BLD 435 FINAL

3. Discuss and also compare and contrast the following AIHA types: DAT-neg

1^o 2^o: SLE, lymphoma Ab is below threshold of detection but is still not below immune system so causes anemia

11. Define HLA identical, two haplotype match, one haplotype match and what are the odds of each within a family.

HLA identical: all alleles match perfectly - seen in between identical twins - Two siblings have a 25% chance of being genotypically HLA identical two haplotype match: (DOUBLE CHECK) one haplotype match: - a 50% chance of being HLA haploidentical (sharing one haplotype).

2. Determine the applicability of further testing based on history and diagnosis.

pregnancy and transfusions - altered RBC - drug:anti-drug - heterophile - NIPA (non-immune binding of protein)

Explain all pre-transfusion testing needed

ABO and Rh Typing - The patient's ABO grouping is performed by ascertaining concordance with both cell typing (forward grouping) and serum testing (reverse typing). - Testing two samples collected at different times is required before finalizing the ABO typing report. - Rh D typing is performed by testing the recipient's red cells with anti-D sera. Weak D testing is not required in the RhD-negative samples unless the recipient is a neonate born to RhD negative mother. Antibody Screening - These are serological tests designed to detect clinically significant antibodies to blood group antigens using an Indirect antiglobulin test (IAT). Antibody identification - If the screening is positive, the next step is to identify the specificity of the antibody and for which an extended panel of unpooled reagent red blood cells is used (11-20 cell panel). - The specificity of the antibody is determined based on the reactivity pattern of the antibody against the cell panel. - Additional testing strategies may be required, namely the use of enhancement media (albumin, polyethylene glycol, low ionic strength solution, or chemical/enzyme treatment of the panel cells to aid identification.) Antigen phenotyping: - Whenever an antibody is identified in a patient, confirmation can be done by phenotyping the recipient for the corresponding antigen to confirm he is negative for that antigen. - Antigen phenotyping also needs to be performed on the donor units to select negative units for the antibody identified in the patient or to give phenotype-matched blood as in thalassemia or pregnancy. Compatibility Testing Immediate spin method: The recipient serum/plasma is mixed with saline-suspended donor cells (major crossmatch) and vice-versa (minor crossmatch) at room temperature. Immediate spin can be the only crossmatch method when the recipient does not have current or previously detected clinically significant antibodies.[6] Antiglobulin crossmatch: In the liquid phase, i.e., tube method, 2 to 5% saline-suspended red cells of the donor unit are mixed with recipient serum and incubated at 37 C for about 45 minutes and washed to remove unbound antibodies. The AHG serum is added to the remaining cell button after washing, centrifuged, and read for agglutination. This can also be done using CAT or solid-phase systems. Computer / electronic crossmatch: In this method, the patient is screened for antibodies and entered into the electronic system, which contains all the donor units in the inventory with their phenotype reports. The system picks the compatible units for the particular patient and displays them from the inventory; compatibility may be verified by computer. Electronic crossmatch can be used as the sole method only after ensuring that the recipient has no clinically significant antibodies present historically or currently. However, the validation of the system is to be done on-site. The system should recognize and correlate anti body detection results, compare previous records, concordant ABO on the recipient from at least two determinations, donor component and unit number, and ABO/Rh retype results, as well as have logic to alert the user to discrepancies between donor unit labeling and confirmatory test interpretation, and ABO incompatibilities between the recipient and the donor unit

6. Define linkage disequilibrium

Conversely, alleles are in linkage disequilibrium (LD) when they do not occur randomly. Under linkage disequilibrium, haplotypes do not occur at the frequencies expected when the alleles were independent.

9. Define what the term CREG and cross-reactive groups.

Cross-reactive groups (CREGs) are defined by cross-reactive antibodies that react with different HLA-A and -B antigens with shared epitopes. In the context of renal transplantation, it has been reported that matching for CREGs results in improved graft outcome and reduces the requirement for rejection treatment.

3. state how HLA can influence platelet transfusion and the selection of platelet products

HLA match between donor and recipient: single donor apheresis units used when recipient is refractory to concentrates - characterization of antibody profile: + known which antigens to avoid

20. state indications for RhIg use other than pregnancy.

Immune thrombocytopenic purpura (ITP) - Rh(D)-positive, non-splenectomized: +++ Chronic ITP in children and adults +++ Acute ITP in children +++Improving platelet counts secondary to HIV infection Rh isoimmunization suppression - Rh(D)-negative, non-sensitized patient with Rh-incompatible fetus +++ Prophylaxis in the following clinical situations: +++ Routine antepartum and postpartum mothers +++ Obstetric complications (ectopic pregnancy, threatened abortion, fetal demise in second or third trimesters) +++ Spontaneous or induced abortions +++ Following invasive obstetric procedures [1] +++ Abdominal trauma involving fetal-maternal hemorrhage Incompatible transfusions of Rh(D)-positive RBCs in Rh(D)-negative individuals

35. explain how ABO incompatibility between mother and fetus may provide protection against development of HDN due to other blood group antibodies.

