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RENAL DISEASE
GLOMERULAR DISORDERS
GLOMERULONEPHRITIS
- Refers to sterile, inflammatory process that
affects the glomerulus and associated with
the finding of blood, protein and casts in
urine.
Acute Poststreptococcal Glomerulonephritis (AGN)
o A disease marked by sudden onset of
symptoms consistent with damage to the
glomerular membrane.
o May include:
o Fever
o Edema (noticeably in eyes)
o Fatigue
o Hypertension
o Oliguria
o Hematuria
o Symptoms: children and young adults ff.
respiratory infections by certain strains of
group A streptococcus that contain M protein
in cell wall.
o During infection: nephrogenic strains of
streptococci from immune complexes with
their corresponding circulating antibodies
and become deposited on glomerular
membrane
o Secondary complications: hypertension and
electrolyte imbalance, until immune
complexes is cleared from blood and
inflammation subsides resulting to no kidney
damage.
o Primary urinalysis: marked hematuria,
proteinuria, and oliguria, accompanied by red
blood cell (RBC) casts, dysmorphic RBCs,
hyaline and granular casts, and white blood
cells (WBCs)
o As toxicity in glomerular membrane subsides
– urinalysis results return to normal
o Blood Urea Nitrogen (BUN): elevate during
acute stages but urinalysis returns normal
o Demonstration of positive anti–group A
streptococcal enzyme tests provides
evidence that the disease is of streptococcal
origin.
Rapidly Progressive (Crescentic) Glomerulonephritis
o More serious form of acute glomerular
disease
o Has a much poorer prognosis often
terminating in renal failure
o Symptoms: initiated by deposition of immune
complexes in the glomerulus – complication
of another form of glomerulonephritis –
Systemic Lupus Erythematosus (SLE).
o Damage by macrophages to the capillary
walls releases cells and plasma into
Bowman’s space, and the production of
crescentic formations containing
macrophages, fibroblasts, and polymerized
fibrin, causes permanent damage to the
capillary tufts.
o Initial laboratory results similar to acute
glomerulonephritis but become abnormal as
the disease progresses – marked elevated
protein levels and very low glomerular
filtration rates.
Goodpasture Syndrome
o Morphologic changes to the glomeruli
resembling those in rapidly progressive
glomerular nephritis are seen in conjunction
with the autoimmune disorder
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o A cytotoxic autoantibody can appear against o Incubating patient’s serum with
the glomerular and alveolar basement either ethanol or formalin
membranes after viral respiratory infections. o Examining preparation using indirect
o Attachment of this autoantibody to the immunofixation – detect serum
basement membrane, followed by antibodies attached to neutrophils
complement activation, produces the  Neutrophils fixed in ethanol:
capillary destruction. antibodies form a
o Antiglomerular basement membrane perinuclear pattern called p-
antibody - an antibody that can be ANCA.
detected in patient serum  Neutrophils fixed with
o Initial pulmonary complaints – Hemoptysis formalin: pattern is granular
and Dyspnea, followed by development throughout the cytoplasm
hematuria called c-ANCA
o Urinalysis Results:
o Proteinuria
o Hematuria
o Presence of RBC casts
o Progression to Chronic
glomerulonephritis and end stage
renal failure is common
Wegener Granulomatosis
o causes a granuloma-producing inflammation
of the small blood vessels primarily of the
kidney and respiratory system.
o Key to diagnosis Wegener Granulomatosis –
demonstration of antineutrophilic cytoplasmic Henoch-Schönlein Purpura
antibody (ANCA) in patient’s serum.
o Binding of these autoantibodies to the
neutrophils located in the vascular walls may
initiate the immune response and the
resulting granuloma formation.
o Patients present first:
o Pulmonary symptoms Henoch-Schönlein purpura disease occurs
o Later develop renal involvement, primarily in children after upper respiratory
includes hematuria, proteinuria, RBC infections.
casts and elevated serum creatinine Initial symptoms
and BUN  appearance of raised, red patches on the
o Testing for ANCA: skin.
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RENAL DISEASE
Respiratory and gastrointestinal symptoms
 blood in the sputum and stools,
The most serious complication
 Renal involvement is the most serious
complication of the disorder and may range
from mild to heavy proteinuria and
hematuria with RBC casts.
Complete recovery with normal renal function is
seen in more than 50% of patients. In other
patients, progression to a more serious form of
glomerulonephritis and renal failure may occur.
Urinalysis and renal function assess - for
monitoring the disease
Membranous Glomerulonephritis
pronounced thickening of the glomerular basement
membrane resulting from the deposition of
immunoglobulin G immune complexes.
Disorders associated
 systemic lupus erythematosus,
 Sjögren syndrome,
 secondary syphilis,
 hepatitis B
 malignancy.
Many cases of unknown etiology have been
reported. As a rule, the disease progresses
slowly, with possible remission; however,
nephrotic syndrome symptoms frequently
develop.There may also be a tendency toward
thrombosis
Laboratory findings include
 Microscopic hematuria and elevated urine
protein excretion that may reach
concentrations similar to those in the
nephrotic syndrome. Demonstration of one of
the secondary disorders through blood tests
can aid in the diagnosis.
Membranoproliferative Glomerulonephritis
Marked by two different alterations in the cellularity
of the glomerulus and peripheral capillaries.
 Type 1 displays increased cellularity in the
subendothelial cells of the mesangium
(interstitial area of Bowman’s capsule),
causing thickening of the capillary walls,
whereas
 Type 2 displays extremely dense deposits in
the glomerular basement membrane. Many
of the patients are children, and the disease
has a poor prognosis: type 1 patients
progress to the nephrotic syndrome and
