BB Lab Final Exam (copy)

The pipette always needs to be held at the same angle; a vertical angle will result in smaller drops, while a 45° angle will result in larger drops- always be consistent and keep the same angle.

How can you assure consistency of volume dispensed when using controlled drop pipettes?

Place tubes in the centrifuge in the same order they were in the rack. This helps to avoid mixups. Also, don't mix and match patients in the centrifuge. If you have time try to only spin one patient at a time

What technique can you use when centrifuging in blood bank immunofuges to increase technologist speed and avoid mix-ups?

Washing: 60 seconds
Reactions: 20 seconds

What is the centrifugation time (at 3400 rpm) for washing cell suspensions ______________ and reaction tubes _______________?

Mother tubes: Patient name and one other identifier (birthday or MRN)
Reaction tubes: Patient last name first initial and what reaction is in tube (anti-A, etc)

What general information should appear on labels of tubes used in blood bank testing (not the tube used to draw the blood). Mother tubes should include______. Reaction tubes should include___________.

Use reagent cells to compare suspensions

How do you judge a 3-5% cells suspension (without measuring precisely)?

This ensures that reactions are performed the same way every time between all techs. Also if you drop dead in the middle of setting up a reaction, someone else could hop right in and know where to continue.

Why is it important to have a specific strategy (that is used consistently) to organize a rack for reaction tubes in blood bank?

1) Never have two lids off at once
2) Never leave uncapped bottles on the table
3) Don't touch tips of the droppers to the reaction tubes

What are 3 rules for handling bottled reagents (antisera or cells)?

Clear stuff first (antisera, plasma) then cells last. This is because it is easier to visualize that all reagents were dropped into their appropriate reaction tubes

In what order should reagents be added to reaction tubes and why?

Front type--Commercial antisera and patient cells. Looking for antigens on patient cells
Back type--Patient plasma and commercial cells. Looking for antibodies in patient plasma

List the two main parts (phases) to a blood type and describe what you are testing for in each part

A person will not make ABYs to antigens they have, this is because the possessed antigens are considered "self". The only exception is autoimmune ABYs to RBC antigens

Describe Landsteiner's Law

For the front type, the patient's cells must be in a 3% solution. From there, it is a 1:1 ratio between patient 3% cell solution and anti-sera (1 drop of each). For the back type, you do a ratio of 2:1, with 2 drops of plasma and 1 drop of commercial A1 or B cells

What volume of anti-sera and patient's cells do you add in the Front Type? How much patient plasma and commercial cells do you add in the back type?

Reactive at 37C. Will cause harm to a patient

Explain what is meant by clinically significant. Are ABO antibodies clinically significant?

We have to use a 3% solution of patient cells to create a "zone of equivalence" between patient AGN and ABY. If the suspension is too light, there will be excess ABY compared to AGN, and create a "prozone" effect. If the suspension is too heavy, it'll create a "post-zone", where there is too much AGN compared to ABY

Why do we use a 3% suspension of patient cells. What do you think could happen if our suspension is too light? Too heavy?

Hemolytic disease of the newborn (HDN), transfusion reactions (HTR), and autoimmune (or drug induced) hemolytic anemias (AIHA/DIHA). A positive DAT result would infer that the patient is making sensitized RBCs to IgG or complement CD3.

What are the 3 situations where a DAT would be appropriate to order? What would a positive result tell you?

Bpos, weak D pos

The following results were obtained on a patient workup. Interpret the ABO and Rh type

If unbound antibodies are not washed away, they remain in the reaction tube and can bind anti-IgG that should be binding to antibody of the cells. You will get reagent neutralization (of anti-IgG), no agglutination, and a false negative result.

What type of error is caused by insufficient washing of cells in a reaction tube ? Provide a reason why.

Coombs control cells (check cells) are IgG-sensitized or complement coated RBCs that are added to reactions to check that negative results. If it is a true negative, agglutination will occur when Check cells are added, if AHG reagent was forgotten then agglutination will not take place even with check cells

What are Coombs control cells? What are they used for?

A pos DAT invalidates all IAT testing. DAT pos cells will react with the anti-IgG in weak D testing regardless of whether or not D antigen is present. Monoclonal anti-D with a neg Rh control at IS. If you really really wanted to know weak D, genetic testing or elution.

How does a positive DAT affect typing for Weak D? Explain the mechanism behind your answer. How can you get an accurate Rh type on the patient?

