SEMEN ANALYSIS , THYROID

FUNCTION TESTS, RENAL

FUNCTION TESTS OR LIVER
FUNCTION TESTS

Students should Describe and interpret the Abnormalities in a panel

containing Semen analysis, thyroid function tests, Renal function tests
A)SEMEN ANALYSIS :
INDICATIONS FOR SEMEN ANALYSIS :
• Investigation of infertility: Semen analysis is the first step in the investigation
of infertility. About 30% cases of infertility are due to problem with males.
• To check the effectiveness of vasectomy by confirming absence of sperm.
• To support or disprove a denial of paternity on the grounds of sterility.
• To examine vaginal secretions or clothing stains for the presence of semen in
medicolegal cases.
• For selection of donors for artificial insemination.
• For selection of assisted reproductive technology, e.g. in vitro fertilization,
gamete intrafallopian transfer technique.
 COLLECTION OF SEMEN

• Semen specimen is collected after about 3 days of sexual abstinence.

• The sample is obtained by masturbation, collected in a clean, dry, sterile, and
leakproof wide-mouthed plastic container, and brought to the laboratory
within 1 hour of collection.
• The entire ejaculate is collected, as the first portion is the most concentrated
and contains the highest number of sperms.
• During transport to the laboratory, the specimen should be kept as close to
body temperature as possible (i.e. by carrying it in an inside pocket).
• Ideally, the specimen should be obtained near the testing site in an adjoining
room.
• Two semen specimens should be examined that are collected 2-3 weeks apart.
Tests done on seminal fluid :
• Physical examination: Time to liquefaction, viscosity, volume, pH, color
• Microscopic examination: Sperm count, vitality, motility, morphology, and
proportion of white cells
• Immunologic analysis: Antisperm antibodies (SpermMAR test,
Immunobead test)
• Bacteriologic analysis: Detection of infection
• Biochemical analysis: Fructose, zinc, acid phosphatase, carnitine.
• Sperm function tests: Postcoital test, cervical mucus penetration test,
Hamster egg penetration assay, hypoosmotic swelling of flagella, and
computer-assisted semen analysis.
 Terminology in semen analysis
• Normozoospermia: All semen parameters normal
• Oligozoospermia: Sperm concentration <20 million/ml (mild to moderate: 5-20 million/ml;
severe: <5 million/ml)
• Azoospermia: Absence of sperms in seminal fluid
• Aspermia: Absence of ejaculate
• Asthenozoospermia: Reduced sperm motility; <50% of sperms showing class (a) and class
(b) type of motility OR <25% sperms showing class (a) type of motility.
• Teratozoospermia: Spermatozoa with reduced proportion of normal morphology (or
increased proportion of abnormal forms)
• Leukocytospermia: >1 million white blood cells/ml of semen.
• Oligoasthenoteratozoospermia: All sperm variables are abnormal
• Necrozoospermia: All sperms are non-motile or non-viable
NORMAL VALUES OF SEMEN
ANALYSIS
BIOCHEMICAL VARIABLE OF SEMEN
ANALYSIS
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
B)THYROID FUNCTION TEST :
• Biochemical tests for diagnosis of a thyroid disorder are called as thyroid function tests.
• The first-line tests are serum TSH, total T4 or free T4 , and total T3 and free T3.
Thyroid Stimulating Hormone (TSH) :
• Most important single test to assess thyroid function and to monitor thyroid hormone
replacement therapy is a sensitive TSH assay.
• TSH is a hormone secreted by anterior pituitary gland.
• A normal TSH level excludes thyroid disease.
• Normal reference range in adults is 0.5 –5.0 mU/L and in newborns < 20 mU/L. In adults,
borderline increase is 5-10 mU/L, while values >10 mU/L are considered as high. Values
less than 0.1 mU/L are low.
• Third and fourth generations TSH assays have detection limits of 0.01 to 0.02 mU/L and
0.001 to 0.002 mU/L,respectively.
Low TSH Increased TSH

