HAD Final Study Guide

Blood Bank Responsibilities

Blood Bank Responsibilities

• Identifying blood types and antibodies

• Testing blood for viruses that might be transmitted during transfusion

• Investigating harmful responses of the body to blood transfusion

• Supervising the collection, separation, delivery and storage of blood components

• Supporting physicians and nurses in blood transfusion therapy

What does a Blood Bank Technologist do?

• test for blood group antigens, compatibility and antibody identification

• investigate abnormalities such as hemolytic diseases of the newborn, hemolytic anemias, and adverse reactions to transfusion

• supporting physicians physicians in transfusion therapy for patients with coagulopathies

• Performing blood collection and processing, including selecting donors, collecting blood, typing blood, and molecular testing

• Performing viral marker testing to ensure patient safety

• Managing patient blood

Blood components

• RBCs

• Plasma

• Cryoprecipitated Antihemophilic Factor

• Platelets

• Granulocytes

• Mononuclear cells

• Hematopoietic progenitor cell

Whole Blood Derived Components

• RBCs

• Platelet Concentrate

• Fresh Frozen Plasma

• Cryoprecipitate

Whole Blood

• Trauma, Surgery

• Most common type of blood donation

• requires minimal processing before it is ready to be transfused into a patient

• Used to treat patients who need all the components of blood, such as those who have sustained significant blood loss due to trauma or surgery

Red cells

• Trauma, surgery, anemia, any blood loss, blood disorders (sickle cell)

Platelets

• cancer treatments, organ transplants, surgery

Fresh Frozen Plasma

• Burn patients, shock, bleeding disorders

Blood Plasma

• Helps maintain satisfactory blood pressure and volume

• Supplies critical proteins for blood clotting and immunity

• Carries electrolytes such as sodium and potassium to muscles

• Helps to maintain a proper pH (acid-base balance)

Plasma derivatives

• Concentrates of specific plasma proteins obtained through process known as fractionation

• Treated with heat and/or solvent to kill certain viruses

Cryoprecipitate

• Hemophilia

• Von Willebrand disease (most common hereditary coagulation abnormality)

• Rich source of Fibrinogen

• Help reduce blood loss by helping to slow or stop bleeding due to illness injury

• Used to prevent or control bleeding in people who own blood does not clot properly

Blood Donation to Transfusion Steps

• Donation

• Processing

• Testing

• Storage

• Distribution

• Transfusion

Infection Disease testing on donations

• Treponema pallidum antibody (Syphilis)

• Hepatitis B surface antigen (HBsAg)

• Hepatitis B core antibody (anti-HBc)

• Hepatitis C virus antibody (anti-HCV)

• HIV-1 and HIV-2 antibody (anti-HIV-1 and

• anti-HIV-2)

• HTLV-I and HTLV-II antibody (anti-HTLV-I

• and anti-HTLV-II)

• HIV, HCV, West Nile Virus RNA (NAT)

• Typanosoma cruzi antibody

• Zika Virus (until 2021)

• Babesia antibody

Blood Type Testing - pretransfusion

• ABO typing

  • A and B antigen test

  • Anti-A and Anti-B antibody test

• Rh typing

  • Rh(D) antigen test

  • Red Cell antibody

  • EDTA purple top blood sample

Blood Types

• A±

• B±

• O±

• AB±

Blood Type- Details

• There are 4 major blood groups determined by the presence or absences of two antigens, A and B, on the surface of RBCs

• In addition to the A and B antigens, there is a protein called the Rh factor, which can be either present (+) or absent (-), creating the 8 most common blood types

Blood Type Chart

Group A Only A antigen on RBCs B antibody in plasma	Group B Only B antigen on RBCs A antibody in plasma
Group AB [Universal Acceptor] Has A and B antigen on RBCs Neither A or B antibody in plasma	Group O [Universal Donor] Neither A and B antigen on RBCs Both A and B antibody in plasma

Selection of Compatible Blood Products

• ABO type

• Rh Type

• Unexpected antibodies

  • antibody identification

  • phenotype negative donors

Crossmatch

check the recipient’s blood for a reaction to the donor blood

Emergency Transfusion

• Group O red cells

• Group AB plasma

• Rh negative preferable

  • Women of childbearing potential

  • if Rh+, then the first pregnancy is generally unaffected but the Anti-Rh antibodies remain in the mother and will attack the Rh factors in the blood of the second Rh+ fetus

