MICROSCOPIC EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY

Published in Clinical Pathology
Monday, 14 August 2017 22:02
The most important test in semen analysis for infertility is microscopic examination of the semen.
 
SPERM MOTILITY
 
The first laboratory assessment of sperm function in a wet preparation is sperm motility (ability of the sperms to move). Sperm motility is essential for penetration of cervical mucus, traveling through the fallopian tube, and penetrating the ovum. Only those sperms having rapidly progressive motility are capable of penetrating ovum and fertilizing it.
 
Principle: All motile and non-motile sperms are counted in randomly chosen fields in a wet preparation under 40× objective. Result is expressed as a percentage of motile spermatozoa observed.
 
Method: A drop of semen is placed on a glass slide, covered with a coverslip that is then ringed with petroleum jelly to prevent dehydration, and examined under 40× objective. Atleast 200 spermatozoa are counted in several different microscopic fields. Result is expressed as a percentage of (a) rapidly progressive spermatozoa (moving fast forward in a straight line), (b) slowly progressive spermatozoa (slow linear or non-linear, i.e. crooked or curved movement), (c) non-progressive spermatozoa (movement of tails, but with no forward progress), and (d) immotile spermatozoa (no movement at all) (WHO critera). Sperms of grades (c) and (d) are considered to be poorly motile (asthenospermia). Normally, ≥ 25% of sperms show rapid progressive motility, or ≥ 50% of sperms show rapid progressive and slow progressive motility.
 
If the proportion of motile spermatozoa is < 50%, then proportion of viable sperms should be determined by examining an eosin preparation.
 
SPERM VIABILITY OR VITALITY
 
Principle: A cell with intact cell membrane (a vital or viable cell) will not take up the eosin Y and will not be stained, while a non-viable or dead cell will have damaged cell membrane, will take up the dye, and will be stained pink-red (Figure 832.1). Another stain (e.g. nigrosin) may  be used to stain the background material. The test is performed if motility is abnormal.
 
Figure 832.1 Eosin nigrosin stain
Figure 832.1 Eosin-nigrosin stain. Dead sperms are stained pink-red, while live sperms are stained white
 
Method
 
  1. Mix one drop of semen with 1 drop of eosin-nigrosin solution and incubate for 30 seconds.
  2. A smear is made from a drop placed on a glass slide.
  3. The smear is air-dried and examined under oilimmersion objective. White sperms are classified as live or viable, and red sperms are classified as dead or non-viable. At least 200 spermatozoa are examined.
  4. The result is expressed as a proportion of viable sperms against non-viable as an integer percentage.
 
Seventy-five percent or more of sperms are normally live or viable.
 
SPERM COUNT
 
Principle: The sperm count is done after liquefaction in a counting chamber following dilution and the total number of spermatozoa is reported in millions/ml (106/ml).
 
Method
 
  1. Semen is diluted 1:20 with sodium bicarbonateformalin diluting fluid (Take 1 ml liquefied semen in a graduated tube and fill with diluting fluid to 20 ml mark. Mix well).
  2. A coverslip is placed over the improved Neubauer counting chamber and the counting chamber is filled with the well-mixed diluted semen sample using a Pasteur pipette. The chamber is then placed in a humid box for 10-15 minutes for spermatozoa to settle.
  3. The chamber is placed on the microscope stage. Using the 20× or 40× objective and iris diaphragm lowered sufficiently to give sufficient contrast, number of spermatozoa is counted in 4 large corner squares. Spermatozoa whose heads are touching left and upper lines of the square should be considered as ‘belonging’ to that square.
  4. Sperm count per ml is calculated as follows:

    Sperm count =                Sperms counted × correction factor             × 1000
                              Number of squares counted × Volume of 1 square
                           = Sperms counted × 20 1000
                                        4 × 0.1
                           = Sperms counted × 50, 000

  5. Normal sperm count is ≥ 20 million/ml (i.e. ≥ 20 × 106/ml). Sperm count < 20 million/ml may be associated with infertility in males.
 
SPERM MORPHOLOGY
 
A smear is prepared by spreading a drop of seminal fluid on a glass slide, stained, and percentages of normal and abnormal forms of spermatozoa are counted. The staining techniques used are Papanicolaou, eosinnigrosin, hematoxylin-eosin, and Rose Bengal-toluidine blue stain. Atleast 200 spermatozoa should be counted under oil immersion. Percentages of normal and abnormal spermatozoa should be recorded.
 
Normal morphology: A spermatozoon consists of three main components: head, neck, and tail. Tail is further subdivided into midpiece, main (principle) piece, and end piece (Figure 832.2 and Box 832.1).
 
Figure 832.2 Morphology of spermatozoa
Figure 832.2 Morphology of spermatozoa
 
Head is pear-shaped. Most of the head is occupied by the nucleus which has condensed chromatin and few areas of dispersed chromatin (called nuclear vacuoles). The anterior 2/3rds of the nucleus is surrounded by acrosomal cap. Acrosomal cap is a flattened membranebound vesicle containing glycoproteins and enzymes. These enzymes are required for separation of cells of corona radiata and dissolution of zona pellucida of ovum during fertilization.
 
Neck is a very short segment that connects the head and the tail. Centriole in the neck gives rise to axoneme of the flagellum. Axoneme consists of 20 microtubules (arranged as a central pair surrounded by 9 peripheral doublets) and is surrounded by condensed fibrous rings.
 
Middle piece is the first part of the tail and consists of central axoneme surrounded by coarse longitudinal fibers. These are surrounded by elongated mitochondria that provide energy for movement of tail.
 
Principle or main piece constitutes most of the tail and is composed of axoneme that is surrounded by 9 coarse fibers. This central core is surrounded by many circularly arranged fibrous ribs.
 
Endpiece is the short tapering part composed of only axoneme.
 
Normally, > 30% of spermatozoa should show normal morphology (WHO, 1999). The defects in morphology that are associated with infertility in males include defective mid-piece (causes reduced motility), an incomplete or absent acrosome (causes inability to penetrate the ovum), and giant head (defective DNA condensation).
 
Box 832.1 Normal sperm morphology
• Total length of sperm: About 60 μ
• Total length of sperm: About 60 μ
• Head:
   – Length: 3-5 μ
   – Width: 2-3 μ
   – Thickness: 1.5 μ
• Neck: Length: 0.3 μ
• Middle piece:
   – Length: 3-5 μ
   – Width: 1.0 μ
• Principal piece:
   – Length: 40-50 μ
   – Width: 0.5 μ
• End piece: 4-6 μ
 
Abnormal morphology (Figure 832.3): WHO morphological classification of human spermatozoa (1999) is given 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
 
Figure 832.3 Abnormal morphological sperm forms
Figure 832.3 Abnormal morphological sperm forms: (1) Normal sperm, (2) Large head, (3) Small head, (4) Tapered head, (5) Pyriform head, (6) Round head, (7) Amorphous head, (8) Vacuoles in head, (9) Round head without acrosome, (10) Double head, (11) Pin head, (12) Round head without acrosome and thick midpiece, (13) Coiled tail, and (14) Double tail

ROUND CELLS
 
Round cells on microscopic examination may be white blood cells or immature sperm cells. Special stain (peroxidase or Papanicolaou) is required to differentiate between them. White blood cells >1 million/ml indicate presence of infection. Presence of large number of immature sperm cells indicates spermatogenesis dysfunction at the testicular level.

