Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

FECALYSIS
Specimen collection

Clean dry container, leak-proof

Not contaminated by urine*

Macroscopic screening
1.
Color changes
a)
brown = normal
b)
gray=fecal obstruction or barium
c)
red=blood or food dyes
d)
black=blood form upper git, iron tx, charcoal tx
e)
green=vegetables/biliverdin
2.

Consistency
a)
formed=normal
b)
hard=constipated
c)
watery=diarrhea/steatorrhea (fat excretion >3g/day)

3.

Form
a)
b)
c)
d)
e)

cylindrical=normal
ribbon-like=intestinal strictures
small, round=constipation
bulky=steatorrhea
mucus=colitis, constipation

Chemical examination

Test for blood:

Melena
large amounts of fecal blood
i.e. 50-100 ml/day

Occult blood
small amounts of fecal blood
i.e. 30-50ml/day

Testing principles for occult blood:

1)
Hemoglobin reduction method
Based on the reaction of Hgb with H2O2 and an indicator
usually: blue green color (indicator)
usual indicators:
a)
benzidine (carcinogenic)
b)
Orthotoluidine
c)
Guaiac - most common
Interfering factors:
False (+) - turnips,brocolli, cantaloupes, banana, aspirin
False (-) - vitamin C
2)
HemoQuant test
detects total fecal Hgb
higher specificity than #1 (not affected by interfering substances)
3)
Immunodiffusion method
using antihuman Hb
most sensitive method
MICROSCOPIC EXAMINATION

Fecal white blood cells

a)
Ulcerative colitis
b)
Dysentery (bacterial)
1.
Salmonella
2.
Shigella
3.
Campylobacter
4.
Yersinia
5.
Enteroinvasive E. coli
c)
Ulcerative diverticulitis
d)
Intestinal TB
e)
Abscesses

NB:
Staph and Vibrio usually do not cause the appearance of fecal
leukocytes
As few as 3 leukocytes per OIL immersion lens have 70% sensitivity
for the presence of invasive bacteria.

other tests:
1)
Lactoferrin* latex agglutination test
By: Rem Alfelor

A component of leukocytes secondary granules (a neutrophil

protein*) which when detected can also a be an indicator of an
invasive bacteria.
Major cause of fecal neutrophils in patients with chronic diarrhea is
chronic inflammatory bowel disease of the colon.
2)
Muscle fibers
pancreatic insufficiency (cystic fibrosis)
use of eosin to highlight the fibers
Only undigested fibers are counted and if more than 10, it is
reported as increased.
Patients are instructed to eat red meat and specimens must be
examined within 24 hours of collection.
3)
Qualitative fecal fat
for suspected case of steatorrhea
Fats stains: Oil red O, Sudan III, IV
will appear as red orange droplets
confirmatory for steatorrhea
3 day specimen collection
Patient on a regular intake of 100g/day of fat during the collection
period.
4)
APT TEST (Fetal hemoglobin)
Grossly bloody stools and vomitus are sometimes seen in the
neonates as the result of swallowing maternal blood.
the material to be tested is emulsified in water to release hemoglobin,
and after centrifugation + 1% NaOH
Result: if maternal hemoglobin yellow brown supernatant
if fetal, supernatant remains pink (alkali-resistant fetal hemoglobin)
Other chemical tests:
1)
Carbohydrate detection
2)
Enzyme tests

Parasites (Ameba)

E. histolytica/E. dispar
Giardia

Giardia using Iodine as stain

Direct fluorescent antibody assay

Giardia
Others: Helminths

Enterobius.UV light microscope

Enterobiusadult worm

URINALYSIS
History of urinalysis

Middle Ages

Johanes de Ketham

Fasciculus Medicinae 1491

Urine color wheel

Based on four temperaments/humors:

1.
Sanguineous-blood
2.
Choleric-yellow bile
3.
Phlegmatic-phlegm
4.
Melancholic-black bile
BASIC URINALYSIS

key points

Many different diseases can display abnormalities in the urine.

Quickly and economically

Therefore, examination of the urine is an important laboratory function.

Basic urinalysis consists of

gross examination of the urine

dipstick analysis for blood, white cells, sugar, and other substances

Dipstick may be read either manually or by an automated instrument.

Microscopic analysis of urine may be necessary in many cases.

This is to detect cellular elements, casts, and crystals.

Each of these items can be caused by several different disease
states.

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Although microscopic examination of the urine is usually performed

manually, there are several automated instruments that can perform
this analysis.

URINE FLOW CYTOMETRY

Cells are counted based on

1.
light scattering
2.
fluorescence
3.
impedance properties

Considerations

Red blood cells within the urine can come from any point along the urinary
tract.

Dysmorphic red blood cells are often a sign of glomerular disease.

The first voided morning urine, because it is the most concentrated, is often the
best specimen for analysis.

Some procedure may require a 12- or 24-hour urine sample.

Specific gravity and osmolality measurements reflect the concentrating ability of

the kidneys.

After a period of dehydration, the osmolality should be three to four times that of
plasma.

Proteinuria of over 4 g/day is seen in the nephrotic syndrome.

Although nephrotic syndrome is usually seen in primary renal disease, it can

occasionally be seen in a systemic disease which affects the kidneys.

Ketonuria can be seen in diabetics.

It can also be seen in other states, such as febrile illnesses and cachexia.

The dipstick nitrite and leukocyte esterase tests are used to help diagnose
urinary tract infections.

Positive results should be confirmed by microscopic analysis of the urine.

Urinary calculi are most commonly formed from calcium.

