(pt 1) exam #1 - immunohematology (cls 544)

history of blood banking (1400s-1700s)

• Ancient beliefs: blood is the most important of the "four body humors"

• 1492: first recorded blood transfusion--Pope Innocent VIII

• 1628: discovery of the intravenous circulation by Dr. William Harvey

• Late 1700s: bloodletting is common practice

“firsts” within transfusion medicine (1600s-1800s)

• 1667: 1st blood transfusion documented by Dr. Jean-Baptise Denis (France)

  • Sheep to human

• 1818: 1st successful human blood transfusion administered by Dr. James Blundell; patient experienced postpartum hemorrhage (human to human)

• 1869: 1st blood preservation research; Braxton hicks recommended sodium phosphate as a nontoxic anticoagulant

“firsts” within transfusion medicine (1900-1916)

• 1901: Karl Landsteiner discovered the ABO blood group system

• 1914: Dr. Albert Hustin reported use of sodium citrate as an anticoagulant for transfusions

• 1915: Richard Lewisohn determined the minimum amount of sodium citrate needed for anticoagulation

• 1916: First blood transfusion used blood stored and refrigerated by Oswald Robertson

“firsts” within transfusion medicine (1937-1953)

• 1937: The first US hospital blood bank by Bernard Fantus (Chicago, Cook county hospital)

• 1941: Dr. Charles Drew appointed as director of the first American Red Cross blood bank

• 1947: The American Association of Blood Banks founded (AABB)

• 1948: American Red Cross began operating their full-scale blood program

• 1953: Plastic blood bank bag invented by the Fenwal Company

components of blood (4)

• Red blood cells (RBCs/PRBCs)

• White blood cells (Granulocytes)

• Platelets

  • Single donor vs random (pooled)

• Plasma

  • Fresh frozen plasma, frozen plasma, cryoprecipitate AHF

red blood cells

• Contains hemoglobin

• Carries oxygen throughout the body and to the tissues

white blood cells

• Help fight against infection

• Neutrophil, eosinophil, basophil, monocyte, lymphocyte

platelets

• Derived from the cytoplasm of the megakaryocyte

• Plays important role in blood coagulation, hemostasis, and blood thrombus formation

  • Prevent massive blood loss

plasma

• Liquid portion of whole blood

• Contains water, electrolytes, glucose, fats, proteins, and gases throughout the body

• Carries all clotting factors necessary for coagulation in inactive form

• Upon occurrence of coagulation, the fluid converts into serum

shelf life of RBCs & platelets

• RBC shelf life = up to 42 days

• PLT shelf = up to 5 days

**Supply constantly replenished by blood collection facilities

blood statistics/facts

• The average adult has 10.5 pints of blood in their body

• No FDA approved substitute for blood products

• Under normal circumstances, about every 2 seconds someone in the US will need blood

general steps to the blood donation process (5)

1. Pre-donation: hydration

2. Registration

3. Health history and mini-physical

   • e.g. hemoglobin, body temperature

4. Donation

5. Post-donation

   • Refreshments

collection method for whole blood

• unit of whole blood is collected from a volunteer donor

• After donation, the whole blood unit is separated into components

• Process lasts ~1 hour

• Can donate every 56 days, up to 6 times per year

eligibility requirements for whole blood donation

• At least 16 years old

• Weigh at least 110 lbs

• Be in good "general" health

apheresis

automated process in which whole blood is removed from the body and passed through an apparatus that separates out one (or more) particular blood components

• RBCs, WBCs (granulocytes), plasma and/or platelets

• The remaining blood components are returned to the donor

(apheresis donation) platelet donation eligibility

• 1.5-2.5 hour process

• Can donate up to 24 times a year

• Shelf life = 5 days

• Same general donation requirements as whole blood

  • Exception--no aspirin in the past 48 hrs

(apheresis donation) double RBCs donation eligibility

• Process may last up to 1.5 hours

• Can donate every 112 days up to 3 times per year

• Shelf life = 42 days

• Age, weight, and height requirement (ARC)

  • Males: 17 yrs, 130 lbs or more, 5'1" or taller

  • Females: 19 yrs, 150 lbs or more, 5'3" or taller

• Be in good health

where does blood go after a blood donation?

