BLD 435 FINAL
endogenous - cancer or viral
MHC class 1 (on all nucleated cells) presents what type of antigen?
class 1 - A,B,C,D,E,F,G class 2 - DQ, DP, DR, DM
MHC class 1 and class 2 genes
exogenous - bacterial, parasite, etc.
MHC class 2 (on APC's - B cells, dendritic cells, macrophages) presents what type of antigen?
store at 1-6 C the "storage lesion" refers to changes in RBC's/blood during storage - glucose decreased - decreased 2,3-BPG - decreased ATP - increased potassium/K+ - decreased pH
what temp to store whole blood at? what happens to blood in storage?
ABO forward and reverse Rh D testing, including weak D unexpected AB screen bacterial/viral serology (Ag/Ab detection)
what testing does the collection site do?
look at history ABO and Rh type do unexpected Ab screen
what testing to do to predict if HDFN will occur?
peptide antigen (9-15 AA) antigen must be complementary to the MHC anchor motif (AA sequence in binding pocket) - what determines what antigen can be presented by that MHC or not!!
what type of antigen is presented in MHC? what does the antigen bind to in MHC?
type 2
what type of hypersensitivity is HDFN?
contaminated blood transfusion reactions are DELAYED examples: HIV Hepatitis HTLV CMV EBV WNV Dengue Parovirus Rabies Prions Malaria Syphilis Lyme disease Parasitic worms
what type of transfusion reaction occurs with a pathogen-contaminated unit? what are some examples?
HIGH - bone marrow LOW - solid organs
what types of transplants need to be high resolution (more closely HLA matched) and which can be low resolution (less closely HLA matched)?
donor has an auto-antibody/sensitization happening in their own body by their own antibody! would expect a positive DAT
what would a positive auto-control indicate?
since sensitization and hemolysis can be caused by IgG or C3 binding, we need to know which one AHG that binds only IgG will tell us if sensitization is caused by antibody or complement!
what would be the benefit of using monospecific AHG?
whenever you suspect there is a transfusion reaction occurring!! BE QUICK DON'T DELAY
when do you call for a transfusion reaction work up?
WBC's in the donor blood get stimulated by different MHC proteins on the recipients RBC's, and they can mount an immune response against the recipient MHC ** MHC has to be a match between donor and recipient for a graft!! or can give leukocyte reduced blood (no donor WBC's to recognize recipient MHC as "foreign")
why can a recipient get Transfusion Associated Graft-Versus-Host disease?
** these people are sick, hemolysis could be the reason they are in the hospital OR the drug they are prescribed could cause it OR something else they put into their body
why does it make sense that 15% of hospitalized people will have a +DAT, but only 0.1% of a random/non-hospitalized population?
1) fetal RBCs express less of the ABO blood group antigens compared with adult levels 2) Fetal RBCs express antigens A and B poorly, making them less of a target, whereas the endothelial and epithelial lining of the placenta expresses A and B antigens profusely, potentially clearing IgG from circulation and lowering the titer within fetal circulation (whereas Rh antigen is only on RBC's, making them more of a target!)
why is ABO HDFN less severe than Rh HDFN?
AHG will bind soluble Ag a lot faster than fixed Ag on RBC surface, can give us FALSE NEGATIVE how do we check we got a true negative? CHECK CELLS (IgG coated RBC's) add check cells to any negative AHG result - if clump forms with check cells, means AHG was free floating and not bound to any Ab, so it could bind check cells - this was a true negative! - if no clump forms with check cells, means AHG bound free Ab and you didn't wash well enough - this was a false negative!
why is is so important to wash the cells before adding AHG in a DAT/IAT?
transfusion related immunomodulation (TRIM)
** every transfusion modulates the recipients immune system (suppresses it)
Hydrops fetalis
Heart failure and generalized edema in multiple body sites in the fetus caused by severe anemia resulting from destruction of RBC's usually results in miscarriage
lower bilirubin in infant blood remove sensitized RBC's provide compatible RBC's reduce level of maternal antibody in the infants blood ** this is essentially a big dilution! won't fix all the problems, but will definitely help
what are the goals of an exchange transfusion (after birth)?
OLD: serology NEW: molecular (SSP, SSO, PCR, sequencing)
what are the old and new ways for HLA typing?
febrile non-hemolytic transfusion reactions neonatal platelet problems platelet refractoriness **platelets are chunks of RBC's, so they do contain some RBC antigen on their surface!
what kind of problems to anti-HLA antibodies cause?
citrate !! or any calcium chelating compound because Ca++2 is needed to activate complement (so complement can't get activated and destroy RBC's in bag)
anticoagulant used in blood bag?
if the clinically significant unexpected Ab titer is 1/16 or higher then monitor over the course of pregnancy - if titer increases 2 tubes/4 fold = ACTIVE IMMUNIZATION OCCURING
based on the titer, when should you be concerned HDFN might be present?
