immuno 2

serologic testing, an assay involving

serum constituents

Immunologic testing, testing related to

antigens and antibodies

blood specimen preparation, preanalytical variables specimen types

most immunology assays are done on serum; tube should be allowed to clot before centrifugation occurs; some assays are done on body fluids (cerebrospinal fluid or urine)

blood specimen preparation, preanalytical variables delay in testing

in most cases, immunology testing should be done within 72 hours; if not, serum should be frozen

blood specimen preparation, preanalytical variables excessive heat

coagulates proteins

blood specimen preparation, preanalytical variables bacterial contamination

alters protein molecules

blood specimen preparation, preanalytical variables strong acid or alkali solutions

must be avoided because these substances have a denaturing effect on serum proteins and make the specimens useless for serologic testing

blood specimen preparation, preanalytical variables lipemia, hemolysis, or icterus

can make a specimen unacceptable

complement in body fluids can be inactivated by heating at

56C for 30 min

manual pipettes - graduated pipette

delivers the amount of liquid contained between two calibration marks on the pipette, required draining between two calibration marks (less accurate)

manual pipettes - serologic pipette

similar to the graduated pipette, but it is calibrated to the tip, contains a frosted ring at the non-calibrated end

Manual pipettes - to contain (TC)

glassware designed to contain like graduated cylinders and volumetric flasks are usually marked with a TC, when liquid is poured from a piece of glassware a small amount remains behind clinging to the slides of the vessel, a 100 ml volumetric flask is designed to hold exactly 100 mL but if the liquid is poured out it will actually deliver a little less than 100 mL

manual pipettes - to deliver (TD)

glassware designed to deliver like pipettes and burets are marked with a TD, these pieces of glassware account for the small amount of liquid that remains behind, a 100 mL pipette contains a little more than 100 mL of liquid but when the liquid is drained from a pipette exactly 100 mL is delivered

steps in using piston type automatic micropipette

A: attached proper tip size for range of pipette volume and twisting tip as it is pushed onto pipette to give airtight continuous seal B: holding pipette before use C: follow instructions for filling and emptying pipette tip

If testing cannot be done without _____ hours of collection, a serum specimen should be frozen at -20C

72

complement can be inactivated in human serum by heating to

56C

solution (in dilutions)

the mixture of one substance (solute) into another (solvent)

dilution (in dilutions)

reduces the concentration of the original solute in the solution

dilutions

a dilution is the number of parts of the substance in the total number of parts of the final diluted solution

if we make a ½ dilution of blood using saline

1 mL of blood + 1 mL of saline =1/2

a patient blood sample is ran on the instrument and reads > 500 mg/dL for glucose. A 1/3 dilution with saline is required in order to get the reading in the dynamic range. How many mL of patient’s blood would be used to prepare a 1/3 dilution in which the total volume is 6 mL

2 mL

a patient blood sample is ran on the instrument and reads >500 mg/dL for glucose. A 1/3 dilution with saline is required in order to get the reading in the dynamic range. If the analyzer gives a result of 300 mg/dL how should the patient’s glucose level be reported

900 mg/dL

serial dilutions

the stepwise decreasing strength of a substance in solution

example of serial dilution

each sample gets 1 ml of saline, 1 patient serum is 1:2 final dilution, take 1 mL of first into second (1 of 1:2) to get final dilution of 1:4, (1:8, 1:16, 1:32 etc)

testing for antibody levels - acute phase specimen

serum from one with a current infection for which bacterial or viral specific immunoglobulins are measured

testing for antibody levels - convalescent phase specimen

serum from one who has recovered from an infectious disease and considered to be especially rich in antibodies against the infectious agent

antibody titer refers to

the concentration of an antibody, the antibody titer is defined as the reciprocal of the highest dilution of the patients serum which the antibody is still detectible (if serum is being tested for antibody levels of specific infection one should be drawn during acute phase and the other during convalescent)

determination of titer

for most pathogenic infections, an increase in the patient’s titer of two double dilutions, or from a positive result of 1:8 to a positive result of 1:32 over several weeks, is an infection of a current infection (this is known as the four-fold rise in the antibody titer)

Stretpozyme testing is preformed on an initial patient serum dilution of 1:100 the dilution procedure is

10 ul of patient serum is added to 990 ul of saline diluent

serum for detection of antibodies should be drawn during

acute and convalescent phases of illness

precipitation and agglutination are

examples of unlabeled immunoassays, visible expressions of the aggregation of antigen particles or molecules alternating with antibody molecules

precipitation

aggregation of soluble test antigens; combination of soluble antigen with soluble antibody to produce a visible insoluble complex

agglutination

specific antigens (eg RBCs) aggregate to form larger visible clumps when the corresponding specific antibody is present in the serum

