ABO discrepancies
Transcript summary in points
ABO Discrepancies
Understanding Forward and Reverse Typing:
Forward Typing: Determines antigens present on the patient’s red blood cells using known antisera (anti-A, anti-B).
Reverse Typing: Detects antibodies present in the patient’s plasma using known reagent red blood cells (A1 cells, B cells).
Expected Concordance: In a correct ABO type, the forward and reverse typing results should be concordant and confirm the same blood type.
Types of ABO Discrepancies:
Definition of Discrepancy: An ABO discrepancy occurs when the forward and reverse typing results do not agree, indicating a potential problem with the ABO typing.
Four Categories of Discrepancies:
Missing Reaction in Forward Typing (Cells): An expected agglutination reaction is absent or weaker than expected in the forward typing.
Extra Reaction in Forward Typing (Cells): An unexpected agglutination reaction is present in the forward typing.
Missing Reaction in Reverse Typing (Plasma): An expected agglutination reaction is absent or weaker than expected in the reverse typing.
Extra Reaction in Reverse Typing (Plasma): An unexpected agglutination reaction is present in the reverse typing.
Discrepancy Resolution – General Principles:
Lab Manual for Directions: Utilize the lab’s standard operating procedure (SOP) manual for specific steps in resolving ABO discrepancies.
Assuming Stronger Reaction is Correct: In discrepancy investigation, generally assume that the stronger agglutination reactions are more likely to be correct. This serves as a starting point for investigation.
Homework Example Context: Referencing the discrepancy homework as a tool to think about the reverse scenario, where reactions are given, and you need to determine the cause.
Discrepancy 1: Weak Reverse Reactions (Missing Reaction in Reverse)
Problem Identification: Missing or weak reaction observed in the reverse typing, specifically with reagent B cells in this example.
Forward Type Interpretation: Forward typing with strong anti-A reaction suggests the patient is likely blood type A.
Discrepancy Focus: The weak reaction with B cells in the reverse is the discrepancy needing resolution.
Possible Cause – Weak Reverse Reactions: Consider causes for weak or missing antibody reactions in the reverse type.
Immunocompromised Patients: Patients with weakened immune systems may produce lower levels of ABO antibodies, leading to weak reverse reactions.
Elderly Patients: Older individuals may have diminished antibody production due to age-related immune senescence.
Age as a Factor: Age is a significant consideration; weak reverses are more common and expected in older patients (e.g., 96-year-old example).
Importance of Patient Demographics: Emphasizing the need to consider patient information when investigating discrepancies.
Patient Diagnosis: Underlying medical conditions, especially those affecting the immune system.
Patient Age: Crucial for interpreting weak reverse reactions.
Pregnancy Status: Relevant in some blood banking contexts.
Resolution Strategy – Enhancing Antibody Reaction: To strengthen weak IgM antibody reactions (ABO antibodies are IgM), consider methods to enhance IgM agglutination.
Extended Incubation: Prolonging incubation time can enhance weak reactions.
Room Temperature Incubation (15 minutes): Incubate reverse typing reactions for an additional 15 minutes at room temperature.
Cold Incubation (4°C for 15 minutes): Incubate reverse typing reactions for 15 minutes at 4°C (refrigerator temperature) to enhance IgM reactions, which are often “cold-loving.”
Documentation on Worksheet: Record any additional incubation steps taken on the ABO discrepancy worksheet or ADL (Automated Data Logger) worksheet.
Example Documentation: “15 minutes at 4 degrees”.
ABO Discrepancy Worksheet Use: Highlighting that ABO discrepancy worksheets often have sections to document extended incubation procedures.
Worksheet Sections: Sections for “Immediate Spin,” “15 Minutes,” “4 Degrees,” “15 Minutes,” to document reactions at different stages of resolution.
Reaction Strength Improvement: Extended incubation, especially at 4°C, may strengthen weak reactions (e.g., from 1+ to 2+ or 3+).
