4.11 - Immunohematology Problem Solving

Is there a discrepancy between the following blood typing and Secretor study results?

Blood Typing Results

Anti-A: 4+, Anti-B: 0, A1 Cells: 0, B Cells: 4+

Secretor Results

Anti-A + saliva + A1 cells = 0

Anti-B + saliva + B cells = 4+

Anti-H + saliva + O cells = 0

A. No problem, the sample is from a group A secretor

B. Blood types as A and saliva types as B

C. Blood types as A, but the secretor study is inconclusive

D. No problem, the sample is from a group A nonsecretor

A. No problem, the sample is from a group A secretor

• Forward/Reverse type: Anti-A 4+, Anti-B 0, A1 cells 0, B cells 4+ → consistent with group A.

• Secretor inhibition:
  • Anti-A + saliva + A1 cells = 0: No agglutination → anti-A neutralized by A substance in saliva → A substance present.
  • Anti-B + saliva + B cells = 4+: Agglutination → anti-B not neutralized, still active → no B substance in saliva.
  • Anti-H + saliva + O cells = 0: No agglutination → H substance present in saliva.

• Secretors with group A secrete A and H (not B) → results are consistent.

Summary
Blood type A; saliva contains A and H → A secretor, no discrepancy.

What is the best course of action given the following test result? (Assume the patient has not been transfused recently.)

Anti-A: Mixed Field

Anti-B: 0

A1 Cells: 1+

B Cells: 4+

A. Nothing, typing is normal

B. Type patient cells with Anti-A1 lectin and type serum with A2 cells

C. Retype patient cells; type with Anti-H and Anti-A,B; use screen cells or A2 cells on patient serum; run patient autocontrol

D. Wash patient cells four times with saline; then repeat the forward type

C. Retype patient cells; type with Anti-H and Anti-A,B; use screen cells or A₂ cells on patient serum; run patient autocontrol

• Mixed-field Anti-A and weak 1+ reaction with A₁ cells suggest A₃ or A subvariant — not recent transfusion (ruled out).

• Comprehensive retyping helps confirm a weak subgroup of A or cold alloantibody.

• Anti-H helps assess H antigen strength (increased in A subgroups).

• Anti-A,B aids in detecting weak A antigen expression.

• Reverse grouping with screen or A₂ cells differentiates A₃ vs. A₂ with Anti-A₁.

• Autocontrol rules out autoagglutination.

• A/B/D: Too limited — they fail to characterize a possible subgroup of A.

Summary:
Mixed-field Anti-A and weak reverse grouping indicate A subgroup discrepancy.
Best approach: repeat typing with Anti-H and Anti-A,B, test with A₂ or screen cells, and run an autocontrol.

The following results were obtained on a 41 yo female:

Anti-A 4+

Anti-B 0

A1 Cells 3+

B Cells 3+

O Cells 3+

Due to the discrepancy reverse grouping, a panel was performed on patient serum revealing Anti-M. How can the reverse grouping be resolved?

A. Repeat reverse grouping with a 10-min incubation at room temp

B. Repeat reverse grouping using A1 cells that are negative for M antigen

C. Repeat reverse grouping using A1 cells that are positive for M antigen

D. No further work is necessary

B. Repeat reverse grouping using A1 cells that are negative for M antigen

• Anti-M (cold IgM) reacts with M+ reagent cells in reverse grouping → false positives (A₁, B, O cells 3+)

• Resolve by using M– reverse cells (A₁ M– and B M–) or by prewarming to suppress cold IgM

• A: Room-temp incubation maintains Anti-M reactivity → still discrepant

• C: Using M+ A₁ cells worsens interference

• D: Discrepancy present; further work is necessary

Summary:
Reverse-grouping discrepancy from Anti-M is corrected by testing with M-negative reverse cells (and/or prewarm technique).

The following results were obtained on a 51 yo male with Hepatitis C:

Anti-A 4+

Anti-B 4+

Anti-D 3+

A1 cells 0

B cells 0

What should be done next?