Primary hyperthyroidism Primary hypothyroidism

T3 toxicosis Secondary hyperthyroidism

Secondary and tertiary hypothyroidsim

TOTAL THYROXINE (T4) :
• Total serum thyroxine includes both free and protein bound thyroxine and
is usually measured by competitive immunoassay. Normal level in adults
is 5.0-12.0 g/dl.
• Test for total thyroxine or free thyroxine is usually combined with TSH
measurement and together they give the best assessment of thyroid
function
Increased Total T4 Decreased Total T4

Hyperthyroidsim Primary hypothyroidism

Increase thyroxin- binding globulin Secondary hypothyroidism

Factitious hyperthyroidism Tertiary hypothyroidism

Pituitary TSH-secreting Tumor Hypoproteinaemia

Drugs : oestrogen, danazol

Severe non thyroidal illness

Free Thyroxine (FT4)
• FT4 comprises of only a small fraction of total T4, is unbound to proteins,
and is the metabolically active form of the hormone. It constitutes about
0.05% of total T4.
• Normal range is 0.7 to 1.9 ng/dl.
• Measurement of FT4 is helpful in those situations in which total T4 level is likely
to be altered due to alteration in TBG level (e.g. pregnancy, oral
contraceptives,nephrotic syndrome).
Total and Free Triiodothyronine (T3)
USES

1. Diagnosis of T3 thyrotoxicosis: Hyperthyroidism with low TSH and elevated T3,

and normal T4/FT4 is termed T3 thyrotoxicosis.
2. Early diagnosis of hyperthyroidism: In early stage of hyperthyroidism, total T4
and free T4 levels are normal, but T3 is elevated.
• For routine assessment of thyroid function, TSH and T4 are measured. T3
is not routinely estimated because normal plasma levels are very low.
• Normal T3 level is 80-180 ng/dl.
Thyrotropin Releasing Hormone (TRH) Stimulation Test :
Uses
1. Confirmation of diagnosis of secondary hypothyroidism
2. Evaluation of suspected hypothalamic disease
3. Suspected hyperthyroidism
• This test is not much used nowadays due to the availability of sensitive TSH assays.
Procedure :
• A baseline blood sample is collected for estimation of basal serum TSH level.
• TRH is injected intravenously (200 or 500 μg) followed by measurement of serum
TSH at 20 and 60 minutes.
• INTERPRETATION
1. Normal response: A rise of TSH > 2 mU/L at 20 minutes, and a small decline at 60
minutes.
2. Exaggerated response: A further significant rise in already elevated TSH level at
20 minutes followed by a slight decrease at 60 minutes; occurs in primary
hypothyroidism.
3. Flat response: There is no response; occurs in secondary (pituitary)
hypothyroidism.
4. Delayed response: TSH is higher at 60 minutes as compared to its level at 20
minutes; seen in tertiary (hypothalamic) hypothyroidism.
Thyroid autoantibodies
• Useful for diagnosis and monitoring of autoimmune thyroid diseases.
• Antimicrosomal or antithyroid peroxidase antibodies: Hashimoto’s
thyroiditis
• Anti-TSH receptor antibodies: Graves’ disease
Interpretation of thyroid function tests
Test Results Interpretations

TSH Normal, FT4 Normal Euthyroid

Low TSH, Low FT4 Secondary hypothyroidism

High TSH , Normal FT4 Subclinical hypothyroidism

High TSH , Low FT4 Primary hypothyroidism

Low TSH , Normal FT4 , Normal FT3 Subclinical hyperthyroidism

Low TSH, Normal FT4, High FT3 T3 toxicosis

Low TSH, High FT4 Primary hyperthyroidism

Terminology in thyroid disorders
• Primary hyper-/hypothyroidism: Increased or decrease of function of thyroid gland due to
disease of thyroid itself and not due to increased or decreased levels of TRH or TSH.
• Secondary hyper-/hypothyroidism: Increased ordecreased function of thyroid gland due
to increased or decreased levels of TSH.
• Tertiary hypothyroidism: Decreased function of thyroid gland due to decreased function
of hypothalamus.
• Subclinical thyroid disease: A condition with abnormality of thyroid hormone levels in
blood but without specific clinical manifestations of thyroid disease and without any history of
thyroid dysfunction or therapy.
• Subclinical hyperthyroidism: A condition with normal thyroid hormone levels but with low
or undetectable TSH level.
• Subclinical hypothyroidism: A condition with normal thyroxine and triiodothyronine level
along with mildly elevated TSH level.
Causes of hyperthyroidism :

