I. Introduction

The ABO blood group system is a cornerstone of transfusion medicine, bridging centuries of scientific inquiry with modern clinical practice. From Karl Landsteiner’s revolutionary discovery of agglutination reactions to the biochemical and genetic mechanisms that define blood compatibility, the ABO system has transformed our understanding and management of blood transfusions. This article traces the evolution of the ABO blood group from its historical roots and molecular foundations to the laboratory techniques used for blood typing and the life-threatening consequences of incompatibility. It also explores rare phenotypes like Bombay and para-Bombay. Finally, this article concludes with a look toward the future of blood typing, where emerging technologies promise greater precision, portability, and safety in transfusion medicine.

II. History of the ABO Blood Group

The discovery of the ABO blood group paved the way to the modern blood bank and the discipline of transfusion medicine. Karl Landsteiner observed unique reactions that would occur when he mismatched serum and red blood cells (RBCs) from different patients. The reaction he observed, agglutination, was the explanation behind past transfusion failures. It took centuries of failed transfusions before Landsteiner’s groundbreaking discovery finally explained why. (1)

Physicians had long postulated transfusions as a potential treatment. In 1667, Jean Baptiste Denys attempted the first recorded transfusion. He used calf’s blood to transfuse a man suffering from psychosis. History remembers the event because the transfusion failed, the man died, and Denys was put on trial for murder. Denys was acquitted, but everyone was left afraid, and transfusion as medicine was not only abandoned but prohibited. (2)

Nearly 200 years, in 1818, Dr. James Blundell entered the scene. Blundell suggested transfusing blood to save a woman dying from postpartum hemorrhage. Blundell selected her husband as the donor. Needles and tubing connected the two, and a direct transfusion commenced. To everyone’s relief, the woman survived the hemorrhage and transfusion. Blundell proved that transfusion could be safe and effective for treating blood loss. (2)

At the turn of the 20th century, Karl Landsteiner noted unique reactions that sometimes occurred when he mismatched RBCs and serum from different patients. Some combinations of serum and RBCs would agglutinate into visible clots. He began tracking these reactions and eventually grouped them into the ABO groups. Landsteiner hypothesized that antibodies in the serum reacted with antigens on the surface of RBCs, triggering agglutination. The antigens included A, B, and O (O-Ohne: German for none), where O lacked both A and B antigens. (1)

Landsteiner’s discovery provided the spark that ignited a burst of studies which formed the discipline of transfusion medicine. Researchers proved the inheritability of the ABO blood group in 1910. Berenstein, in 1924, figured out the inheritance pattern and suggested 3 alleles determine which ABO antigens are expressed on the RBC surface. He identified group O as recessive, while A and B were codominant. (1)

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III. Biochemistry - Genetics - Immunology

Biochemical Foundations

The ABO blood group antigens are carbohydrates present on the surface of RBCs and organ tissues. All ABO antigens originate from the H antigen, which the FUT1 and FUT2 genes regulate. People with type O blood display only the unmodified H antigen on their cell surfaces. Group A, B, and AB are the result of additions that modify the H antigen. A glycosyltransferase adds N-acetylgalactosamine to the H antigen, forming group A. The B antigen arises from the addition of D-galactose by B glycosyltransferase. Individuals with group AB express both A and B antigens simultaneously. Interestingly, the A and B enzymes differ by 4 amino acids. (3) With the biochemical groundwork laid, the genetic architecture of ABO blood types reveals even deeper complexity.

Genetic Foundations

Researchers generally believe that group A was the original ABO blood type. Groups O and B then arose from genetic modifications due to exogenous pressure. For any ABO antigen to be present, a person must have functioning alleles for the foundational antigen H. The FUT1 gene controls H antigen expression on RBCs and tissue cells. Some people can secrete antigens into select body fluids such as saliva. Known as secretors, these individuals carry the FUT2 gene. FUT1 and FUT2 genes, which are located on chromosome 19, code for fucosyltransferase 1 and 2, respectively. These enzymes affect the production of the H antigen. (3)

The ABO gene on chromosome 9 determines whether a person has A, B, or O blood type. Since A is considered the primal blood group, both O and B are modified versions of the A allele. The A alleles code for A glycosyltransferase, which is the enzyme responsible for transforming the H antigen into an A antigen. The B allele codes for B glycosyltransferase, which is the enzyme responsible for converting the H antigen into the B antigen. A and B alleles are codominant, meaning if both sets of alleles are present, a person will produce both enzymes, resulting in the AB blood group. A single nucleotide mutation distinguishes the A and B alleles, resulting in four amino acid differences. The overall effect is the B transferase enzyme. (1)

The O blood group is recessive to both A and B, meaning alleles from both parents must be inherited for the blood group to be expressed. O blood group alleles are similar to A alleles except for a single deletion of guanine. This small change disables the enzyme, preventing conversion of the H antigen. (1)

A small subgroup of individuals produces confounding results during blood typing. This subgroup of people has the Bombay and para-Bombay phenotype. This subgroup lacks genes for FUT1 and FUT2. The result is the inability to make the foundational H antigen. (3) Para-Bombay individuals lack FUT1 but retain FUT2, allowing them to produce only the secreted form of the H antigen. These people are only able to produce the secreted form of the H antigen. (3) Individuals must inherit both recessive alleles to express either Bombay phenotype. (1) The nature of their typing results and challenges will be discussed more later in this article. With the genetic blueprint of ABO blood groups established, the next layer of complexity lies in how our immune system responds to these antigens.

Immunology

Antigens are the foundational units for blood compatibility. Equally important to antigens are the antibodies a person produces against absent antigens. These antibodies, produced as early as 6 months of age, are thought to arise from exposure to ABO-like antigens in food and pathogens. Antibodies to ABO blood group antigens are produced based on the antigens a person lacks. For instance, group O lacks A and B antigens and will produce anti-A, anti-B, and anti-A,B antibodies. (1)

The ABO antibodies are primarily IgM antibodies; however, there are IgG and IgA antibodies in lesser amounts. Group O produces the greatest portion of IgG ABO antibodies. (3) Understanding the interplay between antigens and antibodies sets the stage for practical application. Laboratory testing becomes the critical tool for ensuring safe and compatible transfusions.

IV. Lab Testing & Compatibility Assessment

ABO Typing Techniques

No matter the level of technological sophistication used to determine ABO group, all blood typing methods rely on the same basic principle to identify a person’s ABO group. Two tests are employed to confirm and resolve possible discrepancies. The first test, forward typing, uses manufactured antibodies to bind with potential antigens on the patient’s RBCs. Anti-A and anti-B antisera serve as the reagents for antigen verification. Across manufacturers, anti-A is standardized as blue and anti-B as yellow. The second test, reverse typing, uses reagent RBCs with single ABO antigens. Reverse typing confirms forward typing or reveals discrepancies that suggest a rare blood type. In reverse typing, the patient’s serum is mixed with reagent RBCs. If a person produces antibodies against the RBC antigen, the reagent RBCs will agglutinate.

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Hello, subscribers new and old! I wanted to take a moment to explain my silence over the past few months. I am a full-time mom to three awesome kids. My youngest is a whopping 7 months old! I prefer to write at night, once everyone is asleep, or early in the morning before everyone wakes up. Even though this is my third child, watching their development never ceases to amaze me. It’s simply amazing watching your little human grow from the tiny gummy bear at the first ultrasound, meeting them fully on the day of their birth, and the wild journey to adulthood. Nothing compares to the early formative years from birth to four years old.

During these years, your child is on fire, learning and mastering skills. I am a huge fan of the Wonder Weeks by Frans X. Plooj and his wife Hetty van de Rijt. This husband and wife duo studied babies and found predictable periods of upset, clinginess, and sleep disruption that correlated with neural development. During the first 20 months of a child’s life, they learn vital skills that we take for granted every day. We often forget that we once had to learn different sensations and how to recognize patterns. Before mastering rolling over, we first had to gain control over our movements and then learn to coordinate them. Our lovely little girl has just passed through one of the toughest periods where she recognizes when her parents leave her. In colloquial terms, she is going through the “6-month sleep regression”.

I believe regression is really the wrong term for the situation. What is happening isn’t a regression at all, but a massive burst in neural development. It is progress. She is becoming more coordinated and more aware. Calling it regression only puts parents at odds with their baby, leading them to mistakenly view normal development as “bad behavior”.

Right now, I have less time for myself because, in her infant mind, the world feels brand new. At times, she finds it upsetting and needs to be close to me. Other times, she is fully confident in her new skills and gets into things she shouldn’t. All things considered, she is achieving her potential, and I couldn’t be happier to be absorbed in it.

Introduction

Series Recap:

Welcome to part 3 of the innate immune system review. Part 1, The Innate Immune System, introduced its core components, including phagocytic cells, the initiation of inflammation, and the complement system. This foundational overview set the stage for understanding the body’s frontline defense against pathogens. Part 2, Inflammation: It’s not Just Swelling, took a deeper dive into the process of inflammation. Part 2 also explored the biomarkers and their assays that help to diagnose acute and chronic inflammation. Part 3, The Complement System, explores how complement interacts with both the innate immune system and the inflammatory response. This article will also review the process of testing the function and presence of the complement system.