• Obtain pre transfusion sample asap

Clinical Considerations

• Cause of bleeding or red cell loss

• Rate of blood loss

• Underlying diseases

• Risks of future bleeding

• Physiologic compensations

Indications for transfusion

• Symptomatic anemia

• Bleeding > 15% total blood volume

• Chronic hypoproliferative anemia

• Sickle cell anemia

  • Hemolytic crisis

  • Acute chest syndrome

  • Stroke prophylaxis

• Uremia

Red Blood Cell Antibodies

• Rh (D, C, c, E, e)

• Kell

• Lewis

• Kidd

• Duffy

Transfusion Reactions

• Acute hemolytic

• Delayed hemolytic

• Febrile non-hemolytic

• Simple allergic

• Anaphylactic

• Septic (bacterial contamination)

• Transfusion-related acute lung injury (TRALI)

• Transfusion-associated circulatory overload (TACO)

Cytology

The study of the structure and function of individual cells

• Used in the diagnosis and screening of cancer, pap smears, fetal abnormalities, and for the diagnosis of infectious organisms

• Commons samples include:

  • Pleural fluid, pericardial fluid, peritoneal fluid, an cerebrospinal fluid (CSF)

Cytotechnologists work with pathologists by:

• Using a microscope to examine the cells of the body

• interpreting cells collected from cytological techniques as normal or abnormal

• Detecting changes in human cells that indicate cancer, disease, or other abnormalities

• Collaborating with a pathologist to provide a timely diagnosis to allow physicians to provide appropriate treatment

Cytoplasm

Formative material within the cell that suspends all organelles

Organelles

Tiny structures in the cytoplasm which perform various jobs for the cell

Cytosol

The fluid part of the cytoplasm

Lysosome

An organelle with digestive enzymes that protects the cell from invasion

Centrioles

Organelle that prepares for cell morphing before mitosis

Prokaryotic cells

• Lack a nuclear membrane as well as mitochondria and membrane bound organelles

• Always unicellular

• Considered “primitive cells”

• Bacteria and blue-green algae

Eukaryotic cells

• Clearly defined nucleus

• Possess bound organelles in their cytoplasm

• May be unicellular or multicellular

• Cell volume much larger than prokaryotes (10000 times greater)

• Plant, animal, and fungus cells

Cytotechnologist

• Typically examine human cells

• One of the main functions of a cytopathology lab is that of cancer diagnosis

Cell specimens may be obtained from various body sites:

• Cervical

• Oral cavity

• Lung

• Breast

Cell collection

• Cells that spontaneously exfoliate (scrape off) are typically used (PAP)

• Cells can also be obtained from washings of sites or organs (ex: bronchial, stomach)

• Cells may be obtained from Fine Needle Aspirate Techniques (FNA)

How cells are processed

• Cells are chemically fixed on a slide or in a container with alcohol or formalin

• Cells are stained to distinguish cellular components

Abnormal cell features

• Cellular enlargement

• Enlargement of nucleus

• Irregular nuclear borders

  • it is important to have detailed information about the specific body site, patient information, medications, therapy, etc.

Stains in cytopathology

• Papanicolaou (PAP) stain - a modified hematoxylin and eosin (H&E) stain revealing structures such as the nucleus, cytoplasm, and cellular granules

• Romanowsky stain - allows for a greater size evaluation of the nucleus, cytoplasm and overall cell area. This is used on air-dried cells

Histology

• The study of the structure, function, and composition or normal and abnormal tissue

• May be referred to as microscopic anatomy or microanatomy

• Histotechnology is a science centering on the microscopic detection of tissue abnormalities for diease diagnosis and the treatment of diseases

How tissues are studied in the lab by histotechnologists

• Sections of body tissue are processed in the histology lab by histotechnologists →

• Tissue is fixed in alcohol and placed in a tissue processor to remove fluids →

• Tissue is then made transparent by chemicals →

• Tissue is then immersed with paraffin or wav to become a solid block in order to slice thin-layers of tissue →

• Thin layers are mounted on a glass-slide, and coverslipped so the pathologist can view, to rule out malignancy 

Histology technologist responsibilites

• Grossing and Fixation

• Processing

• Embedding

• Sectioning

• Staining

Histology Specimen Preparation

• Fixation, dehydration, embedding

• Sectioning

• Looking for:

  • Size

  • shape

  • location

  • number

  • nucelolus

  • density (euchromatic [less dense] vs. heterochromatic [denser])

Stains in Histology

• Hematoxylin cationic dye (+) that binds to negatively charged (acidic) structures in the cell