1. Graves’ disease (Diffuse toxic goiter)

2. Toxicity in multinodular goiter
3. Toxicity in adenoma
4. Subacute thyroiditis
5. TSH-secreting pituitary adenoma (secondary hyperthyroidism)
6. Trophoblastic tumours that secrete TSH-like hormone: choriocarcinoma,
hydatidiform mole
7. Factitious hyperthyroidism
Thyroid function tests in hyperthyroidism
• Thyrotoxicosis:
– Serum TSH low or undetectable
– Raised total T4 and free T4.
• T3 toxicosis:
– Serum TSH undetectable
– Normal total T4 and free T4
– Raised T3
Causes of hypothyroidism :

1. Primary hypothyroidism (Increased TSH)

• Iodine deficiency
• Hashimoto’s thyroiditis
• Exogenous goitrogens
• Iatrogenic: surgery, drugs, radiation
2. Secondary hypothyroidism (Low TSH): Diseases of pituitary
3. Tertiary hypothyroidism (Low TSH, Low TRH): Diseases of
hypothalamus
C)RENAL FUNCTION TEST :
INDICATIONS :
1. Early identification of impairment of renal function in patients with increased
risk of chronic renal disease.
2. Diagnosis of renal disease
3. Follow the course of renal disease and assess response to treatment.
4. Plan renal replacement therapy (dialysis or renal transplantation) in advanced
renal disease.
5. Adjust dosage of certain drugs (e.g. chemotherapy) according to renal function.
Tests to evaluate glomerular function
1. Clearance tests to measure glomerular
filtration rate: Inulin clearance, 125I-iothalamate
clearance, 51Cr-EDTA clearance, Cystatin C
clearance, Creatinine clearance, and Urea
clearance
2. Calculation of creatinine clearance from
prediction equations
3. Blood biochemistry: Serum creatinine,
Blood urea nitrogen (BUN), and BUN/serum creatinine ratio
4. Microalbuminuria and albuminuria
Tests to evaluate tubular function
1. Tests to assess proximal tubular
function:
• Glycosuria, phosphaturia, uricosuria
• Generalized aminoaciduria
• Tubular proteinuria
• Fractional sodium excretion
2. Tests to assess distal tubular functions:
• Specific gravity and osmolality of urine
• Water-deprivation test and water-
loading test
• Ammonium chloride loading test
 Glomerular filtration rate (GFR)
• Best test for assessment of excretory renal function
• Varies according to age, sex, and body weight of an individual; a normal GFR also
depends on normal renal blood flow and pressure.
• Normal GFR in young adults is 120-130 ml/min per 1.73 m2.
• Creatinine clearance is commonly used as a measureof GFR.
Equations can be used to estimate GFR from serum creatinine value.
• GFR declines with age (due to glomerular arteriolosclerosis)
• GFR <60 ml/min per 1.73 m2 indicates loss of ≥50% of kidney function.
• Fall in GFR leads to accumulation of waste products of metabolism in blood. GFR <15
ml/min per 1.73 m2 is associated with uremia
 Clearance Tests to Measure Glomerular
Filtration Rate (GFR)
• 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). Clearance of a substance refers to the volume of plasma, which is completely
cleared of that substance per minute; it is calculated from the following formula:
Clearance = UV/P
U = concentration of a substance in urine
V = volume of urine excreted
P= concentration of the substance in plasma
 Cystatin C Clearance
• This is a cysteine protease inhibitor of MW 13,000, which is produced at a
constant rate by all the nucleated cells.
• It is not bound to protein, is freely filtered by glomeruli and is not
returned to circulation after filtration.
• It is a more sensitive and specific marker of impaired renal function than
plasma creatinine.
• Its level is not affected by sex, diet, or muscle mass.
• It is thought that cystatin C is a superior marker for estimation of GFR
than creatinine clearance.
• It is measured by immunoassay.
Inulin Clearance
• Inulin, an inert plant polysaccharide (a fructose polymer), is filtered by
the glomeruli and is neither reabsorbed nor secreted by the tubules;
therefore it is an ideal agent for measuring GFR.
• A bolus dose of inulin (25 ml of 10% solution IV) is administered
followed by constant intravenous infusion (500 ml of 1.5% solution at
the rate of 4 ml/min).
• Timed urine samples are collected and blood samples are obtained at
the midpoint of timed urine collection.
• This test is considered as the ‘gold standard’ (or reference method)
for estimation of GFR.
 Creatinine Clearance
• This is the most commonly used test for measuring GFR.
• Creatinine is being produced constantly from creatinine in muscle.
• It is completely filtered by glomeruli and is not reabsorbed by tubules;
however, a small amount is secreted by tubules.
• A 24-hour urine sample is preferred to overcome the problem of
diurnal variation of creatinine excretion and to reduce the inaccuracy
in urine collection.
• Creatinine clearance is calculated from
(1) concentration of creatinine in urine in mg/ml (U)
(2) volume of urine excreted in ml/min (V) (this is calculated by the formula:
volume of urine collected/collection time in minutes e.g. volume of urine collected
in 24 hours ÷ 1440), and
(3) concentration ofcreatinine in plasma in mg/dl (P). Creatinine clearance in
ml/min per 1.73 m2 is then derived from the formula

