Workshop 1
Transcript summary in points
Workshop 1: Blood Banking Fundamentals
Introduction & Safety
Ideal Workshop Start:
Ideally, workshops would begin with a formal introduction and a review of safety protocols.
This program is still under development, and a formal intro is not yet implemented.
Safety Familiarity:
By this point in the program, participants should already have a good understanding of laboratory safety.
However, it’s crucial to reinforce and keep safety in mind throughout the workshop.
Key Safety Practices:
Hand Washing: Frequent and thorough hand washing is essential to prevent contamination and infection.
Personal Protective Equipment (PPE):
Proper use of PPE is mandatory to protect oneself from biohazards.
This includes gloves, lab coats, and eye protection when necessary.
Sharps Containers:
Used glass tubes and any sharp objects must be disposed of in designated sharps containers.
This prevents accidental needlestick injuries and ensures safe disposal.
Biohazard Trash (Bio Trash/Bio Bin):
Materials contaminated with biological substances (like blood) must be discarded in biohazard bins.
General Lab Rules:
No Food or Drink: Consuming food or beverages is strictly prohibited in the lab environment.
No Applying Makeup: Applying makeup is not allowed in the lab to avoid contamination and maintain a sterile environment.
Hair Restraint: Long hair must be tied back or kept up to prevent it from becoming a contaminant or hazard.
Glass Tubes Disposal:
In this class, all glass tubes, regardless of whether they are sharp or not, must be placed in sharps bins for disposal.
Non-Glass Waste:
Items that are not made of glass can be disposed of in sharp bins as well, as sharp bins are the primary disposal in this lab setting.
Gloves Disposal:
Unsoiled Gloves: Gloves that have not been contaminated with blood or biohazardous materials can be disposed of in regular trash bins.
Soiled Gloves: Gloves that have come into contact with blood must be treated as biohazardous waste and disposed of in biohazard bins (bio bins).
Questions on Safety: Participants are encouraged to ask any questions they may have about safety procedures.
Professionalism Grade
Inclusion of Professionalism Grade: A component of the grading in this course will be based on professionalism.
Information Location: Details about the professionalism grade are outlined and posted within the course materials.
Key Professionalism Aspects:
Cleaning Up After Yourself: Maintaining a clean workspace and cleaning up after experiments is a crucial aspect of professionalism.
Preparedness for Class: Coming to class prepared, with necessary materials and having reviewed pre-class information is essential.
Following Safety Rules: Adhering to all established safety rules and guidelines demonstrates professionalism and responsibility.
General Professional Conduct: This encompasses a range of positive behaviors expected in a laboratory and professional learning environment.
Centrifuge Usage
Centrifuge Type: The centrifuges being used in this workshop are described as older models and not the most modern.
Blood Bank Lab Standard – Immediate Spin Read:
In a blood bank lab setting, a common procedure is the “immediate spin read”.
This involves mixing reagents with a blood sample and then centrifuging for a short duration (20 seconds).
RPM Setting:
Most centrifuges are pre-programmed to spin at 3500 RPM (revolutions per minute).
However, it’s noted that sometimes the centrifuges might require reprogramming upon taking samples out, so always verify and set to 3500 RPM if needed for this class.
3500 RPM is the standard speed for centrifugation in this class.
Spin Durations:
Immediate Spin Read: 20 seconds of centrifugation.
Cell Washing: 60 seconds (1 minute) of centrifugation when washing cells.
Centrifuge Balancing:
It’s assumed that participants are already familiar with the process of balancing a centrifuge.
Balancing is essential to prevent damage to the centrifuge and ensure accurate results.
Clarification Request: The instructor asks if everyone is familiar with centrifuge balancing, indicating its importance.
Documentation
Importance in Blood Banking: Documentation is critically important in blood banking due to the high volume of tubes and tests being processed.
Concurrent Documentation:
Definition: Concurrent documentation means recording information immediately as an action is performed.
Procedure: As soon as a tube is taken out and the reaction is read, it must be documented right away.
Rationale:
Memory is Unreliable: Relying solely on memory is not acceptable in a lab setting.
SOP Requirement: Standard Operating Procedures (SOPs) in hospitals universally mandate concurrent documentation.
Good Practice: Concurrent documentation is considered good laboratory practice to ensure accuracy and traceability.
Indelible Ink Requirement:
Ink Type: All documentation must be done using indelible ink, specifically black or blue ink.
Prohibition of Pencil: Pencil is not permitted for any documentation in this class, as it is erasable and not considered permanent.
Error Correction Protocol:
No Scribbling Out: Erasing or scribbling out errors is strictly prohibited.
Rationale: Scribbling out raises suspicion of data manipulation or hiding information, even if unintentional.
Proper Correction Method:
Draw a single line through the incorrect entry.
Initial and date the correction adjacent to the strike-through.
Write the correct information clearly next to the correction.
Example: If you document something incorrectly, strike through it, initial and date, and then write the correct information next to it.
Antigens vs. Antibodies Terminology
Importance of Terminology: Correct terminology is crucial in blood banking to avoid miscommunication and errors that can impact patient care.
Antigen Notation:
When referring to an antigen, write only the name of the antigen itself.
Example: For the Kidd blood group system, if discussing the ‘Jka’ antigen, simply write “Jka antigen” or “Jka”.
Antibody Notation:
When referring to an antibody, you must indicate it is an antibody.
Methods to Indicate Antibody:
“Anti-” Prefix: Write “anti-” followed by a hyphen and then the antigen name (e.g., anti-Jka).
Hyphen Prefix (Short Form): Use a hyphen as a shorthand prefix to indicate antibody (e.g., -Jka).
Assumption of Antigen: If neither “anti-” nor a hyphen prefix is used, it will be assumed you are referring to the antigen, not the antibody.
Exam Implications:
Example Scenario: If an exam question asks “What antibody is present?” and you write “Jka”, it will be marked as incorrect if the intended answer was the antibody.
Correct Answer Example: To correctly answer, you must write “anti-Jka” or “-Jka” to clearly indicate you are talking about the antibody to Jka.
Importance of Clarity: Understanding and using the correct terminology is extremely important to convey the intended meaning accurately.
Work Up Example – Duffy A Case:
Scenario: Blood bank workups can be lengthy, sometimes exceeding 8 hours, potentially requiring shift changes.
Handover Miscommunication: If you start a workup but don’t finish it and pass it on to a colleague, precise communication is essential.
Incorrect Terminology Consequence:
If you tell your colleague “This patient has a Duffy a,” but you actually mean “anti-Duffy a,” it can lead to significant errors.
Potential Errors:
Unnecessary Extra Work: Your colleague might perform extra tests that are not needed because they are looking for the antigen, not the antibody.
Issuing Wrong Unit: Incorrect terminology could lead to issuing a blood unit that is incompatible for the patient, which is dangerous.
Terminology is Critically Important: Emphasized again that precise terminology is extremely important for patient safety and efficient lab work.
Course Intensity and Extra Credit
Course Pace: This class is described as intense and fast-paced, with a significant amount of information to learn.
Availability of Extra Credit: There is extra credit available in the course.
Midterm Performance:
Don’t Panic: Students are advised not to worry if they don’t perform well on the midterm exam.
Opportunity for Improvement: There is still time to improve their grade through extra credit opportunities and subsequent assessments.
Clinical Lab Importance
Statistical Significance of Clinical Labs:
Statistically, clinical laboratories play a crucial role in healthcare.
It’s estimated that between 70% and 80% of diagnoses are supported or based on laboratory results.
Importance of Lab Work: This statistic highlights the significant impact and importance of the work performed in clinical labs.
Seriousness of the Job: Students are urged to take their job seriously due to its direct impact on patient diagnosis and care.
Potential for Harm:
Consequences of Errors: Mistakes made in the lab can directly harm patients.
Patient Impact: Every test performed in the lab has the potential to affect someone’s health and well-being.
Mindful Lab Work: Students are asked to keep in mind the patient impact of their work every time they are working in the lab.
Questions Before Moving On: The instructor asks if there are any questions before proceeding to the ABO blood group system.
Lecturer Grade Comment: An aside comment is made, seemingly unrelated to the immediate topic, mentioning “the same grade as the lecturer,” possibly a side conversation or a reminder for later.
ABO Blood Group System – Landsteiner’s Laws
Foundation: The ABO blood group system and its principles are based on Landsteiner’s Laws.
Landsteiner’s First Law:
Antigen Absence & Antibody Presence (For ABO System Only): If an antigen is absent on a person’s red blood cells, the corresponding antibody must be present in their plasma.
System Specificity: This law is specifically true for the ABO blood group system.
Naturally Occurring Antibodies: ABO antibodies are naturally occurring, meaning they are produced without prior exposure to foreign red blood cells.
Landsteiner’s Second Law:
Antigen Presence & Antibody Absence (Universal Blood Groups): If an antigen is present on a person’s red blood cells, the corresponding antibody must be absent in their plasma.
Universality: This law is true for all blood group systems, not just ABO.
Importance for Antibody Identification (IDs): This principle is vital to remember when performing antibody identifications.
Self-Recognition: The body should recognize its own antigens as “self” and not produce antibodies against them.
Immune Response Avoidance: The body should not mount an immune response against antigens that are present on its own cells.
Group A Example – Applying Landsteiner’s Laws:
Group A Antigen: If a person is blood group A, it means they have the A antigen on their red blood cells.
Expected Antibody: According to Landsteiner’s first law, a group A person should have anti-B antibodies in their plasma.
Antibody Production Rule: The body produces antibodies against the antigens that it is negative for.
Group AB Example:
Both Antigens Present: Group AB individuals have both A and B antigens on their red cells.
