Displaying items by tag: Diseases and Disorders

The immunologic analysis of semen is a component of the investigation of infertility, and it aims to assess the presence of antibodies that may affect sperm function. Antisperm antibodies (ASA) are immune system proteins that can target and potentially impair sperm function, leading to fertility issues. There are different methods and tests employed in immunologic analysis of semen to detect the presence of these antibodies:

1. Direct Sperm Antibody Test (SAT): This test directly measures the presence of antibodies on the surface of sperm. It involves incubating sperm with a substance that binds to antibodies, and the resulting reaction can be detected under a microscope.
2. Indirect Sperm Antibody Test (ISAT): This test detects antibodies in the seminal plasma (the fluid part of semen). Serum (blood) is mixed with sperm, and if antibodies are present, they will attach to the sperm. The remaining solution is then tested for the presence of antibodies.
3. Mixed Antiglobulin Reaction (MAR) Test: This is another indirect method where sperm are mixed with antibodies that can bind to human antibodies. After washing, the solution is tested to see if any antibodies are attached to the sperm.
4. Immunobead Binding Test: In this test, latex beads coated with antibodies are mixed with semen. If there are antisperm antibodies in the semen, they will bind to the latex beads. The bead-sperm complexes can be detected and quantified.
5. Flow Cytometry: This technique uses a flow cytometer to analyze individual sperm cells tagged with fluorescent markers. It can provide information on the percentage of sperm affected by antibodies and the degree of binding.

It's important to note that the presence of antisperm antibodies does not always correlate with infertility, and the impact of antibodies on fertility can vary. Additionally, the causes of antisperm antibodies can be diverse, including infections, trauma, or autoimmune disorders. Treatment options may include assisted reproductive technologies (ART) such as in vitro fertilization (IVF) or intrauterine insemination (IUI).

Antisperm Antibodies

The role of antisperm antibodies in the etiology of male infertility remains a subject of debate. Immunological assessments conducted on seminal fluid encompass the mixed antiglobulin reaction (MAR test) and the immunobead test.

Antibodies directed against sperm have the capacity to either immobilize or terminate them, impeding their journey through the cervix to reach the ovum. These antibodies can be examined in the serum, seminal fluid, or cervical mucus. In cases where antibodies are affixed to the head of the sperm, they hinder the sperm from penetrating the egg. Conversely, when antibodies are attached to the tail of the sperm, they impede its motility. This intricate interplay underscores the potential impact of antisperm antibodies on various aspects of sperm function, contributing to the complexities surrounding male infertility.

SpermMAR™ test

The SpermMAR™ test is designed to identify IgG and IgA antibodies targeting the surface of sperm in a semen sample. In the direct SpermMAR™ IgG test, a drop of fresh and unwashed semen is combined with IgG-coated latex particles and anti-human immunoglobulin on a glass slide. A thorough examination of at least 200 motile spermatozoa is conducted. If antibodies are present on the sperm surface, antihuman immunoglobulin binds IgG-coated latex particles to the IgG on the spermatozoa surface. This results in the attachment of latex particles to spermatozoa, observable as motile, swimming spermatozoa with attached particles. Conversely, in the absence of antibodies on the sperm surface, swimming spermatozoa without attached particles are observed, and the latex particles demonstrate clumping due to the binding of their IgG to antihuman immunoglobulin.

In the direct SpermMAR™ IgA test, a drop of fresh unwashed semen and IgA-coated latex particles are mixed on a glass slide. The latex particles bind to spermatozoa if they are coated with IgA antibodies.

The indirect SpermMAR™ tests involve examining fluid without spermatozoa (e.g., serum) for the presence of antisperm antibodies. Initially, antibodies are bound to donor spermatozoa, which are then mixed with the fluid under analysis. These antibodies are subsequently detected as described in the direct tests.

A minimum count of 200 motile spermatozoa is recommended. If more than 50% of spermatozoa exhibit attached latex particles, an immunological issue is likely. This meticulous evaluation through the SpermMAR™ test provides valuable insights into the presence of antisperm antibodies, aiding in the diagnosis of potential immunological factors impacting sperm function.

Immunobead test

The Immunobead test is a diagnostic method aimed at identifying antibodies affixed to the surface of spermatozoa. This is achieved by utilizing immunobeads, which are plastic particles with attached anti-human immunoglobulin (IgG, IgA, or IgM). The procedure involves counting the percentage of motile spermatozoa with two or more attached immunobeads within a sample of 200 motile spermatozoa. An abnormal result is indicated when more than 50% of spermatozoa exhibit attached beads.

This meticulous assessment provides valuable information about the presence of antibodies on the surface of sperm, contributing to a comprehensive understanding of potential immunological factors affecting sperm function. The Immunobead test serves as a crucial tool in the diagnostic arsenal for evaluating male fertility and identifying abnormalities in the immune response against spermatozoa.

Microscopic evaluation of semen stands out as the paramount test for assessing infertility in men. This meticulous analysis, focusing on sperm morphology, motility, and concentration, provides crucial insights into reproductive health. By scrutinizing these parameters, clinicians can identify potential impediments to successful conception. The microscopic assessment not only gauges sperm quality but also reveals any abnormalities that may hinder fertilization. This method is instrumental in acquiring precise data for developing targeted interventions and personalized treatment strategies to address male infertility.

Sperm Motility

In the initial laboratory evaluation of sperm function within a wet preparation, the primary focus is on sperm motility, which denotes the sperms' ability to move. This motility is crucial for tasks such as penetrating cervical mucus, navigating the fallopian tube, and ultimately fertilizing the ovum. It is noteworthy that only sperms exhibiting rapid progressive motility possess the capability to successfully penetrate the ovum and facilitate fertilization.

Principle

In the assessment of sperm within a wet preparation, both motile and non-motile spermatozoa are quantified across randomly selected fields using a 40× objective. The outcome is then articulated as the percentage of observed motile spermatozoa.

Method

On a glass slide, a drop of semen is carefully deposited and overlaid with a coverslip, which is then encircled with petroleum jelly to avert dehydration. Subsequently, the specimen is examined under a 40× objective lens. A minimum of 200 spermatozoa is meticulously enumerated across multiple microscopic fields. The findings are expressed as a percentage, delineating (a) rapidly progressive spermatozoa, characterized by swift linear forward movement; (b) slowly progressive spermatozoa, displaying gradual linear or non-linear motion, such as crooked or curved trajectories; (c) non-progressive spermatozoa, manifesting tail movement without concomitant forward progress; and (d) immotile spermatozoa, exhibiting a complete lack of movement (in accordance with WHO criteria). Spermatozoa falling within grades (c) and (d) are classified as poorly motile (asthenospermia). Typically, a minimum of ≥25% of sperm demonstrates rapid progressive motility, or alternatively, ≥50% collectively showcase rapid progressive and slow progressive motility.

If the percentage of motile spermatozoa is less than 50%, it is essential to assess the proportion of viable sperm by examining an eosin preparation.

Sperm Viability or Vitality

Principle

A cell with an intact cell membrane, considered vital or viable, will remain unstained as it does not take up eosin Y. Conversely, a non-viable or dead cell, with a compromised cell membrane, will absorb the dye, resulting in a pink-red stain (refer to Figure 1). An additional stain, such as nigrosin, may be applied to color the background material. This test is conducted when motility is found to be abnormal.

Method

1. Mix one drop of semen with one drop of eosin-nigrosin solution and allow it to incubate for 30 seconds.
2. Make a smear from the drop deposited on a glass slide.
3. Air-dry the smear and inspect it under an oil-immersion objective. Sperms appearing white are categorized as live or viable, whereas red sperms are classified as dead or non-viable. A minimum of 200 spermatozoa are scrutinized.
4. Express the findings as a ratio of viable sperms to non-viable sperms, presented as an integer percentage.

Typically, seventy-five percent or more of spermatozoa are considered to be alive or viable under normal circumstances.

Sperm Count

Principle

The sperm count is conducted post-liquefaction using a counting chamber following appropriate dilution, and the total number of spermatozoa is documented in millions per milliliter (10^6/mL).

Method

1. Dilute semen at a ratio of 1:20 with sodium bicarbonate-formalin diluting fluid. To achieve this, take 1 ml of liquefied semen in a graduated tube and fill it with diluting fluid up to the 20 ml mark. Ensure thorough mixing.
2. Place a coverslip over the improved Neubauer counting chamber, filling the chamber with the well-mixed diluted semen sample using a Pasteur pipette. Subsequently, position the chamber in a humid box for 10-15 minutes to allow spermatozoa to settle.
3. Position the chamber on the microscope stage. Utilizing the 20× or 40× objective and lowering the iris diaphragm adequately for optimal contrast, count the number of spermatozoa in four large corner squares. Consider spermatozoa whose heads touch the left and upper lines of the square as 'belonging' to that square.
4. Calculate sperm count per milliliter using the formula:

   

   • Sperm count = Sperms counted × correction factor × 1000 ÷ Number of squares counted × Volume of 1 square
     Sperm count = Sperms counted × 20 × 1000 ÷ 4 × 0.1
     Sperm count = Sperms counted × 50, 000
5. A normal sperm count is equal to or greater than 20 million per milliliter (i.e., ≥ 20 × 10^6/ml). Sperm counts below 20 million per milliliter may be indicative of male infertility.

Sperm Morphology

To create a smear, a glass slide is used to evenly spread a droplet of seminal fluid, followed by staining. The subsequent step involves counting the percentages of normal and abnormal forms of spermatozoa. Staining techniques employed include Papanicolaou, eosin-nigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. It is essential to count a minimum of 200 spermatozoa under oil immersion, with the recorded results encompassing the respective percentages of normal and abnormal spermatozoa.

Normal Morphology

A spermatozoon comprises three primary components: the head, neck, and tail. The tail is intricately subdivided into the midpiece, main (principle) piece, and end piece (refer to Figure 2 and Box 1 for visual representation).

The head of the spermatozoon exhibits a pear-shaped structure. Predominantly, the nucleus occupies most of the head, characterized by condensed chromatin with scattered areas known as nuclear vacuoles. The anterior two-thirds of the nucleus is enveloped by the acrosomal cap, a flattened, membrane-bound vesicle containing glycoproteins and enzymes. These enzymes play a crucial role in the separation of corona radiata cells and the dissolution of the ovum's zona pellucida during the process of fertilization.

The neck serves as a brief segment connecting the head and the tail of the spermatozoon. Within the neck, the centriole gives rise to the axoneme of the flagellum. The axoneme is comprised of 20 microtubules arranged in a specific pattern, with a central pair surrounded by 9 peripheral doublets, and it is enclosed by condensed fibrous rings.

The middle piece, which constitutes the initial portion of the tail, features a central axoneme surrounded by robust longitudinal fibers. These fibers, in turn, are enveloped by elongated mitochondria that play a vital role in supplying energy for the movement of the tail.

The primary or main piece forms the majority of the tail and is comprised of an axoneme surrounded by nine robust fibers. This central core is further encased by numerous circularly arranged fibrous ribs.

The endpiece, a short and tapering section, is exclusively composed of the axoneme.

Typically, more than 30% of spermatozoa should exhibit normal morphology (according to WHO, 1999). Morphological abnormalities associated with male infertility encompass defects such as a faulty mid-piece leading to reduced motility, an incomplete or absent acrosome resulting in an inability to penetrate the ovum, and a giant head indicating defective DNA condensation.

Abnormal Morphology

The World Health Organization's morphological classification of human spermatozoa, as of 1999, is outlined below:

1. Normal sperm
2. Defects in head:
   • Large heads
   • Small heads
   • Tapered heads
   • Pyriform heads
   • Round heads
   • Amorphous heads
   • Vacuolated heads (> 20% of the head area occupied by vacuoles)
   • Small acrosomes (occupying < 40% of head area)
   • Double heads
3. Defects in neck: Bent neck and tail forming an angle >90° to the long axis of head
4. Defects in middle piece:
   • Asymmetric insertion of midpiece into head
   • Thick or irregular midpiece
   • Abnormally thin midpiece
5. Defects in tail:
   • Bent tails
   • Short tails
   • Coiled tails
   • Irregular tails
   • Multiple tails
   • Tails with irregular width
6. Pin heads: Not to be counted
7. Cytoplasmic droplets: > 1/3rd the size of the sperm head
8. Precursor cells: Considered abnormal

Round Cells

Upon microscopic examination, round cells may be identified as either white blood cells or immature sperm cells. To distinguish between the two, a special stain, such as peroxidase or Papanicolaou, is necessary. An elevated count of white blood cells exceeding 1 million/ml suggests the presence of an infection. Similarly, the detection of a substantial number of immature sperm cells indicates dysfunction in spermatogenesis at the testicular level.

