SOP-H-005 — ABO and Rh(D) Blood Typing (Slide and Tube Method) SOP

1.0 Purpose

This SOP sets the procedure for determining human ABO and Rh(D) blood groups at Tarangnan Community Hospital using monoclonal Anti-A, Anti-B, and Anti-D typing reagents. It covers rapid slide screening alongside mandatory immediate-spin tube method confirmation for weak or discrepant reactions, and forward blood group identification for patient clinical records, prenatal care, and pre-referral assessment. It also covers ABO reverse (serum/plasma) grouping by the tube method, performed alongside forward grouping, with forward and reverse results verified for concordance before any blood group is released.

2.0 Scope

This procedure applies to all Medical Technologists performing diagnostic blood typing in the Hematology Section. It covers forward erythrocyte grouping by rapid slide and immediate-spin test tube methods, and ABO reverse (serum/plasma) grouping by the immediate-spin tube method using known A1 and B reagent red cells. TCH performs no crossmatching, no compatibility testing, and no blood bank or transfusion services; all testing is strictly diagnostic forward grouping for patient clinical documentation.

3.0 Definitions

• ABO Forward Grouping: Determination of ABO blood group antigens present on the patient's red blood cell membrane using known commercial Anti-A and Anti-B monoclonal reagents.
• ABO Reverse Grouping: Determination of the ABO antibodies present in the patient's serum or plasma using known A1 and B reagent red cells. The reverse pattern must agree with the forward grouping before the blood group is released.
• Rh(D) Typing: Qualitative determination of the presence or absence of the Rh(D) antigen on erythrocytes using commercial Anti-D monoclonal/polyclonal blend reagents.
• Agglutination: Macroscopic or microscopic clumping of red blood cells caused by specific cross-linking antigen-antibody complexes.
• Immediate-Spin Tube Method: Confirmatory serological technique where washed erythrocyte suspensions are mixed with antisera, centrifuged immediately for 15–20 seconds, and gently resuspended to evaluate macroscopic agglutination.
• Discrepant Result: Any blood typing reaction that is weak (≤ 2+), mixed-field, ambiguous, or inconsistent with known patient history.

4.0 Responsibility

4.1 Hospital Director

• Approves this SOP and ensures the laboratory has cold-chain refrigeration for typing antisera and certified serological equipment.

4.2 Pathologist

• Reviews and authorizes all blood typing protocols, quality control schemes, and discrepancy resolution procedures.
• Reviews and issues final authorization for unresolved serological discrepancies or atypical reactions before clinical release.

4.3 Chief Medical Technologist / Laboratory Quality Manager

• Ensures technical compliance with this SOP, verifies daily typing sera quality control records, and monitors reagent inventory.
• Manages technologist competency assessments in blood group serology per Competency Assessment Policy.

4.4 Medical Technologists

• Verify patient identification on collection tubes, perform daily antisera quality control, and execute slide screening typing.
• Prepare 2%–5% red cell suspensions and perform immediate-spin tube testing whenever slide reactions are weak (≤ 2+) or ambiguous.
• Record reaction grades, interpret ABO/Rh(D) groups, and escalate unresolved discrepancies to the Pathologist.

4.5 Laboratory Aides

• Clean and decontaminate typing tiles, wash glassware, and maintain neatness of the blood grouping bench.
• Retrieve labeled specimens from accessioning and store completed tubes refrigerated.
• Shall never perform blood typing reactions, read agglutination patterns, interpret blood groups, or report results.

5.0 Policy / Procedure

Diagnostic blood grouping, rapid slide screening, immediate-spin tube confirmation, and serological discrepancy investigation follow the sequence below (see Figure 1).

Figure 1 — ABO and Rh(D) slide screening, tube confirmation, and discrepancy resolution flow

5.1 Principle of ABO and Rh(D) Typing

• Specific monoclonal antibodies directed against human red blood cell surface antigens will bind to their corresponding epitopes.
• In the presence of sufficient antigen and antibody density, antibody molecules bridge adjacent erythrocytes, causing visible cellular agglutination.
• ABO Forward Grouping: - Agglutination with Anti-A indicates presence of Antigen A (Group A). - Agglutination with Anti-B indicates presence of Antigen B (Group B). - Agglutination with both Anti-A and Anti-B indicates presence of Antigens A and B (Group AB). - Absence of agglutination with both Anti-A and Anti-B indicates absence of A and B antigens (Group O).
• Rh(D) Typing: - Agglutination with Anti-D indicates Rh(D)-Positive status. - Absence of agglutination with Anti-D indicates Rh(D)-Negative status. - At TCH's primary diagnostic level, specimens non-reactive with Anti-D on both slide and immediate-spin tube testing are classified and reported as Rh(D)-Negative; weak D testing requiring 37 °C incubation and anti-human globulin (AHG) is referred to the tertiary referral laboratory if clinically ordered.