No Antibodies Expected: Since they have both A and B antigens, they should not produce either anti-A or anti-B antibodies.
Group O Example:
Neither Antigen Present: Group O individuals have neither A nor B antigens on their red cells.
Both Antibodies Expected: They should produce both anti-A and anti-B antibodies in their plasma according to Landsteiner’s first law.
Concept Comprehension Check: The instructor asks if this concept makes sense to everyone, ensuring understanding before moving forward.
ABO Blood Group Chart & Testing Reactions
Chart Explanation: A chart is presented that summarizes the expected reactions for each ABO blood group when tested.
Chart Purpose: The chart illustrates what reactions to expect during ABO blood typing tests.
Reactions & Blood Groups: The chart details the typical reactions for groups A, B, AB, and O.
Testing Application: After today’s testing exercise, participants are instructed to refer back to this chart to compare their results and understanding.
Carbohydrate Antigens Module
Grouping Rationale: Carbohydrate antigens are grouped together in one module despite seeming unrelated because they share a common biochemical nature – they are all carbohydrates.
Indirect Gene Products: Carbohydrate antigens are not direct gene products.
Trick Question Alert: Students are warned about trick questions related to gene-antigen relationships.
Example Trick Question: “The A gene makes the A antigen – True or False?” – The answer is False.
Mechanism of Carbohydrate Antigen Synthesis:
Gene to Transferase: For carbohydrate antigens, the gene does not directly code for the antigen.
Transferase Enzyme Production: Instead, the gene translates into a transferase enzyme.
Sugar Addition: This transferase enzyme adds a specific sugar molecule to an existing carbohydrate chain on the red cell surface.
Antigen Formation: This sugar addition is what creates the specific carbohydrate antigen.
Enzyme, Not Direct Antigen Production: Genes for carbohydrate antigens code for enzymes (transferases), not the antigens themselves.
H Antigen – Precursor for A and B Antigens
H Antigen Necessity: The H antigen is a precursor necessary for the formation of A and B antigens.
FUT1 Gene & H Transferase: The FUT1 gene (Fucosyltransferase 1) codes for a transferase enzyme.
Type 2 Structure Modification: This enzyme adds a fucose sugar to a type 2 carbohydrate precursor structure on the red cell surface.
H Antigen Formation: The addition of fucose creates the H antigen.
A and B Gene Dependency on H:
H Antigen Requirement: To express A or B antigens, the H antigen must be present first.
A and B Gene Action: If a person has the H antigen and also possesses the A or B genes, these genes will then act on the H antigen.
Sugar Addition by A and B Genes: The A and B genes will add further sugars to the H antigen to create the A and B antigens respectively.
Sugar Additions for A and B Antigens:
A Gene – GalNAc Addition:
The A gene’s transferase adds N-acetylgalactosamine (GalNAc) to the H antigen.
This GalNAc addition creates the A antigen.
B Gene – Galactose Addition:
The B gene’s transferase adds galactose to the H antigen.
This galactose addition creates the B antigen.
Bombay Phenotype – Lack of H Gene (hh):
h Gene Absence: The Bombay phenotype occurs in individuals who are homozygous for a non-functional ‘h’ allele (hh).
No H Antigen Production: They do not produce the H transferase and therefore cannot produce the H antigen.
A and B Gene Ineffectiveness: Even if a person has the A or B genes, they will not express A or B antigens because they lack the H antigen precursor.
Antigen Expression Block: The absence of H antigen prevents the formation of A and B antigens, regardless of the presence of A or B genes.
Visual Representations: Different pictures are used to visually demonstrate the process of adding GalNAc to form A antigen and galactose to form B antigen.
Questions on H Antigen: The instructor asks if there are any questions about the H antigen and Bombay phenotype.
Lab Advice – Understanding Steps & Troubleshooting
Importance of Understanding Processes: Understanding the underlying principles of each step in lab procedures is crucial.
Memorization Aid: Comprehending the ‘why’ behind each step will aid in memorizing the procedures more effectively.
Troubleshooting Capability: Understanding the purpose of each step is essential for effective troubleshooting when things go wrong in the lab.
Step-by-Step Thinking:
Encouragement: Participants are encouraged to think about the purpose and action of each step as they perform it.
Example – Cell Washing:
Step Purpose: When washing cells, understand that the goal is to remove plasma from the red blood cells.
Troubleshooting Application: This understanding will be helpful later if troubleshooting issues related to washing efficiency arise.
Purposeful Practice: Make a conscious effort to understand the goal of every step in the lab process.
Blood Draw and Sample Preparation – ABO Typing Procedure
Starting Procedure – Blood Draw: The first step in the ABO typing procedure is obtaining a blood sample.
Tube – HOPE Tube 1: A tube labeled “HOPE 1” will be used for the blood sample.
Spinning Down Samples:
Pre-Spun Samples: The samples used in the workshop should already be spun down.
No Need to Spin Initially: Participants will not need to initially spin down the provided samples.
Tube Labeling – Patient Name & RBC/Plasma:
Two Tubes Required: Take two clean tubes and label them for each patient sample.
Labels: One tube should be labeled “RBCs” and the other labeled “Plasma”.
Patient Name/Donor ID: For today’s exercise, “patient name” is actually your “donor ID”.
Donor ID Source: The donor ID is the last six digits of the number printed on your sample tube.
Use Donor ID as Patient ID: Use these last six digits as the patient identifier for labeling the tubes.
Donor ID Explanation (General Blood Unit):
DIN – Donor Identification Number: For a real blood unit, the label will include a DIN.
DIN Structure: The DIN is comprised of several sections, each with meaning:
Facility ID (First Section):
Represents the FDA-approved facility where the blood was collected.
Each approved collection facility is assigned a unique ID.
Facility Lookup: Facility IDs allow tracking back to the collection source.
Example – LifeSouth: “0300” is given as an example, representing LifeSouth.
Example – Stanford: “05 07 5 0” is given as an example for Stanford.
Multiple IDs: Some blood centers may have multiple facility IDs.
Database for Lookup: A database exists where facility IDs can be entered to identify the collection center (e.g., Kentucky Blood Center, Blood Center of the Ozarks, etc.).
Year of Collection: The next section represents the year the blood was collected.
Unique Number: Follows the year, serving as a unique sequential number for that collection.
BIM Flag:
Some blood centers use a BIM (Blood Information Management) flag.
Other centers may not utilize BIM flags.
Sticker System: Blood collection involves multiple stickers on a sheet (pad).
Sticker Usage:
Some stickers are applied directly to the blood unit bag itself.
Others go onto tubes sent for donor testing for infectious diseases.
Some remain on the pad for paperwork or other uses.
BIM Flag Function (If Used): In centers using BIM flags, each sticker on a pad may have a different, unique ID.
Check Digit (Last Digit):
The final digit is a check digit.
Calculation: It’s generated by a calculation involving summing the preceding digits and producing a check value.
Check Digit Usage: Some donor centers use check digits, others do not.
Verification Function: Check digits are used to verify the accuracy of the DIN.
Meaningful and Unique: DINs are designed to be meaningful and uniquely identify each blood donation.
Using DIN for Paperwork: When working with donor tubes, the DIN (or part of it, like the BIM flag section) can be used as the patient or donor ID on paperwork.
Plasma Separation:
Transfer Pipette Use: Use a transfer pipette to carefully remove the plasma from the original sample tube.
Plasma Transfer: Transfer all the removed plasma into the tube labeled “plasma”.
Action Encouragement: Participants are encouraged to perform these steps as they are being described.
RBC Preparation – Washes:
RBC Transfer: After plasma removal, take two drops of the red blood cells (RBCs) from the original tube.
Transfer to RBC Tube: Place these two drops of RBCs into the tube labeled “RBC”.
Saline Addition for Washing: Fill the “RBC” tube approximately three-fourths full with saline.
Saline Bottle Awareness: Be aware of other bottles on the benches, especially “eye wash” which contains ethanol.
Ethanol Caution: Be extremely careful not to accidentally use ethanol instead of saline for washing cells, as ethanol will lyse (kill) the red blood cells.
Ethanol Bottle Removal: If there are ethanol bottles nearby that look similar to saline bottles, move them away to prevent accidental use.
Saline Bottle Identification: Ensure you are using the correct saline bottle for cell washing.
Cell Washing Steps:
Saline Addition: Add saline to the RBC tube (already containing 2 drops of RBCs) until it is about three-fourths full.
Centrifugation – First Wash: Centrifuge the RBC tube for one minute.
Balanced Centrifuge Use: Ensure the centrifuge is balanced during spinning.
Saline Source: Saline is provided at the benches.
Reusing Transfer Pipettes – Supply Shortages:
Supply Shortage Awareness: Be mindful of potential supply shortages in labs.
Pipette Reuse (When Appropriate): If a transfer pipette is used for the same reagent or sample type, it’s acceptable to reuse it to conserve supplies.
Waste Reduction: Avoid throwing away pipettes after every single drop to minimize waste, especially during shortages.
Transfer Pipette Type: A specific type of transfer pipette is mentioned, likely referring to a Pasteur pipette or similar.
Same Reagent/Sample Reuse: Reusing a pipette for the same reagent or same patient sample is generally acceptable.
RBC Mixing During Washing:
Importance of Mixing: It’s important to mix the RBCs thoroughly with the saline during washing.
Sedimented RBCs: RBCs tend to settle at the bottom of the tube.
Inadequate Washing Without Mixing: If not mixed properly, the cells at the bottom might not be effectively washed, leaving residual plasma.
Mixing Purpose: Mixing ensures all cells are in contact with the saline for efficient plasma removal.
Purpose of Washing – Plasma Removal:
Objective: The primary purpose of washing the red cells is to remove and clean off the plasma.