UV/P.
 Disadvantages of Creatinine clearance
Creatinine clearance is not an ideal test for estimation of GFR because of
following reasons:
1. A small amount of creatinine is secreted by renal tubules that increase
even further in advanced renal failure.
2. Collection of urine is often incomplete.
3. Creatinine level is affected by intake of meat andmuscle mass.
4. Creatinine level is affected by certain drugs like cimetidine, probenecid,
and trimethoprim (which block tubular secretion of creatinine).
 Urea Clearance
• Urea is filtered by the glomeruli, but about 40% of the filtered amount is
reabsorbed by the tubules.
• The reabsorption depends on the rate of urine flow. Thus it underestimates GFR,
depends on the urine flow rate, and is not a sensitive indicator of GFR.
• BUN and serum creatinine, by themselves, are not sensitive indicators of early renal
impairment since values may be normal e.g. if baseline values of serum creatinine is
0.5 mg/dl, then 50% reduction in kidney function would increase it to 1.0 mg/dl.
• Thus clearance tests are more helpful in early cases. If biochemical tests are normal
and renal function impairment is suspected, then creatinine clearance test should
be carried out.
• If biochemical tests are abnormal, then clearance tests need not be done.
 Tests to Evaluate Tubular Function
Tests to Assess Proximal Tubular Function
1. Glycosuria: In renal glycosuria, glucose is excreted in urine, while blood
glucose level is normal.
This is because of a specific tubular lesion which leads to impairment of
glucose reabsorption.
Glycosuria can also occur inFanconi syndrome
2.Generalized aminoaciduria: In proximal renal tubular dysfunction, many
amino acids are excreted in urine due to defective tubular reabsorption.
3)Tubular proteinuria (Low molecular weight proteinuria):
• Normally, low molecular weight proteins (β2 – microglobulin, retinol-binding protein,
lysozyme, and α1-microglobulin) are freely filtered by glomeruli and are completely
reabsorbed by proximal renal tubules.
• With tubular damage, these low molecular weight proteins are excreted in urine and
can be detected by urine protein electrophoresis.
• Increased amounts of these proteins in urine are indicative of renal tubular damage.
4) Urinary concentration of sodium:
• If both BUN and serum creatinine are acutely increased, it is necessary to distinguish
between prerenal azotemia (renal underperfusion) and acute tubular necrosis.
• In prerenal azotemia, renal tubules are functioning normally and reabsorb sodium,
while in acute tubular necrosis, tubular function is impaired and sodium
absorption is decreased.
Therefore, in prerenalazotemia, urinay sodium concentration is < 20 mEq/L while in
acute tubular necrosis, it is > 20 mEq/L.
5. Fractional excretion of sodium (FENa):
• Measurement of urinary sodium concentration is affected by urine
volume and can produce misleading results. Therefore, to avoid this,
fractional excretion of sodium is calculated.
• This refers to the percentage of filtered sodium that has been absorbed
and percentage that has been excreted.
• Measurement of fractional sodium excretion is a better indicator of
tubular absorption
of sodium than quantitation of urine sodium alone.
• This test is indicated in acute renal failure.
• In oliguric patients, this is the most reliable means of early distinction
between pre-renal failure and renal failure due to acute tubular necrosis.