BIOCHEMICAL ANALYSIS OF SEMEN FOR INVESTIGATION OF INFERTILITY

Published in Clinical Pathology
Monday, 14 August 2017 13:42
Biochemical markers (Table 831.1) can be measured in semen to test the secretions of accessory structures. These include fructose (seminal vesicles), zinc, citric acid or acid phosphatase (prostate), and α-glucosidase or carnitine (epididymis).
 
Table 831.1 Biochemical variables of semen analysis (World Helath Organization, 1992)
1. Total fructose (seminal vesicle marker) ≥13 μmol/ejaculate
2. Total zinc (Prostate marker) ≥2.4 μmol/ejaculate
3. Total acid phosphatase (Prostate marker) ≥200U/ejaculate
4. Total citric acid (Prostate marker) ≥52 μmol/ejaculate
5. α-glucosidase (Epididymis marker) ≥20 mU/ejaculate
6. Carnitine (Epididymis marker) 0.8-2.9 μmol/ejaculate
 
TEST FOR FRUCTOSE
 
Resorcinol method is used for detection of fructose. In this test, 5 ml of resorcinol reagent (50 mg resorcinol dissolved in 33 ml concentrated hydrochloric acid; dilute up to 100 ml with distilled water) is added to 0.5 ml of seminal fluid. The mixture is heated and brought to boil. If fructose is present, a red-colored precipitate is formed within 30 seconds.
 
Absence of fructose indicates obstruction proximal to seminal vesicles (obstructed or absent vas deferens) or a lack of seminal vesicles. In a case of azoospermia, if fructose is absent, it is due to the obstruction of ejaculatory ducts or absence of vas deferens, and if present, azoospermia is due to failure of testes to produce sperm.

PHYSICAL EXAMINATION OF SEMEN FOR INVESTIGATION OF INFERTILITY

Published in Clinical Pathology
Monday, 14 August 2017 13:04
Examination is carried out after liquefaction of semen that occurs usually within 20-30 minutes of ejaculation.
 
1. VISUAL APPEARANCE
 
Normal semen is viscous and opaque gray-white in appearance. After prolonged abstinence, it appears slightly yellow.
 
 
Immediately following ejaculation, normal semen is thick and viscous. It becomes liquefied within 30 minutes by the action of proteolytic enzymes secreted by prostate. If liquefaction does not occur within 60 minutes, it is abnormal. The viscosity of the sample is assessed by filling a pipette with semen and allowing it to flow back into the container. Normal semen will fall drop by drop. If droplets form ‘threads’ more than 2 cm long, then viscosity is increased. Increased semen viscosity affects sperm motility and leads to poor invasion of cervical mucus; it results from infection of seminal vesicles or prostate.
 
3. VOLUME
 
Volume of ejaculated semen sample should normally be > 2 ml. It is measured after the sample has liquefied. Volume < 2.0 ml is abnormal, and is associated with low sperm count.
 
4. pH
 
A drop of liquefied semen is spread on pH paper (of pH range 6.4-8.0) and pH is recorded after 30 seconds. Normal pH is 7.2 to 8.0 after 1 hour of ejaculation. The portion of semen contributed by seminal vesicles is basic, while portion from prostate is acidic. Low pH (< 7.0) with absence of sperms (azoospermia) suggests obstruction of ejaculatory ducts or absence of vas deferens. Low pH is usually associated with low semen volume (as most of the volume is supplied by seminal vesicles).

EXAMINATION FOR THE PRESENCE OF SEMEN IN MEDICOLEGAL CASES

Published in Forensic Pathology
Sunday, 13 August 2017 17:08
This includes examination of material obtained from vagina, stains from clothing, skin, hair, or other body parts for semen. This is carried out in cases of alleged rape or sexual assault.
 
Collection of Sample
 
  • Vagina: Direct aspiration or saline lavage
  • Clothing: When scanned with ultraviolet light, semen produces green white fluorescence. A small piece (1 m2) of clothing from stained portion is soaked in 1-2 ml of physiologic saline for 1 hour. A similar piece of clothing distant from the stain is also soaked in saline as a control.

LABORATORY PROCEDURES
 
1. MICROSCOPIC EXAMINATION FOR SPERMS
 
Presence of motile sperms in vaginal fluid indicates interval of < 8 hours. Smears prepared from collected samples are stained and examined for the presence of sperms.
 
2. ACID PHOSPHATASE
 
Acid phosphatase is determined on vaginal or clothing samples. Due to the high level of acid phosphatase in semen, its presence indicates recent sexual intercourse. Level of ≥50 U/sample is considered as positive evidence of semen.
 
3. DETERMINATION OF BLOOD GROUP SUBSTANCES
 
When semen is positively identified in vaginal fluid or other sample, test can be carried out for the presence of blood group substances in the same sample. The ‘secretor’ individuals (80% individuals are secretors) will secrete the blood group substances in body fluids, including semen.
 
4. FLORENCE TEST
 
This test detects the presence of choline found in high concentration in semen. To several drops of sample, add equal volume of reagent (iodine 2.54 g, potassium iodide 1.65 g, distilled water 30 ml); in positive test rhombic or needle-like crystals of periodide of choline form. False-positive tests can occur due to high choline content of some other body fluids.

TESTS FOR DETECTION OF BLOOD IN URINE

Published in Clinical Pathology
Sunday, 13 August 2017 02:34
1. MICROSCOPIC EXAMINATION OF URINARY SEDIMENT
 
Definition of microscopic hematuria is presence of 3 or more number of red blood cells per high power field on microscopic examination of urinary sediment in two out of three properly collected samples. A small number of red blood cells in urine of low specific gravity may undergo lysis, and therefore hematuria may be missed if only microscopic examination is done. Therefore, microscopic examination of urine should be combined with a chemical test.
 
2. CHEMICAL TESTS
 
These detect both intracellular and extracellular hemoglobin (i.e. intact and lysed red cells) as well as myoglobin. Heme proteins in hemoglobin act as peroxidase, which reduces hydrogen peroxide to water. This process needs a hydrogen donor (benzidine, orthotoluidine, or guaiac). Oxidation of hydrogen donor leads to development of a color (Figure 828.1). Intensity of color produced is proportional to the amount of hemoglobin present.
 
Chemical tests are positive in hematuria, hemoglobinuria, and myoglobinuria.
 
Figure 828.1 Principle of chemical test for red cells
Figure 828.1 Principle of chemical test for red cells, hemoglobin, or myoglobin in urine
 
Benzidine Test
 
Make saturated solution of benzidine in glacial acetic acid. Mix 1 ml of this solution with 1 ml of hydrogen peroxide in a test tube. Add 2 ml of urine. If green or blue color develops within 5 minutes, the test is positive.
 
Orthotoluidine Test
 
In this test, instead of benzidine, orthotoluidine is used. It is more sensitive than benzidine test.
 
Reagent Strip Test
 
Various reagent strips are commercially available which use different chromogens (o-toluidine, tetramethylbenzidine).
 
Causes of false-positive tests:
 
  • Contamination of urine by menstrual blood in females
  • Contamination of urine by oxidizing agent (e.g. hypochlorite or bleach used to clean urine containers), or microbial peroxidase in urinary tract infection.
 
Causes of false-negative tests:
 
  • Presence of a reducing agent like ascorbic acid in high concentration: Microscopic examination for red cells is positive but chemical test is negative.
  • Use of formalin as a preservative for urine
 
Evaluation of positive chemical test for blood is shown in Figure 828.2.
 