Plasma Interference: Plasma can interfere with subsequent testing steps if not removed.
Second Centrifugation (After First Wash): Centrifuge the tube again for one minute after the first saline wash.
Supernatant Removal (After Centrifugation): After centrifugation, carefully decant or aspirate the supernatant (the liquid on top) to remove the saline and washed-away plasma.
Repeating Washes (Typically 3 Washes): The washing process with saline, centrifugation, and supernatant removal is typically repeated three times to ensure thorough plasma removal.
Drop Technique – Height and Angle:
Dropping from Above: Always dispense liquids (reagents, saline, etc.) by dropping them from a slight height above the tube.
Angle Pouring: Pouring at an angle can also be beneficial when adding saline or decanting supernatant to minimize cell disturbance.
Dumping Supernatant: When removing the supernatant after washing, it is referred to as “dumping” the saline, implying careful decantation or aspiration without disturbing the cell pellet at the bottom.
3% Cell Suspension Preparation:
Objective: The next step is to prepare a 3% red cell suspension.
Procedure: Add saline to the washed RBCs to achieve an approximately 3% suspension.
Visual Comparison: Compare the turbidity (cloudiness) of your prepared suspension to a pre-made reagent red cell suspension as a visual guide for concentration.
Familiarity with Process – Time Efficiency:
Process Familiarity Benefits: As you become more familiar with the ABO typing process, you’ll become more efficient.
Time Management Example: While the centrifuge is spinning for one minute, use that time to think ahead to the next step and begin labeling tubes for the next stage of the procedure.
Time-Saving Strategies: Proactive planning and preparation during waiting times can significantly save time in the lab.
Tube Orientation in Rack: Pay attention to the orientation of tubes in the rack to ensure they are processed in the correct order.
Up and Down Mixing – Resuspension: During washing, ensure you are properly resuspending the cells by mixing up and down, getting all cells into suspension.
Rocking Centrifuge Buckets: When placing tubes into the centrifuge, gently rock the buckets to ensure they are properly seated and balanced.
Labeling Tubes – ABO Typing:
Tube Labels: Once the 3% suspension is ready, start labeling tubes for the ABO typing tests.
Required Labels: Prepare tubes labeled for:
Anti-A
Anti-B
Anti-D
A1 cells (for reverse typing)
B cells (for reverse typing)
Sample Number Consideration: Labeling strategy depends on whether working with a single sample or multiple samples.
Multiple Samples: If working with multiple samples, label tubes in a way that clearly identifies each sample to avoid mix-ups.
Screening Test Tubes Preparation:
Additional Tubes: In addition to ABO typing tubes, prepare tubes for antibody screening.
Screen Tube Labels: Label tubes for:
S1 (Screen Cell 1)
S2 (Screen Cell 2)
S3 (Screen Cell 3)
AC (Auto Control)
Incubation Preparation: Screening tubes will be set up for incubation after initial steps.
Reagent Addition – Clear Liquids First:
Order of Reagent Addition: Always add clear liquids (antisera, plasma, etc.) to tubes before adding red blood cell suspensions or reagent red cells.
Forward Typing – Antisera Addition: Add the following antisera to the appropriately labeled tubes:
Anti-A
Anti-B
Anti-D
Reverse Typing & Screen – Plasma Addition: Add patient plasma (from the “plasma” tube) to the following tubes:
A1 cells tube
B cells tube
S1 tube
S2 tube
S3 tube
AC tube
Reagent Rack Setup: Reagent rack should be organized with the front row containing: Anti-A, Anti-B, Anti-D, A1 cells, and B cells.
Reverse and Screen Reagents – Plasma: For reverse typing and antibody screen, the “clear liquid” being added is the patient’s plasma.
Confirmation of Liquid Addition – Visual Check:
Post-Clear Liquid Check: After adding all clear liquids (antisera, plasma), visually inspect all tubes in the rack.
Purpose of Check: Ensure that every tube intended to receive liquid actually has liquid in it.
Error Prevention: This visual check helps to catch mistakes like skipping a tube or forgetting to add plasma or antisera to any tube.
Confirmation of Completion: Once all tubes are visually confirmed to contain liquid, proceed to the next step.
Red Cell Suspension Addition – Forward Typing & Auto Control:
Patient Cell Suspension Addition: In the forward typing tubes (Anti-A, Anti-B, Anti-D) and the auto control (AC) tube, add one drop of the 3% patient red cell suspension.
Auto Control Composition: The auto control tube contains patient plasma and patient red blood cells.
Auto Reaction Detection: Auto control is used to detect any autoagglutination or autoantibody reactions occurring within the patient’s own sample.
Reagent Red Cell Addition – Reverse Typing:
A1 Reagent Cell Addition: Add one drop of A1 reagent red blood cells to the tube labeled “A1 cells”.
B Reagent Cell Addition: Add one drop of B reagent red blood cells to the tube labeled “B cells”.
Screen Cell Addition – Antibody Screen:
Screen Cell Vials: Screen cells are provided in vials labeled S1, S2, and S3.
Lot Number Importance: Note down the lot number of the screen cells used.
Anagram Usage: The lot number is crucial because reaction interpretations will be based on the anagram (antigen profile sheet) specific to that lot of screen cells.
Screen Cell Addition Procedure:
Add one drop of Screen Cell 1 (S1) to the tube labeled “S1”.
Add one drop of Screen Cell 2 (S2) to the tube labeled “S2”.
Add one drop of Screen Cell 3 (S3) to the tube labeled “S3”.
Passing Screen Cells: Pass the vials of screen cells around to each participant once you have added your drop to your tubes.
Anagram Retrieval: Everyone needs to get the anagram sheet corresponding to the lot number of the screen cells being used.
Worksheet for Recording Results:
Worksheet Usage: A worksheet will be used to record the reactions observed.
Donor ID Recording: Record the donor ID (patient ID) on the worksheet.
Reaction Recording: Reactions will be recorded in designated areas of the worksheet as they are read.
Worksheet Distribution: Worksheets are distributed to each participant.
Screen Cell Anagram Retrieval (Again Emphasized): Ensure everyone obtains an anagram sheet for the specific lot number of the screen cells they are using.
Anagram Information: The anagram provides the antigen profile for each cell in the screen, necessary for interpretation.
Anagram Sets: Three sets of anagrams are available.
Lot Number Matching: Match the lot number on the screen cell vials to the lot number on the anagram sheet.
Next Steps Discussion (After Cell Addition): Once all cells are added, the instructor will discuss the next steps in the procedure.
Mixing Tubes:
Post-Addition Mixing: After adding all reagents and cells to the tubes, gently mix each tube.
Mixing Method: Flick the tubes lightly with your wrist to ensure thorough mixing of cells and reagents.
Centrifuge Loading: After mixing, tubes are ready to be loaded into the centrifuge.
Centrifuge Loading Order – System Development:
Importance of Consistent Order: Develop a consistent practice for loading and unloading tubes into the centrifuge.
ABO Typing Criticality: ABO typing is the most critical test in blood banking due to the high risk of fatal errors.
Error Consequence: Mistakes in ABO typing are the easiest way to cause a fatal transfusion reaction.
Consistent Loading/Unloading Order Benefits:
Systematic Approach: A consistent order creates a systematic approach, reducing errors.
Interruption Handling: If interrupted (e.g., phone call from hospital), a consistent order allows you to easily resume where you left off without confusion.
System Setup: Establish a system for loading and unloading tubes in the same sequence every time to minimize mistakes and ensure traceability.
Centrifugation – Immediate Spin:
Centrifugation Duration: Centrifuge all prepared tubes (ABO typing, reverse, screen, auto control) for 20 seconds.
Lot Number Importance (Reiteration): The importance of the screen cell lot number is emphasized again, as it will be discussed further during incubation.
Tube Appearance Before Reading – Red Color:
Expected Appearance: All tubes, after mixing, should appear red due to the presence of red blood cells.
Mixing Before Spin (Reiteration): Give the tubes another quick flick with your wrist to ensure everything is well-mixed before spinning.
Tube Rack Order Recommendation:
Order Recommendation: It’s recommended to keep the tubes in a logical order in the rack.
Reading Order Facilitation: This order should facilitate reading the reactions in a consistent and correct direction after centrifugation.
Reading Procedure Demonstration & Supervision:
Reading Importance: Reading reactions is one of the most challenging aspects of blood banking, requiring practice and familiarity.
Instructor Supervision: The instructor will come around and watch participants read their reactions to provide guidance and ensure proper technique.
Reaction Familiarity: Practice and observation are necessary to become familiar with the visual appearance of different reaction strengths.
Illumination Viewer and Mirror Use for Reading Reactions:
Illumination Viewer Usage: Use an illumination viewer to provide backlighting for better visualization of agglutination.
Mirror Angle Adjustment: Angle the mirror of the illumination viewer to optimally view the tube contents.
Tube Shaking and Mirror View: When shaking the tube to read the reaction, view it in the mirror of the illumination viewer.
Initial Tube Observation – Before Shaking (Tail):
Pre-Shake Observation: Before shaking the tube, bring it over the mirror of the viewer and observe.
“Tail” Check: Look for a “tail” or streaming of cells as they settle.
Negative Reaction Indication (Tail): If a slight “tail” of cells trailing down is visible, it often indicates a negative or very weak reaction.
Shaking and Reading Negative Reactions:
Gentle Flicking (If Tail Present): If a “tail” is observed, gently start flicking the tube to resuspend cells.
Cell Breakup in Negative Reaction: For a truly negative reaction, all the cells will break up and become a smooth suspension again.