NO
can you predict what type of HDFN it is based on the strength of the DAT?
YES - hemolysis can be happening but sensitization is not enough where we can detect it with the AHG test can do an elution to run off the antibody if you suspect it
can you still have AIHA if you have a negative DAT?
DAT and IAT
coomb's test is the same as what?
1) adsorption drug absorbs onto RBC surface, anti-drug antibody is made which binds drug, see a positive DAT 2) immune complexes drug:anti-drug complexes form and activate complement - the complexes are then coated w complement - RBC's just passing by also get coated with complement by chance and end up lysed 3) non-immune adherence drugs alter RBC's, make them more sticky = more proteins (including antibody) stick to their surface, but they aren't bound to a specific antigen! 4) autoimmune drug truly alters RBC antigen and an antibody is made that targets that RBC antigen
drug induced AIHA mechanisms
allergic febrile TRALI TACO
examples of AHTR's?
Polygeny
expression of multiple different genes at once
Rouleaux
false positive caused by cells stacking because of high protein how to resolve it? use saline replacement technique
1) T negative/B negative likely no anti-MHC antibody against those isotypes! 2) T positive/B positive OR T negative/B positive can't know for sure if it is anti-class 1 or anti-class 2 because B cells express both class 1 & 2 while T cells only express class 1 3) T positive/B negative not an HLA antibody, not sure what the specificity is
how are the results from the HLA crossmatch flow cytometry interpreted?
IAT (indirect antiglobulin test) mix sample cells with known serum OR mix sample serum with known cells incubate, wash, add AHG, spin, look for agglutination
how do we detect in vitro sensitization?
DAT (direct antiglobulin test) make 3% cell suspension, wash, add AHG, spin, look for clump
how do we detect in vivo sensitization?
Intrauterine transfusion (IUT) - through umbilical cord or into fetal abdominal cavity
how do you help the fetus that can't be taken out of the womb because it is still to young?
IgG anti-D - also called "RhoGAM" if it is suspected the baby could be Rh positive and mom is Rh negative, one dose of RhoGAM will be given at 28 weeks of pregnancy. If your baby is found to be Rh positive at birth, mom will receive an additional dose within 72 hours after delivery (to help protect future pregnancies!)
how do you prevent mom from making antibody in the first place (to prevent HDFN)?
blood must be: - antigen negative for moms antibody - crossmatch compatible with baby cells sample: maternal serum (highest concentration of Ab), or can use eluate from cord sample
how do you select blood for intrauterine or exchange transfusion? what sample do you use?
HLA presents antigen! these present foreign but also self antigen if your HLA are more or less likely to present a certain self antigen (based on what alleles you have = what you can fit in the pocket), that can affect the likelihood your immune cells will mount a response
how does HLA play a role in chronic disease and autoimmune disease?
RhoGAM prevent NAIVE D-antigen sensitive B cells from becoming activated by killing them - prevent any antibody or memory B cells from being made!
how does RhoGAM work?
1) mix donor lymphocytes (that have the MHC isotypes) with the recipient serum (could have anti-HLA antibodies) incubate these together add conjugated anti-HLA AHG, add conjugated anti-CD3 and anti-CD19/20 (to differentiate B and T cells) do flow cytometry !!! 2) virtual crossmatch donor is typed so we know what MHC they have recipient unacceptable are listed (MHC isotypes they have antibody against) compare these two!!
how is donor/recipient HLA crossmatch done? (2)
1) perform Rosette test (qualitative) - use moms blood (lots of Rh negative cells, few Rh positive cells from fetus) and incubate it with anti-D -- then incubate this mixture with D positive cells, see "rosettes" - all bound together more rosettes = more fetal blood present = more fetal-maternal hemorrhage 2) Kleihauer-Betke acid elution stain or flow cytometry (quantitative)
how much RhoGAM to give to mom after birth? ** rule of thumb: 1 dose RhoGAM for every 30 mL of fetal RBC's in mom (if you get decimal, <.5 round down, >.5 round up)
1) amniocentesis (colorless = healthy, color = HDFN) 2) spectrophotometric exam to detect bilirubin
how to confirm HDFN during pregnancy?
Complement-dependent cytotoxicity (CDC) ELISA flow cytometry
how to determine the specificity of HLA antibodies?
evaluate for hemolysis by: hemoglobin (↓) hematocrit (↓) bilirubin (↑) haptoglobin (↓) reticulocyte count (↑)
if DAT is positive, what do you do next?
collect cord sample (EDTA) measure bilirubin and hemoglobin to evaluate severity (if bilirubin is too high , do an exchange transfusion) do ABO and Rh typing, perform DAT (if DAT is positive, eluate and ID the antibody)
if you suspected HDFN during pregnancy, what do you do once the baby is born?
