Comprehensive Study Notes on Fundamentals of Blood Group Serology and Donor Screening

Fundametals of Donor Screening, Blood Banking, and Serology

• Donor screening is a systematic, exhaustive process of evaluating prospective donors to ensure:

  • The safety of the donor, ensuring the donation does not cause them harm.

  • The safety of the recipient, ensuring the blood does not transmit disease or cause adverse reactions.

  • The quality and adequacy of the blood collected for therapeutic clinical use.

Stages of the Donor Screening Process

• a) Registration: Involves donor identification, documentation of demographic data, and recording donation history.

• b) Health History and Questionnaire: A comprehensive survey covering travel history, medication use, past illnesses (e.g., hepatitis, malaria, STIs), high-risk behaviors, pregnancy or lactation status, recent tattoos/piercings, recent surgery, and previous transfusion history.

• c) Physical Examination:

  • General Appearance: The donor must look well, be alert, and show no signs of acute illness.

  • Weight: Minimum requirement is typically $\approx 50\,kg$ in most standard guidelines.

  • Age: Typical range is $18–65$ years.

  • Temperature: Must be normal, $\approx 37\,^{\circ}C$, and must not exceed $37.5\,^{\circ}C$.

  • Pulse Rate: Range of $60–100$ beats/min; must be regular.

  • Blood Pressure: Systolic should be $100–180\,mmHg$; Diastolic should be $60–100\,mmHg$ approximately.

  • Skin Inspection: The venipuncture site must be free of lesions or scars indicating IV drug use.

• d) Haemoglobin/Haematocrit Estimation: Performed to rule out anaemia in the donor.

• e) Mini-physical/Laboratory Pre-screen: May include ABO/Rh grouping and screening for Transfusion-Transmissible Infections (TTIs) on the collected unit.

• f) Final Decision: The donor is either accepted for donation or deferred.

Haemoglobin Estimation: The Copper Sulphate ($CuSO_4$) Method

• The $CuSO_4$ method is the most widely utilized rapid screening tool for donor haemoglobin in resource-limited blood banks.

• Principle: Based on specific gravity. A drop of blood is placed into a copper sulphate solution of a known specific gravity (usually $1.053$, which corresponds to an Hb of $\approx 12.5\,g/dL$).

  • Almost instantly, the copper sulphate forms a sac of copper proteinate around the blood drop, preventing its dilution.

  • If the drop sinks within $15$ seconds, its specific gravity (and Hb concentration) is equal to or greater than the solution.

• Reagent: Copper sulphate solution at specific gravity $1.053$ (for females, $\approx 12.5\,g/dL$ Hb). Some centers use $1.055$ for males ($\approx 13.5\,g/dL$ Hb).

• Procedure:

  1. Clean the donor's fingertip or earlobe with antiseptic and prick for a free-flowing drop.

  2. Allow the drop to fall gently from a fixed height into the $CuSO_4$ container (at room temperature) using a dropper or capillary tube, without touching the sides.

  3. Monitor the drop for $15$ seconds.

• Interpretation:

  • Sinks within $15$ seconds: "Passes" (Hb is adequate).

  • Floats, hovers, or sinks slowly (after $15$ seconds): "Fails" (Hb is likely low); the donor is deferred and referred for definitive testing.

• Advantages: Simple, rapid results (seconds), inexpensive, no electricity required, no sophisticated equipment, ideal for mass donation drives.

• Disadvantages/Limitations: Qualitative only (pass/fail); subjective interpretation (observer-dependent); affected by solution temperature and technique (height/size of drop); requires regular calibration with a hydrometer; risk of cross-contamination.

Alternative Methods for Haemoglobin/Anaemia Screening

• a) Sahli's (Acid Haematin) Method: Blood is mixed with $\text{N/10 HCl}$ to convert Hb into brown acid haematin. It is then diluted with distilled water to match a standard comparator block. It is subject to observer error and considered a classical/teaching technique.

• b) Cyanmethaemoglobin (Haemiglobincyanide, $HiCN$) Method: The gold-standard/reference method. Blood is lysed and Hb is converted using Drabkin’s reagent (potassium ferricyanide + potassium cyanide). Absorbance is read on a spectrophotometer at $540\,nm$. It is highly accurate but involves hazardous cyanide disposal.

• c) Digital/Photometric Haemoglobinometers (e.g., HemoCue®): A microcuvette with dry sodium azide reagent draws capillary blood, converting Hb to azidemethaemoglobin. Provides a direct digital quantitative reading in seconds.

