Immunohematology Lab Midterm Exam

What engineering controls are present within the blood bank lab?

Sharps disposal containers & biohazardous waste containers

What is the golden rule in blood bank?

When in doubt, throw it out

What is the difference between a blood bank and a blood center?

Bank: Tests patient blood products to prepare for transfusions

Center: Collects blood products for distribution

What tests are performed in a blood bank lab?

Blood group typing

Antibody screening

Antibody panels/ID

Crossmatching

Fetal screening

Other advanced techniques

What blood bank products are given to patients?

Whole blood

Packed RBCs

Leukocyte-poor RBCs

Irradiated blood

Fresh frozen plasma (FFP)

Cryoprecipitate

Platelets

Rh immune globulin

What is the primary goal of the blood bank?

Transfusion of a safe unit of blood

What must a requisition form include?

Patient's full name

DOB

Hospital MRN

Doctor's name

Date and tiem of order

Specific nature of the order

Reason for transfusion

Total number of blood components

What must a specimen be labelled with?

Patient's name

DOB

Unique identifier (MRN)

Phlebotomists initials

Date & time of collection

Under what conditions will a specimen be discarded?

Grossly hemolyzed

Unlabeled

Questionable integrity

Discrepancies in patient information

When must a specimen be labelled?

Prior to leaving the patient's room

What tubes are used for testing in blood bank?

EDTA

How many days after collection can patient specimens be used?

3 days typically

14 days for pre-admission testing

What are the blood bank pre-analytical rules?

1. Examine tubes to make sure they are free of debris

2. Hold saline bottle slightly away from tube to avoid contamination

3. Always label tubes fully and clearly

4. NEVER place any specimen or reagent in an unlabeled tube

What are the blood bank analytical rules?

1. Perform all tests according to manufacturer's directions

2. Add serum first and RBCs after (excluding gel)

3. Do not interchange dropper tops or pipettes, avoid cross-contamination

4. Hold all pipettes at same angle to get same drop size

5. Do not touch top of tube or pipette with bare hands (neutralizes Coombs reagent)

6. Always transport tubes in a test tube rack

7. Inspect each tube individually against a well-lit background

8. Use an optical aid to examine reactions that appear negative to the naked eye

What are the blood bank post-analytical rules?

1. Reactions must be read, graded and recorded immediately

2. Always hold the tube in your hand in front of the column while recording

3. Do not place tube in the rack until you have recorded the results

4. All transcription errors must have a single line through them with date and initials of technologist

5. Replace all specimens and equipment, wipe down area

What components are necessary in blood bank compliance for achieving good quality?

Quality control

Quality assurance

Utilization review

Continuous quality improvement

Well constructed SOPs (and personnel who adhere to them)

How are tubes labelled in blood bank testing?

With patient initials and test performed

How is quality control performed on the forward typing reagents?

Expecting 2-4+ reactions when adding

Anti-A: A cells

Anti-B: B cells

Anti-D: Screen cells I, II

Expecting negative reactions when adding

Anti-D: A or B cells

Albumin (D control): Screen cells

How is quality control performed on the reverse typing reagents?

Expecting negative reactions when adding

A1 cells: anti-B

B cells: anti-A

What is the ideal RBC suspension percentage for tube testing?

~3%

How do you prepare a 3% RBC suspension?

1-2 drops of RBCs, fill tube 3/4ths with saline

What might a red cell suspension that is too heavy or light produce?

False-positive or false-negative results

What factors affect hemagglutination?

Length of antibody

Zeta potential

Site of antigen on RBC surface

Incubation time

Temperature

Concentration of antigen/antibody

pH

What are antigens of the surface of RBCs made of?

Glycoproteins, complex carbohydrates, lipoproteins

How do genes determine one's ABO blood type?

They code for transferases which add a monosaccharide molecule onto a precursor substance on the RBC

What antibodies are in each blood type (A, B, AB, O)?

A: anti-B

B: anti-A

AB: none

O: anti-A & anti-B

Why is ABO blood typing the most significant test?

Because ABO incompatibility and transfusion can result in a severe/fatal hemolytic transfusion reaction

What does forward typing test for?