Cautious Approach: While investigation is important, avoid excessive manipulation when a clear cause (like age) is apparent.
Initial Patient Encounters – Thoroughness: For a patient’s first encounter, more extensive investigation is generally warranted to ensure accurate typing and competency in resolution.
Discrepancy 2: Extra Reaction in Forward Typing (Extra Reaction in Cells)
Problem Identification: Extra reaction observed in the forward typing, specifically with anti-B antisera.
Reverse Type Interpretation: Reverse typing suggests blood type A (no reaction with A1 cells, strong reaction with B cells).
Discrepancy Focus: The extra reaction with anti-B in the forward typing is the discrepancy.
Possible Cause – Acquired B Antigen: Consider Acquired B phenomenon as a common cause of extra forward reactions with anti-B.
Acquired B – Board Exam Relevance: Acquired B is a classic board exam topic.
Diagnosis Association: Associated with certain medical conditions, particularly:
Colon Cancer.
Intestinal Bacterial Infections.
Any condition involving intestinal bacteria.
Mechanism: Bacterial enzymes can modify the A antigen on red cells to resemble B antigen, causing false-positive reactions with some anti-B reagents.
Resolution – Clinical Context: Resolution primarily involves clinical correlation and understanding the transient nature of Acquired B.
Diagnosis Review: Check patient diagnosis for conditions associated with Acquired B.
Transient Nature: Acquired B is typically temporary and resolves once the underlying infection or condition is treated.
Limited Lab Resolution: There’s not much lab intervention to “resolve” Acquired B while it’s present.
Documentation: Document the discrepancy and the suspected cause (Acquired B) in lab records.
Current Reagent Impact – Monoclonal Anti-B: Mentioning that Acquired B is less frequently seen in modern labs due to the use of monoclonal anti-B reagents.
Monoclonal vs. Polyclonal Reagents: Contrasting monoclonal anti-B (current) with polyclonal anti-B (older).
Polyclonal Reagent Sensitivity: Polyclonal anti-B reagents were more prone to reacting with the weakly modified “B-like” antigen in Acquired B.
Monoclonal Specificity: Monoclonal anti-B reagents are generally more specific and less likely to cause false-positive reactions in Acquired B.
Decreased Clinical Significance: Acquired B is less of a practical problem in current transfusion practice due to reagent improvements.
Board Exam Importance Remains: Despite reduced clinical relevance, Acquired B remains important knowledge for board exams due to its historical significance and conceptual understanding of ABO variants.
Polyclonal Reagent Issue – Historical Context: Acquired B was more of a problem historically when polyclonal anti-B reagents were in common use.
Polyclonal Reagent Unspecificity: Polyclonal reagents, by nature, contain a mixture of antibodies that can have broader reactivity, including with weakly cross-reacting antigens like Acquired B.
Discrepancy 3: Extra Reaction & Weak Reaction in Forward Typing (Subgroup of A)
Slide Typing Example: Using a slide typing method example to illustrate reactions (common for quick ABO typing, donor centers, or educational kits).
Slide Typing Procedure: Drop of blood on slide, add antisera (anti-A, anti-B), mix with stick, observe for agglutination.
Problem Identification: Extra reaction in forward typing (with anti-B) and a weak reaction with anti-A.
Reaction Pattern Analysis:
Strong Anti-B Reaction: Suggests presence of B antigen.
Weak Anti-A Reaction: Weaker than expected reaction with anti-A.
Stronger Reaction Guidance: Following the principle of assuming stronger reactions are more likely correct, the strong anti-B reaction is taken as a primary indicator.
Possible Cause – Subgroup of A (with anti-A1): Consider a subgroup of A phenotype, specifically one that produces anti-A1 antibody.
Subgroups of A and Anti-A1: Subgroups of A can sometimes produce antibodies against A1 antigen.
A1 Antibody Production Variability: Some subgroups of A make anti-A1, others do not.
