URINE SCREENING FOR METABOLIC DISORDERS

Overflow Vs. Renal Disorders

Overflow disorders result from the disruption of a normal metabolic pathway that causes plasma
concentration of the nonmetabolized substances. Abnormal accumulations of the renal type are caused by
malfunctions in the tubular reabsorption mechanism.

Causes of disruption of enzyme function:

1. Inborn error of metabolism failure to inherit the gene that produces that particular enzyme
2. Organ malfunction from disease
3. Toxic reactions

AMINO ACID DISORDERS

A. Phenylalanine-Tyrosine Disorders
1. Phenylketonuria (PKU)
Occurs when normal conversion of phenylalanine to tyrosine is disrupted,
caused by failure to inherit the gene to produce the enzyme phenylalanine hydroxylase
Occurs in 1 out of every 10,000-20,000 births; most well-known of the
aminoacidurias; inherited as and autosomal recessive trait
Results in mental retardation and albinism
Urine odor: mousy
Once discovered, phenylalanine (a major constituent of milk) is eliminated
from the diet. As the child matures, alternate pathways of phenylalanine develop and the dietary
restrictions can be eased.
Initial screening: Guthries blood test. Procedure: blood from heelstick is
absorbed into filter paper disks then placed on culture media streaked with microorganism Bacillus
subtilis. Presence of phenylalanine in the blood will counteract the action of beta-2-thienylalanine,
an inhibitor of B. subtilis present in the media, and growth will be observed around the paper disks.
Modifications of the Guthrie test will also detect maple syrup urine disease,
homosytinuria, tyrosinemia, histidinemia, valinemia, and galactosemia
Urine test: The addition of ferric chloride to urine containing phenylpyruvic
acid produces a permanent blue-green color. (Nonspecific test)
2. Tyrosyluria either inherited or metabolic defect. Urine may contain tyrosine or its degradation
products -hydroxyphenylpyruvic acid and -hydroxyphenyllactic acid
Most frequently seen is a transitory tyrosemia in premature infants which is
caused by underdevelopment of liver function necessary to complete tyrosine metabolism. Seldom
results in permanent damage but may be confused with PKU because ferric chloride test produces a
green color (rapidly fades).
Acquired sever liver disease is a more serious condition resembling transitory
newborn variety. In both cases tyrosine and leucine crystals are seen in the urine sediment.
Hereditary disorders are usually fatal and results in both liver and renal
disease and in the appearance of a generalized aminoaciduria.
Screening test for tyrosine and its metabolites is the nitroso-naphthol test.
Like ferric chloride it is nonspecific but the presence of an orange-red color shows a positive
reaction and indicates further testing.
3. Alkptonuria
Alkaptonuria was derived from the observation that urine from patients with
this condition darkened after becoming alkaline from standing at room temperature.
Occurs from failure to inherit the gene to produce the enzyme homogentisic
acid oxidase. Without this enzyme homogentisic acid accumulates in the blood, tissues, and urine.
Doesnt usually manifest clinically in early childhood but observations of
brown- or blacked-stained cloth diapers and reddish-stained disposable diapers have been reported.
In later life brown pigment becomes deposited in body tissues (ears). Deposits in cartilage lead to
arthritis and a high percentage develop liver and cardiac disorders
Screening tests:
- ferric chloride: transient deep blue
- Benedicts or Clinitest: yellow precipitate
- Addition of alkali to freshly voided urine (more specific)= darkening of urine; (ascorbic acid )
- Addition of silver nitrate and ammonium hydroxide will produce black urine
- Spectrophometric and chromatography procedures are available
4. Melanuria
A second metabolic pathway exists for tyrosine which is responsible for the
production of melanin, thyroxine, epinephrine, protein, and tyrosine-sulfate.
Like homogentisic acid, increased urinary melanin will produce a darkening of
urine which happens after urine is exposed to air.
Elevation of urinary melanin indicates an overproliferation of the melanocytes,
producing a malignant melanoma.
Screening tests:
- ferric chloride: gray or black precipitate
 - Sodium nitroprusside: red color. Interference from acetone and creatinine can be avoided by
adding glacial acetic acid: melanin green-black color; acetone purple; creatinine amber.