 Tests to Assess Distal Tubular Function
1. Urine specific gravity
• Normal specific gravity is 1.003 to 1.030. It depends on state of hydration and fluid intake.
i. Causes of increased specific gravity: photo urinometer table
a. Reduced renal perfusion (with preservation of concentrating ability of tubules)
b. Proteinuria,
c. Glycosuria,
d. Glomerulonephritis.
e. Urinary tract obstruction.
ii. Causes of reduced specific gravity:
a. Diabetes insipidus
b. Chronic renal failure
c. Impaired concentrating ability due to diseases of tubules.
2. Urine osmolality
• The most commonly employed test to evaluate tubular function is measurement of
urine/ plasma osmolality.
• This is the most sensitive method for determination of ability of concentration.
• Osmolality measures number of dissolved particles in a solution.
• When solutes are dissolved in a solvent, certain changes take place like lowering of
freezing point, increase in boiling point, decrease in vapor pressure, or increase of
osmotic pressure of the solvent.
• These properties are made use of in measuring osmolality by an instrument called as
osmometer.
• Urine/plasma osmolality ratio is helpful in distinguishing pre-renal azotemia (in which
ratio is higher) from acute renal failure due to acute tubular necrosis (in which ratio is
lower).
• If urine and plasma osmolality are almost similar, then there is defective tubular
reabsorption of water.
3. Water deprivation test
• If the value of baseline osmolalityof urine is inconclusive, then water
deprivation test is performed. In this test, water intake is restricted for a
specified period of time followed by measurement of specific gravity or
osmolality.
• Normally, urine osmolality should rise in response to water deprivation.
• If it fails to rise, then desmopressin is administered to differentiate between
central diabetes insipidus and nephrogenic diabetes insipidus.
• Urinary concentration ability is corrected after administration of desmopressin
in central diabetes insipidus, but not in nephrogenic diabetes insipidus.
• If urine osmolality is > 800 mOsm/kg of water or specific gravity is ≥1.025
following dehydration, concentrating ability of renal tubules is normal.
• However, normal result does not rule out presence of renal disease.
• False result will be obtained if the patient is on low-salt, low-protein diet or is
suffering from major electrolyte and water disturbance.
4. Water loading antidiuretic hormone suppression test
• This test assesses the capacity of the kidney to make urine dilute after water
loading.
• After overnight fast, patient empties the bladder and drinks 20 ml/kg of water in
15-30 minutes.
• The urine is collected at hourly intervals for the next 4 hours for
measurements of urine volume, specific gravity, and osmolality.
• Plasma levels of antidiuretic hormone and serum osmolality should be measured
at hourly intervals
• Normally, more than 90% of water should be excreted in 4 hours and specific
gravity should fall to 1.003 and osmolality should fall to < 100 mOsm/kg.
• Plasma level of antidiuretic hormone should be appropriate for serum osmolality.
• In renal function impairment, urine volume is reduced (<80% of fluid intake is
excreted) and specific gravity and osmolality fail to decrease.
• The test is also impaired in adrenocortical insufficiency, malabsorption, obesity,
ascites, congestive heart failure, cirrhosis, and dehydration..