In ABO-incompatible, D-negative mothers, the alloimmunization rate for the D antigen is markedly diminished, thus demonstrating a partially protective effect of ABO in-compatibility.

4. Discuss the following mechanisms of drug induced AIHA, list common drugs for each type and how to prove that it is the causative agent: Autoimmune

alpha-methyl dopa (aldomet) - get IgG directed to RBC usually anti-Rh maybe anti-e if drug alters RBCs, you make Ab against your RBCs

3. Discuss and also compare and contrast the following AIHA types: warm

WAIHA 1^o: a problem 2^o: SLE or lymphoma (problem with something else causing warm autoimmune hemolytic anemia) specificity: usually anti-Rh ocasionally anti-hr' normally all Rhs

3. State what a parent antigen is and how "splits" relate.

Linkage equilibrium - Antigens occur in the same individual more frequently than the frequency of the antigens in the population. Conversely, alleles are in linkage disequilibrium (LD): Where alleles (DNA markers) occur together more often than can be accounted for by chance because of their physical proximity on a chromosome. Also called LD. HLA alleles have linkage disequilibrium: Don't get a lot of recombination they move as blocks A1 - 10% (0.1) B7 - 10% (0.1) So A1, B7 should be (0.1 x 0.1 = 0.01) 1% In reality you see it 6% - way higher

6. describe the pathophysiology of HDFN.

Maternal immunoglobulin G (IgG) antibody crosses the placenta into the fetal circulation, where it binds to fetal red cells or erythroid pre-cursors. The immunoglobulin subclasses IgG1 and IgG3 are more likely than IgG2 or IgG4 to cause early and/or severe hemolytic disease.'2 The resulting increased hematopoietic drive causes a condition termed erythroblastosis feta-lis, with liver and spleen enlargement secondary to extramedullary hematopolesis, and portal hy. pertension. Liver enlargement can lead to decreased production of albumin and associated decreased plasma oncotic pressure, with generalized edema, ascites, and effusions known as hydrops fetalis. Severe HDFN can occur as early as 18 to 20 weeks' gestation or earller with antibodies to K1 (of the KEL system), but may be difficult to detect; severity usually increases in subsequent pregnancies. Untreated, hydrops fe-talis, with its associated high-output cardiovascular failure secondary to anemia, can lead to fetal death. The destruction of red cells also leads to elevated bilirubin levels. After birth, the infant's immature liver enzymatic pathways cannot metabolize the unconjugated bilirubin, which can increase to dangerous levels. The hyperbilirubinemia can cause permanent brain damage, known as kernicterus.' The maternal antibody typically decreases in the neonate over 12 weeks, with a half-life of about 25 days. Some antibodies may cause more prolonged anemia or delayed-onset anemia.

11. describe how to identify the specificity of the antibody causing HDFN.

May want an: unexpected antibody identification panel may want to eluate and then a panel to ID causative antibody specificity if DAT is positive, eluate evaluate for hemolysis: - hgb/hct - bilirubin - haptoglobin - retic count (if no evidence no transfusion is needed) if there is evidence of hemolysis ask: - recent transfusion - Ab screen info - are they on drugs -- if possible transfusion reaction work it up -- all alloimmune should get full attention ——————————————————————————————————— During the fist pre natal visit, the maternal ABO and RhD type and an antibody screen to detect IgG-phase antibod. jes 137 C with antihuman globulin (AHG) should be performed. If a D-negative woman does not have and-u, she is a candidate for RhIG administration to prevent RhD allolmmu itation (see below). A positive antibody screen repuires antibody identification and titration. antibodies against minor carbohydrate blood group antigens such as anti-1, -PI, -Le', and -Le", whether IgM or IgG, do not cause HDFN and may be ignored because these antigens are poor. y developed at birth. Homozygous fathers have a 100% chance of offspring expressing the red cell antigen; het erozygous fathers portend a 50% chance. During an alloimmunized pregnancy, monthly to biweekly maternal antibody titers can be used to deduce if the fetus is the source of ongoing maternal immune stimulation and is at risk for clinically significant HDFN. Once an antibody titer reaches a level above which clinically significant HDFN could occur (the critical titer), further titration may not be of benefit and noninvasive clinical monitoring for fetal anemia should commence.?3 The AABB-recommended method for titration is a saline AHG test incubated for 60 minutes at 37 C

2. state the two events that may lead to immunization of women.

Mom immunized during first pregnancy or by transfusion ABO seen in first pregnancy Second and then subsequent pregnancies get more severe. ABO can always be mild

1. state the rational for performing unexpected antibody screen on pregnant women.

Mom makes antibody to antigen on fetus rbc's she doesn't have mom's IgG binds to fetus rbc's, rbc's destroyed. need to be replaced and mess cleaned up Generalizations: ABO common but mild weak positive DAT Rh and other: rare but more severe and stronger DAT

3. Discuss and also compare and contrast the following AIHA types: PCH

Paroxysmal Cold hemoglobinuria 1^o 2^o: syphilis, viral infections specificity: anti-P

19. determine candidate for RhIg prophylaxis given serological information and history on the mother and neonate.

