Comprehensive Guide to Clinical Biochemistry and Laboratory Medicine

Clinical Biochemistry Course Information and Grading

Clinical biochemistry, also referred to as clinical chemistry or chemical pathology, is a fundamental laboratory service essential for modern medical practice. It involves the application of chemical and biochemical methods to the study of disease. The course is taught by Assist. Prof. Dr. Fehmi B. Alkas from the Faculty of Pharmacy, representing the Department of Toxicology and the Department of Biochemistry. The grading breakdown for this course is structured as follows: quizzes account for $30\%$, the midterm examination accounts for $30\%$, and the final examination accounts for $40\%$.

Introduction to the Role of Clinical Biochemistry

Clinical biochemistry deals with the applications of the biochemistry laboratory to determine the cause of various diseases. The results derived from biochemical investigations serve two primary roles for clinicians: diagnosis, which is the determination of the disease, and prognosis, which involves the follow-up of treatment and the monitoring of recovery from illness. Successful medical practice is considered unimaginable without the services provided by a clinical biochemistry laboratory, as these investigations are involved in every branch of clinical medicine.

Biochemical tests are utilized for several critical purposes: the diagnosis and monitoring of treatment, screening for disease, assessing prognosis, conducting research into the biochemical basis of disease, and performing clinical trials for new drugs. These investigations are specifically key for the diagnosis and prognosis of conditions such as diabetes mellitus, jaundice, myocardial infarction, gout, pancreatitis, rickets, cancers, and acid-base imbalances.

Disease Screening and Diagnostic Abnormalities

Screening in clinical biochemistry allows for the discovery of uncommon or unexpected diseases and the early requesting of a battery of tests which might expedite patient management, although it is noted that most studies have not definitively shown this to be the case. Common unexpected abnormal test results include raised plasma calcium in cases of hyperparathyroidism, and raised plasma $TSH$ and/or a low $T4$ in cases of hypothyroidism. Diabetes mellitus is characterized by high random plasma glucose, while renal tract disease is indicated by raised plasma creatinine or urea. Liver disease often presents as increased plasma $ALT$ and $AST$.

Core and Specialized Biochemical Tests

Clinical biochemical tests comprise over $1/3$ of all hospital laboratory investigations. Laboratories provide "core analyses," which are commonly requested tests of value for many patients on a frequent basis. Core biochemical tests include electrolytes such as sodium ($Na^+$), potassium ($K^+$), chloride ($Cl^-$), and bicarbonate. Other core tests include urea, creatinine, calcium, phosphate, total protein, albumin, bilirubin, alkaline phosphatase ($ALP$), alanine aminotransferase ($ALT$), aspartate aminotransferase ($AST$), glucose, and amylase.

Specialized tests are not performed by every laboratory. Large departments often act as reference centres where less common requests are handled. These specialized investigations include the analysis of hormones, specific proteins, trace elements, vitamins, drugs, lipids and lipoproteins, and various $DNA$ analyses.

Units, Reference Ranges, and Interpretation of Results

Biochemical parameters are expressed in specific units: metabolites such as glucose and urea are measured in $mg/dL$ or $mmol/L$; electrolytes such as $Na^+$ and $K^+$ are expressed in $mmol/L$ or $meq/L$ (the latter being earlier terminology); and enzymes are expressed as $Units/L$. Results are interpreted as being increased, decreased, or within a normal reference range.

Reference values are obtained from healthy individuals as judged by clinical and laboratory parameters after statistical analysis under definite laboratory conditions. The normal or reference range is defined as the values within which $95\%$ of normal healthy persons fall. Cut-off values are typically set as the mean reference value $\pm$ $N$ times the standard deviation ($SD$) of a healthy population, where $N$ varies between $1$, $2$, and $3$.

Common Sources of Error in Laboratory Use

Several errors can arise when using the laboratory, each with distinct consequences. The crossover of addressograph labels can lead to two patients receiving each other's results, necessitating an investigation into both cases. Timing errors are critical; for example, sampling blood too early after drug administration can lead to misleadingly high values in therapeutic monitoring. Furthermore, the interpretation of results like cortisol is dependent on the specific time of day the blood was sampled.

Sample collection tube errors are also significant. Using a lithium-heparin tube invalidates the sample for measurement of therapeutic lithium levels. Serum electrophoresis requires serum; if plasma is used, fibrinogen interferes with the detection of monoclonal bands. Contaminating a biochemistry tube with a hematology sample (such as potassium-$EDTA$) will lead to artificially high potassium and low calcium values.

Sampling blood from near an intravenous ($IV$) infusion site results in dilution, causing most test results to be low, except for those affected by the infusion fluid itself. For instance, normal saline infusion would raise sodium and chloride results while lowering others. Analytical errors are rare but occur; transcription errors are decreasing due to electronic downloads, but errors at reception due to mislabeling remain a common source of internal laboratory error.

Specimen Collection and Blood Types

Biological fluids used in clinical biochemistry include blood, urine, saliva, sputum, feces, tissues, cells, cerebrospinal fluid ($CSF$), peritoneal fluid, synovial fluid, pleural fluid, and stones. Among these, blood is most frequently used, either as whole blood, plasma, or serum. Venous blood is the most common and is usually drawn from the elbow. Capillary blood, in volumes less than $0.2\,ml$, is obtained from a finger or thumb. Arterial blood, often drawn under local anesthesia, is reserved for blood gas determinations. Precautions for collection include the use of sterile disposable needles, cleaning patient skin, and using clean, dry vials.