Work-up of habitual stone formers should include both analysis of the urine and
of the stone.
THE NORMAL URINE

Water

Electrolytes

Bacterial toxins

Drugs

Vitamins

Pigments

Hormones

Urea

Uric acid

Ammonia

Creatinine

Trace of cells

Crystals
URINE

The result of :

glomerular filtration

tubular reabsorption

selective secretion

Normal

Color--Pale yellow to amber

Appearance--Clear

Sp. Gravity---1.003-1.030

pH---4.5-8.0

Glucose, Protein, Nitrates, Blood, Crystals, Bilirubin, Leukocyte Esterase

and Ketones---None

RBCs---0-3/HPF

WBCs---0-4/HPF

Urobilinogen---<1.0

COLOR

Normally pale yellow to amber

The color indicates the concentration of the urine:

Dilute urine=straw colored

Concentrated urine=dark amber colored

APPEARANCE

Normally clear
By: Rem Alfelor

Cloudy urine may be caused by the

presence of pus, RBCs, or bacteria Cloudy urine may be normal due to
ingestion of certain foods

Large amounts of fats and phosphates

SPECIFIC GRAVITY

A measurement of the concentration of particles in the urine

Includes waste and electrolytes

Used to evaluate the concentrating and excretory power of the kidney

Low: Overhydration, diabetes insipidus, renal tubular damage, renal

failure, K+ deficiency, hypercalcemia

High: Dehydration, diabetes mellitus, proteinuria, glycosuria, eclampsia,

lipoid nephrosis, radiographic dye
pH

indicates acid-base balance

Alkaline urine
Alkalemia, bacteria, UTI, or diet high in citrus fruits or vegetables

Acidic urine
Acidemia, starvation, dehydration, or diet high in meat products of
cranberries

Useful in identifying crystals in the urine

PROTEIN

Urine is normally protein free

A sensitive indicator of renal function

Any abnormal results needs 24 hour urine

Most dip sticks are more sensitive to albumin than to globulin,

mucoproteins or Bence-Jones proteins

A negative result does not exclude their presence

High: Renal disease, contamination from menstrual or prostatic secretions

Trace can be seen in healthy adults who do strenuous exercise

BENCE-JONES PROTEIN
Lightweight immunoglobulins found in half of the patients with
multiple myeloma
May also be associated with tumor metastases to the bone, chronic
lymphocytic leukemias, lymphoma, microglobulinemia and
amyloidosis
Rapidly cleared by the kidney and excreted into the urine
Dilute urine may yield a false-negative result
Urine electrophoresis and immunophoresis are the procedures of
choice
GLUCOSE

Indicates the likelihood of diabetes mellitus

Usually see glycosuria when blood glucose levels exceed 180 mg/dl
(Renal threshold)

Increased levels may indicate diabetes mellitus, Cushings syndrome,

severe stress, infection, drug therapy, pregnancy, low renal threshold
KETONES

Usually associated with poorly controlled diabetes

Glucose + Ketones = Diabetic Ketoacidosis

Useful as an early warning sign for a more serious buildup of ketones in

the blood

Also important in evaluating ketoacidosis with alcoholism, fasting,

starvation, high-protein diets, and isopropanol ingestion

May occur in acute febrile illnesses, especially in infants and children

LEUKOCYTE ESTERASE

A screening test used to detect leukocytes in the urine

The positive result is associated with UTI and vaginal contamination

-Urine culture needed

NITRITES

A screening test for the identification of UTIs

Many bacteria produce an enzyme called REDUCTASE

Reduces urinary nitrates to nitrites

A positive results would indicate the need for a urine culture

Indicates gram-negative bacteria

Enhances the sensitivity of the leukocyte esterase test

BILIRUBIN

Not stable in urine, especially when exposed to light

Can be detected in urine before other clinical symptoms are present or

recognizable
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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Also useful in differentiating between obstructive (+) and hemolytic (-)

jaundice

The presence of bilirubin is associated with liver disease, Gallstones,

extra-hepatic duct obstruction, extensive liver metastasis, cholestasis from
drugs
UROBILINOGEN

As with bilirubin, differentiates between obstructive and hemolytic

Low: Cholelithiasis, inflammatory disease, neoplasms, antibiotic therapy

High: Increased bilirubin, hemolytic anemia, pernicious anemia, malaria,

hepatitis, cirrhosis, CHF
BLOOD/RBCs

Increases numbers are present in renal disease, lower urinary tract

disease, and extra-renal disease

May also be present in acute febrile episodes, malignant HTN, blood

dyscrasia, toxic drug reactions, and anticoagulant therapy

BACTERIA

FUNGUS (CANDIDA)

DYSMORPHIC RED BLOOD CELLS

CANDIDA: PSEUDOHYPHAE

WBCs

Increased numbers are seen in almost all renal diseases and diseases of
the urinary tract

May be transiently increased during fevers and following strenuous

exercise

50/HPF and or clumps are suggestive of acute infection

WHITE BLOOD CELLS ON DILUTED ACETIC ACID

MUSCLE FIBERS

EOSINOPHILS

POLLEN GRAIN

Bacteria

May or may not be significant

If UTI is present, there will usually be the presence of WBCs

MISCELLANEOUS

Yeast

May indicate UTI in diabetics

May also be a contaminant from skin, hair or vaginal discharge

By: Rem Alfelor

MUCUS

Presence in small amounts is normal

Increased amounts may be found in chronic inflammation of the urethra

and bladder

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test
STARCH GRANULE

RENAL TUBULAR CELLS WITH WBCs

EPITHELIAL CELLS

Squamous
Normally line the female urethra and 0.5-1.0 cm of the male urethra
Many cells accompanied by WBCs are indicative of contamination

Transitional
The presence of a few is normal
They line the urinary tract from the renal pelvis to the trigone of the
female and to the distal urethra in the male
The presence of large clumps or sheets suggest need for cytology
because of possible transitional cell carcinoma

Renal
The presence of rare to occasional may be seen in normal urine
Normally, newborns may have up to a moderate number of renal
epithelial cells
Increased numbers suggest acute tubular damage
Renal tubular cells which absorbed lipids are called oval fat bodies
seen in Nephrotic Syndrome
Large number of unidentifiable cells should be followed up with
resubmission for cytology

EPITHELIAL CELLS - Squamous

CASTS

CLASSIFICATION
Matrix
Hyaline-variable size
Waxy- often broad
Inclusions
Granules-protein, cell debris
Fat globules-triglycerides, cholesterol esters
Hemosiderin granules
Crystals-uncommon
Melanin granules-rare
Pigments
Hemoglobin, myoglobin, bilirubin, drugs
Cells
RBCs and its remnants
WBCs-neutrophils, lymphocytes, monocytes and histiocytes
Renal tubular epithelial cells
Mixed cells-rbc,wbc & renal tubular cells
TAMM-HORSFALL PROTEIN
a glycoprotein secreted normally by the thick part of the
ascending loop of Henle (and possibly the distal tubule)
this constitute about 1/3 of the total protein in urine in normal
individuals.
This forms the matrix of all casts acting as a mesh trapping
cells, particulate debris etc