RhIG is Indicated after any event that increases the risk for FMH. D-negative females who have been previously Immunized to the D antigen, D positive females, and D-negative females whose infants are known to be D negative are not candidates for RhiG. Women with apparent antibodies to both D and C should be investigated for presence of ant-G before determining their candidacy for RhIG. Unless specific reactivity to D is demon-strated, a pregnant D-negative female with ant.G should receive RhIG.

4. Recognize and diagram the following molecular based methods for HLA typing: PCR-SSOP

Sequence Specific Oligonucleotide Probe: - mostly performed using microbead array (luminex) Sequence-specific oligonucleotide probes (SSO or SSOP) use arrays of labeled oligonucleotide probes to detect HLA nucleotide sequences present in immobilized DNA." Reverse SS0 (rSSO) has been widely adopted and uses probesindividually attached to a solid-phase matrix (for example, each probe may be attached to a differ. ent microbead). DNA from a target locus is then amplified and labeled in PC reactions, and the binding to the different probes is evaluated. Commercially available microbead array assays use rSSO methods for HLA Class I and Class II low-to-high-resolution tissue typing and computer proprietary algorithms to match binding patterns to allele databases.

32. define a replacement transfusion.

Replacement transfusion if you think about it, is you're replacing something that's been lost. So this is really along the lines of standard transfusion practice. If someone needs RBC's or Plasma, or Platelets, or Cryo

4. Recognize and diagram the following molecular based methods for HLA typing: PCR-RT

Reverse transcription PCR, or RT-PCR, allows the use of RNA as a template. An additional step allows the detection and amplification of RNA. The RNA is reverse transcribed into complementary DNA (cDNA), using reverse transcriptase. The quality and purity of the RNA template is essential for the success of RT-PCR. The first step of RT-PCR is the synthesis of a DNA/RNA hybrid

1. list the main criteria used in organ allocation:

blood type and size of the organ(s) needed, time spent awaiting a transplant, the relative distance between donor and recipient, the medical urgency of the recipient, the degree of immune-system match between donor and recipient whether, the recipient is a child or an adult

23. calculate how many doses of RhIG are needed given the FMH in either mLs of WB or PC. 34. calculate the number of vials of Rh immune globulin needed when the FMH is given or when the number of fetal and maternal cells is given.

For example for 6 fetal cells out of 2000 cells, and an estimated maternal blo volume of 5000 mL, the FMH Is calculated to be 15 mL. The estimated FMH volume is used to calcu late the dose of RhIG. A 300-48 dose of RaG will suppress alloimmunization by 30 ml of fetal whole blood. In the above example, the FMH is 15 mL, so the number of 300-ug RhIG vials to administer can be calculated as 15 ml 30 mL RhIG/vial = 0.5 vial. the RhIG dose is rounded up to the next whole number if the number to the right of the decimal point is >5 (if <5, it is rounded down). An additional vial should be added to all calculations.

4. Discuss the following mechanisms of drug induced AIHA, list common drugs for each type and how to prove that it is the causative agent: Non-Immune Adsorption

drugs alter RBC such that all proteins stick including IgG ++ cephalosporins ++ see IgG and C3

25. state two reasons why a donor unit must be < 7 days old when it is used for neonatal transfusion.

ensure low K+ and high 2,3 DPG

if stumble upon a positive DAT during pre-tx testing

is anti-D cause false + due to a DAT most likely A/C is positive or Ab screen remember we want to use an EDTA sample: and we want monospecific AHG

22. state how a FMH can be quantitated, how a massive one can be detected and why it needs to be detected.

(Fetal cells/total cells counted) × maternal blood volume (mL) = FMH, whole blood (mL) It needs to be detectedThe estimated FMH volume is used to calculate the dose of RhIG. A 300-ug dose of Rhic will suppress alloimmunization by 30 mL of fetal whole blood.

3. State the advantages and disadvantages of molecular testing and serological methods of testing

(double check)

4. define PRA and CPRA

- PRA- percent/ panel reactive antibody...estimate of success in finding compatible donor at random in the population: of the 50 cells how many reacted - cPRA: calculated PRA, USA now uses cPRA + calculated using population frequencies for the unacceptable antigens listed - takes into account both class I and II specificites

4. Recognize and diagram the following molecular based methods for HLA typing:NextGen Sequencing

- being adopted/adapted for the HLA lab - adopted meaning few centers - Adapted meaning not done with traditional NextGen protocols • Traditional NextGen is a few hundred passes across whole genome • HLA is adapted by a few thousand reads across just the areas of interest con: * Expensive + takes a long time Levels of Molecular Typing: High Resolution = SBT + other methods Bone Marrow Low Resolution = SSP/SSO/Realime PCR Solid Organ Massively parallel sequencing ("next-generation sequencing," or NGS) has allowed for sequencing of whole genes and reduced the frequency of the ambiguities that occur with Sanger-chemistry SBT of HLA because NGS sequences single strands of DNA. HLA typing kits are commercially available for both clinical and re. search instruments.!* NGS methods obtain se-quences, from libraries, formed from fractured pre- or post-PCR cellular DNA. The very large number of sequences obtained allow for identification of overlapping sequences and arrange. ment of the resulting sequences through computer analysis (requiring very powerful processors, large sequence databases, and complex programming).