Semen analysis involves the assessment of biochemical markers, as outlined in Table 1, to examine the secretions from various accessory structures. These markers encompass fructose, associated with seminal vesicles; zinc, citric acid, or acid phosphatase, linked to the prostate; and α-glucosidase or carnitine, indicative of epididymal contributions. This comprehensive approach allows for a nuanced evaluation of the diverse components contributing to semen composition, providing valuable insights into the functionality of the reproductive system.

Test for Fructose

The Resorcinol method serves as a means for detecting fructose within biological samples. In this analytical procedure, 5 ml of the resorcinol reagent is employed. This reagent is prepared by dissolving 50 mg of resorcinol in 33 ml of concentrated hydrochloric acid, followed by dilution up to 100 ml with distilled water. The resulting solution is then added to 0.5 ml of seminal fluid. Subsequently, the mixture is subjected to heat and brought to a boil. The emergence of a red-colored precipitate within a brief span of 30 seconds signifies the presence of fructose.

The absence of fructose in the test results points towards potential obstructions proximal to the seminal vesicles, which may manifest as either obstructed or absent vas deferens, or an absence of seminal vesicles altogether. In instances of azoospermia, the absence of fructose may be indicative of the obstruction of ejaculatory ducts or the absence of vas deferens. Conversely, the presence of fructose in cases of azoospermia suggests a failure of the testes to produce sperm. This nuanced interpretation allows for a more comprehensive understanding of the underlying factors contributing to the observed test results.

Examination is conducted subsequent to the liquefaction of semen, typically occurring within 20-30 minutes of ejaculation.

Visual Appearance

Normal semen exhibits a viscous and opaque gray-white appearance. Following extended periods of abstinence, it may display a slight yellow tint.

Viscosity

Immediately after ejaculation, normal semen is characterized by thickness and viscosity. Liquefaction, facilitated by proteolytic enzymes secreted by the prostate, occurs within 30 minutes. Failure of liquefaction within 60 minutes is considered abnormal. The sample's viscosity is evaluated by filling a pipette with semen and observing its flow back into the container. In normal semen, it drips drop by drop. If droplets form ‘threads’ longer than 2 cm, viscosity is heightened. Increased semen viscosity adversely impacts sperm motility, leading to poor invasion of cervical mucus. This condition often results from infections of the seminal vesicles or prostate.

Volume

The volume of ejaculated semen should typically exceed 2 ml and is measured post-liquefaction. A volume less than 2.0 ml is abnormal and is associated with a low sperm count.

pH

A drop of liquefied semen is applied to pH paper with a range of 6.4-8.0, and the pH is recorded after 30 seconds. Normal pH ranges from 7.2 to 8.0 one hour post-ejaculation. The portion of semen contributed by seminal vesicles is basic, while the portion from the prostate is acidic. A low pH (< 7.0) in conjunction with the absence of sperm (azoospermia) suggests an obstruction of ejaculatory ducts or the absence of the vas deferens. Low pH is typically linked to low semen volume, as most of the volume is supplied by seminal vesicles.

The presence of gross hematuria imparts a pink, red, or brown hue to the urine. While the alteration in color may be disconcerting, it's noteworthy that even a minute quantity of blood in the urine can bring about this perceptible change. In most instances, gross hematuria does not elicit pain or other associated symptoms. However, the occurrence of blood clots in the urine may lead to discomfort. Passing blood clots during urination can be a painful experience, and if these clots obstruct the urinary flow, they may result in bladder or back pain.

Conversely, microscopic hematuria, despite not influencing the visible coloration of urine, generally transpires without noticeable symptoms.

Microscopic Examination of Urinary Sediment

Microscopic hematuria is defined as the identification of 3 or more red blood cells per high-power field upon microscopic analysis of urinary sediment in two out of three appropriately collected samples. It is crucial to note that a limited number of red blood cells in low specific gravity urine might undergo lysis, leading to the potential oversight of hematuria if solely relying on microscopic examination. Hence, it is recommended to complement microscopic urine examination with a chemical test for a comprehensive evaluation.

Chemical Tests for the Detection of Blood in Urine

These assays identify both intracellular and extracellular hemoglobin, encompassing intact and lysed red blood cells, as well as myoglobin. Heme proteins within hemoglobin serve as peroxidases, facilitating the reduction of hydrogen peroxide to water. This enzymatic process requires a hydrogen donor, such as benzidine, orthotoluidine, or guaiac. The oxidation of these hydrogen donors initiates the development of color (refer to Figure 1). Importantly, the intensity of the resultant color is directly proportional to the quantity of hemoglobin present.

Positive results from chemical tests are indicative of conditions such as hematuria, hemoglobinuria, and myoglobinuria. These tests play a crucial role in detecting and distinguishing these pathologic states.

Benzidine Test

Prepare a saturated solution of benzidine in glacial acetic acid. Combine 1 ml of this prepared solution with an equal volume of hydrogen peroxide in a test tube. Introduce 2 ml of urine into the mixture. A positive result is indicated by the development of a green or blue color within 5 minutes.

Orthotoluidine Test

In this assay, orthotoluidine is employed in lieu of benzidine, imparting a heightened sensitivity compared to the benzidine test. This modification enhances the precision and accuracy of the test, making it a more discerning method for the detection of specific reactions.

Reagent Strip Test

Several commercially available reagent strips utilize diverse chromogens, including o-toluidine and tetramethylbenzidine. These strips serve as valuable tools in diagnostic processes, each employing distinct chemical compounds to facilitate precise and reliable results.

Causes of false-positive tests:

• Urine contamination with menstrual blood in females
• Urine contamination due to the presence of oxidizing agents (such as hypochlorite or bleach used for cleaning urine containers) or microbial peroxidase in the context of urinary tract infections.

Causes of false-negative tests:

• Elevated concentrations of a reducing agent, such as ascorbic acid: Microscopic examination reveals the presence of red cells, but the chemical test yields a negative result.
• Utilization of formalin as a urine preservative

Refer to Figure 2 for the illustration of the assessment of a positive chemical test for blood.

Urine serves as a diagnostic medium for detecting both physical and biochemical irregularities. This analysis aids in screening and diagnosing conditions such as urinary tract infections, kidney disorders, liver problems, diabetes, and various metabolic conditions. Prior to examination, the specimen's acceptability is assessed.

The chemical examination encompasses the analysis of the following substances in urine:

• Proteins
• Glucose
• Ketones
• Bilirubin
• Bile salts
• Urobilinogen
• Blood
• Hemoglobin
• Myoglobin
• Nitrite or leukocyte esterase

Proteins in Urine

The kidneys typically eliminate a minimal amount of protein in the urine, not exceeding 150 mg in a 24-hour period. These proteins encompass those originating from plasma, such as albumin, as well as proteins derived from the urinary tract, including Tamm-Horsfall protein, secretory IgA, and proteins originating from tubular epithelial cells, leucocytes, and other desquamated cells. Importantly, this level of proteinuria falls below the detection threshold of routine tests.

It's noteworthy that Tamm-Horsfall protein is a normal mucoprotein secreted by the ascending limb of the loop of Henle.

In adults, the term "proteinuria" denotes the excretion of protein in the urine exceeding 150 mg in a 24-hour period.

Causes of Proteinuria

The etiologies of proteinuria can be categorized, as illustrated in Box 1.

1. Glomerular Proteinuria: Proteinuria arising from an augmented permeability of the glomerular capillary wall is termed glomerular proteinuria. Within this category, two distinct types exist: selective and nonselective. In the initial stages of glomerular disease, there is an elevated excretion of lower molecular weight proteins such as albumin and transferrin. Selective proteinuria occurs when glomeruli can retain larger molecular weight proteins but allow passage of relatively lower molecular weight proteins. As glomerular damage progresses, selectivity is lost, resulting in the excretion of larger molecular weight proteins, including γ globulins, alongside albumin—termed nonselective proteinuria. The differentiation between selective and nonselective proteinuria can be achieved through urine protein electrophoresis. In selective proteinuria, distinct bands of albumin and transferrin are observable, while in the nonselective type, the pattern mirrors that of serum (Figure 1). Glomerular proteinuria is instigated by diseases affecting the glomerular basement membrane's permeability. The extent of proteinuria corresponds to the severity of the disease and its prognosis. Monitoring the response to treatment is facilitated by serial estimations of urinary protein. The most severe manifestation of proteinuria is observed in nephrotic syndrome (Box 2).
2. Tubular Proteinuria: Under normal circumstances, the glomerular membrane, impermeable to high molecular weight proteins, allows the passage of low molecular weight proteins like β2-microglobulin, retinol-binding protein, lysozyme, α1-microglobulin, and free immunoglobulin light chains. These low molecular weight proteins are actively reabsorbed by proximal renal tubules. In diseases predominantly affecting the tubules, these proteins are excreted in urine while albumin excretion remains minimal. Urine electrophoresis reveals prominent α- and β-bands, representing the migration of low molecular weight proteins, and a faint albumin band (Figure 1). Tubular proteinuria is commonly observed in acute and chronic pyelonephritis, heavy metal poisoning, tuberculosis of the kidney, interstitial nephritis, cystinosis, Fanconi syndrome, and kidney transplant rejection. Purely tubular proteinuria is not detectable through reagent strip tests, sensitive to albumin. However, positive results are obtained with the heat and acetic acid test, as well as the sulphosalicylic acid test.
3. Overflow Proteinuria: Overflow proteinuria occurs when the concentration of a low molecular weight protein rises in plasma, leading to its "overflow" into the urine. Proteins involved in this type include immunoglobulin light chains or Bence Jones proteins (associated with plasma cell dyscrasias), hemoglobin (resulting from intravascular hemolysis), myoglobin (due to skeletal muscle trauma), and lysozyme (linked to acute myeloid leukemia type M4 or M5).
4. Hemodynamic Proteinuria: Changes in blood flow through the glomeruli cause increased protein filtration, although protein excretion is transient. This phenomenon is observed in conditions such as high fever, hypertension, heavy exercise, congestive cardiac failure, seizures, and exposure to cold. Postural (orthostatic) proteinuria occurs when the subject is standing or ambulatory, but is absent in the recumbent position. Common in adolescents (3-5%), it is likely due to a lordotic posture causing inferior vena cava compression between the liver and vertebral column. This condition usually disappears in adulthood, with proteinuria levels below 1000 mg/day. Periodic testing for proteinuria is recommended in such individuals to rule out renal disease.
5. Post-renal Proteinuria: This type is induced by inflammatory or neoplastic conditions in the renal pelvis, ureter, bladder, prostate, or urethra.

Further reading: Methods for the Detection of Protein in Urine.

Glucose

The primary purpose of conducting urine glucose testing is to identify undiagnosed diabetes mellitus or to monitor known diabetic patients during follow-up.

Virtually all glucose that undergoes filtration in the glomeruli is reabsorbed by the proximal renal tubules and subsequently returned to the circulation. Under normal circumstances, only a minute quantity of glucose is excreted in the urine (typically < 500 mg/24 hours or < 15 mg/dl), a level that remains undetectable through routine tests. The presence of discernible quantities of glucose in the urine is termed glucosuria or glycosuria (Box 3). Glycosuria occurs when the filtered glucose load surpasses the reabsorptive capacity of the renal tubules, with hyperglycemia resulting from diabetes mellitus constituting the most prevalent cause.

Causes of Glycosuria

Glycosuria with hyperglycemia

• Endocrine Diseases: Conditions encompassing diabetes mellitus, acromegaly, Cushing's syndrome, hyperthyroidism, and pancreatic disease fall within the realm of endocrine disorders.
• Non-Endocrine Diseases: Central nervous system diseases and liver disorders are among the non-endocrine conditions associated with relevant glycosuric manifestations.
• Drug-Induced Causes: The administration of adrenocorticotrophic hormone, corticosteroids, and thiazides represents drug-related factors contributing to glycosuria.
• Alimentary Glycosuria (Lag-Storage Glycosuria): Following a meal, the swift intestinal absorption of glucose leads to a transient elevation of blood glucose levels beyond the renal threshold. This phenomenon may manifest in individuals with gastrectomy or gastrojejunostomy, as well as those experiencing hyperthyroidism. A glucose tolerance test reveals a peak at 1 hour exceeding the renal threshold, resulting in glycosuria, while fasting and 2-hour glucose values remain within normal limits.