type 2 patients experience symptoms of
chronic glomerulonephritis. The laboratory
findings vary, but hematuria, proteinuria, and
decreased serum complement levels are
usual findings. There appears to be an
association with autoimmune disorders,
infections, and malignancies
Chronic Glomerulonephritis
 Depending on the amount and duration of
the damage to the glomerulus in the
previously discussed glomerular disorders,
progression to chronic glomerulonephritis
and end-stage renal disease may occur.
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RENAL DISEASE
Gradually worsening symptoms include
 fatigue,
 anemia,
 hypertension,
 edema, and
 oliguria.
Examination of the urine reveals hematuria,
proteinuria, glucosuria as a result of tubular
dysfunction, and many varieties of casts, including
broad casts.
Immunoglobulin A Nephropathy
Also known as Berger disease,
IgA nephropathy, in which immune complexes
containing IgA are deposited on the glomerular
membrane, is the most common cause of
glomerulonephritis.
Patients have increased serum levels of IgA, which
may be a result of a mucosal infection.
Most frequently seen in
 children and young adults.
Patients usually present with an episode of
macroscopic hematuria following an infection or
strenuous exercise.
Recovery from the macroscopic hematuria is
spontaneous; however,
asymptomatic microhematuria and elevated serum
levels of IgA remain.Except for periodic episodes of
macroscopic hematuria, a patient with the disorder
may remain essentially asymptomatic for 20 years or
more; however, there is agradual progression to
chronic glomerulonephritis and end-stage renal
disease
Tubular Disorders- Disorders affecting the renal
tubules such as:
1. Tubular function is disrupted as a result of
actual damage to the tubules
2. Metabolic or hereditary disorder affecting the
intricate functions of the tubules
Acute Tubular Necrosis (ATN)- primary disorder
associated with damage to the renal tubules
a. Produced by decreased blood flow that
causes a lack of oxygen presentation to the
tubules (ischemia)
Disorders causing ischemic ATN:
a. Shock- cause by cardiac failures, sepsis
involving toxigenic bacteria, anaphylaxis,
massive hemorrhage, and contact with
high-voltage electricity.
b. trauma (such as crushing injuries),
c. surgical procedures.
Disorders associated with ATN
Acute glomerulonephritis:
Laboratory Testing :
Primary Urinalysis Result: Macroscopic hematuria,
Proteinuria , RBC casts, Granular casts
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RENAL DISEASE
Other Significant Tests: Anti–group A
streptococcal enzyme tests
Clinical Information:
Etiology: Deposition of immune complexes,
formed in conjunction with group A Streptococcus
infection, on the glomerular membranes.
Clinical Course: Rapid onset of hematuria
and edema, permanent renal damage seldom occurs
Rapidly progressive glomerulonephritis
Laboratory Testing
Primary Urinalysis Result: Macroscopic
hematuria , Proteinuria , RBC casts
Other Significant Tests: BUN , Creatinine
,eGFR
Clinical Information
Etiology: Deposition of immune complexes
from systemic immune disorders on the glomerular
membrane
Clinical Course: Rapid onset with
glomerular damage and possible progression to end-
stage renal failure
Goodpasture syndrome
Laboratory Testing
Primary Urinalysis Result: Macroscopic
hematuria , Proteinuria , RBC casts
Other Significant Tests: Antiglomerular
basement membrane antibody
Clinical Information:
Etiology: Attachment of a cytotoxic antibody
formed during viral respiratory infections to
glomerular and alveolar basement membranes
Clinical Course: Hemoptysis and dyspnea
followed by hematuria. Possible progression to end-
stage renal failure
Wegener granulomatosis
Laboratory Testing
Primary Urinalysis Result: Macroscopic
hematuria ,Proteinuria ,RBC casts
Other Significant Tests: Antineutrophilic
peripheral or cytoplasmic antibody
Clinical Information
Etiology: Antineutrophilic cytoplasmic
autoantibody binds to neutrophils in vascular walls
producing damage to small vessels in the lungs and
glomerulus
Clinical Course: Pulmonary symptoms
including hemoptysis develop first followed by renal
involvement and possible progression to end-stage
renal failure
Henoch-Schönlein purpur
Laboratory Testing
Primary Urinalysis Result: Macroscopic
hematuria, Proteinuria , RBC casts
Other Significant Tests: Stool occult blood
Clinical Information
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RENAL DISEASE
Etiology: Occurs primarily in children
following viral respiratory infections; a decrease in
platelets disrupts vascular integrity
Clinical Course: Initial appearance of
purpura followed by blood in sputum and stools and
eventual renal involvement. Complete recovery is
common, but may progress to renal failure
Membranous glomerulonephritis
Laboratory Testing
Primary Urinalysis Result: Microscopic
hematuria, Proteinuria
Other Significant Tests: Antinuclear antibody,
Hepatitis B surface antigen , Fluorescent
treponemal antibodyabsorption test (FTA-ABS)
Clinical Information
Etiology: Thickening of the glomerular
membrane following IgG immune complex deposition
associated with systemic disorders
Clinical Course: Slow progression to the
nephrotic syndrome or possible remission
Membranoproliferative glomerulonephritis
Etiology: Cellular proliferation affecting the
capillary walls or the glomerular basement
membrane, possibly immune-mediated
Clinical Course: Slow progression to
chronic glomerulonephritis or nephrotic syndrome
Chronic glomerulonephritis
Laboratory Testing
Primary Urinalysis Result: Hematuria ,
Proteinuria, Glucosuria Cellular and granular casts ,
Waxy and broad casts
Other Significant Tests: BUN. Serum
creatinine , eGFR, Electrolytes
Clinical Information
Etiology: Marked decrease in renal function
resulting from glomerular damage precipitated by
other renal disorders
Clinical Course: Noticeable decrease in
renal function progressing to renal failure
IgA nephropathy (early stages)