HYALINE CASTS
Formed in the distal convoluted tubule or collecting ducts.
Most commonly seen cast (N 0-2/lpf)
Narrow casts are generally a normal finding while broad casts are
more significant
Transient increases may be noted during fevers, after strenuous
exercises and following diuretic therapy
HYALINE CASTS

EPITHELIAL CELLS - TRANSITIONAL EPITHELIAL CELLS

EPITHELIAL CELLS - RENAL TUBULAR EPITHELIAL CELLS

By: Rem Alfelor

GRANULAR CASTS
Almost always indicates significant renal disease
May be present in pyelonephritis, viral diseases, chronic lead
intoxication, acute allograft rejection
Normal following strenuous exercise or pure carbohydrate diet
RBC/HEMOGLOBIN CASTS
Diagnostic of glomerular disease
Acute glomerulonephritis, lupus endocarditis, renal infarct,
malignant HTN, sickle cell disease, vasculitis, Goodpastures
syndrome
WBC CASTS
Primarily indicates pyelonephritis

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test
May also be found in lupus nephritis, acute glomerulonephritis,
interstitial nephritis, and nephrotic syndrome
WAXY CASTS
Reflects final phase of dissolution of the fine granules of granular
casts
Implies localized nephron obstruction and oliguria
Observed most frequently in chronic renal failure

MIXED (WBC & RENAL EPITHELIAL) CAST

WAXY CASTS

HEMOGLOBIN CAST

FINE GRANULAR CASTS

RBC CAST

BROAD WAXY CASTS

The most ominous of all casts
Indicates severe renal disease
Caused from renal tubular dilation and epithelial atrophy
FATTY CASTS
Seen in nephrotic syndrome

FATTY CAST

WBC CAST
FATTY CAST (OIL RED O)

RENAL TUBULAR EPITHELIAL CELL CAST

By: Rem Alfelor

CRYSTALS
Indicate that renal stone formation is imminent, if not already
present
Formed as a result of precipitation of urinary solutes out of solution
by:
The concentration of the solute in the urine
The urinary pH
The flow of urine through the tubules
Temperature
Acidic urine is associated with xanthine, cystine, uric acid, and calcium oxalate
stones
To treat or prevent these stones, urine should be kept alkaline

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Alkaline urine is associated with calcium carbonate, calcium phosphate, and

magnesium phosphate stones

To treat or prevent these stones, urine should be kept acidic

CALCIUM OXALATE CRYSTALS

ACID URATES

CALCIUM OXALATE (RARE OVAL FORM)

URIC ACID CRYSTALS

CALCIUM PHOSPHATE CRYSTALS - LARGE PLATE

LARGE URIC ACID CRYSTAL PLATE

CALCIUM PHOSPHATE CRYSTALS - LARGE PLATE (PICTURE MODIFIED)

HEXAGONAL URIC ACID CRYSTAL

TRIPLE PHOSPHATE CRYSTALS

POLARIZED URIC ACID CRYSTALS

CALCIUM PHOSPHATE

By: Rem Alfelor

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

AMMONIUM BIURATE

CYSTINE

TYROSINE CRYSTALS

LEUCINE CRYSTALS

AMPICILLIN

RENOGRAFIN

RENOGRAFIN POLARIZED

LABORATORY DIAGNOSIS OF ACUTE CORONARY SYNDROME

Dennis Macapagal, MD
Objectives

By the end of this session you will be able to

1.
Discuss the biochemical markers that are used to detect acute myocardial
infarction.
2.
Discuss the sensitivity and specificity of CK-MB and the Troponins for the
detection of AMI
3.
Discuss other tests that are indirectly helpful in diagnosing an acute
coronary syndrome.

SULFADIAZINE CRYSTALS

Overview:

3 hours of ischemia=80% risk of cell death

6 hours=100% cell death

For this reason, early recognition of persistent ischemia and intervention to

restore blood flow are needed to minimize cell death.
Angioplasty, stenting, coronary artery bypass graft

By: Rem Alfelor

Heart disease is an affliction intimately tied to high technology. Technology has

had a causal role, in part by allowing people to live longer and in part by
enabling a sedentary and overly consumptive lifestyle.
During the 20Th century, heart disease rose from obscurity to become the
leading cause of morbidity and mortality in developed nations.
Diagnosis and treatment of heart disease also depend heavily on advanced
technology, including electrophysiologic, imaging, catheterization, surgical and
clinical laboratory testings.
Heart disease statistics in the US:
1.
80M (about 1 in 3) has some form of CVD inc HPN.
2.
17M have a history of CHD
3.
8M have had an MI
4.
10M have had an angina pectoris
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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test
5.
6.

6M have HF
7M have experienced stroke

7.

CVD is the underlying cause in 35% of all deaths (860K/year) versus

cancer, chronic LRTD, accidents and DM combined.
annual cost is about USD500Billion
There is an alarming increase in obesity and type 2 DM which will fuel the
epidemic for many years to come.

8.
9.

Non-laboratory risk factors for CHD:

1.
Smoking (any smoking in the past month)
2.
HPN (>140/90mmHg)
3.
Age (men over 45 years;F>55)
4.
Obesity
5.
DM
6.
Sedentary lifestyle
Key points:
1.
Measurement of proteins present in cardiac myocytes indicates recent damage
to cardiac muscle.
2.
Measurement of substances that are damaging to the coronary arteries, or at
least have been proven association with coronary heart disease, is used to
assess risk and select appropriate preventive measures. The most important lab
risk factors are LIPIDS.
Serial sampling for cardiac markers

time window from death of muscle to release markers in blood

ESC & ACC>>

1.
on admission
2.
6-9 hours
3.
12-24 hours (if earlier specimens were negative) and the clinical
suspicion for MI is high

POC testing must be available ALL the time

Historically: LDH & CK

20+ years ago Troponin antibodies

currently, the markers assay include:

Myoglobin
Troponins
CK isoenzyme (MB)

CARDIAC ENZYME MARKERS:

Historical development:

1950s-La Due et al investigating transaminases (AST)

Very simple principle:
when heart muscle diesrelease proteins in blood (as markers)

By: Rem Alfelor

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Transaminases have not endured as cardiac markers because they are

not specific to the heart. And soon afterLDH and CK.