Glycosuria without hyperglycemia

• Renal Glycosuria: Constituting approximately 5% of glycosuria cases in the general population, renal glycosuria is characterized by a renal threshold—the highest blood glucose level at which glucose becomes detectable in urine through routine laboratory tests. The typical renal threshold for glucose is 180 mg/dl. Substances reaching the threshold necessitate a carrier for transport from the tubular lumen to the blood. Once the carrier becomes saturated, the threshold is attained, leading to excretion of the substance. Up to this point, glucose filtered by the glomeruli undergoes efficient reabsorption by the tubules. Renal glycosuria represents a benign condition where the renal threshold is set below 180 mg/dl, yet glucose tolerance remains normal. This disorder is inherited as an autosomal dominant trait. Other instances of glycosuria with blood glucose levels below 180 mg/dl occur in renal tubular diseases, such as Fanconi's syndrome, characterized by diminished glucose reabsorption, and in cases of toxic renal tubular damage. Pregnancy induces a reduction in the renal threshold for glucose, underscoring the importance of blood glucose estimation when the initial detection of glucose in urine occurs.

Further reading: Methods for the Detection of Glucose in Urine.

Ketones

The elimination of ketone bodies (specifically, acetoacetic acid, β-hydroxybutyric acid, and acetone) through urine is termed ketonuria. Ketones, derived from the breakdown of fatty acids, appearing in urine signals an elevated level of fatty acid metabolism as a source of energy.

Causes of Ketonuria

Typically, ketone bodies are undetectable in the urine of individuals in good health. When the metabolism of glucose is compromised due to issues such as defective carbohydrate metabolism, insufficient carbohydrate intake, or heightened metabolic demands, the body turns to the breakdown of fats for energy. This metabolic shift results in the production of ketone bodies, as illustrated in Figure 2.

1. Reduced Carbohydrate Utilization:
   1. Uncontrolled Diabetes Mellitus with Ketoacidosis: In the context of diabetes, inadequate glucose utilization triggers compensatory heightened lipolysis. This process elevates the levels of free fatty acids in the plasma. The liver's degradation of these free fatty acids results in the formation of acetoacetyl CoA, subsequently giving rise to ketone bodies. These ketone bodies, potent acids, generate H⁺ ions, neutralized by bicarbonate ions. A decrease in bicarbonate levels (alkali) leads to ketoacidosis. Ketone bodies also augment plasma osmolality, inducing cellular dehydration. Individuals, particularly children and young adults with type 1 diabetes, are predisposed to ketoacidosis during acute illnesses and periods of stress. Presence of glycosuria necessitates ketone body testing. Concurrent presence of glucose and ketone bodies in urine signifies diabetes mellitus with ketoacidosis. In certain diabetes cases, blood ketone levels may rise without manifesting in urine. Detection of ketone bodies in urine can serve as a warning sign of an impending ketoacidotic coma.
   2. Glycogen Storage Disease (von Gierke’s Disease)
2. Insufficient Carbohydrate Availability in the Diet:
   1. Starvation
   2. Persistent Vomiting in Children
   3. Weight Reduction Program (Severe Carbohydrate Restriction with Normal Fat Intake)
3. Elevated Metabolic Demands:
   1. Fever in Children
   2. Severe Thyrotoxicosis
   3. Pregnancy
   4. Protein-Calorie Malnutrition

Further reading: Methods for the Detection of Ketones in Urine.

Bile Pigment (Bilirubin)

Bilirubin, a byproduct of hemoglobin breakdown, is typically absent in the urine of healthy individuals. The presence of bilirubin in urine is termed bilirubinuria.

Two distinct forms of bilirubin exist: conjugated and unconjugated. Following its generation from hemoglobin within the reticuloendothelial system, bilirubin circulates in the bloodstream, bound to albumin—referred to as unconjugated bilirubin. Being insoluble in water and bound to albumin, unconjugated bilirubin cannot traverse the glomeruli, and as a result, it does not manifest in the urine.

The liver plays a crucial role in processing unconjugated bilirubin. Here, it combines with glucuronic acid, forming bilirubin diglucuronide, which is categorized as conjugated bilirubin. Unlike its unconjugated counterpart, conjugated bilirubin is water-soluble, undergoes filtration by the glomeruli, and consequently, is excreted in the urine.

The identification of bilirubin in urine, coupled with the presence of urobilinogen, proves valuable in distinguishing various causes of jaundice (refer to Table 1).

During acute viral hepatitis, bilirubin manifests in urine even prior to the clinical onset of jaundice. In cases of an unexplained fever, the presence of bilirubinuria suggests a potential hepatitis etiology.

The detection of bilirubin in urine signifies the presence of conjugated hyperbilirubinemia, indicative of obstructive or hepatocellular jaundice. This is attributable to the water-solubility of conjugated bilirubin. Conversely, bilirubin does not appear in urine in hemolytic jaundice due to the water-insolubility of unconjugated bilirubin.

Further reading: Methods for the Detection of Bilirubin in Urine.

Bile Salts

Bile salts comprise salts derived from four distinct types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with either glycine or taurine, forming intricate salts or acids. Transported through the bile, bile salts enter the small intestine, serving as detergents that emulsify fat and reduce surface tension on fat droplets. This action facilitates the enzymatic breakdown of fat by lipases. Following absorption in the terminal ileum, bile salts enter the bloodstream, undergo hepatic uptake, and are subsequently re-excreted in bile, constituting the enterohepatic circulation.

Further reading: Methods for the Detection of Bile Salts in Urine.

Urobilinogen

Conjugated bilirubin, excreted into the duodenum via bile, undergoes bacterial conversion to urobilinogen within the intestine. The majority of this urobilinogen is expelled through feces. A fraction is absorbed into the bloodstream, initiating recycling through the enterohepatic circulation, while a minor quantity, not reabsorbed by the liver, is excreted in urine. Initially colorless, urobilinogen transforms into urobilin upon oxidation, displaying an orange-yellow hue. Typically, 0.5-4 mg of urobilinogen is expelled in urine over 24 hours, resulting in the normal, detectable presence of a small urobilinogen quantity.

The diurnal variation of urobilinogen urinary excretion peaks in the afternoon, emphasizing the preference for a 2-hour post-meal sample for accurate assessment.

Causes of Increased Urobilinogen in Urine

1. Hemolysis: The excessive breakdown of red blood cells results in hyperbilirubinemia, leading to an elevated production of urobilinogen in the gastrointestinal tract. Bilirubin, predominantly unconjugated in this scenario, remains absent in urine. Elevated urobilinogen in the absence of bilirubin is characteristic of hemolytic anemia. This phenomenon is also observed in megaloblastic anemia, attributed to the premature destruction of erythroid precursors within the bone marrow—a manifestation of ineffective erythropoiesis.
2. Hemorrhage in tissues: The increased bilirubin formation results from the breakdown of red blood cells during tissue hemorrhage.

Causes of Reduced Urobilinogen in Urine

1. Obstructive jaundice: When there's an obstruction in the biliary tract, the transport of bilirubin to the intestine is hindered, resulting in minimal or no urobilinogen formation. Consequently, this leads to pale or clay-colored stools.
2. Reduction of intestinal bacterial flora: The decrease in the population of intestinal bacterial flora hampers the conversion of bilirubin to urobilinogen within the intestine. This phenomenon is particularly observed in neonates and following antibiotic treatment.

The analysis of urine for both bilirubin and urobilinogen proves to be valuable in assessing a case of jaundice (refer to Table 1 for details).

Further reading: Methods for the Detection of Urobilinogen in Urine.

Blood

The identification of an abnormal quantity of intact red blood cells in urine is termed hematuria. This indicates the existence of a bleeding lesion within the urinary tract. Observable bleeding in urine, either apparent to the naked eye or through macroscopic examination, is referred to as gross hematuria. In cases where bleeding is detectable only through microscopic analysis or chemical tests, it is designated as occult, microscopic, or hidden hematuria.

Causes of Hematuria

1. Diseases of urinary tract:

• Glomerular diseases: Conditions within this category include glomerulonephritis, Berger’s disease, lupus nephritis, and Henoch-Schonlein purpura.
• Nonglomerular diseases: This encompasses a range of conditions such as calculus, tumor, infection, tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney disease, trauma, occurrences after strenuous physical exercise, and diseases of the prostate (benign hyperplasia of the prostate, carcinoma of the prostate).

2. Hematological conditions:

In the context of coagulation disorders and sickle cell disease, the presence of red cell casts, along with proteinuria and hematuria, indicates a glomerular origin of the hematuria.

Further reading: Methods for the Detection of Blood in Urine.

Hemogobin

The condition characterized by the presence of free hemoglobin in the urine is referred to as hemoglobinuria.

Causes of Hemoglobinuria

1. Hematuria accompanied by subsequent lysis of red blood cells in urine of low specific gravity.
2. Intravascular hemolysis: Hemoglobin becomes evident in the urine when haptoglobin, the plasma protein binding hemoglobin, is fully saturated with hemoglobin. Intravascular hemolysis manifests in various conditions, including severe falciparum malaria, clostridial infections, E. coli septicemia, trauma to red cells (such as march hemoglobinuria, extensive burns, prosthetic heart valves), glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant drugs, immune hemolysis (resulting from mismatched blood transfusion, paroxysmal cold hemoglobinuria), paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and disseminated intravascular coagulation.

Tests for Detection of Hemoglobinuria

Methods employed to identify hemoglobinuria include the benzidine test, ortho-toluidine test, and reagent strip test.

Hemosiderin

The occurrence of hemosiderin in urine, known as hemosiderinuria, signifies the presence of free hemoglobin in the plasma. Visualization of hemosiderin is achieved through staining urine sediment with Prussian blue stain, revealing blue granules (refer to Figure 3). These granules are situated within tubular epithelial cells or may be present independently if cellular disintegration has occurred. Hemosiderinuria is a characteristic finding in cases of intravascular hemolysis.

Myoglobin

Myoglobin, a protein found in striated muscles (both skeletal and cardiac), serves the function of oxygen binding. Myoglobinuria, the presence of myoglobin in urine, is associated with conditions causing injury to skeletal or cardiac muscles, such as crush injuries, myocardial infarction, dermatomyositis, severe electric shock, and thermal burns.

Chemical tests designed for the detection of blood or hemoglobin also yield a positive reaction with myoglobin, given that both hemoglobin and myoglobin exhibit peroxidase activity. The ammonium sulfate solubility test is employed as a preliminary screening test for myoglobinuria. Notably, myoglobin is soluble in an 80% saturated solution of ammonium sulfate, while hemoglobin remains insoluble and precipitates. A positive chemical test for blood conducted on the supernatant indicates the presence of myoglobinuria.

A comprehensive differentiation between hematuria, hemoglobinuria, and myoglobinuria is detailed in Table 2.

Chemical Tests for Significant Bacteriuria (Indirect Tests for Urinary Tract Infection)

In addition to the direct microscopic examination of urine samples, there are commercially available chemical tests in a reagent strip form designed to detect significant bacteriuria. These tests, namely the nitrite test and leucocyte esterase test, prove valuable in settings where urine microscopy is unavailable. A positive result on these tests warrants further investigation through urine culture.

Nitrite Test: Normal urine does not contain nitrites; ingested nitrites are converted to nitrate and excreted. In the presence of gram-negative bacteria (such as E. coli, Salmonella, Proteus, Klebsiella, etc.), these bacteria, through the action of the bacterial enzyme nitrate reductase, reduce nitrates to nitrites. Reagent strip tests then detect the presence of nitrites in urine. Given that E. coli is the predominant organism causing urinary tract infections, the nitrite test serves as a useful screening tool for such infections.

Certain organisms like Staphylococci or Pseudomonas do not convert nitrate to nitrite, resulting in a negative nitrite test in these infections. It's crucial to retain urine in the bladder for a minimum of 4 hours for the conversion of nitrate to nitrite to occur; hence, a fresh early morning specimen is preferred. Adequate dietary intake of nitrate is necessary. Therefore, a negative nitrite test does not conclusively indicate the absence of a urinary tract infection, as the test detects approximately 70% of cases.

Leucocyte Esterase Test: This test identifies the esterase enzyme released in urine from the granules of leucocytes, indicating pyuria. A positive result on this test suggests the need for urine culture. The test is not sensitive to leucocytes fewer than 5 per high-power field.

The microscopic analysis of urine is often referred to as the “liquid biopsy of the urinary tract.”