• Eosin: anionic dye (-) adheres to basic structures in the cell

• (NH+) on proteins make cytoplasm pink

Features that help differentiate cells

• Ribosomes

• Golgi apparatus

• mitochondria

• endoplasmic reticulum

• microfilaments

• secretory vesicles

• pigment granules

• lipid droplets

Cell specilizations

• Microvilli

• Cilia

• Stereocilla

Microvilli

Found on many epithelial cells

Cilia

Motile structures found in trachea, bronchi, oviducts, and flagella of sperm

Stereocilia

Very long microvilli found in epididymis and on cells of inner ear hairs

Technologists & Teamwork

• Technologists in these specialties typically do not release results, but prepare specimens for review by the pathologist who then determines if the sample is normal or abnormal

• Having good communication and teamwork skills is imperative for these departments

• Histology and cytology technologists require special certification to operate in NY

Immunology

The study of the immune system

Clinical Immunology Laboratories

• Specialize in the evaluation of autoimmune disease, immunodeficiencies, immunoproliferative disorders, allergy, and some aspects of infectious disease serology

• Investigates novel aspects of the cellular immune response to pathogens

• The lab performs and offers expert interpretation on a broad array of laboratory tests and comprehensive consultation in clinical and diagnostic immunology

The Immune System

• complex system of tissues, cells, cell products, and biologically active chemicals

• Produces an immune response

• Defense mechanism against foreign substances called antigens (ag)

Natural Resistance

• Includes physical barriers (skin mucous membranes, etc.) white blood cells like neutrophils, and proteins that cause inflammation

• Non-specific

• Does not require exposure to an antigen

Specific Immunity

• Recognition: recognizes foreign antigens and distinguishes them from “self”

• Specificity: reacts with a specific antigen without reacting with others

• Memory: anamnestic response, a renewed rapid production of an antibody following second or later contact with the provoking antigen or with related antigens

Primary Lymphoid organs

• Lymphocytes (wbcs) are produced in the bone marrow

• B-cells/T-cells

Secondary Lymphoid organs

• Lymph nodes

• Spleen

• Tonsils

• Certain tissue in various mucous membrane layers in the body (for instance in the bowel)

• It is in these organs where the cells of the immune system do their actual job of fighting off germs and foreign substances

Humoral Immunity

• B lymphocytes produce antibodies against specific antigens

• Good protection against bacteria, toxins, and circulating antigens

Cell-mediated immunity

T-lymphocytes protect against viruses, fungi, tumor cells, and intracellular organisms

Immunoglobins (Ig) - Antibodies (Ab)

• Named the antibodies by placing the prefix anti- before the name of the antigen with which the anitbody reacts

• Recognize and bind to specific antigens, such as bacteria or viruses, and aid in their destruction

Primary Immune Response

• Occurs after first exposure to an antigen

• First antibody produced is mainly IgM

• Amount of antibody depends on nature of antigen. Usually produced in low amount and declines rapidly

• Naive B and T-cell response

• Leads to production of memory cells with high specificity to the exposed antigen

Secondary Immune Response

• Lymphocytes remember the antigen, memory cells respond

• Immunizations or vaccinations are effective b/c of the secondary immune response

• Mainly IgG antibody is produced.

  • Other IgA and in the case of allergy IgE are produced

• Usually, 100-1000 times more antibodies are produced, and levels remain high for a longer period

• Affinity for antibody of antigen is high

• Knowing this pattern of antibody response is basis for the immunology lab

Tests of immune function

• Based on antigen-antibody reactions

  • May be qualitative (+ or -)

  • May be quantitative ( Titer=reciprocal of the highest dilution of patient’s serum showing a positive reaction with antigen)

Agglutination and agglutination inhibition

visible clumping of cells or particles due to their reaction with an antibody

Categories of conditions associated with immune system abormalities

• Autoimmune disease

• Hypersensitivities

• Malignancies

• Acquired immunodeficiencies

• Congenital immunodeficiencies

Agglutination

Antibodies cause the cross-linking of particulate antigen, usually found on a cell

Direct Agglutination

• Test patient serum against large cellular antigens to screen for the presence of antibodies

• Antigen is naturally present on the surface of cells

• Ag-Ab reaction forms directly visible agglutination

Indirect Agglutination

• When identifying serovars of bacteria and viruses, to improve visualization of agglutination, the antibodies may be attached to inert latex beads

• The agglutination of the beads is a marker for antibody binding to some other antigen. Indirect assays can be used to detect the presence of either antibodies or specific antigens

Neutralization Tests

• The presence of an antibody prevents the antigen from functioning properly

• Neutralization tests are based on the binding of antibodies to the pathogen or to toxins, resulting in the inhibition of their biological properties

• This method can be applied to detect pathogens (with defined immunosera) or their toxins or to identify antibodies (with defined pathogens)

• These antibodies, named neutralizing antibodies (NAbs), play a vital role in the antiviral response and prevent viral entry