Purpose & Overview

1. Goal: explore complement system and provide an in-depth look as lab test methods and implications

2. Highlight the need for complement testing for diagnosis and management.

The Complement System: An Overview

Historical and Biological Significance:

Intonations of the complement system were first noticed in the late nineteenth century as scientists began to notice that serum possessed bactericidal attributes. Because of its complexity, multiple scientists made ‘complementary’ discoveries that together shaped early the early understanding of the complement system. These scientists included Buchner and colleagues, Jules Bordet, and Paul Erlich. Erlich pioneered the idea that complement functioned alongside antibodies to kill bacteria. By the mid-twentieth century, all component proteins and pathways as we know today were ironed out. ⁽²⁾

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Complement, often referred to as a cascade, is composed of enzymatic proteins called zymogens. ⁽¹⁾ Similar to the coagulation cascade (click here for more information), each activated protein becomes the activating enzyme for the next protein in the cascade. ⁽¹⁾ Complement proteins interact to kill bacteria and promote inflammation. ⁽¹⁾ There are three pathways complement is executed based on the initiator: classical, alternative, and lectin pathways. ⁽²⁾ The classical pathway is activated when C1 protein either binds with antibodies attached to the bacterial cell surface or directly to the bacterial surface. ⁽¹⁾ The alternative pathway begins with the spontaneous hydrolysis of C3 protein. ⁽¹⁾ This pathway also provides positive feedback, ramping up the activity of the classic and lectin pathways. Finally, the lectin pathway begins when collectins (multimeric lectin complexes) bind to mannose, a sugar present only on bacteria. ⁽²⁾ To a lesser extent, several coagulation factors can initiate the complement system. These include factors Xa, XIa, VIII, and plasmin. ⁽²⁾ All three pathways converge to the activation of C3 and the formation of pores (membrane attack complex - MAC) on the bacterial cell surface. This weakens the bacteria by allowing the internals to be extruded. See figure 1 depicting the mechanics of the various pathways.

Figure 1

Role in Innate Immunity:

Complement strengthens innate immunity through three key functions: ⁽¹⁾

1. It binds to pathogens, opsonizing them for phagocytosis.

2. Fragments leftover from complement activation recruit and activate phagocytes.

3. Terminal complement components form large pores on bacterial membranes, leading to cell lysis.

Activated complement proteins, specifically C5a and C3a, promote local inflammation. ⁽¹⁾ C5a dominates this function due to the ubiquitous presence of C5a receptors on inflammatory cells, such as dendritic cells, and non-inflammatory cells. ⁽²⁾ The following actions reflect C5a’s ability to promote inflammation: ⁽²⁾

· Promotes neutrophil mobility and aggregation.

· Promotes release of inflammatory cytokines and lysozymes by monocytes and macrophages.

· Improves adhesion between neutrophils and endothelial cells.

C3a promotes inflammation to a lesser extent. It promotes eosinophil and mast cell migration. ⁽²⁾

Relevance to Disease:

Diseases linked to complement span two basic categories:

1. Deficiencies of one or more complement proteins.

2. Dysregulation of the complement cascade.

Deficiencies can be inherited or acquired. Genetic deficiencies follow a recessive inheritance pattern, requiring both parents to carry the affected gene. Acquired deficiencies occur when protein synthesis malfunctions (such as in liver disease), protein loss (such as in severe burns), overconsumption, or autoimmunity against complement components. Either inherited or acquired deficiencies result in recurrent infections and pyogenic infections, especially infections of Neisseria species. ⁽³⁾

Dysregulation is commonly encountered in many autoimmune diseases and direct damage to the kidneys. Complement contributes to excessive tissue damage in lupus erythematosus, vasculitides, Sjogren’s syndrome, antiphospholipid syndrome, systemic sclerosis, dermatomyositis vasculitides, and rheumatoid arthritis. ⁽⁴⁾ The kidneys are often the target of unregulated complement, resulting in atypical hemolytic uremic syndrome, C3 glomerulopathy, and paroxysmal nocturnal hemoglobinuria. ⁽⁵⁾

Lab Testing

Why Test Complement

Complement testing is not always an immediate consideration, but it can help solve complex medical cases. The best reason to test for complement is to discover the “why” behind recurrent infections and explore the inflammation and damage associated with autoimmune diseases. Once a clinician knows the “why”, targeted therapeutics are deployed to help treat a disease.

Objectives of Testing:

1. Establish the functionality by exploring qualitative and quantitative aspects of the complement cascade.

2. Assist in tailoring effective treatments for patients.

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Intro

One of the most exciting aspects of working in the clinical laboratory is identifying the microorganism responsible for an infection. The immunology, serology, and microbiology departments are responsible for naming microorganisms. Each department uses unique techniques designed to identify microorganisms. Let’s explore the dedicated laboratorians who play a crucial role in patient care.

Identifying with Antibodies

The primary role of the immunology department is to detect antibodies that indicate exposure to pathogens. Enzyme-linked immunosorbent assays (ELISA) are the most common test method employed by the immunology department. In these tests, patient serum or plasma interacts with the target antigen - the microorganism being investigated. If antibodies against the target antigen are present, the antibodies will bind to the antigen, activating an enzyme, and causing a color change or expression of fluorescence. Identifying infectious agents is not the only testing performed in the immunology department. Depending on the facility, allergy testing, electrophoresis, and autoimmune disease screening may also occur in the immunology department. While many techniques are automated, skilled laboratorians carefully analyze patterns to ensure accurate results.

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There is Probably a Kit for that

Serology is a unique department. The majority of serological testing is performed by manual kit tests. Common serology tests include flu, strep, and COVID. If you are swabbed for something, it will likely go to the serology department. Other serology tests include RPRs for syphilis and meningitis panels on spinal fluid. This department is typically small and is sometimes broken up between the larger departments in the lab.

The Smell of Cultures in the Air

Microbiology is one of the most recognizable laboratory departments, alongside blood banking. The microbiology department has the means to identify bacteria, viruses, parasites, and fungi. The identification process begins with a culture. Once colonies grow, molecular techniques allow laboratory scientists to rapidly identify organisms. In addition to molecular techniques, organisms are identified based on microscopic features, metabolic activity, and chemical interactions.

Antibiotic susceptibility is critical to treating infections for optimal outcomes. Selecting the best antibiotic is crucial. Not only to eliminate the infection but also to minimize unnecessary exposure and reduce the risk of antibiotic resistance. Laboratorians provide this vital information to physicians either through diffusion disc cultures or molecular methods. The latter technique allows for rapid results compared to cultures.

Wrapping up

From common infections to rare conditions, microbiology, immunology, and serology departments provide the answers needed for effective treatment. Thank you, everyone, for reading along this week. Thank you for helping celebrate Laboratory Professionals Week.

Intro

Although Blood Bank and Coagulation (Coag) are separate departments, they share a vital goal: ensuring proper circulatory function. For many, the blood bank is the most recognizable laboratory department. The blood bank specializes in matching and transfusing blood products to patients in need. The Coag department may also be familiar. If you or a family member takes anticoagulants, you may require monitoring for coagulation function. Your blood sample is sent to the Coag department. Both departments are crucial to helping patients in crisis.

Blood Bank: A Puzzle of Matching and Transfusing

Blood bank is a department that laboratorians either love or dread working in. Despite its high-pressure environment, blood banking is one of the most mentally stimulating areas in the lab. I am sure you are familiar with the importance of matching ABO blood group for receiving blood. You probably don’t know that matching units of blood can be much more complicated. Most laboratorians serious about blood banking receive the Specialist in Blood Banking (SBB) credentials and exclusively work in transfusion medicine.

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Red Blood Cells: The Other Fingerprint

The surface of red blood cells is covered with hundreds of identification markers called antigens. These antigens, uniquely determined by your genetics, are akin to a fingerprint of the red blood cells. This greatly complicates matching units of blood, especially when someone has been exposed to multiple outside blood groups. This can occur mostly through repeat transfusions or multiple pregnancies. In pregnancy, one critical consideration is Rhogam, a treatment designed to prevent complications from incompatible blood types. Rhogam is an infusion of antibodies against a specific antigen that may be present on fetal red blood cells. The Rhogam works to cloak the fetal red blood cells from the maternal immune system. This action prevents fetal anemia in utero. Besides units of blood, the blood bank also provides plasma and platelets for transfusion.

Coagulation: The Science Behind Clots and Bleeding

If you’ve read my article on the mechanics of coagulation, you may already be familiar with the Coag department. The tests performed in the Coag department target specific molecules of the coagulation cascade or the remnants of a dissolving clot. These tests are primarily used for anticoagulant drug monitoring, such as Coumadin or heparin. Coag tests play a crucial role in detecting deep vein thrombosis and pulmonary embolism. Coagulation tests can even diagnose coagulation disorders such as hemophilia.

Wrapping it Up

The blood bank and coagulation departments provide crucial services to physicians and patients. Laboratorians in both departments work tirelessly to maintain balance and stability in the circulatory system. The next article will delve into the world of immunology, serology, and microbiology.

