CLS 306 - Immunohematology (Blood Banking) Notes

Introduction to Immunohematology (Blood Banking)

CLS 306 2025, Professor Won, Clinical Science Dept. Chapters 1, 2, 3, & Plate 1.

Healthcare World Overview

• The healthcare world is heavily regulated by different regulatory and accrediting agencies.

• Regulating government agencies:

  • Federal (e.g., CMS/CLIA, FDA)

  • State

  • Local (commonly, the County)

• Accrediting agencies (National Level):

  • The Joint Commission (TJC)

  • College of American Pathologists (CAP)

  • Association for the Advancement of Blood & Biotherapies (AABB)

• CMS: Centers for Medicare & Medicaid Services

• CLIA: Clinical Laboratory Improvement Amendments

New Systems in Healthcare

• When entering the healthcare industry, you will begin using two "new" systems:

  • The metric system for measurements:

    • Length: millimeter (mm), centimeter (cm), etc. US 1 inch ≈ 2.54 cm

    • Weight: milligram (mg), gram (gm), kilogram (kg), etc. US 110 lb. ≈ 50 kg

    • Volume: milliliters (ml), liters (L), etc. US cup (8.4 oz) ≈ 250 ml, 1 qt ≈ 1 Liter

    • Temperature: centigrade (C) US 34 - 43°F ≈ 1-6°C

  • The 24-hr clock for time:

    • Examples:

      • 0800 = 8:00 am

      • 0945 = 9:45 am

      • 1730 = 5:30 pm

      • 2145 = 9:45 pm

      • 2200 = 10:00 pm

      • 2335 = 11:35 pm

      • 1200 = 12:00 pm (noon)

      • 2400 = 12:00 (midnight)

      • 00:05 = 12:05 am

24-Hour Clock Examples

• Conversion between standard and 24-hour time formats:

  • 12 Midnight = 2400

  • 12 Noon = 1200

  • 12:01 AM = 0001

  • 12:01 PM = 1201

  • 12:15 AM = 0015

  • 12:15 PM = 1215

  • 12:30 AM = 0030

  • 12:30 PM = 1230

  • 12:45 AM = 0045

  • 12:45 PM = 1245

  • 1 AM = 0100

  • 1 PM = 1300

  • 2 AM = 0200

  • 2 PM = 1400

  • 3 AM = 0300

  • 3 PM = 1500

  • 4 AM = 0400

  • 4 PM = 1600

  • 5 AM = 0500

  • 5 PM = 1700

  • 6 AM = 0600

  • 6 PM = 1800

  • 7 AM = 0700

  • 7 PM = 1900

  • 8 AM = 0800

  • 8 PM = 2000

  • 9 AM = 0900

  • 9 PM = 2100

  • 10 AM = 1000

  • 10 PM = 2200

  • 11 AM = 1100

  • 11 PM = 2300

Laboratory Medicine

• The Healthcare Lab is officially called the "Clinical Laboratory".

• Clinical Laboratory vs.

  • Crime Lab

  • Pharmaceutical Lab

  • Biotech Lab

• The Clinical Laboratory is regulated by:

  • Federal agency

  • State agency

  • National Accreditation agency

• The Blood Bank has an option to be certified by another National Certification agency (AABB), but can operate without this certification.

• The Donor Center (connected to the Blood Bank) is regulated by another Federal agency.

California Clinical Laboratory Regulation

• The State of California regulation defines a "clinical laboratory" as a facility that tests any human sample for the purposes of diagnosis, prevention, treatment, or monitoring of disease.

• The State of California "clinical laboratory" regulation requires both:

  • Personnel license

  • Facility license

• A CLS license is NOT required to work in a Crime Lab or Pharmaceutical Lab.

• It is not required in a Biotech Lab but could be a benefit to the employer because you have knowledge of diagnostic testing principles.

CSUDH and CLS Licensure

• CSUDH is linked to the State-approved training program for the California Clinical Laboratory Scientist (CLS) licensure because of the personnel license requirement.

• CSUDH Clinical Science BS program is also accredited/regulated by the National Accrediting Agency for Clinical Laboratory Sciences (NAACLS).

• CSUDH offers, specifically, the Blood Banking course in its BS curriculum program…other schools do not.