A2 Phenotype and Anti-A1: Specifically mentioning A2 phenotype in relation to anti-A1.
A2 Prevalence: A2 is a relatively common A subgroup, accounting for about 20% of A phenotype individuals.
A2 Anti-A1 Variability: Not all A2 individuals make anti-A1; some do, some do not.
“Lesser Minus” Answer in Homework: Referencing “lesser minus” reaction strength in the homework as representing the variable presence of anti-A1 in A2 phenotypes.
A2 Phenotype – Variable Anti-A1: A2 phenotype can be A2 with or without anti-A1 antibody.
Resolution – Subgroup Investigation with Additional Reagents: Use additional reagents to characterize A subgroups.
A2 Cells (Reagent Cells): Test patient plasma with reagent A2 cells.
Rationale: If patient has anti-A2 antibody (unlikely in A subgroups, but tested for completeness), it would react with A2 cells.
Normal A2 Phenotype Reaction: A normal A2 phenotype should not have anti-A2. Therefore, reaction with A2 cells should be negative (or weak/negative).
A2 vs. A1 Reaction Comparison: Compare reaction strength with A2 cells (should be 0 or weak) to reaction with A1 cells (expected 1+ in example).
Anti-A1 Lectin: Use anti-A1 lectin reagent.
Lectins – Seed-Derived Reagents: Lectins are serum-like reagents derived from plant seeds (or other sources).
Historical/Empirical Discovery: Lectins were discovered through empirical testing of seed extracts with blood cells to observe specific reactions.
Anti-A1 Lectin Specificity: Anti-A1 lectin is specific for A1 antigen and will agglutinate A1 red cells but not A2 red cells (or weaker A subgroups).
A1 Lectin Use in Subgrouping: Anti-A1 lectin helps differentiate A1 from A2 and weaker A subgroups.
Reagent Rack Availability: Checking if anti-A1 lectin is currently available in the lab’s reagent inventory.
A1 Lectin Reaction with A1 Phenotype: Anti-A1 lectin will react with A1 phenotypes (positive agglutination).
A1 Lectin Reaction with A2 Phenotype: Anti-A1 lectin will not react with A2 phenotypes (negative agglutination).
Confirming A2 Subgroup with Anti-A1 and A2 Cells: Combined results of A2 cell testing (negative/weak) and anti-A1 lectin testing (negative) would confirm A2 subgroup phenotype.
Reporting Anti-A1 in A2 Subgroups – Clinical Significance: When an A2 subgroup is identified and anti-A1 is present, it is clinically important to report the anti-A1.
Clinical Relevance of Anti-A1: Presence of anti-A1 in A2 individuals has transfusion implications.
A2 to A Red Blood Cell Transfusion Avoidance: While A2 individuals are blood type A, they should not receive A red blood cells for transfusion (ideally).
Anti-A1 Reaction with A Red Cells: A red blood cell units are typically A1 or a mix of A1 and A2. Anti-A1 in an A2 patient can react with transfused A1 red cells.
Transfusion Reaction Risk: Transfusing A1 red cells to an A2 patient with anti-A1 can cause a transfusion reaction (though often mild).
O Blood for A2 Subgroups: For A2 subgroup patients with anti-A1, it is safest to transfuse O blood red cells instead of A red cells to avoid potential reactions.
Thinking Ahead – Safe Transfusion Practice: Emphasizing proactive consideration of antibody specificity for safe transfusion.
Discrepancy 4: Missing Reverse Reaction (Missing Reaction in Reverse – with A1 Cells)
Problem Identification: Missing reaction in the reverse typing, specifically with reagent A1 cells.
Forward Type Interpretation: Forward typing suggests blood type O (no reaction with anti-A, no reaction with anti-B).
Reverse Type Inconsistency: Reverse typing should show reactions with both A1 and B cells for type O, but reaction with A1 cells is missing in this discrepancy.