How do NAbs prevent viral entry

• Blocking receptor engagement

• Arresting the membrane fusion process

• Accelerating the decay of viral particles

• Can be used to detect streptolysin or rabies or monitor the neutralizing effect of vaccines

Complement Fixation

• RBCs are artificially coated with the desired antigen and then mixed with patient serum (as a source of antibodies)

• Patient antibodies that have bound to antigen on the red cells activate complement, and the red cells are lysed

Parts of a labeled assay:

• Analyte (labeled and unlabeled)

• Specific antibody

• Separation of bound and free components

• Detection of label

• Standards/calibrator

Antibody Titers

Can help determine antibody concentration levels

• Two-fold serial dilutions of serum containing an antibody are made, then tested against cells possessing the target antigen

• The titer is the reciprocal of the greatest dilution in which agglutination is observed

Titer results

• Titers provide more valuable information when tested in parallel with a previous titer specimen

• A comparison of the current specimen’s results and previous specimen’s current results should be made

• A change in titer of 2 or more tubes is considered significant

Why is a titer performed?

• Acute and convalescent

• Prenatal (Rh)

• Verify past infection

• Confirm vaccination

Types of pathogen tests in immunology

• Rubella

• Syphilis

• Lyme

• Hepatitis A (outbreak situations)

• Hepatitis B

• Hepatitis C

• HIV

• Chlamydia

• Gonorrhea

Flow Cytometry

• A technique used to detect and measure physical and chemical characteristics of a population of cells or particles.

  •  In this process, a sample containing cells or particles is suspended in a fluid and injected into the flow cytometer instrument

• Count, examine, and sort cells

• Light scatter is dependent on internal structure, size, and shape of the cells

• Uses 3 systems: fluidics, optics, and electronics

Common uses for flow cytometry

• Counting the number of CD4 T-cells in the blood of someone with HIV. This helps to determine how healthy their immune system is and to track any damage caused by the virus. This is generally done on a blood sample.

• Counting the number of reticulocytes (immature red blood cells) in the bone marrow. This can be used to determine the cause of anemia. It can also be used toc heck the health of the bone marrow after a transplant or after chemotherapy. This is generally done on a bone marrow sample.

• Histocompatibility (HLA) testing prior to an organ transplant, whether someone is intended to be a donor or a recipient. This is generally done on a blood sample.

• Checking the number of sperm in a semen sample. This may be done either as part of an infertility workup or to see if a vasectomy has been successful.

• Diagnosing and classifying leukemia or lymphoma. This may require a blood sample, bone marrow, or a different type of tissue sample.

• Making certain your platelets are working correctly

Molecular technologist

• Analyzes extracted DNA and RNA

• Extract DNA from tissue and blood specimens

• Perform molecular assays on specimens

• Maintain instruments and perform quality control

• Review results for accuracy

Analysis of DNA/RNA

• Gel electrophoresis (separates DNA fragments)

• Hybridization (e.g. Southern blot)

• Sequencing

• Polymerase Chain Reaction (PCR)

Applications of Nucleic Acid-Based Tests

• Unique sequences of various bacterial, fungal, or viral organisms

• defective genes found in inherited disease such as cystic fibrosis

• mutations in certain cancer cells

• translocations in cancer cells from solid tissues

DNA sequencing

• A technique that determines the sequence of the nucleotides in the patient’s genomic DNA

• “mutation” is a change in the genomic DNA sequence

  • Can cause changes in protein expression or function

Hybridization

• hybridization probe bind a specific sequence in the specimen DNA only if the sequences match

  • Can be used to detect the presence of a specific target sequence

  • can detect the presence of mutation

Polymerase Chain Reaction (PCR)

• A segment of DNA is amplified using various types of thermal cyclers until enough DNA is produced for analysis

• For RNA, reverse transcription (RT) converts RNA into DNA, and then PCR can be performed (RT-PCR)

Virology

The identification of pathogenic viruses

Enteric

Fecal oral route

• Rotavirus, Hepatitis A, Enterovirus

Respiratory

Respiratory or salivary routes

• Measles

• Influenza

Zoonotic

Vectors, animal to human

• Rabies, Cowpox, West Nile

Sexually transmitted

Sexual contact

• Herpes simplex virus-2 HIV

Detection of Virus

• Tissue culture, bird embryos, live mammals

• Electron microscope

• Antibody Detection - IgM, IgG, used for HepA, B, EBV

Enzyme-Linked Immunosorbent Assays(ELISA)

enzyme reacts with substrate forming colored product

Western Blotting

viral proteins are separated using electrophoresis

Molecular Assays

PCR - DNA viruses

RT-PCR - RNA viruses