11. Applications of BB Flashcards
3 general functions of HLA
- immune regulation
- recognition of self vs nonself
- coordination of cellular and humoral immunity
Class I HLA
nomenclature
location
HLA-A, B, C
platelets and most nucleated cells
Bg Ag on RBCs
Class II HLA
nomenclature
location
HLA-D-related
immune cells, interstitial epithelium, early hematopoietic cells
HLA on chromosome…
6
HLA gives a 3D configuration to each molecule to form a unique …
function?
peptide binding groove
holds processed peptides for presentation to T-cells
MHC I function
- present peptides from intracellular proteins (self, cancer, viral)
- present to CD8 T-cells
MHC II function
- present peptides from extracellular/cytosolic proteins (self, bacterial)
- present to CD4 T-cells
immune cells are only activated when…
antigenic peptide is displayed in context with the self HLA antigens
2 types of HLA nomenclature
conventional (serological)
DNA sequencing
HLA
capital letter denotes…
number denotes…
locus
antigenic specificity
HLA
“w” indicates… (3)
- C locus specificities (distinguish from complement)
- DP and D specificities (cellular methods)
- Bw4 and Bw6, public antigens found on multiple B specificities
HLA
—- shows allele is defined by DNA techniques
asterisk
HLA
set of two digit numbers separated by colons describe…
gene variants
HLA
low resolution
high resolution
serologic results
genetic variants
2 HLA genes inherited as a ———
haplotype
HLA
parents and children always share…
one haplotype match
best source of HLA-identical matches, 25% probability
siblings
HLA phenotype represents…
the combined expression of both haplotypes
HLA genes codominant
HLA
dash represents…
blank where an antigen is not detected
required to determine haplotypes
family studies
testing used to find HLA matches
histocompatibility
historically HLA Abs for testing were from…
multiparous women
transplant/tranfusion recipients
Ab recognizing a single HLA gene product
private epitopes/splits
Ab recognizing multiple HLA gene products
public epitopes/crossreactive
CREG
cross-reactive group
HLA antigens that consistently demonstrate cross-reactivity
———- antigens are less immunogenic when transplanted, allowing mismatch
within CREG
source for HLA DNA typing
any nucleated cell
3 ways to analyze amplified DNA for HLA
- sequence-specific oligonucleotides
- sequence-specific primers
- DNA sequence-based typing
HLA Ab testing indications
- determine presence of Ab in a transplant or platelet transfusion recipient
- find HLA Ab in donor plasma, which could cause TRALI
HLA Ab testing techniques (3)
- complement dependent lymphocytotoxicity (CDC)
- ELISA
- microbead array assay
standard serological HLA Ab typing method
CDC
CDC reagent cells
- T cells for class I
- B cells for class II
CDC principle
utilizes biological function of Ab to activate complement and disrupt cell membrane
dead cells are stained for visualization
HLA ELISA principle
HLA Ag bound to wells
HLA Ab binds, visualized with enzyme-conjugated substrate that changes color
microbead assay principle
beads labelled with ratio of 2 different fluorescent markers
beads of specific HLA type assigned to different color ratio
anti-IgG used to ID Ab binding to certain beads
explain HLA XM
recipient serum + donor purified WBCs
HLA XM incompatibility is a contraindication for….