Figure 828.2 Evaluation of positive chemical test for blood in urine
Figure 828.2 Evaluation of positive chemical test for blood in urine

Telomere Indicator of Physiological Age

Published in Genetics
Saturday, 12 August 2017 13:06
Have you ever wondered, what is your physiological age? Is it more or less than your chronological age? Physiological age determines a person’s health condition. Are we able to determine physiological age? You would think the answer is NO. but it can be done by determining telomere’s length. “Telomere is a repetitive nucleotide sequence (having no meaningful information) at each end of chromosome to protect DNA from deterioration and or from fusion with other chromosomes.” This sequence is about 3000-15000 base pairs in length. In vertebrates this repeated sequence is TTAGGG.
 
Significance of Telomeres
 
Cells divide and increase their number, DNA duplication also occurs. Enzymes involved in this duplication process, can’t continue duplication all the way to the end so some part of DNA is lost and chromosome is shortened. This lost part is some base pairs of telomere. Somatic cells lose about 50-100 nucleotides on each cell division. In this way, telomeres, having no meaningful information, act as CAPS preventing the important information (DNA) from deterioration and preserve the critical information. Telomeres are never tied to each other which allows chromosomes to remain segregate. Without telomeres, chromosomes would fuse with each other. Telomere Shortening Telomeres shorten because of the two major factors:
 
  1. End replication problem in eukaryotes accounts for loss of 20 base pairs per cell division.
  2. Oxidative stress accounts for loss of 50-100 base pairs per cell division.
 
Figure 827.1
 
Oxidative stress in the body depends on lifestyle factors. Smoking, poor diet and stress can cause increase in oxidative stress. With each cell division telomeres shorten, so there are limited number of divisions that a cell can undergo, this limit is called Hayflick Limit. This is to prevent the loss of vital DNA information and to prevent production of abnormal cells. When a cell reaches this limit it undergoes apoptosis that is a programmed cell death. Telomere Lengthening to reverse telomere shortening, there is an enzyme named Telomerase that adds telomere sequence nucleotides and replenish the lost telomere nucleotides. Telomerase activity is not present in all cells. It is almost absent in somatic cells including; lung, liver, kidney cells, adult tissues, cardiac and skeletal muscles etc. In the presence of telomerase enzyme, a cell can divide to unlimited extent without ageing giving rise to tumors. That’s why it is found only in some cells in considerable concentration including germline cells and stem cells. These cells don’t show signs of ageing.
 
Figure 827.3
 
Relation between Telomere’s Shortening and Ageing
 
Figure 827.2It is still controversial that whether telomere shortening is a reason of ageing or is a sign of ageing just like grey hair. Whatever it is, the thing is, it determines your physiological age because ageing cells mean an ageing body. Telomere shortening is related with poor lifestyle. People who are active and have a healthy lifestyle have the same telomere length as someone 10 years younger than them has. Depression causes increase in oxidative stress in the body so the higher the stress, the shorter the telomere is Link between Telomeres and Cancer “Cancer in general is defined as an uncontrollable rapid growth of cells.”
 
What causes these cells to grow uncontrollably?
 
These cells have active telomerase enzyme, which doesn’t let the telomere to shorten, so no Hayflick limit reaches and cell continues to divide. This is the reason why telomerase is not used as an anti-ageing medicine because it has potential to turn normal body cells into cancerous cells. Without telomerase activity cancer cells activity would stop, which is an under research treatment for cancer. However, drugs inhibiting telomerase activity, can interfere with normal functioning of cells that require telomerase. In healthy female breast there is a portion of cells named, luminal progenitors, with critically short telomere length. In these cells telomerase becomes active causing these cells to turn into cancer cells on higher activity. To tackle breast cancer, use of telomerase inhibiting drugs should be practiced. Telomere biology is very important for understanding cancer biology and scientists are working hard on it.
 
 
Reviewed by Dr. Nida Hayat Khan
Editor @ BioScience.pk 

CHEMICAL EXAMINATION OF URINE

Published in Clinical Pathology
Thursday, 10 August 2017 23:29
The chemical examination is carried out for substances in urine are listed below:
 
  • Proteins
  • Glucose
  • Ketones
  • Bilirubin
  • Bile salts
  • Urobilinogen
  • Blood
  • Hemoglobin
  • Myoglobin
  • Nitrite or leukocyte esterase
 
PROTEINS

Normally, kidneys excrete scant amount of protein in urine (up to 150 mg/24 hours). These proteins include proteins from plasma (albumin) and proteins derived from urinary tract (Tamm-Horsfall protein, secretory IgA, and proteins from tubular epithelial cells, leucocytes, and other desquamated cells); this amount of proteinuria cannot be detected by routine tests. (Tamm-Horsfall protein is a normal mucoprotein secreted by ascending limb of the loop of Henle).
 
Proteinuria refers to protein excretion in urine greater than 150 mg/24 hours in adults.
 
Causes of Proteinuria
 

Box 826.1: Causes of proteinuria

• Glomerular proteinuria
• Tubular proteinuria
• Overflow proteinuria
• Hemodynamic (functional) proteinuria
• Post-renal proteinuria
Causes of proteinuria can be grouped as shown in Box 826.1.
 
  • Glomerular proteinuria: Proteinuria due to increased permeability of glomerular capillary wall is called as glomerular proteinuria.

    There are two types of glomerular proteinuria: selective and nonselective. In early stages of glomerular disease, there is increased excretion of lower molecular weight proteins like albumin and transferrin. When glomeruli can retain larger molecular weight proteins but allow passage of comparatively lower molecular weight proteins, the proteinuria is called as selective. With further glomerular damage, this selectivity is lost and larger molecular weight proteins (γ globulins) are also excreted along with albumin; this is called as nonselective proteinuria.

    Selective and nonselective proteinuria can be distinguished by urine protein electrophoresis. In selective proteinuria, albumin and transferrin bands are seen, while in nonselective type, the pattern resembles that of serum (Figure 826.1).

     
    • Massive proteinuria (>3.5 gm/24 hr)
    • Hypoalbuminemia (<3.0 gm/dl)
    • Generalised edema
    Hyperlipidemia (serum cholesterol >350 mg/dl)
    • Lipiduria
    Causes of glomerular proteinuria are glomerular diseases that cause increased permeability of glomerular basement membrane. The degree of glomerular proteinuria correlates with severity of disease and prognosis. Serial estimations of urinary protein are also helpful in monitoring response to treatment. Most severe degree of proteinuria occurs in nephrotic syndrome (Box 826.2).

  • Tubular proteinuria: Normally, glomerular membrane, although impermeable to high molecular weight proteins, allows ready passage to 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 involving mainly tubules, these proteins are excreted in urine while albumin excretion is minimal.

    Urine electrophoresis shows prominent α- and β-bands (where low molecular weight proteins migrate) and a faint albumin band (Figure 826.1).

    Tubular type of proteinuria is commonly seen in acute and chronic pyelonephritis, heavy metal poisoning, tuberculosis of kidney, interstitial nephritis, cystinosis, Fanconi syndrome and rejection of kidney transplant.

    Purely tubular proteinuria cannot be detected by reagent strip test (which is sensitive to albumin), but heat and acetic acid test and sulphosalicylic acid test are positive.

  • Overflow proteinuria: When concentration of a low molecular weight protein rises in plasma, it “overflows” from plasma into the urine. Such proteins are immunoglobulin light chains or Bence Jones proteins (plasma cell dyscrasias), hemoglobin (intravascular hemolysis), myoglobin (skeletal muscle trauma), and lysozyme (acute myeloid leukemia type M4 or M5).