No Clumps in Negative: In a negative reaction, there will be no visible clumps or agglutination – it will look uniformly red and smooth.
Negative Reaction Recording: This smooth, clump-free appearance is interpreted and recorded as a negative reaction.
Positive Reaction – 4+ (Four Plus) Grading:
4+ Reaction Appearance: A 4+ reaction is a strong positive.
Single Solid Clump (4+): In a 4+ reaction, all the cells will agglutinate into a single, solid clump.
Immediate Clump Release (4+): When shaken, this solid clump will come off the bottom of the tube as one chunk.
4+ Recording: This strong, single-clump agglutination is recorded as a 4+ reaction.
Reaction Grading Scale (Beyond 4+):
Grading Scale: Reaction strengths are graded on a scale from 4+ (strongest positive) down to 0 (negative).
3+ Reaction:
Multiple Large Clumps: Characterized by several large clumps of agglutinated cells.
2+ Reaction:
Bunch of Smaller Clumps: Consists of many smaller, but distinct, clumps.
1+ Reaction:
Really Little Clumps: Very small, fine clumps, just barely visible.
Grading Subjectivity: Reaction grading involves some degree of subjectivity and requires practice to become proficient.
Mixed Field Reaction – Two Cell Populations:
Definition: Mixed field reactions indicate the presence of two distinct populations of red blood cells.
Causes of Mixed Field:
Transfusion: Recent blood transfusion can result in a mixed field if the patient has cells from their original blood type and the transfused donor cells.
Example – Group A Patient Transfused with Group O Cells: If a group A patient receives group O red cells, they will have both A cells (their own) and O cells (transfused).
Differential Reaction with Anti-A (Example): When testing with anti-A, the patient’s A cells will agglutinate (positive reaction), but the transfused O cells will not react (negative reaction).
Visual Appearance of Mixed Field:
Suspension with Clumps and Unagglutinated Cells: A mixed field reaction will show a suspension where some cells are agglutinated into clumps (e.g., like a 1+ or 2+ reaction) while other cells remain unagglutinated (appearing as a negative reaction background).
Key Takeaway – Two Cell Populations: The most important interpretation of a mixed field reaction is that it signifies the presence of two distinct red cell populations.
Expected Mixed Field in Current Exercise: It is stated that mixed field reactions should not be expected in this particular exercise, as samples are from single donors.
Clinical Significance of Mixed Field: Mixed field reactions are clinically significant and require further investigation to determine the cause.
Recording Reactions – Numerical Grading & Zero for Negative:
Recording Scale: Reaction strengths are recorded using the numerical grading scale (4+, 3+, 2+, 1+, 0).
Zero for Negative: In blood banking, negative reactions are typically recorded as “0” (zero) instead of “negative” or “-“.
Enhancement Reagent Addition – PEG or LISS:
Post-Immediate Spin Reading: After reading reactions at the immediate spin phase, enhancement reagents are added to screening tubes and auto control tube (S1, S2, S3, AC).
Enhancement Reagent Options: Two common enhancement reagents are:
PEG (Polyethylene Glycol): Specifically gamma PEG is mentioned.
LISS (Low Ionic Strength Saline): One brand is mentioned as “Muon,” but it’s identified by its function as “Low Ionic Strength Medium” (white LISS).
Reagent Selection: Either PEG or LISS can be added, protocols in different labs may vary.
Volume of Reagent: Add two drops of either PEG or LISS to each screening tube and auto control tube.
Incubation at 37°C: After adding enhancement reagent, incubate the tubes at 37°C (body temperature).
Incubator Location: A 37°C incubator is available in the lab.
Incubation Duration: Incubate for 15 minutes.
37°C Incubator Indicators: Incubators have temperature indicators (digital display or side indicators) to confirm they are at 37°C.
Tube Holders in Incubator: Tube holders are available for placing tubes upright in the incubator.
Re-reading Reaction Example – Satellite Agglutination:
Example Reaction: An example is given of a 2+ reaction that, upon closer inspection, is described as having a “tiny little satellite” agglutination.
Interpretation: This type of reaction, even with a small satellite clump, is still considered a positive reaction.
Solid Clump Absence: It’s not a solid, 4+ clump, but it is still agglutination.
Recording: This would likely be graded and recorded as a 2+ reaction, acknowledging the weaker, satellite-like nature of the agglutination.
Incubation Time – 15 Minutes at 37°C: Reiterates the 15-minute incubation at 37°C after adding enhancement reagent.
Forward Typing – Antigen Detection on Patient Cells:
Forward Typing Purpose: Forward typing is designed to determine which ABO antigens are present on the patient’s red blood cells.
Known Antisera: Forward typing uses known antisera (anti-A, anti-B, anti-D reagents).
Antisera Colors:
Anti-A – Blue: Anti-A antiserum is always colored blue.
Anti-B – Yellow: Anti-B antiserum is always colored yellow.
FDA Regulation: These color-coding conventions are mandated by the FDA for patient safety in blood banking.
Mistake Prevention: Standardized colors help to prevent errors in reagent usage.
Forward Type Interpretation – Antigen Presence: Reactions in forward typing directly indicate which antigens are present on the patient’s cells.
Reaction Example – Donor Results (Anti-A 0, Anti-B 0, Anti-D 4+):
Anti-A Reaction: 0 (negative)
Anti-B Reaction: 0 (negative)
Anti-D Reaction: 4+ (positive)
Interpretation – ABO Group: Based on negative reactions with both anti-A and anti-B, this donor does not have A or B antigens.
ABO Group Determination – Group O: Absence of both A and B antigens indicates blood group O.
Rh Factor Determination – D Positive: Positive reaction with anti-D indicates D antigen presence, making the Rh type positive.
Full Blood Type – O Positive: Combined ABO and Rh results lead to a blood type of O positive.
Reverse Typing – Antibody Detection in Patient Plasma:
Reverse Typing Purpose: Reverse typing checks for the presence of ABO antibodies (anti-A, anti-B) in the patient’s plasma.
Reagent Red Cells (Known Antigens): Reverse typing uses reagent red cells with known ABO antigens (A1 cells and B cells).
Reaction Interpretation – Antibody Presence: Reactions in reverse typing indicate which ABO antibodies are present in the patient’s plasma.
Expected Reverse Reactions for Group O – Landsteiner’s Law:
Group O Antibody Expectation: According to Landsteiner’s law, a group O person should have both anti-A and anti-B antibodies in their plasma.
Expected Reactions – A1 and B Cells: For a group O person, reactions with both A1 reagent cells and B reagent cells should be positive (indicating anti-A and anti-B antibodies reacting with the antigens on reagent cells).
Expected Reaction Strength – 4+ in Both: For a typical group O individual, reactions with both A1 and B cells should be strong, like 4+.
Reverse Typing and Forward Typing Match – Confirmation: The results of forward and reverse typing must be concordant (match) according to ABO blood group rules.
ABO Discrepancy – Definition and Implications:
Discrepancy Definition: An ABO discrepancy occurs when the forward and reverse typing results do not match according to expected ABO rules.
Not Necessarily “Wrong”: A discrepancy does not automatically mean there is a testing error or that reagents were used incorrectly.
Investigation Required: A discrepancy signals the need for further investigation to determine the cause of the mismatch between forward and reverse typing.
Types of ABO Discrepancies:
Missing Reactions in Forward Typing: e.g., weaker or absent reactions in forward typing compared to expected.
Extra Reactions in Forward Typing: e.g., unexpected positive reactions in forward typing.
Missing Reactions in Reverse Typing: e.g., weaker or absent reactions in reverse typing.
Extra Reactions in Reverse Typing: e.g., unexpected positive reactions in reverse typing.
Discrepancy Troubleshooting: The type of discrepancy observed will guide the troubleshooting steps and further investigations needed to resolve it.
Antibody Screens – Status Update: The antibody screens that were set up earlier are now in progress.
37°C Read – PEG vs. LISS Enhancement:
37°C Read Frequency: Most hospitals no longer routinely perform a 37°C read in tube testing, especially when using PEG. Some facilities may still include it in their protocols.
PEG and 37°C Spin – Contraindication: When using PEG as an enhancement reagent, it is crucial to never spin the tubes at 37°C after the 15-minute incubation.
False Positive Risk with PEG and 37°C Spin: Spinning PEG at 37°C can cause false positive reactions due to PEG’s sticky nature.
37°C Read Procedure with PEG (If Performed): If a 37°C read is performed with PEG, do not spin. Instead:
Remove tubes from the 37°C incubator after 15 minutes.
Allow results to settle naturally (as most reactions will have occurred during incubation).
Examine the plasma for hemolysis.
37°C Read Recording with PEG: If a 37°C read is performed with PEG, record the result as either:
“H” – Hemolysis present
“NH” – No hemolysis
Current Trend – Immediate Spin and Final Read: Most modern hospital protocols use an immediate spin read and a final read (after AHG) and often omit the 37°C read, particularly with PEG.
Purpose of 37°C Incubation (General):
Incubation Rationale: Incubating at 37°C (body temperature) is done to allow time for antibody-antigen binding to occur if antibodies are present.
Facilitating Antibody Binding: The 37°C incubation provides optimal conditions for clinically significant antibodies (primarily IgG) to bind to red blood cell antigens.
Post-Incubation Washing – Antibody Removal (Unbound):
Washing After Incubation: After the 15-minute incubation, tubes need to be washed.
Cell Washer Availability: Cell washers (automated washing devices) are common in labs, but they are not available in this workshop lab.
Manual Washing Importance: It’s still important to learn manual washing techniques, even with cell washers, because manual washing may be necessary if a cell washer breaks down or in smaller labs.