kidney and pancreas transplant
types of HLA XM used most
virtual
flow
HLA XM that utilizes fluorescence
flow cytometry
advantage of HLA’s polymorphism
ID of individuals for parentage or forensic testing
GVHD occurs when ——- are transfused into a ——- patient and…
HLA-homozygous donor cells
heterozygous patient
patient does not recognize them as foreign
causes platelet refractoriness
pt sensitized to HLA Ags on platelets
leukocyte reduced product count
< 5 x 10^6
nonimmune causes of platelet refractoriness (not true refractoriness)
(3)
- sequestration —splenomegaly
- consumption —sepsis, DIC, meds, fever, bleeding
- prothrombotic conditions —HIT, TTP, HUS
HIT
heparin-induced thrombocytopenia
TTP
Thrombotic Thrombocytopenic Purpura
HUS
hemolytic uremic syndrome
alloimmune causes of platelet refractoriness
ABO
HLA
HPA
ITP
idiopathic thrombocytopenia purpura
platelet transfusion contraindications (2)
- prothrombotic conditions (HIT, TTP, HUS)
- acute ITP (bleeding complications are rare)
essential to detect platelet refractoriness & ID successful donor-recipient matches
one hour post transfusion platelet ct
required for HLA-matched platelets
irradiation
anti-A/B titer of —– associated with incompatible platelet XM
> 64
best way to avoid platelet refractoriness
pt HLA ABS and ABID
absence of B * 27
ankylosing spondylitis
presence of DQ6
narcolepsy
absence of DQB1 * 02
celiac
presence of DRB1 * 03/04
T1DM
acute noncardiogenic bilateral pulmonary edema
TRALI
cause of TRALI
donor HLA or HNA Ab fix complement and cause tissue damage leading to capillary leakage and pulmonary edema
TRALI prevention (3)
- use male plasma/platelets
- use female plasma/platelets = for HLA Ab
- donors implicated in TRALI deferred
organization charged with development of an equitable scientifically and medically sound organ allocation system
united network for organ sharing
established to meet the needs of pateints who do not have a matched, related CTP donor
national marrow donor program
UNOS responsibilities
- maximize use of organs
- ensuring quality of care
- addressing ethical issues
- evaluates potential donors
- obtains appropriate consent
- prepares organs for transportation
goal of NMDP
recruit a large number of HLA-typed volunteers to be listed as potential donors
establish national coordinating center for facilitating donor searches
NMDP uses ——- to assist in targeted recruitment of volunteer CTP donors
geo-coding
serologic testing to prevent disease transmission for all donors of tissue
- all testing required on blood donors
- pretransfusion specimen preferred
ABH antigens are concerns for ——- transplantation because they are expressed on vascular endothelium (5)
kidneys
heart
lungs
livers
pancreases
ABH not important in these tissue grafts (5)
fascia
bone
heart valve
skin
cornea
goal of CTP transplant HLA match
6-antigen match
A, B, DR most important
required before kidney & pancreas transplant
ABO compatibility
HLA Xm
required before liver, heart, lung, and heart-lung transplant
ABO compatibility
HLA XM only if recipient has a presensitization
shown to tolerate ABO-mismatched heart and liver
pediatric patients with weak isoagglutinins
tx acute rejection of heart or kidney
plasmapheresis
immunosuppressive therapy
ECMO
function
extracorporeal membrane oxygenation
heart-lung machine removes Co2 and oxygenates blood
ECMO indications
allows heart, lungs to rest and heal
severe illness of heart, lungs
waiting for or recovering from heart transplant
ECMO pts require…
a current TS
4-6 units XMed available at all times
often transfused with 1 platelet and 2-5 pRBCs per day
reduce risk of developing HLA Ab
leukocyte reduction
BB support during liver surgery
preparedness, supply and responsiveness during 6-12 hr surgery
blood loss and hypocoagulability occur during liver transplant because…
preexisting liver disease (coag factors)
ahepatic & early reperfusion periods
products often used during liver transplant
6 pRBCs, 6 FFP, 2 platelets
why blood product use has declined during liver transplant (6)
- preop iron
- erythropoietin therapy
- surgical technique
- interop blood recovery
- antifibrinolytic drugs
- acute normovolemic hemodilution
CTP contain…
hematopoietic progenitor cells (HPC)
or
stem cells
2 stem cell populations in CTP
- capable of self-renewal and differentiation into all blood cell lineages
- committed to certain blood cell lineage
HPC are infused after…
lethal doses of radiation or chemo, killing existing marrow
does not require a 6/6 match
cord blood
naive
cord blood CTP indications
- tx blood diseases in pediatric pts
- second choice for adults lacking HLA-matched donor (may need 2)
autologous CTP transplant indication
malignancies without marrow involvement (ie lymphoma) and metastatic or recurrent solid tumors
allogeneic CTP transplant indication
CML, AML, ALL, aplasic anemia, malignancies with marrow involvement, hemoglobinopathies
newly developing autologous CTP method
chimeric antigen receptor T-cell therapy (CART)
preventive measures for GVHD
- irradiation of blood products
- HLA matching
- T-cell depletion (CTP)
- immunosuppressive drug therapy