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Introducing the Chemistry Department

The chemistry department has the largest scope of testing of all the departments. Tests that once required hours of manual techniques can now be completed in under an hour with automated analyzers. This has earned those of us who enjoy chemistry the nickname “button pushers”. Chemistry goes beyond simply running a high volume of tests each shift. Chemistry laboratorians are immersed in numbers and quickly develop a keen eye for statistical discord. They also become fairly skilled at fixing robotics.

Wide Horizons

The chemistry department covers a wide range of tests. Metabolic panels monitor key organ function. Cholesterol panels and cardiac markers assess cardiac health. Therapeutic and drugs of abuse are screened to determine levels. Inflammation markers help uncover infection and sepsis. Even hormone level dynamics are determined within the chemistry department.

Urinalysis: A Closer Look at the Secrets in Urine

Related to both chemistry and hematology is urinalysis. Urine is surprisingly full of information about the body. Urinalysis is a unique blend of chemistry and hematology for two key reasons. The first part of testing urine is chemical testing with a dipstick. The second part is to qualitatively assess urine sediment under the microscope. Under the microscope, one might find kidney stone crystals, bacteria, red and white blood cells, parasites, and epithelial cells from the urinary tract. Even the color of urine is influenced by more than just hydration status. Maybe it’s bright orange or teal from UTI medication, or it’s dark amber due to liver failure. Urinalysis may be a smelly business, but it plays a crucial role in uncovering vital patient information.

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Wrapping it up

Next time you have to fast for labs or pee in a cup, the chemistry and urinalysis department has got you covered. Tomorrow, I will introduce you to the coagulation and blood bank departments.

Intro

The hematology department primarily focuses on the study and analysis of red blood cells (RBCs), white blood cells (WBCs) and platelets. Complete blood counts (CBCs) are the cornerstone of the hematology department. A CBC begins on a cell counter analyzer that counts and classifies all cells within a whole blood sample. When a deviation from normal is detected, the analyzer tags the sample for a microscopic review. A blood smear is carefully prepared on a glass slide and stained with Wright’s stain for microscopic examination. The laboratorian reviews the smear for the shape and color of the RBCs, the numbers and maturity of WBCs, and the platelet count.

Unmasking Health Clues

Collectively, the CBCs allow the laboratorian to uncover clues about a wide scope of diseases. These diseases include anemias, infections, and cancer. Laboratorians can also use CBCs to classify the type and underlying causes of anemia, infections, and cancers. For instance, the shape of an RBC should resemble a round donut. In anemias, the shape of the RBC may be disfigured, and the color may be different. An infection can be determined as bacterial, parasitic, or viral based on which WBC is most prevalent.

Beyond CBCs

CBC assessments are the most common test performed by the hematology department, but it is certainly not the only one. The hematology department also performs sedimentation rates, sickle cell screening, and preliminary bone marrow studies. Body fluid cell counts and WBC differentiation are also performed by the hematology lab.

Specialized Analysis

The flow cytometry department complements the work of hematology by specializing in advanced analysis of white blood cells. Flow cytometry is a highly specialized hematology lab focusing on classifying WBCs based on cell markers present on the cell surface. WBCs are isolated, interacted with antibodies, and passed through a detector. The flow cytometry lab can determine sub-populations of WBCs, which aids in determining the type and origins of cancer. Flow cytometry can be performed on whole blood, bone marrow, body fluids, and even fine needle aspirates.

Wrapping Up the Mystery

Though their test menu is limited, hematology and flow cytometry provide essential clues, helping physicians identify the root of a patient’s illness. Tomorrow, I will cover one of my favorite departments, chemistry.

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Happy Lab Week, everyone! It’s laboratory professionals’ week to shine! We uncover hidden clues for physicians to solve the mystery of disease. Each day this week, I will cover the departments within the lab, highlighting the unique tests performed within each.

The laboratory is a very diverse department that is split into multiple subdisciplines. Most laboratories include hematology, chemistry, urinalysis, coagulation, blood bank, immunology/serology, and microbiology. Depending on the size of the facility, there may be more subdisciplines, such as molecular and flow cytometry. Each department provides key clues influencing diagnosis, treatment, and monitoring patients.

Stay tuned as we highlight each subspecialty within the lab. Follow along to learn about all of the testing we perform every day.

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*This article is not intended to diagnose or treat any disease. Refer to your physician for information concerning your specific health status.*

When the body sustains an injury or encounters a pathogen, a swarm of molecules, such as enzymes, cytokines, adhesion molecules, and chemokines, are released, causing an array of symptoms such as heat, swelling, and redness. We know this process as inflammation, and it's part of the body’s response to mitigate infection and tissue damage. In today’s article, part two of the innate immune system review, we will discuss the process of inflammation from initiation to resolution. Inflammation often faces criticism, so this article aims to highlight its benefits in the appropriate context.

Stages of Inflammation

Five general stages actuate the inflammatory process. Pattern recognition receptors (PRRs) initiate inflammation by binding to pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs). This interaction triggers an intracellular cascade that activates and transcribes inflammatory genes. Cells release inflammatory markers into the extracellular space. Inflammatory markers promote fluid exchange and direct phagocytic cells to the target area. Finally, as the cleanup of pathogens and damage to tissues resolves, anti-inflammatory molecules are released to end the process. Over the next several sections, we will go deeper into the mechanics of the stages of inflammation.

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1) Cell Surface Receptors Interact with Stimuli

All processes must have a beginning, and inflammation is no exception. Pattern recognition receptors on the surface of macrophages, dendritic cells, monocytes, neutrophils, mast cells, and natural killer cells are specially designed to recognize ubiquitous bacterial molecules – PAMPs - as well as common molecules released by damaged native cells -DAMPs. Several surface receptors fall under the PRR category, including Toll-like receptors (TLRs), C-Type lectin receptors (CLRs), retinoic acid-inducible gene I-like receptors (RLRs), and NOD-like receptors (NLRs). Upon interacting with PAMPs and DAMPs, PRRs transport these molecules into the cytoplasm. This action kicks off an intracellular cascade, taking us to the next step of inflammation: activation of inflammatory genes. ⁽¹⁾

2) Activation and Transcription of Inflammatory Genes

The internalized PAMPs and DAMPs initiate an activation cascade crucial for actualizing inflammation. Three pathways trigger the transcription of genes coding for inflammatory molecules: Mitogen-activated protein kinase (MAPK), Nuclear factor kappa-B (NF κB), and Janus Kinase – signal transducer and activator of transcription (JAK–STAT) pathways. Different stimuli activate each pathway, which regulates distinct aspects of inflammation. ⁽¹⁾

• MAPK Pathway: ⁽³⁾

o Stimulated by osmotic stress, mitogens, heat shock, and inflammatory cytokines such as TNF-α and IL1β.

o Promotes transcription of proinflammatory cytokines.

o Integral in cell proliferation, differentiation, survival, and apoptosis.

• NF κB Pathway: ⁽¹⁾

o Stimulated by PAMPs, cytokines, and enzymes.

o Promote transcription of cytokines.

o Contributes to cell recruitment.

• JAK–STAT Pathway: ⁽¹⁾

o Stimulated by cytokines, growth factors, interferons, leptin, and growth hormone.

o Promotes transcription of inflammatory cytokines.

o Notable pathway for extracellular signals to directly trigger transcription of inflammatory genes.

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Intro

The innate immune system is the body’s first line of defense against pathogens and injury – an integral mechanism providing non-stop defense to keep us healthy. This article is the first of three articles covering the innate immune system. After this overview of the innate immune system as a whole, parts two and three will cover inflammation and complement in greater detail. Whether you're refreshing your knowledge or learning for the first time, you'll gain a deeper understanding of the vital processes that protect our bodies every day.

Physical Barriers

The body's immune defenses are multifaceted, relying on both physical barriers and cellular responses to combat pathogens. The frontlines are protected by an array of armor from the skin's physical and chemical makeup to the antimicrobial properties of tears. The innate immune system begins with physical barriers. The skin's tightly packed epidermal and dermal layers block pathogens from entering the body. The cells of the skin are tightly fitted together, creating an impenetrable covering for the body. The skin also provides antimicrobial chemical defenses. The pH of skin and proteins such as psoriasin prevent pathogens from colonizing the skin surface. Beneficial microbiota on the skin and in organ systems outcompete pathogens, providing an additional layer of defense. The normal flora also provides the host with immune system support by synthesizing some vitamins and modulating the immune system through host cell interactions. ⁽¹⁾

Epidermal layers inside the body, such as the respiratory, digestive, and genitourinary tracts, use additional defenses. Mucus membranes trap and expel pathogens, forming a crucial defense within these specialized organ systems. The mucus is specially designed with surfactants to prevent bacterial adhesion to epithelial cells. The respiratory tract is lined with hair-like projections called cilia that are capable of physically moving pathogens out. The reflex actions of coughing and sneezing are dramatic modes of physically moving pathogens out of the body. (1) In the digestive tract, stomach acid and lysozymes eliminate many ingested pathogens. Lysosomes, which are found in tears and saliva, break down the cell walls of gram-positive bacteria. Finally, the genitourinary tract expels pathogens in urine and vaginal pH prevents pathogenic bacterial colonization. ⁽¹⁾