Washing Purpose – Unbound Antibody Removal: The purpose of washing after incubation is to remove any unbound antibodies from the reaction mixture.
Isolating Antibody-Cell Complexes: Washing leaves only the antibodies that have bound to red blood cell antigens.
AHG (Anti-Human Globulin) Reagent Addition – IgG Antibody Detection:
Post-Wash Reagent: After washing the cells three times, the next step is to add AHG reagent.
AHG Acronym and Meaning: AHG stands for Anti-Human Globulin.
AHG Nature – Antibody to Antibody: AHG is literally an antibody directed against human antibodies (specifically IgG). It is an “antibody to an antibody”.
Patient IgG Antibodies (If Present): If the patient has IgG antibodies that have bound to red blood cells during incubation:
IgG Binding to Red Cells: These patient IgG antibodies will be attached to the red blood cell surface.
Antigen-Binding (Fab Region): The antigen-binding region (Fab) of the IgG is bound to the red cell antigen.
Fc Region Exposure: The Fc region (constant region) of the IgG antibody will be sticking out from the red cell surface.
AHG Reaction Mechanism:
AHG Introduction: When AHG reagent (anti-IgG) is added, it acts as a “bridge”.
AHG Binding to IgG Fc Region: The AHG reagent (which is also an IgG antibody) specifically binds to the Fc portion of the patient’s IgG antibodies that are already attached to the red blood cells.
Cross-linking RBCs: AHG cross-links the red blood cells that are coated with IgG antibodies.
Agglutination Visualization: This cross-linking action of AHG causes visible agglutination (clumping) that can be seen and graded.
IgG Antibody Size Limitation: IgG antibodies are relatively small and may not be able to cause visible agglutination on their own because they can only bind to one or two red cells at a time, not enough to form large clumps visible to the naked eye.
AHG as Agglutination Enhancer: AHG provides the “cross-linking” needed to enhance agglutination and make it visually detectable for IgG antibodies.
No Patient Antibody Scenario – No Agglutination: If the patient does not have any IgG antibodies against the red cells being tested, then:
No IgG antibodies will be bound to the red cells after incubation.
When AHG is added, there will be no IgG for it to bind to on the red cells.
Therefore, no cross-linking and no agglutination will occur.
Sense Check Question: The instructor asks if this mechanism of AHG action is making sense to participants to ensure understanding.
Check Cells – Verification of Negative AHG Reactions:
Check Cell Purpose: Check cells are added to all tubes that show a negative reaction after AHG testing.
Check Cell Function: Check cells serve as a control to verify that the AHG test system is working correctly, especially when a reaction is negative.
Check Cell Composition: Check cells are:
Group O RBCs: They are always group O red blood cells.
IgG Sensitized: They are pre-sensitized with IgG antibodies (meaning they are coated with IgG).
Check Cell Addition Procedure:
Add check cells to any tube with a 0 (negative) reaction after the AHG step.
Expected Reaction with Check Cells: After adding check cells to a truly negative AHG reaction, you should observe agglutination.
Positive Check Cell Reaction Interpretation: A positive agglutination reaction after adding check cells indicates:
AHG Was Added and is Active: It confirms that AHG reagent was added to the tube.
AHG Was Not Neutralized: It demonstrates that the AHG reagent was not neutralized or inactivated in the reaction.
Valid Negative AHG Result: A positive check cell reaction validates the initial negative AHG result as a true negative.
Negative Check Cell Reaction – Problem Indication: If, after adding check cells, you do not get agglutination in a tube that was initially AHG negative, it indicates a problem.
Possible Causes of Negative Check Cells (Invalid Test):
AHG Not Added: The most common reason is that AHG reagent was accidentally not added to the tube during the AHG step.
No AHG in Vial: In rare cases, the vial of AHG reagent may be defective and contain no active AHG.
Inadequate Washing: If cell washing was not done adequately before adding AHG:
Residual Patient IgG: If there was a large amount of patient IgG in the plasma that was not washed away, this free IgG in the solution can neutralize the AHG reagent.
Neutralization Mechanism: The free IgG in the plasma will bind up the AHG reagent, preventing it from being available to cross-link any IgG-coated red cells that might have been present in the original test.
False Negative Result: This neutralization can lead to a false negative result in the AHG test, even if the patient actually does have IgG antibodies.
False Negative Risk – Patient Harm: False negative results are dangerous because they can lead to missing clinically significant antibodies, potentially causing hemolytic transfusion reactions.
Positive Check Cell Reaction – Test Validity: A positive reaction with check cells confirms that the AHG test procedure was valid and that the initial negative AHG reading is trustworthy.
Agglutination Grading with Check Cells: Agglutination with check cells is typically graded as a positive reaction (e.g., 1+ to 4+), similar to other agglutination reactions.
Solid Clump (4+) Example for Check Cells: A 4+ reaction with check cells would be a single, solid clump.
Reaction Strength Grading (Check Cells): Reaction strengths are graded down from 4+ (solid clump) based on clump size and dispersion, just like with other agglutination reactions.
Hemolysis Observation – Meaningful Finding:
Hemolysis as a Positive Sign: If hemolysis (rupture of red blood cells, indicated by red color in the supernatant/plasma) is observed in a tube, it is a meaningful positive observation.
Note Hemolysis: If hemolysis is seen, it should be noted in the documentation.
Clear Plasma Start, Hemolyzed Plasma End: If the plasma was initially clear before testing, but becomes hemolyzed after testing, it is a significant finding.
Antibody-Induced Hemolysis: Some blood group antibodies are known to cause hemolysis.
Directional Clue: Observing hemolysis can provide a clue in identifying the type of antibody present.
Antibody Screens – Purpose and Clinical Significance
Main Purpose of Antibody Screens: The primary purpose of antibody screens is to detect clinically significant red blood cell antibodies before transfusion.
Transfusion Reaction Prevention: Detecting these antibodies is crucial to prevent hemolytic transfusion reactions.
Pre-Transfusion Testing Component: Antibody screens are a standard part of pre-transfusion testing protocols.
FDA Regulation (Screening Mandate): Performing antibody screens is mandated by the FDA for blood transfusions.
Clinically Significant Antibodies – Target Detection: Antibody screens are specifically designed to detect clinically significant antibodies.
Non-Clinically Significant Antibodies: There are some antibodies that are not clinically significant.
Non-Significant Antibody Issues: Non-significant antibodies can still cause complexities and frustrations in blood bank testing, making it more challenging and time-consuming.
Transfusion Reaction Risk Antibodies – Clinically Significant Examples: The clinically significant antibodies that screens aim to detect are those known to cause transfusion reactions, including antibodies in these blood group systems:
Rh system
MNS system
Kell system
Duffy system
Kidd system
P1 system
Lewis system
Screen Cell Antigen Profile – Comprehensive Coverage: Every antibody screen cell preparation must contain antigens from all of these clinically significant blood group systems to ensure adequate antibody detection.
Antibody Screen Anagram – Interpretation Guide
Anagram Definition: An anagram (or antigen profile sheet) is provided for each lot of screen cells.
Cell Screen Types: Screen cells are available in 3-cell and 2-cell formats.
Lot Number Matching (Critical): Always ensure you are using the anagram that matches the lot number printed on the screen cell vials.
Anagram Information Content: Anagrams provide detailed information about the antigen profile of each cell in the screen:
Blood Group System: The name of the blood group system (e.g., Rh, Kidd, Duffy) is listed at the top of each column.
Antigens: Below the system name, the specific antigens within that system that are tested for are listed (e.g., D, C, c, E, e, for Rh system).
Cell Designation (Vial/Cell Number): Anagrams refer to each screen cell donor as either “vial,” “cell one,” “cell two,” “cell three,” or similar. The terminology is not standardized, but it refers to each individual donor cell in the screen.
Donor Specificity: Each vial (or cell designation) in the screen contains red blood cells from a different donor. This is a key characteristic of antibody screens.
Anagram Example Interpretation – Vial 1:
Donor Profile for Vial 1: For “Vial 1” in a specific lot, the anagram will list the antigen profile of that specific donor.
Example Profile: e.g., “Positive for D, Negative for little c, and so on” for all the antigens tested.
Reaction Record: The anagram lists the reactions (positive or negative) for each antigen for that specific donor in Vial 1.
Anagram Example Interpretation – Vials 2 and 3: The anagram provides similar antigen profiles and reaction records for “Vial 2” and “Vial 3,” each representing a different donor.
Lot-to-Lot Donor Variation: Each lot of screen cells will contain cells from different donors.
Donor Profile Similarity Across Lots: While donor profiles in subsequent lots may be similar, they will not be identical because they are different individuals.
Lot Number Importance (Reiteration – Antibody ID): The lot number is crucial because the specific donor antigen profiles vary from lot to lot.
Anagram Mismatch Error: Using the wrong anagram for your screen cell lot number can lead to significant errors in antibody identification.
Example Lot Variation – Duffy Antigens: An example is given of how antigen profiles can change slightly between lots. For example, comparing Lot 502 to a hypothetical Lot 503:
Lot 502 & 503 Similarity: Most antigen reactions might be the same between Lot 502 and 503.
Lot 502 & 503 Duffy Difference: However, for Duffy antigens, the profile might change, e.g., in Lot 502, Duffy a and Duffy b might be listed in a certain order, but in Lot 503, they could be switched (Duffy a negative, Duffy b positive in one lot, and vice versa in the next).
Antibody ID Consequence of Anagram Error: If a patient has an anti-Duffy antibody and you use the wrong anagram, you could misinterpret the reactions and go down the wrong path in antibody identification.
Two-Cell Screens – Fewer Donors, Same Information: Two-cell screens contain cells from only two different donors. The anagram for a two-cell screen provides the same type of information (blood group systems, antigens, donor profiles) but for only two cells instead of three.