4. Discuss the following mechanisms of drug induced AIHA, list common drugs for each type and how to prove that it is the causative agent: Adsorption

- penicillin and cephalosporins + drugs absorb to RBC, anti- drug binds see positive DAT no pos Ab screen because screen ce;;s arent coated in penicillin - crossmatch is neg and screen cells are negative

4. Discuss the following mechanisms of drug induced AIHA, list common drugs for each type and how to prove that it is the causative agent: Immune complex mediated

- phenacetin - quinidine - 3rd generation: cephalosporins * drug and anti-drug interact and activate complement - may be reaction on rbc or in plasma ++++ not going to impact Ab screen and crossmatch

2. discuss immunization events that lead to HLA antibodies

- pregnancy - transfusion - vaccination - disease - previous graft

3. Discuss and also compare and contrast the following AIHA types: mixed

1^o: a problem 2^o: SLE or lymphoma specificity: anti-I antibody reacts with cold or warm

21. state the length of time RhIg remains circulating after administration and for how large a fetal-maternal hemorrhage (FMH) one dose will compensate.

30 mL whole blood and 15 mL packed RBCs

26. define neonate.

A neonate is also called a newborn. The neonatal period is the first 4 weeks (28 days) of a child's life. It is a time when changes are very rapid.

23. define intrauterine transfusion.

An Intrauterine transfusion (IUT) is a procedure that provides blood to a fetus, most commonly through the umbilical cord.[1][2] It is used in cases of severe fetal anemia, such as when fetal red blood cells are being destroyed by maternal antibodies. IUTs are performed by perinatologists at hospitals or specialized centers.[3] for intrauterine transfusion, the blood should be irradiated, cytomegalovirus (CMV), a reduced-risk. hemoglobin S negative, and generally group O, and less than 7 days old

24. define and describe the process of exchange transfusion and state the circumstances that justify its performance.

An exchange transfusion is a blood transfusion in which the patient's blood or components of it are exchanged with (replaced by) other blood or blood products.[1] The patient's blood is removed and replaced by donated blood or blood components. This exchange transfusion can be performed manually or using a machine (apheresis). exchange transfusion we mostly refer to neonates or sickle cell patients, where the process is literally exchanging the blood volume they have invivo with products from the blood bank. FOR INFANT: If doing an exchange transfusion decision based on: positive DAT cord hgb (decreased hgb) rate of rise in indirect bilirubin blood selection: - must be Ag negative for maternal Ig's - must be crossmatch compatible with moms serum (if not available use eluate form cord or neonate serum) Blood must be: - less than 7-14 days old to ensure low K+ and high 2,3 DPG - CMV neg - Hgb S neg - irradiated * institution policy FOR A SICKLE CELL PATIENT: - a sickle cell patient and in addition to their disease, they also have some RBC antibodies. So think about what that patient would need (non neonate).

7. state the class of immunoglobulin that is responsible for HDFN.

Antibodies that cause HDN are of the IgG class. The most common cause of HDN is ABO incompatibility, in which cases the haemolysis is usually mild. More severe cases of HDN can be caused by anti-D, anti-c and anti-K. CANT BE IgM because they don't cross the placenta

4. Recognize what Bw4, Bw6, DR51, 52, 53, DRB3*, DRB4* and DRB5* represent and how they are associated with other HLA antigens/genes.

Bw4 and Bw6 Bw4 and Bw6 epitopes are centered on α-1 α-helix amino acid residues 77-83, which have diverse amino acid sequences. HLA-DR51/52/53: donor-specific antibody (DSA) might be correlated with antibody-mediated rejection (ABMR). The unbound anti-HLA-DR51/52/53 might be more easily detectable than other DSAs after ABMR has begun. HLA-DR51/52/53 typing is recommended for donors and recipients in kidney transplant. HLA-DRB3 HLA-DRB3 affects type 1 diabetes risk and islet autoantibodies HLA- DR4 The HLA- DR4 gene was clearly associated with susceptibility to rheumatoid arthritis. HLA-DRB5 HLA-DRB5 was associated with the risk of systemic lupus erythematosus

5. evaluate given prenatal test results and offer a decision on the level of risk of HDFN.