CREATINE PHOSPHOKINASE

By: Rem Alfelor

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Newer tests:

Troponinshighly specific

A complex of three proteins that resides at regular intervals in the thin

filament of striated muscle.
TnT, TnI, TnC
TnC=identical forms in Type 2 and cardiac muscle
TnI & TnT=cardio specific

In contrast with other markers, their levels are almost undetectable or zero
in normal serum. (detection limits of 0.01ng/ml)

The 99th percentile of the healthy population is around 0.04ng/ml

depending on the assay.

Levels above this threshold are almost certainly indicative of myocyte

damage but possibly reveal a much lesser amount of damage than was
detectable with earlier cardiac markers such as CK-MB.

Note that elevation maybe found in pericarditis, myocarditis, PE, renal

failure, sepsis, and other critical illness.

Prolonged intense exercise, such as marathon can cause small

elevations.

NB: in healthy newborns, levels as high as 3.0ng/ml has been found!

By: Rem Alfelor

Other markers for coronary risk:

C-REACTIVE PROTEIN

First isolated in 1930 in patients with pneumococcal pneumonia

so named because it binds to the C-polysaccharide of the pneumococcus

Later it was found that it appeared in plasma during infectious or

inflammatory conditions.

It is the original acute phase reactant protein.

Normal level=1mg/ml

in acute illness, levels may reach as high as 300mg/ml

Baseline average was 1.5mg/ml for those who developed MI.

It is pro inflammatory.

Still, CRP has an unclear role in the pathogenesis of vascular disease.

But the AHA suggested:

Asymptomatic men >50 years and women >60 years be screened
even if LDL cholesterol is not elevated.

HOMOCYSTEINE

Hcy is sulfur containing amino acid and a metabolic intermediate.

HOMOCYSTINURIA:
homozygous defect in the enzyme cystathionine-Beta-synthase
lens dislocation, osteoporosis, MR, psychiatric disturbance,
thromboembolic disease (e.g. CHD)

High levels of Hcy:

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

1.
endothelial injury
2.
platelet activation
To name a few damaging effects
as a CHD marker, it parallels that of a high cholesterol level
Folate supplements can bring down elevated levels of Hcy.

Summary:
1.
The most important disease affecting heart is coronary heart disease, which is
atherosclerosis affecting the coronary arteries.

CHD can lead to thrombotic occlusion of coronary blood flow, causing an

acute coronary syndrome or ACS. ACS with frank necrosis of any amount
is MI.
2.
The primary tests for diagnosing ACS are ECG, lab measurements of cardiac
markers which are proteins released in circulation from damaged heart muscle.
The most important cardiac marker today is cardiac troponin (cTn).
3.
Troponin is a complex of three proteins, two of which are suitable as specific
cardiac marker tests: cTnI and ctnT
4.
There is a delay of a few hours following MI before cTn is detected in the
circulation. It peaks about 24 hours and then declines over several days
5.
We can also measure risk factors associated with the development and
progression of CHD: Lipids, homocysteine, CRP
Assignment (got from Henrys):
Read on
1.
BRAIN NATRIURETIC PEPTIDE (BNP)

Circulating BNP derives from a 108 amino acid prohormone, proBNP,

which is cleaved within the cardiac myocyte by the endoprotease furin to a
32 amino acid C-terminal fragment, the active BNP, and an inactive Nterminal fragment, N-BNP or NT-proBNP

Secretion of both fragments is enhanced by ventricular wall stretch and

volume overload, as occur in HF

BNP is removed from the circulation by binding to a clearance receptor

and also through the action of endopeptidases; its circulating half-life is
approximately 22 minutes.

The circulating half-life of N-BNP is considerably longer (60120 min), and

its mechanism of clearance is not well understood.

BNP and the other natriuretic peptides exert their effects through two
types of G-proteincoupled receptors, resulting in release of the second
messenger cyclic guanosine monophosphate. They downregulate the
renin-angiotensin-aldosterone system, decrease sympathetic nerve
activity in the heart and kidney, increase renal blood flow, and increase
sodium excretion via a direct effect on the renal collecting duct

Plasma levels of BNP are less than 100 pg/mL in most healthy individuals;
reference ranges depend on age and gender.

The best established application of BNP measurement is for diagnosing

acutely ill patients presenting to emergency service with shortness of
breath.

Distinguishing HF from lung disease, such as emphysema, in these

patients is occasionally difficult, and until now no laboratory test has been
specifically applicable.

The multinational Breathing Not Properly study enrolled patients who

presented to emergency centers with dyspnea and used a point-of-care
method for measurement of BNP. At a decision point of 100 pg/mL, the
BNP test had the following characteristics for diagnosis of HF: sensitivity
90%, specificity 76%, positive predictive value 79%, and negative
predictive value 89%.

Among patients with a history of ventricular dysfunction, BNP was higher

in those whose current symptoms were thought to be caused by HF; it
was also higher in patients with more severe failure.

BNP levels decline when effective therapy for HF is instituted, and so the
test may be used to monitor the course of treatment

Other proposed applications include risk stratification of patients with

ACS; monitoring disease severity in patients with stable CHD; screening
for ventricular dysfunction in selected populations; and testing for drug
cardio toxicity

However, the test is relatively expensive, so routine application that leads

to a large volume of testing needs to be carefully considered.

Some medical centers have introduced restrictions to limit the use of BNP
for monitoring inpatients
By: Rem Alfelor

2.

A major limitation of BNP is that a wide range of values is observed in

patients with and without HF, and all of the determinants of the circulating
BNP level have not yet been well established.

