ABO Problem Solving & False Negatives

ABO Problem Solving

• Give O negative or O positive blood if the ABO type is unknown.
• Give AB plasma if the exact blood type is unknown.
• Adhere to institutional guidelines for blood product administration.
• Administering cells involves a mix of cells and plasma, which dilutes the recipient's plasma.
• Forward typing reactions should ideally be 4+, while reverse typing should be 3+ or 4+.
• Subgroups of A exist like A1 and A2.
• 80% have A1.
• 20% have A2.
• A2 can produce anti-A1 antibodies.
• A2 differs qualitatively and quantitatively from A1.
• 1-8% of A2 people make anti-A1.
• 22-35% of A2B people make anti-A1.
• A1 lectin or absorbed anti-A1 used to distinguish between A1 and subgroups.
• A3 causes mixed field agglutination.
• Anti-A sera might not agglutinate A3 cells entirely at once.

False Negatives

• Forgetting to add reagent.
• Missing hemolysis.
• Incorrect Ig/Ab.
• Poor technique when testing.
• Improper incubation which must be at room temperature (70).
• Bad interpretation.

False Positives

• Poor testing technique.
• Contaminated reagents.
• Dirty glassware.
• Bad interpretation of results.

Specimen-Related Problems

• Recent transfusion.
• Mixed field agglutination occurs in Type A individuals with O cells due to a combination of A and O cells.
• After a bone marrow transplant a mismatch in ABO can occur, also known as a true chimera.
• There will be 90% donor cells and 10% of the original cells after trasnplant.
• Someone who has a3 subgroups, is lucky they aren't worse.
• A1 subgroup constitutes 80% of the population.
• A1 RBCs react with both anti-A and anti-A1.
• A2 subgroup represents 20% of the population.
• A2 is both qualitatively and quantitatively different from A1 so they can make anti- A1.
• Individuals with group A whose RBCs react with anti-A but not anti-A1 are classified as A2.
• A1 Lectin made from seeds of the plant "dolicous biflorus" agglutinates human cells with specificity.

Problems in Red Cell Testing

• Mixed field agglutination is a challenge
• This is due to bone marrow transplants
• Recent transfusions

Weak/Missing Antigens

• -A3 variant poses challenges.
• Weak reactions are the forward of reverse.
• Due to weak reactions, a twin might test as group O, while having 10% A cells.
• Patient make antibodies against reagents.
• Polyagglutinable cells are those with multiple agglutinations with everything.
• May have a high level of soluble antigens in the sera, which will inhibit agglutination.
• Cold reacting Ig in the serum, which can cause false positives.

Problems in Serum Testing

• Fibrin clots can be mistaken for agglutination.
• Rouleaux affects the reverse type.
• Antibodies other than ABO antibodies may be detected during reverse typing.
• Antibody-related issues can happen when mixing patient serum with reverse cells.
• Immunodeficient patient, old or young patients can affect the reading.
• Decreased levels of anti-A and anti-B antibodies may affect readings.
• Kids may not have reverse typing when they are first born.
• Complement blocking, presence of a large complex, may block the reaction.
• Mismatch between forward and reverse typing, might be homozygous (weak) forward/ (strong) hetero reverse
• This usually occurs with bone or arrow transplan
• Recent plasma transfusions can cause issues.
• After an injury where we don't know the group type and administering O cells, a mixed field with anti-A and anti-B may be observed.

Resolving Issues: Absence of Ag

• Incubating at lower temperatures strengthens cold reactions.
• Enzymes, such as salic acids, enhance antigen detection.
• Check saliva for secreted antigens.
• Polyagglutinable cells show multiple agglutinations.
• Cells that agglutinate with everything may have a high level of soluble antigen sera, can neutralize reagents.
• Anti-A dye is royal blue to prevent screwups.
• Anti-B dye is yellow.
• Rh dye is green.
• People can develop antibodies against dyes.
• When dyes are present, agglutination may occur due to dye-antibody reactions, causing RBCs to appear caught up as innocent bystanders.
• Cold-reacting Immunoglobulins may be present.
• A1 is negative, D negative.
• High protein levels cause cells to become sticky, leading to Rouleaux, affecting the reverse type.
• Young or old age can make reverse typing difficult, with weaker antibody production.
• All sites are blocked by complement, especially C1qrs.

