and also to evaluate the effectiveness of antiviral therapy in patients with chronic hepatitis B
 The progression of liver disease in HBV infection is fostered by active virus replication, manifested by the presence of an HBV
DNA level above a threshold of approximately 1000 to 10,000 IU/mL

COMPLETE HEPATITIS SEROLOGICAL MARKERS

1. HBsAg  First maker to appear

 Early Indicator of active infection
 Important marker in Screening blood donors
 The presence of HBsAg indicates active HBV infection, acute or chronic.

Tests Available for HBsAg Detection

Third Generation
 Radioimmunoassay
 Reversed passive Hemagglutination
 Enzyme-linked Immunosorbent assay
 Reversed passive latex agglutination
Second Generation
 Counterelectrophoresis
 Rheophoresis
 Complement fixation
First Generation
 Ouchterlony Double Diffusion

2. HBeAg  Present during periods of active replication of the virus

 Indicates a high degree of infectivity when present
3. HBcAg  Not detectable in serum because of the viral envelope that masks it
 Detected only through biopsy of the infected liver
4. Anti-HBc  Indicator of current or recent infection
IgM  Useful in detecting infection during the “Core Window” period
5. Anti-HBc  Persists for the lifetime of the individual
IgG  Serves as a lifelong marker of HBV infection
6. Anti-HBe  Indicates that the patient is recovering from HBV infection
 Marker of convalescence and favorable prognosis
7. Anti-HBs  Apperas during the recovery period of acute hepatitis B, weeks to months after HBsAg disappears
 Persists for years and provide protective immunity
 It is the Serologic marker of recovery and immunity
 Also produced after immunization with the hepatitis B vaccine, which consists of recombinant HBsAg
produced from genetically engineered yeasts or mammalian cells.
 Protective titers of the antibody in the serum are considered to be 10 mIU/mL or higher

BLOOD BANKING

1- ABO
2- MNs
3- P
4- Rh
 5- Lutheran
6- Kell
7- Lewis
8- Duffy
9- Kidd
10- Diego

DISCUSS THE PROCESS OF WHOLE BLOOD CROSSMATCHING IN BDH LAB INCLUDING THE MATERIALS
USED

IMMUNOGLOBULINS