Urine comprises diverse microscopic, insoluble, solid components in suspension. These components fall into two categories: organized and unorganized. Organized substances encompass red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. On the other hand, unorganized substances consist of crystalline and amorphous material. These elements remain suspended in urine, settling down and forming sediment at the container's bottom over time. Consequently, they are termed urinary deposits or urinary sediments. The examination of these deposits proves valuable in diagnosing urinary tract diseases, as outlined in Table 1.

Various categories of urinary sediments are illustrated in Figure 1. The primary objective of the microscopic analysis of urine is to discern distinct types of cellular elements and casts. It's noteworthy that the majority of crystals observed have minimal clinical significance.

Specimen

Cellular elements exhibit optimal preservation in acid, hypertonic urine, while they degrade swiftly in an alkaline, hypotonic solution. A mid-stream, freshly voided, first-morning specimen is the preferred choice due to its heightened concentration. It is crucial to examine the specimen within 2 hours of voiding, as cells and casts undergo degeneration when left standing at room temperature. If a preservative is necessary, the addition of 1 crystal of thymol or 1 drop of formalin (40%) to approximately 10 ml of urine is recommended.

Method

A thoroughly mixed urine sample (12 ml) undergoes centrifugation in a centrifuge tube for 5 minutes at 1500 rpm, and the supernatant is carefully poured off. To resuspend the sediment, the tube is tapped at the bottom, utilizing 0.5 ml of urine. A small droplet of this resuspended sediment is placed on a glass slide and covered with a cover slip, as depicted in Figure 2. Subsequently, the slide is promptly examined under the microscope, initially using the low-power objective and then transitioning to the high-power objective. For enhanced visualization of the elements, it is advisable to lower the condenser, thereby reducing the illumination.

Cells

Figure 3 illustrates the cellular elements present in urine.

Red Blood Cells

Normally, urine contains either no red blood cells or only an occasional one. In a fresh urine sample, red cells manifest as small, smooth, yellowish, anucleate biconcave disks measuring approximately 7 μ in diameter, commonly referred to as isomorphic red cells. However, in dilute or hypotonic urine, these red cells may appear swollen, taking on the form of thin discs with a larger diameter (9-10 μ). Conversely, in hypertonic urine, they may exhibit a crenated appearance, characterized by a smaller diameter and a spiky surface.

In cases of glomerulonephritis, red cells are often described as dysmorphic, indicating a marked variability in size and shape. This distortion arises from the passage of red cells through damaged glomeruli. The presence of over 80% dysmorphic red cells strongly suggests glomerular pathology.

The quantification of red cells can be reported as the number of red cells per high-power field. The causes of hematuria have been previously outlined.

White Blood Cells (Pus Cells)

White blood cells typically exhibit a spherical form, measuring 10-15 μ in size, and display a granular appearance where nuclei may be discernible. Degenerated white cells, on the other hand, appear distorted, smaller, and possess fewer granules. In instances of infection, clumps of numerous white cells become evident. The presence of a substantial number of white cells in urine is termed pyuria. In hypotonic urine, white cells swell, and their granules become highly refractile, exhibiting Brownian movement. These cells, referred to as glitter cells, indicate injury to the urinary tract, particularly when present in large numbers.

Under normal conditions, 0-2 white cells may be observed per high-power field. However, the detection of pus cells exceeding 10 per high-power field or the presence of clumps suggests a potential urinary tract infection.

Elevated white cell counts are associated with conditions such as fever, pyelonephritis, lower urinary tract infection, tubulointerstitial nephritis, and renal transplant rejection.

In cases of urinary tract infection, a combination of the following is typically observed:

• Clumps of pus cells or pus cells exceeding 10 per high-power field
• Presence of bacteria
• Albuminuria
• Positive nitrite test

The simultaneous presence of white cells and white cell casts is indicative of renal infection, specifically pyelonephritis.

Eosinophils, constituting more than 1% of urinary leucocytes, serve as a characteristic feature of acute interstitial nephritis due to a drug reaction, a detail better appreciated with Wright's stain.

Renal Tubular Epithelial Cells

The identification of renal tubular epithelial cells holds significant diagnostic value. Elevated counts of these cells are indicative of conditions causing tubular damage, such as acute tubular necrosis, pyelonephritis, viral kidney infections, allograft rejection, and poisoning from substances like salicylate or heavy metals.

These cells exhibit characteristics of being small, approximately the same size or slightly larger than white blood cells. They can take on a polyhedral, columnar, or oval shape, featuring granular cytoplasm. Often, a single, large, refractile, eccentric nucleus is observable.

Distinguishing renal tubular epithelial cells from pus cells in unstained preparations can pose a challenge due to their similarity in appearance. This makes the identification process in such conditions a meticulous task requiring close examination.

Squamous Epithelial Cells

Squamous epithelial cells form the lining of the lower urethra and vagina. Optimal visibility is achieved under a low-power objective (×10). When a substantial quantity of squamous cells is detected in urine, it suggests contamination of the urine with vaginal fluid. These cells are characterized by their size, being large and rectangular, with a flat morphology, abundant cytoplasm, and a small, central nucleus.

Transitional Epithelial Cells

Transitional cells form the lining of the renal pelvis, ureters, urinary bladder, and upper urethra. These cells, often referred to as caudate cells due to their diamond- or pear-shaped appearance, are notably large. The presence of abundant transitional cells or the observation of cell sheets in urine is commonly associated with scenarios such as catheterization procedures and cases of transitional cell carcinoma.

Oval Fat Bodies

These represent degenerated renal tubular epithelial cells laden with highly refractile lipid, primarily composed of cholesterol droplets. When examined under polarized light, these cells exhibit a distinctive “Maltese cross” pattern. To facilitate identification, they can be stained using a fat stain like Sudan III or Oil Red O. This phenomenon is commonly observed in nephrotic syndrome, a condition characterized by the presence of lipiduria.

Spermatozoa

Occasionally, these elements may be observed in the urine of males.

Telescoped Urinary Sediment: This term denotes a urinary sediment composition comprising red blood cells, white blood cells, oval fat bodies, and various types of casts in approximately equal proportions. This phenomenon is associated with conditions such as lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis.

Organisms

The organisms identifiable in urine are depicted in Figure 4.

Bacteria

Detection of bacteria in urine can be accomplished through microscopic examination, reagent strip tests assessing for significant bacteriuria (utilizing the nitrite test and leucocyte esterase test), as well as culture.

Significant bacteriuria is confirmed when the bacterial colony-forming units/ml of urine surpass specific thresholds:

• In a clean-catch midstream sample, it is present when there are more than 10^5 bacterial colony-forming units per milliliter (cfu/ml) of urine.
• In a catheterized sample, the threshold is set at over 10^4 cfu/ml of urine.
• For a suprapubic aspiration sample, significant bacteriuria is considered when there are more than 10^3 cfu/ml of urine.

Tests for the Detection of Bacteria in Urine

1. Microscopic Examination: In a wet preparation, the documentation of bacteria presence is pertinent only when the urine is freshly collected. Bacteria are typically observed in conjunction with pus cells. A Gram's-stained smear of uncentrifuged urine revealing one or more bacteria per oil-immersion field suggests the likelihood of over 10^5 bacterial colony-forming units per milliliter (cfu/ml) of urine. If numerous squamous cells are present, there's a probability of urine contamination with vaginal flora. Additionally, the sole presence of bacteria without pus cells indicates potential contamination with vaginal or skin flora.
2. Chemical or Reagent Strip Tests for Significant Bacteriuria: These tests utilize strips saturated with specialized chemical reagents that interact with components linked to bacterial existence in urine. These strips commonly evaluate the presence of nitrites, signaling bacterial metabolism, and leukocyte esterase, an enzyme released by white blood cells in response to infection.
3. Culture: Upon culturing, a colony count exceeding 10^5/ml strongly indicates a urinary tract infection, even in asymptomatic females. A positive culture is subsequently followed by sensitivity testing. The majority of infections are attributed to Gram-negative enteric bacteria, notably Escherichia coli.

Identification of three or more bacterial species in a culture typically suggests contamination by vaginal flora.

A lack of bacterial growth in the presence of pyuria, often referred to as ‘sterile’ pyuria, is associated with various conditions. These include prior antibiotic therapy, renal tuberculosis, prostatitis, renal calculi, catheterization, fever in children (regardless of the underlying cause), female genital tract infection, and non-specific urethritis in males.

Yeast Cells (Candida)

These structures exhibit a round or oval morphology, closely mirroring the size of red blood cells. Unlike red cells, they display a distinctive budding pattern, possess an oval and highly refractile appearance, and are insoluble in 2% acetic acid.

The detection of Candida in urine may indicate an immunocompromised condition, the presence of vaginal candidiasis, or diabetes mellitus. Typically, the presence of pyuria accompanies Candida infection. However, it's crucial to note that Candida could also be a potential contaminant in the sample. Therefore, the urine sample must be examined in a fresh state to ensure accurate interpretation.

Trichomonas vaginalis

These microorganisms exhibit motility and possess a distinctive pear shape, featuring an undulating membrane on one side and four flagellae. Their presence is associated with vaginitis in females, making them contaminants in urine samples. Notably, their motility renders them easily detectable in freshly collected urine.

Eggs of Schistosoma haematobium

The presence of Schistosoma haematobium eggs is notably widespread in Egypt. This parasitic organism, responsible for causing schistosomiasis, particularly affects the urinary tract. The eggs of Schistosoma haematobium are commonly found in human urine, indicating an established infection.

Schistosomiasis, also known as bilharzia, is a waterborne disease prevalent in regions with inadequate sanitation and limited access to clean water. In Egypt, where conditions conducive to the transmission of this parasitic infection exist, the prevalence underscores the public health challenges associated with waterborne diseases in the region.

Effective control measures, including improved water sanitation and targeted medical interventions, are crucial to mitigating the impact of Schistosoma haematobium infections in affected populations.

Microfilariae

Microfilariae, minute larvae of filarial parasites, may be observed in urine, particularly in cases of chyluria. Chyluria arises from the rupture of urogenital lymphatic vessels, allowing the infiltration of chyle, a milky fluid rich in lymphocytes and lipids, into the urinary system.

The presence of microfilariae in urine samples is a diagnostic marker for certain filarial infections, with different species exhibiting distinct characteristics. Common culprits include Wuchereria bancrofti and Brugia malayi, both known to cause lymphatic filariasis in affected individuals.

Clinical assessment and laboratory analysis of urine containing microfilariae play a crucial role in confirming the diagnosis and guiding appropriate treatment strategies. Management often involves antiparasitic medications and supportive care to address the underlying lymphatic pathology.

Casts

Urinary casts represent cylindrical, cigar-shaped microscopic structures originating within the distal renal tubules and collecting ducts. These formations mirror the shape and diameter of the lumina, essentially serving as molds or ‘casts’ of the renal tubules. Exhibiting parallel sides and rounded ends, the dimensions of these casts can vary. Composed primarily of a precipitate of Tamm-Horsfall protein, a protein secreted by tubules, their formation is confined to the renal tubules. Consequently, the presence of casts serves as an indicative marker of renal parenchymal disease.

While various types of casts exist, all urine casts fundamentally possess a hyaline composition. The diverse types emerge as different elements deposit onto the hyaline material, as illustrated in Figure 5. Optimal visualization of casts is achieved under a low-power objective (×10), with the condenser lowered to diminish illumination.

Casts constitute the exclusive components within the urinary sediment that distinctly originate from the renal system.

There are two primary categories of casts (refer to Figure 6):

1. Noncellular: This includes hyaline, granular, waxy, and fatty casts.
2. Cellular: Encompassing red blood cell, white blood cell, and renal tubular epithelial cell casts.

Hyaline and granular casts may manifest in both normal and pathological conditions. In contrast, casts featuring red blood cells, white blood cells, or renal tubular epithelial cells are indicative of kidney diseases.

Non-cellular Casts

Hyaline Casts: This type of cast is the most prevalent in urine, characterized by a homogenous, colorless, transparent, and refractile appearance. Hyaline casts present as cylindrical structures with parallel sides, blunt rounded ends, and a low refractive index. Occasional hyaline casts are considered normal, but an increased presence, known as “cylinduria”, is indicative of abnormal conditions. Composed primarily of Tamm-Horsfall protein, they may transiently appear after strenuous exercise or during fever. Elevated numbers are associated with conditions causing glomerular proteinuria.

Granular Casts: The presence of degenerated cellular debris imparts a granular appearance to these cylindrical structures. Featuring coarse or fine granules representing degenerated renal tubular epithelial cells embedded in a Tamm-Horsfall protein matrix, granular casts are observed after strenuous exercise, in cases of fever, acute glomerulonephritis, and pyelonephritis.