Possible Cause – Subgroup of A or B (Weak A Subgroup): Consider a very weak subgroup of A or B that may not express antigens strongly enough for typical forward reactions and may have atypical antibody patterns.
Anti-A,B Reagent Use: Utilize anti-A,B reagent (anti-A,B serum) to investigate weak subgroups.
Anti-A,B – IgG and IgM: Anti-A,B reagent contains both IgM and IgG antibodies against A and B antigens.
IgM for Immediate Spin, IgG for Crosslinking: IgM component allows for immediate spin agglutination; IgG component can enhance reactions and detect weaker antigens.
Stronger Reactions with Anti-A,B: Anti-A,B often gives stronger reactions with weak A or B subgroups compared to standard anti-A or anti-B reagents alone.
Subgroup Detection Aid: Anti-A,B can help detect weakly expressed A or B antigens that might be missed by routine anti-A and anti-B.
Anti-A,B Reaction in Subgroup Scenario: If it’s a weak A subgroup, anti-A,B is likely to show a positive reaction, even if anti-A is negative or weak.
Ruling Out O Type: Positive reaction with anti-A,B would argue against true blood type O, as type O red cells should not react with anti-A,B.
Questioning O Type Diagnosis: Considering if the initial interpretation of type O (based on negative forward reactions) was incorrect.
O Type Antibody Expectation: True type O individuals should produce both anti-A and anti-B antibodies, which should react strongly with both A1 and B reagent cells in reverse typing (unless there are other issues like weak antibodies, as in Discrepancy 1).
Ax Subgroup Example: Mentioning Ax subgroup as a very weak A subgroup.
Ax Reaction Variability: Ax reactions can be very weak and variable, sometimes reacting with anti-A,B, sometimes not.
Very Weak Subgroups – Absorption/Elution: Extremely weak subgroups may require absorption and elution techniques to detect A antigen presence.
Genotype for Definitive Resolution: Genotyping (DNA-based ABO typing) is the most definitive method to resolve complex ABO discrepancies, especially involving weak subgroups.
Genotyping Cost: Acknowledging that genotyping is more expensive than routine serological testing.
Hospital Cost Consideration: Hospitals may be reluctant to use genotyping routinely due to cost.
Serology Limitations: Highlighting the limitations of serological methods in definitively characterizing very weak or variant ABO types.
Considering O Type with Atypical Antibodies: Acknowledging the possibility of a true O type patient with an unusual antibody profile, but emphasizing that subgroup investigation is crucial first.
Normal O Antibody Production: Typical type O individuals produce both anti-A and anti-B in comparable titers, unless there is an underlying immune issue.
Discrepancy 5: Mixed Field Reaction in Forward Typing (Mixed Field in Cells)
Problem Identification: Mixed Field (Mf) agglutination observed in the forward typing, specifically with anti-A and anti-B.
Reverse Type Interpretation: Reverse typing suggests blood type B (strong anti-A, no anti-B).
Discrepancy Focus: Mixed Field reaction in forward typing is the key discrepancy.
Mixed Field Reaction Significance: Mixed Field agglutination indicates the presence of two distinct red cell populations in the patient’s sample.
Causes of Mixed Field Populations: Various clinical scenarios can lead to mixed cell populations.
Transfusion: Recent blood transfusion, especially of a different ABO type.
Chimera: Rare condition where an individual has two genetically distinct cell lines, arising from the merging of two embryos in early development.
Stem Cell Transplant: Post-stem cell transplant, especially if the donor and recipient have different ABO types; can be seen during ABO conversion.
Traumatic Delivery (Maternal-Fetal Mix): In mothers post-traumatic delivery, fetal blood cells can enter maternal circulation, causing transient mixed field.
Board Exam Association – A3 and B3 Subgroups: For board exams, remember the association of Mixed Field reactions with A3 and B3 subgroups.
A3 and B3 – Mixed Field Phenotype: A3 and B3 subgroups typically exhibit Mixed Field agglutination with anti-A and/or anti-B reagents.