O cells--ABO antibody or another blood group.
AC--Auto antibody

When resolving ABO discrepancies, how would O cells and an autocontrol help you? When would you use them?

Anti-A,B used to distinguish from O from A or B. Also can pick up weak subgroups of A

When resolving ABO discrepancies, when would you use Anti-A,B?

Using Anti-A,B in a discrepancy helps distinguish between Type A/B or O. Not useful if you have strong front type reactions because you use Anti-A,B to make weak reactions stronger. If it's already 4+, A,B won't help. O patient shouldn't have strong front type.

Would using Anti-A,B help you if you have strong reactions with Anti-A and/or Anti-B? Explain your answer

Trust the strongest reactions. NO not always the best approach because sometimes you have evidence of something else going on (patient history, etc). "Cold antibody" can look strong.

What's generally the "first rule" when beginning to troubleshoot a discrepancy? Is this always the best approach?

A2 cells used to distinguish between A or O/B.
A1-lectin pos, A2 cells neg--Type A1
A1-lectin neg, A2 cells neg--Subgroup of A
A1-lectin neg, A2 cells pos--Type O or B
Not react--A or AB

Describe when you would use A2 cells. What type of patient do you expect to react with A2 cells? Not react?

anti-A,B

What reagent would you use to perform QC on reagent A2 cells?

anti-A1 lectin is used to differentiate A1 individuals from other A subgroups in the ABO grouping system. anti-A1 lectin will react with A1 cells only, whereas other A subgroups will all be non-reacting (0). This could help solve an unexpected reverse type discrepancy.

What is the purpose of anti-A1 lectin? Explain when you would choose to use it and what results you would expect in your scenario. How would it help you in solving a discrepancy?

a. in vivo

DAT detects ______ sensitization of RBCs

a. in vivo
b. in vitro

washed patient cells and saline to rule out spontaneous agglutination

what control is used for a DAT?

- increase incubation time, re-spin, and re-read
- add 2 extra drops of serum
- if neither of those work, you can do AC or O cells

how do you handle missing reactions in the reverse type?

- run AC and O cells
- if both neg, run anti-A1 lectin and A2 cells
- rule out rouleaux

how do you handle unexpected positives in the reverse type?

rule out spontaneous agglutination with DAT
- rule out rouleaux
- double check patient history and diagnosis
- polyagglutination, acquired B, cord blood, etc.

how do you handle unexpected reactions in the forward type?

What cells are used in Antibody Screens?

Commercial single-donor O cells

Clinically significant antibodies react strongest at which phase?

IAT

What is the goal in performing an antibody screen on the patient? Why is this important in transfusions?

Goal is to identify UNEXPECTED, non- ABO antibodies that a patient may have in plasma. It is important to transfuse antigen neg blood to patient with corresponding antibody to avoid transfusion reactions.

Explain at what point in the antibody screen procedure you will add enhancement reagents. How much will you add?

Immediately prior to 37 incubation. Add same drops enhancement as plasma. (2)

Explain why you never stop an antibody screening procedure at IS.

Clinically significant antibodies react at IAT and may not react at IS.

What percent of cell suspensions are used in antibody screening using the gel method? How much volume of cells and plasma are added to the gel card?

0.8%.
50uL cells 25uL plasma.

Why is it recommended to wait for the results of E typing before typing for e?

Conservation of anti-e sera due to expenses.

What two requirements must be met for a donor unit of pRBC's selected for transfusion to patients with clinically significant antibodies?

1. crossmatch compatible at IAT
2. antigen negative for antibodies

What is the importance of antigen testing a patient for antigens to which you think a patient has antibodies to?

Antigen test patient to see if they are capable of making suspected antibodies.

Explain the difference between abbreviated and full crossmatches and what type of patients qualify for each type.

A full crossmatch tests recipient plasma against donor cells using an IAT method. This is commonly for people who have a positive screen and are getting transfusions.
The abbreviated version is for people who test negative during the screen and record review. This version only carries the crossmatch through the immediate spin phase.

Compare the procedural differences between PeG and LISS methods for screen/ID.

PeG: there is no 37°C centrifugation step; only IS and IAT.
LISS: Includes IS, 37°C, and IAT phase.

Why would a tech choose to use PeG instead of LISS in an antibody workup?

PeG more sensitive at pickup of weak, warm, Kidd antibodies.

In the DAT gel procedure, what is the percent cell suspension used? What is the source of cells? How does the procedure differ from the Antibody Screen done in gel?