Effector Cells and Functions

The white blood cells (leukocytes) are widely known to be the effector cells of the immune system. There are two general groups of leukocytes responsible for two different types of immune responses. This article focuses on the innate immune cells, distinct from those of the humoral response. The innate response of leukocytes is responsible for a general nonspecific destruction of pathogens and dysfunctional native cells. These leukocytes phagocytose, initiate cellular destruction, and present antigens to the leukocytes responsible for the humoral response. The humoral response functions as the immune system's specialized unit, targeting specific antigens. These leukocytes produce highly specific antibodies to the antigens they are presented with. These leukocytes maintain a memory of past antigens, which allows for a quicker response in future infections. ⁽²⁾

For the humoral response to work, the leukocytes of the innate response must act first. Innate response leukocytes are divided by the compartment in which they reside (tissue or circulation) and by function (phagocytes and apoptosis-inducers). ⁽²⁾ Within the tissues of the body, macrophages (once circulating monocytes), fibroblasts, mast cells, and dendritic cells engage with pathogens and recruit circulating leukocytes. he circulating leukocytes include neutrophils, monocytes, and natural killer cells. Neutrophils and monocytes are the primary phagocytic cells, while natural killer cells initiate apoptosis. ⁽³⁾

Both tissue and circulating leukocytes recognize common patterns associated with pathogens. Pattern recognition receptors (PRRs) on innate immune cells detect chemoattractants and immunostimulants, triggering immune responses. Common chemoattractants include formylmethionine (a bacteria-specific amino acid), ATP, interleukin-1α, and uric acid released by native necrotic cells. ^(2,3) PRRs include Toll-like receptors, C-type lectin receptors, retinoic acid-inducible gene, I-like receptors, and NOD-like receptors. ⁽⁴⁾ Inflammation and the activation of complement are the cumulative effects of PRRs interacting with chemoattractants and immunostimulants.

Inflammation

Inflammation is the body's first responder, addressing pathogens and tissue damage with precision. It provides the signal and optimal conditions to recruit leukocytes to the designated site. It consists of enzymes, chemokines, cytokines, adhesion molecules, and regulators. The sole purpose of inflammation is the recruitment of leukocytes to mitigate the damage. As described earlier, PRRs' interaction with chemoattractants and immunostimulants is also identified as pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs). As the names imply, some molecules are distinctly from pathogens while others originate from damaged native cells. ⁽³⁾

The inflammatory process can be divided into five stages, culminating in resolution. Inflammation begins when PRRs and PAMPs/DAMPs interact. Next, inflammatory genes are activated to transcribe inflammatory markers. Inflammatory markers are subsequently released from the cells. These markers signal innate immune cells to the specified area. Finally, the inflammation process resolves. ⁽⁴⁾

Complement

Inflammation provides the signals and environment necessary to import the innate immune cells. Once at the designated site, the complement system marks and weakens the pathogens and dying cells for phagocytosis. The 3 goals of complement are to mark and prime pathogens for phagocytosis, recruit and activate phagocytes, and create transmembrane pores on bacteria to weaken them. ⁽⁵⁾

There are 3 complement pathways known as the classical pathway, mannon-binding lectin pathway, and alternative pathway. Each pathway is initiated by a unique promoter. The classical pathway starts when C1 binds to bacterial surfaces or antibodies attached to bacteria. Mannon-binding lectin pathway is initiated when complement binds with mannon lectin on the bacterial surface. The alternative pathway begins with the spontaneous hydrolysis of C3. t amplifies the classical and lectin pathways through positive feedback. All three pathways converge to a common enzyme, C3, that ultimately facilitates the goals of complement. ⁽⁵⁾

Wrapping it up

The innate immune system is a multifaceted defense system providing the initial defenses against pathogens and laying the foundations for adaptive immunity. Physical barriers, immune cells, and molecular defenses work tirelessly to protect us from pathogens and tissue damage. Parts two and three will provide you with a deeper understanding of the inflammatory process and the complement system. Stay tuned for a special glimpse into how these processes are monitored in the clinical setting.

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References

1. C BG. Innate immunity: characteristics, external and internal defense mechanisms. The Science Notes. Published August 30, 2024. https://thesciencenotes.com/innate-immunity-key-characteristics-external-internal-defense/

2. Uzman A. Molecular biology of the cell (4th ed.): Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., and Walter, P. Biochemistry and Molecular Biology Education. 2003;31(4):212-214. doi:10.1002/bmb.2003.494031049999

3. Newton K, Dixit VM. Signaling in innate immunity and inflammation. Cold Spring Harbor Perspectives in Biology. 2012;4(3):a006049. doi:10.1101/cshperspect.a006049

4. Chen L, Deng H, Cui H, et al. Inflammatory responses and inflammation-associated diseases in organs. Oncotarget. 2017;9(6):7204-7218. doi:10.18632/oncotarget.23208

5.Janeway CA Jr, Travers P, Walport M, Shlomchik MJ. The complement system and innate immunity. Immunobiology - NCBI Bookshelf. Published 2001. https://www.ncbi.nlm.nih.gov/books/NBK27100/

Bacteremia, a bacterial infection of the blood and circulatory system, requires rapid and accurate identification of the causative organism. Blood cultures provide medical laboratory scientists with the means to supply physicians with critical information to treat their patients. Cultures are collected in broth media bottles that promote bacterial growth. If growth occurs, microbiologists utilize a range of simple and advanced testing methods to identify the organism. In this article, we will discuss collection best practices, culture processing, identification techniques, and the most common organisms isolated from blood cultures.

Collection Best Practices

Reliable blood culture results depend on proper collection techniques. Blood samples are collected aseptically by thoroughly cleaning the venipuncture site with chlorhexidine, tincture of iodine, or povidone iodine. To further minimize contamination, the tops of blood culture bottles must be wiped with alcohol before use. Blood culture collection requires a specialized venipuncture adapter designed specifically for these bottles or syringes can be used.⁽¹⁾

The amount of blood drawn varies based on the patient’s age and weight. An ideal blood-to-broth ratio is between 1:5 and 1:10. To optimize turnaround time, collected bottles should be returned to the laboratory at room temperature within two hours. For adult patients, a typical set consists of two bottles—one aerobic and one anaerobic. Pediatric cultures, on the other hand, typically use a single small-draw aerobic bottle. Collecting a series of two or three sets significantly increases the likelihood of isolating the responsible organism. ⁽¹⁾

Culture Processing

Blood culture bottles are specifically formulated to support a broad range of organisms. The broth contains tryptic soy medium and sodium polyanethol sulfonate (SPS) as an anticoagulant. For adult patients, aerobic and anaerobic bottles are collected. Aerobic bottles contain ambient air supplemented with carbon dioxide, while anaerobic bottles contain a mix of carbon dioxide and nitrogen gas. Pediatric blood culture bottles may include resins to neutralize antibiotics and are designed for smaller sample volumes. ⁽⁵⁾

Once received in the lab, blood culture bottles are placed in an incubator that promotes bacterial growth. Most modern labs employ continuous blood culture monitoring systems, which incubate and track samples around the clock. These systems monitor blood cultures for up to five days. If growth occurs, the system flags the bottle, prompting scientists to remove it for analysis. ⁽⁵⁾

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Identification Techniques

The first step in processing a positive blood culture is a Gram stain. Under the microscope, Gram-positive bacteria appear purple due to the thick peptidoglycan layer in their cell walls. Gram-negative bacteria lack significant peptidoglycan and stain pink. These Gram stain results and bacterial morphology serve as vital initial clues in determining appropriate treatment. ⁽²⁾

Following classification via Gram stain and morphology, precise identification and antibiotic susceptibility testing (AST) are conducted. Rapid identification techniques include molecular-based methods and mass spectrometry. Subculturing the bacteria onto agar plates for brief incubation can further improve the accuracy of these tests. ⁽⁵⁾

Molecular testing employs genetic probes to detect bacterial DNA. Using the polymerase chain reaction (PCR), the bacterial DNA is amplified, producing a signal that identifies the organism. Mass spectrometry, particularly matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF), is another advanced method. This technology identifies bacteria and yeast by analyzing unique spectral patterns emitted after laser interaction with the sample. ⁽⁶⁾

Antibiotic Susceptibility Testing

Knowing which antibiotic will effectively target the bacteria is as crucial as identifying the organism itself. While traditional disc diffusion methods remain in use, rapid antimicrobial susceptibility testing (AST) systems now deliver results within hours. Two primary approaches dominate: fluorescence in situ hybridization (FISH) and colorimetric testing. Examples of FDA-approved systems include the Accelerate PhenoTest BC Kit, which utilizes FISH, and platforms like BD Phoenix and BioMérieux VITEK2, which rely on colorimetric methods. ⁽⁵⁾

Common Organisms in Blood Cultures

Contaminants: Contamination typically results in growth within only one bottle of a series. Common contaminants include coagulase-negative Staphylococcus, Bacillus species, Cutibacterium species, diphtheroids, and Micrococcus species. These organisms are part of the skin's normal flora and often enter samples due to inadequate cleansing or improper sterile technique. However, they may become pathogenic if they colonize catheters or implanted devices. ⁽¹⁾