Laboratory Testing Laboratory Testing

Primary Urinalysis Result: Hematuria Primary Urinalysis Result: Macroscopic or
Proteinuria microscopic hematuria
Other Significant Tests: Serum complement Other Significant Tests: Serum IgA
levels
Clinical Information
Clinical Information
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RENAL DISEASE
Etiology: Deposition of IgA on the glomerular
membrane resulting from increased levels of serum
IgA
Clinical Course: Recurrent macroscopic
hematuria following exercise with slow progression
to chronic glomerulonephritis
IgA nephropathy (late stages)
Laboratory Testing
Primary Urinalysis Result: See Chronic
glomerulonephritis
Other Significant Tests: ------------
Clinical Information
Etiology: Deposition of IgA on the glomerular
membrane resulting from increased levels of serum
IgA
Clinical Course: Recurrent macroscopic
hematuria following exercise with slow progression
to chronic glomerulonephritis
Nephrotic syndrome
Laboratory Testing
Primary Urinalysis Result: Heavy proteinuria
, Microscopic hematuria, Renal tubular cells, Oval
fat bodies , Fat droplets , Fatty and waxy casts
Other Significant Tests: Serum albumin ,
Cholesterol , Triglycerides
Clinical Information
Etiology: Disruption of the shield of
negativity and damage to the tightly fitting podocyte
barrier resulting in massive loss of protein and lipids
Clinical Course: Acute onset following
systemic shock
Gradual progression from other glomerular
disorders and then to renal failure
Minimal change disease
Laboratory Testing
Primary Urinalysis Result: Heavy proteinuria
, Transient hematuria, Fat droplets
Other Significant Tests: Serum albumin,
Cholesterol, Triglycerides
Clinical Information
Etiology: Disruption of the podocytes
occurring primarily in children following allergic
reactions and immunizations
Clinical Course: Frequent complete
remission following corticosteroid treatment
Focal segmental glomerulosclerosis
Laboratory Testing
Primary Urinalysis Result: Proteinuria ,
Microscopic hematuria, Macroscopic or microscopic
hematuria
Other Significant Tests: HIV tests
Clinical Information
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RENAL DISEASE
Etiology: Disruption of podocytes in certain
areas of glomeruli associated with heroin and
analgesic abuse and AIDS
Clinical Course: May resemble nephrotic
syndrome or minimal change disease
Alport syndrome (early stages) (late stages)
Laboratory Testing
Primary Urinalysis Result
( early stages ) - See Nephrotic syndrome ( late
stages ) - Microalbuminuria
Other Significant Tests: Genetic testing
Clinical Information
Etiology: Genetic disorder showing
lamellated and thinning glomerular basement
membrane
Clinical Course: Slow progression to
nephrotic syndrome and end-stage renal disease
Diabetic nephropathy (late stages)
Laboratory Testing
Primary Urinalysis Result: See Chronic
glomerulonephritis
Other Significant Tests: Blood glucose
GENERALLY: Correcting the ischemia, removing
the toxic substances, and effectively managing
the accompanying symptoms of acute renal
failure frequently result in a complete recovery.
HEREDITARY AND METABOLIC TUBULAR
DISORDERS
Fanconi Syndrome
-may be inherited in association with cystinosis and
Hartnup disease, acquired through exposure to toxic
agents including heavy metals and outdated
tetracycline or seen as a complication of multiple
myeloma and renal transplant.
Alport Syndrome
-an inherited disorder of collagen production
affecting the glomerular basement membrane. The
glomerular basement membrane has a lamellated
appearance with areas of thinning.
Uromodulin-Associated Kidney Disease
-is a glycoprotein and is the only protein produced by
kidney.
It is produced by the proximal and distal convoluted
tubules.
An inherited disorder caused by an autosomal
mutation in the gene produces uromodulin.
Diabetic Nephropathy
-the most common cause of end-stage renal
disease. This glomerular damage is believed to be
associated with the deposition of glycosylated
proteins resulting from poorly controlled blood
glucose levels.
Nephrogenic Diabetes Insipidus
-inherited as a sex-linked recessive gene or acquired
from medications, including lithium and amphotericin
B. It is also may be seen as a complication of
polycystic kidney disease and sickle cell anemia.
Renal Glycosuria
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RENAL DISEASE
-either the number of glucose transporters in the
tubules is decreased or the affinity of the
transporters for the glucose is decreased.
Acute Pyelonephritis
Interstitial Disorders
Close proximity between the renal tubules and the
renal interstitium, disorders affecting the interstitium
also affect the tubules, resulting the condition
commonly called tubulointerstitial disease.
Urinary Tract Infection
 most common renal disease
 infection may involve lower urinary tract
(urethra and bladder) or the upper urinary
tract (renal pelvis, tubules, and interstitium)
 Cystitis
 most frequently encountered is
infection of the bladder
 progress to a more serious upper
UTI if left untreated
 seen more often in women and
children, who present with
symptoms of urinary frequency and
burning
 Urinalysis reveals presence of WBCs and
bacteria, often accompanied by the mild
proteinuria and hematuria and an
increased pH
 infection of the upper urinary tract,
including both the tubules and interstitium,
termed as pyelonephritis and can occur in
both acute and chronic forms.
 most frequently occurs as a result of
ascending movement of bacteria from a
lower UTI into the renal tubules and
insterstitium
 rapid onset of symptoms, including urinary
frequency, burning on urination, and
lower back pain
 These includes obstructions such as renal
calculi, pregnancy, and reflux of urine
from the bladder back to the uterus
(vesicoureteral reflux)
 Urinalysis results are similar to cystitis,
including numerous leukocytes and
bacteria with mild proteinuria and
hematuria.
 Additional finding of WBC casts, signifying
infection within the tubules, is of primary
diagnostic value for both acute and
chronic pyelonephritis
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RENAL DISEASE
 Sediments also should be carefully  symptoms tend to develop approximately 2
observed for the presence of bacterial weeks following administration of
casts medications
 penicillin, methicillin, ampicillin,
Chronic Pyelonephritis cephalosporins, sulfonamides,
 serious disorder that can result in NSAIDs, and thiazide diuretics
permanent damage to the renal tubules  urinalysis results include hematuria,
and possible progression to chronic renal possibly macroscopic, mild to moderate
failure proteinuria, numerous WBCs, and WBC
 congenital urinary structural defects casts without bacteria.
producing reflux nephropathy are the most
frequent cause of chronic pyelonephritis
 often diagnosed in children and may not be
suspected until tubular damage has become
advanced
 urinalysis results are similar to those seen in
acute pyelonephritis, particularly in the
early stages
 as the disease progress, a variety of
granular, waxy, and broad casts are
present, accompanied by increased
proteinuria and hematuria, and renal
concentration is decreased