A. Retype the patient's sample to confirm group AB positive

B. Repeat the Rh typing

C. Run a saline control in forward grouping

D. Report the patient as group AB, Rh positive

C. Run a saline control in forward grouping

• In hepatitis and other liver diseases, plasma proteins or abnormal globulins can cause false-positive agglutination in forward typing (especially with high-titer anti-A and anti-B).

• A saline control (Rh control) is used to detect false agglutination due to rouleaux or nonspecific protein coating of cells.

• If the saline control is positive, results must be interpreted cautiously or repeated with washed cells.

• A & D: Don’t confirm whether agglutination is real or nonspecific.

• B: Rh typing already clear; not the issue.

Summary:
Viscous plasma or abnormal globulins can cause false-positive forward grouping; confirm results by running a saline control to detect rouleaux or protein interference.

An Rh phenotyping shows the following results:

Anti-D 4+

Anti-C 2+

Anti-E 0

Anti-c 0

Anti-e 3+

What is the most likely Rh genotype?

A. R1r'

B. R0r

C. R1R1

D. R1r

C. R1R1

• Phenotype: D+, C+, E−, c−, e+ → fits DCe/DCe (R1R1)

• c− excludes haplotypes carrying c (e.g., r[dce], R0[Dce])

• Most likely choice: R1R1 is far more common than R1r′ (DCe/dCe)

• A (R1r′ = DCe/dCe): Also yields D+, C+, E−, c−, e+ but is much rarer

• B (R0r = Dce/dce): Would be c+ and C− → mismatch

• D (R1r = DCe/dce): Would be c+ → mismatch

Summary:
The D+C+E−c−e+ phenotype is best explained by R1R1 (DCe/DCe); R1r′ could mimic it but is significantly less common.

An OB patient, 34 weeks pregnant, shows a positive ABSC at the IAT phase of testing in screening cells I and II; screening cell III was neg. She is group B, Rh neg. This is her first pregnancy. She has no prior hx of transfusion. What is the most likely explanation for the positive ABSC?

A. She has developed an Ab to fetal RBCs

B. She probably does not have antibodies because this is her 1st pregnancy

C. She received an antenatal dose of RhIg

D. Impossible to determine without further testing

C. She received an antenatal dose of RhIg

• Rh– OB patients receive RhIg at ~28 wks → passive anti-D detectable at IAT (AHG)

• Timing fits (patient at 34 wks) and no transfusion history

• Passive anti-D often causes screen positive with no hemolysis/HDFN

• A: Possible allo-anti-D, but first pregnancy + typical 28-wk prophylaxis makes passive anti-D more likely

• B: Incorrect—first pregnancy ≠ no antibodies (RhIg or true alloimmunization can be present)

• D: Further testing helps, but clinical context strongly favors RhIg

Summary:
At 34 weeks in an Rh-negative primigravida, a new IAT-positive ABSC is most likely passive anti-D from 28-week RhIg prophylaxis.

A patient's serum contains a mixture of antibodies. One of the antibodies is identified as Anti-D. Anti-Jka or Anti-Fya and possibly another antibody are present. What technique(s) may be helpful to identify the other antibody(s)?

A. Enzyme panel; select cell panel

B. Thiol reagents

C. Lowering the pH and increasing the incubation time

D. Using albumin as an enhancement medium in combination with selective adsorption

A. Enzyme panel; select cell panel

• Enzyme panel (e.g., ficin, papain) enhances or destroys certain antigens, helping separate overlapping antibody reactions.

  • Enhances: Rh, Kidd, Lewis, P1, I

  • Destroys: MNS, Duffy

• Select cell panels allow use of RBCs with known antigen profiles to isolate which antibodies remain reactive.

• This combination is ideal for sorting multiple alloantibodies (e.g., Anti-D + Anti-Jka + Anti-Fya).

• B: Thiol reagents (DTT, 2-ME) destroy Kell antigens—useful for anti-K, not here.

• C/D: Altering pH or albumin use has minimal value for resolving mixed antibodies.