Reaction Strength Recording & Phases
Reaction Strength Scale (Reiteration): Reaction strengths are recorded as: 4+, 3+, 2+, 1+, or 0.
Microscopic Reading (M or +/-): Some labs may record “M” (microscopic) or “+/-” if agglutination is only seen microscopically after a weak reaction.
Microscope Reading Policy – SOP Dependent: Whether microscopic readings are performed and recorded depends on the specific Standard Operating Procedure (SOP) of the lab. Some facilities may prohibit microscope reading.
“Weak” Reaction Recording – Not Preferred: Recording “weak” as a reaction strength is discouraged.
Positive/Negative Call – Necessity: You must make a definitive call: is it positive (even if weak, record 1+) or is it negative (record 0)?
Phase of Reading – Documentation Requirement: It is crucial to record the phase at which each reaction is read.
Phases of Reading: Common phases include:
IS (Immediate Spin): Reactions read immediately after centrifugation, before incubation or AHG.
37°C Read: Reactions read after 37°C incubation (if performed).
AHG (Anti-Human Globulin) / IAT (Indirect Antiglobulin Test): Reactions read after AHG reagent addition and centrifugation.
IAT/AHG Terminology Variation: Different facilities may use different terminology for the AHG phase: IAT (Indirect Antiglobulin Test), AHG, or sometimes even “IgG” (though “IgG” is less accurate as it is the reagent anti-IgG, not the antibody IgG itself being measured).
Special Testing Phases – Documentation: If any special testing phases are performed (e.g., incubation at room temperature, 4°C), these must also be documented, including temperature and duration.
Example of Special Testing Documentation: e.g., “4°C for 15 minutes,” “Room Temp for 30 minutes.”
Documentation Examples – Worksheet Recording
Example Worksheet Setup: An example worksheet is shown to illustrate how to record results.
Auto Control Recording: The auto control (AC) reaction is recorded on the fourth line of the worksheet.
Phase Recording – Worksheet Columns/Rows: The worksheet has columns or rows to indicate the phase of testing (IS, 37°C, AHG).
Enhancement Media Recording: Document the enhancement media used (e.g., LISS, PEG) on the worksheet.
Example – LISS AHG Notation: If LISS was used as the enhancement, it would be noted as “LISS AHG” on the worksheet, indicating LISS enhancement followed by AHG reading.
Check Cell Recording – “Check” Notation: For negative AHG reactions, check cell results are recorded. A simple “check” mark in the same box as the 0 reaction is suggested as a concise way to document a positive check cell result.
Check Cell Recording – Alternative Methods: Some labs might use a separate row for check cells and record reaction strength for check cells, but a simple “check” mark is presented as a more efficient approach.
Invalid Test – No Check Cells for Negative AHG: A zero (negative) AHG reaction without a documented positive check cell result is considered an invalid test in tube testing.
Check Cell Reminder: Always remember to add and check cells for all negative AHG reactions in tube tests.
Additional Testing Documentation: If any additional or special testing is performed (e.g., DTT-treated cells, ficin-treated cells), document these treatments on the anagram or worksheet.
Consistent Anagram Usage for All Phases: As long as the same cells are used, all different treatments or phases can be documented on the same anagram or worksheet.
Documentation Questions: The instructor asks if there are any questions about how to document results.
Check Cell Recording Clarification:
Question: A question is asked about the process of reading reactions and recording check cell results in each column.
Clarification of AHG and Check Cell Recording:
AHG Reading First: Yes, first read the reaction after AHG addition.
Zero Reaction after AHG: If the AHG reaction is zero (negative).
Check Cell Addition: Then, add check cells to that tube.
Check Cell Agglutination: If agglutination is observed after adding check cells.
“Check” Notation – Confirmation: Record “check” (or a checkmark) to indicate that the check cells reacted positively, confirming the validity of the negative AHG result.
Gel Test Check Cell Difference: In gel testing, check cells are not performed.
Gel Test Validity – Pre-Incorporated AHG: Gel cards for AHG testing already contain the AHG reagent within the gel matrix.
No AHG Reagent Omission Risk in Gel: There is no risk of accidentally omitting the AHG reagent in gel testing because it’s pre-incorporated.
Gel Test Check Cell Not Needed: Therefore, check cells are not needed or performed for gel tests, and you will not see check cell results documented for gel tests.
Gel Test Documentation – No Check Cell Expectation: When documenting gel test results, you should not expect to see check cell results recorded.
IgG in Gel Matrix: The AHG reagent (anti-IgG) is already present within the gel matrix in gel cards designed for AHG testing.
Information Overload Acknowledgment: The instructor acknowledges that this is a large amount of information.
Resource Availability: Reassures students that all materials will be posted later.
Gel Testing – ABO and Antibody Screens
Gel Testing for ABO and Screens: ABO typing and antibody screens can also be performed using gel technology.
Gel Cards – Reagent Incorporation: Gel cards come with antisera (anti-A, anti-B, anti-D, etc.) already pre-dispensed into the wells.
Reagent RBCs Separate: Reagent red blood cells (A1 cells, B cells, screen cells) are not pre-dispensed in the gel cards and must be added separately.
RBC Instability: Reagent red cells are not pre-loaded because they are not stable for long periods and would degrade within the gel card.
Cell Settling in Gel Cards: Even reagent red cells added to gel cards will settle over time (as observed after 15 minutes of setup).
Reagent Addition to Gel Cards – Patient Cells and Plasma:
Patient Cells for Forward Typing: Add patient red blood cells (3% suspension) to the gel card wells designated for forward typing (anti-A, anti-B, anti-D wells).
Patient Plasma for Reverse Typing & Screens: Add patient plasma to gel card wells for reverse typing (A1 and B cell wells) and antibody screens (screen cell wells).
Product Demonstration – Gel Cards (Ortho): Gel cards (specifically Ortho brand) are passed around for participants to examine.
Card Labeling: Gel cards are labeled to indicate the type of test they are designed for.
Pre-dispensed Reagents: Gel cards come with reagents already in the wells (e.g., anti-A, anti-B, anti-D in ABO cards, anti-IgG in AHG cards).
Buffered Wells: The last two wells on some cards are “buffered” and may be used as controls or for adding A1 and B cells for reverse typing.
Unlabeled Wells – A1/B Cell Wells: The last two wells of the gel card are not pre-labeled but are intended for A1 and B cells for reverse typing.
Reverse Typing in Gel – Matching Forward Type: In gel ABO typing, the reverse type reactions (A1 and B cells) are used to confirm the forward type results.
Gel Card Circulation – Type Interpretation Exercise: Gel cards are circulated, and participants are asked to examine them and determine the ABO type based on the reactions observed.
Gel Reaction Interpretation – Lecture Review Reminder: A reminder is given to review the lecture materials on gel reaction interpretation if participants are unsure.
Gel Reaction Appearance – Negative vs. Positive
Negative Gel Reaction:
No Agglutination: Negative reaction means no agglutination has occurred.
Cell Pellet at Bottom: When centrifuged, all the red blood cells will pass through the gel matrix and form a pellet at the very bottom of the well.
Appearance: A negative gel reaction looks like a small, compact red cell pellet at the bottom of the well.
Positive Gel Reaction:
Agglutination Present: Positive reaction means agglutination has occurred.
Cells Trapped in Gel Matrix: Agglutinated red blood cells are too large to pass through the gel matrix.
Cell Distribution: Agglutinated cells will be trapped at the top or dispersed throughout the gel matrix, not forming a pellet at the bottom.
Appearance: A positive gel reaction shows red blood cells at the top of the well or dispersed throughout the gel, not a pellet at the bottom.
Gel Reaction Grading Scale: Gel reactions are also graded, but the grading scale is different from tube testing.
4+ Gel Reaction:
Solid Band of Cells at Top: In a 4+ gel reaction, almost all red blood cells are trapped at the top of the gel well, forming a solid band above the gel matrix.
3+ Gel Reaction:
Majority of Cells at Top: Most of the red blood cells are at the top, but some may have passed partway into the gel matrix.
2+ Gel Reaction:
Cells Throughout Gel Matrix: Red blood cells are dispersed throughout the gel matrix, from top to bottom, indicating a moderate level of agglutination.
1+ Gel Reaction:
Mostly Cells at Bottom, Some Above: Most of the red blood cells have passed through to the bottom, forming a pellet, but there is still some red cell material visible above the pellet, indicating a weak positive reaction.
Mixed Field Gel Reaction:
Two Cell Populations Visible: Mixed field in gel shows two distinct populations: agglutinated cells trapped at the top or within the gel matrix, and unagglutinated cells that have passed through and formed a pellet at the bottom.
Clear 4+ and 0 Appearance: It appears as a combination of a clear 4+ positive reaction (cells at top) and a clear 0 negative reaction (pellet at bottom) within the same well.
AHG Types – Polyclonal vs. Monoclonal
AHG Reagent Types: There are two main types of AHG reagents:
Polyclonal AHG: Contains antibodies against multiple epitopes of human globulins (IgG and complement).
Monoclonal AHG (Anti-IgG): Specifically contains antibodies only against IgG.
Poly AHG Composition: Polyclonal AHG contains:
Anti-IgG: Antibodies against IgG.
Anti-Complement: Antibodies against complement components (like C3b, C3d).
Laboratory Policy – AHG Selection: Different labs have different policies regarding which type of AHG to use.
Anti-IgG Only Rationale: Some labs prefer to use anti-IgG (monoclonal AHG) only.
Complement Detection Issue – Cold Antibodies: Complement detection in AHG can sometimes detect “cold antibodies,” which are often clinically insignificant.