Acute Interstitial Nephritis

 marked by inflammation of the renal
interstitium followed by inflammation of
the renal tubules.
 rapid onset of symptoms, renal
dysfunction including oliguria, edema,
decreased renal concentrating ability, and
a possible decrease in the glomerular
filtration rate.
 fever and the presence of a skin rash are
frequent initial symptoms
 primarily associated with an allergic
reaction to medications that occurs within
the renal interstitium, possibly caused by the
medication binding to the interstitial protein
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RENAL DISEASE
RENAL FAILURE as acute pyelonephritis or septicemia; urinary tract
obstruction (obstructive uropathy); autoimmune
Kidney failure, also called end-stage renal disease
kidney disease such as interstitial nephritis or acute
(ESRD), is the last stage of chronic kidney disease.
nephritic syndrome; disorders that cause clotting
When your kidneys fail, it means they have stopped
within the thin blood vessels of the kidney; transfusion
working well enough for you to survive without dialysis
reaction; and many more.
or a kidney transplant.
Renal failure can occur rapidly or over time posing a
serious or even life-threatening risk to the patient.
This situation requires measures such as dialysis or
kidney transplant to rid the body of toxic nitrogenous
wastes that the kidney is no longer able to remove. CAUSES OF ACUTE RENAL FAILURE
PRERENAL POSTRENA
In most cases, kidney failure is caused by other health RENAL
L
problems that have done permanent damage (harm)
Decreased Acute Renal calculi
to your kidneys little by little, over time. When your blood glomerulonephrit Tumors
kidneys are damaged, they may not work as well as pressure/cardi is
they should. If the damage to your kidneys continues ac output Acute tubular
to get worse and your kidneys are less and less able Hemorrhage necrosis Acute
to do their job, you have chronic kidney disease. Burns pyelonephritis
Kidney failure is the last (most severe) stage of Surgery Acute interstitial
chronic kidney disease. This is why kidney failure is Septicemia nephritis
also called end-stage renal disease, or ESRD for
short.
NOTE:
RENAL FAILURE EXISTS IN BOTH ACUTE AND
Patients may present with many different symptoms
CHRONIC FORMS.
relating to the particular disorder involved; however, a
ACUTE RENAL FAILURE decreased glomerular filtration rate, oliguria,
edema, and azotemia are general characteristics
Acute (sudden) kidney failure is the sudden
of renal failure.
loss of the ability of the kidneys to remove waste and
concentrate urine without losing electrolytes. There
are many possible causes of kidney damage that
HEPATORENAL SYNDROME
precipitate this condition.
This syndrome usually occurs with fulminant hepatitis
They include decreased blood flow, which
or advanced cirrhosis of the liver with ascites fluid
may occur with extremely low blood pressure caused
buildup. Hepatorenal syndrome has an acute onset in
by trauma, surgery, serious illnesses, septic shock,
these patients with severe liver disease. This
hemorrhage, burns, or dehydration; acute tubular
condition is characterized by a progressively intense
necrosis, infections that directly injure the kidney such
vasoconstriction which leads to oliguria, elevated
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RENAL DISEASE
BUN and creatinine, and renal failure. Despite renal
failure, little renal morphologic changes can be seen
in biopsy. The kidneys are still able to produce a
smaller amount of hypertonic urine. This urine is very
low in sodium due to hyperaldosteronism. The
characteristic proteinuria of kidney disease is absent
and the urine does not contain abnormal sediment. If
the hepatic function can be restored the kidney
function can also improve.
CHRONIC RENAL FAILURE
The progression to end-stage renal disease is
characterized by a marked decrease in the glomerular
filtration rate (less than 25 mL/min); steadily rising
serum BUN and creatinine values (azotemia);
electrolyte imbalance; lack of renal concentrating
ability producing an isosthenuric urine; proteinuria;
renal glycosuria; and an abundance of granular,
waxy, and broad casts, often referred to as a
telescoped urine sediment.
The rate of chronic renal failure
progression varies from several months to many
years. This progression occurs in these four
stages:
(a) diminished renal reserve (GFR drops to
about 50% of normal),
(b) renal insufficiency (GFR drops from 20 to
50%, azotemia, anemia, and hypertension begin),
(c) renal failure (GFR is less than 20%,
kidneys cannot regulate volume and solute
concentration, and metabolic acidosis, edema, and
hyperkalemia develop), and
(d) end stage renal disease (GFR less than
5% of normal, glomerular scarring and reduction of
renal capillaries, tubular atrophy and fibrosis, and loss
of kidney mass and dialysis or transplantation may be
required for survival. Critical renal functions are lost.)
END STAGE RENAL DISEASE—DIABETES
Currently, diabetes mellitus and related diabetic
nephropathy (Kimmelstiel–Wilson disease) are the
most common cause of end stage renal disease.
Monitoring patients with diabetes for the presence of
microalbuminuria is important for early detection of
the onset of diabetic nephropathy. Control of blood
sugar and blood pressure is critical for patients with
diabetes to control damage of the kidney.
SYMPTOMS OF KIDNEY FAILURE
You may notice one or more of the following
symptoms if your kidneys are beginning to fail:
 Itching
 Muscle cramps
 Nausea and vomiting
 Not feeling hungry
 Swelling in your feet and ankles
 Too much urine (pee) or not enough urine
 Trouble catching your breath
 Trouble sleeping
If your kidneys stop working suddenly (acute kidney
failure), you may notice one or more of the following
symptoms:
 Abdominal (belly) pain
 Back pain
 Diarrhea
 Fever
 Nosebleeds
 Rash
 Vomiting
LABORATORY FINDINGS IN RENAL FAILURE
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RENAL DISEASE
OTHER  the calculi vary in size from barely visible to
TYPICAL
DISEASE/DISOR LABORATO large, staghorn calculi resembling the shape
URINALYSI
DER RY of the renal pelvis and smooth, round bladder
S FINDINGS
FINDINGS stones with diameters of 2 or more inches.
↓urine
Proteinuria, Lithotripsy
output with
hemoglobinu
inability to  a procedure using high-energy shock waves,
ria, RTEs
vary specific
and RTE can be used to break stones located in the
Acute renal failure gravity,
casts, WBC upper urinary tract into pieces that can then
↑BUN,
and other be passed in the urine.
↑creatinine,
casts,
↓creatinine  Stones can also be removed surgically.
crystals
clearance
↓Urine Conditions favoring the formation of renal
output with calculi are similar to those favoring formation
Proteinuria, inability to of
hemoglobinu vary specific
Chronic renal  urinary crystals
ria, waxy and gravity,
failure
broad casts, ↑BUN,  including pH,
crystals ↑creatinine,
↓creatinine  chemical concentration, and
clearance
 urinary stasis
NOTE: Analysis can be performed chemically, but
Similar to clinical symptoms, urinalysis findings are examination using x-ray crystallography
varied, but because they relate to the primary cause provides a more comprehensive analysis.
of the ARF, they can be diagnostically valuable. For  Approximately 75% of the renal calculi are
example, the presence of RTE cells and casts composed of calcium oxalate or calcium
suggests ATN of prerenal origin; RBCs indicate phosphate.
glomerular injury; WBC casts with or without bacteria
indicate interstitial infection or inflammation of renal  Magnesium ammonium phosphate
origin; and postrenal obstruction may show normal- (struvite), uric acid, and cystine are the
and abnormal appearing urothelial cells possibly other primary calculi constituents.
associated with malignancy.  Calcium calculi are frequently associated
with metabolic calcium and phosphate
disorders and occasionally diet.
Renal Lithiasis
 Magnesium ammonium phosphate calculi
 Renal calculiIs (kidney stones) are frequently accompanied by urinary
infections involving urea-splitting bacteria.
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RENAL DISEASE
 The urine pH is often higher than 7.0.
 Uric acid calculi may be associated with
increased intake of foods with high purine
content and with uromodulin-associated
kidney disease.