Can we get the fetus/kid out????? look at creatinine for renal maturity: <1.5 mg/dI <35 weeks 1.8-2.9 mg/dl intermediate >2.0 mg/di go for It lecithin/sphingomyelin ratio L/S ratio for pulmonary maturity <1.5 immature 1.8 or > mature enough to go for it If you can't get it out transfuse it so it has some RBI's intrauterine transfusion may be through umbilical may just be IP use fresh O cells neg for offending antigen mom has antibody to and probably AB plasma or 6% Albumin less than 7-14* days old, CMV & Hgb S negative and irradiated *institution policy If no indication until birth or no emergency, evaluate when the kid hits the ground

6. describe a lymphocyte crossmatch in terms of target cells and reagents used

Donor cells/target: HLA expressed on lymphocytes (T cells and B cells) recipient serum: source of anti-HLA antibodies

8. discuss GVHD in terms of temporal placement to transplant, immunology involved and the positive and negative outcomes.

GvHD means the graft reacts against the host. The graft is the marrow or stem cells from the donor. The host is the person having the transplant. GvHD happens when particular types of white blood cell (T cells) in the donated stem cells or bone marrow attack your own body cells. This is because the donated cells (the graft) see your body cells (the host) as foreign and attack them. Normally T cells don't attack our own body cells, because they recognise proteins on the cells called HLA (human leukocyte antigens). We inherit our HLA from our parents. Apart from identical twins, HLA is unique to each person. Before a stem cell transplant or bone marrow transplant you and your donor have blood tests to check how closely your HLA matches. This test is called tissue typing. If you and your donor have very similar HLA this lowers the chance of GvHD. The more differences there are between your HLA and your donor's, the more likely you are to get GvHD. After a transplant your bone marrow starts making new blood cells from the donor stem cells. These new blood cells have the donor's HLA pattern. They recognise the HLA pattern on your body cells as different (foreign) and may begin to attack some of them. Balancing the benefits and disadvantages of GvHD The symptoms of GvHD can be difficult to cope with. In some cases GvHD is serious, and can even life threatening. But having mild GvHD can be a good thing. As well as attacking your body cells, the donor T cells will also attack any remaining cancer cells. Doctors call this the graft versus disease effect. You have treatment to prevent GvHD. The aim is to try to lower the risk of serious GvHD as far as possible, but still keep some benefits of GvHD. This might help to stop the disease coming back.

3. suggest and discuss the information that must be investigated when a pregnant woman has a detectable unexpected antibody and select appropriate laboratory tests to gather some/all of the needed information.

How can we predict?? laboratory testing history ABO and Rh unexpected Ig screen if unexpected antibody screen is negative?? if unexpected antibody screen is positive?? Is Ig clinically significant? will Ig become clinically significant? How much Ig is there??: titer If you think HDN: do ABO: Rh including weak D do DAT: if DAT pos: Eluate and I.D.

14. state the long term problems of an infant who has suffered HDFN.

Hyperbilirubinemia and severe jaundice - High levels of bilirubin can result in hearing problems and brain damage, if left untreated. Heart failure - Severe untreated anemia can lead to heart failure since the heart is not getting enough oxygen-rich hemoglobin. cardiovascular failure secondary to anemia, can lead to fetal death. The destruction of red cells also leads to elevated bilirubin levels. After birth, the infant's immature liver enzymatic pathways cannot metabolize the unconjugated bilirubin, which can increase to dangerous levels. The hyperbilirubinemia can cause permanent brain damage, known as kernicterus.'

4. describe a "titer" and state when its performance is indicated. Interpret results of titers and describe precautions to insure proper interpretation.

If titer is 1/16 or higher maybe HDN (if anti-K 1/8) if less that 1/16, want to monitor over course of pregnancy titer increase 2 tubes or 4 fold active immunization, be afraid titers hot bed for technical errors, run controls, be careful If problem: Amniocentesis normal amniotic fluid colorlesswith HDN we see color... do spectrophotometric exam to detect bilirubin and quantitate. read at 375, 450 & 525nm absorbance for bili max at 450 plot 375 and 525 readings draw straight line. plot 450, see how far off expected the greater the deviation, the more the bilirubin. The more bilirubin present the greater the red cell destruction, the more the rbc destruction the greater the danger

17. list the criteria for administration of post-partum RhIg prophylaxis for women who have received antenatal treatment and those who have not.

Postpartum evaluation for RhiG prophylaxis:: 1. Rh negative mom 2. Rh positive neonate or ? 3. Mom not actively producing anti-D

13. state how to detect HDFN serologically.