• Blood center/manufacturing center

  • Blood processed into separate components and stored based on product type

• Laboratory

  • Donor samples sent for infectious disease testing to ensure safety of blood for transfusion

• Blood center

  • Blood components labeled and distributed to hospitals as needed

• Hospital

  • Healthcare providers determine patient needs for transfusion

  • Lab responsible for compatibility testing

  • Blood components are transfused to the patient

US Food and Drug Administration (FDA)

• Regulates the donor screening process

• Code of Federal Regulations (CFR)

• Blood treated as both a biologic and a drug

• Ensure compliance in all aspects of transfusion medicine

• Provides licenses for:

  • Collection and processing facilities

  • Blood products and derivatives

  • Reagents used in the processing and testing of those products

Center for Biologics Evaluation and Research (CBER)

• Regulates the collection of blood and blood components

  • Used for transfusion

  • Manufactured pharmaceuticals derived from blood

• Enforces quality standards

• Inspects blood establishments

• Monitors errors, accidents, & adverse clinical events

  • Any fatality or adverse events need to be documented to this organization

Centers for Medicare and Medicaid Services (CMS)

part of the Department of Health and Human Services (HHS)

• provides health coverage through Medicare, Medicaid, and the Children's Health Insurance Program (CHIP), while also overseeing the Health Insurance Marketplace

Clinical Laboratory Improvement Amendments of 1988 (CLIA ‘88)

• Regulate laboratory testing and require clinical laboratories to be certificated by their state (and CMS) before they can accept human samples for diagnostic testing

• Three federal agencies are responsible for CLIA

  • FDA, CMS, and CDC

American Association of Blood Banks (AABB)

• International association of blood centers, transfusion and transplantation services and transfusion medicine

• Provides the highest standard of care for transfusion medicine

• Voluntary inspection and accreditation program for member institutions

• Approved by CMS and CLIA requirements

• Resources for donor screening procedures

  • AABB standards for blood banks and transfusion services

  • AABB technical manual

the College of American Pathologists (CAP)

• Provides voluntary inspection and accreditation program for member institutions

• Blood bank is usually included in CAP inspections along with general lab

• Approved by CMS and meet the CLIA requirements

blood bank history (antigen/antibody discoveries)

• 1901: Landsteiner discovers the ABO blood group system

• 1927-1947: M, N, PI antigens were discovered

• 1945: the antihuman globulin (AHG) testing technique was developed by Coombs, Mourant, and Race

  • Prior to the AHG test, only IgM antibodies were detected

  • First used for Rh antibody detection

  • Later, used for antibody detection in other blood groups

• 1946: Kell blood group system discovered

antihuman globulin (AHG)

derived from serum in rabbits or other animals (e.g. mice) that were previously immunized with purified human globulin to produce antibodies against human globulin

• AHG is used in the direct and indirect antiglobulin tests

• AHG binds to human globulins such as IgG or complement

• In blood bank, the primary focus is on IgG and IgM blood group antibodies

immunoglobulins

• Two functions:

  • Bind w antigens

  • Mediate various biological effects by binding to host tissues, various cells of the immune system, phagocytic cells, and the first component of the classical complement system

• The ability to recognize antigens is the product of the adaptive immune system

• Immunoglobulins are proteins composted of two heavy chains and two light chains

  • Five isotypes: IgA, IgD, IgE, IgG, IgM

IgG

• Predominant class of antibody produced in the secondary immune response (non-agglutinating antibodies)

• Comprises 75% of immunoglobulins in plasma = most abundant

• Monomer → bivalent antibody molecule → capable of binding to two antigen sites that are very close together

  • due to its smaller size, IgG are not as detectable in "non-enhanced" testing environments

subclasses of IgG

• IgG1 and IgG3 = activate complement, readily recognized and bound by macrophages

• IgG2 = weakly activates complement and not easily recognized

• IgG4 = does not activate complement and not recognized at all (in most cases)

• Ability to cross the placenta (IgG1 is best at doing this, then 4, 3, & 2))

IgM

• First immunoglobulin class produced in a primary immune response

• Comprises 6% of immunoglobulins in the plasma

• Pentamer with a valency of 10 (10 potential antigen-binding sites)

• Considered a direct agglutinate (or complete antibody)

  • Can bind to multiple RBCs and agglutinate in the absence of AHG reagent

• Very efficient at complement activation

• Does NOT cross the placenta

(general) direct antiglobulin test (DAT)