Presence or absence of A and B antigens

What does reverse typing test for?

Presence of antibodies for confirmation of the forward type

After A and B antigens, which is the next most important antigen in transfusion practice?

D

What does D typing test for?

Presence of D antigen; no anti-D antibodies are expected unless previously exposed

What does immunogenic mean?

Capable of producing an immune response

How does formation of anti-D occur?

Through exposure to D positive cells either through pregnancy or transfusion

What are the naturally occurring antibodies?

Anti-A and Anti-B

What are the possible mechanisms which might produce weak D?

Position effect

Weak D

Partial D

Why is a D control necessary?

It is a high protein media (similar to the anti-D reagent) and tests to confirm that agglutination with anti-D is not due to the high protein content, and is in fact due to presence of D antigen on RBCs

What should be done if a D control is positive?

Retesting or alternative testing; hold patient results

In what scenarios must a D control be run?

When the patient is typing as AB+ or when the patient is D negative

What are the five alleles in the Rh blood group system?

D, C, c, E, e

What portion of testing is the D control QC for with AB+ and D- patients?

AB+: IS phase

D-: AHG phase

What is the procedure for weak D testing?

1. Incubate D and D control tubes for 10-15 mins at 37 degrees

2. Wash 3x with saline

3. Add two drops AHG to both tubes

4. Spin 15 sec and read

What are the reagents used in IAT?

AHG (Antihuman globulin)

Check cells

What might cause a false negative with check cells?

Delay in adding AHG

Failure to add AHG

Inadequate washing

What blood can a patient with weak D vs partial D receive?

Weak D: D positive

Partial D: D negative

Which antibody isotype is largest?

IgM

Which antibody isotype can cross the placenta?

IgG

What are alloantibodies?

Antibodies found in the absence of the corresponding antigen

What are autoantibodies?

Antibodies found in the presence of the corresponding antigen

What are unexpected antibodies?

Any antibodies that aren't anti-A and anti-B (naturally occurring)

What makes an antibody clinically significant?

Reacts at 37 C and/or the AHG phase of testing, causes HTR, HDFN and/or shortened RBC survival

What are the limitations of antibody screening tests?

Cannot detect all antibodies of clinical significance if they correlate with low incidence antigens, or have a low titer

What are the different phases for screen testing?

IS

37 C

AHG

What is an ABO discrepancy?

A reaction in which the results obtained in the forward type do not match those found in the reverse type

What can cause unexpected negative or weak reactions in the forward type?

Elderly/newborn

Immunocompromised (leukemia)

Weak subgroups of A or B

How do you resolve unexpected negative or weak reactions in the forward type?

Incubate at room temperature for 15-30 mins

Check patient history

What can cause unexpected positive reactions in the forward type?

Acquired B

Rouleaux

Cold autoantibody

How do you resolve unexpected positive reactions in the forward type?

Check patient history

Acidify anti-B

Wash cells/ saline replacemnt

Run an autocontrol (pt RBC & plasma)

Perform DAT

What can cause unexpected negative or weak reactions in the reverse type?

Age

Immunodeficiency/Immunosuppression

Dilution of antibodies (transfusion, plasma exchange)

How do you resolve an unexpected negative or weak reaction in the reverse type?

Incubate reverse at room temp or at 4 C with an autocontrol

Check patient history

What can cause an unexpected positive in the reverse type?

Subgroup of A with anti-A1

Cold autoantibody/alloantibidy

Rouleaux

How do you resolve an unexpected positive in the reverse type?

Test patient cells with A1 lectin

Pre-warm technique

Saline replacement

Perform antibody ID

What is mixed field?

Reaction during typing when two distinct cell populations are present

When might you see mixed field?

Post transfusion

Bone marrow transplant

Chimerism

What is used to differentiate agglutination from rouleaux?

Microscopic detection and saline replacement

How do you perform saline replacement?

1. Combine patient plasma with reagent/donor RBCs

2. Centrifuge 15 sec

3. Remove plasma

4. Add 2 drops of saline and re-suspend

5. Spin 15 sec and read

How is the quantity of A antigen different between people with A1 vs A2 type blood?