Autoimmune Hemolytic Anemia (AIHA)
immune based (antigen-antibody) hemolytic anemias with intravascular or extravascular hemolysis
intravascular: complement coated RBC's - lysis by MAC complex extravascular: antibody (IgG) or complement (C3) coated RBC's get lysed by macrophages in spleen or liver
intravascular vs extravascular hemolysis mechanisms
fresh frozen plasma (FFP) stored at -18 C for 12 months prepared within 8 hours of collection thaw at 30-37 C in water bath
most common type of plasma given? storage/shelf-life/thawing requirements?
prepared by centrifuging harvested within 8 hours of collection stored at room temp for 72 hours with agitation --> shorter shelf life because storing at room temp is more susceptible to bacterial growth!
platelet storage and preparation?
primary - Ag:Ab binding is the problem secondary - Ag:Ab complex caused by an underlying health problem (ex. SLE) is the problem
primary vs secondary AIHA
decrease Na+ (LISS) - less Na+ allows more IgG to bind, which will decrease the overall net negative charge as more binds, and ultimately reduce zeta potential so cells can agglutinate! remove negative sialic acid (Ag) on RBC surface with proteolytic enzymes - but then there is less Ag for Ab to bind to suspend cells in albumin
the net negative charge between two RBC's impedes agglutination! two negatives don't want to be next to each other in the clump! the distance between the two cells is called "zeta potential" IgM is so large it can connect the two cells, but IgG is small and in order for it to bind to two cells, the zeta potential must be reduced, HOW?
Polymorphism
the number of variants (alleles) for a given gene
DECREASE ionic strength more ions around = higher ionic strength = less room for Ab to bind! how do you do this? use LISS (low ionic strength saline) - less ions than regular saline! get faster and more efficient antibody uptake
to maximize Ab:Ag reaction strength, should you increase or decrease ionic strength?
Mixed field agglutination
two populations of cells - get weird graded reaction because some cells react and some don't
warm AIHA (usually anti-Rh), seen in SLE or lymphoma cold AIHA (usually anti-I), lymphoma, infectious mononucleosis mixed cold and warm (usually anti-I) paroxysmal cold hemoglobinuria (PCH) (anti-P), syphilis or viral infection drug induced (anti-Rh)
types of AIHA
Kernicterus
unconjugated bilirubin buildup in the brain
platelet concentrates are from a single donor and can be prepared from whole blood (less concentrated) or platelet apheresis (more concentrated) random - platelets selected based on ABO and Rh only typed - platelets selected based on HLA also people become refractory (unresponsive) to platelet therapy and it stops being effective - this is because the recipient starts to make antibody against platelet antigens (HPA) or HLA antigens SO you want to start with random platelets first then switch to typed once they become refractory! this will prolong the window in which platelet therapy can be used effectively
what are platelet concentrates? what does "random" and "typed" mean? should you use a random or typed platelet concentrate first in a platelet donation? why?
auto-antibody allo-antibody (Ab made from exposure to foreign antigen, such as from transfusion or pregnancy) anti-drug antibody binding to drug altered RBC surface heterophile Ab non-immune binding of protein (NIPA)
what are possible causes of a positive DAT?
antigen testing compatibility testing antibody screens
what are some uses of an IAT?
renal maturity = creatinine (> 2.0) pulmonary maturity = lecithin/sphingomyelin ratio (> 1.8)
what are the criteria that need to be met to deem the fetus "mature" enough to be removed from the womb early (in cases of severe HDFN)?
chills, fever, vomiting, diarrhea, septic shock
what symptoms do you see with bacterial contamination of a blood unit?
1) urticaria (hives) - itching, not life threatening 2) anaphylactic - life threatening!! occurs if blood has a component they don't have (ex. if the recipient does not have any IgA, and gets blood with IgA, they make anti-IgA IgE which activates mast cells next time they are exposed to IgA!) CAUSE: allergy-causing antigens within the donor's blood, or transfusion of antibodies from a donor who has allergies, followed by antigen exposure
what are the two types of allergic transfusion reactions? what causes these allergic reactions?