Resolving Reactions with Extra Rxn in Forward Typing

• Something in a blood group A patient causes:
• Reduced anti-A grade.
• Elevated anti-B grade.
• They're losing A antigen and acquiring B antigen.
• The A antigen is N-acetyl galactosamine, while the B antigen is D-galactose.
• An acetyl group distinguishes the two at the front end of the A antigen.
• Microbes produce enzymes that can alter cell surface antigens, leading to acquired B antigen characteristics. Certain bacteria make acetylase, which cleaves acetyl groups, causing it look like a B ANTIGEN.
• Removal of acetyl groups by bacteria can cause it to look like a B antigen. The The more you see B and the less you see A antigen.
• This can happen in patients with a deep festering abscess that was previously unknown.
• The acetylase chewing up the acetyl group causes the patient to appear to have a b-antigen.
• Even though they have a "b-antigen," there isn't anti-B in their serum.

Acquired A

• Tn activated a blood group antigen that we DON'T type for.
• Tn is a blood group antigen not routinely typed for. When Tn is activated on RBCs, it typically indicates an ongoing infection, suddenly making it prevalent.
• The reagents used will include anti-Tn.
• It can be regarded as a "poly agglutinable cell" because anything combined with it will elicit a reaction

Resolving Reverse Typing Issues

• Immunodeficient.
• Insufficient antibody for detection.
• Resulting in a Weak reaction.
• Too much Antibody is present, leading to the prozone effect.
• The cold reacting antibodies are usually autoantibodies.
• To resolve, add more sera and incubate at lower temperatures, like in the refrigerator because some people react outside of the ABO.

Addressing Rouleaux

• Detected to shaking the tube to get it resuspended.
• When it comes off, swirling is negative or chunks positive.
• Chunks are stringy because of the high protein.
• Resolve it using “saline replacement technique". You have to put serum in a tube, put in 2 drops of serum, 1 drop 3% cells, spin/read it.
• After spinning and reading, spin it down, discard supernatant, add back in 2-3 drops of saline and resuspend.

Weak/Missing Antigens

• If we take away the serum/protein that should show us if there's true agglutination or if there's just rouleaux we have to dilute it.
• Leukemia can cause weak or missing antigens. This happens when the production of WBCs interferes with the production of necessary components.
• Transfusions can result in mixed-field reactions.
• Intrauterine fetal transfusions necessitate typing of the infant to identify odd variations.
• Bone marrow transplant neutralizes the serum.

Troubleshooting When Problems Arise

• Identify whether it's a really bad ABO mismatch.
• Always Draw several tubes, EDTA and more tubes.
• There is a lot of time involved figuring it out.
• Secure blood immediately before there's a transfusion, avoid a mixed sample due to donor cells, because you're fighting against those donor cells
• Immediately resolve all ABO/RH discrepancies.
• A lot of other blood groups affect typing result,
• Stronger Ahg if reacting at 37= immediate spin.
• Good if reacting at AHG because it can detect antibodies bound to RBC.
• This suggests the presence of igG antibodies (anti-D anti-K) which can cause HDFN
• Ahg= sensitized at 37.
• This is important, since it impacts the safety of transfusing.

Clinically Relevant Antibodies

• When patient plasma against reagent RBCs has a reaction to AHG suggests the presence of IgG antibodies like anti-D/ anti-K.

Kell System Antibodies

• Frequenting can impact the test and.
• You should assume that at 37 it reacts, that it reacts with ahg, causes hdn, cause htr.
• How easy will it be to find the antigen?
• If we treat with enzymes will it be okay?
• The blood group system can have an impact. Also IVIG= therapeutic treatment plan which effects your reverse type
• Further investigation becomes necessary in order to become a detective to track their history and dig something deeper!
• For tranfusion you must know the specific tranfusion needs.
• Administer A2 subgroups with anti-A1 antibodies and mainly A cells to give them their real type.
• Consider its original Ig type:
• Think about its orgional Ig.
• Consider the reaction temperature at 37 or 4.

When Unsure About Blood Group

• Assume IgG presence.
• Assume reactivity at 37°C.
• Assume codominance.
• Assume HDNF potential
• Determine the antibody type that reacts at 37 has IgG, or if it causes has HDFN.

Other Blood Groups

• Lewis system genetics and characteristics:
• In the Lewis system, the lower the number the earlier it was recognized.
• Lewis gene (Le/le) exhibits codominance.
• Influence determined by H/Se genes;
• Three genes influence Lewis expression and it not used by the red cell.
• Le a+b-: non-secretors (sese).
• le/le leads to Lewis negative expression.
• Le a-b+: secretors (Se).
• If you have just ONE big Le= you will be Lewis positive.
• Lewis substance is independently secreted, NOT linked to secretor gene, depends on the lewis gene itself!
• The Se influences Le A+ or Le B+ along with H.
• ONLY LEWIS gene controls secretion
• They are mostly IgM and can be hemolytic.
• Usually you have to have red ell stimuli to get it.