Waxy Casts: Easily recognizable, waxy casts form when hyaline casts linger in renal tubules for an extended period (prolonged stasis). Exhibiting a homogenous, smooth, glassy appearance with cracked or serrated margins and irregular, broken-off ends, waxy casts are distinct. Unlike other casts, their ends are straight and sharp. Typically light yellow in color, waxy casts are frequently observed in end-stage renal failure.

Fatty Casts: Comprising cylindrical structures filled with highly refractile fat globules, mainly triglycerides and cholesterol esters within a Tamm-Horsfall protein matrix, fatty casts are characteristic of nephrotic syndrome.

Broad Casts: These casts form in dilated distal tubules and are associated with chronic renal failure and severe renal tubular obstruction. Both waxy and broad casts are linked to a poor prognosis.

Cellular Casts

To be classified as cellular, casts must contain a minimum of three cells within the matrix. The nomenclature of cellular casts is contingent upon the specific type of cells entrapped in the matrix.

Red Cell Casts: These cylindrical structures exhibit red blood cells embedded in a Tamm-Horsfall protein matrix. Their coloration may range to brown due to hemoglobin pigmentation. Red cell casts hold paramount diagnostic significance, particularly in distinguishing hematuria stemming from glomerular disease as opposed to other causes. The presence of RBC casts typically indicates glomerular pathology, such as acute glomerulonephritis.

White Cell Casts: These cylindrical formations feature white blood cells encased in a Tamm-Horsfall protein matrix. Leukocytes generally traverse into tubules from the interstitium; thus, the presence of white cell casts is indicative of tubulointerstitial disease, such as pyelonephritis.

Renal Tubular Epithelial Cell Casts: Comprising sloughed-off renal tubular epithelial cells, these casts are observed in conditions like acute tubular necrosis, viral renal disease, heavy metal poisoning, and acute allograft rejection. Even the occasional detection of renal tubular casts holds significant diagnostic value.

Crystals

Crystals represent refractile structures characterized by a distinct geometric shape, resulting from the orderly three-dimensional arrangement of atoms and molecules. In contrast, amorphous material lacks a definite shape and typically presents as granular aggregates or clumps.

Urine crystals, as illustrated in Figure 7, can be classified into two primary types: (1) Normal, observed in the urinary sediment of healthy individuals, and (2) Abnormal, prevalent in states of disease.

Nevertheless, crystals identified in normal urine may manifest in elevated quantities in certain diseases.

While the majority of crystals, especially phosphates, urates, and oxalates, generally lack clinical significance, their increased presence can be indicative of underlying conditions. The identification of crystals in urine relies on their distinctive morphology. However, before confirming the existence of any abnormal crystals, it is imperative to validate their presence through chemical tests.

Normal Crystals

Crystals found in acidic urine:

1. Uric Acid Crystals: Displaying variable shapes such as diamond, rosette, and plates, these crystals exhibit a yellow or red-brown coloration attributed to urinary pigments. Soluble in alkali but insoluble in acid, increased numbers are associated with conditions like gout and leukemia. It's noteworthy that flat hexagonal uric acid crystals may be mistaken for cysteine crystals, which also form in acidic urine.
2. Calcium Oxalate Crystals: Manifesting as colorless, refractile, and envelope-shaped structures, these crystals may also assume dumbbell-shaped or peanut-like forms. Soluble in dilute hydrochloric acid, an elevation in their numbers, known as oxaluria, may suggest the presence of oxalate stones. Certain food intake, such as tomatoes, spinach, cabbage, asparagus, and rhubarb, can contribute to increased crystal numbers. Moreover, a substantial quantity is observed in cases of ethylene glycol poisoning.
3. Amorphous Urates: Comprising urate salts of potassium, magnesium, or calcium in acidic urine, these crystals typically appear as yellow, fine granules in compact masses. Their solubility in alkali or saline at 60°C distinguishes them within the crystalline spectrum.

Crystals found in alkaline urine:

1. Calcium Carbonate Crystals: Small, colorless, and often found in pairs, these crystals demonstrate solubility in acetic acid, releasing gas bubbles upon dissolution.
2. Phosphates: Phosphates may manifest as crystals, such as triple phosphates and calcium hydrogen phosphate, or as amorphous deposits.
   • Triple Phosphates (Ammonium Magnesium Phosphate): Colorless and exhibiting a shiny, 3-6 sided prismatic structure with oblique surfaces at the ends, often resembling “coffin lids”, or presenting a feathery fern-like appearance.
   • Calcium Hydrogen Phosphate (Stellar Phosphate): Colorless and displaying variable shapes, such as star-shaped formations, plates, or prisms.
   • Amorphous Phosphates: Occurring as colorless, small granules, often dispersed. All phosphates exhibit solubility in dilute acetic acid.
3. Ammonium Urate Crystals: Presenting as cactus-like structures covered with spines, these crystals, often referred to as ‘thornapple’ crystals, are yellow-brown and soluble in acetic acid at 60°C.

Abnormal Crystals

Although rare, the presence of these crystals indicates an underlying pathological process.

These crystals typically emerge in an acidic pH, often in substantial quantities. However, reporting abnormal crystals based solely on microscopy is insufficient; additional chemical tests are essential for confirmation.

1. Cysteine Crystals: Colorless, transparent, and hexagonal with six sides, these highly refractile plates are commonly found in acid urine. Frequently occurring in layered formations, they dissolve in 30% hydrochloric acid. Cysteine crystals are associated with cystinuria, an inborn metabolic error, and are often linked to the formation of cysteine stones.
2. Cholesterol Crystals: Colorless and refractile, these flat rectangular plates feature notched corners, arranged in a stair-step pattern. Soluble in ether, chloroform, or alcohol, they are observed in lipiduria, such as nephrotic syndrome and hypercholesterolemia. Identification can be confirmed with a polarizing microscope.
3. Bilirubin Crystals: Small (5 μ) and brown, these crystals exhibit variable shapes, including square, bead-like, or fine needles. Confirmation of their presence is achieved through reagent strip or chemical tests for bilirubin. Soluble in strong acid or alkali, they are indicative of severe obstructive liver disease.
4. Leucine Crystals: Refractile and yellow or brown, these crystals manifest as spheres with radial or concentric striations. Soluble in alkali, they are commonly found in urine alongside tyrosine in cases of severe liver disease, such as cirrhosis.
5. Tyrosine Crystals: Appearing as clusters of fine, delicate, colorless, or yellow needles, these crystals are observed in liver disease and tyrosinemia, an inborn metabolic error. They dissolve in alkali.
6. Sulfonamide Crystals: Exhibiting variable shapes but often appearing as sheaves of needles, these crystals arise following sulfonamide therapy. They are soluble in acetone.

Bile salts encompass salts derived from four distinct types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids form intricate salts or acids by binding with either glycine or taurine. Originating from the bile, these bile salts make their way into the small intestine, where they serve as detergents. Their primary function is to emulsify fat, thereby reducing the surface tension on fat droplets. This reduction in tension enables enzymes, specifically lipases, to effectively break down the fat. Subsequently, in the terminal ileum, bile salts are absorbed and enter the bloodstream. Once in the blood, they are taken up by the liver and subsequently re-excreted in bile, participating in the enterohepatic circulation.

In instances of obstructive jaundice, both bile salts and bilirubin can be identified in urine. In this condition, increased intrabiliary pressure causes the regurgitation of bile salts and conjugated bilirubin into the bloodstream from biliary canaliculi. The subsequent excretion of these elements in urine is detectable through a test known as the Hay’s surface tension test. This examination capitalizes on the ability of bile salts to lower surface tension.

To conduct the Hay’s surface tension test, collect a sample of fresh urine in a conical glass tube at room temperature. Sprinkle sulphur particles onto the urine's surface. If bile salts are present, the sulphur particles will sink to the bottom due to the surface tension-lowering effect of bile salts. Conversely, if the sulphur particles remain on the surface, it indicates the absence of bile salts. Notably, the presence of thymol, a preservative, can yield false positive results in this test.

When exposed to light, whether it be daylight or fluorescent light, bilirubin undergoes a conversion into the non-reactive biliverdin. This transformation also occurs when the substance is left standing at room temperature. It is crucial to note that biliverdin remains undetectable through tests designed for bilirubin. Hence, the necessity for a fresh sample, meticulously protected from light. The consequential findings associated with bilirubinuria are outlined below.

• Jaundice
• Urine Color: Presents as dark yellow with a noticeable yellow foam
• Elevated levels of serum conjugated bilirubin

Various methods are available for the detection of bilirubin in urine. These include the foam test, Gmelin’s test, Lugol iodine test, Fouchet’s test, Ictotest tablet test, and the reagent strip test.

1. Foam Test: Introduce approximately 5 ml of urine into a test tube, shake it vigorously, and observe for the formation of yellowish foam. It is essential to note that a similar outcome may occur with proteins and highly concentrated urine. In standard urine, the foam typically appears white.
2. Gmelin’s Test: In a test tube, combine 3 ml of concentrated nitric acid with an equal quantity of urine. Gently shake the tube, and the appearance of a spectrum of colors (yellow, red, violet, blue, and green) indicates a positive test (refer to Figure 1).
3. Lugol Iodine Test: Utilize a test tube to mix 4 ml of Lugol iodine solution (comprising 1 gm of iodine, 2 gm of potassium iodide, and distilled water to a total volume of 100 ml) with 4 drops of urine. A positive test is signaled by the development of a green color.
4. Fouchet’s Test: This test is both simple and sensitive.
   1. Combine 5 ml of fresh urine with 2.5 ml of 10% barium chloride in a test tube, ensuring thorough mixing. A precipitate of sulfates, to which bilirubin is bound (forming a barium sulfate-bilirubin complex), will appear.
   2. Filter the mixture to isolate the precipitate on a filter paper.
   3. To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent (composed of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water to a volume of 100 ml).
   4. The immediate development of a blue-green color around the drop indicates the presence of bilirubin (see Figure 2).
5. Reagent Strips or Tablets Impregnated with Diazo Reagent: These tests rely on the reaction between bilirubin and the diazo reagent. The resulting color change is directly proportional to the concentration of bilirubin. Ictotest tablets can detect bilirubin levels as low as 0.05-0.1 mg/dl of urine, whereas reagent strip tests exhibit lower sensitivity at 0.5 mg/dl.

The examination of ketone bodies in urine is employed to assess the ketone levels in your urine. Typically, the body utilizes sugar as its primary energy source. However, when there is an insufficient supply of sugar for energy, the body shifts to burning fat and generates compounds known as ketones. These ketones find their way into both the bloodstream and urine.

While it is normal to have a minimal quantity of ketones in the body, elevated levels can lead to severe illness or even fatalities.

This test may be necessary if you exhibit elevated blood sugar levels, a condition often associated with diabetes. Individuals with diabetes commonly experience heightened ketone levels due to insufficient insulin production or inadequate responsiveness to insulin, leading to an inability to utilize blood sugar for energy. Regular monitoring of ketone levels becomes imperative for those with elevated blood sugar levels and either type 1 or type 2 diabetes.

However, it's worth noting that individuals without diabetes can also display ketones in their urine, especially when the body relies on fat for energy instead of glucose. This metabolic shift may occur with factors such as chronic vomiting, intense physical exertion, adherence to low-carbohydrate diets (such as ketogenic or keto diets), or in the presence of eating disorders.

Particular attention to ketone levels is necessary if you have diabetes and experience:

• Blood sugar levels surpassing 300 mg/dL
• Alcohol misuse
• Diarrhea
• Cessation of carbohydrate intake, like rice and bread
• Pregnancy
• Periods of fasting
• Episodes of vomiting
• Presence of an infection

Your healthcare provider may prescribe or recommend this test if you manifest symptoms such as frequent urination, increased thirst, muscle aches, fatigue, weight loss, shortness of breath, nausea, vomiting, abdominal pain, confusion, or if there is a detectable fruity smell on your breath. Regular monitoring serves as a proactive measure in managing and maintaining optimal health.

Test Procedure

The concentration of ketone bodies in urine during ketosis can vary: β-hydroxybutyric acid comprises approximately 78%, acetoacetic acid constitutes around 20%, and acetone makes up roughly 2%.

No single method for detecting ketonuria reacts to all three ketone bodies. The Rothera’s nitroprusside method, along with methods derived from it, identifies acetoacetic acid and acetone, with the test demonstrating a sensitivity that is 10-20 times higher for acetoacetic acid compared to acetone. The ferric chloride test exclusively identifies acetoacetic acid. Notably, none of the screening tests are capable of detecting β-hydroxybutyric acid.