The direct Coombs test detects maternal anti-D antibodies that have already bound to fetal RBCs.: First, a sample of fetal RBCs is washed to remove any unbound antibody (Ig). When the test antibodies (anti-Ig) are added, they agglutinate any fetal RBCs to which maternal antibodies are already bound. This is called the direct Coombs test because the anti-Ig binds "directly" to the maternal anti-D Ig that coats fetal RBCs in HDN. Indirect Coombs test: used in the prevention of HDN The indirect Coombs test finds anti-D antibodies in the mother's serum. If these were to come into contact with fetal RBCs they would hemolyse them and hence cause HDN. By finding maternal anti-D before fetal RBCs have been attacked, treatment can be given to prevent or limit the severity of HDN. For this test, the mother's serum is incubated with Rh D-positive RBCs. If any anti-D is present in the mother's serum, they will bind to the cells. The cells are then washed to remove all free antibodies. When anti-Ig antibodies are added, they will agglutinate any RBCs to which maternal antibodies are bound. This is called the indirect Coombs test because the anti-Ig finds "indirect" evidence of harmful maternal antibodies, requiring the addition of fetal RBCs to show the capacity of maternal anti-D to bind to fetal RBCs.

15. given results of neonatal serological tests, judge the presence or absence of HDN.

The most important serologic test for the diagnosis of HDFN is the DAT with IgG reagent. A positive DAT indicates sensitization of fetal red cells and is in itself not diagnostic of HDFN. The DAT results must be interpreted in the clinical context. Where the infant has a positive DAT with suggestive clinical findings; a red cell eluate confirms the antibody specificity, and presence of the corresponding antigen on cord cells confirms the diagnosis of HDFN. In Rh D hemolytic disease, the DAT may be strongly positive without clinical signs of disease; whereas in ABO hemolytic disease, clinical features may exist with only a weak /negative DAT.

6. state the types of graft rejection

There are three major types of allograft rejection: Hyperacute, acute, and chronic rejection

8. describe the events which must have occurred in order for HDFN to develop.

There are two main mechanisms by which maternal antibodies target fetal or newborn RBC antigens: - ABO incompatibility - Fetomaternal hemorrhage ABO incompatibility is a congenital, inherent mismatch between maternal and fetal blood types.[4] Conversely, alloimmunization due to fetomaternal hemorrhage (FMH) is an acquired immune-mediated mechanism that typically affects subsequent pregnancies rather than the pregnancy in which the FMH happens. ABO incompatibility is currently the most common cause of HDFN. When present, ABO HDEN is typically mild. I° ABO HDFN is defined by maternal/fetal ABO Incompatibility, an elevated bilirubin (corrected for gestational age), and a positive DAT result. If treatment is need-ed, phototherapy is usually sufficient, and the need for neonatal exchange transfusion is un-common.

28. define a two volume exchange.

To exchange all or part of an infant's blood supply for certain medical conditions. A double volume exchange transfusion is replacing the baby's total blood volume twice, leaving the intravascular amount the same. A partial exchange is either increasing or decreasing an infant's hematocrit, while maintaining a constant blood volume. Double Volume Exchange: 1. Hyperbilirubemia 2. Hyperammonimia 3. To remove bacterial toxins 4. To correct life-threatening electrolyte and fluid imbalance Partial Exchange Tranfusion 1. Severe anemia in the face of normal or excess blood volume. 2. Clinical polycythemia

1. State reasons and mechanisms causing a positive DAT

autoantibody alloantibody: -- rcpt vs donor -- donor vs rcpt -- maternal (HDFN) anti-drug

8. Describe how the polymorphism seen in the HLA system is beneficial for the immune system but a hurdle for transplantation.

beneficial for the immune system because the more polymorphic the more antigens the HLA system can respond too. But it is a hurdle for transplantation because have to find a match but there is so much variability that it can be difficult

18. describe the proposed means by which RhIg prevents sensitization.

cells it does this by marking your baby's red blood cells for clearance before your body can the cells are subsequently removed by the body's natural immune response without the need to form antibodies this blocks the sensitization process The mechanism of action of RhIG has not been completely elucidated. D-positive red cells are opsonized by RhiG and cleared by macrophages in the spleen, which results in cytokine secretion and immunomodulation. Similar antigen-specific suppression of the immune response has been observed in a murine system where anti-K1 prevented immunization against transfused K1-positive cells but not against other antigens.

1. Define and categorize HLA alleles into class I or class II classical antigens and identify those that are not classical.

class I: endogenous • Class I - all nucleated cells class II: exogenous • Class II - antigen presenting cells (APCs) ++ B cells, Macrophages, Dendrtic cells Major Histocompatibility Complex (MHC) • Originally identified as the target of graft rejection • Named as Human Leukocyte/Lymphocyte Antigens (HLA) • Class I and Class II • Found in all vertebrate species • Differences across different species

3. Discuss and also compare and contrast the following AIHA types: cold

cold agglutinin disease: 1^o 2^o: lymphoma, mycoplasma pneumonia, mono specificity: anti-I

33. compare and contrast exchange transfusions and replacement transfusions in terms of specimens for pretransfusion testing, age of units to be used., components needed and predisposing events.

exchange transfusions: are transfusing whole blood volume specimen: blood and plasma pretransfusion testing: If doing an exchange transfusion decision based on: (use moms serum) - positive DAT - cord hgb (decreased hgb) - rate of rise in indirect bilirubin blood selection: - must be Ag negative for maternal Ig's - must be crossmatch compatible with moms serum (if not available use eluate form cord or neonate serum) Blood must be: - less than 7-14 days old to ensure low K+ and high 2,3 DPG - CMV neg - Hgb S neg - irradiated * institution policy replacement transfusions: specimens for pretransfusion testing: recipents serum age of units to be used: must use high quality specimen - no color issues