  • Hemodynamic proteinuria: Alteration of blood flow through the glomeruli causes increased filtration of proteins. Protein excretion, however, is transient. It is seen in 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 recumbent position. It is common in adolescents (3-5%) and is probably due to lordotic posture that causes inferior venacaval compression between the liver and vertebral column. The condition disappears in adulthood. Amount of proteinuria is <1000 mg/day. First-morning urine after rising is negative for proteins, while another urine sample collected after patient performs normal activities is positive for proteins. In such patients, periodic testing for proteinuria should be done to rule out renal disease.

  • Post-renal proteinuria: This is caused by inflammatory or neoplastic conditions in renal pelvis, ureter, bladder, prostate, or urethra.
 
 
Figure 826.1 Glomerular and tubular proteinuria
Figure 826.1 Glomerular and tubular proteinuria. Upper figure shows normal serum protein electrophoresis pattern. Lower part shows comparison of serum and urine electrophoresis in (1) selective proteinuria, (2) non-selective proteinuria, and (3) tubular proteinuria
 
GLUCOSE
 
The main indication for testing for glucose in urine is detection of unsuspected diabetes mellitus or follow-up of known diabetic patients.
 
Box 826.3: Urine glucose
 
• Urine should be tested for glucose within 2 hours of collection (due to lowering of glucose by glycolysis and by contaminating bacteria which degrade glucose rapidly)
• Reagent strip test is a rapid, inexpensive, and semi-quantitative test
• In the past this test was used for home-monitoring of glucose; the test is replaced by glucometers.
• Urine glucose cannot be used to monitor control of diabetes since renal threshold is variable amongst individuals, no information about level of blood glucose below renal threshold is obtained, and urinary glucose value is affected by concentration of urine.
Practically all of the glucose filtered by the glomeruli is reabsorbed by the proximal renal tubules and returned to circulation. Normally a very small amount of glucose is excreted in urine (< 500 mg/24 hours or <15 mg/dl) that cannot be detected by the routine tests. Presence of detectable amounts of glucose in urine is called as glucosuria or glycosuria (Box 826.3). Glycosuria results if the filtered glucose load exceeds the capacity of renal tubular reabsorption. Most common cause is hyperglycemia from diabetes mellitus.
 
Causes of Glycosuria
 
1. Glycosuria with hyperglycemia:
 
  • Endocrine diseases: diabetes mellitus, acromegaly, Cushing’s syndrome, hyperthyroidism, pancreatic disease
  • Non-endocrine diseases: central nervous system diseases, liver disorders
  • Drugs: adrenocorticotrophic hormone, corticosteroids, thiazides
  • Alimentary glycosuria (Lag-storage glycosuria): After a meal, there is rapid intestinal absorption of glucose leading to transient elevation of blood glucose above renal threshold. This can occur in persons with gastrectomy or gastrojejunostomy and in hyperthyroidism. Glucose tolerance test reveals a peak at 1 hour above renal threshold (which causes glycosuria); the fasting and 2-hour glucose values are normal.
 
2. Glycosuria without hyperglycemia
 
  • Renal glycosuria: This accounts for 5% of cases of glycosuria in general population. Renal threshold is the highest glucose level in blood at which glucose appears in urine and which is detectable by routine laboratory tests. The normal renal threshold for glucose is 180 mg/dl. Threshold substances need a carrier to transport them from tubular lumen to blood. When the carrier is saturated, the threshold is reached and the substance is excreted. Up to this level glucose filtered by the glomeruli is efficiently reabsorbed by tubules. Renal glycosuria is a benign condition in which renal threshold is set below 180 mgs/dl but glucose tolerance is normal; the disorder is transmitted as autosomal dominant. Other conditions in which glycosuria can occur with blood glucose level remaining below 180 mgs/dl are renal tubular diseases in which there is decreased glucose reabsorption like Fanconi’s syndrome, and toxic renal tubular damage. During pregnancy, renal threshold for glucose is decreased. Therefore it is necessary to estimate blood glucose when glucose is first detected in urine.
 
 
KETONES
 
Excretion of ketone bodies (acetoacetic acid, β-hydroxybutyric acid, and acetone) in urine is called as ketonuria. Ketones are breakdown products of fatty acids and their presence in urine is indicative of excessive fatty acid metabolism to provide energy.
 
Causes of Ketonuria
 
Box 826.4: Urine ketones in diabetes
 
Indications for testing
 
• At diagnosis of diabetes mellitus
• At regular intervals in all known cases of diabetes, and in gestational diabetes
• In known diabetic patients during acute illness, persistent hyperglycemia (>300 mg/dl), pregnancy, clinical evidence of diabetic acidosis (nausea, vomiting, abdominal pain)
Normally ketone bodies are not detectable in the urine of healthy persons. If energy requirements cannot be met by metabolism of glucose (due to defective carbohydrate metabolism, low carbohydrate intake, or increased metabolic needs), then energy is derived from breakdown of fats. This leads to the formation of ketone bodies (Figure 826.2).
 
  1. Decreased utilization of carbohydrates:
    a. Uncontrolled diabetes mellitus with ketoacidosis: In diabetes, because of poor glucose utilization, there is compensatory increased lipolysis. This causes increase in the level of free fatty acids in plasma. Degradation of free fatty acids in the liver leads to the formation of acetoacetyl CoA which then forms ketone bodies. Ketone bodies are strong acids and produce H+ ions, which are neutralized by bicarbonate ions; fall in bicarbonate (i.e. alkali) level produces ketoacidosis. Ketone bodies also increase the plasma osmolality and cause cellular dehydration. Children and young adults with type 1 diabetes are especially prone to ketoacidosis during acute illness and stress. If glycosuria is present, then test for ketone bodies must be done. If both glucose and ketone bodies are present in urine, then it indicates presence of diabetes mellitus with ketoacidosis (Box 826.4).
    In some cases of diabetes, ketone bodies are increased in blood but do not appear in urine.
    Presence of ketone bodies in urine may be a warning of impending ketoacidotic coma.
    b. Glycogen storage disease (von Gierke’s disease)
  2. Decreased availability of carbohydrates in the diet:
    a. Starvation
    b. Persistent vomiting in children
    c. Weight reduction program (severe carbohydrate restriction with normal fat intake)
  3. Increased metabolic needs:
    a. Fever in children
    b. Severe thyrotoxicosis
    c. Pregnancy
    d. Protein calorie malnutrition
 
 
Figure 826.2 Formation of ketone bodies
Figure 826.2 Formation of ketone bodies. A small part of acetoacetate is spontaneously and irreversibly converted to acetone. Most is converted reversibly to β-hydroxybutyrate
 
BILE PIGMENT (BILIRUNIN)
 
Bilirubin (a breakdown product of hemoglobin) is undetectable in the urine of normal persons. Presence of bilirubin in urine is called as bilirubinuria.
 
There are two forms of bilirubin: conjugated and unconjugated. After its formation from hemoglobin in reticuloendothelial system, bilirubin circulates in blood bound to albumin. This is called as unconjugated bilirubin. Unconjugated bilirubin is not water-soluble, is bound to albumin, and cannot pass through the glomeruli; therefore it does not appear in urine. The liver takes up unconjugated bilirubin where it combines with glucuronic acid to form bilirubin diglucuronide (conjugated bilirubiun). Conjugated bilirubin is watersoluble, is filtered by the glomeruli, and therefore appears in urine.
 
Detection of bilirubin in urine (along with urobilinogen) is helpful in the differential diagnosis of jaundice (Table 826.1).
 
Table 826.1 Urine bilirubin and urobilinogen in jaundice
Urine test Hemolytic jaundice Hepatocellular jaundice Obstructive jaundice
1. Bilirubin Absent Present Present
2. Urobilinogen Increased Increased Absent
 
In acute viral hepatitis, bilirubin appears in urine even before jaundice is clinically apparent. In a fever of unknown origin bilirubinuria suggests hepatitis.
 