The techniques employed for identifying ketone bodies in urine encompass Rothera’s test, the Acetest tablet method, the ferric chloride test, and the reagent strip test.

Method 1: Rothera’s’ Test (Classic Nitroprusside Reaction)

When acetoacetic acid or acetone comes into contact with nitroprusside in an alkaline solution, a distinctive purple-colored complex is produced (refer to Figure 1). Rothera's test exhibits sensitivity in the range of 1-5 mg/dl for acetoacetate and 10-25 mg/dl for acetone.

Method

1. Begin by taking 5 ml of urine and placing it into a test tube, then saturate it with ammonium sulfate.
2. Introduce a small crystal of sodium nitroprusside and ensure thorough mixing.
3. Gradually add liquor ammonia along the side of the test tube to create a layered effect.
4. Observe for an immediate formation of a distinct purple permanganate-colored ring at the junction of the two fluids; this signifies a positive test (refer to Figure 2).

It's essential to note that a false-positive result may occur in the presence of L-dopa in urine and in cases of phenylketonuria.

Method 2: A Acetest Tablet Test

Presented here is Rothera's test in tablet form, known as the Acetest tablet. Comprising sodium nitroprusside, glycine, and an alkaline buffer, the tablet exhibits a purple lavender discoloration when acetoacetate or acetone is present (at levels ≥ 5 mg/dl). An approximate quantification of ketone bodies can be derived by comparing the tablet's color with the provided manufacturer's color chart.

It's noteworthy that this test surpasses the reagent strip test in terms of sensitivity for detecting ketones.

Method 3: Ferric Chloride Test (Gerhardt’s)

Upon introducing a 10% ferric chloride solution to urine, the solution undergoes a color change, turning reddish or purplish in the presence of acetoacetic acid. It's crucial to note that the test lacks specificity, as certain drugs, such as salicylate and L-dopa, can yield a similar reaction. The sensitivity of this test is set at a range of 25-50 mg/dl.

Method 4: Reagent Strip Test

Reagent strip tests represent adaptations of the nitroprusside test, as illustrated in Figures 1 and 2. These tests exhibit a sensitivity level of 5-10 mg/dl for acetoacetate. It's important to note that reagent strips can yield false-negative results when exposed to moisture. The ketone pad on the strip test is particularly susceptible to damage due to improper storage.

A urine protein test checks the amount of protein in urine. Normally, healthy people don't have much protein in their urine. But if the kidneys aren't working well or if there are high levels of certain proteins in the bloodstream, protein might show up in the urine.

Qualitative Estimation of Proteins

Method 1: Heat and Acetic Acid Test (Boiling Test)

This test operates on the principle that when proteins are heated in an acidic solution, they undergo precipitation.

Method

Ensure clarity in urine; if not, either filter it or utilize the supernatant from a centrifuged sample.

Confirm urine acidity (verify with litmus paper); if not acidic, gradually add 10% acetic acid until the blue litmus paper transitions to red.

Take a test tube and fill it two-thirds with urine. Incline the tube at an angle and heat the upper portion over a flame. (Specifically, only heat the upper portion to avoid disrupting the precipitate with convection currents generated by heat, allowing for a clear comparison between the upper and lower segments). Compare the heated part with the lower part. The presence of cloudiness or turbidity indicates the existence of either phosphates or proteins (refer to Figure 1). Introduce a few drops of 10% acetic acid, then boil the upper portion again. Turbidity originating from phosphates disappears, whereas that arising from proteins persists.

This test may yield a false-positive outcome in the presence of tolbutamide and elevated levels of penicillins.

Method 2: Reagent Strip Test

The reagent section of the strip is covered with an indicator and buffered to an acidic pH, displaying a color change in the presence of proteins (refer to Figures 821.2 and 821.3). This phenomenon is referred to as the "protein error of indicators" principle.

The reagent region is saturated with bromophenol blue indicator buffered to pH 3.0 using citrate. As the dye adheres to proteins, there is a shift in ionization (and consequently, pH) of the indicator, resulting in a color change. The intensity of the produced color is directly proportional to the protein concentration. This test is semi-quantitative in nature.

The reagent strip test primarily reacts to albumin but may yield false-negative results in the presence of Bence Jones proteins, myoglobin, and hemoglobin. If solely relying on the reagent strip method, there is a risk of overlooking overload (Bence Jones) proteinuria and tubular proteinuria. To ensure accuracy, it is advisable to follow this test with the sulphosalicylic acid test, which serves as a confirmatory measure. It's important to note that highly alkaline urine, significant hematuria, and contamination with vaginal secretions can potentially cause false-positive reactions in this test.

Method 3: Sulphosalicylic Acid Test

Introduction of sulphosalicylic acid into urine leads to the precipitation of a white substance if proteins are present. Organic acids denature proteins, causing them to precipitate out of the solution.

To perform the test, take 2 ml of clear urine in a test tube. If the urine's pH is neutral or alkaline, introduce a drop of glacial acetic acid. Following this, add 2-3 drops of sulphosalicylic acid (3 to 5%) and observe for turbidity against a dark background (refer to Figure 3). This method proves to be more sensitive and dependable compared to the boiling test.

However, it's crucial to note that false-positive results may arise due to various factors such as gross hematuria, highly concentrated urine, radiographic contrast media, excess uric acid, tolbutamide, sulphonamides, salicylates, and penicillins. On the contrary, a false-negative outcome may occur when dealing with very dilute urine.

This test is proficient in detecting a range of proteins, including albumin, hemoglobin, myoglobin, and Bence Jones proteins. For a comprehensive understanding, Table 1 illustrates a comparison between the reagent strip test and the sulphosalicylic acid test.

Quantitative Estimation of Proteins

The indications for quantifying protein levels in urine encompass several key aspects within the realm of clinical diagnosis and therapeutic monitoring. These include:

1. Diagnosis of Nephrotic Syndrome: Determining the presence of nephrotic syndrome stands as a primary motive for conducting quantitative estimations of proteins in urine. This serves as a crucial diagnostic parameter, aiding in the identification and characterization of renal conditions associated with abnormal protein excretion.
2. Detection of Microalbuminuria or Early Diabetic Nephropathy: Another pivotal application is the identification of microalbuminuria, a subtle yet significant indication of early-stage diabetic nephropathy. This underscores the proactive role of protein quantification in recognizing incipient renal complications in diabetic patients, facilitating timely intervention.
3. Monitoring Response to Therapy in Renal Disease: Quantitative assessment of proteins in urine plays a vital role in monitoring the efficacy of therapeutic interventions in renal diseases. This ongoing evaluation provides valuable insights into the patient's response to treatment, guiding adjustments and refinements in the therapeutic approach as needed.

In the context of proteinuria levels, specific thresholds offer diagnostic distinctions:

• Proteinuria exceeding 1500 mg in a 24-hour period signals the presence of glomerular disease.
• When proteinuria surpasses 3500 mg in 24 hours, it is categorized as nephrotic range proteinuria. Conversely, in tubular, hemodynamic, and post-renal diseases, proteinuria typically falls below 1500 mg in a 24-hour timeframe.

The grading of albuminuria, a subset of proteinuria, is systematically presented in Table 2. This classification aids in stratifying the severity of albumin excretion, providing a standardized framework for clinical interpretation.

Two distinct methodologies are employed for the quantitative assessment of proteins in urine:

1. Estimation of Proteins in a 24-hour Urine Sample: This method involves collecting urine over a complete day to analyze the total protein excreted during that period.
2. Estimation of Protein/Creatinine Ratio in a Random Urine Sample: An alternative approach involves determining the ratio of protein to creatinine in a random urine sample. This method offers a more immediate assessment, providing valuable information without the need for a full-day urine collection.

Quantifying Proteins in a 24-Hour Urine Sample: Accurate assessment of protein levels begins with the collection of a 24-hour urine sample, a procedure outlined earlier. The reliability of this sample is ensured by verifying its adequacy through the calculation of the expected 24-hour urine creatinine excretion. The daily output of urinary creatinine, a marker influenced by muscle mass, tends to remain relatively stable within an individual. For adult males, the range of creatinine excretion falls between 14-26 mg/kg/24 hours, while in females, it spans from 11-20 mg/kg/24 hours. Diverse methods are available for the precise quantification of proteins, encompassing Esbach’s albuminometer method, turbidimetric methods, the biuret reaction, and immunologic methods.

Assessing Protein/Creatinine Ratio in a Random Urine Sample: Recognizing the challenges associated with incomplete 24-hour urine collections, many laboratories opt for measuring the protein/creatinine ratio in random urine samples. A normal protein/creatinine ratio is < 0.2. In cases of low-grade proteinuria, the ratio ranges from 0.2 to 1.0; in moderate cases, it falls between 1.0 and 3.5; whereas in instances of nephrotic-range proteinuria, it surpasses 3.5. This method offers a pragmatic alternative, addressing the practical limitations of the 24-hour collection process and providing valuable insights into protein excretion levels.

Microalbuminura

This is characterized by the urinary excretion of 30 to 300 mg/24 hours (equivalent to 2-20 mg/dl) of albumin in the urine.

Significance of Microalbuminuria

1. Microalbuminuria stands as the initial indicator of renal impairment in diabetes mellitus, specifically in the context of diabetic nephropathy. This phenomenon signifies an elevation in capillary permeability to albumin, marking the onset of microvascular disease. Notably, microalbuminuria precedes the onset of diabetic nephropathy by several years. Rigorous management involving aggressive control of blood glucose levels and hypertension during this stage can significantly impede or forestall the progression toward irreversible renal damage and eventual renal failure.
2. Microalbuminuria emerges as an autonomous risk factor for cardiovascular disease in individuals with diabetes mellitus.

Detection of Microalbuminuria

Microalbuminuria eludes detection through routine proteinuria tests. Various methods employed for its identification include:

• Assessing the albumin-creatinine ratio in a random urine sample
• Measuring albumin levels in an early morning or random urine sample
• Determining albumin levels in a 24-hour urine sample

Commercially available test strips are designed to screen for microalbuminuria. For precise quantification, immunologic assays such as radioimmunoassay or enzyme-linked immunosorbent assay (ELISA) can be employed.

Bence Jones Proteinuria

Bence Jones proteins, integral to the realm of plasma cell dyscrasias like multiple myeloma and primary amyloidosis, represent monoclonal immunoglobulin light chains—either κ or λ—synthesized by neoplastic plasma cells. The heightened production of these light chains leads to their excess presence, resulting in overflow proteinuria as they are excreted in urine.

These proteins exhibit a distinctive thermal behavior. Upon heating, Bence Jones proteins precipitate within the temperature range of 40°C to 60°C— a phenomenon setting them apart, as other proteins typically precipitate between 60-70°C. Notably, the precipitate vanishes upon further heating at 85-100°C, a behavior not shared by other proteins. Upon cooling within the range of 60-85°C, the precipitate of Bence Jones proteins reappears. It is crucial to note that while this test provides insights, it lacks specificity for Bence Jones proteins, and both false-positive and -negative results can arise. Consequently, the contemporary standard involves protein electrophoresis of a concentrated urine sample (see Figure 4).

Figure 5 illustrates the subsequent assessment of enduring overt proteinuria.

It is important to note that quantifying proteins and assessing creatinine clearance are conducted for all patients experiencing persistent proteinuria.

Glucose, a primary source of energy for the body, is meticulously regulated to maintain optimal physiological functioning. However, when the delicate balance is disrupted, as seen in diabetes, glucose levels in the blood can spill into the urine, signaling an underlying issue. The tests designed for the detection of glucose in urine play a pivotal role in diagnosing and managing diabetes and other metabolic disorders.

1. Copper Reduction Methods

A. Benedict’s qualitative test

When we boil urine in Benedict's solution, a blue copper sulfate turns into a red-brown cuprous oxide, indicating the presence of a reducing agent (see Figure 1). The intensity of this color change relates to how much of the reducing substance is present. It's important to note that while this test provides valuable information, it isn't exclusive to glucose—it can detect various reducing agents in urine.

Some sugars (such as lactose, fructose, galactose, and pentoses), specific body substances (like glucuronic acid, homogentisic acid, uric acid, and creatinine), and various medications (including ascorbic acid, salicylates, cephalosporins, penicillins, streptomycin, isoniazid, para-aminosalicylic acid, nalidixic acid, etc.) also cause a reduction in alkaline copper sulfate solution.