Regulating Agencies

• Centers for Medicare & Medicaid Services (CMS) / CLIA (Clinical Laboratory Improvement Amendments) regulations - Federal

• Clinical & Laboratory Standards Institute (CLSI) - National

• California Dept of Public Health, Laboratory Field Services (CDPH LFS) regulations - State

• Donor Center - Food & Drug Administration (FDA)

• The Clinical Laboratory must follow regulations, implement policies & procedures to remain compliant.

• Failure to be compliant results in disciplinary actions, up to and including loss of facility license.

Accrediting Agencies

• The Joint Commission (TJC) - Hospital & Laboratory Healthcare Organization

• College of American Pathologists (CAP) - Clinical Lab as a whole entity; also covers the Donor Center, when applicable

• Association for the Advancement of Blood & Biotherapies (AABB) - applicable to the Blood Bank & Donor Center only; certification is optional

Clinical Rotation

• Once you enter into the CLS training program, aka, Clinical Rotation, you are beginning your "professional" career. You are no longer an "academic student."

• You are an "intern professional!"

• You will also be responsible & held accountable for your test results that you release…which will play a vital role in the diagnosis and treatment of the patient you just tested!

• As a licensed CLS, you are bound by certain rules and regulatory requirements.

• Negligence in testing and/or failure to follow the required rules/regulation or the facility's policies & procedures could also result in disciplinary actions, up to and including employment termination and/or loss of your CLS license.

• So…please be ACCURATE AND PRECISE in your testing.

Clinical Laboratory Departments

• The Clinical Lab has a variety of departments:

  • Laboratory Information Service (LIS)

  • Phlebotomy including phlebotomy drawing stations

  • Processing

  • Hematology / Coagulation / Urinalysis

  • Chemistry / Immunology / Serology / Flowcytometry

  • Microbiology / Virology / Mycology / Parasitology

  • Blood Bank / Transfusion Medicine (may include a Donor Center & its oversight)

  • Histology / Cytology

  • Anatomical Pathology

  • Administration

What is Immunohematology?

• Taking the definition and applying it to the Clinical setting, Immunohematology is commonly called "Blood Banking" (BB, hereafter).

• It can be divided into 2 subgroups:

  • Transfusion Medicine / BB – Giving Blood (to Patients)

  • Donor Center (Blood Establishment) – Taking Out Blood (from Donors)

The Blood Bank (BB)

• Since the BB is a department within the Clinical Lab, it is regulated by the State, CLIA, & CAP.

• It can also be regulated by an additional regulatory agency, e.g., Food & Drug Administration (FDA) when there is a Donor Center.

• Accreditation from AABB, a BB-specific organization, is optional.

• These groups protect both the patient receiving blood products and the donor who gives blood.

The Patient

From now on…It's all about "the patient" in Transfusion Medicine
That patient is depending on your knowledge to ensure proper and accurate test results for treatment.

The Blood Bank (BB) - Regulations

• Of all the various clinical laboratory departments, the BB is the most regulated department.

• There are many regulations/rules that exist that must be followed and a Quality Management System (QMS) must be in place.

• The BB QMS is slightly different than the Clinical Lab's QMS…hence, essentially, there are 2 QMSs functioning within the whole laboratory.

• There must also be a Patient Blood Management (PBM) program in place.

Blood Banking is Transfusion Medicine

• Involves testing the patient (who is about to receive a blood transfusion) to ensure that:

  • The unit of blood/blood component is compatible with the patient's blood AND

  • There is no intentional adverse reaction(s) toward the patient from the transfused blood/blood component

• Testing of the patient involves:

  • Blood typing (ABO & Rh)

  • Antibody Screening (ABS)

  • Crossmatching (xmatch or xmat) of blood

    • Physical vs Electronic

Blood Banking is Transfusion Medicine - Anemia

• Blood is transfused into "anemic" patients (an - without; emia - blood; ic - pertaining to)

• Specifically, the word "Blood" means red blood cells (RBCs) and there are 2 types of blood components containing RBCs:

  • Whole Blood (WB) - vol approx. 500 mls; provides both plasma for volume and Red Blood Cells (RBCs) for oxygen-carrying capacity to vital organs, e.g., brain, liver, heart, etc.