B. Branched-Chain Amino Acid Disorders

The branched-chain amino acids differ from other amino acids by having a methyl group that branches
from the main aliphatic carbon chain. A significant lab finding in branched-chain amino acid disorders is
the presence of ketonuria in a newborn.
1. Maple Syrup Urine Disease
Inborn error of metabolism, inherited as an autosomal recessive trait. The amino acids involve are
leucine, isoleucine, and valine. The metabolic pathway begins normally with the transamination of
the 3 amino acids in the liver to the keto acids (-ketoisovaleric, -ketoisocaproic, -keto--
methylvaleric). Failure to inherited the gene for the enzyme necessary to produce oxidative
decarboxylation of these keto acids result in their accumulation in the blood and urine
Newborns fail to thrive after 1 week
Urine odor: strong odor resembling maple syrup
Screening test: 2,4-dinitrophenylhydrazine (DNPH) reaction. Adding DNPH to urine that contains
keto acids will produce a yellow turbidity or precipitate
Confirmatory test: chromatography
2. Organic Acidemias
General symptoms: severe illness with vomiting accompanied by metabolic acidosis; hypoglycemia;
ketonuria; and increased serum ammonia.
Isovaleric acidemia sweaty feet urine odor caused by accumulation of isovalerylglycine due to
deficiency of isovaleryl CoA in the leucine pathway. No screening test available. Identification of
isovalerylglycine is by chromatography.
Propionic and methylmalonic acidemias result from errors in the metabolic pathway converting
isoleucine, valine, threonine, and methionine to succinyl CoA. Propionic acid is the immediate
precursor to methylmalonic acid in this pathway.
- screening test: -nitroaniline to produce an emerald green color in the presence of
methylmalonic acid

C. Tryptophan Disorders
1. Indicanuria - blue diaper syndrome ; inherited disorder
Caused by intestinal obstruction, abnormal bacteria, malabsorption syndromes, and Hartnup
disease,
Indican is colorless until oxidized by exposure to air to indigo blue
Urinary indican will react with acidic ferric chloride to form a deep blue or violet color that can be
extracted into chloroform
2. 5-Hydroxyindoleacetic acid
A second metabolic pathway for tryptophan is the production of serotonin used in stimulation of
smooth muscles
Serotonin is produced by argentaffin cells in the intestine and is carried in the body primarily by the
platelets. Cancinoid tumors involving argentaffin results in excess amounts of the degradation
product 5-HIAA
Addition of nitrous acid and 1-nitroso-2-naphthol to urine that contains 5-HIAA causes the
appearance of purple to black color, depending on amount present
Normal daily excretion is 2-8 mg, with argentaffin cell tumors it will be 160-628 mg/24 hours so
random or first morning urine can be used. If 24-hour sample is used, preserve with hydrochloric
acid or boric acid
Dietary instructions: No bananas, pineapples, and tomatoes. Medications should be withheld 72
hours before specimen collection.

D. Cystine Disorders
1. Cystinuria Inherited; inability of renal tubules to reabsorb cystine filtered by glomerulus
Two modes of inheritance: 1- cystine, lysine, arginine, and ornithine are not reabsorbed; 2- cystine
and lysine are not reabsorbed
Persons not able reabsorb the 4 amino acids have the tendency to form renal calculi. Only cystine
forms calculi, the rest have to be identified using chromatograhy procedures.
Screening test: sodium cyanide-nitroprusside test. Reduction of cystine by sodium cyanide followed
by addition of nitroprusside will produce a red-purple color in specimen that contains excess
cystine. False + occur with ketones and homocystine
2. Cystinosis Genuine error of metabolism
Cystine deposits in cornea, bone marrow, lymph nodes and internal organs. Major tubular
reabsorption defect also occurs
Lab findings: polyuria, generalized aminoaciduria, positive test for reducing substances, and lack of
urinary concentration
3. Homocystinuria
Defects in the metabolism of homocystine can result in failure to thrive, cataracts, mental
retardation, thromboembolic problems, and death
Additional screening test: silver nitroprusside
PORPHYRIN DISORDERS
Porhyrins are the intermediate compounds in the production of heme. Disorders of porphyrin
metabolism are collectively called porphyrias. Acquired porphyrias: lead poisoning, iron deficiency, liver
and renal disease
Urine color: red or port wine (red discoloration of infants diaper)
Screening tests: Erhlichs reaction and fluorescence under ultraviolet light in 550-600 nm
Ehrlich: detection of ALA and porphobilinogen. (ALA to porphobilinogen w/ acetylacetone)
Fluorescence: uroporphyrin, coproporhyrin, protoporhyrin. Negative: blue fluorescence;
Positive: violet, pink, or red depending on concentration
Free erythrocyte protoporphyrin (FEP): protoporphyrin