Pathogens: Many bacteria can cause bacteremia, with common pathogens including Staphylococcus aureus, Staphylococcus epidermidis, Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. ⁽⁴⁾ Certain conditions, like endocarditis, are associated with specific bacteria. Gram-positive pathogens such as Staphylococcus aureus, Viridans streptococci, and Enterococcus faecalis, as well as gram-negative HACEK bacilli, are frequent culprits. The HACEK bacteria include Haemophilus species, Actinobacillus actinomycetemcomitans, Cardiobacterium hominis, Eikenella corodens, and Kingella kingae. These bacteria often enter the bloodstream during dental procedures, body piercings, catheterization, or wound infections. ⁽²⁾

Brucella: This gram-negative coccobacillus is rare but significant in veterinarians and farmers. Brucella commonly infects livestock such as cows, pigs, and goats. It causes recurrent fevers in humans and necessitates biosafety level 3 precautions for handling due to its highly infectious. ⁽²⁾

Nutritionally Variant Streptococci: Abiotrophia and Granulicatella species, known as satelliting streptococci, are fastidious gram-positive cocci. They form small colonies near Staphylococcus aureus and occasionally near Enterobacteriaceae or other streptococci. Found in the human mouth, these bacteria require specific nutrients like cysteine or pyridoxal (active vitamin B6) for in vitro growth. ⁽³⁾

Final Thoughts

This concludes the review of blood cultures from collection to reporting results. What are your thoughts on blood cultures? Are there any topics you wish to hear more about? Drop your thoughts in the comments or Med Lab Mastery official Chatroom. Please subscribe and share so we can spread knowledge together! Thanks for reading!

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References

1. Dien Bard J, Children’s Hospital Los Angeles, University of Southern California. PIONEERING DIAGNOSTICS BLOOD CULTURE: A key investigation for the diagnosis of bloodstream infections.; 2022. https://www.biomerieux.com/content/dam/biomerieux-com/medical-affairs/educational-booklets/2022%20BLOOD%20CULTURE%20BOOKLET%20PROVIDED%20BY%20BIOMERIEUX.pdf

2. Parker N, Schneegurt Ma, Thi Tu AH, et al. Microbiology. Openstax; 2016. https://openstax.org/details/books/microbiology?Book%20details

3. Yacoub AT, Krishnan J, Acevedo IM, Halliday J, Greene JN. NUTRITIONALLY VARIANT STREPTOCOCCI BACTEREMIA IN CANCER PATIENTS: A RETROSPECTIVE STUDY, 1999-2014. Mediterranean Journal of Hematology and Infectious Diseases. 2015;7:e2015030. doi:10.4084/mjhid.2015.030

4. Pechorsky A, Nitzan Y, Lazarovitch T. Identification of pathogenic bacteria in blood cultures: Comparison between conventional and PCR methods. Journal of Microbiological Methods. 2009;78(3):325-330. doi:10.1016/j.mimet.2009.07.008

5. Gonzalez MD, Chao T, Pettengill MA. Modern blood culture. Clinics in Laboratory Medicine. 2020;40(4):379-392. doi:10.1016/j.cll.2020.07.001

Clark AE, Kaleta EJ, Arora A, Wolk DM. Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry: a Fundamental Shift in the Routine Practice of Clinical Microbiology. Clinical Microbiology Reviews. 2013;26(3):547-603. doi:10.1128/cmr.00072-12

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Intro:

Today, I have an interview with Elizabeth Hewett. She is a senior application specialist and technical sales specialist with Thermo Fisher Scientific. We discuss her career transition from the lab, the lab skills she uses every day in sales, what she finds challenging and rewarding, the wisdom she has gained, and advice to you if you are considering a similar transition. If you are a student or a career scientist, I hope you find this helpful. I hope you enjoy this interview with a “Labie outside the Lab.”

1. Career Transition:

- What inspired you to transition from a career in medical laboratory science to sales in the in vitro diagnostic industry?

Throughout my MLS career, I have always been responsible for training new employees, Medical Residents, and MLS students. Being a Technical Sales Specialist allows me to teach, troubleshoot, and provide valuable information to a customer deciding to bring in new testing. It is the perfect trifecta for me.

2. Skill Transfer:

- How have the skills and knowledge you acquired as a medical laboratory scientist helped you in your current role in sales?

I don’t believe selling is for everyone, but if you enjoy introducing customers to new testing, it can be very rewarding. Many times, Sales personnel come into a lab trying to sell a new test but may not be able to answer all the technical questions. This is where my MLS knowledge comes in handy. I believe that because I have been in the MLS field for 36 years, my experience may help give more weight to the decision to bring in new testing. Selling isn’t the forefront of my role; I verify analyzers, train, troubleshoot, and support workflow also.

3. Challenges and Rewards:

- What were the biggest challenges and rewards of this career transition?

Some of the biggest challenges for this career is the travel. The last several years, I have been on the road over 45+ weeks a year. Work life balance can suffer.

The biggest rewards include watching a customer who has been doing testing manually or with an analyzer that is labor intensive move to an analyzer that improves their workflow. I also enjoy introducing laboratories to testing that is not done by all laboratories. I find this brings excitement to everyone in the lab. I enjoy meeting people from all areas of our country and visiting unique places in the United States that many people will never see.

4. Industry Insight:

- Can you share any insights into the current trends and innovations in the in-vitro diagnostic industry that you find exciting?

I am fortunate to work for an in-vitro diagnostic company that believes in research. I have been with this company for almost 5 years and have seen improvements in our assays and additional assays added. I am always learning, even after 36 years!

I believe advancements in molecular testing will continue to be at the forefront of trends and innovations. Advancements in technology and AI will continue to move our profession forward in the decades to come.

5. Advice for Others:

- What advice would you give to other medical laboratory professionals considering a similar career transition into the sales or business side of the healthcare industry?

My advice for anyone wanting to enter the commercial side of laboratory diagnostics is to get to know your Service Engineers and Technical Application Specialists. Most people hired into the commercial side have shown an ability to solve problems, work under stress, and work well with others. This is a very competitive field, and if at first you don’t succeed, keep trying. The role I currently have required five interviews, and I had to deliver a presentation. Have confidence in your knowledge! You must also be willing to travel a lot! I waited until my youngest was in high school to transition into the commercial side, but I also know many who have young children and are able to do this role well.

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Intro

Hemostasis serves as the body's mechanism to prevent excessive bleeding. The process begins with primary hemostasis, resulting in platelet aggregation and the formation of a platelet plug. The final step, stabilizing the plug with fibrin, is achieved through the coagulation cascade. This article will review the entire process of hemostasis from injury to stabilized platelet plug for optimal healing. We will also spend a short time on different native and synthetic anticoagulants to finish this discussion.

Primary Hemostasis

When our bodies sustain an injury, the first set of actions encompassed in primary hemostasis results in a platelet plug. Primary hemostasis consists of four distinct actions: vasoconstriction, platelet adhesion, platelet activation, and platelet aggregation. Vasospasm and vasoconstriction are initiated when a blood vessel is damaged. The damaged endothelium exposes endothelin-1, collagen, von Willebrand factor (vWF), ATP, and inflammatory molecules. These various molecules, in turn, activate platelet adhesion. ⁽¹⁾

Platelets enter the injured area and adhere to exposed endothelium and vWF. Platelets bind to vWF with G protein receptors Ib-IX and become activated. Activated platelets undergo conformational change from smooth spheres to spiked with pseudopods. This activation also prompts platelets to release cytoplasmic granules. The cytoplasmic granules contain serotonin, platelet-activating factor, and adenosine diphosphate. Activated platelets begin synthesizing thromboxane A_2, which promotes vasoconstriction and platelet recruitment. ⁽¹⁾

The cumulative effect of platelet activation is platelet aggregation. The GpIIb/IIIa receptors of activated platelets bind with fibrinogen and vWF. The fibrinogen begins forming cross-links, trapping platelets. This final step produces an unstable platelet plug and provides the environment to begin the coagulation cascade. ⁽¹⁾

Secondary Hemostasis

Secondary hemostasis aims to produce a stable platelet plug surrounded by a fibrin mesh. This process is accomplished through the intrinsic and extrinsic pathways, which converge to the common pathway. ⁽²⁾ Secondary hemostasis involves proteolytic enzyme factors (zymogens) and calcium ions. ⁽³⁾ The zymogens are designated as “factor” plus a Roman numeral but also have conventional names. Each activate factor (designated by “a” after the Roman numeral) becomes the enzyme that activates the next factor in the cascade. ⁽³⁾ See Table 1. The liver is responsible for producing most of the coagulation factors except III, IV, and VIII. ⁽³⁾ The coagulation factors can be classified by the pathway they participate in, which will be discussed later in this article. They can also be classified by unique characteristics such as the fibrinogen family, vitamin K dependent, and contact family. ⁽³⁾ See Table 1.