A1 individuals presents more A antigen than A2 individuals

What conditions can rouleaux be associated with?

Multiple myeloma

Chronic inflammation

Waldenstrom's macroglobulinemia

How do you differentiate rouleaux from agglutination?

Rouleaux is a stacking of cells while agglutination is clumping of cells

What is the ABO type of screening cells? Which are D positive?

O-type (1 & 2 are positive, 3 is negative)

What are the components of an antibody panel identification test?

Patient plasma and 10 different reagent red cells

What must be run alongside each antibody panel?

Autocontrol

What can you determine if the autocontrol is positive?

There is an autoantibody present

When can you rule out an antigen on an antibody panel?

When the serum does not react with it (0), and the antigen is either homozygously positive, heterozygously positive twice, or heterozygously positive once if it is a low frequency antigen (excluding K)

What is the rule of three?

To rule in an antibody, the patients plasma must be positive on 3 cells with the antigen and negative on 3 cells lacking the antigen

What are the low frequency antigens?

K, Kpa, Jsa, Lua

When can anti-C and anti-E be ruled out on a heterozygous cell on an antibody panel?

When anti-D is present

Which antibodies need to be ruled out, and which don't?

Clinically significant antibodies must be rules out, low frequency antibodies do not need to be ruled out (unless there is evidence of a low frequency antibody)

What causes a positive reaction in gel to remain closer to the top?

Agglutination inhibits movement through the gel

What do the gel cards contain?

Microtubules with dextran acrylamide gel containing IgG

What cell suspension is used for gel testing?

0.8%

What could cause a false negative reaction in gel testing?

Too many or too few RBCs

How does rouleaux and mixed field reaction present in gel testing?

Agglutination at the top and free cells at the bottom

What is the solid phase method used for?

Antibody screening, identification and crossmatching

What components are used in solid-phase testing?

Microplate wells coated with red cell antigens, patient serum and indicator red cells (coated with IgG) for detecting bound antibodies

What do positive and negative reactions look like in solid phase testing?

Positive: Adherence of indicator cells to well surface

Negative: indicator cells settle at the bottom as a button

What are the advantages of solid phase testing?

Increased sensitivity

Standardized & automated

Requires smaller sample volumes

Reduces subjectivity

What are the limitations of solid phase testing?

Higher sensitivity may lead to false positives

Some antibodies react less than in other methods

Equipment and reagent costs

Requires staff training

What are the steps in gel testing?

1. Label gel cards with reagent red cell numbers (and an autocontrol)

2. Add 50 microliters of each red cell suspension to its corresponding microtube on the gel card (patient 0.8% suspension in autocontrol)

3. Add 25 microliters of patient plasma to each microtube in the gel card

4. Incubate at 37 C for 15-30 mins

5. Spin for 10 minutes and read

What results might an autoantibody produce on an antibody panel?

All positive results, including the autocontrol

How do cold autoantibodies react at each phase of testing?

Strongest at IS, weakens with 37 C and AHG

Are cold autoantibodies clinically significant?

No, but might interfere with detection of clinically significant antibodies

What happens if cold autoantibodies react at the AHG phase of testing?

They could interfere with the detection of clinically significant antibodies

What isotype is a cold autoantibody?

IgM

How do you resolve a cold autoantibody in antibody panel testing?

Warm the sample and testing reagents (including saline) to 37 C. Bypass IS testing and read reactions at AHG phase

How do you resolve a warm autoantibody in antibody panel testing?

Autoadsorption; incubate patient's serum with their own red cells to allow the RBCs to adsorb the autoantibodies, then use the serum for testing

When are select cells chosen?

When they have an antigen corresponding to an antibody that is trying to be ruled in or out

What does it mean if an antibody exhibits dosage?

It exhibits a weaker reaction with heterozygous antigen expression and stronger reactions with homozygous expression

What antigens are in the Kell system?

K, k, Kpa, Kpb, Jsa, Jsb

What antigens are in the Duffy system?

Fya and Fyb

What antigens are in the Kidd system?

Jka and Jkb