REFFERENCE:
Urinalysis-and-Body-Fluids-Strasinger-
Susan-King-Di-Lorenzo-Marjorie-Schaub-
6th-Edition

GROUP 1
AMILASAN, IRISH
BUNAYOG,ZESA
DECUNA, ALEXANDRA
DUMAPADAT, JILLIAN JOY
OMAR, HANNAH
ORMIDO, KEITH
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URINE SCREENING FOR

METABOLIC DISORDERS

Overflow Versus Renal Disorders
The appearance of abnormal metabolic substances in
the urine can be caused by a variety of disorders that can
generally be grouped into two categories, termed the
overflow type and the renal type.
sample for specific substances associated with
particular IEMs.
Figure 8–1 shows the standard form collected for testing
using MS/MS. Methods for specific gene testing are also
rapidly being developed.

1. Overflow type disorders - Result from

disruption of a normal metabolic pathway that
causes increased plasma concentrations of the
nonmetabolized substances.
- These chemicals either override the
reabsorption ability of the renal tubules or are
not normally reabsorbed from the filtrate
because they are present in only minute
amounts.
2. Renal type disorder - Abnormal accumulations
of the renal type are caused by malfunctions in
the tubular reabsorption mechanism.

 The most frequently encountered abnormalities are AMINO ACID DISORDERS

associated with metabolic disturbances that produce
urinary overflow of substances involved in protein,
fat, and carbohydrate metabolism.
 Disruption of enzyme function can be caused by
- failure to inherit the gene to produce a
particular enzyme, referred to as an Inborn
Error of Metabolism (IEM),
- organ malfunction from disease or toxic
reactions.
 The most frequently encountered abnormal urinary
metabolites
NEWBORN SCREENING TEST
 Many of the urine tests traditionally were performed
primarily to detect and monitor newborns for IEMs.
 Levels of substances are elevated more rapidly in
blood than urine. Therefore, blood collected by infant
heel puncture is initially tested.
 Testing for many substances is now performed using
tandem mass spectrophotometry (MS/MS).
MS/MS is capable of screening the infant blood
The amino acid disorders with urinary screening tests
include phenylketonuria (PKU), tyrosyluria, alkaptonuria,
melanuria, maple syrup urine disease, organic
acidemias, indicanuria, cystinuria, and cystinosis.
Phenylalanine and tyrosine metabolic pathway including
the normal pathway (blue), enzymes (orange), and
disorders caused by failure to inherit particular enzymes
(green).
Phenylketonuria (PKU)
 The most well-known of the aminoacidurias, PKU
is estimated to occur in 1 of every 10,000 to 20,000
births and, if undetected, results in severe mental
retardation.
 It was first identified in Norway by Ivan Følling in
1934, when a mother with other mentally retarded
children reported a peculiar mousy odor to her
child’s urine.
 Analysis of the urine showed increased amounts of
the keto acids, including phenylpyruvate.
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URINE SCREENING FOR

METABOLIC DISORDERS

lowered from 4 mg/dL to 2 mg/dL, the presence of

PKU should be detected. Tests may need to be
repeated during an early visit to the pediatrician.
 More girls than boys escape detection of PKU during
early tests because of slower rises in blood
phenylalanine levels.
 Urine testing using ferric chloride may be used as
a follow up test to ensure proper dietary control in
previously diagnosed cases and as a means of
monitoring the dietary intake of pregnant women
known to lack phenylalanine hydroxylase.
 Urine tests for phenylpyruvic acid are based on the
ferric chloride reaction performed by tube test. The
addition of ferric chloride to urine containing
phenylpyruvic acid produces a permanent blue-
green color.

Ferric Chloride Tube Test

PROCEDURE
 This occurs when the normal conversion of 1. Place 1 mL of urine in a
phenylalanine to tyrosine is disrupted. tube.
 PKU is caused by failure to inherit the gene to 2. Slowly add five drops of
produce the enzyme phenylalanine hydroxylase. 10% ferric chloride.
The gene is inherited as an autosomal recessive 3. Observe color for a
trait with no noticeable characteristics or defects permanent blue-green
exhibited by heterozygous carriers. color.

 Dietary changes that eliminate phenyl alanine, a Tyrosyluria

major constituent of milk, from the infant’s diet can
 Tyrosinemia – accumulation of excess tyrosine
prevent excessive buildup of serum phenylalanine.
in the plasma
 The initial screening for PKU does not come under
 Tyrosyluria – urinary overflow of excess
the auspices of the urinalysis laboratory, because
tyrosine or its degradation products, p-
increased blood levels of phenylalanine must, of hydroxyphenylpyruvic acid and p-
course, occur before urinary excretion of
hydroxyphenyllactic acid
phenylpyruvic acid, which may take 2 to 6 weeks.
 result from either inherited or metabolic defects
 State laws require that blood be collected after 24
 Transitory tyrosinemia (inherited) – caused by
hours after birth and before the newborn leaves the
underdevelopment of the liver function required
hospital.
to produce the enzymes necessary to complete
 Studies have shown that in many cases
the tyrosine metabolism
phenylalanine can be detected as early as 4 hours
after birth and, if the cutoff level for normal results is
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URINE SCREENING FOR