B3 Subgroup Example: In this example, considering B3 subgroup as a possible cause based on Mixed Field and reverse type suggesting type B.
Transfusion History Review: Check patient’s transfusion history.
No Transfusion History – Subgroup Likelihood: If no recent transfusion, a subgroup (like B3) becomes more likely.
“Three Goes with Mixed Field” Mnemonic: Remember “three” (as in A3, B3) is associated with “Mixed Field” reactions.
A3, B3 – Only Mixed Field Subgroups: A3 and B3 are the only ABO subgroups that typically present with Mixed Field agglutination in routine testing.
Definitive Resolution – Genotyping: Genotyping is the definitive method to resolve Mixed Field ABO types and confirm subgroups like A3 or B3.
Limited Serological Resolution: Serological methods alone may not fully resolve Mixed Field cases beyond identifying the presence of mixed populations.
Discrepancy 6: Mixed Population – D Typing (Mixed Field in D Typing)
Problem Identification: No ABO discrepancy (forward and reverse type concordant), but Mixed Field reaction observed in the D typing (Rh typing).
Forward and Reverse Concordance: ABO typing (anti-A, anti-B, reverse typing) is consistent and does not show a discrepancy.
D Typing Discrepancy – Mixed Field: Mixed Field reaction observed with anti-D reagent.
D Typing – Not ABO: Remember that D typing (Rh) is separate from ABO typing; D typing discrepancies are not ABO discrepancies.
Mixed Population in D Typing Cause: Mixed Field in D typing suggests a mixed population regarding RhD antigen status.
Possible Cause – Transfusion with D-Positive Cells in D-Negative Person (or vice versa): Transfusion of RhD-positive red cells to an RhD-negative individual (or vice versa) is a common cause of Mixed Field D typing.
Transfusion Scenario Example: D-negative person transfused with D-positive cells would result in a mixed population of D-negative (patient’s own cells) and D-positive (transfused cells).
Transfusion or Chimera – Mixed D Population: Mixed Field in D typing typically points to recent transfusion or, less commonly, a chimera situation.
Monoclonal vs. Blend Anti-D Reagents: Mentioning different types of anti-D reagents and their impact on Mixed Field interpretation.
Monoclonal Anti-D: Monoclonal anti-D reagents are highly specific, often targeting a single epitope on the D antigen.
Blend Anti-D: Blend anti-D reagents are mixtures of monoclonal antibodies or polyclonal and monoclonal antibodies, targeting multiple D epitopes.
Epitope Specificity and Partial D: Relevance to weak D and partial D phenotypes (to be discussed in more detail in a future session).
Weak D and Partial D Phenotypes – RH System Complexity: Briefly introducing the complexity of the Rh system, including weak D and partial D phenotypes.
Multi-Pass Protein – RhD: RhD antigen is a multi-pass transmembrane protein, with complex epitope structure.
Weak D – Quantitative Difference: Weak D phenotypes have a reduced quantity of normal D antigen epitopes on the red cell surface.
Normal D Epitopes, Fewer in Number: All D epitopes are present, just fewer copies per cell.
No Anti-D Antibody Production: Weak D individuals typically do not produce anti-D antibody because they possess all D epitopes (albeit fewer).
Partial D – Qualitative Difference: Partial D phenotypes are missing some D antigen epitopes; they have an altered D antigen structure.
Missing Epitopes: Some D epitopes are absent or altered.
Anti-D Antibody Production Risk: Partial D individuals can produce anti-D antibody if exposed to red cells with the complete D antigen (i.e., epitopes they lack).
Discrepancy Resolution – Transfusion History: Review patient’s transfusion history to assess recent transfusions that could explain Mixed Field D typing.
Immediate Reaction – Transfused Cells: Mixed Field reaction due to transfusion is typically immediate because donor cells are directly present in the recipient’s circulation.