Method

1. Get a test tube and put 5 ml of Benedict's qualitative reagent in it. (Benedict's reagent is made up of copper sulfate, sodium carbonate, sodium citrate, and distilled water.)
2. Add 0.5 ml (or 8 drops) of urine to the test tube and mix it well.
3. Boil the mixture over a flame for 2 minutes.
4. Let it cool to room temperature.
5. Observe any color change.

The test is sensitive to about 200 mg of reducing substance per dl of urine. While Benedict's test reacts positively with carbohydrates other than glucose, it's also used to screen for inborn errors of carbohydrate metabolism in infants and children, detecting galactose, lactose, fructose, maltose, and pentoses in urine.

If you're only checking for glucose in urine, it's preferable to use reagent strips.

Results are reported in grades as follows:

• Nil: No change from blue color.
• Trace: Green without precipitate.
• 1+ (approx. 0.5 grams/dl): Green with precipitate.
• 2+ (approx. 1.0 grams/dl): Brown precipitate.
• 3+ (approx. 1.5 grams/dl): Yellow-orange precipitate.
• 4+ (> 2.0 grams/dl): Brick-red precipitate.

B. Clinitest tablet method (Copper reduction tablet test):

This test is like the Benedict’s test, but the chemicals are in tablet form (copper sulfate, citric acid, sodium carbonate, and anhydrous sodium hydroxide). It can detect glucose at a sensitivity of 200 mg/dl.

2. Reagent Strip Method

This test is special for checking glucose and is better than Benedict’s and Clinitest methods. It works by using the glucose oxidase-peroxidase reaction. The strip has two enzymes (glucose oxidase and peroxidase) and a color-changing substance. When glucose is present, it reacts with glucose oxidase to make hydrogen peroxide and gluconic acid. The color-changing substance reacts with the hydrogen peroxide, causing a color change (see Figure 3). Different strips have different color substances and buffers.

You dip the strip in the urine, wait for a bit, and then compare the color to a chart (see Figure 2).

This test is better than Benedict’s for finding glucose, and it only reacts to glucose, not other substances.

It can detect about 100 mg of glucose in a deciliter of urine.

However, be aware that the test might show a false positive if there's bleach or hypochlorite (used for cleaning urine containers) around, as they can directly affect the color change.

On the flip side, a false negative might happen if there are lots of ketones, salicylates, ascorbic acid, or a strong Escherichia coli infection. The catalase produced by the bacteria can deactivate the hydrogen peroxide used in the test.

In the analysis of urinary urobilinogen, it is imperative to utilize a freshly collected urine sample. This crucial choice is rooted in the fact that, over time, urobilinogen undergoes conversion into urobilin, rendering it undetectable through routine tests. For a more nuanced examination, particularly when timing is a factor, a timed sample, such as a 2-hour postprandial collection, proves to be a valuable alternative for urobilinogen testing.

To quantify increased levels of urobilinogen in urine, two notable methods come to the forefront: the Ehrlich’s aldehyde test and the reagent strip test. These methodologies serve as reliable tools in the diagnostic arsenal, offering distinct advantages in discerning elevated urobilinogen concentrations.

Ehrlich's aldehyde test stands as a venerable technique, capitalizing on the reaction between urobilinogen and Ehrlich's reagent. This chemical interplay results in a characteristic color change, enabling precise quantification of urobilinogen levels. This method, steeped in scientific tradition, provides a meticulous approach for discerning subtle variations in urobilinogen concentration.

Complementing this, the reagent strip test emerges as a modern and efficient means of urobilinogen detection. This method capitalizes on advanced chemical indicators incorporated into specialized strips. These strips undergo specific reactions with urobilinogen, yielding color changes that can be quantified to gauge the concentration accurately. The reagent strip test, with its rapid and user-friendly nature, aligns with contemporary demands in clinical settings.

Ehrlich’s Aldehyde Test

Ehrlich’s reagent, also known as pdimethylaminobenzaldehyde, engages in a distinctive reaction with urobilinogen within urine, culminating in the manifestation of a discernible pink color. The intensity of this coloration serves as a direct correlate to the concentration of urobilinogen present in the specimen. It is crucial to note that the presence of bilirubin can impede this reaction. Consequently, in cases where bilirubin is detected, it becomes imperative to eliminate its interference.

To address the interference posed by bilirubin, a meticulous process is employed. Equal volumes of the urine specimen and a 10% solution of barium chloride are thoroughly amalgamated and subsequently subjected to filtration. The ensuing filtrate serves as the medium for conducting the urobilinogen test. This strategic filtration step aids in the removal of bilirubin, ensuring the fidelity of the urobilinogen reaction.

However, it is essential to exercise caution and discernment in the interpretation of results, as a parallel reaction can be induced by porphobilinogen. Porphobilinogen, a substance excreted in the urine of individuals afflicted by porphyria, shares a similar reactivity with Ehrlich's reagent, leading to analogous color changes. Thus, in the clinical assessment of urobilinogen, it becomes imperative to distinguish between the contributions of urobilinogen and porphobilinogen to the observed reactions.

Methodology for Urobilinogen Assessment

In the pursuit of accurate urobilinogen analysis, a meticulous method is prescribed to ensure precision and reliability. Commencing with a 5 ml aliquot of freshly collected urine, the protocol unfolds by introducing 0.5 ml of Ehrlich’s aldehyde reagent into a designated test tube. This reagent formulation comprises hydrochloric acid (20 ml), distilled water (80 ml), and paradimethylaminobenzaldehyde (2 gm). Following this amalgamation, the concoction is allowed to stand at room temperature for a judicious period of 5 minutes.

The interpretive nuances lie in the observed color changes. The emergence of a pink hue signifies a normal concentration of urobilinogen, offering a visual cue to the discerning analyst. Conversely, a transition into a dark red coloration serves as a pivotal indicator of an elevated urobilinogen presence, as elucidated in Figure 1.

Given the potential for overlapping reactions between urobilinogen and porphobilinogen, a supplementary evaluation becomes imperative. Enter the Watson-Schwartz test, a discriminating assay designed to delineate between these chemically akin entities. With the introduction of 1-2 ml of chloroform and a subsequent two-minute agitation period, the solution undergoes a distinctive phase separation. A telltale pink coloration in the chloroform layer signifies the exclusive presence of urobilinogen, while a corresponding manifestation in the aqueous portion signals the dominance of porphobilinogen. This critical differentiation is pivotal for accurate diagnosis and is depicted in Figure 2.

To fortify the diagnostic accuracy, the pink layer is meticulously decanted and subjected to further scrutiny through interaction with butanol. A persistent pink color in the aqueous layer unequivocally indicates the presence of porphobilinogen, thus offering a conclusive resolution to the diagnostic conundrum.

Potential Factors Influencing False-Negative Reactions in Urobilinogen Testing

It is crucial to be cognizant of potential confounding factors that may lead to false-negative reactions. Two notable circumstances warrant consideration in this context.

Firstly, the presence of a urinary tract infection (UTI) introduces a dynamic that can obscure the accuracy of urobilinogen assessments. Specifically, the oxidation of urobilinogen to urobilin can occur in the presence of nitrites, a phenomenon commonly associated with UTIs. This chemical transformation during the testing process may yield a false-negative outcome, necessitating a judicious interpretation of results in the context of suspected or known UTIs.

Secondly, the impact of antibiotic therapy on urobilinogen testing integrity is noteworthy. Antibiotics, while instrumental in combatting infections, possess the capacity to disrupt the delicate balance of gut bacteria responsible for urobilinogen production. Consequently, the destruction of these bacteria can lead to a reduction in urobilinogen levels, potentially resulting in false-negative reactions during testing.

Urobilinogen Assessment via Reagent Strip Method

In the pursuit of urobilinogen quantification, the Reagent Strip Method emerges as a targeted and specialized approach. This method intricately focuses on the specific detection of urobilinogen, underscoring its precision in elucidating this particular component within urine samples.

The method hinges on the strategic impregnation of the test area with either p-dimethylaminobenzaldehyde or 4-methoxybenzene diazonium tetrafluoroborate. These meticulously chosen reagents serve as discriminating agents, interacting selectively with urobilinogen to yield discernible reactions. The deliberate incorporation of these compounds into the test area ensures a high level of specificity, facilitating the accurate identification and quantification of urobilinogen amidst the complex milieu of urinary components.

Porphyrias, derived from the Greek word "porphura," meaning purple pigment, encompass a diverse array of rare disorders arising from disruptions in the heme biosynthetic pathway. This disturbance leads to the abnormal accumulation of red and purple pigments known as porphyrins within the body. Heme, an integral component of hemoglobin, undergoes synthesis through a series of distinct steps, as illustrated in Figure 1. Each step is facilitated by a specific enzyme. In instances where any of these enzymatic processes falter due to hereditary or acquired causes, precursor molecules of heme, referred to as porphyrin intermediates, amass in the blood, deposit in the skin and various organs, and are excreted in urine and feces.

The categorization of porphyrias is contingent upon the location of the defect, resulting in different types with diverse clinical manifestations, severity levels, and the specific nature of accumulated porphyrin.

Historically, porphyria has been postulated as a potential explanation for medieval tales of vampires and werewolves. This conjecture arises from notable parallels between the behaviors exhibited by individuals afflicted with porphyria and the characteristics found in folklore. These similarities include light aversion, skin mutilation upon sunlight exposure, red teeth, psychiatric disturbances, and the consumption of blood to obtain heme.

Detection and accurate diagnosis of porphyrias pose significant challenges, often leading to oversight or misdiagnosis. Many porphyrias lack definitive physical findings, screening tests may produce false-negative results, diagnostic criteria are ambiguously defined, and mild disorders may yield enzyme assay results within the ostensibly 'normal' range.

Heme plays a crucial role in both bone marrow, where it is essential for hemoglobin synthesis, and the liver, where it is vital for cytochromes. Consequently, porphyrias are classified into erythropoietic and hepatic types based on the site of disease expression. Hepatic porphyrias predominantly impact the nervous system, while erythropoietic porphyrias primarily affect the skin. Furthermore, porphyrias are categorized as acute and nonacute (or cutaneous) types based on their clinical presentations, as outlined in Table 1. This systematic classification enhances our understanding of porphyrias, aiding clinicians, researchers, and medical professionals in comprehending the nuances of these complex disorders.

The inheritance patterns associated with porphyrias manifest as either autosomal dominant or recessive. The majority of acute porphyrias follow an autosomal dominant inheritance, signifying the transmission of a single abnormal gene copy. Consequently, the activity of the deficient enzyme is halved, resting at 50%. In instances where the hepatic heme level diminishes due to various factors, the activity of ALA synthase is spurred, resulting in an escalation of heme precursors up to the point of the enzyme defect. This surge in heme precursors precipitates the onset of symptoms characteristic of acute porphyria. Notably, as the heme level normalizes, the associated symptoms subside.

The accumulation of porphyrin precursors presents a noteworthy occurrence in lead poisoning, attributed to the inhibition of the enzyme aminolevulinic acid dehydratase within the heme biosynthetic pathway. This phenomenon can simulate the clinical presentation of acute intermittent porphyria.

Understanding the genetic underpinnings and enzymatic intricacies of porphyrias is crucial for clinicians, researchers, and medical professionals. The autosomal dominant inheritance pattern underscores the significance of a singular abnormal gene copy in the manifestation of acute porphyrias. Additionally, the intricate interplay between heme levels and enzymatic activity elucidates the nuanced triggers and resolution of acute porphyria symptoms. Moreover, the correlation between lead poisoning and the disruption of the heme biosynthetic pathway highlights the diagnostic challenges posed by conditions that mimic porphyric presentations, necessitating a keen clinical acumen for accurate differentiation.

Clinical Features

The clinical manifestations of porphyrias exhibit variability contingent upon the specific type of the disorder. Acute porphyrias, for instance, manifest with a spectrum of symptoms, including acute and intense abdominal pain, vomiting, constipation, chest pain, emotional and mental disturbances, seizures, hypertension, tachycardia, sensory deficits, and muscular weakness. This diverse array of clinical presentations underscores the complexity of acute porphyrias and the multisystemic impact they can exert.

In contrast, cutaneous porphyrias present a distinctive set of clinical features. Patients with cutaneous porphyrias commonly experience photosensitivity, characterized by the development of redness and blistering of the skin upon exposure to sunlight. Additional symptoms include itching, necrosis of the skin and gums, and an observable increase in hair growth over the temples. This distinctive clinical profile, outlined in Table 2, provides a comprehensive overview of the characteristic features associated with cutaneous porphyrias.