BNP is increased in conditions of fluid imbalance other than HF,

particularly renal insufficiency, which commonly coexists with HF.

In individuals without HF, higher levels are associated with female gender,
advanced age, and lower body mass index

Also, patients with symptomatic HF, especially when it is chronic and

stable, can have normal levels.

Intraindividual variability is relatively high; in a group of patients with

stable, chronic HF, week-to-week variability for BNP and N-BNP was
30%40%

At this time, it appears that the most appropriate use of the BNP test is as
an adjunctive

test to rule out HF in the acute setting; it must not be used as a sole
criterion for establishing the diagnosis of HF

it should be used judiciously as more information becomes

Available.

The earliest assay for BNP commercially available in the United States
was an immunoassay using an instrument most suitable for point-of-care
measurement.

Recently, the test has become available on large, automated

immunoassay platforms.

Assays for both BNP and N-BNP are available; a clear advantage of one
biomarker over the other for any particular application has not been
established

Besides being a biomarker for HF, BNP has natriuretic, vasodilatory, and
other effects that are ameliorative for the syndrome, and in fact is
available as the drug nesiritide (Natrecor) for the treatment of HF.

Because of the short half-life of BNP, measured levels several hours after
its administration would reflect endogenous secretion. However, the utility
of BNP measurement in the context of its therapeutic administration has
not been established at this time.
D-DIMER

Measures plasmin-cleaved, insoluble, cross-linked fibrin that originally

arose from thrombin cleavage of fibrinogen

can be performed by immunonephelometric assay or by latex

agglutination assay, and results can be obtained rapidly, facilitating the
use of this test in clinical decision-making

As would be anticipated, the ddimer is elevated in a wide variety of

thrombotic conditions, including DIC

By careful establishment of a cutoff value, the d-dimer may be judiciously

used in concert with clinical findings as a negative predictive test to
exclude deep vein thrombosis or pulmonary embolism

Level of d-dimer may decrease in response to anticoagulation.

In the past, fibrin degradation (split) products (FDPs) were used to

recognize DIC (see Fig. 39-4, A). However, depending on the specificity of
epitopes recognized by antibodies employed in the particular test
systems, FDP may variably represent plasmin-cleaved fibrinogen, soluble
fibrin, or insoluble fibrin.

The finding of increased FDPs accordingly does not necessarily translate

into a measure of plasmin-cleaved, insoluble, cross-linked fibrin equivalent
to that obtained with the d-dimer assay.

Fibrin monomer is the large molecular mass protein of fibrinogen that

remains after fibrinopeptides A and B are liberated. It can be elevated in
DIC, but if DIC is severe, it may be absent. Thus, it is an unreliable assay
for use in recognizing DIC.

A scoring system that combines in a quantitative fashion the results of four

commonly employed laboratory tests that may be helpful, in the
appropriate clinical setting, to evaluate the likelihood of DIC

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

From Book:

SEMEN ANALYSIS
Dennis Macapagal 2014
Reasons for doing semen analysis
1.
For fertility testing
2.
To determine the need for IVF
3.
As post vasectomy testing
4.
Forensic analysis
Outline
I.
Semen analysis
II.
Sperm preparation
III.
Quality assurance
SEMEN ANALYSIS

Normal physiology:

During ejaculation, semen is produced from a concentrated suspension of

spermatozoa, stored in the paired epididymides, mixed with, and diluted
by, fluid secretions from the accessory sex organs.

It is emitted in several boluses.

During sexual intercourse, the initial, sperm-rich prostatic fraction of the

ejaculated semen may come into contact with cervical mucus extending
into the vagina with the rest of the fluid remaining as a pool in the vagina.

In contrast, in the laboratory setting, the entire ejaculate is collected in one

container, where spermatozoa are trapped in a coagulum developed from
proteins of seminal vesicular origin.

This coagulum is subsequently liquefied by the action of prostatic

proteases
By: Rem Alfelor

There is some evidence that the quality of semen specimens varies

depending on how the ejaculate is produced.
Ejaculates produced by masturbation and collected into containers in a
room near the laboratory can be of lower quality than those recovered
from non-spermicidal condoms used during intercourse at home.
This difference may reflect a different form of sexual arousal.
Sperm concentrations in semen from young and old men may be the
same, but total sperm numbers may differ, as both the volume of seminal
fluid and total sperm output decrease with age, at least in some
populations.
In the absence of ejaculation, spermatozoa accumulate in the
epididymides, then overflow into the urethra and are flushed out in urine.
Steps:
1.
In the first 5 minutes:
Placing the specimen container on the bench or in an
incubator (37 C) for liquefaction.
2.
Between 30 and 60 minutes:
Assessing liquefaction and appearance of the semen.
Measuring semen volume.
Measuring semen pH (if required).
Preparing a wet preparation for assessing microscopic
appearance, sperm motility and the dilution required for
assessing sperm number.
Assessing sperm vitality (if the percentage of motile cells is
low).
Making semen smears for assessing sperm morphology.
Making semen dilutions for assessing sperm concentration.
Assessing sperm number.
3.
Within 3 hours:
Sending samples to the microbiology laboratory (if required).
4.
After 4 hours:
Fixing, staining and assessing smears for sperm morphology.
5.
Later on the same day (or on a subsequent day if samples are
frozen):
Assaying accessory gland markers (if required).

SAMPLE COLLECTION
1.
Dedicated private room for collection near the lab or within the lab.
2.
Minimum of 2 days and max of 7 days abstinence
3.
Verbal and or written instructions. (If a part is lost, it should be documented if
possible).

Note: Collection at home is possible

A. Submit within an hour
B. 20-37 degrees C
C. Condom collection:

nontoxic types

latex laden condoms

affect motility

Safe handling of specimens:

semen is a biohazard:
1. HIV
2. Hepatitis virus
3. Herpes simplex virus
I.

ANALYSIS
Initial microscopic exam:

soon after liquefaction, around 30 mins but not >1 hr from ejaculation
LIQUEFACTION:

initially as a coagulum becomes thinner and watery thereafter

Usually, in 15 minutes, rarely in 1 hour

Condom specimens: If more than 1 hr, record.