  • Red Blood Cells (RBCs) - vol approx. 250 - 300 mls; provides oxygen-carrying capacity to vital organs, e.g., brain, liver, heart, etc.

    • Most common blood component that is transfused into anemic patients

    • Oxygen-carrying capacity is related to the Hemoglobin-Oxygen Dissociation Curve

Blood Components

• Whole Blood (WB)

• Red Blood Cells (RBC)

Oxygen-Carrying Mechanism

• Human Hemoglobin

  • Oxygen molecules

  • Heme

  • Hemoglobin molecule

  • Red blood cell

Hemoglobin-Oxygen Dissociation Curve

• Curve illustrating the relationship between hemoglobin saturation and oxygen partial pressure.

• Important factors include:

  • Decreased P50 (increased affinity) shifting the curve to the left:

    • Temperature ↓

    • PC02 ↓

    • 2,3-DPG ↓

    • pH ↑

  • Increased P50 (decreased affinity) shifting the curve to the right:

    • Temperature ↑

    • PC02 ↑

    • 2,3-DPG ↑

    • pH ↓

Hemoglobin-Oxygen Dissociation Curve - Importance

• This curve shows the red cells' affinity to oxygen or its ability to release (dissociation) oxygen (to the vital organs).

• The Hemoglobin-Oxygen Dissociation Curve shifts to the "left", the longer blood is stored.

• This is an important, recognized factor when choosing RBCs for a neonate transfusion.

Blood Banking - Challenges

• Working in the BB department, there are challenges that you will face:

  • You MUST pay very close attention to detail ALL THE TIME…EVEN DURING AN EMERGENCY!! You cannot make any writing/transcription errors on your documentation!

  • You have to make the correct decision immediately – your decision could result in either benefiting the patient.

  • If you make a bad decision/mistake, there is the potential that you could actually cause a patient's demise…related to a blood transfusion.

  • You can be under a lot of pressure during a massive hemorrhage (hemo-blood; rrhage-rapid flow) (e.g., Trauma, surgery gone bad, etc.) when doctors/nurses are screaming for more blood. The phone could be ringing "off the wall"! Or, if the dept has an intercom box, they will call all the time.

Blood Banking - Rewards and Risks

• Not many CLSs choose to work in the BB because of the:

  • Intense pressure during emergencies/traumas

  • Constant necessity to pay close attention to detail ALL the time

  • Potential that you could "kill" someone, if you make a bad mistake.

• On the other hand, you can actually save a life immediately…because of a blood transfusion. You can feel an immediate gratification when you’ve saved that person's life!

• The department's staff are the "Navy Seals" of the lab…during an emergency, you are under great pressure to test & release blood, you cannot make a mistake or make the wrong decision, but you've "correctly accomplished your mission!"

Transfusion History

First Human Blood Transfusion

• June 15, 1667 using sheep blood

First Successful Human-to-Human Transfusion

• 1870's

  • Old Time blood TYANFUSION

  • The scene is of an "old time blood transfusion" at Bellevue Hospital in the 1870's.

  • There are several nurses and doctors in attendance, and the man giving blood is seated upright in a chair. This image is not an original from the 19th century. It is a modern print of an original that is either lost or no longer exists.

Blood Components as Pharmaceutical Drugs

• The FDA considers Blood/Blood Components as Pharmaceutical Drugs.

• Because of this, a prescription must be written to receive any blood or blood component.

• During an emergency, verbal orders (VOs) are acceptable, however, when the emergency is over, all documentation must be completed in the patient's chart.

• Pharmaceutical drugs are heavily regulated for the protection of the consumer - the FDA is the consumer advocate.

Blood/Blood Component Storage Requirements

• Red Blood Cells (RBCs)

  • Stored in a Blood refrigerator (1-6°C) for specific time frames dependent on type of anticoagulant preservative found in the donation bag.

    • CPD = 21 days

    • CPDA-1 = 35 days

    • Adsol (additive solution) = 42 days.

• Platelets (Plts)

  • Stored at Room Temperature (20-24°C) for 5 days.

• Fresh Frozen Plasma (FFP)

  • Stored (≤ ─ 18°C) frozen for up to 1 yr.

• Liquid Plasma (LP)

  • Stored in the Blood refrigerator (1-6°C) for up to 5 days.

