Lecture 6 - Genetic Concepts: Application in HLA and Blood Group Typing, Flow Cytometry

Gene - Mendelian definition

Basic unit of heredity

Gene - Molecular definition

A specific sequence of DNA which is transcribed to produce a protein

Locus

Specific location of a gene on c chromosome (i.e. RHD gene locus is on chromosome 1, and ABO is on chromosome 19)

Genotype

Combination of alleles for a specific trait

Phenotype

Anything that is produced by the genotype, could be an enzyme to control a blood group, or a protein to regulate RBC production

Allele

Different forms of a gene at the same locus

Autosomal

The chromosomes with number (1-22) are autosomes, every individual has 22 pairs of autosomal chromosomes and one pair of sex chromosomes, any trait/phenotype carried on any of the 22 pairs will have an ________________________ pattern of inheritance

X - linked

AA = Homozygous

AA = heterozygous

Any trait carried on X chromosome

AA =

AO =

Dominant gene or allele

it will be expressed in both homozygous and heterozygous states, in AA and AO, A will be expressed (A and B are dominant over O), usually denoted by capital letter

Recessive gene or allele

Usually denoted by lowercase, for instance h is recessive vs H, it can be expressed only in a homozygous state (hh, OO)

Amorph

Gene is inherited but cannot produce any product, O gene in the ABO system is...

Hemizygous

One allele is normal with the other is mutated or deleted (i.e. XY males with hemophilia A (FVIII) deficiency), chromosome X carries the mutated allele

Co-dominant

Both alleles inherited from both parents will be expressed, one gene is not dominant over the other

ABO, Rh, Kell, KIdd

What blood group antigens are inherited in co-dominant fashion?

Antithetical antigens

In the Kidd blood group sysem where the alleles JKA and JKB encode the Jka and Jkb antigens

Jka and Jkb are considered

cis position

trans position

When alleles found on the same chromosome, they are:

When alleles found on opposite chromosomes, they are

O

Which blood group cannot be inherited by any offpsring of parents with AA and BO genotypes?

A, AB, O, or B

OO

If the father's blood group is O, what is his most likely genotype?

AO, BO, AB, or OO

Both as they are co-dominant

If a person inherits K and k alleles (Kell blood group system), what antigens will be expressed on their RBCs

a) Only K because it is dominant over K

b) Only k because it is dominant over K

c) None of them, as both are amorph

d) Both, as they are co-dominant

Chromosome 6

Genes encoding HLA antigens are part of MHC gene system located on chromosome __?

MHC system

What system is first recognized and named from experiment in tissue transplantation?

Class I - A, B, C loci

Class II - D region; DR, DR, DQ

Class III - code for complement proteins and cytokines

Inheritance of HLAs

MHC genes are divided into 3 classes, name each class and the located

HLA genes

Genes that are closely linked and is inherited as one haplotype from each parent

Both haplotypes are expressed by offspring

Serologic or molecular methods

Each antigen with unique number are determined by what types of methods?

LOW

What is the probability that any two individuals will express the same HLA antigens

MHC region

Most polymorphic system of genes in humans, many possible alleles at each location

Letter - designates the locus

Number - indicates the antigen

HLA Nomenclature

Letter designates:

Number indicates:

At C locus

"W" distinguishes HLA C-locus antigens from complement components

HLA Nomenclature

At what locus is "w" included for?

What does it distinguish?

number of antigens

In HLA nomenclature, the number of alleles determined continues to increase. What stayed the same?

More accurate and specific molecular typing assays

When HLA nomenclature began, the only test method was the lymphocytotoxicity assay. What is there now in present day?

Serologic testing

Low-resolution typing

High-resolution testing

HLA Nomenclature

A2 for antigen level resolution is determined by?

HLA-A*02 is for:

HLA-A*02:01 is for:

HLA class I

Found on surface of platelets, leukocytes, and most nucleated cells

Mature RBCs

reticulocytes

What lacks HLA antigens?

In blood, what type of cells express HLA class I antigens?