This is the time when the sperms obtain the ability to move and is the best
time to examine them microscopically.

It is helpful to shake gently the specimen during liquefaction to produce a

homogenous sample

If there are no moving sperms, wait until full liquefaction.

Delayed liquefaction:
impossible to asses motility
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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Dulbeccos phosphate buffered saline

Proteolytic digestion by bromelain
Note: affects motility and morphology and use must be recorded
SEMEN VISCOSITY:

after liquefaction:

use a 1.5mm bore disposable pipette

Normally, it leaves the tip of the pipette in small dropsif abnormal; they
will form thread more than 2 cm long.

Alternatively, one may use a glass rod.

NB: high viscosity affects motility

APPEARANCE OF THE EJACULATE:

after liquefaction, it is homogeneous, grey-opalescent

It may appear less opaque if the sperm concentration is very low.

The color may also be different, i.e. red-brown when red blood cells are
present (haemospermia), or yellow in a man with jaundice or taking
certain vitamins or drugs.
VOLUME:

Collect the sample in a pre-weighed, clean, disposable container.

Weigh the vessel with semen in it.

Subtract the weight of the container.

Calculate the volume from the sample weight, assuming the density of
semen to be 1 g/ml.

Note:
Empty specimen containers may have different weights, so each
container should be individually pre-weighed.
The weight may be recorded on the container before it is given to
the client.
Use a permanent marker pen on the vessel itself or on a label.
If a label is used for recording the weight, it should be attached
before the empty container is weighed.
Alternatively:
Collect the sample directly into a modified graduated glass
measuring cylinder with a wide mouth.
Read the volume directly from the graduations (0.1 ml
accuracy).
Measuring volume by aspirating the sample from the specimen
container into a pipette or syringe, or decanting it into a measuring
cylinder, is not recommended, because not all the sample will be
retrieved and the volume will therefore be underestimated.
The volume lost can be between 0.3 and 0.9 ml

Comments on volume:
1.
Low semen volume is characteristic of obstruction of the ejaculatory
duct or congenital bilateral absence of the vas deferens, a condition
in which the seminal vesicles are also poorly developed.
2.
Low semen volume can also be the result of collection problems
(loss of a fraction of the ejaculate), partial retrograde ejaculation or
androgen deficiency.
3.
High semen volume may reflect active exudation in cases of active
inflammation of the accessory organs.

Lower reference limit: 1.5 ml (5th centile, 95% confidence interval)

pH

after liquefaction preferably after 30 minutes

use pH paper between 6-10

Spread a drop of semen evenly onto the pH paper.

Wait for the color of the impregnated zone to become uniform
(<30 seconds).
Compare the color with the calibration strip to read the pH.

Note: The accuracy of the pH paper should be checked against known

standards.

For viscous samples, the pH of a small aliquot of the semen can be

measured using a pH meter designed for measurement of viscous
solutions.

Reference values:
There are currently few reference values for the pH of semen from
fertile men.
Pending more data, a consensus value of 7.2 as a lower threshold
value.

Comments:

By: Rem Alfelor

1.

if less than 7.0 in a semen sample with low volume and low sperm
numbers:
There may be ejaculatory duct obstruction or congenital
bilateral absence of the vas deferens, a condition in which
seminal vesicles are also poorly developed.
2.
Semen pH increases with time, as natural buffering decreases, so
high pH values may provide little clinically useful information.
Initial microscopic investigation:

use a phase contrast microscope

This provides an overview of the sample, to reveal:

mucus strand formation;
sperm aggregation or agglutination;
The presence of cells other than spermatozoa, e.g. epithelial cells,
round cells (leukocytes and immature germ cells) and isolated
sperm heads or tails.

The preparation should then be observed at 200 or 400 total

magnifications (i.e. a combination of a 20 or a 40 objective with a 10
ocular).
This permits:
assessment of sperm motility
determination of the dilution required for accurate assessment
of sperm number
A. Making a wet preparation
B. Aggregation of spermatozoa

AGGLUTINATION OF SPERMATOZOA:
Agglutination specifically refers to motile spermatozoa sticking to
each other, head-to-head, tail-to-tail or in a mixed way.
The motility is often vigorous with a frantic shaking motion, but
sometimes the spermatozoa are so agglutinated that their motion is
limited.
grade 1: isolated <10 spermatozoa per agglutinate, many free
spermatozoa
grade 2: moderate 1050 spermatozoa per agglutinate, free
spermatozoa
grade 3: large agglutinates of >50 spermatozoa, some
spermatozoa still free
grade 4: gross all spermatozoa agglutinated and agglutinates
interconnected
Note: Motile spermatozoa stuck to cells or debris or immotile
spermatozoa stuck to each other (aggregation) should not be
scored as agglutination.
A. Head-to-head
B. Tail-to-tail
C. ail-tip-to-tail-tip
D. Mixed
E. Tangle
Comment 1: The presence of agglutination is not sufficient
evidence to deduce an immunological cause of infertility, but
is suggestive of the presence of anti-sperm antibodies; further
testing is required.
Comment 2: Severe agglutination can affect the assessment
of sperm motility and concentration.

Cellular elements other than spermatozoa

epithelial cells from the genitourinary tract, as well as leukocytes
and immature germ cells, the latter two collectively referred to as
round cells
They can be identified by examining a stained smear at 1000
magnification
These cells can be more precisely identified and quantified by
detecting peroxidase activity or the antigen CD45.
SPERM MOTILITY

The extent of progressive sperm motility is related to pregnancy rates

computer-aided sperm analysis

Sperm motility within semen should be assessed as soon as possible after

liquefaction of the sample, preferably at 30 minutes, but in any case within
1 hour, following ejaculation, to limit the deleterious effects of dehydration,
pH or changes in temperature on motility.

Mix the semen sample well.

Wait for the sample to stop drifting (within 60 seconds).

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test

Examine the slide with phase-contrast optics at 200 or 400

magnifications.

Assess approximately 200 spermatozoa per replicate for the percentage

of different motile categories.

Note: examination should be at room temperature or at 37 C with a

heated microscope stage, but should be standardized for each laboratory.