IgM is the best

at agglutination and precipitation and is almost always exclusively used as reagents

precipitation assays - double immunodiffusion technique

over time, antibody from the antibody well and antigen from the antigen from the antigen well will migrate toward each other to form a precipitation line at the zone of equivalence, used in fungal serology to detect antibodies in response to aspergillus, Blastomyces, Candida, coccidioides, histoplasma

precipitation assay - this precipitation band is called

line of identity (if the unknown serum contains antibody to the known antigen, a precipitation band forms at a point of optimal concentration - zone of equivalence - of each component)

precipitation assays - double immunodiffusion

patient samples must always be adjacent to positive control, rate of diffusion is affected by size - igG diffuses well in gel, igA is the predominate immunoglobulin in chronic cases and diffuses moderately well, igM does not diffuce well due to size and will negative in gel

what is A’s reaction

reaction of identity (positive)

what is B’s reaction

reaction of nonidentity (negative)

what is C’s reaction

reaction of partial identity (spur, multiple infections)

precipitation is the term applied to

aggregation of soluble test antigens

agglutination

observable clumping of insoluble antigen particles with soluble antibody, forms a framework in which antigen particles or molecules alternate with antibody molecules

Agglutination is the clumping of particles that have antigens on their surface, such as erythrocytes, by antibody molecules that form bridges between the antigenic determinants, this is the end point for

most tests involving erythrocyte antigens

particle agglutination reactions

artificial carrier particles such as latex or charcoal may be needed to indicate visibly that an antigen-antibody agglutination reaction has taken place, when carrier particles are used the assay is known to be passive, quality of results depends on: time of incubation with the antibody source, amount and avidity of an antigen conjugated to the carrier, conditions of the test environment

immunologic assays preformed by latex particle agglutination

C-reactive protein, igG rheumatoid factor, igM rheumatoid factor, rubella antibody

latex particles in C reactive protein assay results

if CRP is present in the serum, an antigen-antibody reaction takes place. This reaction causes a change in the uniform appearance of the latex suspension and a clear agglutination results

Stabilized sheep erythrocytes in Rheumatoid factor (RF) results

if gamma globulin is attached to a particular carrier the reaction of RF with gamma globulin becomes a visible agglutination

agglutination - liposome-enhanced testing

is a variation of latex agglutination to enhance visibility or agglutination

agglutination - direct bacterial agglutination

detects patient antibody; antigen is naturally on the particle itself (bacteria, fungus); the binding of patients antibody to antigen in a bacterial suspension causes the bacteria to clump together in visible aggregates, also called bacterial agglutination

agglutination - inhibitation agglutination

detects patient antigen (hCG - pregnancy), the blocking of the formation of clumps (agglutination) is the principle of the test

agglutination - passive agglutination

(passive hemaagglutination) detects patient antibody; a carrier such as red blood cells is used to absorb soluble antigen onto their surface; the red blood cells than aggluation in the presence of antiserum specific for the absorbed antigen

reverse passive agglutination

detects antigen (CRP fibrinogen); antibody is attached to the carrier particle

agglutination - flocculation tests for antibody detection

based on interaction of soluble antigen with antibody, which results in the formation of a precipitate of fine particles of visible masses (aggregates) indicating an antigen antibody reaction, these particles are macroscopically or microscopically visible only because the precipitated product is forced to remain in a confined space, examples include veneral disesase research labs (VDRL) test and rapid plasma reagin (RPR) for syphillus testing

hemagglutination

method of testing that detects antibodies to erythrocyte antigens

Hemagglutination direct

direct antiglobulin test (DAT or Coombs): detects antibodies directly bound to the RBC surface antigens; helps diagnose hemolytic disease of the newborn (HDN)

Hemagglutination indirect

Indirect antiglobulin test (IAT): detects circulating antibodies not yet bound but have potential to do harm

Hemagglutinin is influences by a number of factors and is believed to occur in two stages:

1. sensitization: physical attachment of antibody molecules to antigens on erythrocytes, which is influenced by: particles charge, electrocytes concentration and viscosity, antibody type, antigen-to-antibody ratio, antigenic determinants, physical conditions such as pH, temperature, and incubation