Clinical Significance of Cold Antibodies: Cold antibodies are typically not clinically significant in transfusion reactions.
Focus on Clinically Significant IgG: Using anti-IgG only focuses detection on clinically significant IgG antibodies, which are more likely to cause transfusion reactions.
Anti-IgG in Workshop: The AHG reagent used in the workshop is anti-IgG (monoclonal AHG).
AHG Reagent Color – Green (Typical but Not FDA Mandated): AHG reagent is often colored green.
Green Color – Visual Confirmation: The green color is helpful as a visual confirmation that AHG reagent has been added to the tubes.
Color Not FDA Mandated: However, the green color is not an FDA regulatory requirement.
Clear AHG Option: Clear (uncolored) AHG reagents are also available, but green is commonly preferred for visual confirmation.
Check Cell Procedure – Detailed Steps
Check Cell Addition Time: Check cells are added after reading the AHG reactions.
Check Cell Volume: Add one drop of check cells to each tube that had a negative (0) AHG reaction.
Mixing Check Cells: Gently flick the tubes to mix the added check cells with the reaction mixture.
Centrifugation – Check Cell Spin: Centrifuge the tubes again for 20 seconds after adding check cells.
Reading Check Cell Reactions: Read the reactions after the 20-second spin following check cell addition.
Check Cell Stability and Expiration – Potential for False Negative Check Cells:
Check Cell Instability: Check cells (IgG sensitized red cells) are not very stable and have a limited shelf life.
IgG Dissociation: Over time, the IgG antibodies sensitized to the check cells can start to dissociate (come off) the red cells.
Expired Check Cells – Negative Check Reaction: If check cells are expired, they may not give a positive reaction, even if the AHG test was performed correctly.
Negative Check Reaction in Class – Potential Expiration: In class, it is possible to observe negative reactions with check cells, which might be due to the check cells being slightly expired.
Expired Check Cells – Not Necessarily Error: A negative check cell reaction in class may not necessarily indicate a procedural error by the student if the check cells are expired.
Real-World Check Cell Expectations (Fresh Reagents): In a real lab setting, using in-date (unexpired) check cells, a positive reaction must be observed for a valid negative AHG test.
Negative Check Reaction in Class – Acceptable Interpretation: If negative check cell reactions are seen in class, it is acceptable to attribute it to possible expiration of the check cells, not necessarily a procedural error.
Check Cell Identification – “IgG Sensitized by Biorad” Vials:
Vial Labeling: Check cells are typically provided in vials labeled as “IgG Sensitized” and may specify the manufacturer (e.g., “by Biorad”).
Rack Location: Check cell vials are typically kept in a separate location from other reagents, not in every reagent rack.
Negative AHG Reaction with Negative Check Cells – Interpretation:
Negative AHG, Negative Check Cells: If you observe a negative AHG reaction and a negative reaction after adding check cells.
Possible Expiration – Most Likely Cause: The most likely explanation in a class setting is that the check cells are slightly expired or have degraded.
Procedure Not Necessarily Wrong: It doesn’t automatically mean the student performed the procedure incorrectly if check cells are negative.
Real-World Implication – Investigate (If Fresh Reagents): In a real lab with fresh reagents, a negative check cell reaction would require investigation to rule out procedural errors (like not adding AHG).
Anagram Usage – Reaction Recording and Interpretation
Anagram Importance (Again): Emphasizes the importance of the anagram sheet for screen cell interpretation.
Anagram – Reaction Interpretation Key: The anagram is essential for interpreting screen cell reactions because it provides the antigen profile of each cell.
Anagram – Not Going to Tell Anything Without it: Without the correct anagram, the screen cell reactions are meaningless because you don’t know which antigens are present or absent on each cell.
Worksheet Submission – Study Questions and Lab Worksheet: For all workshops, when submitting study questions from the lab manual, also upload your lab worksheets where you recorded your results from today’s ABO and screen testing.
Worksheet Upload Requirement: Lab worksheets must be uploaded along with study questions for grading.
Worksheet Content – Donor ID and Screen Anagram: Worksheets should include your ABO/Rh results and the anagram sheet (or a clear record of the lot number) for the screen cells you used.
AHG Reading Completion and Check Cell Addition Status: Instructor checks if everyone has read their AHG reactions and added check cells to negative tubes.
Check Cell Reaction Reading – Timing: Read the reactions after adding check cells, spinning, and allowing a brief settling time.
Check Cell Reaction Strength – Weak Positive (Check Mark): If the check cell reaction is weak (e.g., 1+), it is still considered a positive check. You can simply record “check” or a checkmark to indicate a positive reaction.
Crypto No/Crypto Yes – Reading Check Cell Reactions: “Crypto no” and “Crypto yes” are mentioned, likely referring to a quick way to assess if check cells are reacting (yes/no), rather than detailed grading in this context.
Gel Card Usage – ABO Typing (Alternative Method):
Gel Cards for ABO Typing: Gel cards can also be used for ABO typing, providing an alternative method to tube testing.
Patient Name Labeling: Label gel cards with the patient name (or donor ID).
Additional Labels (Optional): Additional labels, like “A1 cells,” “B cells,” etc., can be added to the gel card if desired for clarity, though the wells are often pre-designated.
Anti-IgG Cards – AHG Testing in Gel: Specifically mention “anti-IgG cards,” indicating gel cards designed for AHG testing (like antibody screens and antibody IDs). Gel cards are available with different reagents pre-loaded.
Screening with Gel Cards: Gel cards can also be used for antibody screening.
Antisera Cards: “Antisera cards” are also mentioned, likely referring to gel cards specifically for forward typing (anti-A, anti-B, anti-D).
Mass Screening with Gel Cards: Gel cards can be used for mass screening, such as screening blood donors or large patient populations.
Little e Antigen Example – Mass Screening for Specific Antigen: Example: if a patient has anti-little e antibody, gel cards with anti-little e can be used to quickly screen all available blood units in the refrigerator for little e antigen negativity.
Gel Card Setup – Patient Suspension and Plasma:
Patient Suspension: Add patient red cell suspension to appropriate wells (forward typing wells).
Patient Plasma: Add patient plasma to appropriate wells (reverse typing and screen wells).
Positive/Negative Control in Gel – Feed Control (Outdated Term): “Feed control” is mentioned as a type of control in gel testing, but described as an “old thing.” It is likely referring to a cell control well on the gel card.
AB Positive Control Rationale – Spontaneous Agglutination Check: For AB positive patients, a control well is important to ensure that any agglutination seen in the anti-A and anti-B wells is due to true antigen-antibody reaction, and not spontaneous agglutination of the patient’s cells.
Control Well Necessity for AB Positive: A control well is particularly needed for AB positive patients because their cells can sometimes exhibit spontaneous agglutination due to high antigen density.
Negative Control Function: The control well should be negative, indicating no spontaneous agglutination, and thus validating the positive reactions in the anti-A and anti-B wells as true positives.
Meter Pipettes for Gel Card Reagent Addition: Meter pipettes (adjustable volume pipettes) are useful for accurately dispensing reagents into gel card wells.
Gel Card Reagent Volumes: Typical volumes for gel card reagents are in the 12.5, 25, or 50 microliter range, depending on the specific gel card and manufacturer.
Multi-Channel Pipettes for Panel Cells: For antibody panels (which use many different cells), multi-channel pipettes are efficient for adding panel cells to multiple wells simultaneously.
Gel Card Centrifugation – Immediate Spin for ABO: For ABO typing in gel cards, it is often an “immediate spin” procedure, meaning centrifugation is performed immediately after setup.
Gel Card Incubation for Screens/Panels: For antibody screens and panels in gel, incubation at 37°C is required to allow time for antibody-antigen reactions to occur before centrifugation.
Gel Stations – Manual and Electric Options: Gel card centrifuges (“gel stations”) are available in manual and electric (automated) versions.
Manual Gel Station: Manual gel stations may have a manual timer and require manual initiation of centrifugation.
Electric Gel Station: Electric gel stations are pre-programmed with RPM and spin times, automating the centrifugation process.
Virtual Lab – Rh Phenotyping: A virtual lab exercise on Rh phenotyping will be part of the course.
Antisera – Reagents with Known Antibodies: Antisera are reagents that contain known antibodies.
Phenotyping – Antigen Detection: Phenotyping is the process of determining which antigens are present on a person’s red blood cells.
Rh Phenotyping Antisera – Standard Set: For Rh phenotyping, a standard set of antisera is used, including:
Anti-D (already done in ABO/Rh typing)
Anti-C (big C)
Anti-c (little c)
Anti-E (big E)
Anti-e (little e)
Rh Phenotype – Standard Set of 5 Antigens: This set of 5 antisera (including anti-D) is used to determine the standard Rh phenotype.
Zygosity – Homozygous vs. Heterozygous
Genetics Lecture Reminder: Concepts from the genetics lecture are relevant to blood banking.
Zygosity Definition – Homozygous/Heterozygous: Zygosity refers to whether an individual is homozygous or heterozygous for a particular gene or antigen.
Importance of Zygosity in Blood Banking: Zygosity is important to understand in blood banking, particularly for antibody rule-outs and reagent QC.
Allelic Pairs – Rh System Example: In the Rh blood group system, certain antigens are allelic pairs (meaning they are encoded by alleles at the same gene locus):
Big C and little c are allelic pairs.
Big E and little e are allelic pairs.
Allelic Pair Explanation: For each allelic pair, an individual inherits one allele from each parent. At a given gene locus, a person can have alleles for either big C or little c (but not both at the same locus on one chromosome).
Heterozygous Example – Big C and Little c:
Scenario: If one chromosome carries the allele for big C, and the other chromosome carries the allele for little c.