If sperm motility is to be assessed at 37 C, the sample should be

incubated at this temperature and the preparation made with pre-warmed
slides and coverslips.

Categories of sperm movement:

1.
PROGRESSIVE MOTILITY (PR): spermatozoa moving actively,
either linearly or in a large circle, regardless of speed.
2.
NON-PROGRESSIVE MOTILITY (NP): all other patterns of motility
with an absence of progression, e.g. swimming in small circles, the
flagellar force hardly displacing the head, or when only a flagellar
beat can be observed.
3.
IMMOTILITY (IM): no movement.

Lower reference limit: Motility

1.
The lower reference limit for total motility (PR + NP) is 40%.
2.
The lower reference limit for progressive motility (PR) is 32%.

Comment:
The total number of progressively motile spermatozoa in the
ejaculate is of biological significance.
This is obtained by multiplying the total number of spermatozoa in
the ejaculate by the percentage of progressively motile cells.
SPERM VITALITY

Comment 1: It is clinically important to know whether immotile

spermatozoa are alive or dead.

Comment 2: The presence of a large proportion of vital but immotile cells

may be indicative of structural defects in the flagellum; a high percentage
of immotile and non-viable cells (necrozoospermia) may indicate
epididymal pathology.

Vitality test using eosinnigrosin stain

Tally the number of stained (dead) or unstained (vital) cells with the
aid of a laboratory counter.

Eosinnigrosin smear observed in bright field optics

Lower reference limit: Vitality

The lower reference limit (membrane-intact spermatozoa) is 58%

Comment: The total number of membrane-intact spermatozoa in the

ejaculate is of biological significance. This is obtained by multiplying the
total number of spermatozoa in the ejaculate by the percentage of
membrane-intact cells.
SPERM NUMBERS

Comment 1: The terms total sperm number and sperm concentration

are not synonymous.

SPERM CONCENTRATION refers to the number of spermatozoa per unit

volume of semen and is a function of the number of spermatozoa emitted
and the volume of fluid diluting them.

TOTAL SPERM NUMBER refers to the total number of spermatozoa in

the entire ejaculate and is obtained by multiplying the sperm concentration
by the semen volume.

Counting at least 200 spermatozoa.

Calculating the concentration in spermatozoa per ml.

Calculating the total number of spermatozoa per ejaculate.

Use of Neubauer haemocytometer

Using the haemocytometer grid:

Count only whole spermatozoa (with heads and tails).
Whether or not a spermatozoon is counted is determined by the
location of its head; the orientation of its tail is unimportant.
Lower reference limit for sperm concentration
The lower reference limit for sperm concentration is 15 106
spermatozoa per ml.
Lower reference limit for total sperm number
The lower reference limit for total sperm number is 39 106
spermatozoa per ejaculate
Counting of cells other than spermatozoa
The presence of non-sperm cells in semen may be indicative of
testicular damage (immature germ cells), pathology of the efferent

By: Rem Alfelor

II.
III.

ducts (ciliary tufts) or inflammation of the accessory glands

(leukocytes).
SPERM MORPHOLOGY

Morphologically normal spermatozoa

Spermatozoa consist of a head, neck, middle piece (midpiece), principal

piece and endpiece.

As the endpiece is difficult to see with a light microscope, the cell can be
considered to comprise a head (and neck) and tail (midpiece and principal
piece).

For a spermatozoon to be considered normal, both its head and tail must
be normal.

All borderline forms should be considered abnormal.

Lower reference limit

The lower reference limit for normal forms is 4%
Assessment of leukocytes in semen

Leukocytes, predominantly polymorphonuclear leukocytes (PMN,

neutrophils), are present in most human ejaculates

Note: Leukocytes can impair sperm motility and DNA integrity through an
oxidative attack
Assessment of immature germ cells in semen

Germ cells include round spermatids and spermatocytes, but rarely

spermatogonia.

They can be detected in stained semen smears, but may be difficult to

distinguish from inflammatory cells when the cells are degenerating.
Testing for antibody coating of spermatozoa

If spermatozoa demonstrate agglutination (i.e. motile spermatozoa stick to

each other head-to-head, tail-to-tail or in a mixed way), the presence of
sperm antibodies may be the cause.

Anti-sperm antibodies (ASAs) in semen belong almost exclusively to two

immunoglobulin classes: IgA and IgG.
Others:

Optional procedures
Interaction between spermatozoa and cervical mucus In-vivo
(postcoital) test
Capillary tube test
The test measures the ability of spermatozoa to penetrate a
column of cervical mucus in a capillary tube.
SPERM PREPARATION

Cryopreservation of spermatozoa
QUALITY ASSURANCE

Equipment and safety

Pregnancy Testing
Introduction

Most pregnancy tests used today, whether home urine test, a physician's office
urine or blood test, or a clinical laboratory blood test are "sandwich assays".

Sandwich assays use two or more animal antibodies raised against different
sites on Human Chorionic Gonadotropin (hCG).

Usually a mouse monoclonal antibody against one site on the hCG molecule,
and a mouse monoclonal, or a sheep, rabbit or a goat polyclonal antibody
against a second distant site on the hCG molecule.
Simple to Perform

But understanding the concepts of the principle may be challenging.

SUMMARY AND EXPLANATION OF THE TEST

Human chorionic gonadotropin (hCG) is a glycopeptide hormone produced by

the placenta during pregnancy.

The appearance and rapid rise in the concentration of hCG in the woman's urine
makes it a good pregnancy marker.

Usually, concentration of hCG in urine is at least 25 mIU/ml as early as seven to

ten days after conception.

The concentration increases steadily and reaches its maximum between the
eighth and eleventh weeks of pregnancy.

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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test
Fertilization

Figure 3: Excess tracer antibody is washed away. Amount of label or tracer (red star)
is measured. This is proportional to amount of hCG.
Illustration of Principle
PRINCIPLE OF THE TEST

The pregnancy testing device contains a unique set of dye-conjugated and

immobilized antibodies used to produce a distinctive visual pattern indicating
elevated concentration of hCG (=25 mIU/ml) in the test sample.