HLA class II

Found on antigen-presenting cells (i.e. macrophages, dendritic cells, and B cells)

Greater than

In the circulation, the number of cells expressing class I antigens is _______________________ the number of cells with class II antigens

Because class II antigens are not found on platelets

Why is it NOT necessary to match class II antigens when HLA-compatible platelets are requested

Lymphocytotoxicity test method aka complement-dependent cytotoxicity (CDC)

What type of test does serologic HLA identification require?

What is it also known as?

Antibodies in micro-liter plates (96 well)

Serologic HLA Identification (typing)

Suspension of B or T cells is incubated with what?

Complement from rabbit serum

Vital exclusion dye

Serologic HLA Identification (typing)

What is added with additional incubation? What is it followed by?

Activation of complement

Takes in the dye -- the cells are stained

Serologic HLA identification (typing)

If Ab-Ag reaction occurs, what causes lysing of the cell? What does it take in?

Reaction Intensity

Serologic HLA identification (typing)

What is each well score based on?

Cells that tape up the dye are positive

Cells that have no color (white) are negative

Serologic HLA Identification (typing)

What is a positive result vs a negative result?

Antibodies cant react to more than one epitope (or cross-reactive bodies), and specific antibodies have not been developed to recognize many different HLA antigens

Why is serologic typing of HLA antigens being replaced by molecular methods?

ABO comptability

careful matching of HLA antigens in patients with existing antibodies

What do successful (viable) transplants require?

Pregnancy

Blood transfusion

Previous transplants

Patients may become sensitized to HLAs by the following exposures:

HLA-A1

A negative test in serologic HLA identification indicates that the patient does NOT possess

Serologic tests on microtiter trays (30-60 antigens)

% Panel-reactive antibody (PRA)

What are the tests that are administered prior to organ transplant for HLA antibodies?

% Panel-reactive antibody (PRA)

The proportion of lymphocytes in the panel that are killed by the patient's serum or the number of antibodies

A high PRA means that the recipient patient has many reactions, making it less likely compatible with available donor organs

If the patient has a high PRA, what is the probability that the patient will be compatible with available donor organs?

Calculated panel-reactive antibody (CPRA)

Newer solid-phase method/more sensitive and specific/determines probability of compatibility to donor organ

Antigens that the organ candidate is reactive against

Unacceptable antigens

What is the CPRA number based on?

What is this called?

Determining reactivity within the general donor population

What does the CPRA provide a statistical guide for?

Genetic relatedness of the donor and the recipient

What is the transplantation of cells or tissues classified by?

Autograft

Transfer of tissue from one area of the body to another of the same individual

Allograft

Transfer of cells or tissue between two genetically disparate individuals of the same species

Syngeneic graft aka isograft

Transfer of cells or tissues between individuals of the same species who are genetically identical

Ex) identical twins

Xenograft

Transfer of tissue between two individuals of different species

Allografts and xenografts

What tissue transfers results in a vigorous cellular and humoral immune response to the foreign MHC antigens? What does this primarily stimulate?

Direct Allorecognition

Indirect Allorecognition

The immune system of the recipient recognizes foreign HLA proteins by what mechanisms?

Direct allorecognition

Cytotoxic T cells from the host bind directly to foreign HLA (MHC I) proteins on graft cells

Indirect allorecognition

APCs from the host present foreign MHC I or MHC II antigens to CD4+ Th cells, which produce cytokines that stimulate graft rejection

T cells proliferate in response to foreign HLA-D antigens

In mixed lymphocyte reaction (MLR)-an in vitro assay, if the HLA-D antigens (class II antigens) of two cell populations differ, what occurs?

Incorporation of thymidine into the DNA of the proliferating cells

How is the T cell response in mixed lymphocyte reaction (MLR)-an vitro assays quantitated?

Donor is more likely to simulate graft rejection

A high level of radioactivity indicates what in the mixed lymphocyte reaction (MLR)?

Kidney and pancreas transplants

Crossmatching recipient serum with T and B cells from the potential donor to avoid rejection caused by antibodies to the donor tissue is possible for

compatibility and D (class II) antigen typing

Mixed lymphocyte (MLC) crossmatching was historically used for...

Immuno-fluorescent flow cytometry techniques

What has replaced MLC crossmatching?