METABOLIC DISORDERS

 Acquired severe liver disease - produces Melanuria

tyrosyluria resembling that of the transitory;
 Second metabolic pathway of tyrosine -
more serious
responsible for the production of melanin,
 Tyrosine and leucine crystals may be observed
thyroxine, epinephrine, protein and
in microscopic examination
tyrosine sulfate
Types of hereditary tyrosinemia
 Major concern of urinalysis laboratory is
Enzyme defect Effects
melanin
Type 1 Fumarylacetoacetat Generalized
 Albinism – deficient production of melanin
tyrosinemia ehydrolase (FAH) renal tubule
disorder and  Melanuria – increased urinary melanin which
progressive liver darkens urine; darkening appear after exposure
failure to air
Type 2 Tyrosine Corneal erosion  Indicates proliferation of melanocyte =
tyrosinemia aminotransferase and lesions on malignant melanoma
the palms,  5,6-dihydroxyindole – colorless precursor of
fingers, and on melanin; secreted by tumors
the sole of the (5,6-dihydroxyindole  melanogen 
feet melanin)
(crystallization of
 Melanin may react with sodium nitroferricyanide
tyrosine)
Type 3 p- Mental (acetone reagent strip), producing a red color.
tyrosinemia hydroxyphenylpyruvi retardation Alkaptonuria
c acid dioxygenase
 Described by Garrod in 1902
 Alkaptonuria – alkali lover; urine darkened after
becoming alkaline from standing at room
temperature
 Occurs from failure to inherit gene to produce
homogentisic acid oxidase
 Causes brown-stained or black-stained cloth
and reddish-stained disposable diapers
 In later life, brown pigment deposited in body
tissue (in the ear)
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URINE SCREENING FOR

METABOLIC DISORDERS

Routine screening test

 Ferric chloride test – transient deep blue is
produced
 Clinitest – yellow precipitate is produced
 Addition of alkali to freshly voided urine –
darkening of the color (ascorbic acid
interfere)
Paper and thin chromatography – for quantitating
homogentisic acid

 Two major groups of disorders are associated

with errors in the metabolism of the branched-
chain amino acids.

 Maple Syrup Urine Disease

 Organic Acidemias

 Maple Syrup Urine Disease

-is caused by an IEM (Inborn error of

metabolism).

- inherited as an autosomal recessive trait.

- Amino acids: leucine, isoleucine, and valine.

Branched-Chain Amino Acid - The metabolic pathway begins normally, with

 differ from other amino acids by having a methyl
group that branches from the main aliphatic
carbon chain.
the transamination of the three amino acids in the liver to
the keto acids (a-ketoisovaleric, a-ketoisocaproic,
and a -keto-b -methylvaleric).
-

 Organic Acidemias

- Generalized symptoms of the organic acidemias

include early severe illness, often with vomiting
accompanied by metabolic acidosis; hypoglycemia;
ketonuria; and increased serum ammonia.
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URINE SCREENING FOR
METABOLIC DISORDERS
- The three most frequently encountered disorders
are isovaleric, propionic, and
methylmalonicacidemia.
 Isovaleric acidemia
- is a rare disorder in which the body is unable
to properly break down a particular protein
building block (amino acid ).
- possess a characteristic odor of “sweaty
feet”.
 Propionic acidemia
- is an inherited disorder in which the body is
unable to process certain parts of proteins and
lipids (fats) properly.
- is the immediate precursor to methylmalonic
acid
 Methylmalonic acidemia
- is a disorder in which the body cannot break
down certain proteins and fats.
NOTE:
The presence of isovaleric, propionic, and
methylmalonic acidemias can be detected by
newborn screening programs using MS/MS.
Tryptophan Disorders
 The major concern of the urinalysis laboratory in
the metabolism of tryptophan is the increased
urinary excretion of the metabolites indican and
5-hydroxyindoleacetic acid (5-HIAA).
 Indicanuria
- the presence of an abnormally high
concentration of indican in the urine.
5-Hydroxyindoleacetic Acid
•A breakdown product of a hormone called serotonin.
•Serotonin is produced from tryptophan by the
argentaffin cells in the intestine and is carried through
the body primarily by the platelets.
•Argetaffin (enterochromaffin) cell tumors developed,
increased of 5-HIAA in urine from excess serotonin
produced.
•URINE TEST: Silver Nitroprusside Test
Adding nitrous acid and 1-nitroso-2-naphthol to urine
that contains 5-HIAA causes the urine to turn
purple to black, depending on the amount of 5-HIAA
present.
• NORMAL: 2-8 mg/day
• >25 mg/day (indication of argentaffin cell tumors)
PATIENT INSTRUCTIONS
• Patients must be given explicit dietary instructions
before collecting any sample to be tested for 5-HIAA,
because serotonin is a major constituent of foods such
as bananas, pineapples, and tomatoes.
• Medications, including phenothiazines and
acetanilids, also interfere with results.
• Patients should be directed to withhold medications
for 72 hours before specimen collection.
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URINE SCREENING FOR

METABOLIC DISORDERS

CYSTINE DISORDERS • Cystinosis can occur in three variations, ranging from

a severe fatal disorder developed in infancy to a benign
• Cystinuria - defect in the renal tubular transport of
form appearing in adulthood.
amino acids
• 2 categories:
• Cystinosis - IEM
(1) NEPROPATHIC
A noticeable odor of sulfur may be present in the urine
- Infantile
of people with cystine metabolism disorders.
- Late – on set
(2) NON – NEPHROPATHIC

CYSTINURIA
• Elevated amounts of the amino acid cystine in the • Cystinosis can occur in three variations, ranging from
urine. a severe fatal disorder developed in infancy to a benign
form appearing in adulthood.
• 2 modes of inheritance:
• Deposits: cornea, bone marrow, lymph nodes, organs
(1) Cystine, lysine, arginine, and ornithine are not
reabsorbed. • Renal tubules are affected by deposits causing
(2) Cystine, lysine are not reabsorbed. Fanconi syndrome.
• Approximately 65% of the people in whom all four • Renal transplants and the use of cystine-depleting
amino acids are affected can be expected to produce medications.
calculi early in life.
HOMOCYSTINURIA
• Cystine is much less soluble than the other three - Defects in the metabolism of amino acids
amino acid. methionine produce an increase in homocystine.
• Cystine is also the only amino acid found during the - Increased homocystine can result in failure to
analysis of calculi from these patients. thrive, cataracts, mental retardation,
thromboembolic problems, and death.
URINE SCREENING TEST: Cyanide- Nitroprusside
Test
- Reduction of cystine by sodium cyanide
followed by the addition of nitroprusside
produces a red-purple color in a specimen that
contains excess cystine.
- False positive results: ketonuria,
homocystinuria