Presence of bilirubin in urine indicates conjugated hyperbilirubinemia (obstructive or hepatocellular jaundice). This is because only conjugated bilirubin is water-soluble. Bilirubin in urine is absent in hemolytic jaundice; this is because unconjugated bilirubin is water-insoluble.
 
 
BILE SALTS
 
Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).
 
 
UROBILINOGEN
 
Conjugated bilirubin excreted into the duodenum through bile is converted by bacterial action to urobilinogen in the intestine. Major part is eliminated in the feces. A portion of urobilinogen is absorbed in blood, which undergoes recycling (enterohepatic circulation); a small amount, which is not taken up by the liver, is excreted in urine. Urobilinogen is colorless; upon oxidation it is converted to urobilin, which is orange-yellow in color. Normally about 0.5-4 mg of urobilinogen is excreted in urine in 24 hours. Therefore, a small amount of urobilinogen is normally detectable in urine.
 
Urinary excretion of urobilinogen shows diurnal variation with highest levels in afternoon. Therefore, a 2-hour post-meal sample is preferred.
 
Causes of Increased Urobilinogen in Urine
 
  1. Hemolysis: Excessive destruction of red cells leads to hyperbilirubinemia and therefore increased formation of urobilinogen in the gut. Bilirubin, being of unconjugated type, does not appear in urine. Increased urobilinogen in urine without bilirubin is typical of hemolytic anemia. This also occurs in megaloblastic anemia due to premature destruction of erythroid precursors in bone marrow (ineffective erythropoiesis).
  2. Hemorrhage in tissues: There is increased formation of bilirubin from destruction of red cells.
 
Causes of Reduced Urobilinogen in Urine
 
  1. Obstructive jaundice: In biliary tract obstruction, delivery of bilirubin to the intestine is restricted and very little or no urobilinogen is formed. This causes stools to become clay-colored.
  2. Reduction of intestinal bacterial flora: This prevents conversion of bilirubin to urobilinogen in the intestine. It is observed in neonates and following antibiotic treatment.
 
Testing of urine for both bilirubin and urobilinogen can provide helpful information in a case of jaundice (Table 826.1).
 
 
BLOOD
 
The presence of abnormal number of intact red blood cells in urine is called as hematuria. It implies presence of a bleeding lesion in the urinary tract. Bleeding in urine may be noted macroscopically or with naked eye (gross hematuria). If bleeding is noted only by microscopic examination or by chemical tests, then it is called as occult, microscopic or hidden hematuria.
 
Causes of Hematuria
 
1. Diseases of urinary tract:
 
  • Glomerular diseases: Glomerulonephritis, Berger’s disease, lupus nephritis, Henoch-Schonlein purpura
  • Nonglomerular diseases: Calculus, tumor, infection, tuberculosis, pyelonephritis, hydronephrosis, polycystic kidney disease, trauma, after strenuous physical exercise, diseases of prostate (benign hyperplasia of prostate, carcinoma of prostate).
 
2. Hematological conditions:
 
Coagulation disorders, sickle cell disease Presence of red cell casts and proteinuria along with hematuria suggests glomerular cause of hematuria.
 
 
HEMOGLOBIN
 
Presence of free hemoglobin in urine is called as hemoglobinuria.
 
Causes of Hemoglobinuria
 
  1. Hematuria with subsequent lysis of red blood cells in urine of low specific gravity.
  2. Intravascular hemolysis: Hemoglobin will appear in urine when haptoglobin (to which hemoglobin binds in plasma) is completely saturated with hemoglobin. Intravascular hemolysis occurs in infections (severe falciparum malaria, clostridial infection, E. coli septicemia), trauma to red cells (march hemoglobinuria, extensive burns, prosthetic heart valves), glucose-6-phosphate dehydrogenase deficiency following exposure to oxidant drugs, immune hemolysis (mismatched blood transfusion, paroxysmal cold hemoglobinuria), paroxysmal nocturnal hemoglobinuria, hemolytic uremic syndrome, and disseminated intravascular coagulation.
 
Tests for Detection of Hemoglobinuria
 
Tests for detection of hemoglobinuria are benzidine test, orthotoluidine test, and reagent strip test.
 
HEMOSIDERIN
 
Hemosiderin in urine (hemosiderinuria) indicates presence of free hemoglobin in plasma. Hemosiderin appears as blue granules when urine sediment is stained with Prussian blue stain (Figure 826.3). Granules are located inside tubular epithelial cells or may be free if cells have disintegrated. Hemosiderinuria is seen in intravascular hemolysis.
 
Figure 826.3 Staining of urine sediment with Prussian blue stain
Figure 826.3 Staining of urine sediment with Prussian blue stain to demonstrate hemosiderin granules (blue)
 
MYOGLOBIN
 
Myoglobin is a protein present in striated muscle (skeletal and cardiac) which binds oxygen. Causes of myoglobinuria include injury to skeletal or cardiac muscle, e.g. crush injury, myocardial infarction, dermatomyositis, severe electric shock, and thermal burns.
 
Chemical tests used for detection of blood or hemoglobin also give positive reaction with myoglobin (as both hemoglobin and myoglobin have peroxidase activity). Ammonium sulfate solubility test is used as a screening test for myoglobinuria (Myoglobin is soluble in 80% saturated solution of ammonium sulfate, while hemoglobin is insoluble and is precipitated. A positive chemical test for blood done on supernatant indicates myoglobinuria).
 
Distinction between hematuria, hemoglobinuria, and myoglobinuria is shown in Table 826.2
 
Table 826.2 Differentiation between hematuria, hemoglobinuria, and myoglobinuria
Parameter Hematuria Hemoglobinuria Myoglobinuria
 1. Urine color  Normal, smoky, red, or brown  Pink, red, or brown  Red or brown
 2. Plasma color  Normal  Pink  Normal
 3. Urine test based on peroxidase activity  Positive  Positive  Positive
 4. Urine microscopy  Many red cells  Occasional red cell  Occasional red cell
 5. Serum haptoglobin  Normal  Low  Normal
 6. Serum creatine kinase  Normal  Normal  Markedly increased
.
Chemical Tests for Significant Bacteriuria (Indirect Tests for Urinary Tract Infection)
 
In addition to direct microscopic examination of urine sample, chemical tests are commercially available in a reagent strip format that can detect significant bacteriuria: nitrite test and leucocyte esterase test. These tests are helpful at places where urine microscopy is not available. If these tests are positive, urine culture is indicated.
 
1. Nitrite test: Nitrites are not present in normal urine; ingested nitrites are converted to nitrate and excreted in urine. If gram-negative bacteria (e.g. E.coli, Salmonella, Proteus, Klebsiella, etc.) are present in urine, they will reduce the nitrates to nitrites through the action of bacterial enzyme nitrate reductase. Nitrites are then detected in urine by reagent strip tests. As E. coli is the commonest organism causing urinary tract infection, this test is helpful as a screening test for urinary tract infection.
 
Some organisms like Staphylococci or Pseudomonas do not reduce nitrate to nitrite and therefore in such infections nitrite test is negative. Also, urine must be retained in the bladder for minimum of 4 hours for conversion of nitrate to nitrite to occur; therefore, fresh early morning specimen is preferred. Sufficient dietary intake of nitrate is necessary. Therefore a negative nitrite test does not necessarily indicate absence of urinary tract infection. The test detects about 70% cases of urinary tract infections.
 
