Dayyal Dg.

Dayyal Dg.

Clinical laboratory professional specialized to external quality assessment (proficiency testing) schemes for Laboratory medicine and clinical pathology. Author/Writer/Blogger

One can estimate GFR from age, sex, body weight, and serum creatinine value of a person from the following formula (Cockcroft and Gault):
 
 
Creatinine clearance in ml/min = (140 - Age in years) × (Body weight in kg)
                                                              (72 × Serum creatinine in mg/dl)
 
 
In females, the value obtained from above equation is multiplied by 0.85 to get the result.
 
It is recommended by National Kidney Foundation (USA) to calculate creatinine clearance by Cockcroft and Gault or other equation from serum creatinine value rather than estimating creatinine clearance from a 24-hour urine sample. This is because the latter test is inconvenient, time-consuming, and often inaccurate.

Glomerular filtration rate refers to the rate in ml/min at which a substance is cleared from the circulation by the glomeruli. The ability of the glomeruli to filter a substance from the blood is assessed by clearance studies. If a substance is not bound to protein in plasma, is completely filtered by the glomeruli, and is neither secreted nor reabsorbed by the tubules, then its clearance rate is equal to the glomerular filtration rate (GFR).

Renal biopsy refers to obtaining a small piece of kidney tissue for microscopic examination. Percutaneous renal biopsy was first performed by Alwall in 1944.

Diabetes mellitus (DM) is a metabolic group of disorders characterized by persistent hyperglycemia due to deficiency and/or diminished effectiveness of insulin. There are derangements of carbohydrate, protein, and fat metabolism due to failure of insulin action on target cells.

Wednesday, 16 August 2017 07:56

PREGNANCY TESTS

Pregnancy tests detect human chorionic gonadotropin (hCG) in serum or urine. Although pregnancy is the most common reason for ordering the test for hCG, measurement of hCG is also indicated in other conditions as shown in Box 836.1.

Human chorionic gonadotropin is a glycoprotein hormone produced by placenta that circulates in maternal blood and excreted intact by the kidneys. It consists of two polypeptide subunits: α (92 amino acids) and β (145 amino acids) which are non-covalently bound to each other. Structurally, hCG is closely related to three other glycoprotein hormones, namely, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH). The α subunits of hCG, LH, FSH, and TSH are similar, while β subunits differ and confer specific biologic and immunologic properties. Immunological tests use antibodies directed against β-subunit of hCG to avoid cross-reactivity against LH, FSH, and TSH.

Semen (or seminal fluid) is a fluid that is emitted from the male genital tract and contains sperms that are capable of fertilizing female ova. Structures involved in production of semen are:

  • Testes: Male gametes or spermatozoa (sperms) are produced by testes; constitute 2-5% of semen volume.
  • Epididymis: After emerging from the testes, sperms are stored in the epididymis where they mature; potassium, sodium, and glycerylphosphorylcholine (an energy source for sperms) are secreted by epididymis.
  • Vas deferens: Sperms travel through the vas deferens to the ampulla which is another storage area. Ampulla secretes ergothioneine (a yellowish fluid that reduces chemicals) and fructose (source of nutrition for sperms).
  • Seminal vesicles: During ejaculation, nutritive and lubricating fluids secreted by seminal vesicles and prostate are added. Fluid secreted by seminal vesicles consists of fructose (energy source for sperms), amino acids, citric acid, phosphorous, potassium, and prostaglandins. Seminal vesicles contribute 50% to semen volume.
  • Prostate: Prostatic secretions comprise about 40% of semen volume and consist of citric acid, acid phosphatase, calcium, sodium, zinc, potassium, proteolytic enzymes, and fibrolysin.
  • Bulbourethral glands of Cowper secrete mucus.
Monday, 14 August 2017 20:44

SPERM FUNCTION TESTS OR FUNCTIONAL ASSAYS

These tests are available only in specialized andrology laboratories. The tests are not standardized thus making interpretation difficult. If used singly, a sperm function test may not be helpful in fertility assessment. They are more predictive if used in combination.
 