CYSTINOSIS
• Genuine IEM
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URINE SCREENING FOR

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- Early detection of homocystinuria and a change

in diet that excludes foods high in methionine
can alleviate the metabolic problems. Therefore,
screening for homocystine is included in
newborn screening programs. Newborn
screening tests are performed using MS/MS
testing.
- Increased urinary homocystine gives a positive
result with the cyanide-nitroprusside test.
Therefore, an additional screening test for
homocystinuria must be performed by following
a positive cyanide-nitroprusside test result with
a silver-nitroprusside test, in which only
homocystine will react.
- The use of silver nitrate in place of sodium
cyanide reduces homocystine to its
nitroprusside-reactive form but does not reduce
cystine.
- A positive reaction in the silver-nitroprusside test
confirms the presence of homocystinuria.
- Fresh urine should be used when testing for
homocystine.
PORPHYRIN DISORDER
- Porphyrins are the intermediate compounds in
the production of heme.
- The basic pathway for heme synthesis shows - Disorders of porphyrin metabolism are
the three primary porphyrins (uroporphyrin, collectively termed porphyrias. They can be
coproporphyrin, and protoporphyrin) and the inherited or acquired from erythrocytic and
porphyrin precursors (α -aminolevulinic acid hepatic malfunctions or exposure to toxic
[ALA] and porphobilinogen). agents.
- The solubility of the porphyrin compounds varies - Common causes of acquired porphyrias include
with their structure. ALA, porphobilinogen, and lead poisoning, excessive alcohol intake, iron
uroporphyrin are the most soluble and readily deficiency, chronic liver disease, and renal
appear in the urine. disease.
- Coproporphyrin is less soluble but is found in the
urine, whereas protoporphyrin is not seen in the - Inherited porphyrias are much rarer than
urine. acquired porphyrias. They are caused by failure
- Fecal analysis has usually been performed to to inherit the gene that produces an enzyme
detect coproporphyrin and protoporphyrin. needed in the metabolic pathway.
- To avoid false-positive interference, bile is a - An indication of the possible presence of
more acceptable specimen. porphyrinuria is the observation of a red or port
- CDC recommends analysis of whole blood for wine color to the urine after exposure to air
the presence of free erythrocyte protoporphyrin - other inherited disorders, the presence of
(FEP) as a screening test for lead poisoning. congenital porphyria is sometimes suspected
from a red discoloration of an infant’s diapers.
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URINE SCREENING FOR

METABOLIC DISORDERS

- The two screening tests for porphyrinuria use COMMON PORPHYRIAS

the Ehrlich reaction and fluorescence under Porphyria Elevated Clinical Laboratory
ultraviolet light in the 550- to 600-nm range. Compoun Symptoms Testing
- Acetyl acetone must be added to the specimen d(s)
to convert the ALA to porphobilinogen prior to Acute ALA Neurologic Urine/Ehrli
performing the Ehrlich test. intermittent Porphobili /psychiatri ch reaction
- The Ehrlich reaction that is now included in the porphyria nogen c
Multistix urobilinogen pad was originally used for Porphyria Uroporphy Photosens Urine
all urobilinogen testing. cutanea rin itivity fluorescen
- Variations of the Ehrlich reaction include the tarda ce
Watson-Schwartz test for differentiation Congenital Uroporphy Photosens Urine or
between the presence of urobilinogen and erythropoieti rin itivity feces
porphobilinogen and the Hoesch test c porphyria Coproporp fluorescen
- Testing for the presence of porphobilinogen is hyrin ce
most useful when patients exhibit symptoms of Variegate Coproporp Photosens Bile or
an acute attack. This can be done with the porphyria hyrin itivity/ feces
Hoesch test. neurologic fluorescen
- A negative test result is obtained in the presence ce
of lead poisoning unless ALA is first converted Erythropoiet Protoporp Photosens Blood FEP
to porphobilinogen. ic hyrin itivity Bile or
- Fluorescent screening for the other porphyrins protoporphy feces
uses their extraction into a mixture of glacial ria fluorescen
acetic acid and ethyl acetate. ce
- Negative reactions have a faint blue Lead ALA Neurologic Acetoaceti
fluorescence. poisoning Protoporp c acid +
- Positive reactions fluoresce as violet, pink, or hyrin urine/
red, depending on the concentration of Ehrlich
porphyrins. reaction
- The fluorescence method does not distinguish Blood FEP
among uroporphyrin, coproporphyrin, and
protoporphyrin, but it rules out porphobilinogen
and ALA. Mucopolysaccharide Disorders
- Identifying specific porphyrins requires
additional techniques and the analysis of fecal • are a group of large compounds located
and erythrocyte samples. primarily in the connective tissue.
- Increased protoporphyrin is best measured in • They consist of a protein core with numerous
whole blood. polysaccharide branches.
• Inherited disorders in the metabolism of these
compounds prevent complete breakdown of the
polysaccharide portion of the compounds
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URINE SCREENING FOR

METABOLIC DISORDERS

PRODUCTS MOST FREQUENTLY FOUND IN  Hunter syndrome- the skeletal structure is

THE URINE abnormal and there is severe mental
retardation
• Dermatan sulfate
- is inherited as sex-linked
• Keratan sulfate, and recessive and is rarely seen in females
 Sanfilippo syndrome- the only abnormality is
• Heparan sulfate
mental retardation
 Identification of the specific enzyme deficiency  Note: Bone marrow transplants and gene
is necessary to establish a specific diagnosis replacement therapy are the most promising
treatments for these disorders
TYPES OF MUCOPOLYSACCHARIDOSES
 Hurler syndrome- the skeletal structure is THE MOST FREQUENTLY USED
abnormal and there is severe mental SCREENING TESTS
retardation  Acid-albumin- a thick, white turbidity forms
- mucopolysaccharides when these reagents are added to urine that
accumulate in the cornea of the eye contains mucopolysaccharide.
 cetyltrimethylammonium bromide (CTAB)
turbidity tests- a thick, white turbidity forms
when these reagents are added to urine that
contains mucopolysaccharide
 metachromatic staining spot tests
 Turbidity is usually graded on a scale of 0 to 4
after 30 minutes with acid-albumin and after 5
minutes with CTAB