• One-stage procedure that detects in vivo RBCs that have been sensitized by antibodies

• Detects if your cells are coated by antibodies from autoimmune reactions or other processes that may cause agglutination when other RBCs are introduced

**antihuman globulin (AHG) reagent is added to form a "bridge" when antigen-antibody complexes present—allows for visualization of the Ag/Ab reaction (e.g. agglutination)

(general) indirect antiglobulin test (IAT)

• Two stage procedure that demonstrates in vitro reactions between RBCs and corresponding IgG antibodies

• Detects if you have antibodies that can coat other cells and cause agglutination

**antihuman globulin (AHG) reagent is added to form a "bridge" when antigen-antibody complexes present—allows for visualization of the Ag/Ab reaction (e.g. agglutination)

AHG (Coombs) tests overview

• Used to detect RBCs sensitized with IgG alloantibodies, IgG autoantibodies, and complement (e.g. C3d)

• AHG reagents

  • Polyspecific AHG

    • Specificities: contains antibodies to human IgG + the C3d human complement component

  • Monospecific AHG--one specificity

    • Anti-IgG

    • Anti-complement (anti-C3b, C3d)

how are AHG reagents made?

injecting animals with specific human protein and harvesting antibody that animal produces in responses

polyspecific AHG

• contains both anti-IgG and anti-C3d

  • Reagent of choice for DAT on adults

  • It is common practice to perform initial DAT workup with polyspecific AHG reagent

  • If positive, repeat testing of patient sample using monospecific AHG reagent

monospecific AHG

• contains either anti-IgG or anti-C3d

  • Anti-IgG reagent preferred over polyspecific AHG for antibody detection to avoid detecting clinically insignificant cold-reactive antibodies that bind complement

  • Anti-IgG mixture primarily comprised of IgG1 and IgG3 subclasses

polyclonal AHG production

•  prepared by injecting human globulins into rabbits

  • Advantages:

    • Detects many different IgG antibodies

  • Disadvantages

    • Manufacturing--if excess antibody present (IgG), then prozoning may occur resulting in potentially false-negative results

    • Unable to determine potency of anti-C3d, C3b individually

monoclonal AHG production

• prepared by injecting human globulins into mice (e.g. murine reagents)

• Advantages:

  • Produces higher antibody titers with well-defined specificities to IgG and fragments of C3

  • Clonal line produces a single antibody: no need to remove heterospecific antibodies

• Disadvantages:

  • Detects single epitopes: IgG antigens are composed of multiple epitopes

clinical applications of the IAT

• Used for the detection of in vitro sensitization of RBCs

• Use patient plasma/serum

• Clinical applications

  • Antibody screen

  • Antibody identification

  • Crossmatch (or compatibility) testing

  • RBC phenotype (antigen typing)

  • Antibody titration

polyspecific vs monospecific AHG in the IAT

• Most clinically significant antibodies are IgG class

• Use of enhancement media

  • Increase rate and sensitivity of antibody attachment to RBC antigens

  • Reduce incubation period

    • Bovine serum albumin (BSA)

    • Low ionic strength solution (LISS)

    • Polyethylene glycol (PeG)

  • Some clinically significant antibodies are detected with the anticomplement component of AHG but not with anti-IgG

    • e.g. Anti-Jk^a may be detected with polyspecific AHG & LISS but not with albumin  

clinical applications of the DAT

• Used for detection of in vivo sensitization of RBCs with IgG antibodies and/or complement

• Use patient blood

• Clinical applications

  • Hemolytic disease of the fetus and newborn (HDFN)--maternal antibody

  • Hemolytic transfusion reaction (HTR)--recipient antibody

  • Autoimmune hemolytic anemia (AIHA)--patient autoantibody

    • Drug-induced hemolytic anemia--drug/anti-drug complex

testing/sample criteria for DAT

• Patient sample must be collected in an anticoagulant (e.g. EDTA--preferred, ACD, CPD)

• Positive DAT result is not definitive--need further investigation

  • e.g. Patient's diagnosis, blood transfusion history, list of medications

check cells (Coomb’s control cells)

• IgG-sensitized or complement-coated RBCs (should produce agglutination when tested)

  • D positive cells coated with anti-D (IgG)