MUCOPOLYSACCHARIDE DISORDERS
Mucopolysaccharides or glycosaminoglycans are large compounds in connective tissue
Incomplete breakdown of polysaccharide portion accumulates in lysosomes and increased urine
excretion
Best known mucopolysaccharidoses: Hunters, Hurlers and Sanfilippos syndrome
Hunters & Hurlers: skeletal structure abnormal with severe mental retardation (fatal)
Sanfilippos: mental retardation
Screening tests: acid-albumin and cetyltrimethylammonium bromide (CTAB) turbidity test and
metachromatic staining spot test
Spot test: dip filter paper into 0.59% azure A dye in 2% acetic acid and air dry. Urine with
mucopolysaccharide will produce blue spot that cannot be washed away by dilute acidified methanol
solution

PURINE DISORDERS
Lesch-Nyhan disease, inherited as sex-linked recessive results in massive excretion of urinary uric acid
crystals.
Absence of enzyme hypoxanthine guanine phosphoribosyltransferase
Uric acid accumulates throughout the body
Severe motor defects, mental retardation, a tendency to self-destruction, gout, renal calculi
Normal development first 6-8 months
First symptom: uric acid crystals resembling orange sand in diapers

CARBOHYDRATE DISORDERS
Melituria is most frequently due to an inherited disorder. The finding of a positive copper reduction test
(Benedicts or Clinitest) is strongly suggestive of a disorder of carbohydrate metabolism.

Galactosuria
Failure to convert galactose to glucose.
Infant failure to thrive, liver disorders, cataracts, severe mental retardation.
Removal of lactose from the diet can prevent these symptoms.
 CEREBROSPINAL FLUID
Formation and Physiology
Approximately 20 mL of CSF is produced every hour by the choroid plexuses. It flows through the
subarachnoid space between the arachnoid mater and the pia mater. The CSF provides a physiologic
system to supply nutrients to the nervous tissue, to remove metabolic wastes, and to produce a
mechanical barrier to cushion the brain and spinal cord against trauma.
Normal CSF volume: Adults = 140-170 mL
Neonates = 10-60 mL

Specimen Handling and Collection:

CSF is routinely collected by lumbar puncture between the 3 rd, 4th, or 5th lumbar vertebrate. Specimens are
usually collected in 3 sterile tubes:
4. Tube 1 Chemistry/Serology (Frozen)
5. Tube 2 Microbiology (Room temperature)
6. Tube 3 Hematology (Refrigerated)

Appearance
CSF is normally crystal clear. The major terminology used to describe CSF appearance: crystal clear, cloudy
or turbid, milky, xanthochromic, and hemolyzed/bloody.

Table 10-1. Clinical Significance of Cerebrospinal Fluid Appearance

Appearance Cause Major Significance
Crystal clear Normal
Hazy, turbid, WBCs Meningitis
cloudy, milky RBCs Hemorrhage
Traumatic
Microorganisms Meningitis
Protein Disorders that affect blood-brain
barrier
Oily Radiographic contrast Production of IgG within the CNS
Bloody media
RBCs Hemorrhage
Xanthochromic Traumatic tap
Hemoglobin Old hemorrhage
Lysed cells from traumatic tap
Bilirubin RBC degradation
Elevated serum bilirubin level
Carotene Increased serum levels
Protein See Above
Clotted Melanin Meningeal melanosarcoma
Protein See Above
Pellicle Clotting factors Introduced by traumatic tap
Protein Disorders that affect blood-brain
Clotting factors barrier
Tubercular meningitis