• d) Packed Cell Volume (PCV)/Haematocrit: Capillary blood in a heparinized tube is centrifuged at high speed and read via an Hct reader. Requires a microhaematocrit centrifuge.

• e) Automated Haematology Analysers: Used for full blood counts; extremely accurate but expensive and impractical for on-the-spot mass screening.

Comparison Summary of Hb Methods

• CuSO$_4$: Qualitative; Very fast; Low–moderate accuracy; No equipment needed.

• Sahli's: Semi-quantitative; Moderate speed; Low accuracy; Comparator block needed.

• Cyanmethaemoglobin: Quantitative; Slow speed; Very high accuracy; Spectrophotometer needed.

• HemoCue/Digital: Quantitative; Fast speed; High accuracy; Portable photometer needed.

• Microhaematocrit: Quantitative (Hct); Moderate speed; High accuracy; Centrifuge needed.

• Automated Analyser: Quantitative; Fast (batch) speed; Very high accuracy; Haematology analyser needed.

Donor Deferral Criteria

• Temporary Deferral (Donor can return after a period):

  • Recent minor illness (cold/flu): Defer until fully recovered.

  • Recent tattoo/piercing/acupuncture: $6–12$ months.

  • Pregnancy and lactation: $6$ months–$1$ year post-delivery/weaning.

  • Recent vaccination: Days to weeks depending on vaccine.

  • Travel to malaria-endemic areas: $3–12$ months.

  • Low Hb/Hct: Defer until corrected.

  • Minor surgery: Defer until healed.

  • Recent donation: Minimum interval is typically $3$ months for whole blood.

• Permanent Deferral:

  • Confirmed HIV, HBV, HCV, or HTLV infection.

  • History of viral haemorrhagic fever.

  • Chronic severe cardiac, renal, or hepatic disease.

  • Certain types of cancer history.

  • Intravenous drug use history.

  • Confirmed Creutzfeldt-Jakob disease risk factors.

Types of Blood Donors

• Voluntary Non-Remunerated Donor (VNRD): Donates altruistically with no payment; safest category due to low coercion to hide risk factors.

• Replacement/Family Donor: Donates for a specific relative/friend; carries higher risk due to potential pressure to donate.

• Paid/Professional Donor: Donates for money; discouraged/banned due to the highest risk of TTI transmission and concealment of risk history.

• Autologous Donor: Donates for their own planned elective surgery; eliminates alloimmunization risks.

• Directed/Designated Donor: Specifically selected by the recipient or family to donate for a particular patient.

• Apheresis Donor: Donates specific components (platelets, plasma) via cell separator; remaining components are returned.

Donor Counselling and Reactions

• Pre-donation: Covers process explanation, risks, confidential discussion of high-risk behaviors, and informed consent.

• Post-donation: Advice on rest, fluids, and avoiding strenuous activity. Guidance on recognizing delayed reactions (dizziness).

• Post-test: If reactive for a TTI, involves referral for confirmatory testing and clinical care while maintaining confidentiality.

• Adverse Reactions and Management:

  • Vasovagal syncope (fainting): Most common. Management: Stop donation, lay donor flat, raise legs, loosen clothing, give fluids, monitor vitals.

  • Haematoma: Management: Apply firm pressure, ice pack, elevate limb.

  • Arterial puncture: Immediate needle withdrawal, pressure for $\ge 10$ minutes.

  • Citrate toxicity: Tingling/tetany in apheresis. Management: Slow procedure, provide oral calcium.

  • Convulsions: Protect from injury, use recovery position, medical review.

Screening for Transfusion-Transmissible Infections (TTIs)

• Mandatory screening for: HIV, Hepatitis B (HBsAg), Hepatitis C, Syphilis, and Malaria (in endemic regions).

• Testing Platforms:

  • Rapid Diagnostic Test (RDT): Lateral-flow cards; results in $15–20$ minutes; less sensitive but useful in emergencies.

  • ELISA: Mainstay method; detect antigens/antibodies (e.g., 4th Gen HIV ELISA detects p24 antigen and antibody).

  • CLIA/ECLIA: Fully automated, highly sensitive/specific; reduces human error.

  • Nucleic Acid Testing (NAT): Directly detects viral RNA/DNA. Gold standard for donor safety; drastically shortens the window period but is expensive.

• The Window Period: The time between infection and when a test can detect markers. It remains the greatest residual risk in transfusion safety.

Blood Bank Organization and Facilities

• Personnel Roles:

  • Medical Director/Consultant Haematologist: Policy and technical oversight.