  • Cells coated with anti-complement

• positive reaction means that reagent has been added and ensures test works

• Lack of reaction due to improper washing

antibody screen test

determines if patient has unexpected antibodies present in plasma

• Utilizes indirect antiglobulin test (IAT)

  • Immediate spin (IS) phase

  • Incubation/37 deg C phase

  • Antihuman globulin (AHG) phase

levels of detection for DAT/IAT

• overall goal is to detect all clinically significant antibodies for both DAT and IAT and none of the clinically insignificant antibodies

  • e.g. warm and cold reacting autoantibodies

• DAT level of detection

  • 100 to 500 IgG molecules per RBC

  • 400 to 1100 molecules of C3d per RBC

• IAT level of detection

  • 100 to 200 IgG or C3d molecules on the RBC to obtain a positive reaction—more specific

factors affecting the AHG phase of the antibody screen

• plasma:cell ratio ; ionic strength ; pH

• Enhancement media:

  • Albumin, LISS, PeG

• temperature ; incubation time

• Washing of RBCs & saline used

• Addition (or lack thereof) of AHG reagent

• Centrifuge setting for reading purposes

sources of error for AHG test (DAT/IAT)

• most encountered sources of error:

  • Inadequate washing of RBCs

  • Non-reactive AHG reagent used in the procedure

  • Failure to add AHG reagent to the tubes

• False negative reactions: seen most in tube testing and inadequate washing of RBCs

• Note: all negative AHG test reactions must be checked by the addition of check cells!

AHG testing methods (3)

1. Tube Test: the "gold standard" for detection of Ag/Ab reactions

2. Gel: the sensitivity is equivalent to the polyethylene glycol (PeG) test tube method

3. Solid Phase: RBC antigens coat the bottom of microtiter plate

contributions to immunology by edward jenner; robert koch; pasteur; bordet; and kaus?

• Edward Jenner: discovered vaccination (used cowpox to protect against smallpox)

• Robert Koch: proved infections were caused by microorganisms

• Louis Pasteur: developed rabies vaccine

• Bordet: discovered complement

• Robert Kaus: discovered precipitins

  • antigen-antibody reaction that produces a visible precipitate

who developed the idea of the “lock and key” binding for antigens and antibodies?

Paul Ehrlich

• postulation that specialized cells (e.g. plasma cells) carried antibodies and the molecular structure had receptor sites for the antigens that stimulated their formation

general characteristics of the immune response

• occurs when the human body is exposed to foreign substances, organisms, and environmental toxins

• equipped with remarkable immune defense mechanisms

innate/natural immunity

primary line of defense ; acts quickly, nonspecific

• Physical: skin, mucosal linings

• Biochemical: chemical secretions, (e.g. tears, saliva)

• Immune cells: phagocytic cells & NK cells remove invading orgs

• Humoral: cyto/chemokines initiate inflammation process

acquired/adaptive immunity

“learned” immunity; needs time, is specific, and capable of memory

• Cellular: B and T lymphocytes

• Humoral: antibodies

• ability to respond to infinite variety of antigens upon reintroduction

primary vs secondary immune responses

• Primary antibody response: accomplished by B cells with help of antigen-specific T cells; IgM antibodies

• Secondary (anamnestic) antibody response: occurs upon re-exposure to an antigen

  • Involvement of memory B cells

  • Primarily IgG antibodies

antigen

• a foreign substance that can trigger an immune response or damaged host cells

  • Chemically complex

  • Foreign or non-self

  • Usually, high molecular weight

• antigens associated with blood bank testing are proteins that sit on the surface of blood cells

antigen vs immunogen

• Antigen: recognized and bound by antibodies

• Immunogen: initiates and immune response

  • All immunogens are antigens; not all antigens are immunogens

immunogenicity

•  chemical composition and molecular complexity of the antigen

  • in blood bank: ABO > D > K > c > E > k > e

major histocompatibility complex (MHC)

• molecules for presentation to lymphocytes

  • Consists of a group of genes located on chromosome 6

  • MHC Class I antigens: found on nucleated cells in the body

  • MHC Class II antigens: found on antigen presenting cells

functions of antibody molecules (3)

1. Neutralization—antibodies bind to pathogens or toxins/prevent entry into cells

2. Opsonization—antibodies bind to the bacteria coating them, allow phagocytes to recognize Fc portion of the antibody molecule and eliminate bacteria