Intrinsic Pathway

The intrinsic and extrinsic pathways are typically described as occurring independently. The reality is that they occur simultaneously and provide positive feedback for each other. To simplify, we’ll start with the intrinsic pathway. It begins with activating factor XII when it comes in contact with endothelial collagen. Factor XIIa catalyzes factor XI activation. Factor XIa activates factor IX. Factor IXa, along with factor VIII, calcium, and phospholipids, activate factor X. At this point, the intrinsic pathway is complete, and the common pathway begins. Let’s put a pause on the common pathway for a moment and explore the extrinsic pathway. ⁽²⁾

Extrinsic Pathway

The extrinsic pathway is significantly shorter than the intrinsic pathway. After vascular injury, the vessel endothelium releases tissue factor. Tissue factor activates factor VII. Factor VIIa, factor III, and calcium activate factor X. This brings us to the convergence of the intrinsic and extrinsic pathways into the common pathway. ⁽²⁾

Common Pathway

The common pathway takes us to the main objective of the coagulation cascade: converting the fibrinogen surrounding the platelet plug into stable fibrin mesh. Activated factor X and factor Va together activate factor II to thrombin. Factor IIa activates the conversion of fibrinogen to fibrin. Fibrin strands crosslink with factor XIII to form a strong, stable mesh around the platelet plug. ⁽²⁾

Anticoagulation

Managing excessive coagulation is as crucial as hemostasis itself. This is achieved by several negative feedback loops that check the coagulation process at various stages. Thrombin encourages coagulation but also activates plasminogen to plasmin. Plasmin activates antithrombin, whose primary duty is fibrin degradation. Antithrombin also reduces the production of thrombin and the activation of factor X. Proteins C and S are also important internal anticoagulants. They are responsible for inactivating factors V and VIII.⁽²⁾

In some conditions, synthetic anticoagulants are essential to prevent excessive coagulation. These conditions include atrial fibrillation, deep vein thrombosis, major surgery, stroke, heart attack, pulmonary embolism, and unstable chest pain. There are four main classes of anticoagulants, each group acting on different parts of the coagulation cascade. Coumarins, commonly known as coumadin or warfarin, act by removing available vitamin K, thereby reducing the production of vitamin K-dependent coagulation factors. Factor Xa inhibitors, as the name indicates, bind with factor Xa and prevent its participation in the coagulation cascade. Heparins include unfractionated heparin, low molecular weight heparin, and heparinoids. Heparins bind with or inactivate factor Xa and thrombin. Finally, direct thrombin inhibitors bind to thrombin, rendering it ineffective. ⁽⁴⁾

Final Thoughts

Hemostasis is an important process for preventing blood loss and healing. It is an intricate dance of enzymes, proteins, and calcium to form a stable platelet plug. Additionally, the body has innate mechanisms to prevent uncontrolled coagulation. When extra anticoagulant assistance is needed, synthetic anticoagulants can help.

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Riddle Answer:

1. Vitamin K

References:

1. Garmo C, Bajwa T, Burns B. Physiology, Clotting Mechanism. [Updated 2023 Sep 4]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK507795

2. Chaudhry R, Usama SM, Babiker HM. Physiology, Coagulation Pathways. [Updated 2023 Aug 28]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK482253/

3. Palta S, Saroa R, Palta A. Overview of the coagulation system. Indian Journal of Anaesthesia. 2014;58(5):515. doi:10.4103/0019-5049.144643

4. Pope C BPharm, ed. Anticoagulants. https://www.drugs.com/drug-class/anticoagulants.html. Published November 22, 2024. Accessed February 27, 2025. https://www.drugs.com/drug-class/anticoagulants.html

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Disclaimer: This article is for information only. It is not intended to diagnose or treat any diseases.

Intro

Hemoglobin is the primary transporter of oxygen in the blood, with iron bound to the core of the heme porphyrin ring giving red blood cells their iconic color. Hemoglobin consists of the heme porphyrin ring and the globin protein. In this article, we will explore the structure, function, and abnormalities of the hemoglobin molecule, highlighting key findings that distinguish each hemoglobin abnormality.

All about heme

Let us begin with the heme molecule. Heme is produced in the liver and in erythroid progenitor cells within the mitochondria. Heme is a porphyrin ring with a strong affinity to bind (chelate) with iron. Heme Is a vital component to many molecules within the body such as myoglobin, respiratory cytochrome, and cytochrome p450. The majority of heme is dedicated to forming hemoglobin. Heme’s affinity to chelate iron gives hemoglobin the ability to transport oxygen by oxidizing the iron core. ⁽¹⁾

Heme synthesis comprises eight steps mediated by several enzymes. It begins with the combination of glycine and succinyl CoA. The next seven steps result in various porphyrins and terminate at heme production. The heme molecule is secreted into the cytoplasm where it chelates iron. The increased presence of heme in liver cells provides negative feedback on its production. Erythroid precursor cells are conditioned to allow for substantially more heme molecules to be produced. This is essential to providing adequate amounts of building blocks for hemoglobin production. ⁽¹⁾

The special protein: Globin

Globins are proteins transcribed from genes on chromosomes 11 and 16. In erythroid precursor cells, the presence of heme in the cytoplasm promotes globin synthesis. Four globin subunits are used to form hemoglobin at various stages of human development. These include alpha (α), beta (β), gamma (γ), and delta (δ) globin proteins. ⁽²⁾

The making of hemoglobin

Hemoglobin synthesis occurs in the cytoplasm when four heme molecules and four globin subunits combine. More specifically, each globin chain contorts around a heme molecule and four of these bundles come together to form hemoglobin. There are three types of normal hemoglobin molecules found within the body at various stages of development. Each type of hemoglobin derives its name from the globin subunits.

Starting in the first trimester, the dominant hemoglobin is called fetal hemoglobin. ⁽²⁾ It’s produced within the yolk sac and eventually by the fetal liver, spleen, and bone marrow. ⁽³⁾ Fetal hemoglobin consists of two α and two γ globin subunits. Fetal hemoglobin quickly decreases after birth and is replaced by the adult hemoglobin called hemoglobin A. Hemoglobin A is composed of two α and two β globin subunits. In a healthy human being, hemoglobin A makes up 95 to 98% of hemoglobin within the body. There is also a secondary hemoglobin called hemoglobin A2 which is made of two α and two δ globin chains. Hemoglobin A2 concentration is around 5 to 2% of adult hemoglobin. ⁽²⁾

Inherited hemoglobin disorders

Mutations, deletions, and substitutions affecting the globin gene segments are inheritable causes of qualitative and quantitative hemoglobin disorders. The first type, thalassemia, is caused by a deletion or point mutation of the gene segments coding for the globin subunit. The result of an underproduction or zero production of the affected globin subunit. The severity of disease reflects the pattern of inheritance, either heterozygous or homozygous.

Alpha thalassemia is the result of the deletions within the α-globin gene segment. 4 alleles contain the blueprint for α-globin construction. Disease severity is directly related to the number of alleles that are deleted. For instance, the mild form of α-thalassemia called α-thalassemia minor is lacking one allele. Each allele deleted increases disease severity because of a decrease in α-globin subunit production. The most severe expression of the disease, total deletion of the gene segment, is incompatible with life and results in fetal hydrops. ⁽⁴⁾

Beta thalassemia is the result of a point mutation within the beta-globin gene segment. Inheritance patterns dictate the severity of β-thalassemia. Heterozygous inheritance produces asymptomatic to mild symptoms due to a small decrease in β-globin subunit production. It is known as β-thalassemia minor. Homozygous inheritance results in β-thalassemia major which is a total absence of β-globin production. People with β-thalassemia major suffer severe anemia, jaundice, slow growth, hepatosplenomegaly, and endocrine dysfunction. Both α and β-thalassemia can be coinherited or inherited with a qualitative hemoglobin variant. ⁽⁴⁾

Qualitative hemoglobin variants result from amino acid substitutions within the β-globin gene segment.. There are three commonly encountered hemoglobin variants. They are hemoglobin S, C and E. It should be noted that hemoglobin S is the most common, while hemoglobin C and E tend to have a much lower frequency. Similar to thalassemia, the disease severity is directly related to inheritance pattern.

Hemoglobin S, sickle cell, is the most prevalent and well-known hemoglobin variant. Hemoglobin S results from the substitution of valine for glutamic acid in the sixth position within the β-globin subunit. In the homozygous inheritance, hemoglobin S is exclusively produced instead of hemoglobin A. Hemoglobin S prefers to polymerize which distorts the red blood cells into the iconic sickle shape. The deformed red blood cells get lodged in capillaries causing pain, and organ infarction. Heterozygous inheritance, sickle cell trait, is asymptomatic or mild. Anemia and sickle crises can still occur if the person is placed under extreme hypoxic stress. ⁽⁵⁾

Hemoglobin C is another hemoglobin variant with the propensity to form crystals. Hemoglobin C is caused by the substitution of lysine for glutamic acid in the sixth position of the β-globin subunit. Zygosity also dictates the severity of the disease from asymptomatic heterozygotes to severe hemolytic anemia in homozygotes. The crystals produced by the polymerization of hemoglobin C are sometimes observed ion peripheral smear and resemble a prism bar. ⁽⁶⁾

Hemoglobin E is the final most commonly encountered hemoglobin variant, although at a much lower frequency. It is caused by the substitution of lysine for glutamic acid at the twenty-sixth position of the β-globin. The hemoglobin E variant causes structurally abnormal hemoglobin and an overall decrease in hemoglobin production. ⁽⁷⁾

Acquired hemoglobin disorders

Hemoglobin dysfunction isn’t always inherited. Occasionally, the hemoglobin of an otherwise healthy person can be altered. This can happen when the person is exposed to toxins in the environment. Carboxyhemoglobin occurs when carbon monoxide binds with hemoglobin instead of oxygen. The bonds formed with carbon monoxide out compete oxygen essentially suffocating body tissues. Methemoglobin is the result of the oxidation of iron to the ferric state when the body is thrown into an alkaline state. The heme molecules are changed to hematin which will irreversibly bind with oxygen. This typically occurs in the presence of toxic agents such as nitrates, aniline dyes, chlorates, quinones, phenacetin, procaine, benzocaine, and lidocaine. Methemoglobin can be reversed if reduced by the NADH cytochrome reductase system. The last instance of conversion of normal hemoglobin is sulfhemoglobin. This is an irreversible reaction of adding sulfur to the porphyrin rings of heme. This can occur when a person ingests sulfur-containing substances. Sulfhemoglobin is unable to transport oxygen. ⁽¹⁾

Conclusion

The hemoglobin molecule plays a vital role in oxygen transport within the blood. Its structure and function are uniquely designed to accomplish this ability. Understanding the various abnormalities, from thalassemias to hemoglobin variants, highlights the intricacies of genetic and acquired conditions affecting hemoglobin. Through continued research and education, we can better diagnose, treat, and manage these hemoglobin disorders, improving patient outcomes.