27. state the four goals of an exchange transfusion.

goal: - lower bilirubin - remove sensitized fetal RBCs - provide compatible RBC's - reduce level of maternal Ig in neonate

3. Discuss and also compare and contrast the following AIHA types: drug induced

going to behave as a warm antibody specificity: anti-Rh, maybe anti-e may have test requested bcs of suspected hemolysis -> but need to check medication

31. list the source of units for an exchange transfusion when the antibody involved is against a high incidence antigen.

if high incidence antigen we can wash moms cells until plasma is gone or sometimes we might have to use Ag + cells because we are still getting rid on moms antibody and decrease bilirubin

12. compare and contrast ABO HDN with Rh or other HDFN in terms of relative severity, which pregnancy is at risk and what the usual immunizing event is.

in terms of relative severity: it is about hemoglobin and bilirubin Mom immunized during first pregnancy or by transfusion ABO seen in first pregnancy Second (at higher risk) and then subsequent pregnancies get more severe. ABO can always be mild more common HDFN is ABO than Rh BUT.... HDN due to ABO incompatibility is usually less severe than Rh incompatibility. One reason is that fetal RBCs express less of the ABO blood group antigens compared with adult levels. RhD is the most potent immunogenic red cell antigen. It remains the most important cause of HDFN in nations without robust access to prenatal care. Anti-KI is also an important cause of HDFN, and a potent immunogen. Other antibodies that have been less commonly reported to cause moderate or severe disease include antibodies against E, c, C, k, K', Kp°, Ku, js*, 15, JK', Pr, Fy, S, s, and U.2021 (See Table 13-5 in Chapter 13. The presence of multiple antibodies may lead to more severe HDFN.

16. describe Rh Immune Globulin.

is made from pooled human plasma from individuals either naturally or intentionally immunized to the D antigen; recombinant products are in development. The product contains IgG subtype anti-D. It is available in 300, 120, and 50-ug doses. Appropriate ante- and postpartum administration of RhIG reduces the risk of a D-negative mother becoming immunized by a D-positive fetus from about 16% to <0.1%. The American College of Obstetricians and Gynecologists recommends the first dose of RhIG be given at 28 weeks' gestation because 92% of women who develop anti-D during pregnancy do so at or after 28 weeks. Administration of RhIG during pregnancy typically leads to a positive antibody screening result in the mother, but the anti-D titer is low and thus poses no risk to the fetus. Occasionally, RhIG will cause a positive DAT result in the newborn. 300 ugs of human IgG anti-Rho(D) 1 vial prevents immunization from 15 mls of Rh pos rbc' s ** given postpartum to Rh negative women after delivery of an Rh positive neonate or if unknown.. must be given within 72 hours of delivery incidence of Rh HDN dropped from 13% to 1-2% (significant) Why still a 1-2% occurrence???: fetal-maternal hemorrhage all during pregnancy But if Prevent by using RhIG antenatal at 28 weeks of gestation incidence drops to 0.1% also comes in a 50ug dose.... takes care of 2.5mls of Rh pos rbc': neonate may have pos DAT at delivery and may detect maternal anti-D for up to 12 weeks post delivery.

1. Describe microlymphocytotoxicity testing, NIH and AHG methods.

microlymphocytotoxicity: Serology - also known as CDC (complement dependent cytotoxicity) • Patient's serum run against 30-50 cells of differing HLA types • Reaction patterns analyzed looking for patterns of specificity • Use r value, ×2, and classic Bayes analysis • PRA based on % of cells reacting • PRA- percent/ panel reactive antibody...estimate of success in finding compatible donor at random in the population NIH method: (double check) AHG methods: (double check)

2. Describe cellular typing both lymphoblastogenesis and mixed lymphocyte cultures, MLC's

mixed lymphocyte cultures (do-The first cellular approach used for recognition of additional alleles (to figure out why "compatible" individuals could still reject transplants)-lymphocytes from two individuals are co-incubated in tissue culture. If the individuals are different for something other than serologically defined alleles, both cell types undergo lympho-blastogenesis, synthesize DNA, and proliferate,uble check)

30. list in descending order of choice the specimens used for pretransfusion testing for an exchange transfusion.

moms serum --> if not available use eluate from cord cells --> neonate serum (3-5 drops)