Summary:
Use enzyme and select cell panels to distinguish coexisting antibodies (e.g., Anti-D, Anti-Jka, Anti-Fya) by exploiting differences in antigen sensitivity to enzymes.

An Anti-M reacts strongly through all phases of testing. Which of the following techniques would NOT contribute to removing this reactivity so that more clinically significant antibodies may be revealed?

A. Acidifying the serum

B. Prewarmed technique

C. Adsorption with homozygous cells

D. Testing with enzyme-treated RBCs

A. Acidifying the serum

• Anti-M often shows variable reactivity; when it reacts at 37 °C and AHG, it can mask other antibodies.

• Acidifying the serum (lowering pH to ~6.5) enhances Anti-M reactivity rather than weakening it — making it harder to remove.

• The following do help minimize Anti-M interference:

  • Prewarm technique — prevents cold IgM binding.

  • Adsorption with M+ cells — removes Anti-M from serum.

  • Enzyme-treated RBCs — destroy M antigen, eliminating reactivity.

Summary:
Acidification enhances Anti-M, so it would not help reduce its reactivity when trying to uncover other clinically significant antibodies.

The reactivity of an unknown antibody could be Anti-Jka, but the antibody identification panel does not fit this pattern conclusively. Which of the following would NOT be effective in determining if the specificity is Anti-Jka?

A. Testing with enzyme-treated cells

B. Select panel of homozygous cells

C. Testing with 2-aminoethylisothiouronium bromide (AET)-treated cells

D. Increased incubation time

C. Testing with 2-aminoethylisothiouronium bromide (AET)-treated cells

• Kidd (Jka) features: enhanced by enzymes; shows dosage; can be weak/variable → benefits from longer 37 °C incubation/PEG

• AET primarily denatures Kell antigens; doesn’t aid in confirming Anti-Jka specificity

• Enzyme-treated cells: ↑ reactivity of Kidd → supportive for Anti-Jka

• Select homozygous cells: demonstrates dosage (Jk(a+a−) vs Jk(a−b+))

• Increased incubation time: helps detect weak Kidd antibodies

Summary:
AET targets Kell, not Kidd—so it’s not effective for confirming Anti-Jka.

A cold-reacting antibody is found in the serum of a recently transfused patient and is suspected to be Anti-I. The antibody ID panel shows reactions with all cells at room temperature, including the autocontrol. The reaction strength varies from 2+ to 4+. What procedure would help to distinguish this antibody from other cold-reacting antibodies?

A. Autoadsorption technique

B. Neutralization using saliva

C. Autocontrol using ZZAP reagent-treated cells

D. Reaction with cord blood cells

D. Reaction with cord blood cells

• Anti-I reacts with adult (I+) RBCs but not with cord (i+) cells, since I antigen is weak or absent at birth.

• Testing with cord blood cells differentiates Anti-I from other cold-reactive antibodies like Anti-IH or Anti-H.

• Pattern: Adult cells = positive, cord cells = negative → confirms Anti-I.

• A: Autoadsorption removes autoantibody but doesn’t identify specificity.

• B: Neutralization with saliva is for Lewis or P1, not I.

• C: ZZAP removes autoantibody but doesn’t confirm specificity.

Summary:
To confirm Anti-I, test serum with cord (i+) cells—lack of reactivity verifies the antibody as Anti-I.

An antibody identification panel reveals the presence of Anti-Leb and a possible second specificity. Saliva from which person would best neutralize the Leb antibody?

Genes (Pattern):

Lewis, ABO, Secretor

A. Le, H, sese

B. Le, hh, Se

C. Le, H, Se

D. lele, hh, sese

C. Le, H, Se

• Lewis system: soluble Leᵃ and Leᵇ antigens form in secretions of individuals with both Le and Se genes.

  • Le, H, Se → Le(b+) — saliva contains Leᵇ substance that can neutralize Anti-Leᵇ.

  • Le, H, sese → Le(a+), contains only Leᵃ, not Leᵇ.