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Riddle answers:

1. Hemoglobin

2. Methemoglobin

3. Carboxyhemoglobin

References:

1. Sunheimer RL, Graves L. Clinical laboratory chemistry.; 2010. http://ci.nii.ac.jp/ncid/BB0638763X

2. Farid Y, Bowman NS, Lecat P. Biochemistry, Hemoglobin Synthesis. [Updated 2023 May 1]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK536912/

3. Sankaran VG, Orkin SH. The Switch from Fetal to Adult Hemoglobin. Cold Spring Harbor Perspectives in Medicine. 2012;3(1):a011643. doi:10.1101/cshperspect.a011643

4. Bajwa H, Basit H. Thalassemia. [Updated 2023 Aug 8]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK545151/

5. Washington State Department of Health Newborn Screening Program. Health Care Provider Hemoglobinopathy Fact Sheet.; 2011. https://doh.wa.gov/sites/default/files/legacy/Documents/5220/HbSFactSheet.pdf

6. Karna B, Jha SK, Al Zaabi E. Hemoglobin C Disease. [Updated 2023 May 29]. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK559043/

7. Washington State Department of Health Newborn Screening Program. Health Care Provider Hemoglobinopathy Fact Sheet.; 2011. https://doh.wa.gov/sites/default/files/legacy/Documents/5220/HbEFactSheet.pdf

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I'd like to share the mission and vision of Med Lab Mastery. This publication began as a companion to a tutoring service website, which I hope to launch soon. Stay tuned for its debut and thank you for reading my articles so far. I will be adding a paid tier soon where I will really get into the weeds on specific laboratory tests. Articles on career development and more general laboratory topics will remain free. In the meantime, consider pledging to the publication. There are monthly, yearly, and founding member options. I really appreciate the support! Join me as we continue to explore the world of clinical laboratories.

Mission

1. Medical Laboratory Science Students: Med Lab Mastery is here for students pursuing an associate or bachelor’s degree in medical laboratory science. The articles offer easy-to-digest reviews of topics you'll encounter in school and on the BOC. The riddles are fun practice questions based on the article topics—take a chance and post your answers!

2. Seasoned Medical Laboratory Scientists: For seasoned professionals, these articles can serve as a reminder of the foundational knowledge behind your practical skills, turning muscle memory back into conscious competence.

3. Healthcare Workers: Med Lab Mastery provides insight into our operations and how we support patient care, especially for healthcare workers who rarely visit laboratories hidden deep within hospitals.

1. Curious Public: For the curious public, these articles offer valuable information to help you make informed health decisions. While not intended for diagnosis or treatment, they explain the reasoning behind various lab tests.

Vision

I envision Med Lab Mastery providing useful, actionable knowledge to everyone. One major goal is to get my articles approved for continuing education credits for fellow medical laboratory scientists. Additionally, I aim to educate the next generation of medical laboratory scientists, helping them become confident and knowledgeable in their field.

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Intro

This article describes the kidney anatomy and the physiology of producing urine. It covers the meat of what a medical laboratory scientist needs to know to perform their job. The kidneys are essential organs, encountering more blood than even the brain and liver. They remove toxins, regulate fluid and electrolytes, and maintain acid-base balance. The result is around one to two liters of urine per day. The kidneys are even able to generate vitamin D if the skin and liver are unable to. Read on to find out how your kidneys function.

Anatomy

The kidneys, two fist-sized organs, are the first part of the larger urinary tract. On the macroscopic level, the kidneys consist of an outer tissue layer called the cortex and an inner tissue space called the medulla. The medulla houses the primary functional unit called the nephron. Each kidney contains 1 to 2 million nephrons which are the site of urine formation. The kidneys contain a large network of blood vessels that bring blood to individual nephrons, where excess nutrients and toxins are filtered out. The resulting filtrate leaves the kidneys as urine, while the filtered blood returns to general circulation.

The nephron is akin to an intricate plumbing system. Blood is brought to the nephron by the afferent arteriole which splits into a capillary cluster called the glomerulus. The glomerulus sits within a cup-like structure of the nephron called the Bowman's capsule. The blood exits the glomerulus and enters the efferent arteriole. The efferent arteriole splits into a network of capillaries that surround the entire nephron structure. Immediately following the bowman’s capsule is the proximal convoluted tubule. The proximal tubule narrows abruptly into the hairpin-shaped loop of Henle. The loop of Henle is composed of a descending and ascending leg. It is surrounded by a mesh-like network of capillaries called the peritubular capillaries. The ascending loop of Henle opens abruptly again into the distal convoluted tubule. This section is also surrounded by the peritubular capillaries which consolidate into the arcuate vein. The nephron comes to an end as the distal convoluted tubule empties into the collecting duct.

The overall shape of the nephron and its surrounding vessels is important to its function. The convolutions maximize the length and surface area needed to filter the blood. Narrowing and expanding of different sections helps generate pressure to improve passive transport and drive fluid within the nephron.

Physiology

Urine formation involves three main processes: glomerular filtration, tubular reabsorption, and tubular secretion. Glomerular filtration drives low molecular weight (<66,000 daltons) molecules such as albumin, other low molecular weight proteins, glucose, amino acids, urea, and creatinine into the Bowman’s capsule. Pressure differences enable glomerular filtration as blood travels from afferent to efferent arterioles. The glomerular basement membrane is negatively charged, which repels large negatively charged molecules to stay within the blood circulation.

The next two activities, tubular reabsorption, and tubular secretion, occur together primarily in the proximal convoluted tubule but also throughout the nephron. In tubular reabsorption fluid and electrolytes exit the nephron and reenter the surrounding blood vessels. This occurs through active and passive transport. Sodium is actively transported against its concentration gradient with the help of transmembrane carrier proteins, using energy in the form of ATP. When the concentration of an actively transported substance exceeds the carrier protein capacity, it is “spilled into” the urine. The best example of this is glucose in diabetic patients. The threshold of glucose is between 160 to 180 mg/dL. Substances that are passively transported down their concentration gradients include chloride (only in the proximal convoluted tubule), water, and urea.

Complementary to tubular reabsorption is tubular secretion. This is the process of moving substances from the peritubular capillaries into the nephron. Secretion occurs predominantly in the proximal and distal convoluted tubules. Secretion enhances the elimination of waste products and drives normal acid-base balance in the body. Substances that are not able to be filtered by the glomerulus because of charge are now able to leave circulation to be excreted. Substances such as drugs become unbound from carrier proteins and become small enough to pass through capillary walls and into the filtrate in the tubules. Commonly secreted substances include hydrogen ions, ammonia, potassium, and weak acids and bases.

The prepared urine leaves the nephron by the collecting duct which coalesces into the renal pelvis. As is commonly known, urine travels from the kidney, down the ureters, to the bladder, and exits the body by the urethra.

Urinalysis

Urinalysis is a test protocol that combines chemical and microscopic examination of urine. Routine urinalysis may be the first test to uncover urinary tract dysfunction. The urine is observed for color and appearance. A dipstick with cellulose reagent pads is dipped into the urine to chemically test for protein, hemoglobin, leukocytes (white blood cells), red blood cells, nitrites, specific gravity, glucose, bilirubin, urobilinogen, and pH. Each pad is impregnated with unique chemicals to react with specific substances in the urine.

After dipstick testing, the urine is centrifuged to concentrate any solids present. Microscopic examination of urine is used to confirm the presence or absence of leukocytes, red blood cells, epithelial cells, casts, mucus, crystals, and bacteria. A deeper dive into routine urinalysis will be covered in a future article.

Wrapping it up

The kidneys are amazingly busy and efficient organs. This article covered the basics of kidney anatomy and physiology. Future articles will discuss urine testing and specific kidney diseases in more detail. I hope this article was helpful.