Aplastic anemia

Leukemia

Lymphoma

Hodgkin's disease

What does can be treated by HPC transplants?

HLA matching at the allelic level

What is important in HPC transplants in order to avoid rejection and graft-versus-host disease (GVHD)?

Graft-versus-host Disease (GVHD)

Occurs when grated immunocompetent cells from donor mount immune response against host tissue

- can occur in any organ transplant

When recipients of HPC transplants become immunosuppressed to allow for donor cell growth

When is GVHD more likely to occur?

Platelets do not contain class II HLA antigens

Case Study 1: HLA Graft/Donor Selection

A patient who is not responding to platelet transfusions requires HLA matched platelet transfusion. The patient's HLA type is determined and is reported as A2, A11, B8, B35. No report of the HLA C antigen is given. A donor with a compatible type is called on to donate for this patient.

Why were the HLA class II antigens not considered for matching?

A2, B50, DR17

Sibling 1

Case Study 2: HLA Graft/Donor Selection

Parents of a kidney transplant candidate were tested for a potential compatible kidney match within the family because the patient had produced HLA antibodies. The patient's HLA antigens were typed and found to be:

A1, A2, B27, B50, DR11, DR17.

Antibodies identified were specific for:

A3, B18, and DR7 antigens.

The mother's HLA typing is:

A1, A3, B35, B27, DR4, DR11

The father's HLA typing is:

A2, A24, B50, B44, DR1, DR17

Siblings HLA Typing:

Sibling 1: A1, A24, B27, B44, DR11, DR1

Sibling 2: A3, A2, B35, B50, DR4, DR17

Sibling 3: A3, A24, B35, B44, DR4, DR1

What HLA antigens did the father contribute?

Select the sibling with the best match.

Flow Cytometry

Technique that involves measuring the physical and chemical properties of cells which are flowing in a stream of fluid

A. Lymphocytes

B. basophils

C. Monocytes

D. Neutrophils

Indicate what A-D is

Fluidics - sheath fluid tank, flow cell

Optics - lasers, filters

Detectors - FSC, SSC, and FL

Electronics - ADC system, data processing system

Flow Cytometry components

Cluster of differentiation

The term "CD" stands for

Identification and characterization of leukocytes

Cell surface markers are useful for....

mAbs conjugated with FL

What is used to detect CD markers?

A - lymphocytes (small size, low complexity)

B - monocytes (largest size, low/medium complexity)

C - granulocytes (medium size, highest complexity)

D - dead cells, nRBCs, debris (smallest size, lowest complexity)

Indentify A-D

Friend 2

A 40-year-old mother of three needs to have a second kidney transplant. Her first transplant was lost because of chronic rejection. The mother's HLA type, HLA antibodies, and ABO blood group status were determined. The patient was found to have antibodies to HLA-B35 by flow cytometric testing with HLA-B35-coated beads. The HLA type and blood group were also determined for two of her siblings and two close friends who are interested in donating a kidney to the patient.

From the available donors, who would most likely be the most compatible patient?

(Percentage fetal cells x 1800 x 1.22)mL

FMH volume formula

6 lasers

21 detectors

23 parameters

96-well plate loader

CytoFLEX Lx Flow cytometry components

How many lasers?

How many detectors

How many parameters

What type of well is it equipped with?

X-axis - increasing green fluorescence

Y-axis - increasing red fluorescence

1 - cells not expressing either A or B

2 - cells expressing only B

3 - cells expressing only A

4 - cells expressing both A and B

Identify the X and Y axis

Identify 1-4 and what it tells us

1) SCID/HIV

2) CD34

3) CD55/CD59

4) RNA binding dyes

5) Ab against HbF, RHD, CA

6) DNA content

7) HLA, flow cross match

Other applications of Fc in Hematology

List what pertains to each:

(Word Bank: CD34, CD55/CD59, SCID/HIV, RNA binding dyes, DNA content, HLA/flow cross match, Ab against HbF/RhD/CA)

1) immune deficiency

2) stem cell enumeration

3) PNH

4) Reticulocytes

5) FMH

6) Cell cycle analysis

7) Transplantation