Antibody Formation – Delayed Reaction: If Mixed Field was due to antibody production against donor cells, the reaction would not be immediate; antibody formation takes time.
Transfusion Size and Visibility of Mixed Field: The visibility of Mixed Field due to transfusion depends on the transfusion volume relative to the patient’s blood volume.
Large Transfusion in Small Person – Obvious Mf: Large transfusion volume in a small individual (e.g., baby) makes Mixed Field more obvious.
Small Transfusion in Large Person – Subtle Mf: Small transfusion volume in a large adult may result in subtle or undetectable Mixed Field.
Tube vs. Gel – Mixed Field Visibility: Mixed Field reactions are often more visually apparent in gel testing compared to tube testing.
Gel Sensitivity to Mf: Gel methods can concentrate agglutinated cells, making Mixed Field easier to detect.
Tube Testing – More Subtle Mf: Mixed Field in tube testing can be more subtle and require careful reading.
Tube and Gel – Routine Testing: Questioning whether labs routinely perform both tube and gel testing or if they use one method preferentially (depends on lab SOP).
Discrepancy 7: Missing Reactions – All Reactions Negative (No Reactions in Forward or Reverse)
Problem Identification: Missing reactions in both forward and reverse typing; all reactions are negative (or very weak).
Forward and Reverse Type – No Interpretation: No clear ABO type can be determined from the completely negative reactions.
Possible Cause – Technical Error, Reagent Issues, or Very Weak Sample: Consider possible technical or sample-related issues first.
Redo Testing – Initial Step: Repeat the entire ABO typing procedure.
Rule Out Technical Error: Redoing testing helps rule out simple errors like reagent omission, incorrect procedure, or mislabeling.
Reagent Addition Check: Verify that antisera and reagent cells were added to all appropriate tubes or wells.
Plasma and Cell Addition Check: Ensure patient plasma and red cells were correctly added.
Extended Incubation – Next Step if Redo is Negative: If repeat testing still shows no reactions, perform extended incubation.
Room Temperature Incubation (15 minutes): Incubate all reactions at room temperature for 15 minutes and reread.
Cold Incubation (4°C for 15 minutes): If still negative after room temperature incubation, incubate at 4°C for 15 minutes and reread.
4°C Incubation – Expect Some Reaction: Expect that cold incubation should elicit some reaction if there is any weak ABO antigen or antibody present, or if there is a cold autoantibody contributing to weak reactions.
True “No Reaction” – Rare: Completely negative reactions across ABO typing are very uncommon in clinical practice.
Weak Reactions More Common: Weak reactions are more frequently encountered than complete absence of reactions.
Severely Diluted Sample – Plasma Dilution: Consider if the sample is severely diluted, which could weaken reactions.
Saline Dilution Example: Sample drawn into saline, significantly diluting plasma and cells.
Plasma Concentration Impact: Dilution reduces antibody and antigen concentrations, potentially weakening reactions, especially in reverse typing (plasma-based).
Forward Reactions Less Affected: Forward reactions (cell-based) may be less dramatically affected by moderate dilution compared to reverse typing.
No Plasma Scenario – Extreme Dilution:** Complete absence of plasma in a tube is highly unlikely in routine blood banking.
Most Likely Cause – Human Error: Human error is the most probable cause of completely negative ABO reactions, such as reagent omission or procedural mistakes.
Discrepancy 8: Extra Reactions in Reverse Typing (Extra Reaction in Plasma – with A1 and B Cells)
Gel Card Example: Using a gel card (example mentioned is “Grifols,” though picture may be another brand) to illustrate reactions.
Gel Card Types: Mentioning that different gel cards exist with varying configurations (reverse typing, multiple D reagents, etc.).
Gel Card with Reverse Typing Example: Using a gel card that includes reverse typing wells (A1 cells, B cells).
Problem Identification: Extra reactions observed in the reverse typing with both A1 cells and B cells.