Understanding the nuanced clinical features associated with different types of porphyrias is pivotal for accurate diagnosis and effective management. The delineation of symptoms, ranging from abdominal pain and neurological manifestations in acute porphyrias to the distinctive photosensitivity and dermatological changes in cutaneous porphyrias, enhances the knowledge base for clinicians, researchers, and medical professionals. Such precision in characterizing the clinical landscape facilitates targeted interventions and improves patient outcomes.

The manifestation of symptoms in porphyrias is intricately linked to various triggers, encompassing pharmaceutical agents and lifestyle factors. Certain drugs, such as barbiturates, oral contraceptives, diazepam, phenytoin, carbamazepine, methyldopa, sulfonamides, chloramphenicol, and antihistamines, can serve as catalysts for symptoms. Emotional or physical stress, infections, dietary changes, fasting, substance abuse, premenstrual periods, smoking, and alcohol consumption also constitute potential triggers, amplifying the complexity of porphyria management.

Autosomal dominant porphyrias comprise a diverse group of disorders, including acute intermittent porphyria, variegate porphyria, porphyria cutanea tarda, erythropoietic protoporphyria (predominantly), and hereditary coproporphyria. In contrast, autosomal recessive porphyrias encompass congenital erythropoietic porphyria, erythropoietic protoporphyria (in a limited number of cases), and ALAdehydratase porphyria, also known as plumboporphyria.

Understanding the array of triggers and the genetic underpinnings of different porphyria types is pivotal for comprehensive patient care. The intricate interplay between pharmaceutical agents, lifestyle factors, and the distinct genetic characteristics of autosomal dominant and recessive porphyrias underscores the necessity for a nuanced approach in diagnosis and management. This multifaceted perspective equips medical professionals, and researchers with the knowledge required to navigate the complexities of porphyria, facilitating enhanced patient outcomes and advancing scientific understanding in the field.

Laboratory Diagnosis

The diagnosis of porphyria involves conducting tests on blood, urine, and feces, particularly during symptomatic periods. Achieving a timely and precise diagnosis is crucial for the effective management of porphyrias. Given the diverse and extensive clinical features associated with porphyrias, they are often considered in the differential diagnosis of various medical conditions. Most routine hospital laboratories are equipped to perform initial investigations when porphyria is suspected. However, for the identification of specific porphyria types, specialized laboratories with advanced testing capabilities become essential.

In the diagnostic process, blood, urine, and fecal analyses play a pivotal role, allowing for the detection of characteristic abnormalities during symptomatic phases. This comprehensive approach ensures a more accurate identification of porphyria subtypes, facilitating targeted and tailored therapeutic strategies.

Considering the intricate nature of porphyria diagnoses, collaboration with specialized laboratories becomes imperative. These facilities possess the advanced technologies and expertise required for in-depth analyses, enabling the discrimination between various porphyria types. This collaboration enhances the precision of the diagnostic process, contributing to improved patient outcomes and informed decision-making in the realm of porphyria management.

In essence, the diagnostic journey for porphyria demands a synergistic effort between routine hospital laboratories and specialized facilities, with a focus on leveraging advanced testing methodologies to unravel the complexities inherent in these conditions. This collaborative approach underscores the significance of accurate diagnostics in paving the way for effective porphyria management strategies.

Initial Studies

In the assessment of suspected acute porphyrias, specifically during acute neurovisceral attacks, it is imperative to submit a freshly collected, randomly obtained urine sample (10-20 ml) for the detection of heightened urinary excretion of porphobilinogen (PBG) (refer to Figure 2). Notably, in acute intermittent porphyria (AIP), the urine may exhibit a reddish or brown hue upon standing, as illustrated in Figure 3.

For cases where cutaneous porphyrias are suspected, with manifestations such as acute photosensitivity lacking skin fragility, a comprehensive diagnostic approach is warranted. This includes analyzing free erythrocyte protoporphyrin (FEP) in EDTA blood for the diagnosis of erythrocytic protoporphyria. Additionally, for all other cutaneous porphyrias characterized by skin fragility and bullae, an examination of fresh, random urine (10-20 ml) and either feces (5-10 g) or plasma is indispensable for identifying excess porphyrins. Detailed guidance on this diagnostic protocol is provided in Figure 4 and Table 2.

In essence, the diagnostic strategy for suspected porphyrias involves specific tests tailored to the clinical presentation. The utilization of various specimen types, including urine, blood, feces, and plasma, is crucial for the accurate identification of porphyrin abnormalities. The distinct visual cues, such as the color changes in urine associated with AIP, serve as valuable indicators in the diagnostic process. This meticulous and multifaceted approach ensures a comprehensive evaluation, facilitating precise diagnoses in the intricate landscape of porphyrias.

In addition to aiding in the diagnosis, the identification of specific heme intermediates excreted in urine or feces plays a pivotal role in pinpointing the site of defect in porphyria. Heme precursors, extending up to coproporphyrinogen III, exhibit water solubility, enabling their detection in urine. Conversely, protoporphyrinogen and protoporphyrin, being insoluble in water, are excreted through bile and can be identified in fecal samples. It is essential to shield all samples from light to preserve their integrity.

The requisite samples for a comprehensive porphyria assessment include a 10-20 ml fresh, random urine sample devoid of any preservatives, 5-10 g of wet fecal weight, and blood anticoagulated with EDTA. Each specimen type offers distinct insights into the porphyrin profile, contributing to a thorough evaluation of the disorder. The meticulous collection and analysis of these samples serve as critical steps in unraveling the intricacies of porphyria, ensuring a comprehensive understanding of the disorder's manifestations and facilitating targeted interventions.

In essence, the choice of sample type is guided by the solubility characteristics of specific heme intermediates, aligning with their excretion pathways. This systematic approach, coupled with stringent measures to protect samples from light-induced alterations, underscores the importance of precision and accuracy in the diagnostic process.

Test for Porphobilinogen in Urine

The Ehrlich’s aldehyde test serves as a diagnostic tool for detecting porphobilinogen (PBG) in urine. In this assay, Ehrlich’s reagent, composed of p-dimethylaminobenzaldehyde, reacts with PBG, resulting in the formation of a distinctive red color. The characteristic red product exhibits an absorption spectrum with a peak at 553 nm and a shoulder at 540 nm. It's noteworthy that both urobilinogen and porphobilinogen elicit a similar reaction, necessitating additional tests to discern between the two substances. To distinguish PBG from urobilinogen, solvent extraction can be employed, as outlined in the Watson-Schwartz test. This additional step is crucial for achieving specificity in the diagnostic process.

It is essential to recognize that levels of PBG might appear normal or near normal between attacks. Consequently, to mitigate the risk of false-negative results, it is imperative to conduct the test during an acute attack. This strategic timing ensures a more accurate and reliable assessment of PBG levels, contributing to a precise diagnosis and informed clinical decision-making.

Test for Total Porphyrins in Urine

The quantification of total porphyrins within an acidified urine sample is achievable through spectrophotometry, leveraging the distinct absorbance peak of porphyrins around 400 nm. This method allows for a semiquantitative estimation of porphyrin levels, providing valuable insights into the porphyrin profile present in the urine sample. Spectrophotometry, with its ability to measure the absorption of light at specific wavelengths, offers a precise and objective means of assessing the concentration of porphyrins in the given sample.

In this analytical approach, the intense absorbance peak observed around 400 nm serves as a reliable marker for the presence of porphyrins. This peak reflects the characteristic absorption spectrum of porphyrins, allowing for their identification and quantification in a systematic and accurate manner. The semiquantitative nature of the estimation enhances the diagnostic utility of the method, providing clinicians and researchers with valuable information regarding the porphyrin content in the urine.

The utilization of spectrophotometry in conjunction with acidified urine samples offers a robust technique for detecting and estimating total porphyrins. This method, marked by its precision and objectivity, contributes to the comprehensive assessment of porphyrin levels, facilitating diagnostic and research endeavors within the realm of porphyria evaluation.

Test for Total Porphyrins in Feces

The quantification of total porphyrins in fecal samples involves the use of spectrophotometry on an acidic extract of the specimen. However, a crucial preparatory step is required to eliminate interference from dietary chlorophyll, which also absorbs light around 400 nm. This interference is addressed through diethyl ether extraction, ensuring a more accurate and specific assessment of total porphyrin levels.

In the analytical process, an acidic extract of the fecal sample serves as the basis for spectrophotometric analysis. Spectrophotometry, with its ability to measure the absorption of light at specific wavelengths, allows for the precise determination of total porphyrins. The focus on an acidic environment facilitates optimal conditions for the assessment of porphyrin content.

To enhance the specificity of the analysis, the extraction of dietary chlorophyll using diethyl ether becomes imperative. This preparatory step is essential as chlorophyll absorbs light in a similar range as porphyrins, potentially leading to inaccurate measurements. By effectively removing this interference, the spectrophotometric analysis can provide more reliable and meaningful data regarding the concentration of total porphyrins in the fecal sample.

The process of determining total porphyrins in feces through spectrophotometry necessitates a meticulous approach, including the removal of dietary chlorophyll through diethyl ether extraction. This method ensures the accuracy and specificity of the analysis, contributing to the reliability of results in the evaluation of porphyrin levels in fecal samples.

Evaluation of Porphyrins in Erythrocytes and Plasma: Modern Techniques

Contemporary methodologies have supplanted traditional approaches such as visual examination for porphyrin fluorescence, as well as solvent fractionation and spectrophotometry. The advent of fluorometric methods represents a significant advancement in the analysis of porphyrins in erythrocytes and plasma.

Fluorometric techniques have emerged as the preferred means of assessing porphyrins, offering heightened precision and sensitivity compared to their predecessors. These methods leverage the unique fluorescence properties of porphyrins, providing a more refined and objective approach to their detection and quantification.

By utilizing fluorometry, researchers and medical professionals can achieve a clearer understanding of porphyrin levels in erythrocytes and plasma. This modernized approach enhances both the accuracy and efficiency of porphyrin testing, aligning with the evolving standards of scientific analysis in hematology and related fields.

This transition to fluorometric methods underscores the commitment to staying at the forefront of scientific advancements, ensuring that diagnostic and research practices remain not only rigorous but also in harmony with the latest technological innovations.

Comprehensive Assessment for Porphyria Diagnosis

Upon obtaining a positive result in the initial porphyria screening, a more in-depth analysis becomes imperative. This involves the quantification of porphyrin concentrations in urine, feces, and blood, a crucial step in establishing a precise and specific diagnosis. The tables below (Tables 3 and 4) provide detailed insights into the diagnostic criteria and reference values for this comprehensive evaluation.

By meticulously measuring porphyrin levels in these biological samples, healthcare professionals can discern the specific type and severity of porphyria, facilitating targeted and effective management strategies. This comprehensive approach not only confirms the presence of porphyria but also aids in tailoring treatment plans to the individual nuances of the patient's condition.

As with any diagnostic process, the accuracy and reliability of results depend on the proficiency of the laboratory techniques employed and the adherence to standardized protocols. This commitment to precision ensures that the gathered data is not only scientifically robust but also forms the cornerstone for informed clinical decisions and patient care.

Diagnostic Challenges in Latent Porphyrias and Remission Periods

During latent porphyrias and periods of remission, individuals may exhibit normal porphyrin levels, posing a diagnostic challenge. In such instances, a comprehensive approach involving enzymatic and DNA testing becomes imperative for an accurate diagnosis. These advanced testing methods delve into the genetic and enzymatic aspects, providing a more nuanced understanding of porphyria manifestation even in the absence of elevated porphyrin levels.

Upon confirming a diagnosis of porphyria, a critical next step involves the systematic examination of close family members for the presence of the disorder. This proactive approach is essential for early detection and intervention. Family members testing positive for porphyria should undergo counseling, equipping them with valuable insights into potential triggering factors and empowering them to make informed lifestyle choices that mitigate the risk of symptomatic episodes.

This meticulous diagnostic and familial investigation strategy not only aids in individual patient management but also contributes to the broader goal of familial health and well-being. By addressing latent cases and involving family members, healthcare professionals can establish a more comprehensive and effective framework for the understanding and management of porphyrias.

Fresh platelets should always be used. Storing platelets dramatically changes the level of transmembrane proteins. The best way is to follow one of standardized protocols defined in: Immunophenotypic analysis of platelets. Krueger LA, Barnard MR, Frelinger AL 3rd, Furman MI, Michelson AD.Curr Protoc Cytom. 2002 Feb;Chapter 6:Unit 6.10. 

• TEST FOR FIBRINOGEN/FIBRIN DEGRADATION PRODUCTS (FDPs)
• PLATELET GLYCOPROTEIN ANALYSIS