Xanthochromia is a term used to describe CSF supernatant that is pink, orange, or yellow.
Pink very slight amount of oxyhemoglobin
Orange heavy hemolysis
 Yellow conversion of oxyhemoglobin to unconjugated bilirubin
Other causes of xanthochromia: elevated serum bilirubin, presence of pigment
carotene, markedly increased protein concentrations, and melanoma pigment.
Xanthochromia is also commonly seen in premature infants due to immature
liver function.

Visual Determination to Differentiate Intracranial Hemorrhage or Traumatic Tap

Visual Exam Intracranial hemorrhage Traumatic Tap
Distribution of Evenly distributed throughout Uneven distribution: heaviest in tube 1
blood the 3 CSF tubes and diminishing amounts in tubes 2 &
3. Streaks of blood may also be seen.

Clot formation Does not contain enough May form clots due to the introduction
fibrinogen to clot of plasma fibrinogen into the specimen

Xanthochromic Xanthochromic (RBCs must Clear (introduction of serum protein

Supernatant remain in CSF for 2 hours from traumatic tap can cause
before noticeable hemolysis xanthochromia)
begins; recent hemorrhage
produces clear supernatant)

Additional
tests: Macrophages containing
Microscopic ingested RBCs or hemosiderin
granules

D-dimer test Detection of D-dimer indicates the

formation of fibrin at a anemorrhage
site

Cell Counts (Hematology)

Only the WBC count is performed routinely on CSF. RBC counts are usually determined only
when there is a traumatic tap, therefore a correction for leukocytes or protein is needed.
Formulas:
RBC Count = Total Cell Count WBC Count
No. of cells counted X dilution
Cells/uL = No. of squares counted X volume of 1
square
1 uL
Cells/uL = No. of cells counted X dilution X
1 uL (0.1 X 10)

WBC (added) = WBC (blood) X RBC

(CSF)
RBC
***The third formula is used for (blood)
CONTAMINATION CORRECTION

Total cell count

Clear specimens may be counted undiluted, provided no overlapping of cells is seen during the
microscopic examination.
Dilutions for total cell counts are made with normal saline and loaded into the hemocytometer with
a Pastuer pipette.

Sample dilution:
Amount of
Clarity Dilution Amount of Sample
Diluent
 Slightly hazy 1:10 30 L 270 L
Hazy 1:20 30 L 570 L
Slightly cloudy 1:100 30 L 2970 L
Slightly bloody 1:200 30 L 5970 L
Cloudy
Bloody 1:10,000 0.1 mL of a 1:100 9.9 mL
Turbid dilution

Lysis of RBCs must be obtained before performing WBC counts either in diluted or undiluted
specimens. For specimens requiring dilution, use 3% acetic acid as diluent. For undiluted specimens:
place 4 drops of mixed specimen in a clean tube. Rinse Pasteur pipette with glacial acetic acid, draining
thoroughly, and draw the four drops of CSF into the rinsed pipette, discard the first drop and load the
hemocytometer.
Normal values for WBC in CSF:
Adults: 0-5 WBCs/uL
Newborns: up to 30 mononuclear cells/uL
Differential count should be performed on a stained smear (concentrated specimen/
Cytocentrifuge) and not from the cells in the counting chamber so that if abnormal cells are
present, they can be observed. 100 cells should be counted, classified and reported in %.
The cells found in normal CSF are primarily lymphocytes and monocytes. Adults have predominance
of lymphocytes to monocytes (70:30) whereas monocytes are more prevalent in children. Occasional
neutrophils are also found in normal CSF.
Pleocytosis increased number of WBCs in CSF (lymphocyte, monocyte, neutrophil) is considered
abnormal as is the finding of immature leukocytes, eosinophils, plasma cells, macrophages, increased
tissue cells, and malignant cells.