  • Le, hh, Se → no H substance, thus no Leᵇ expression.

  • lele → no Lewis antigens at all.

• Therefore, only Le, H, Se individuals have Leᵇ in saliva capable of neutralizing Anti-Leᵇ.

Summary:
Saliva from Le, H, Se individuals contains Leᵇ antigen, which will neutralize Anti-Leᵇ in antibody testing.

The automated blood bank analyzer does not detect weak forms of D antigen. Why would running type and screens on the analyzer prevent a patient with a weak D phenotype from forming Anti-D?

A. Weak D persons cannot form Anti-D

B. The analyzer would show the sample as Rh negative; the patient would receive Rh-negative blood

C. The analyzer would show the sample as Rh positive; the patient would receive Rh-positive blood

D. A and C

B. The analyzer would show the sample as Rh negative; the patient would receive Rh-negative blood

• Automated analyzers may miss weak D expression, typing such patients as Rh negative.

• As a result, they’re transfused with Rh-negative blood, preventing exposure to D antigen and avoiding Anti-D formation.

• Therefore, even though the analyzer doesn’t detect weak D, the transfusion practice protects against alloimmunization.

• A: Incorrect — some weak D variants can make Anti-D.

• C/D: Incorrect — analyzer won’t show Rh positive; it reads as Rh negative.

Summary:
If a weak D patient is mistyped as Rh negative, they’ll receive Rh-negative blood, preventing Anti-D formation despite the analyzer’s limitation.

A cord blood workup was ordered on baby boy Jones. The mother is O neg. Results on the baby are as follows:

Anti-A 4+

Anti-B 0

Anti-A,B 4+

Anti-D 0

DAT (poly) 2+

The test for Weak D on the baby was positive at the AHG phase. Is the mother an RhIg candidate?

A. No, the baby is Rh positive

B. Yes, the baby's Rh type cannot be determined because of the positive DAT result

C. No, the baby is Rh negative

D. Yes, the mother is Rh negative

B. Yes, the baby’s Rh type cannot be determined because of the positive DAT result

• A positive DAT indicates antibody coating on the infant’s RBCs, which can interfere with Rh typing by blocking D antigen sites and causing false negatives or variable reactions.

• Even though the weak D test was positive, results may not be reliable when a DAT is positive.

• In this situation, the infant’s true D status is uncertain, and RhIg should be administered to the Rh-negative mother only if D typing is inconclusive or negative.

• A/C: Cannot definitively call the baby Rh positive or negative because of DAT interference.

• D: Although mother is Rh negative, the uncertain infant Rh type drives the decision — not maternal status alone.

Summary:
A positive DAT can invalidate weak D results; therefore, the baby’s Rh type cannot be confirmed, and the mother is treated as an RhIg candidate pending clarification.

RBCs from a recently transfused patient were positive on DAT when tested with Anti-IgG. Screen cells and a panel performed on a patient's serum showed very weak reactions with inconclusive results. What procedure would help to identify the antibody?

A. Elution followed by a panel on the eluate

B. Adsorption followed by a panel on the adsorbed serum

C. Enzyme panel

D. Antigen typing the patient's RBCs

A. Elution followed by a panel on the eluate

• Recently transfused + DAT (IgG+) → likely alloantibody coating donor RBCs

• Elution recovers bound IgG from patient RBCs; test eluate against a panel to ID specificity

• Serum screen/panel weak/inconclusive → eluate often reveals the culprit Ab

• B (Adsorption): Complicated post-transfusion (mixed donor cells); risks removing the clinically significant Ab from serum

• C (Enzyme panel): May change reactivity patterns but won’t extract the bound IgG needed here

• D (Antigen typing patient RBCs): Unreliable after transfusion (mixed field); may type donor antigens

Summary:
In a recently transfused, DAT (IgG+) patient with weak serum reactivity, elute the patient RBCs and panel the eluate to identify the alloantibody coating donor cells.

A patient types as O positive. All three screening cells and RBCs from two O-positive donor units show agglutination after incubation at 37C and increase in reactivity at the IAT phase of testing. What action should be taken next?