Whole blood mixed with an anticoagulant is utilized for estimating hemoglobin, carboxyhemoglobin, $pH$, urea, non-protein nitrogen, pyruvate, lactate, and ammonia. While glucose was previously estimated from whole blood, plasma is preferred in recent years. Whole blood consists of $55\%$ acellular plasma ($91\%$ water, $8\%$ protein, and $1\%$ other) and $45\%$ cellular elements. Of the cellular elements, $99.1\%$ are red blood cells and $0.9\%$ are leukocytes and platelets.

The Physiology of Blood Coagulation

Coagulation is the process of forming a blood clot, known as secondary hemostasis. Primary hemostasis involves vasoconstriction and platelet aggregation at the site of vessel injury. Clinical clotting involves $13$ principal coagulation factors designated by Roman numerals ($I$ to $XIII$). The process occurs via two pathways: the extrinsic and the intrinsic. Both pathways eventually lead to the production of Factor $X$, which initiates the common pathway of coagulation.

The extrinsic pathway is usually the first activated and is stimulated by tissue factor from cells external to blood vessels. Tissue factor activates Factor $VII$ to $VIIa$, which triggers reactions producing Factor $X$. The intrinsic pathway is activated by internal vessel injury, starting with the activation of Factor $XII$ (Hageman factor) upon contact with injured internal surfaces. Elements of the intrinsic pathway can be cross-activated by the extrinsic pathway; for example, Factor $VIIa$ can activate Factor $IX$ to amplify the process.

The Coagulation Cascade and Clotting Factors

The cascade involves the sequential activation of factors. In the intrinsic system, Factor $XII$ becomes $XIIa$, which activates $XI$ to $XIa$, then $IX$ to $IXa$. Factor $IXa$ (with $VIII$ and calcium) activates $X$ to $Xa$. In the extrinsic system, tissue factor and $VII$ activate $X$ to $Xa$. Factor $Xa$ (with $V$, calcium, and phospholipid) converts prothrombin ($II$) to thrombin ($IIa$). Thrombin then converts soluble fibrinogen ($I$) into insoluble fibrin ($Ia$). Factor $XIII$ acts on fibrin monomers to create a stable, cross-linked fibrin clot.

Clinical Transfusions and Anticoagulants

Deficiencies in specific factors require the transfusion of specific blood components:

• Fibrinogen deficiency: Cryoprecipitate or stored plasma.
• Factor $V$ deficiency: Fresh frozen plasma ($FFP$).
• Factor $VII$ deficiency: Factor $IX$ complex ($II$, $VII$, $IX$, $X$) or stored plasma.
• Factor $VIII$ deficiency: Factor $VIII$ concentrate ($AHF$), cryoprecipitate, or $FFP$.
• von Willebrand's disease: Cryoprecipitate or $FFP$.
• Factor $IX$ deficiency: Factor $IX$ complex or stored plasma.
• Factor $XIII$ deficiency: Stored plasma.

Anticoagulants are classified by their use. In vivo parenteral options include heparin and low molecular weight heparins ($LMWH$); oral options include coumarin and indandione derivatives. In vitro anticoagulants include heparin and calcium complexing agents like sodium citrate, sodium oxalate, and sodium edetate ($EDTA$). Specific drug inhibitors include Factor $Xa$ inhibitors (Apixaban, Betrixaban, Edoxaban, Rivaroxaban) and direct thrombin inhibitors (Argatroban, Dabigatran). Monitoring is performed via Prothrombin Time ($PT$) and International Normalized Ratio ($INR$), which compare patient results to a normal average.

Characteristics of Plasma and Serum

Plasma is obtained by centrifuging whole blood collected with an anticoagulant. It contains all clotting factors and involves parameters like fibrinogen, glucose, bicarbonate, and ascorbic acid. Serum is the supernatant fluid collected after centrifuging clotted blood; it is effectively plasma without clotting factors (as they are consumed during the irreversible clotting process where fibrin strands entrap cells). Serum is the most frequently used specimen in chemistry labs and for serology, used for measuring proteins, creatinine, bilirubin, electrolytes ($Na^+$, $K^+$, $Cl^-$), enzymes ($ALT$, $AST$, $LDH$, $CK$, $ALP$, $ACP$, amylase), and vitamins.

Blood Collection Tube Standards

Standardized tube cap colors indicate the additive and function:

• Light-blue: contains $3.2\%$ Sodium citrate; binds calcium to prevent clotting. Used for coagulation tests.
• Red or Gold: Serum tube with/without clot activator or gel. Used for chemistry, serology, and immunology.
• Green: Sodium or lithium heparin; inhibits thrombin/thromboplastin. Used for stat and routine chemistry.
• Lavender or Pink: Potassium $EDTA$; binds calcium. Used for hematology and blood banking.
• Gray: Sodium fluoride and sodium/potassium oxalate. Fluoride inhibits glycolysis; oxalate precipitates calcium. Used for glucose, blood alcohol, and lactic acid.

Age-Specific PSA Reference Ranges and Fluid Methods

Reference ranges for Prostate-Specific Antigen ($PSA$) vary by age and ethnicity ($ng/mL$):

• 40 to 49 years: Asian Americans ($0-2.0$), African Americans ($0-2.0$), Caucasians ($0-2.5$).
• 50 to 59 years: Asian Americans ($0-3.0$), African Americans ($0-4.0$), Caucasians ($0-3.5$).
• 60 to 69 years: Asian Americans ($0-4.0$), African Americans ($0-4.5$), Caucasians ($0-4.5$).
• 70 to 79 years: Asian Americans ($0-5.0$), African Americans ($0-5.5$), Caucasians ($0-6.5$).

Other body fluids are collected via specific methods: Urine is collected directly or via catheter for investigating sugar, protein, bile salts, and steroids. Cerebrospinal fluid ($CSF$) is obtained via lumbar puncture from the subarachnoid space to investigate sugar, protein, and chloride.