Chemistry Tests
CSF Protein
The most frequently performed chemical test on CSF is protein determination.
Normal Values = 15-45 mg/dL
Elevated total protein values are seen in pathologic conditions. The causes are: damage to blood
brain barrier (meningitis and hemorrhage), production of immunoglobulins within the CNS, decreased
clearance of normal protein from the fluid, and degeneration of neural tissue
In general CSF contains fractions similar to those found in serum although the ratio varies among
the fractions. Tau is a carbohydrate-deficient transferrin fraction that is seen only in CSF and not in
serum.
Correction for protein contamination

[serum protein mg/dL X (1.00 Hct)] X CSF RBCs/uL

mg/dL protein added =
(plasma volume)
blood RBCs/uL
Methods for measuring protein
o Turbidimetric methods
- Reagent of choice is trichloroacetic acid because it will precipitate both albumin and globulin
equally. SSA is more sensitive to albumin unless combined with sodium sulfate.
o Dye-binding ability
- Coomassie brilliant blue G250 is based on the principle of protein error of indicators. The
color change of the pH-stabilized dye from red to blue occurs when protein binds to the dye.
This dye binds to a variety of proteins, not just to albumin.
o Automated chemistry analyzers

Protein Fractions
Diagnosis of neurologic disorders associated with abnormal CSF protein often requires
measurement of individual protein fractions
To determine whether IgG is increased because it is produced within the CNS or is elevated as a
result of a defect in the blood-brain barrier comparison between serum and CSF levels of albumin and
IgG must be made.
CSF albumin (mg/dL)
CSF/serum albumin indexSerum
= albumin (mg/dL)

An index value of 9 represents an intact blood-brain barrier. The index increases relative to the degree
of damage to the barrier.
CSF IgG (mg/dL) / serum IgG (g/dL
IgG index = CSF albumin (mg/dL) / serum albumin
(g/dL)
IgG values greater than 0.77 are indicative of IgG production within the CNS

CSF Glucose
Glucose enters the CSF by selective transport across the blood-brain barrier, which results in a
normal value that is approximately 60-70% that of plasma glucose.
 For accurate evaluation of blood glucose, a blood test must be run for comparison. The blood
glucose must be drawn 2 hours before the spinal tap.
Elevated CSF glucose values are always a result of plasma elevations. Low CSF glucose values can
be of diagnostic value in determining the causative agents in meningitis

CSF Lactate
The determination of CSF lactate levels can be a valuable aid in the diagnosisi and management of
menigitis cases because levels >25 mg/dL occurs much more consistently than does the depression of
glucose and provides more reliable information when the initial diagnosis is difficult. CSF lactate levels
remain elevated during initial treatment but fall rapidly when treatment is successful, thus offering a
sensitive method for evaluating the effectiveness of antibiotic therapy.
Also used to monitor severe head injuries since destruction of tissue within the CNS due to hypoxia
causes the production of increased CSF lactic acid levels
Falsely elevated results may be obtained on xanthochromic or hemolyzed fluid since RBC contains
high concentrations of lactate.

CSF Glutamine
Glutamine is produced in the CNS by brain cells from ammonia and -ketoglutarate. This serves to
remove the toxic metabolic waste product ammonia from the CNS.
Normal values = 8-18 mg/dL
Frequently requested test for patient with coma of unknown origin.

Microbiology Tests
Gram stain
Routinely performed on CSF from all suspected cases of meningitis for the detection of bacterial
and fungal elements
All smears and cultures should be performed on concentrated specimens; centrifuged at 1500g for
15 minutes.

Acid Fast Stain and or Fluorescent antibody stain

Not routinely performed unless tubercular meningitis is suspected

India Ink
Performed for the detection of Cryptococcus neoformans
In the Gram stain the classic starburst pattern produced by Cryptococcus is seen more often than a
positive India Ink.

Latex Agglutination and ELISA methods

Provide a rapid means for detection and identification of microorganism in CSF but some tests are
not sensitive to some organisms. The Gram stain is still the recommende method for detection of
organisms.