5.2 Reagents, Materials, and Storage

• Monoclonal Typing Antisera: - Anti-A: Clear blue-colored monoclonal IgM antibody reagent. - Anti-B: Clear yellow-colored monoclonal IgM antibody reagent. - Anti-D: Clear, colorless monoclonal/polyclonal blend IgM/IgG reagent formulated for slide and rapid tube testing. - Storage: Store unopened and opened vials refrigerated at 2–8 °C. Never freeze. Do not use past the stated expiration date. Protect from microbial contamination.
• Reagent Red Cells for Reverse Grouping: Commercial known Group A1 and Group B reagent red cell suspensions. Store at 2–8 °C. Never freeze. Do not use past the stated expiration date, and bring to room temperature before use.
• Reagent Diluent: Fresh, sterile 0.9% isotonic saline (pH 6.5–7.5).
• Materials: - Clean, dry, grease-free white porcelain tiles or glass slides with distinct partitioned reaction wells. - 10 × 75 mm or 12 × 75 mm clean glass test tubes. - Serological centrifuge (calibrated to 1,000 × g or 3,400 rpm for 15 seconds). - Disposable wooden applicator sticks. - Transfer pipettes and calibrated micropipettes (50 µL, 100 µL). - Agglutination viewing lamp / concave mirror.

5.3 Specimen Requirements

• Whole blood collected into a K2EDTA (lavender-top) vacuum tube is the preferred specimen.
• Fresh whole blood collected in a plain tube (red-top without clot activator) or capillary blood may be used immediately.
• Every specimen must carry two verified patient identifiers. Grossly hemolyzed or severely clotted specimens shall be rejected and recollected per SOP-H-001.
• Specimens shall be tested within 48 hours of collection when maintained refrigerated at 2–8 °C.

5.4 Rapid Slide Screening Method

• Equilibrate typing sera and whole blood to room temperature (18–25 °C) for 10 minutes before testing.
• Label a clean glass slide or porcelain tile with the patient accession number and divide it into three distinct zones labeled A, B, and D.
• Place 1 drop of well-mixed patient whole blood (approximately 40–50 µL) into each labeled zone.
• Add 1 drop of commercial Anti-A reagent adjacent to the blood drop in zone A.
• Add 1 drop of commercial Anti-B reagent adjacent to the blood drop in zone B.
• Add 1 drop of commercial Anti-D reagent adjacent to the blood drop in zone D.
• Using a separate, clean wooden applicator stick for each zone, mix the blood and reagent thoroughly, spreading the mixture over an area approximately 20 mm in diameter.
• Gently rock and tilt the slide back and forth for exactly 2 minutes (or per the reagent manufacturer's specified reaction window). Do not heat the slide.
• Observe macroscopically for visible agglutination against an indirect white background within the 2-minute window. Do not read or record reactions after drying has begun, as evaporation mimics agglutination.
• Interpretation Criteria: - Definite, strong agglutination (3+ or 4+) with a clear background indicates a positive reaction. - Complete smooth, homogenous suspension without clumps indicates a negative reaction. - Any weak reaction (≤ 2+), delayed clumping, grainy texture, or discrepancy mandates immediate confirmation by the Immediate-Spin Tube Method.

5.5 Confirmatory Immediate-Spin Tube Method

• The Immediate-Spin Tube Method shall be performed: - Whenever slide testing yields weak (≤ 2+), doubtful, or delayed agglutination. - Whenever an unexpected or discrepant ABO/Rh pattern is observed. - For all pre-operative or maternity patients requiring confirmed baseline grouping.
• Preparation of 2%–5% Red Cell Suspension: - Label a clean 12 × 75 mm test tube with patient identification. - Add 3 to 4 drops of patient whole blood. - Fill the tube three-quarters full with fresh 0.9% isotonic saline. - Centrifuge in a serological centrifuge for 1 minute at 3,400 rpm (or until red cells are cleanly packed). - Decant the supernatant saline completely. - Repeat the saline wash two additional times (total of 3 washes) to remove plasma proteins, soluble blood group substances, and cold autoantibodies. - After the final decantation, add approximately 2.0 mL of fresh saline to the packed cell button to achieve a light cherry-red 2% to 5% cell suspension.
• Stepwise Tube Test Procedure: - Label three clean 10 × 75 mm test tubes: A, B, and D. (Include a fourth tube labeled Ctrl [Rh Control: 1 drop 6% albumin or patient serum + 1 drop cell suspension] if testing AB-positive blood). - Dispense 1 drop of Anti-A reagent into tube A. - Dispense 1 drop of Anti-B reagent into tube B. - Dispense 1 drop of Anti-D reagent into tube D. - Add 1 drop of the prepared 2% to 5% patient red cell suspension to each tube. - Mix the contents of all tubes gently by shaking. - Centrifuge all tubes immediately in a serofuge at 3,400 rpm for 15 to 20 seconds (or per manufacturer's calibrated centrifugal force). - Gently dislodge the cell button from the tube bottom by tilting and rolling the tube over an agglutination viewing mirror against a white background. - Observe macroscopically for agglutination and grade the reaction strength.
• ABO Reverse (Serum/Plasma) Grouping (Tube Method): - Label two additional clean tubes: A1 and B. - Use serum from the plain-tube specimen, or plasma separated from the EDTA specimen. - Add 2 drops of patient serum or plasma to each tube. - Add 1 drop of A1 reagent red cells to tube A1. - Add 1 drop of B reagent red cells to tube B. - Mix gently, centrifuge immediately at 3,400 rpm for 15 to 20 seconds. - Gently resuspend the cell buttons and observe macroscopically for agglutination; grade each reaction using Section 5.6. - Record the reverse reactions beside the forward reactions and verify concordance under Section 5.7 before releasing the blood group.