A 0.8% cell suspension is taken from packed RBCs. from patient, in both the DAT and screening gel procedures.
- For the DAT, the 0.8% suspension is added into the gel cartridge (which includes anti- C3d and anti- IgG). There is no incubation step that the gel card goes directly to centrifuging step.
- For the ABY screen, a 0.8% is used again. The suspension is added to the anti-IgG cartidge (theres no anti-C3d). The gel card must incubate at 37°C for 15-40 minutes, then centrifuge.

How would you perform donor reconfirmation on a Bneg unit? What results would you expect?

A Bneg unit would receive confirmation with anti-A, anti-B, and anti-D. You would expect a strong reaction (4+) with anti-B, and a negative (0) reaction with anti-A and anti-D.

Provide 2 reasons why you would get variable reactivity strength on an antibody ID panel

1) multiple antibodies
2) one antibody displaying dosage

Describe the differences in grading procedures/interpretation between Gel and Solid Phase technologies when performing antibody screens

GEL
- Gels are graded based on where the RBCs fall in terms of the tube. If at the very top, this is a strong positive and is a 4+. When there are some cells at the top, and some in the upper portion, this is a 3+. a 2+ is when the cells and suspended throughout the gel. a 1+ is when the cells are suspended in the lower portion of the gel with a visible button at the bottom. Negative (0) is when all cells are suspended at the bottom. Mf can occur when there is cells at the top and cells at the bottom

SOLID
- a positive reaction (4+) is shown when there is presence of RBC adherence to the monolayer with no visible button. a 3+ is when there is strong degrees of adherence to the monolayer (about 3/4ths), but can still have somewhat of a button in the middle. 2+ reactions resemble a large hole/blister where the RBCs are in the middle. A 1+ looks like a 'fuzzy button' in the middle for the RBCs while only 1/4th RBCs adhere to the monolayer. a negative reaction (0) is shown when there is no RBC adherence

How would you QC anti-Jkb antisera?

Use a known positive Jkb and a known negative Jkb to run alongside patients once every 24 hours. We use CorQC and Saline in lab.

In your own words, describe the principle of Solid Phase technology

Essentially it seems like we are adding RBC antigens to the bottom of the microtiter plate. Then, we are adding in patient serum/plasma. If there is binding to create an ABY/AGN complex, leftover ABY will washed away. If there is no binding (a neg test), the ABY will be washed away (after the incubation step). After the washing step, we are adding in indicator anti-IgG coated cells. When we centrifuge this, it will bring the IC in contact with the patient serum ABYs (that are attached to the RBC membrane-bound AGNS). In a positive test, the IC will be impeded/stopped bc of the anti-IgG and IgG complexes, this causes the indicator cells to bind to the screening cells at the bottom. In a negative test, the ABY from the patient would have never bound to the AGN on the titer plate, been washed away, and then the indicator cells would have centrifuged to the bottom and created a button without a problem

How do PeG and LISS work?

LISS (low ionic strength solution) works by canceling out the negative zeta potential that RBCs have

PeG works by removing H2O molecules which allows small IgG abs and RBCs to come into contact together

why do we not spin PeG after the 37° stage?

can cause anomalous precipitation of plasma proteins (spontaneous agglutination)

List the screens in order from the most sensitive to the least

SP/Gel > PeG > LISS > Albumin

unexpected antibodies are found in _____% of the population

0.3-3.0%

Explain why intact RBC's in urine are not indicative of a HTR?

An HTR is an intravascular hemolytic reaction, therefore intact RBCs would not be in the urine (free hgb would be)

provide 2 circumstances where an elution may be performed

1. identify ABYs that are coating cells of a patient with a positive DAT
2. isolate specific antibodies from plasma

explain the purpose of the last wash in an elution. What do we do with the last wash sample that's collected and why?

The last wash is to assure that the washing was adequate to remove unbound aby . We do this to detach the ABYS from the RBC and then keep the last wash to test it to assure the washing was adequate.

D frequency

85

C frequency

70

E frequency

30

c frequency

80%

e frequency

98%

Jk(a) frequency

77

Kell frequency

9

k frequency

99

Fy(a) frequency

66

Fy(b) frequency

83

A frequency

40

B frequency

11

O frequency

45

AB frequency

4

Major crossmatch

Donor RBC vs recipient serum

Minor Crossmatch

Donor serum vs recipient RBC