2. Leucocyte esterase test: It detects esterase enzyme released in urine from granules of leucocytes. Thus the test is positive in pyuria. If this test is positive, urine culture should be done. The test is not sensitive to leucocytes < 5/HPF.

MICROSCOPIC EXAMINATION OF URINE

Published in Clinical Pathology
Thursday, 10 August 2017 00:57
Microscopic examination of urine is also called as the “liquid biopsy of the urinary tract”.
 
Urine consists of various microscopic, insoluble, solid elements in suspension. These elements are classified as organized or unorganized. Organized substances include red blood cells, white blood cells, epithelial cells, casts, bacteria, and parasites. The unorganized substances are crystalline and amorphous material. These elements are suspended in urine and on standing they settle down and sediment at the bottom of the container; therefore they are known as urinary deposits or urinary sediments. Examination of urinary deposit is helpful in diagnosis of urinary tract diseases as shown in Table 825.1.
 
Table 825.1 Urinary findings in renal diseases
Condition Albumin RBCs/HPF WBCs/HPF Casts/LPF Others
1. Normal 0-trace 0-2 0-2 Occasional (Hyaline)
2. Acute glomerulonephritis 1-2+ Numerous;dysmorphic 0-few Red cell, granular Smoky urine or hematuria
3. Nephrotic syndrome > 4+ 0-few 0-few Fatty, hyaline, Waxy, epithelial Oval fat bodies, lipiduria
4. Acute pyelonephritis 0-1+ 0-few Numerous WBC, granular WBC clumps, bacteria, nitrite test
HPF: High power field; LPF: Low power field; RBCs: Red blood cells; WBCs: White blood cells.
 
Different types of urinary sediments are shown in Figure 825.1. The major aim of microscopic examination of urine is to identify different types of cellular elements and casts. Most crystals have little clinical significance.
 
Figure 825.1 Different types of urinary sediment
Figure 825.1 Different types of urinary sediment
 
Specimen: The cellular elements are best preserved in acid, hypertonic urine; they deteriorate rapidly in alkaline, hypotonic solution. A mid-stream, freshly voided, first morning specimen is preferred since it is the most concentrated. The specimen should be examined within 2 hours of voiding because cells and casts degenerate upon standing at room temperature. If preservative is required, then 1 crystal of thymol or 1 drop of formalin (40%) is added to about 10 ml of urine.
 
Method: A well-mixed sample of urine (12 ml) is centrifuged in a centrifuge tube for 5 minutes at 1500 rpm and supernatant is poured off. The tube is tapped at the bottom to resuspend the sediment (in 0.5 ml of urine). A drop of this is placed on a glass slide and covered with a cover slip (Figure 825.2). The slide is examined immediately under the microscope using first the low power and then the high power objective. The condenser should be lowered to better visualize the elements by reducing the illumination.
 
Figure 825.2 Preparation of urine sediment for microscopic examination
Figure 825.2 Preparation of urine sediment for microscopic examination
 
CELLS
 
Cellular elements in urine are shown in Figure 825.3.
 
Figure 825.3 Cells in urine
Figure 825.3 Cells in urine (1) Isomorphic red blood cells, (2) Crenated red cells, (3) Swollen red cells, (4) Dysmorphic red cells, (5) White blood cells (pus cells), (6) Squamous epithelial cell, (7) Transitional epithelial cells, (8) Renal tubular epithelial cells, (9) Oval fat bodies, (10) Maltese cross pattern of oval fat bodies, and (11) spermatozoa
 
Red Blood Cells
 
Normally there are no or an occasional red blood cell in urine. In a fresh urine sample, red cells appear as small, smooth, yellowish, anucleate biconcave disks about 7 μ in diameter (called as isomorphic red cells). However, red cells may appear swollen (thin discs of greater diameter, 9-10 μ) in dilute or hypotonic urine, or may appear crenated (smaller diameter with spikey surface) in hypertonic urine. In glomerulonephritis, red cells are typically described as being dysmorphic (i.e. markedly variable in size and shape). They result from passage of red cells through the damaged glomeruli. Presence of > 80% of dysmorphic red cells is strongly suggestive of glomerular pathology.
 
The quantity of red cells can be reported as number of red cells per high power field.
 
Causes of hematuria have been listed earlier.
 
White Blood Cells (Pus Cells)
 
White blood cells are spherical, 10-15 μ in size, granular in appearance in which nuclei may be visible. Degenerated white cells are distorted, smaller, and have fewer granules. Clumps of numerous white cells are seen in infections. Presence of many white cells in urine is called as pyuria. In hypotonic urine white cells are swollen and the granules are highly refractile and show Brownian movement; such cells are called as glitter cells; large numbers are indicative of injury to urinary tract.
 
Normally 0-2 white cells may be seen per high power field. Pus cells greater than 10/HPF or presence of clumps is suggestive of urinary tract infection.
 
Increased numbers of white cells occur in fever, pyelonephritis, lower urinary tract infection, tubulointerstitial nephritis, and renal transplant rejection.
 
In urinary tract infection, following are usually seen in combination:
 
  • Clumps of pus cells or pus cells >10/HPF
  • Bacteria
  • Albuminuria
  • Positive nitrite test
 
Simultaneous presence of white cells and white cell casts indicates presence of renal infection (pyelonephritis).
 
Eosinophils (>1% of urinary leucocytes) are a characteristic feature of acute interstitial nephritis due to drug reaction (better appreciated with a Wright’s stain).
 
Renal Tubular Epithelial Cells
 
Presence of renal tubular epithelial cells is a significant finding. Increased numbers are found in conditions causing tubular damage like acute tubular necrosis, pyelonephritis, viral infection of kidney, allograft rejection, and salicylate or heavy metal poisoning.
 
These cells are small (about the same size or slightly larger than white blood cell), polyhedral, columnar, or oval, and have granular cytoplasm. A single, large, refractile, eccentric nucleus is often seen.
 
Renal tubular epithelial cells are difficult to distinguish from pus cells in unstained preparations.
 
Squamous Epithelial Cells
 
Squamous epithelial cells line the lower urethra and vagina. They are best seen under low power objective (×10). Presence of large numbers of squamous cells in urine indicates contamination of urine with vaginal fluid. These are large cells, rectangular in shape, flat with abundant cytoplasm and a small, central nucleus.
 
Transitional Epithelial Cells
 
Transitional cells line renal pelvis, ureters, urinary bladder, and upper urethra. These cells are large, and diamond- or pear-shaped (caudate cells). Large numbers or sheets of these cells in urine occur after catheterization and in transitional cell carcinoma.
 
Oval Fat Bodies
 
These are degenerated renal tubular epithelial cells filled with highly refractile lipid (cholesterol) droplets. Under polarized light, they show a characteristic “Maltese cross” pattern. They can be stained with a fat stain such as Sudan III or Oil Red O. They are seen in nephrotic syndrome in which there is lipiduria.
 
Spermatozoa
 
They may sometimes be seen in urine of men.
 
Telescoped urinary sediment: This refers to urinary sediment consisting of red blood cells, white blood cells, oval fat bodies, and all types of casts in roughly equal proportion. It occurs in lupus nephritis, malignant hypertension, rapidly proliferative glomerulonephritis, and diabetic glomerulosclerosis.
 
ORGANISMS
 
Organisms detectable in urine are shown in Figure 825.4.
 