Forward Type Interpretation: Forward typing suggests blood type AB positive (strong anti-A, strong anti-B, positive anti-D, negative control).
Control Negativity – Trustworthy Forward Reactions: Control well is negative, indicating that spontaneous agglutination of patient’s cells is not occurring, and forward reactions are likely valid.
Discrepancy Focus: Extra reactions in the reverse typing (with both A1 and B cells) are the discrepancy.
Possible Cause – Transfusion with O Plasma or Rouleaux: Consider transfusion of type O plasma to an AB recipient or Rouleaux formation.
Transfusion of O Plasma to AB Recipient: Transfusion of type O plasma to an AB patient can introduce anti-A and anti-B antibodies into the AB recipient’s circulation.
O Plasma Antibody Content: Type O plasma contains both anti-A and anti-B antibodies.
Transient Antibodies in AB Recipient: Transfused anti-A and anti-B can cause transient positive reactions in the AB recipient’s reverse type.
Rouleaux Formation: Rouleaux is a phenomenon of red cell “stickiness” due to abnormal plasma proteins, causing false-positive agglutination in reverse typing.
Rouleaux – Plasma Protein Effect: Caused by increased or abnormal plasma proteins.
Conditions Associated with Rouleaux: Multiple myeloma, Waldenström macroglobulinemia, and other conditions with high plasma protein levels.
Plasma Protein Stickiness: Excess proteins in plasma make red cells “sticky,” causing them to stack together like coins, mimicking agglutination.
Rouleaux Confirmation – Tube Method and Microscopy: If Rouleaux is suspected, switch to tube testing and examine reactions microscopically.
Tube Test for Rouleaux: Repeat reverse typing in tubes, as tube testing is more suitable for Rouleaux detection.
Microscopic Examination: Examine tube reactions microscopically to differentiate true agglutination from Rouleaux.
“Stack of Coins” Appearance: Rouleaux has a characteristic “stack of coins” or linear arrangement of red cells under the microscope, distinct from true agglutination clusters.
Saline Replacement for Rouleaux Differentiation: Perform saline replacement to confirm Rouleaux.
Saline Replacement Procedure:
Spin down the reverse typing reaction tubes.
Remove plasma: Carefully aspirate off the plasma from above the red cell button.
Saline Addition: Replace the removed plasma with an equal volume of physiological saline.
Mix and Respin: Mix the saline-cell mixture gently and respin.
Reread Reaction: Reread the agglutination reaction after saline replacement.
Rouleaux Dispersal with Saline: In Rouleaux, the false-positive reactions will typically disperse or become negative after saline replacement because the sticky plasma proteins are diluted and removed.
True Agglutination Persistence: In true antibody-mediated agglutination, the positive reactions will generally persist or remain positive after saline replacement because antibodies are bound to red cell antigens and are not removed by saline washing.
Saline Replacement – Differentiating Rouleaux: Saline replacement is a key technique to distinguish Rouleaux from true antibody agglutination.
Saline Replacement Procedure Details – Practical Considerations:
Volume Replacement: Replace the volume of plasma removed with an equal volume of saline to maintain reaction volume.
Two Drops Example: If using two drops of plasma initially, remove two drops of plasma and replace with two drops of saline.
No Additional Reagents: Saline replacement involves only removing plasma and adding saline; no other reagents are added during this step.
Saline Replacement – Mechanism of Action: Explaining why saline replacement works to differentiate Rouleaux from true agglutination.
Rouleaux – Plasma Protein-Mediated: Rouleaux is caused by sticky plasma proteins, not antibody-antigen binding.
Plasma Removal – Protein Removal: Removing plasma during saline replacement removes the excess proteins causing Rouleaux.
Saline – Non-Sticky Medium: Saline is a non-sticky medium that does not cause red cell aggregation.
Antibody Binding – Unaffected by Saline: True antibodies bound to red cells are not removed by saline washing.