One antibody, the capture antibody, is in a solid phase permanently attached to

a tube, plate, membrane, or bead.

Conjugate pad contains the label reagent, i.e. antibody labeled with either red,
gold or blue latex particles.

Sample is applied and dissolves the label mixture and migrates to the zone of
immobilized antibody lines. If hCG is present, labeled antibody-dye conjugate
binds it, forming an antibody-antigen complex.

Positive, that is hCG containing, sample causes the formation of a colored test
line, which indicates a positive test result.

As the reaction mixture continues to flow along the test membrane, the complex
binds to the anti-hCG antibody in the test zone of the membrane, and produces
a color band.

Unbound conjugate binds to the reagents immobilized in the control zone

producing a color band, demonstrating proper performance of the test.

TEST PROCEDURE

NOTE: Bring test components and specimens to room temperature prior to

testing.

Remove a Testing Device from the foil pouch by tearing at the "notch" and place
it on a level surface.

Holding a Sample Dropper vertically, add exactly four drops of the urine
specimen to the sample well. NOTE: Picture shows incorrect orientation of
dropper to test area, must be completely vertical to ensure adequate sample.

Read results at time indicated in procedure.

Interpretation

If two color bands are visible the test is positive.

The presence of a Control Band only indicates a negative test.

Results

Figure 1: Device with solid phase captures antibody to one site on hCG, and liquid
phase tracer antibody (label shown by red star) to second or distant site on hCG. In
this way the label becomes immobilized.

Function of the Control Band

The Control Band is used as a reference and built in quality control check.

If the Test Band is darker or similar to the Control band, the test result is
considered positive.

The Control Band is used for procedural control to check whether the test
reagents are working properly and that a sufficient amount of urine sample has
been applied to the test area.
Figure 2: Serum or urine containing hCG (shown as ab) added to device. The hCG
forms a sandwich or bridge between capture and tracer antibody. After a short
incubation period the hCG binds both the solid phase and liquid phase antibodies
linking them.

By: Rem Alfelor

Invalid Tests

If, after performing the test, no purple color band is visible anywhere within the
Results Window, the result is considered invalid.

If a color appears in the test area but NO color appears in the control area, the
test is invalid.
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Clinical Pathology LE # 1: Fecalysis, Urinalysis, Acute Coronary Syndrome, Semen Analysis, Pregnancy Test
Ovulation Test

CHORIOCARCINOMA

Choriocarcinoma is a highly malignant germ cell tumor which usually follows an

abnormal pregnancy with a hydatidiform mole. It may also occur after a
spontaneous abortion, and rarely, may follow a normal pregnancy.

The tumor metastasizes early, by means of vascular invasion and blood spread.
Picture of liver.
CHORIOCARCINOMA OF TESTES

Another Example of Results

HYDATIFORM MOLE
Causes of Invalid Results

The directions may not have been followed correctly.

Inadequate amount of sample has been exposed to the test system.

The test may have deteriorated.

PRECAUTIONS

Do not use test kit components after the expiration dates.

Dispose of all used test components in a proper biohazard container.

If specimens or test components have been stored in a refrigerator, allow them

to warm to room temperature before performing the test.

Human specimens should be handled as if capable of transmitting infectious

agents.
Limitations of the Procedure

Besides pregnancy, elevated concentrations of hCG may be found in patients

with both gestational and non-gestational trophoblastic diseases. These
conditions should be ruled out in the interpretation of hCG levels to establish a
diagnosis of pregnancy.

A low incidence of false results can occur. Consult with a physician if

unexpected or inconsistent results.

A normal pregnancy cannot be distinguished from an ectopic pregnancy based

on hCG levels alone.

A spontaneous miscarriage may cause confusion in interpreting the test results.

A definitive diagnosis should not be based on the results of a single test, but
should only be made by the physician after all clinical and laboratory findings
have been evaluated.

A negative result from a specimen collected from a woman in very early

pregnancy may be due to an unusually low concentration of hCG. In such cases,
the test should be repeated on a fresh specimen obtained approximately two
days later.

A urine sample may be too diluted and thus may not contain a representative
concentration of hCG. If a negative result is obtained with a urine specimen and
pregnancy is still suspected, obtain a first morning urine specimen and re-test.
ECTOPIC PREGNANCY

Hydatidiform mole is a tumor of the placenta that is usually benign. It develops

from placental tissue during an early pregnancy in which the embryo fails to
develop normally. The tumor consists of many small vesicles (sacs) and
resembles a large cluster of grapes. Although the condition is fairly rare in the
USA, it is common in the Orient and other parts of the world. In the United
States, molar pregnancy occurs in 1 of every 1,000-1,200 pregnancies.

SEMINOMA

50% of all testicular tumors.

Human chorionic gonadotropin-beta is a better marker than hCG. For earlier
detection of recurrence, both markers should be examined.

SPECIMEN COLLECTION AND STORAGE

First morning urine usually contains the highest concentration of hCG and is
therefore the best sample when performing the urine test. However, randomly
collected urine specimens may be used.

Collect a urine specimen in a clean glass, plastic, or wax coated container. Do

not use preservatives.

If the test is not run immediately following collection of the sample specimen, but
is to be run within 48 hours following collection, the specimen should be
refrigerated (2-8C), and brought back to room temperature (15-28C) before
testing.

If testing is delayed more than forty-eight hours, the specimen should be frozen.
A frozen specimen should not be used if stored more than two weeks.

Prior to testing, the frozen specimen must be completely thawed, thoroughly

mixed, and brought to room temperature.
QUALITY CONTROL

The use of controls is recommended to verify proper kit performance.

Quality control reagents (positive and negative) should be tested according to

quality control requirements established by the testing laboratory.

Use controls in the same procedure as specimens.

This may be required with each test or only when a new lot number is being put
into use.

This unfortunate patient came in with a late cycle. The pregnancy test was
positive but we couldn't see a pregnancy on her ultrasound scan. We suspected
an ectopic and this is what we found during laparoscopy.
The pregnancy was lodged in the middle of her left tube. A small slit was made
over this and the pregnancy was removed without removing her tube

By: Rem Alfelor

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