  • Blood Bank Scientist/Technologist: Grouping, screening, cross-matching, component prep, QC.

  • Phlebotomist/Collection Staff: Blood collection.

  • Quality Assurance Officer: SOP compliance, accreditation.

• Physical Facilities:

  • Donor area/lounge for waiting and rest.

  • Counselling room for private history-taking.

  • Laboratory/Testing area with biosafety cabinets.

  • Storage: Refrigerators ($2–6\,^{\circ}C$), Plasma freezers ($-18\,^{\circ}C$ to $-30\,^{\circ}C$), Platelet agitators ($20–24\,^{\circ}C$).

• Records in Blood Banking: Required for "vein to vein" traceability. Must be accurate, legibility, and contemporaneous. Retention is usually $5–10$ years.

Blood Component Preparation and Storage

• Packed Red Blood Cells (PRBC): Heavy spin centrifugation; stored at $2–6\,^{\circ}C$ for $35–42$ days. Used for anaemia and oxygen-carrying capacity.

• Fresh Frozen Plasma (FFP): Separated within $6–8$ hours of collection; stored at $\le -18\,^{\circ}C$ for $1$ year. Used for clotting factor deficiencies.

• Platelet Concentrate: Prepared by light-spin or apheresis; stored at $20–24\,^{\circ}C$ with continuous agitation for $5$ days. Used for thrombocytopenia.

• Cryoprecipitate: Slowly thawing FFP at $1–6\,^{\circ}C$; rich in Factor VIII, vWF, and fibrinogen; stored at $\le -18\,^{\circ}C$ for $1$ year.

• Whole Blood: Stored at $2–6\,^{\circ}C$ for $21–35$ days. Used for massive haemorrhage.

• Cold Chain Management: An unbroken sequence of controlled temperatures. Units removed from storage for $>30$ minutes are typically not returned to stock without validation.

Blood Group Specific Substances (BGSS)

• Definition: Water-soluble glycoproteins/glycolipid-derived antigens carrying ABO/Lewis specificity found in body secretions.

• Secretor Status: Genetically controlled by the $FUT2$ ($Se$) gene.

  • Secretors ($\approx 80\%$ of population): Genotypes $SeSe$ or $Sese$. Contain ABH substances in secretions.

  • Non-secretors ($\approx 20\%$ of population): Genotype $sese$. Lack soluble ABH substances; red cell expression (controlled by $H$ gene) remains normal.

• Locations: Saliva, gastric secretions, ovarian cyst fluid (rich source), amniotic fluid, semen, milk, tears, bile, and urine. Not found in CSF or sweat.

• Synthesis: Paralells red cell synthesis but on Type 1 precursor chains (secretions) vs Type 2 (red cells).

• Biochemistry of Substances (Terminal Sugars):

  • H substance: L-Fucose added by $\alpha\text{-1,2-fucosyltransferase}$ ($H$ or $Se$ gene).

  • A substance: N-acetylgalactosamine added by $\alpha\text{-1,3-N-acetylgalactosaminyltransferase}$.

  • B substance: D-Galactose added by $\alpha\text{-1,3-galactosyltransferase}$.

  • $Le^a$ substance: L-Fucose added directly to precursor by $\alpha\text{-1,4-fucosyltransferase}$ ($Le$ or $FUT3$ gene).

  • $Le^b$ substance: Requires both $Le$ and $Se$ genes acting together.

Identification and Forensic Application of BGSS

• Hemagglutination Inhibition Test: If a secretion contains substance (e.g., A), it neutralizes corresponding antibody (anti-A).

  • Result: No agglutination when A red cells are added indicates the substance was present (Inhibition).

  • Result: Agglutination indicates substance was absent (No Inhibition).

• Forensic Medicine: Identifying secretor status from crime scene stains (saliva, semen) to exclude or link suspects.

• Bombay Phenotype ($Oh$): Individuals with genotype $hh, sese$. No H substance produced on red cells or in secretions. Red cells react as group O but lack reactivity with anti-H lectin.

• Para-Bombay: General genotype is $hh$ on red cells, but $Se$ is functional. Secretions contain H (and possibly A/B) substance even though red cells lack it.

Quality Control (QC) in Serology

• Physical Equipment QC:

  • Centrifuges: Speed (RPM/RCF) and timing checked periodically.

  • Refrigerators ($2–6\,^{\circ}C$) and Freezers: Monitored daily or twice daily.

  • Water baths ($37\,^{\circ}C$): Temperature verified daily.