Postcoital (Sims-Huhner) Test
 
This is the examination of the cervical mucus after coitus and assesses the ability of the sperm to penetrate the cervical mucus. The quality of the cervical mucus varies during the menstrual cycle, becoming more abundant and fluid at the time of ovulation (due to effect of estrogen); this facilitates penetration of the mucus by the spermatozoa. Progesterone in the secretory phase increases viscosity of the mucus. Therefore cervical mucus testing is scheduled just before ovulation (determined by basal body temperature records or follicular sizing by ultrasonography). Postcoital test is the traditional method to detect the cervical factor in infertility. Cervical mucus is aspirated with a syringe shortly before the expected time of ovulation and 2-12 hours after intercourse. Gross and microscopic examinations are carried out to assess the quality of cervical mucus (elasticity and drying pattern) and to evaluate the number and motility of sperms (Box 834.1). If ≥ 10 motile sperms are observed the test is considered as normal. An abnormal test may result from: (a) poor quality of cervical mucus due to wrong judgment of ovulation, cervicitis or treatment with antioestrogens (e.g. Clomid), and (b) absence of motile sperms due to ineffective technique of coitus, lack of ejaculation, poor semen quality, use of coital lubricants that damage the sperm, or presence of antisperm antibodies. Antisperm antibodies cause immotile sperms, or agglutination or clumping of sperms; they may be present in either partner. If cervical factor is present, intrauterine insemination is the popular treatment. The value of the postcoital test is disputed in the medical literature.
 
Box 834.1 Interpretation of postcoital test
  • Normal: Sperms are normal in amount and moving forward in the mucus; mucus stretches atleast 2 inches (5 cm) and dries in a fern-like manner.
  • Abnormal: Absence of sperms or large number of sperms are dead or sperms are clumped; cervical mucus cannot stretch 2 inches (5 cm) or does not dry in a fern-like manner.
 
This test can be carried out if semen analysis is normal, and the female partner is ovulating and fallopian tubes are not blocked. It is also done if antisperm antibodies are suspected and male partner refuses semen analysis.
 
Cervical Mucus Penetration Test
 
In this test, greatest distance traveled by the sperm in seminal fluid placed and incubated in a capillary tube containing bovine mucus is measured. Majority of fertile men show score >30 mm, while most infertile men show scores <20 mm.
 
Hamster Egg Penetration Assay
 
Hamster oocytes are enzymatically treated to remove the outer layers (that inhibit cross-species fertilization). They are then incubated with sperms and observed for penetration rate. It can be reported as (a) Number of eggs penetrated (penetration rate <15% indicates low fertility), or as (b) Number of sperm penetrations per egg (Normal >5). This test detects sperm motility, binding to oocyte, and penetration of oocyte. There is a high incidence of false-negative results.
 
Hypo-osmotic Swelling of Flagella
 
This test assesses the functional integrity of the plasma membrane of the sperm by observing curling of flagella in hypo-osmotic conditions.
 
Computer-assisted Semen Analysis
 
Computer software measures various characteristics of the spermatozoa; however, its role in predicting fertility potential is not confirmed.

ANTISPERM ANTIBODIES

The role of antisperm antibodies in causation of male infertility is controversial. The immunological tests done on seminal fluid include mixed antiglobulin reaction (MAR test) and immunobead test.

The antibodies against sperms immobilize or kill them, thus preventing their passage through the cervix to the ovum. The antibodies can be tested in the serum, seminal fluid, or cervical mucus. If the antibodies are present bound to the head of the sperm, they will prevent the penetration of the egg by the sperm. If antibodies are bound to the tail of the sperm, they will retard motility.

SpermMAR™ test

This test can detect IgG and IgA antibodies against sperm surface in semen sample. In direct SpermMAR™ IgG test, a drop each of semen (fresh and unwashed), IgG-coated latex particles, and anti-human immunoglobulin are mixed together on a glass slide. At least 200 motile spermatozoa are examined. If the spermatozoa have antibodies on their surface, antihuman immunoglobulin will bind IgG-coated latex particles to IgG on the surface of the spermatozoa; this will cause attachment of latex particles to spermatozoa, and motile, swimming sperms with attached particles will be seen. If the spermatozoa do not have antibodies on their surface, they will be seen swimming without attached particles; the latex particles will show clumping due to binding of their IgG to antihuman immunoglobulin.

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

In indirect SpermMAR™ tests, fluid without spermatozoa (e.g. serum) is tested for the presence of antisperm antibodies. First, antibodies are bound to donor spermatozoa which are then mixed with the fluid to be analyzed. These antibodies are then detected as described above for direct tests.

Atleast 200 motile spermatozoa should be counted. If >50% of spermatozoa show attached latex particles, immunological problem is likely.

Immunobead test

Antibodies bound to the surface of the spermatozoa can be detected by antibodies attached to immunobeads (plastic particles with attached anti-human immunoglobulin that may be either IgG, IgA, or IgM). Percentage of motile spermatozoa with attached two or more immunobeads are counted amongst 200 motile spermatozoa. Finding of >50% spermatozoa with attached beads is abnormal.

The most important test in semen analysis for infertility is microscopic examination of the semen.

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.
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