Other Blood System Antibodies with Significance

• KELL SYSTEM ANTIBODIES: Highly immunogenic, thus second in importance for transfusion -Anti-K, causing HDFN reactions/HTR -IgG (reacts at 37, AHG phase)
• KIDD: delayed hemolytic transfusion rxns -Anti-Jka, anti-Jkb -IgG (reacts at 37/ AHG phase)
• DUFFY: common in African American populations, resistance to plasmodium malaria -Anti- Fya, anti-Fyb -Ig class: IgG (AHG phase)
• MNS: some are clinically significant -Anti-M, anti-N, anti-s, anti-S, anti-U -Ig class: IgG or IgM depending on specificity
• P: linked to PCH (paroxysmal cold hemoglobinuria) -Anti-P1, anti-P -Ig class: IgM or IgG
• LEWIS: naturally occurring antibodies that DONT cause transfusion reactions -Anti-Lea, anti-Leb -No HDFN and this is still controlled by Le gene NOT Se -Ig class: IgM

More on Anti-I

• Higher prevalence in newborns.
• Present on adult RBCs.
• Cord cells lack I antigen and help detect anti-I
• Big I negative, newborns only have i antigens. Suggests presence of anti-l if the patient serum reacts with adult RBCs but not with cord cells.

Compatibility Tests

• Cross transfusions can give to patients with anti-lewis antibody by introducing lewis positie cells: The minute you put the blood in, its going to be plasma thats lewis neg, antigen is going to come off or Give lewis pos cells to lewis neg person.
• lewis pos plasma to lewis neg person will show up lewis ag from the plasma.
• Pregnant women and newborns are typically Lewis negative. Pregnant moms can develop the antibody.
• They can start making more ab even if they have Will igM coss placent = no.
• Newborns are Lewis A /B negative.

Pre-Sample Info

• Be negative to the cells.
• Necessary Details for Blood Samples:
• Physician & orders.
• Two identifiers: First and last name; ID number.
• Transmission can occur by paper, computer, and rarely a phone log
• You can then draw a sample.
• For collections of samples make sure you have the CORRECT person and Name and ID number.
• Also record Date/Time; if it was Pre/post transfusion, and the phlebotomist.
• Sample quality: non-lipemic and -Non-hemolyzed.
• The age of the sample on the patient impacts Dependends on the patent and : If the patient is or has been pregannt/transfused within 3 months.
• You'd like the sample collected within 3 DAYS (you got it three) by time you like to transfuse. If these are not good ask Why? -Good state to reflect the current state and Look for circulating.
• Follow the institution's policies if this hasn't occurred for patient.
• The samples taken the patient must be post 7 days of the transfusion but kep at at 1-6 degrees.

Clinically Antibodies

• NEED TO MAKE SURE YOU HAVE THE RIGHT ABO!!
• Clinically antibodies against red cell antigens.
• The serum OR plasma from the recipient.
• Against: human RCs and reagents that have specific characteristics.
• Incubate at 37 and aren't worried about the cold agglutinants.
• Check AHG.

Polyspecificity

• Use a Polyspecific AHG or anti igG
• Tests use a wide range of blood group antigens. To find what antibody make sure to know the squares

Antibody Identification

• Screen cells are most effective when homozygous.

• A set of screen cells can be purchased.

• What antibodies can be missed? Antibodies against low-frequency antigens

• Is nice because it adds more information as we go

• If somebody has anti-kel 4 years later.

• Still treat them like they have it though since you may not see it.

• anti-H reaction is strongest with O type

• anti-H weakest reaction is Bombay- clinically significant

• Compatibility testing/antibody detection= pre-transfusion testing.

• Also Samples of recipient and donor must be retained for 7 days post-transfusion at 1-6C

• Check with the recipient serum and if are going to miss any antibodies with the antibody screen? For compatibility tests: Also includes the AHG -donor cells with recipient serum & put cells n serum together and do an immediate spin

• Use the Pre-transfusion testing to find COMPATIBILITY TESTS.

• Check If there's a history or antibodies, you HAVE to use antigen- negative blood/ crossmatch through AHG. -Most antibody will be detected but not all.

• Be be sure to crosscheck you blood.

• For a neonate make sure there's not -No anti-a or anti-b

• Do no compatibility test needed if you give o cells!