3. Activation of complement—enhances the bactericidal actions of phagocytes

summary of the five classes of immunoglobulins

• IgA - mucosal secretions, 13%

• IgD - bound to surface of naive B lymphocytes with surface IgM, 1%

• IgE - factor in serum that causes allergies; activates mast cells, <1%

• IgG - principal isotype in blood and extracellular fluid, 80%

• IgM - first antibody produced in primary immune response; found in blood and lymph, 6%

activation pathways of complement (3)

all three lead to formation of membrane attack complex (MAC)

• Classical – presence of antigen-antibody complex activate complement

• Alternative – activated by high molecular weight molecules with repeating units on the surfaces of target cells

• Lectin – activated by the attachment of plasma mannose-binding lectin (MBL) to microbes

**regulated by C1 inhibitor that inhibits formation of C3 convertase

how does complement relate to blood bank?

• Complement system plays role in blood group testing

• Some antigen-antibody complexes activate complement; hemolysis or RBC lysis

• Complement components related to blood group serology

  • Chido/Rodgers blood group (epitopes on C4 protein)

• Fixation tests

  • May be used to detect the amount of antigen or antibody present

  • Fixed via the classical pathway

effects of storage/anticoagulants on complement

• C1 and C2 components inactivated by heating serum to 56 deg C for 30 minutes

• Degrades at room temp and refrigerated storage

• Activity inhibited by EDTA (chelation of calcium)

membrane attack complex

• Final step of complement activation

  • Consists of C5b and C6-C9

  • Classical pathway regulated by two mechanisms in fluid phase and complement control proteins

• IgM is very efficient in activating complement, causes intravascular hemolysis

• IgG is less efficient but may cause extravascular hemolysis

primary factor in the formation of an immune complex?

• antigen's chemical composition plays the biggest role in the formation of an immune complex

  • Determines type of noncovalent bonds

  • Exothermic: released energy appears as heat

    • Enhanced at lower temperature

  • Endothermic: need energy from the environment

    • Enhanced at warmer temperatures

affinity

strength of the interaction between the antigen and the antibody's binding site at ONE individual site

avidity

• SUM of affinities between antigens and antibodies

• Increased avidity can make up for decreased affinity

influence of attraction on AGN-ABY complexes

• fit between antigen and antibody must be high enough to allow for the formation of multiple noncovalent bonds

• complementary nature needs to exist between antigen and antibody (e.g., size, shape, charge), lock and key

  • basis of all blood bank testing is centered around the reaction between antigen and antibody/antibodies

law of mass action

• Antigen and antibody bonds are reversible

• Low repulsion and high attraction = binding is likely to occur

• High repulsion and low attraction = binding is less likely to occur

agglutination

• Visible endpoint of antigen-antibody reactions for blood bank testing

• Particulate matter caused by combination with specific antibody

• reversible reaction between antigen and antibody

stage 1 of agglutination

• Sensitization

  • Antigen and antibody come together to form an immune complex

  • Binding only occurs if antigen and antibody are complementary in nature

• Factors affecting stage 1

  • Temperature

  • Incubation time

  • pH ; Ionic strength

  • Antigen-antibody concentration

stage 2 of agglutination

• Lattice formation

  • Antibody cross links form between RBCs forming a lattice that allows visualization of the antigen and antibody reactions

• Dependent upon the strength of test system, pH, and temperature

• Overcoming forces that keep red blood cells apart (zeta potential)

  • Repulsion may occur in physiologic saline

factors influencing agglutination reactions

• Centrifugation—enhances agglutination

• Antigen-Antibody (serum-to-cell) Ratio

  • Equivalent amount of antigen-antibody bind

• pH

  • Test system, 6.5 -7.5

  • Saline

• Temperature—IgM vs. IgG antibodies

• Enhancement Media

  • 22% Albumin - adjusts for zeta potential between RBCs

  • LISS - reduces zeta potential, RBCs take up antibody more rapidly

  • PeG - increases test sensitivity

    • Aggregates RBCs causing closer proximity of RBCs to each other/enhances antibody cross-linking

genetics

study of inheritance of transmissible characteristics or traits, including red blood cell antigens