Figure 825.4 Organisms in urine
Figure 825.4 Organisms in urine: (A) Bacteria, (B) Yeasts, (C) Trichomonas, and (D) Egg of Schistosoma haematobium
 
Bacteria
 
Bacteria in urine can be detected by microscopic examination, reagent strip tests for significant bacteriuria (nitrite test, leucocyte esterase test), and culture
 
Significant bacteriuria exists when there are >105 bacterial colony forming units/ml of urine in a cleancatch midstream sample, >104 colony forming units/ml of urine in catheterized sample, and >103 colonyforming units/ml of urine in a suprapubic aspiration sample.
 
  1. Microscopic examination: In a wet preparation, presence of bacteria should be reported only when urine is fresh. Bacteria occur in combination with pus cells. Gram’s-stained smear of uncentrifuged urine showing 1 or more bacteria per oil-immersion field suggests presence of > 105 bacterial colony forming units/ml of urine. If many squamous cells are present, then urine is probably contaminated with vaginal flora. Also, presence of only bacteria without pus cells indicates contamination with vaginal or skin flora.
  2. Chemical or reagent strip tests for significant bacteriuria: These are given earlier.
  3. Culture: On culture, a colony count of >105/ml is strongly suggestive of urinary tract infection, even in asymptomatic females. Positive culture is followed by sensitivity test. Most infections are due to Gram-negative enteric bacteria, particularly Escherichia coli.
 
If three or more species of bacteria are identified on culture, it almost always indicates contamination by vaginal flora.
 
Negative culture in the presence of pyuria (‘sterile’ pyuria) occurs with prior antibiotic therapy, renal tuberculosis, prostatitis, renal calculi, catheterization, fever in children (irrespective of cause), female genital tract infection, and non-specific urethritis in males.
 
Yeast Cells (Candida)
 
These are round or oval structures of approximately the same size as red blood cells. In contrast to red cells, they show budding, are oval and more refractile, and are not soluble in 2% acetic acid.
 
Presence of Candida in urine may suggest immunocompromised state, vaginal candidiasis, or diabetes mellitus. Usually pyuria is present if there is infection by Candida. Candida may also be a contaminant in the sample and therefore urine sample must be examined in a fresh state.
 
Trichomonas vaginalis
 
These are motile organisms with pear shape, undulating membrane on one side, and four flagellae. They cause vaginitis in females and are thus contaminants in urine. They are easily detected in fresh urine due to their motility.
 
Eggs of Schistosoma haematobium
 
Infection by this organism is prevalent in Egypt.
 
Microfilariae
 
They may be seen in urine in chyluria due to rupture of a urogenital lymphatic vessel.
 
CASTS
 
Urinary casts are cylindrical, cigar-shaped microscopic structures that form in distal renal tubules and collecting ducts. They take the shape and diameter of the lumina (molds or ‘casts’) of the renal tubules. They have parallel sides and rounded ends. Their length and width may be variable. Casts are basically composed of a precipitate of a protein that is secreted by tubules (Tamm-Horsfall protein). Since casts form only in renal tubules their presence is indicative of disease of the renal parenchyma. Although there are several types of casts, all urine casts are basically hyaline; various types of casts are formed when different elements get deposited on the hyaline material (Figure 825.5). Casts are best seen under low power objective (×10) with condenser lowered down to reduce the illumination.
 
Figure 825.5 Genesis of casts in urine
 Figure 825.5 Genesis of casts in urine. All cellular casts degenerate to granular and waxy casts
 
Casts are the only elements in the urinary sediment that are specifically of renal origin.
 
Casts (Figure 825.6) are of two main types:
 
  1. Noncellular: Hyaline, granular, waxy, fatty
  2. Cellular: Red blood cell, white blood cell, renal tubular epithelial cell.
 
Hyaline and granular casts may appear in normal or diseased states. All other casts are found in kidney diseases.
 
Figure 825.6 Urinary casts
Figure 825.6 Urinary casts: (A) Hyaline cast, (B) Granular cast, (C) Waxy cast, (D) Fatty cast, (E) Red cell cast, (F) White cell cast, and (G) Epithelial cast
 
Non-cellular Casts
 
Hyaline casts: These are the most common type of casts in urine and are homogenous, colorless, transparent, and refractile. They are cylindrical with parallel sides and blunt, rounded ends and low refractive index. Presence of occasional hyaline cast is considered as normal. Their presence in increased numbers (“cylinduria”) is abnormal. They are composed primarily of Tamm-Horsfall protein. They occur transiently after strenuous muscle exercise in healthy persons and during fever. Increased numbers are found in conditions causing glomerular proteinuria.
 
Granular casts: Presence of degenerated cellular debris in a cast makes it granular in appearance. These are cylindrical structures with coarse or fine granules (which represent degenerated renal tubular epithelial cells) embedded in Tamm-Horsfall protein matrix. They are seen after strenuous muscle exercise and in fever, acute glomerulonephritis, and pyelonephritis.
 
Waxy cast: These are the most easily recognized of all casts. They form when hyaline casts remain in renal tubules for long time (prolonged stasis). They have homogenous, smooth glassy appearance, cracked or serrated margins and irregular broken-off ends. The ends are straight and sharp and not rounded as in other casts. They are light yellow in color. They are most commonly seen in end-stage renal failure.
 
Fatty casts: These are cylindrical structures filled with highly refractile fat globules (triglycerides and cholesterol esters) in Tamm-Horsfall protein matrix. They are seen in nephrotic syndrome.
 
Broad casts: Broad casts form in dilated distal tubules and are seen in chronic renal failure and severe renal tubular obstruction. Both waxy and broad casts are associated with poor prognosis.
 
Cellular Casts
 
To be called as cellular, casts should contain at least three cells in the matrix. Cellular casts are named according to the type of cells entrapped in the matrix.
 
Red cell casts: These are cylindrical structures with red cells in Tamm-Horsfall protein matrix. They may appear brown in color due to hemoglobin pigmentation. These have greater diagnostic importance than any other cast. If present, they help to differentiate hematuria due to glomerular disease from hematuria due to other causes. RBC casts usually denote glomerular pathology e.g. acute glomerulonephritis.
 
White cell casts: These are cylindrical structures with white blood cells embedded in Tamm-Horsfall protein matrix. Leucocytes usually enter into tubules from the interstitium and therefore presence of leucocyte casts indicates tubulointerstitial disease like pyelonephritis.
 
Renal tubular epithelial cell casts: These are composed of renal tubular epithelial cells that have been sloughed off. They are seen in acute tubular necrosis, viral renal disease, heavy metal poisoning, and acute allograft rejection. Even an occasional renal tubular cast is a significant finding.
 
CRYSTALS
 
Crystals are refractile structures with a definite geometric shape due to orderly 3-dimensional arrangement of its atoms and molecules. Amorphous material (or deposit) has no definite shape and is commonly seen in the form of granular aggregates or clumps.
 
Crystals in urine (Figure 825.7) can be divided into two main types: (1) Normal (seen in normal urinary sediment), and (2) Abnormal (seen in diseased states).
 
Figure 825.7 Crystals in urine
Figure 825.7 Crystals in urine. (A) Normal crystals: (1) Calcium oxalate, (2) Triple phosphates, (3) Uric acid, (4) Amorphous phosphates, (5) Amorphous urates, (6) Ammonium urate. (B) Abnormal crystals: (1) Cysteine, (2) Cholesterol, (3) Bilirubin, (4) Tyrosine, (5) Sulfonamide, and (6) Leucine
 
However, crystals found in normal urine can also be seen in some diseases in increased numbers.
 
Most crystals have no clinical importance (particularly phosphates, urates, and oxalates). Crystals can be identified in urine by their morphology. However, before reporting presence of any abnormal crystals, it is necessary to confirm them by chemical tests.
 