More about Testing

• Allow streamlining of workload.
• Do lots of a lots of Lots of surgical procedures don't use blood testing, but make sure there's the potential.
• A type and screen should be done.
• If you know you're already know but Allow units to be there but not tied up, which -Reduces inventory-Have blood ready when its neede,.
• If something doesn't line up there'sonly three %
• An unexpected AB screen defects an lg or if match was incompatible you need to FIX IT!!- double-check the expiration date/ color/ documents to who you release the unit
• In a medical emergency the ER can sign off to give patients o postive or Oneg blood if there's an emergency.
• MAKE SURE ALL UNITS LABELED CORRECTLY!!!*

Transfusions

• Transfusions can require a massive or Large volume of blood within 24 hours exchnaged depending on the patient age/ weight.
• *If you've given non-group specific blood nd want to switch back, Base your decison on what your REVERSE TYPE is looking like.
• And can be logic and fast ifyou already know the recip.
• If it is incompatible you need to fix it;

Unexpected AB ID Panel

• The panel usually has ten cells present. The more cells the better characterization of Ab you'll get
• Cord cells are: Lewis a- b- & ii- NO BIG I antigen
• Helpful when working with cold agglutinates by determining which to run the crossmatch and match the pattern
• the little k is ALWAYS positive so be sure there consistent results
• Look under the AHG and see who's positive reaction and if they are strong or weak. If cells aren't negative but “compatible"
• Sometimes you have to interpret it as compatible
• Also Make sure, If gradeing reations

To Conclude

• We need to detect ab from ab screen and Identify the ab.
• Anti-z can always be detected.
• So Anti-X can the the antibody the antibody is present if there are cells and a scrum for it.
• "Unexpected" ID panel:
• Looking at the chart of antigens that may have a we never tested it so we have no real proof those cells have the antigen.
• H (i)and (P )show variations in expression of antigens
• Panel cells are blood group “O” beacuse we dont want to see the expected antibodies!!- Always make Panel cells are blood group “O” because we dont want to see the expected antibodies. Always make sure you are sure. the panel.
• Also Make sure there's multiple positive cells. . There can also be a cross match thats incompatible

Detecting

• If a result is incompatible there can also be a the blood cells and scrurum.
• the next step is, to identify another antibody
• Then run it the same way so we can detect that antibody Replicating that helps us see the antibody the same wayOnly the negative cells are usefulIf we have anti x, and we get positive reaction= X antigen and no reaction and no antigen on that cell
• *Cross off if its positive for antigen on the panel and Whats left after crossing off are our possibilities. We should use cell we got to work on and look into the patient and and make sure there isn't any contamination. To comfirm there can’t be any if the result says theyLack the D antigen meaning they are Rh negative Select the cell panels needed (1pos1neg/1pos/D neg If there's only one anitbody, make sure its that! Other wise it might need to be reviewed and there could be multiple. Make sure there aren’t any and Check of un clear identification if the is- Cold autoagglutinant/Warm autoagglutinant Rouleaux Is the autocontrol going to be positive with cold autoagglutinate? YIS

Panel Cells and Reactions

• Is the autocontrol going to be positive with cold autoagglutinate? YES Rouleaux.
• Use in immediate spin
• Probable specificity- what didn't you cross out
• *Panel cells = Cross of the positive antigens .What is the reaction dose in serum multiple things can cause it, such as technical dificulties multiple. and dosage
• The way test the the antigens show their is different based on how it reads and there reactions and interactions.
• The stronger the results can tell us the the patterns of dosage reaction, what can be positive Use the Look under the AHG and see who's positive reaction.

Testing

• With Panel cells the best to always have good control, with Antigen type the patient (antigen neg or antigen pos). To confirm
• What class of ig is duffy b igG?* is to test the antigen and it's 37 c read Quality control!! if it had: This is the opposite of antibody screening!! Make sure the blood can pick up the weak expression and that DGE.A. We want to make sure our antibody can pick up weak antigen

Enchance testing

• Most just sensitized, you need to incubate at 37 degrees or Wash 3x first as an (IAT). But the results ENCHANCED with: kidd, lewis, rh, P, I when testing in this manner. (AET) a chemical that gets rid of K will always cause. There can be Ab/Ag reactions there always need a if we have anti d/ anti-K we can separate it

• If in serum we conduct there can be Absoption/elution, that are are - Change pH or concentration

• This means the testing: Ag/Ab reaction is reversib/e and Specific Agglutination is stable across*

• Can Also have : aTakeiga serum that has an anti-l and mix it with human breats milk,YOU MAY GET RID OF BIGI Sdis in urineWe mix this antibodiesE granulosa cyst fluid**

Antibodies

• **Cold autoagglutinin antibodies You can get rid of it by running eveyrthng at 37 degrees and is Only effective if it hasn't been transfused.

To Avoid seeing seeing complement being bound

• **We could add (Ca2+), use EDTA plasmaUse monospecific igG then you won't see complement

• To work with anti -I there are 5 ways- Pre-warm Absorption

• NeutralizeEDTA plasmaMonospecifi igG-Antibody screen with big one we keep i positive to see it! How to remove a warm autoantibody? and IgG WARM autoabsorption

• It could take many applications to fix and take a lot of time and have results