2. lattice formation: occurs when antigen particles and antibodies crosslink to form bridges, which is influenced by: factors such as zeta potential: electrostatic charge that repels adjacent particles

enzyme pretreatment of red blood cells - techniques to reduce zeta potential

removes negatively charged sialic acid residues from cell surface membrane

addition of colloids (e.g. albumin) - techniques to reduce zeta potential

increases electrical conductivity of environment

centrifugation - techniques to reduce zeta potential

mechanical process to force red blood cells closer together

other techniques to enhance hemagglutination include the use of

anti-human globulin (AHG), forms cross-links between antibodies that are bound to antigens on a carrier surface, AHG is incorporated into the procedure to link the antigens and antibodies close enough to cross-link

4+, 3+, 2+ 1+ mixed field aggluination reactions

all one solid, several large solids, medium solids clear supernatant, lots of small solids with turbid background, red background

prozone

if excessive antibody concentration is present, the prozone phenomenon occurs and can result in a false-negative reaction

postzone

if an excess of antigens occurs, the postzone phenomenon occurs, in which small aggregates (clumps) are surrounded by excess antigen and no lattice formation is established

nephelometry

antigens and antibody added to a tube. Visualize light scattered at 10-90 (usually 70) determine concentration using a standard curve

turbidometry

antigen and antibody added to a tube visualize that is directly across from the light source. Determine concentration using a standard curve

Immunogenic assays performed by nephelometry

acid a-glycoprotein, albumin, c1-8, transferrin

agglutination can be used to enhance reactions by all the following means

using enzyme treatment to alter the zeta potential, centrifugation, using colloids and anti-human globulin

anti-human globulin (AHG) is used to

form cross-links between antibodies that are bound to antigens on a carrier surface

the appropriate description of the prozone phenomenon is

an excessive antibody concentration produces a false-negative reaction

Major Histocompatibility complex - class I region

genes control HLA-A, B, C antigens found on most nucleated cells, MHC class I proteins functions to present endogenous antigen to CD8 T cytotoxic lymphocytes

Major histocompatibility complex - Class II region

genes code for DP, DQ, and DR antigen expressed primarily on B cells, macrophages, other antigen presenting cells, and activated T cells, MHC class II proteins function to present exogenous antigen to CD 4 T helper lymphocytes

Major histocompatibility complex - class III region

genes code for some of the complement components and cytokines

role of HLAs (human leukocytes antigens)

HLAs are the molecular basis for T cell discrimination of self from non-self, key applications for HLA typing include organ transplantation and disease association

HLA nomenclature - N

null allele (produces a non-functional protein)

HLA nomenclature - L

lower than normal cell surface expression

HLA nomenclature - S

soluble protein not found on cell surface

HLA nomenclature - Q

questionable (allele may affect normal expression)

HLA nomenclature - C

cytoplasm protein not present on the cell surface

HLA nomenclature - A

adherent expression (uncertain if protein is expressed)

HLA haplotypes inheritance

expressed codominantly, example of an HLA type: HLA-A10, A28, B7, Bw52, Cw2 (better donor - homogyzous, person receiving - hetero)

the major histocompatibility complex (MHC) HLA-II region codes for

class I molecules

the _____ antigen is a MHC class II antigen

HLA-DR

application of HLA typing disease association examples

HLA B27 is useful in diagnosing ankylosing spondylitis

reasons for a transplant

organ transplantation is widely viewed as the preferred treatment for end stage organ failure

reasons for kidney recipients

diabetes, glomerulonephritis, hypertensive nephrosclerosis, or polycystic kidneys

reasons for liver recipients

cirrhosis, cholestatic liver disease, biliary atresia, or hepatitis C infection

reasons for heart recipients

cardiomyopathy, congenital heart disease, valvular heart disease, or coronary heart disease

HLA crossmatching methods

the classic method to detect antibodies was complement dependent cytotoxicity, it has been gradually replaced by more sensitive solid phase immunoassays, such as enzyme linked immunosorbent assay (ELISA) and the bead-based technology, flow cytometry

autograph

graft transferred from one position to another in the same individual

syngraft

graft transplanted between different but identical recipient and donor

allograft (homograft)

graft between genetically different recipient and donor the same species; grafted donor tissue or organ contains antigens not present in recipient

xenograft (heterograft)

graft between individuals of different species

allogenic

stem cells come from a donor

syngeneic

stem cells from an identical twin

autologous

stem cells from the patients own stem cells

examples of diseases treatable by stem cell transplantations

acute leukemias, stem cell disorders, other malignancies

application of HLA typing includes all of the following

platelet transfusion, disease association, organ transplantation

graft between genetically different recipient and donor of the same species; the grafted donor tissue or organ contains antigens not present in the recipient

allograft

transplant rejection, role of T cells

graft rejection is primarily regulated by the interaction of the host’s T cells with the antigens in the graft