subtype of GVHD that occurs when lymphocytes from donor make Ab to recipient RBC Ags, causing hemolysis
passenger lymphocyte syndrome (PLS)
PLS onset
1-3 weeks post transplant
transient DAT+
chimerism
persistence of donor cells in allogeneic HSCT recipients demonstrating transplant survival
can indicate CTP graft rejection
lack of/reduced presence of donor cells
allows for analysis of CTP genomes to detect graft rejection
short tandem repeats
AFDC
aid to families with dependent children
reasons for parentage testing
- divorce/custody disputes
- welfare laws/child support
- inheritance questions
- questioned parentage (switched at birth, adoption, rape/incest cases)
necessary steps for parentage testing
- allegation
- sample collection
- chain of custody
- selection of genetic markers
- testing
with antigen parentage testing, children must be —- old
6 months
element of chain of custody (6)
- positively ID, photograph, fingerprint individuals
- proper labelling
- documentation in writing of everyone who handles samples
- all test performed in parallel by 2 techs using 2 lot numbers
- samples locked up when not in use
- reports sent only to ordering physician/court
requirements for genetic markers chosen for parentage testing (6)
- polymorphic
- inherited by simple mendelian genetics
- well developed in children
- not affected by environment
- objectively interpreted
- remain constant through life
4 methods of parentage testing
- RBC antigens
- HLA markers
- enzyme techniques
- DNA fingerprinting
ABO grouping can exclude —–% fathers
73%
BG systems used for parentage testing
ABO
Rh
Kell
Kidd
Duffy
MNSs
problems with RBC parentage testing (10)
- null types
- modified types
- allelic alterations
- weakened Ag expression
- other alterations (cis AB, mosaics, chimera, bombay)
- recent transfusion
- DAT+
- polyagglutination
- ABO subgroups
- acquired B
HLA can exclude —–% fathers
90
parentage test not run in duplicate
HLA
any apparent exclusions repeated from original sample
HLA
problems with HLA in parentage testing (4)
- large number of lymphocytes needed
- rare antisera
- highly technique-sensitive typing
- unknown allele may be mistaken for homozygous state
red cell enzymes tested for parentage (5)
- phosphoglucomutase (PGM)
- esterase (ESD)
- glyoxylase (GLO)
- group-specific component (GC)
- acid phosphatase (ACP)
serum proteins tested for parentage
function
transferrin and haptoglobin
can be used to distinguish siblings
DNA methods used to test for parentage
- restriction fragment length polymorphism (RFLP)
- PCR
RFLP
DNA is harvested from ———-
isolation and purification of DNA is through a process of…
WBCs
cell separation, lysis, enzyme digestion, precipitation of protein with NaCl
RFLP
cut DNA strands at the same sequence every time
restriction enzymes
RFLP
polymorphism results from individual differences in the sequence of —— and —–
VNTR (variable number tandem repeats)
STR (short tandem repeats)
RFLP
separation of DNA fragments (3)
- separated by size in a gel electrophoresis
- negatively-charged DNA fragments move toward anode side
- smaller fragments move farther along
RFLP
interpretation of electrophoresis (5)
- Southern Blot used to transfer DNA bands from gel to membrane filter
- the DNA is denatured to ssDNA
- membrane dried and baked to permanently fix DNA to membrane
- radioactively labelled probes bind the bands, and unbound probe is washed
- x-ray is used to visualize
RFLP
if an AF isn’t exlcuded…
a paternity index is calculated based on gene frequency in general pop
RFLP
required (7)
- good QC of restriction enzymes
- standardized controls
- standardized electrophoretic separation
- population data sufficient to accurately assess gene frequency
- standardized probes
- standardization of nomenclature of probes
- publication of genetic probe data and family studies to assure genetic inheritance patterns
must be used in conjunction with another parental testing method, usually ABO
RFLP
PCR fragments can be visualized ——- on the ———- gel with a ——–
directly
agarose
silver stain
standard sample containing each of alleles run with samples, for direct visual comparison
PCR
RBC excludes —-% fathers
73
RBC + HLA exludes ——% fathers
95
RBC + HLA + enzymes excludes —–% fathers
99
RBC + DNA excludes ——% fathers
99.9
direct exclusion
the child possesses a factor not present in the mother, or the AF
POG
factor the child possesses but the mother does not
indirect exclusion
child lacks a factor that the AF would’ve had to pass on (homozygous)
likelihood of paternity based on —— principle, which states:
Hardy-Weinberg
selection of mates is random, and genetic markers are passed to offspring in a random manner
probabilty calculation in a 2-allele system
p^2 + 2pq + q^2
basis for calculation of the likelihood of paternity
POG
genetic odds ratio which reflects how much more likely it is that an included AF is the father than a random man of same race
paternity index
PI = x/y
x = odds of AF passing on POG
y = gene frequency of POG
will be 0 if any PI value is 0
CPI
combined paternal index
product of all individual system PI values
converts PI into a percentage
likelihood of paternity
W = PI/(PI + 1)