Serologic Testing
In addition to serologic procedures performed for the identification of microorganisms, serologic
testing is also performed to detect the presence of neurosyphilis. VDRL or fluorescent treponemal
antibody-absorption test are recommended for CSF testing.

SEMEN ANALYSIS

Physiology
Semen is composed of four fractions that are contributed individually by:
1. Testes and epididymis 5% of semen volume
2. Seminal vessels 60% of volume which contains fructose. Sperm do not become motile until exposed
to fluid from seminal vessels.
3. Prostate 20-30% (acidic fluid acid phosphatase, citric acid, zinc and proteolytic enzymes which are
responsible for both the coagulation and liquefaction of semen after ejaculation)
4. Bulbourethral glands 5% in the form of thick alkaline mucus

Specimen Handling and Collection:

Specimens are usually collected following a period of sexual abstinence of 3-6 days.
Should be collected in a room provided by laboratory but if this is not appropriate, the specimen should
be kept at body temperature and delivered to the lab within 1 hour.
Collected by masturbation but if not possible, use only nonspermicidal, nonlubricant-containing
polymeric silicone condoms. (Majority of sperm are contained in first portion of ejaculate.)

Semen Analysis
Semen analysis for fertility evaluation consists of both macroscopic and microscopic examination.
Parameters reported include appearance, volume, viscosity, pH, sperm concentration and count, motility,
and morphology. (T11-1)
Appearance
- Normal semen has a gray-white color, appears translucent and a characteristic musty odor.
- Increased white turbidity indicates WBCs and infection within the reproductive tract.
- Red coloration indicates RBCs and is abnormal.
- Yellow coloration may be caused by urine contamination, collection following prolonged
abstinence, or medications.
- Fresh specimens are clotted and should liquefy within 30-60 minutes. Failure of liquefaction
to occur may be caused by a deficiency in prostatic enzymes and should be reported.

Volume
- 2-5 mL
- Increase volume is may be seen after prolonged abstinence.
- Decreased volume is more associated with infertility

Viscosity
- Refers to the consistency of the fluid and may be related to specimen liquefaction
- Ratings: 0 (watery) to 4 (gel-like)
- Normal specimen should be easily drawn into a pipette and form droplets that do not appear
clumped or stringy when discharged form the pipette.
- Increased viscosity and incomplete liquefaction will impede sperm motility

pH
- Normal pH is alkaline (7.2-8.0)
- Increased pH indicates infection within the reproductive tract
- Decreased pH is associated with increased prostatic fluid

Sperm Concentration/Count
- Normal values for sperm concentration: 20-160 million sperm/mL
- Sperm concentration is usually performed using the Neubauer hemocytometer.
- Most common dilution used is 1:20. Dilution of the semen is essential because it immobilizes
the sperm prior to counting. The traditional diluting fluid contains sodium bicarbonate and formalin; but
good results can be achieved with tap water.
- Only fully developed sperm should be counted. Spermatids and WBCs (round cells) must
not be included. A WBC count of >1 million WBCs/mL indicates inflammation of the reproductive
organs.
- Total sperm count for the ejaculate can be calculated by multiplying the sperm concentration
by the specimen volume
- Examples:
1. Using a 1:20 dilution, an average of 60 sperms are counted in the five RBC
counting squares. Calculate the sperm concentration/mL and the total sperm count in a specimen
with a volume of 4 mL.

60 sperm counted X 1M = 60,000,000 sperm/mL

60,000,000 sperm/mL X 4 mL = 240,000,000 sperm/ejaculate
***When you are counting the 5 RBC squares simply multiply by 1million to get the sperm concentration,
and multiply it to the volume to get the sperm count.

2. Using a 1:20 dilution, 600 sperm are counted in two WBC counting squares.
Calculate the sperm concentration/mL and the total sperm count in a specimen with a volume of 2
mL.