3. state what histocompatibility support is needed to support the major organ systems

need immunosuppresives

4. Recognize and diagram the following molecular based methods for HLA typing: PCR-SBT

sequence based typing: PCR-SBT is the only technique that directly detects the nucleotide sequence of an allele, so it can be accurately assigned. It requires very expensive equipment and complex laboratory methods to provide clear, high-resolution HLA typing results. High-resolution typing is necessary for assignment of HLA alleles. Sanger-chemistry sequence-based typing (SBT) can be used to ident. fy known alleles and characterize new alleles it Although SBT is considered the "gold standard" for HLA typing, ambiguities occur when two dir. ferent base pairs are found at the same position and can result in two different possible combinations of alleles. These ambiguities occur because SBT evaluates both maternal and paternal HLA genes (haplotypes) simultaneously. Pairs of single nucleotide substitutions can be encountered in which cis (same parental haplotype) or trans (nucleotide assignment of the second polymorphic site is on the other haplotype) can give ambiguous results when those nucleotide combinations are assigned to different alleles. The haplotype to assign individual base pairs in polymorphic combinations may be determined with additional results from selected SSP or SSO reactions.

4. Recognize and diagram the following molecular based methods for HLA typing: PCR-SSP

sequence specific primer - amplification refractory mutation (ARMS A second major technique uses sequencespecific primer (SSP) pairs that target and amplity a par-ficular DNA sequence. This sequence-specific method requires the performance of multiple PCR assays in which each reaction is selected for a particular allele or group of alleles. The amplification products are directly visualized after agarose gel electrophoresis. Because SSPs have specific targets, the amplified material indicates the presence of the allele or alleles that have that sequence. The pattern of positive and negative PCR amplifications is examined to deter. mine the HLA allele(s) present. Primer pair sets are commercially available that can determine HLA-A, -B, -C, DR, -DOA1, DOB1, and - DPB1 phenotypes and may be combined to determine common alleles.

1. compare and contrast solid phase vs. microlymphocytotoxicity based assays.

solid phase: The current approach to Identity HLA antibodies relles on the use of beads or microparticles lie, solld-phase methodology) coated either with clusters of HLA Class I or Class Il antigens from cultured lymphocytes le, an HLA phenotype; or with individually purified or recombinant Hi antigens (single-antigen beads). 19 Antibody binding is detected by staining with fluorescent ly labeled antihuman globulin (AHG). The presence of antibody is detected with flow go tometry, flow microarrays, or enzyme- linked im munosorbent assay (ELISA). Only an ELISA method is FDA-approved for donor screening (August 2018). Flow cytometry and flow mi croarray methods are more sensitive than lym phocytotoxicity and focus on the detection of 1gG antibodies. The use of single-antigen bead assays are of particular importance for highly sensitized patients where multiple HLA ant-body specificities cannot be reliably distinguished and identified with either cell-based cytotoxic assays or solid-phase assays using clus ters of HLA molecules. Although HLA antibody has been demon strated in transplantation population studies to be detrimental to transplanted organ and patient survival, the clinical significance of low-level antibodies detectable only by solid-phase assays cannot be predicted for individual patients. Newer adaptations of the solid-phase technology can determine whether the antibodies do or do not fix complement and may improve the pre dictive value of testing for indirtdual patens lymphocytotoxicity: supplemental testing DNA-based HLA typings without defined HLA antigen assignments, and research support for HLA allele and antigen sesignation

1. state how HLA plays a role in chronic disease

some conditions especially ones with a autoimmune etiology and association exists between HLA phenotype and the occurrence of or resistance to clinical disease

9. state the source of the "offending" antigen.

the offending antigen would be the babies antigen that is causing a type II hypersensitivity

What are the three test for fmh

the rosette screening test, the Kleihauer-Betke (KB) test, and the flow cytometry test. How much to give?? get sample with in 1 hour of delivery: perform: Rosette test Rosette test: Mom's blood- mostly D neg with a few D pos fetal cells incubate with anti-D ** add incubate with D pos indicator cells — get rosettes The more rosettes seen the more fetal blood present-Perform Kleihauer-Betke acid elution stain Make a blood smear: elute with acid all Adult hemoglobin fix stain for hgb, only hgbF left. count 2000 rbc' s calculate %F Can also enumerate by Flowcytometry: say you get 1.39 take 1.3 x 50* = 65 ml fetal *50 = 5000ml/100 (5L is estimated maternal blood volume) 1 dose RhIG good for 30 mis WB so: 65/30 = 2.2 doses if <0.5 round down + 1 dose if>0.5 round up + 1 dose give no more than 5 at a time rotate cheeks

2. state why pediatric patients may have an advantage in the transplant algorithm

they don't have a lot of antibodies that could interfere with the process of finding a donor

36. select cells with preferred antigenic characteristics to be used in a titer when the antibody specificity is given. example: mom A pos baby: B neg positive DAT: has anti Fya

use O neg Fya- and AB plasma (explanation on pg 591)

7. Detail both at the molecular and protein level where HLA molecules have conserved structure and where they have variability

variability region MHC I: alpha 1 and 2 (and the peptide region that binds to the antigen) MHC II: alpha 1 and beta 1 (and the peptide region that binds to the antigen) conserved structure MHC I: alpha 3 and Beta 2 m MHC II: alpha 2 and beta 2