Basic Genetics

Chapter 2

Human Blood Groups

• Human Blood Group antigens (ags) are found on the surfaces of RBCs & Plts and are associated with genetic make up

• There are 36 Human Blood Groups that result in over 379 different ags, i.e., 346 RBC ags and 33 platelet ags*.

• Of which, approx. 27 ags are what we deal with on a daily basis.

• Transfusion, vol 56, no.3, Mar 2016, pgs 743-754

Human Blood Groups and Immunology

• Familiarity with immunology concepts regarding antigen vs. antibody and the body's immune response is assumed.

• Blood Banking commonly deals with the Class/Type II Hypersensitivity category.

• If a patient lacks a RBC antigen and is transfused with blood containing the patient's lacking RBC antigen, the patient can develop an antibody to that RBC antigen.

• These RBC antibodies can cause subsequent transfusion reactions by attacking the transfused cells and destroying them, either through hemolysis or extravascular sequestration.

Mendel's Law of Inheritance

• Law of Inheritance (based on his study of the sweet pea plant)

  • Dominance

    • Certain genes expressed themselves as dominant

    • Dominant was designated by a capital letter (R = red color); flowers were all seen as Red color.

    • However, there was a white flower that he saw which was designated as a lower case letter (r = white) where the white color was expressed when the recessive genes were only expressed in the homozygous state

  • Segregation

    • When the two different traits come together in one hybrid pair, the two characters do not mix with each other and are independent of each other.

Mendel's Law of Inheritance (cont.)

• Independent Assortment

  • Genes for different traits are inherited separately from each other and allows for all possible combinations of genes to occur in the offspring.

Mendel's Law of Inheritance - Inheritance Pattern

• Autosomal Dominant - gene indicated by using a capital letter

• Autosomal Recessive - gene indicated by using a small letter

• The dominant gene is expressed over the recessive gene, e.g., B = brown eyes; b = blue eyes; Bb = brown eyes are seen

• Homozygous - the presence of the same two genes

• Heterozygous - the presence of two different genes

• Sex-linked (Dominant or Recessive) - linked to x gene

Human Blood Groups - Autosomal Codominant

• Autosomal Codominant - 2 genes are equally expressed when both are present, e.g., A gene & B gene present, AB is expressed. This is the typical gene expression that we see in the Blood Groups that we deal with in the BB.

Hardy-Weinberg Law

• One of the most useful tools in the study of population genetics. It permits the estimation of gene frequencies from phenotype frequencies observed in a population.

• States (in algebraic form) that the genotype frequencies in a population, remain constant or in equilibrium from generation to generation, unless specific influences are introduced. (Certain criteria must be met)

• Equation: p^2 + 2pq + q^2 = 1

  • where: p = gene frequency of the dominant allele

  • q = gene frequency of the recessive allele

Criteria for the use of the Hardy-Weinberg Formula

• Population studied must be large.

• Mating among all individuals must be random.

• Mutations must not occur in parents or offspring.

• There must be no migration, differential fertility, or mortality of genotypes studied.

Genotype vs Phenotype

• Genotype

  • Is an attribute that you can NOT "see"

  • Old thinking - Individual genetic makeup cannot be absolutely determined without family studies, but can be "inferred" from the phenotype, based on gene frequencies in a population

  • New evolution today - the detection of the molecular make-up of the blood group ags; analysis / probe for a single nucleotide polymorphism (SNPs)

• Phenotype

  • A physical attribute that you can "see" i.e., hemagglutination

  • Test results using specific antisera with red blood cells

    • 3 ABO typing sera available: anti-A, anti-B, anti-A,B

    • 5 Rh typing sera available: anti-D, anti-C, anti-c, anti-E, anti-e

Blood Bank Immunoglobulins, Specimens, & Hemagglutination Reactions

• Chapter 3

Performing Lab Tests - Transcription Errors

• Whenever you are performing any manual test result entry(ies) into the computer or onto a document, you MUST ENSURE that you do NOT commit a transcription error…as this could have a detrimental effect on the patient.