• Chemical Reagent QC: Saline (pH $\approx 7.0$), Bovine Serum Albumin (BSA), and Enzymes (Papain, Ficin) must have potency verified.

• Reagent (Antisera/Cells) QC:

  • Potency: Ability to react at defined dilutions.

  • Specificity: No cross-reactivity.

  • Avidity: Speed and strength of agglutination.

• Anti-Human Globulin (AHG) QC: Checked daily using IgG-sensitised (Coombs' control) cells.

• Validation vs. Verification:

  • Validation establishes that a brand-new method works as specified.

  • Verification confirms that a previously validated method continues to work (routine QC).

• Statistical Tools: Levey-Jennings charts plot control values against mean $\pm$ Standard Deviation (SD). Westgard Rules define statistical limits for run acceptance (e.g., $1_{3s}$, $2_{2s}$).

• CAPA (Corrective and Preventive Action): Process for identifying non-conformance, investigating root causes, taking immediate correction, and implementing preventive measures.

• Donor screening ensures donor safety, recipient safety, and blood quality.

• Stages of Donor Screening Process:

  • Registration:

  • Donor identification, demographic data, and donation history.

  • Health History and Questionnaire:

  • Covers travel, medication, past illnesses, high-risk behaviors, pregnancy/lactation, tattoos/piercings, surgery, transfusion history.

  • Physical Examination:

  • General appearance: well, alert, no acute illness.

  • Weight: Minimum $ ext{approx. }50 ext{ kg}$.

  • Age: $18–65$ years.

  • Temperature: Normal $ ext{approx. }37 ext{°C}$ (not exceeding $37.5 ext{°C}$).

  • Pulse Rate: $60–100$ beats/min; must be regular.

  • Blood Pressure: Systolic $100–180 ext{mmHg}$; Diastolic $60–100 ext{mmHg}$.

  • Skin Inspection: No lesions or scars.

  • Haemoglobin/Haematocrit Estimation:

  • Rules out anaemia.

  • Mini-physical/Laboratory Pre-screen:

  • ABO/Rh grouping and screening for TTIs.

  • Final Decision:

  • Donor accepted or deferred.

• Haemoglobin Estimation - Copper Sulphate Method:

  • Most common rapid screening tool.

  • Based on specific gravity; drop of blood in $CuSO_4$ solution.

  • Immediate results; low equipment needs.

  • Alternative Methods:

  • Sahli's Method: Subject to observer error.

  • Cyanmethaemoglobin Method: High accuracy, hazardous.

  • HemoCue: Digital, rapid results.

  • Packed Cell Volume: Requires centrifuge.

  • Automated Analysers: Accurate but impractical for mass screening.

• Donor Deferral Criteria:

  • Temporary: Recent illness, tattoos, pregnancy, travel, low Hb/Hct, minor surgeries.

  • Permanent: Confirmed infections (HIV, HCV), chronic diseases, IV drug use.

• Types of Blood Donors:

  • Voluntary Non-Remunerated Donor (safest).

  • Replacement/Family Donor (higher risk).

  • Paid Donor (highest risk).

  • Autologous Donor (self-donation).

  • Directed/Designated Donor (specific patient).

  • Apheresis Donor (specific components).

• Donor Counselling and Reactions:

  • Pre-donation: Process explanation, risks discussed.

  • Post-donation: Advice on rest and recognizing delayed reactions.

  • Adverse Reactions: Management techniques outlined.

• Screening for Transfusion-Transmissible Infections (TTIs):

  • Mandatory testing for various infections.

  • Testing methods: RDT, ELISA, CLIA/ECLIA, NAT.

  • The window period is crucial for transfusion safety.

• Blood Bank Organization:

  • Personnel roles defined (Medical Director, Scientist, Phlebotomist, etc.).

  • Physical facilities necessary.

• Blood Component Preparation and Storage:

  • PRBC: Stored at $2–6 ext{°C}$ for $35–42$ days.

  • FFP: Stored at $-18 ext{°C}$ for 1 year.

  • Platelet Concentrate: Stored at $20–24 ext{°C}$ for 5 days.

  • Whole Blood: Stored at $2–6 ext{°C}$ for $21–35$ days.

• Blood Group Specific Substances (BGSS):

  • Definition and secretor status explained.

  • Synthesis and locations of BGSS detailed.

• Quality Control in Serology:

  • Equipment checks and reagent QC outlined.

  • Validation vs verification processes.

  • CAPA process for non-conformance management.