Post-Saline Reaction Interpretation:
Negative Reaction Post-Saline – Rouleaux Confirmed: If reaction becomes negative after saline replacement, Rouleaux is confirmed.
Positive Reaction Post-Saline – True Antibody: If reaction remains positive after saline replacement, it indicates true antibody-mediated agglutination, not Rouleaux.
Saline Replacement and Zeta Potential – Misconception: Addressing a potential misconception about saline replacement and Zeta potential.
Zeta Potential – LISS Enhancement Mechanism: Zeta potential reduction is relevant to LISS (Low Ionic Strength Saline) enhancement of antibody reactions, not Rouleaux differentiation in saline replacement.
LISS – Enhancing Antibody Reactions: LISS reagents reduce ionic strength to enhance the rate of antibody-antigen binding.
Saline Replacement – Protein Removal Mechanism: Saline replacement for Rouleaux differentiation works by removing plasma proteins causing stickiness, not by altering Zeta potential.
ABO Typing – No LISS in Routine ABO: Routine ABO typing (forward and reverse) does not typically involve LISS reagents; LISS is more commonly used in antibody detection and identification.
Elution – Not for Rouleaux Differentiation: Elution techniques (acid elution, heat elution) are not used for differentiating Rouleaux.
Elution – Antibody Recovery from Cells: Elution is used to recover antibodies bound to red cells, typically for investigating positive Direct Antiglobulin Tests (DATs) or identifying antibodies coating red cells.
Rouleaux – Not Antibody-Cell Binding: Rouleaux is not caused by antibody binding to cells, so elution is not relevant for its differentiation.
Elution – Time-Consuming and Costly: Elution is a more complex, time-consuming, and costly procedure compared to saline replacement.
Saline Replacement – Simpler and Faster for Rouleaux: Saline replacement is a simpler, faster, and more direct method for differentiating Rouleaux in reverse typing.
22°C Immediate Spin – Routine ABO Phase: Routine ABO typing includes a 22°C immediate spin phase, which is relevant for IgM antibodies, but not directly for Rouleaux differentiation or elution.
Auto Control in Rouleaux: Auto control should also be positive if Rouleaux is causing the extra reactions in reverse typing.
Auto Control – Patient Cells vs. Plasma: Auto control tests patient’s plasma against patient’s own red cells.
Rouleaux Effect on Auto Control: If Rouleaux is plasma protein-mediated, it will affect all reactions containing the patient’s plasma, including the auto control.
Consistent Positive Reactions: If Rouleaux is present, both reverse typing reactions (A1, B cells) and the auto control are likely to be positive due to the sticky plasma.
Non-Specific Protein Effect: Rouleaux is a non-specific protein effect affecting plasma reactions generally, not a specific antibody-antigen reaction.
Screen Cells and Rouleaux: Rouleaux would also affect reactions with screen cells (in antibody screening), causing false-positive reactions in antibody screens as well if Rouleaux is significant.
Lectins and Rouleaux – Not Directly Related: Lectins are used for ABO subgrouping and characterizing A and B antigens, not directly for differentiating Rouleaux.
Lectins – ABO Antigen Specificity: Lectins have specificity for certain ABO antigens (e.g., anti-A1 lectin for A1 antigen).
Rouleaux – Non-ABO Specific: Rouleaux is a non-specific phenomenon unrelated to ABO antigen specificity; it’s a physical effect of plasma proteins.
Lectins – Not Helpful for Rouleaux: Lectins would not be helpful in distinguishing Rouleaux from true antibody agglutination in reverse typing discrepancies.
Lecture Length and Helpfulness: Acknowledging the length of the lecture and asking if it was helpful to participants.
Extensive Topic: Recognizing ABO discrepancies as a complex and extensive topic in blood banking.
📘 New to Blood Bank?
Start your 5-day journey with Immunohematology Made Easy — a simple, beginner-friendly guide with real-life examples!
👉 Get Your Copy Now