• A transcription error(s) could be entering/writing the test result:

  • Into the wrong line/box/computer field

  • On the wrong patient

  • Misinterpreting the test result

  • Marking the wrong bubble on the scantron sheet

Immunoglobulin Types

• IgM

  • Large Ig - pentamer

  • Does not pass through the placenta

  • Readily binds C' (complement)

  • Reacts at RT or lower

  • Usually seen in the first phase of antibody detection testing, i.e., IS

• IgG

  • Small Ig - a monomer (single Y-shaped Ig); accounts for 80% of Igs present in the sera

  • Passes through the placenta barrier

  • Only certain IgG subtypes readily bind C' (IgG3 > IgG1 > IgG2 > IgG4)

  • Reacts at the 37°C or at the Anti-Human Globulin (AHG) test phase of antibody detection testing / ABS (Antibody Screen)

Anamnestic / Secondary Response

• IgM present during First exposure

• IgG present during Second exposure

• Low level remains

Immunoglobulin Types (cont.)

• IgA

  • Usually a dimer or trimer containing a polypeptide called the J chain that joins the monomers together

  • Most IgA exists in mucosal secretions & other body fluids

  • Secretory IgA protects the underlying epithelium from bacterial/viral penetration

  • Does not bind C' like IgM & IgG

  • Rare case: patient may have an anti-IgA which can cause a severe transfusion reaction (anaphylaxis rxn) leading to death if transfused with plasma-containing products.

Laboratory Specimens

• Clot Tube vs Plasma Tube

• Serum = Plasma - Clotting Factors

• Plasma (55% of total blood)

• Buffy Coat - leukocytes & platelets (<1% of total blood)

• Erythrocytes (45% of total blood)

Laboratory Specimen Tube Types

• Various tubes with different additives used for specific tests.

• Examples: Sodium Citrate (Blue), Clot activator and gel (Gold), Silicone coated (Red), Lithium Heparin and gel (Light Green), Lithium Heparin (Green), Sodium Heparin (Green), K EDTA (Lavender), Spray coated KZEDTA (Pink), Sodium Fluoride/K Oxalate (Gray), Clot activator (Royal Blue)

BB Serologic Grading - Ab / ag Reactions

• When reading hemagglutination (either via manual test tube or automated) reactions, the reactions are always graded, i.e., 0, 1, 2, etc. [Refer to the text book, Plate 1]

• Strength of reactions may also be associated with the titer of antibody or the affinity of the antibody

• Also be aware of non-immune hemagglutination reactions, e.g., rouleaux caused by the presence of high amounts of protein seen in Multiple Myeloma

BB Serologic Grading - Ab / ag Reactions - Grading

• Grading of Agglutination Reaction:

  • +4: Single clump of agglutination with no free cells

  • +3: Three or four individual clumps with few free cells

  • +2: Many fairly large clumps with many free cells

  • +1: Fine granular appearance visually, but definite small clumps (10-15 cells) per low power field

  • +W: 2 to 3 cells sticking together per low power field, uneven distribution Visually no agglutination, All cells are free

  • 0: All cells are free

  • H: Hemolysis (partial or total) must be interpreted as positive

BB Serologic Grading (Visual) - Tube

• Grading System for Reactions:

  • 4+ Reaction

  • 3+ Reaction

  • 2+ Reaction

  • 1+ Reaction

  • Negative reaction

  • Hemolysis

BB Serologic Grading (Visual) - Microtiter Plate, Tube, & Gel

• Grading Capture R Hemagglutination Gel/CAT:

  • 4+

  • 3+

  • 2+

  • 1+

  • 0 (negative)

References Used in This Course

1. Harmening, Denise M., Modern Blood Banking & Transfusion Practices, 7th Ed., 2019, F.A. Davis, Philadelphia, PA.

2. ASCP, Quick Compendium of Medical Laboratory Sciences, 2nd Ed., 2024, ASCP Press,

3. AABB, ABC, ARC, ASBP, Circular of Information for the use of human blood and blood components pamphlet, Dec., 2021.

4. AABB, Technical Manual, 20th Ed., 2020, AABB Publisher, Bethesda, MD.

5. AABB, Standards for Blood Banks and Transfusion Services, 32nd Ed., 2020, AABB Publisher, Bethesda, MD.

6. FDA, Guidance, Compliance, & Regulatory Information (Biologics) website: www.fda.gov/vaccines-blood-biologics.

7. Other references (e.g., internet information, pictures, etc.), as appropriate & cited within the presentation slide.