5.6 Agglutination Grading Scale

5.7 Interpretation and Reporting Matrix

Forward / Reverse Concordance (tube method):

Release rule: the ABO group shall be reported only when the forward and reverse results agree. Any forward/reverse discrepancy shall be investigated under Section 5.9 and shall not be released until it is resolved or referred.

5.8 Quality Control and Daily Verification

• On each day of patient testing, the Medical Technologist shall verify the reactivity and specificity of commercial typing sera (Anti-A, Anti-B, and Anti-D) using known donor or retained control blood samples: - Known Group A Red Cells: Must yield ≥ 3+ reaction with Anti-A, and 0 (negative) with Anti-B. - Known Group B Red Cells: Must yield ≥ 3+ reaction with Anti-B, and 0 (negative) with Anti-A. - Known Group O Red Cells: Must yield 0 (negative) with both Anti-A and Anti-B. - Known Rh(D)-Positive Red Cells: Must yield ≥ 3+ with Anti-D. - Known Rh(D)-Negative Red Cells: Must yield 0 with Anti-D.
• On each day of patient testing, verify the A1 and B reagent red cells with known antisera: A1 cells shall agglutinate with Anti-A only, and B cells with Anti-B only.
• If any antisera lot or reagent red cell lot yields an unexpected, weak, or non-specific reaction with control cells, the lot shall be quarantined immediately and taken out of use.
• Record daily QC results in the TCH Daily QC Log before issuing patient reports.

5.9 Investigation of Discrepant and Ambiguous Typing Results

• Potential Causes of Blood Grouping Discrepancies: - Technical Errors: Tube mislabeling, failure to add reagent, incorrect centrifuge speed/time, reading dried slide edges, shaking too vigorously. - Rouleaux Formation (Pseudoagglutination): Occurs in specimens with high fibrinogen or abnormal globulins (multiple myeloma). Differentiated by the saline replacement technique: in tube testing, add 2 drops of saline to the cell button; true agglutination persists, whereas rouleaux disperses into a smooth suspension. - Cold Autoagglutinins: Spontaneous agglutination of patient red cells in their own serum. Resolved by washing red cells three times with warm (37 °C) isotonic saline before re-testing. - Weak Subgroups: A2 with anti-A1, weak D variants, or leukemia-associated antigen loss. - Missing or Weak Reverse Reactions: Newborns, elderly patients, and immunocompromised patients may lack the expected ABO antibodies, producing a forward/reverse discrepancy; resolve under the protocol below, with referral when unresolved. - Mixed-Field Agglutination: Recent infused donor red cells from another hospital, stem cell transplantation, or fetal-maternal hemorrhage.
• Discrepancy Resolution Protocol: - Re-check patient tube label against requisition and rerun testing using freshly washed 2% to 5% cell suspension. - Review patient medical record for recent infused blood products, clinical diagnosis, and historical grouping records. - If the discrepancy cannot be resolved at the bench, do not release the result. - Refer the case with all test tubes and clinical history to the Pathologist for review per SOP-H-001. - If emergency clinical care is urgently required at a receiving facility, report: "ABO/Rh(D) grouping pending confirmation; refer patient for tertiary immunohematology evaluation."

6.0 Records

7.0 References

• DOH Clinical Laboratory QMS Tool, Section 3 — Service Delivery
• DOH Administrative Order No. 2021-0037, Annex B1 — Technical Standards for Clinical Laboratories
• ISO 15189:2022, Clause 7.3 Examination processes
• WHO Guidelines on Basic Blood Group Serology, Module 3 — Blood Group Testing
• Commercial Monoclonal Anti-A, Anti-B, and Anti-D Package Inserts (Current Product IFU)

Companion policies:

• Hematology Section Workflow Quality Control and Result Validation Sop — Hematology Section quality control, interference, and result validation SOP
• Laboratory Reporting Policy — Result review, validation, and diagnostic report release
• Laboratory Retention Schedule Policy — Retention schedules for hematology worksheets and patient reports