***When you are counting in 2 WBC squares you need to solve the sperm concentration using the formula
below, and then multiply it to the volume to solve for the sperm count.
No. of sperm counted X dilution
No. of squares counted X volume of 1 square = = sperm concentration

600 X 20 = 60,000 sperm/L

2 X 0.1 L

60,000 sperm/L X 1,000 = 60,000,000 sperm/mL

60,000,000 sperm/mL X 2 mL = 120,000,000 sperm/ejaculate

***But you can also do the short cut in solving sperm concentration and sperm count using 2 WBC squares.
Simply multiply the sperm counted to 100,000 to get the sperm concentration, and multiply it to the
volume to get the sperm count.

Sperm Motility
- Assessment of sperm motility should be performed on well mixed, liquefied semen within 1
hour of specimen collection.
- Consistent amount of semen under the same size coverslip (10 L under 22 X 22 mm
coverslip)
- The percentage of sperm showing actual forward movement can be estimated after
evaluating 20 high-power fields. (T11-2)
- A minimum motility of 50% with a rating of 2.0 after 1 hour is considered normal. High
percentage of immobile sperm and clumps of sperm requires further evaluation to determine sperm
viability or the presence of sperm agglutinins.
- Computer-assisted semen analysis (CASA)

Sperm Morphology
- Sperm morphology is evaluated with respect to both head and tail appearance.
- Normal sperm has an oval-shaped head approximately 5 m long and 3 m wide and a long,
flagellar tail approximately 45 m long
- Morphology is evaluated from a thinly smeared, stained slide under oil immersion. (Stain:
Wrights, Giemsa, or Papanicolau)
- At least 200 sperm should be evaluated and the percentage of abnormal sperm reported.
- Krugers strict criteria: measurement of head, neck, and tail size; the size of the acrosome,
and the presence of vacuoles
- Differentiation and enumeration of round cells (immature sperm and WBCs) can also be
made during morphology examination. By counting the number of spermatids or neutrophils seen in
conjunction with 100 mature sperm, the amount per milliliter can be calculated using the formula:
- NXS
C = 100

Where N equals the number of spermatids or neutrophils counted per 100 mature
sperm and S equals the sperm concentration in millions per milliliter.

Additional Testing
Should abnormalities be discovered in any of these routine parameters, additional tests may be requested.
(T11-3)

Sperm Viability
- Specimen has normal sperm concentration with markedly decreased motility
- Procedure: Mix specimen with eosin-nigrosin stain, make smear, and count dead cells in 100
sperm. Living cells are not infiltrated by dye and remain bluish-white color while dead cells stain red
against the purple background.
- Normal viability = 75% living cells and should correspond to previously evaluated motility.

Seminal Fluid Fructose

- Decreased sperm count may be caused by lack of support medium produced in seminal
vesicles. This is indicated by a low or absent fructose level in the semen.
- Screening test: resorcinol test. Orange-red color = presence of fructose
- Normal fructose = 13 mol per ejaculate. Specimen must be tested within 2 hours or
frozen to prevent fructolysis.

Antisperm antibodies
- Antisperm antibodies can be present in both men and women in the semen, cervical
mucosa, or serum and are a possible cause of infertility.
- In males: suspected when clumps of sperm are observed during a routine semen analysis
- In females: suspected when normal semen analysis is accompanied by continued infertility
- Tests: Mixed agglutination reaction (MAR) and immunobead test

Microbial testing
The presence of > 1 million WBCs/mL indicates infection within the reproductive system

Chemical testing
- Disorder of epididymis = decreased levels of -glucosidase
- Lack of prostatic fluid = decreased zinc, citrate, and acid phosphatase
- Acid phosphatase = used to determine whether semen is actually present in a specimen.

Postvasectomy Semen Analysis

- Done 2 months postvasectomy until 2 consecutive monthly specimens show no
spermatozoa.
- A negative wet preparation is followed by centrifugation for 10 min and examination of
sediment

Sperm Function Tests

1. Hamster egg penetration sperm are incubated with species-nonspecific hamster eggs and penetration
is observed microscopically.
2. Cervical mucus penetration observation of sperm penetration ability of partners midcycle cervical
mucus.
3. Hypo-osmotic swelling sperm exposed to low-sodium concentrations are evaluated for membrane
integrity and sperm viability
4. In-vitro acrosome reaction evaluation of the acrosome to produce enzymes essential for ovum
penetration.