Heterozygous Genotype: The individual is heterozygous for the C/c antigens (genotype Cc).
Homozygous Example – Big C and Big C:
Scenario: If both chromosomes carry the allele for big C.
Homozygous Genotype: The individual is homozygous for big C (genotype CC).
Blood Banking Relevance – Antibody Rule-Outs and Reagent QC: Zygosity is particularly important for:
Antibody rule-outs (ensuring weak antibodies are not missed).
Quality control (QC) of antisera reagents.
Homozygous Cells Preferred for Antibody Rule-Outs:
Dosage Effect: Some blood group antigens exhibit “dosage,” meaning that homozygous cells express more antigen sites than heterozygous cells.
Weak Antibody Detection Issue: If an antibody is weak or newly developing, it might react weakly or even negatively with heterozygous cells, but more strongly with homozygous cells.
Missing Weak Antibodies – Heterozygous Cells: Using only heterozygous cells for antibody rule-outs could potentially lead to missing weak antibodies.
Homozygous Cell Advantage: Homozygous cells, with their higher antigen expression, are preferred for antibody rule-outs to increase sensitivity and detect even weak antibodies.
Dosage Effect – Antibody Strength Variation:
Antibody Reaction Strength – Dosage Dependent: The strength of an antibody reaction can vary depending on whether the red cells are homozygous or heterozygous for the antigen.
Example – Anti-Big C and Homozygous Cells: If a patient has anti-big C antibody, it will typically react more strongly (e.g., 4+) with red cells that are homozygous for big C.
Example – Anti-Big C and Heterozygous Cells: The same anti-big C antibody might react weaker (e.g., 2+) or even give a negative reaction with red cells that are heterozygous for big C, especially if the antibody is weak.
Dosage and Weak Antibody Detection – Clinical Significance:
Missing Weak Antibodies – Transfusion Risk: Missing weak antibodies can still be clinically significant because even weak antibodies can cause transfusion reactions.
Importance of Sensitivity: It is critical to use testing methods and reagents that are sensitive enough to detect even weak antibodies to ensure patient safety.
Zygosity in Antisera QC – Positive Control Selection: Zygosity is also important when selecting positive controls for quality control (QC) of antisera reagents.
Dosage Phenomenon in Blood Groups
Antigen Dosage – Definition: Dosage refers to the phenomenon where some antigens are expressed more strongly on red blood cells if the individual is homozygous for the gene encoding that antigen compared to heterozygous.
Dosage in Rh System: All Rh antigens exhibit dosage effects.
Dosage in Kidd System: Kidd antigens (Jka and Jkb) exhibit dosage.
Dosage in Duffy System: Duffy antigens (Fya and Fyb) exhibit dosage.
Dosage in MNS System: M, N, S, and s antigens in the MNS blood group system exhibit dosage.
Dosage Example – Kidd System and Hemolytic Transfusion Reactions:
Kidd Antibodies and Delayed Hemolytic Transfusion Reactions: Kidd antibodies (anti-Jka, anti-Jkb) are notorious for causing delayed hemolytic transfusion reactions.
Delayed Reactions – Challenge in Detection: Delayed reactions can be challenging to detect because the initial compatibility testing might be negative, but the reaction develops days after transfusion.
Kidd and Dosage – Clinical Relevance: Dosage effect in Kidd antigens contributes to the difficulty in detecting weak Kidd antibodies, increasing the risk of delayed reactions if weak antibodies are missed.
Sargosti – Inheritance from Parents: “Sargosti” is likely a mispronunciation of “zygosity.” Zygosity (homozygous or heterozygous status) is determined by inheritance from parents.
Parental Genotypes and Offspring Zygosity: If both parents are heterozygous for a particular antigen, their children can inherit genes resulting in homozygous or heterozygous genotypes.
Zygosity Relevance to Antibody Rule Outs and Antisera QC (Reiteration): Zygosity is important to consider in antibody rule-outs (using homozygous cells) and in antisera quality control.
Antisera Quality Control (QC) – Daily Use Controls
Antisera QC – Daily Requirement: Antisera reagents must be quality controlled (QC’d) every “day of use.”
Daily QC – Lab Policy Definition: “Day of use” definition varies by lab policy (e.g., calendar day, shift-based, etc.).
QC Log – Tracking Antisera QC: Most labs maintain a QC log to record when antisera are QC’d and the results.
QC Check Before Patient Testing: Before using an antiserum for patient testing, check the QC log to ensure it has been QC’d within the current “day of use.”
QC with Patient Sample – If Not QC’d: If the antiserum has not been QC’d for the current “day of use” (according to lab policy), QC must be performed concurrently with patient testing.
Antisera QC – Negative and Positive Controls: Antisera QC requires testing both negative and positive controls.
Negative Control for Anti-Big C Example: For QC of anti-big C antiserum, select a red cell sample that is known to be negative for big C.
Panel or Screen Cells for Negative Control: Antibody panel cells or screen cells can be used as negative controls, as their antigen profiles are known and documented in the anagram.
Example – Ortho Panel VSS 400 and Anti-Big C QC: Using Ortho Panel VSS 400 anagram:
Negative Control Cell Selection – Anti-Big C: To QC anti-big C, find a cell in the panel that is negative for big C.
Anagram Review – Big C Negative Cells: Cells 2 and 3 in the example panel are negative for big C.
Cell 2 or 3 – Negative Control Option: Either Cell 2 or Cell 3 could be used as a negative control for anti-big C QC.
Positive Control for Anti-Big C Example: For QC of anti-big C antiserum, select a red cell sample that is known to be heterozygous for big C.
Heterozygous Positive Control Rationale – Weakest Expression Detection: The positive control should be heterozygous because the goal is to ensure the antiserum can detect even the weakest expression of the antigen.
Homozygous Positive Control – Not Preferred: Using a homozygous positive control is not preferred for QC because it might not detect if the antiserum is losing sensitivity to weaker antigen expression.
Allelic Pairs – Key for Heterozygous Identification: To select a heterozygous positive control, you need to know the allelic pairs for the antigen system being tested.
Allelic Pairs – Rh System (C/c, E/e, K/k, Jka/Jkb, Fya/Fyb, M/N, S/s): List of allelic pairs in Rh, Kell, Kidd, Duffy, and MNS systems.
Heterozygous Identification – Allelic Pair Positivity: For allelic pairs (like K/k), if both antigens (big K and little k) are positive on a red cell, it indicates the cell is heterozygous for both.
Homozygous Identification – Allelic Pair Negative for One, Positive for Other: If for an allelic pair (like K/k), one antigen is positive (e.g., little k) and the other is negative (e.g., big K), then the cell is homozygous for the positive antigen (homozygous for little k in this example).
Example – Cell 2 Anagram – Homozygous for Little k: Cell 2 in the example anagram is homozygous for little k (big K negative, little k positive).
Example – Cell 3 Anagram – Heterozygous for Big K: Cell 3 in the example anagram is heterozygous for big K (big K positive, little k positive).
Anti-Big K QC – Negative Control Cell 1 or 2: For QC of anti-big K antiserum, Cell 1 or Cell 2 (negative for big K) could be used as a negative control.
Anti-Big K QC – Positive Control Cell 3 (Heterozygous): For QC of anti-big K antiserum, Cell 3 (heterozygous for big K) should be used as the positive control.
Reason for Heterozygous Positive Control – Weakest Expression Detection (Reiteration): Using a heterozygous positive control ensures that the antiserum is capable of detecting even weak expression levels of the antigen, preventing false negatives in patient testing.
Co-Dominant Expression – Most Blood Group Systems: Most blood group systems exhibit co-dominant expression.
Co-Dominant Inheritance – Both Alleles Expressed: Co-dominant inheritance means that if an individual inherits alleles for two different antigens in an allelic pair (e.g., both Jka and Jkb alleles), both antigens will be expressed on their red blood cells.
Board Exam Question – Co-Dominant Inheritance: Co-dominant inheritance in blood groups is a common topic in board exams.
Example – Anti-Jkb QC: Example of QC for anti-Jkb antiserum.
Anagram Review – Anti-Jkb QC Example: Using the example anagram to select controls for anti-Jkb QC.
Negative Control for Anti-Jkb – Cell 1 (Jkb Negative): Cell 1 in the example anagram is negative for Jkb and can be used as a negative control for anti-Jkb QC.
Positive Control for Anti-Jkb – Cell 2 (Heterozygous Jkb): Cell 2 is heterozygous for Jkb (Jka positive, Jkb positive) and is suitable as a heterozygous positive control for anti-Jkb QC.
Cell 3 – Homozygous Jkb – Not Preferred for QC Positive Control: Cell 3 is homozygous for Jkb (Jka negative, Jkb positive). While it is positive for Jkb, it is not the preferred positive control because it does not test the antiserum’s ability to detect weaker (heterozygous) expression.
Heterozygous Positive Control – Weakest Expression Detection (Reiteration): The goal of using a heterozygous positive control in QC is to ensure the antiserum can detect the weakest clinically relevant expression of the antigen.
Questions on QC and Zygosity: The instructor asks if there are questions about QC and zygosity, acknowledging it can be a difficult concept to grasp.
Bigger Antigen – Not Always Relevant for Heterozygous Selection: The question “Is it always the bigger one that you want to look at?” is asked, likely referring to “big” vs. “little” antigen names (e.g., big K vs. little k). The answer clarifies that it’s about proving the antiserum can detect heterozygous expression, not just focusing on the “bigger” named antigen.
Heterozygous Detection – QC Purpose: The key purpose of the heterozygous positive control is to prove that the antiserum is sensitive enough to detect heterozygous antigen expression.
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