1) ABO - common but mild, does occur in first pregnancy since mom already has these expected antibodies 2) Rh and other unexpected antibodies - less common but much more severe, usually does not cause problems until second pregnancy since first was the primary response (IgM mostly) and second is secondary response (IgG mostly) -- unless mom had a transfusion before and was exposed to D antigen!!
what are the types/severity of HDFN?
fresh O cells that are negative for the antigen mom's antibody is against AB plasma or 6% albumin less than 7-14 days old, CMV (-), HbS (-), irradiated
what blood product should be given to the fetus/baby in an intrauterine or exchange transfusion?
ask yourself: 1) is the Ab clinically significant? 2) will this Ab become clinically significant (is it her second or beyond pregnancy?) 3) how much antibody is there? do titer!!
what do you do if you get a positive unexpected antibody screen on a pregnant woman?
stop transfusion, keep line open with NACL evaluate for signs of hemolysis, anaphylaxis, TRALI, TACO, sepsis --> if you do see signs = get blood sample, get the blood bag used in transfusion, repeat ABO look for hemolysis, perform a DAT, check hemoglobin/haptoglobin *** if there is hemolysis, means the response is IMMUNE MEDIATED if all is negative, probably not hemolytic and proceed as normal if ANYTHING is positive, repeat typings, Ab screen, crossmatch using recipient sample and blood bag
what do you do if you suspect a transfusion reaction?
Transfusion Associated Circulatory Overload (TACO) volume overload - too much blood given this is a concern for patients with cardiac problems (poor circulation), pulmonary disease, anemia, infants
what does TACO stand for?
Transfusion Related Acute Lung Injury (TRALI) sudden acute respiratory distress following transfusion severe shortness of breath (from pulmonary edema), fever and low blood pressure cause? cytokine overload from storage blood bag, anti-HLA antibodies (that causes inflammatory response in the patient and create cytokines anyway!) donor plasma contains antibodies that cause damage to the immune cells in the lungs. This lung damage results in fluid buildup in the lungs and can severely limit the ability of the lungs to supply oxygen to the body. occurs within six hours of receiving blood
what does TRALI stand for?
repeat ABO forward and reverse, repeat Rh on negative units by doing an immediate spin (don't need to recheck weak D)
what does the blood bank do once blood gets there from the collection center?
pregnancy transfusion vaccination disease previous graft
what events lead to sensitization of WBC's and production of anti-HLA antibodies?
too much iron in the body! RBC's contain a lot of iron, and getting a transfusion introduces lots of iron to your body who is at risk? 1) people who get lots of transfusions 2) someone who got a massive transfusion 3) severely anemic patients
what is "iron overload"? who is at risk?
PRA = percent per panel of reactive antibody, tells us how likely you are to find a matching donor for a given recipient you mix the recipient serum with a lot of different donor cells, that all have various HLA - see how reactive the serum is more reactive = antibody against a higher incidence allele = harder to find a donor match ex. PRA = 20% means 20% of donors are incompatible HIGH PRA = less likely to find a donor LOW PRA = more likely to find a donor
what is a PRA? how is it done and what does it tell us about chances of finding successful donor with correct HLA matching?
someone born with the hh phenotype H is the base that is used to make A and B antigens (enzymes associated with these genes convert H to A or H to B, type O makes no enzyme and has lots of H) bombay has NO ABO antigens!!
what is a bombay?
Ag and Ab concentration antibody affinity cold (IgM) or warm (IgG) reacting antibody pH temperature ionic strength incubation period
what is an antigen:antibody reaction affected by? and therefore what is sensitization affected by
mix patient cells (unknown Ag) with known antibody
what is the ABO forward type?
mix patient serum (unknown Ab) with known cells
what is the ABO reverse type?
the auto control (A/C) is patient cells mixed with patient serum should always be NEGATIVE
what is the auto control? what is the expected result?
recipient makes/has antibody against platelets = they are removed and nothing there to stop the bleeding!
what is the cause for post-transfusion purpura (bruising?)
delayed reactions are the secondary/anamnestic response! usually caused by recipient having an unexpected antibody at a very low titer that went unnoticed extravascular hemolysis not deadly, but blood was wasted since it got hemolyzed
what is the cause of DHTR's?
HLA genes are HIGHLY POLYMORPHIC, so most people are heterozygous (have different alleles) at each gene - 6 genes total (A, B, C, DR, DQ, DP) HLA genes are inherited as haplotypes - move across generations as "blocks" (called linkage equilibrium) a child will get a full haplotype (6 alleles) from both mom and dad, so 12 total alleles!! most of the time these alleles are all different, so they end up with 12 isotypes of MHC!
what is the inheritance pattern for HLA genes? how many different HLA genes (and therefore HLA isotypes) does a person usually have?
Febrile nonhemolytic transfusion reaction (FNHTR) very common, not usually severe, NO hemolysis observed symptoms: fever and chills cause? anti-WBC or anti-platelet antibodies in the recipient, or WBC/platelet cytokines that accumulated while the blood was in storage
what is the most common type of transfusion reaction? what causes it?