A. Perform an autocontrol and DAT on the patient

B. Perform an enzyme panel

C. Perform an elution

D. Choose another 2 units and repeat crossmatching

A. Perform an autocontrol and DAT on the patient

• Panreactivity at 37 °C → stronger at IAT suggests warm autoantibody (IgG)

• Next step: Autocontrol + DAT to confirm RBC-bound IgG/C3 (autoimmune hemolysis)

• Guides further workup (adsorptions, phenotype/genotype, least incompatible units)

• B: Enzymes won’t distinguish auto vs allo panagglutination

• C: Elution is premature without confirming DAT+

• D: Trying more units won’t fix autoantibody interference

Summary:
Panreactivity at 37 °C/AHG → suspect warm autoantibody; do autocontrol and DAT first to confirm and guide transfusion strategy.

Four units of blood are ordered for a patient. Blood bank records are checked and indicate that 5 years ago this patient had an Anti-Jkb. What is the next course of action?

A. Antigen type units for the Jkb antigen, and only crossmatch units positive for Jkb

B. Antigen type units for the Jkb antigen, and only crossmatch units negative for Jkb

C. Randomly pull 4 units of blood that are ABO compatible, and perform crossmatching

D. Perform IS crossmatching on 4 Jkb-negative units

B. Antigen type units for the Jkb antigen, and only crossmatch units negative for Jkb

• History of Anti-Jkb (Kidd) = clinically significant even if not currently detectable

• Provide Jkb-negative RBCs and perform AHG crossmatch (history trumps current screen)

• Prevents delayed hemolytic transfusion reactions common with Kidd antibodies

• A: Giving Jkb-positive risks hemolysis

• C: Random units may be Jkb+; unsafe

• D: IS crossmatch is inadequate; must use AHG due to significant antibody history

Summary:
With a prior Anti-Jkb, select Jkb-negative, AHG-crossmatched units—treat as significant regardless of current screen.

A 56 yo patient diagnosed with colon cancer demonstrates a positive antibody screen in all three screening cells at the AHG phase. The panel study shows 10 cells as positive as well as the autocontrol at the AHG phase. The reactions varied from 1+ to 3+. This patient had a history of receiving 2 units of blood approximately 1 month ago. What should be done next?

A. Perform a DAT on patient cells

B. Perform an autoadsorption

C. Perform an alloadsorption

D. Issue O-negative cells

C. Perform an alloadsorption

• All screening and panel cells positive + autocontrol positive → warm autoantibody (IgG).

• History of transfusion 1 month ago → patient’s RBCs are mixed donor + self, so autoadsorption is unreliable (donor cells could adsorb alloantibodies).

• Alloadsorption uses RBCs of known phenotypes (e.g., R1R1, R2R2, rr) to remove the autoantibody and uncover underlying alloantibodies.

• A: DAT already implied by autocontrol reactivity.

• B: Autoadsorption invalid—recent transfusion.

• D: Unsafe; antibody specificity unresolved.

Summary:
In a recently transfused, DAT-positive patient, perform an alloadsorption to remove autoantibody and identify hidden alloantibodies.

An O-neg mother with no record of any previous pregnancies gives birth to her 1st child, a B-pos baby. The baby's DAT is weakly pos & the saline control is neg. The ABSC is also neg. The baby appears healthy but after 2 days develops mild jaundice, which is treated with phototherapy. After 4 days, the baby goes home w/o complications. What is the most likely explanation for the weakly pos DAT result?

A. Technical error

B. A low-titer Anti-D

C. Immune Anti-B from the mother

C. Immune Anti-B from the mother

• O mothers make IgG anti-A,B → crosses placenta; B-positive infant gets weakly + DAT

• ABSC negative because screen cells are group O (no A/B antigens)

• Clinical fit: mild jaundice at 2 days → ABO HDFN (typically mild, phototherapy effective)

• Saline control negative → rules out nonspecific agglutination/rouleaux

• A (Technical error): Less likely with consistent serology + clinical course

• B (Low-titer Anti-D): Primigravida, maternal screen negative; ABO HDFN explains findings better

Summary:
Mild ABO HDFN from maternal IgG anti-A,B coating a B-positive newborn’s RBCs causes a weakly positive DAT with a negative maternal antibody screen.