Normal Crystals
 
Crystals present in acid urine:
 
  1. Uric acid crystals: These are variable in shape (diamond, rosette, plates), and yellow or red-brown in color (due to urinary pigment). They are soluble in alkali, and insoluble in acid. Increased numbers are found in gout and leukemia. Flat hexagonal uric acid crystals may be mistaken for cysteine crystals that also form in acid urine.
  2. Calcium oxalate crystals: These are colorless, refractile, and envelope-shaped. Sometimes dumbbell-shaped or peanut-like forms are seen. They are soluble in dilute hydrochloric acid. Ingestion of certain foods like tomatoes, spinach, cabbage, asparagus, and rhubarb causes increase in their numbers. Their increased number in fresh urine (oxaluria) may also suggest oxalate stones. A large number are seen in ethylene glycol poisoning.
  3. Amorphous urates: These are urate salts of potassium, magnesium, or calcium in acid urine. They are usually yellow, fine granules in compact masses. They are soluble in alkali or saline at 60°C.
 
Crystals present in alkaline urine:
 
  1. Calcium carbonate crystals: These are small, colorless, and grouped in pairs. They are soluble in acetic acid and give off bubbles of gas when they dissolve.
  2. Phosphates: Phosphates may occur as crystals (triple phosphates, calcium hydrogen phosphate), or as amorphous deposits.
    Phosphate crystals
    Triple phosphates (ammonium magnesium phosphate): They are colorless, shiny, 3-6 sided prisms with oblique surfaces at the ends (“coffinlids”), or may have a feathery fern-like appearance.
    Calcium hydrogen phosphate (stellar phosphate): These are colorless, and of variable shape (starshaped, plates or prisms).
    Amorphous phosphates: These occur as colorless small granules, often dispersed.
    All phosphates are soluble in dilute acetic acid.
  3. Ammonium urate crystals: These occur as cactus-like (covered with spines) and called as ‘thornapple’ crystals. They are yellow-brown and soluble in acetic acid at 60°C.
 
Abnormal Crystals
 
They are rare, but result from a pathological process.
 
These occur in acid pH, often in large amounts. Abnormal crystals should not be reported on microscopy alone; additional chemical tests are done for confirmation.
 
  1. Cysteine crystals: These are colorless, clear, hexagonal (having 6 sides), very refractile plates in acid urine. They often occur in layers. They are soluble in 30% hydrochloric acid. They are seen in cysteinuria, an inborn error of metabolism. Cysteine crystals are often associated with formation of cysteine stones.
  2. Cholesterol crystals: These are colorless, refractile, flat rectangular plates with notched (missing) corners, and appear stacked in a stair-step arrangement. They are soluble in ether, chloroform, or alcohol. They are seen in lipiduria e.g. nephrotic syndrome and hypercholesterolemia. They can be positively identified by polarizing microscope.
  3. Bilirubin crystals: These are small (5 μ), brown crystals of variable shape (square, bead-like, or fine needles). Their presence can be confirmed by doing reagent strip or chemical test for bilirubin. These crystals are soluble in strong acid or alkali. They are seen in severe obstructive liver disease.
  4. Leucine crystals: These are refractile, yellow or brown, spheres with radial or concentric striations. They are soluble in alkali. They are usually found in urine along with tyrosine in severe liver disease (cirrhosis).
  5. Tyrosine crystals: They appear as clusters of fine, delicate, colorless or yellow needles and are seen in liver disease and tyrosinemia (an inborn error of metabolism). They dissolve in alkali.
  6. Sulfonamide crystals: They are variably shaped crystals, but usually appear as sheaves of needles. They occur following sulfonamide therapy. They are soluble in acetone.

TEST FOR DETECTION OF BILE SALTS IN URINE

Published in Clinical Pathology
Thursday, 10 August 2017 00:34
Bile salts are salts of four different types of bile acids: cholic, deoxycholic, chenodeoxycholic, and lithocholic. These bile acids combine with glycine or taurine to form complex salts or acids. Bile salts enter the small intestine through the bile and act as detergents to emulsify fat and reduce the surface tension on fat droplets so that enzymes (lipases) can breakdown the fat. In the terminal ileum, bile salts are absorbed and enter in the blood stream from where they are taken up by the liver and re-excreted in bile (enterohepatic circulation).
 
Bile salts along with bilirubin can be detected in urine in cases of obstructive jaundice. In obstructive jaundice, bile salts and conjugated bilirubin regurgitate into blood from biliary canaliculi (due to increased intrabiliary pressure) and are excreted in urine. The test used for their detection is Hay’s surface tension test. The property of bile salts to lower the surface tension is utilized in this test.
 
Take some fresh urine in a conical glass tube. Urine should be at the room temperature. Sprinkle on the surface particles of sulphur. If bile salts are present, sulphur particles sink to the bottom because of lowering of surface tension by bile salts. If sulphur particles remain on the surface of urine, bile salts are absent.
 
Thymol (used as a preservative) gives false positive test.

TEST FOR DETECTION OF BILIRUBIN IN URINE

Published in Clinical Pathology
Wednesday, 09 August 2017 12:21
Bilirubin is converted to non-reactive biliverdin on exposure to light (daylight or fluorescent light) and on standing at room temperature. Biliverdin cannot be detected by tests that detect bilirubin. Therefore fresh sample that is kept protected from light is required. Findings associated with bilirubinuria are listed below.
 

Methods for detection of bilirubin in urine are foam test, Gmelin’s test, Lugol iodine test, Fouchet’s test, Ictotest tablet test, and reagent strip test.
 
  1. Foam test: About 5 ml of urine in a test tube is shaken and observed for development of yellowish foam. Similar result is also obtained with proteins and highly concentrated urine. In normal urine, foam is white.
  2. Gmelin’s test: Take 3 ml of concentrated nitric acid in a test tube and slowly place equal quantity of urine over it. The tube is shaken gently; play of colors (yellow, red, violet, blue, and green) indicates positive test (Figure 823.1).
  3. Lugol iodine test: Take 4 ml of Lugol iodine solution (Iodine 1 gm, potassium iodide 2 gm, and distilled water to make 100 ml) in a test tube and add 4 drops of urine. Mix by shaking. Development of green color indicates positive test.
  4. Fouchet’s test: This is a simple and sensitive test.
    i. Take 5 ml of fresh urine in a test tube, add 2.5 ml of 10% of barium chloride, and mix well. A precipitate of sulphates appears to which bilirubin is bound (barium sulphate-bilirubin complex).
    ii. Filter to obtain the precipitate on a filter paper.
    iii. To the precipitate on the filter paper, add 1 drop of Fouchet’s reagent. (Fouchet’s reagent consists of 25 grams of trichloroacetic acid, 10 ml of 10% ferric chloride, and distilled water 100 ml).
    iv. Immediate development of blue-green color around the drop indicates presence of bilirubin (Figure 823.2).
  5. Reagent strips or tablets impregnated with diazo reagent: These tests are based on reaction of bilirubin with diazo reagent; color change is proportional to the concentration of bilirubin. Tablets (Ictotest) detect 0.05-0.1 mg of bilirubin/dl of urine; reagent strip tests are less sensitive (0.5 mg/dl).
 
Figure 823.1 Positive Gmelins test for bilirubin showing play of colors
Figure 823.1 Positive Gmelin’s test for bilirubin showing play of colors
 
Figure 823.2 Positive Fouchets test for bilirubin in urine
Figure 823.2 Positive Fouchet’s test for bilirubin in urine
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