• genetic material that determines each trait is found in nucleus of cells

• Chromosome--linear thread of DNA

mitosis

• somatic or nonsexual cell division

  • Diploid: human cells, except for sexual cells, contain two sets of chromosomes

  • DNA replication: chromosomes within nucleus duplicated before cell division

meiosis

• sex cell division

  • Daughter cells contain half of number of chromosomes in parent cell

  • Mutations--increase genetic variability; potential to create new phenotypes

  • Crossover--when two genes are near each other on the chromosome

DNA structure

• Usually exists in coiled double strand

• Made up of nucleotides: deoxyribose sugar, phosphate group, one of four bases

• Four bases: cytosine, guanine, adenine, thymine

factors affecting DNA replication

• Errors in the primary nucleotide sequence

• Chemical and environmental factors

• Ionizing radiation and strong oxidants

• Ultraviolet (UV) radiation

• Medications

mutations in DNA

any changes in the structure or sequence of DNA (physical of biochemical)

• various chemicals and conditions that can cause mutations are referred to as mutagens

• original form of the DNA sequence and the organism in which it occurs is called the wild type

• common blood group antigens result from SNPs

(general) DNA translation

• Genetic information carried by DNA transformed into an organism

• In cell nucleus, DNA unwound, and complementary strand of messenger ribonucleic acid (mRNA) produced

• mRNA transported from cells’ nucleus to cells’ cytoplasm

• Set of three base pairs (codon) translate into a particular amino acid or stop codon

define gene, locus, allele, & antithetical

• genes - made of DNA = basic unit of heredity

• locus - site of a gene on a chromosome

• allele - one or more different forms of a gene at specific locus on a chromosome

• antithetical - antigens that represent different forms of a gene product from same locus

define homozygous, heterozygous, hemizygous, and the dosage effect

• homozygous—two identical alleles at a given locus on both chromosomes

• heterozygous—alleles are non-identical at a given locus (two different alleles)

• hemizygous—refers to the condition when one chromosome has a copy of the gene and the other chromosome has that gene deleted or absent

• dosage effect—serologic difference encountered with heterozygous versus homozygous antigen

mendel’s first law of inheritance

aka law of separation ; shows that alleles of genes have no permanent effect on one another when present in the same plant but segregate unchanged by passing into different gametes

• unlike the flower color of many types of plants, most blood group genes are inherited in a codominant manner

  • in codominance, both alleles are expressed, and their gene products are seen at the phenotypic level

  • ex: In the MNSs blood group system, a heterozygous MN individual would type as both M and N antigen positive

mendel’s second law of inheritance

aka law of independent assortment ; genes for different traits are inherited separately from each other

• allows for all possible combinations of genes to occur in the offspring

• Mendel's laws apply to all sexually reproducing diploid organisms

hardy-weinberg principle

allows the study of Mendelian inheritance in detail

• formula states if there are only alleles for a trait in a population, then p + q = 1

  • p = gene frequency of the dominant allele

  • q = frequency of the recessive allele

  • This can be stated as:

    •  p² + 2pq + q² = 1

    • (p + q)² = 1

five assumptions under the hardy-weinberg principle

1. Large population size

2. Random mating

3. No natural selection

4. No mutations in parents or offspring

5. No migration, no new alleles introduced or lost

problems with the five assumptions under the hardy-weinberg principle

• Collecting sample data from a significantly large enough segment of a population is not always feasible

• Mating is not always random

• Mutations happen

• Mixing of populations on a global scale leads to “gene flow” on a constant basis

examples of various inheritance patterns

• Autosomal dominant: ABO blood system

• Autosomal codominant: S/s phenotype

• Autosomal recessive: OO phenotype

• X-linked dominant: Xg^a blood group system

• X-linked recessive inheritance: Hemophilia A

• Linkage: tendency for genes close together on same chromosome to be inherited as unit (haplotypes)

what can we use for genotype predictions?

• Punnett squares

  • Shows different ways that genes can separate and combine

• Pedigree chart

  • Records a family tree in which a trait is mapped through several generations

practical applications for genetics in blood bank

• Paternity testing ; transfusion

• High- and low-prevalence antigens

  • High-prevalence red blood cell antigens present in 98-99% of population

• Genetic testing methods

  • PCR

  • DNA microarrays

  • Restriction fragment length polymorphism analysis (RFLP)