A 59-year-old male came to the emergency department of a community hospital complaining of dizziness and fatigue. History included no transfusions and a positive rheumatoid factor 1 year ago. His complete blood count (CBC) confirmed anemia.
A sample was sent to the blood bank for typing and crossmatching. Upon receipt of the sample in the blood bank, the MLS noticed the EDTA sample appeared very viscous. Fearing that the sample would clog the automated instrument, testing was performed using the tube method.

The initial results revealed the following:

Test	Result
Anti-A	0
Anti-B	0
Anti-D	4+
Rh Control	2+
A₁ cells	4+
B cells	4+

The patient’s RBCs were washed eight times with saline, and testing was repeated, giving the following results:

Test	Result
Anti-A	0
Anti-B	0
Anti-D	4+
Rh Control	0
A₁ cells	4+
B cells	4+

The antibody screen was negative at the IS, 37 °C, and AHG phases; the check cells were positive.
Crossmatch testing using two O-positive donor units revealed 1+ at IS, and negative results at 37 °C and AHG phases. The check cells were positive.

Question:
In light of the crossmatching results, what is the next course of action?

Options:
A. Use other donor cells for crossmatching
B. Perform a saline replacement for crossmatching
C. Run the crossmatch test using the Gel system
D. Result the crossmatch as incompatible

B. Perform a saline replacement for crossmatching

• The 1+ reaction at IS with negative results at 37 °C and AHG, plus a viscous EDTA sample and prior positive rheumatoid factor, suggests rouleaux formation causing false-positive agglutination.

• Saline replacement removes excess plasma proteins, dispersing rouleaux and confirming whether the reaction is true agglutination or artifact.

• After saline replacement, true antibody-mediated agglutination will persist; rouleaux will disappear.

• A: Unnecessary — the issue lies in the plasma, not donor cells.

• C: Gel won’t correct rouleaux interference.

• D: Incorrect — do not report incompatible until rouleaux is ruled out.

Summary:
Viscous plasma + 1+ IS reaction = likely rouleaux.
Next step: perform a saline replacement crossmatch to confirm compatibility.

A 33-year-old maternity patient has blood drawn for a type and screen at 36 weeks’ pregnancy. The following results are found on the automated blood bank analyzer:

Anti-A	Anti-B	Anti-A,B	Anti-D	A₁ cells	B cells
3+	0	4+	4+	2+	4+

SC I	SC II	SC III	A₁ lectin
0	0	0	3+

The reference laboratory identified anti-P₁ in the patient plasma by using enzyme techniques.

Question:
How could the ABO discrepancy be solved?

Options:
A. Wash the patient’s RBCs, and repeat the forward grouping
B. Test the patient’s plasma against A₂ cells
C. Warm the patient’s plasma at 37°C for 10 minutes and repeat the reverse grouping
D. Treat the A₁ cells with DTT, and repeat the reverse grouping

C. Warm the patient’s plasma at 37°C for 10 minutes and repeat the reverse grouping

• Identified anti-P₁ (cold, usually IgM) → reacts in reverse typing at RT

• Reverse shows unexpected 2+ with A₁ cells; B cells 4+ fits group A

• Warming plasma (prewarm technique) suppresses cold antibody reactivity → resolves discrepancy

• Alternative (not in options): neutralize with P₁ substance (pigeon egg albumin/hydatid cyst fluid)

• A: Washing patient RBCs affects forward grouping; problem is in reverse

• B: Testing against A₂ cells doesn’t address anti-P₁

• D: DTT won’t resolve anti-P₁ interference

Summary:
ABO discrepancy from anti-P₁ (cold) is corrected by warming the plasma and repeating reverse typing; results should confirm group A, D positive.