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Riddle Answers

1. Kidneys

2. Glomerular filtration rate

3. Urine production

References

Sunheimer RL, Graves L. Clinical laboratory chemistry.; 2010. http://ci.nii.ac.jp/ncid/BB0638763X

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Electrolytes are essential ions your body uses to maintain electrical neutrality across cell membranes, regulate fluid balance, and produce action potential for nerves and muscles. ⁽¹⁾ Essential electrolytes found in the body include sodium (Na⁺), potassium (K⁺), chloride (Cl^-), magnesium (Mg²⁺), Calcium (Ca²⁺), phosphorous (P^3-), and bicarbonate (HCO₃^-). This article will discuss the unique properties and interplay of sodium, potassium, chloride, and bicarbonate. We will also cover the test method for measuring this particular group of electrolytes and the important information you can infer from electrolyte test results.

Electrolytes are elements and molecules with either a positive or negative charge. Ions with a positive charge are called cations, while negatively charged ions are anions. In the case of the elements (sodium, potassium, and chloride), the accompanying charge indicates the difference between protons and electrons in the element’s nucleus. Bicarbonate is a molecule of hydrogen, carbon, and three oxygen, which produces a net negative charge. This means there is one available electron that interacts in chemical reactions. The charges of these electrolytes influence chemical reactions by the attraction of opposites similar to magnets. The electrons participate in chemical reactions by forming bonds between elements to achieve neutrality.

Electrolyte basics

Sodium

Sodium, the eleventh element on the periodic table, is the most important extracellular ion in the body. Sodium’s primary roles include:

• Fluid Regulation: Sodium regulates fluid balance in the extracellular space.

• Membrane Potential: Along with potassium, sodium regulates membrane potential via active transport across the cell membrane.

• Hormonal Regulation: The hormone aldosterone tightly regulates sodium concentration, with the kidneys acting as hubs of sodium excretion and reabsorption.⁽⁷⁾

Dysregulated Sodium Levels:

• Hyponatremia (Less than 135 mmol/L): Caused by low intake, heart failure, cirrhosis, prolonged hyperglycemia, etc. Symptoms include headaches, confusion, nausea, and delirium.

• Hypernatremia (Greater than 145 mmol/L): Caused by dehydration, hypertonic saline solutions, etc. Symptoms include tachypnea, insomnia, and restlessness. ⁽¹⁾

Potassium

Potassium, the nineteenth element on the periodic table, is a crucial intracellular ion in the body. Potassium’s primary roles include:

• Intracellular Dominance: Potassium is the most important ion inside cells.

• Membrane Potential Regulation: Potassium works with sodium to regulate the exchange of ions across cell membranes via the Na/K ATP pump.

• Hormonal Regulation: Aldosterone is the major driver of potassium excretion in the body. Potassium is reabsorbed in the kidney's proximal convoluted tubule and the thick ascending loop of Henle and secreted in the distal convoluted tubule.

Dysregulated Potassium Levels:

• Hypokalemia (Less than 3.6 mmol/L): Caused by hyperaldosteronism or loop diuretics. Symptoms include weakness, fatigue, muscle twitches, and paralysis.

• Hyperkalemia (Greater than 5.5 mmol/L): Caused by metabolic acidosis, insulin deficiency, hypoaldosteronism, prolonged beta-blocker use, and kidney disease. Symptoms include arrhythmias, muscle cramps and weakness, rhabdomyolysis, and myoglobinuria. ⁽¹⁾

Chloride

Chloride is an anion found within the extracellular space, working in conjunction with sodium and potassium. Chloride’s primary roles include:

• Membrane Neutrality: Chloride helps regulate membrane neutrality alongside sodium and potassium.

• Kidney Regulation: Chloride concentration is controlled within the kidney, being filtered and secreted in the glomerulus, and reabsorbed in the proximal and distal tubules through active and passive transport.

Dysregulated Chloride Levels:

• Hypochloremia: Caused by excessive vomiting or water retention in cases of congestive heart failure.

• Hyperchloremia: Associated with bicarbonate loss due to diarrhea and excessive normal saline infusion. ⁽¹⁾

Bicarbonate

Bicarbonate is the dissolved form of carbon dioxide within the blood. It is the most crucial molecule governing the acid-base balance within the blood. Excess bicarbonate is either exhaled or filtered through the kidneys. Bicarbonate is reabsorbed or filtered to maintain the acid-base balance by forming a titratable acid and ammonia. ⁽¹⁾

The lungs and kidneys manage bicarbonate levels. Diarrhea is the major contributor to rapid excessive loss of bicarbonate, while kidney dysfunction results in increased bicarbonate. Decreased bicarbonate levels are present in metabolic acidosis and compensatory primary respiratory alkalosis. Increased bicarbonate is associated with metabolic alkalosis and compensatory respiratory acidosis. ⁽¹⁾

Electrolyte testing

Electrolyte, specifically sodium, potassium, and chloride, concentrations are determined by interacting patient samples with ion-selective electrodes (ISE). Ion-selective electrodes are potentiometric devices made of glass, liquid, or solid-state electrodes. In healthcare, ISEs are used in automated chemistry analyzers and point-of-care devices. The ISE measures the change in potential caused by the interaction between ions and electrodes. The difference between the measured potential and a reference electrode determines the concentration of the target ion. ⁽²⁾ Bicarbonate concentration is determined by acid titration performed on the chemistry analyzer.

Direct vs. Indirect ISE testing

There are two types of ISE test methods used in healthcare: direct ISE and indirect ISE. The significant difference is how the patient sample interacts with the electrode. In direct ISE testing, the patient sample comes into direct contact with the electrode. Direct ISEs are employed in point-of-care testing devices. In contrast, the indirect ISE method begins by diluting the patient sample 1:16 to 1:34 with a reagent buffer. The diluted sample interacts with the electrode membrane. Finally, the electrolyte concentration is calculated assuming the patient sample is 93% liquid and 7% solids. This version of ISE testing is typically found in the standard chemistry analyzers used by clinical labs. While the indirect ISE method is convenient for large sampling volumes, it does present unique drawbacks. Conditions that raise protein and lipid levels, such as diabetes, liver, and kidney disease, and alcoholism, change the percentage of solids present in the patient sample. This affects the calculation used to determine electrolyte concentrations, skewing patient results. ⁽³⁾

Anion Gap

Determining the electrolyte concentrations is only the beginning. The anion gap is a value calculated from the measured electrolytes used to manage the acid-base balance, fluid balance, and electrolyte balance. The anion gap is calculated by subtracting the sum of cations (Na⁺ & K⁺) and anions (Cl^- & HCO₃^-). The exact calculation is [(Na+K) – (Cl+HCO₃)]. A healthy body is electrically neutral, which is necessary when observing an anion gap. A normal anion gap is 4 to 12 mmol/L. The calculation does not include other anions and cations within the body but is assumed to bring the anion gap to zero. ⁽⁴⁾

Anion gaps are most helpful in determining acid-base balance in the body. An increased anion gap indicates metabolic acidosis caused by diabetic ketoacidosis or salicylate poisoning. ⁽⁴⁾ Albumin, the most influential unmeasured anion, directly affects the anion gap result. ⁽⁴⁾ For every 1g/L decrease in albumin, the anion gap decreases by 0.25 mmol/L. ⁽⁴⁾ This makes identifying metabolic acidosis from the anion gap difficult in hypoalbumin patients. ⁽⁴⁾ Another cause of a decreased anion gap is increased paraproteins, as seen in multiple myeloma. ⁽⁵⁾ When a patient has increased paraproteins, the body will attempt to neutralize the increased positively charged paraproteins by increasing chloride ions. ⁽⁵⁾

Recap

Electrolytes are essential for many functions within the body. By maintaining optimal fluid levels, acid-base control, and electrical neutrality, electrolytes ensure our bodies function at peak performance. Ion-selective electrodes and the anion gap provide physicians with the information they need to keep their patients well.

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Riddle answers!

1. Sodium

2. Bicarbonate

3. Anion Gap

References

1. Shrimanker I, Bhattarai S. Electrolytes. StatPearls - NCBI Bookshelf. Published July 24, 2023. https://www.ncbi.nlm.nih.gov/books/NBK541123/

2. Baranwal J, Barse B, Gatto G, Broncova G, Kumar A. Electrochemical Sensors and their applic

ations: a review. Chemosensors. 2022;10(9):363. doi:10.3390/chemosensors10090363

3. When direct and indirect ion selective electrode results conflict. myadlm.org. Published September 1, 2018. https://myadlm.org/cln/articles/2018/september/when-direct-and-indirect-ion-selective-electrode-results-conflict

4. Pandey DG, Sharma S. Biochemistry, Anion Gap. StatPearls - NCBI Bookshelf. Published July 10, 2023. https://www.ncbi.nlm.nih.gov/books/NBK539757/

5. Haber LA, Dhaliwal G, Lo L, Rizzuto G. Evaluating a low anion gap: A practical approach. Cleveland Clinic Journal of Medicine. 2023;90(10):619-623. doi:10.3949/ccjm.90a.23035

6. Biga LM, Bronson S, Dawson S, et al. 26.3 electrolyte balance. Pressbooks. Published September 26, 2019. https://open.oregonstate.education/aandp/chapter/26-3-electrolyte-balance/