Purine disorders Carbohydrate metabolism

Lesch-Nyhan
 disorder of purine metabolism
 sex-linked recessive
 results in massive excretion of urinary uric acid
crystals
 failure to inherit the gene to produce
hypoxanthine guanine
phosphoribosyltransferase
 causes severe motor defects, mental
retardation, a tendency toward self-destruction,
gout, and renal calculi
 orange sand in diapers – first sign
Melituria
 increased urinary sugar; usually inherited
 Pentosuria – one of Garrod's original six IEMs
 positive copper reduction test + negative
reagent strip glucose oxidase test = strongly
suggestive of a disorder of carbohydrate
metabolism
 Other causes of melituria include lactose,
fructose, and pentose
 Lactosuria may be seen during pregnancy and
lactation.
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URINE SCREENING FOR

METABOLIC DISORDERS

 Fructosuria is associated with parenteral

feeding and pentosuria with ingestion of large
amounts of fruit
 Additional tests including chromatography can
be used to identify other nonglucose reducing
substances.
Galactosuria
 inability to properly metabolize
galactose to glucose
 can lead to failure to thrive, combined
with liver disorders, cataracts, and
severe mental retardation (in infants)

 caused by a deficiency in any of

three enzymes, galactose-1-
phosphate uridyl transferase
(GALT), galactokinase, and UDP-
galactose-4-epimerase
 GALT deficiency that causes the
severe, possibly fatal symptoms
associated with galactosemia; the
enzyme is measured in the red blood
cells as part of the newborn heel
puncture protocol
 Galactose kinase deficiency -
cataracts
 UDP-galactose-4-epimerase
deficiency - asymptomatic or mild
symptoms
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CEREBROSPINAL FLUID
Cerebrospinal fluid (CSF)
• Major fluid of the body.
• Fx: provides a physiologic system to supply
nutrients to the nervous tissue, remove metabolic
wastes, produce a mechanical barrier to cushion
the brain and spinal cord against trauma
I. Formation and Physiology
• The brain and spinal cord are lined by the
meninges
• Meninges are composed of:
 The dura mater (Latin for “hard mother”) – outer
layer.
 The arachnoid (“spiderweb-like”) – inner
membrane
 Pia mater (Latin for “gentle mother”)
• CSF is produced in the choroid plexuses
• Adults - 20 mL of fluid produced per hour
• The fluid flows through the subarachnoid space
• Total Volume of Fluid: Adults – 90-150 mL;
Neonates – 10-60 mL
• Choroid plexuses - are capillary networks that
form the CSF from plasma by mechanisms of
selective filtration under hydrostatic pressure and
active transport secretion.
• The chemical composition of the CSF does not
resemble an ultrafiltrate of plasma.
• Capillary walls throughout the body are lined with
endothelial cells.
• BLOOD BRAIN BARRIER
II. Specimen Collection and Handling
• CSF - collected by lumbar puncture between the
third, fourth, or fifth lumbar vertebra.
• This procedure requires certain precautions such
as: measurement of intracranial pressure and
careful technique to prevent infection or neural
tissue damage.
• Specimens are collected in three sterile tubes.
• The tubes are labeled 1, 2, and 3 in the order in
which they are withdrawn.
• Tube 1 - for chemical and serologic tests.
• Tube 2 – for microbiology laboratory.
• Tube 3 - for the cell count.
• 4th tube may be drawn for the microbiology
laboratory to provide better exclusion of skin
contamination or for additional serologic tests.
• Supernatant fluid - may also be used for additional
chemical or serologic tests.
• Excess fluid should not be discarded and
should be frozen until there is no further use
for it .
• Laboratory personnel should handle CSF
specimens carefully.
• Ideally, tests are performed on a STAT basis.
• If STAT is not possible, specimens are maintained
in the following manner: Hematology tubes are
refrigerated; Microbiology tubes remain at room
temperature; Chemistry and serology tubes are
frozen.
• CSF appearance includes crystal clear, cloudy or
turbid, milky, xanthochromic, and
hemolyzed/bloody.
• Cloudy, turbid, or milky specimen - protein or
lipid concentration, indicative of infection, with the
cloudiness being caused by the presence of
WBCs.
• All specimens should be treated with extreme
care.
• Fluid for centrifugation must be in capped tubes
• Xanthochromia
 is a term used to describe CSF supernatant that
is pink, orange, or yellow.
 due to the presence of RBC degradation products.
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CEREBROSPINAL FLUID
 Other causes of xanthochromia include elevated IV. Traumatic Collection (Tap)
serum bilirubin, presence of the pigment carotene,
markedly increased protein concentrations, and
melanoma pigment. o Grossly bloody CSF can be an indication of
intracranial hemorrhage, but it may also be due to the
 Xanthochromia that is caused by bilirubin due to puncture of a blood vessel during the spinal tap procedure.
immature liver function - commonly seen in
infants, particularly premature infants. o Three visual examinations of the collected
specimens can usually determine whether the blood is the
result of hemorrhage or a traumatic tap.
III. Appearance

1. Uneven Blood Distribution

- Blood from a cerebral hemorrhage will be evenly
distributed throughout the three CSF specimen tubes,
whereas a traumatic tap will leave the heaviest
concentration of blood in tube 1, and gradually diminishing
amounts in Tubes 2 and 3.
- Performing red blood cell (RBC) counts on all
three tubes to measure decreasing or constant blood is not
always reliable.

2. Clot Formation
- Bloody CSF caused by intracranial hemorrhage
does not contain enough fibrinogen to clot. Diseases in
which damage to the blood–brain barrier allows increased
filtration of protein and coagulation factors also cause clot
formation but do not usually produce a bloody fluid.
- These conditions include meningitis, Froin
syndrome, and blocked CSF circulation through the
subarachnoid space.

3. Xanthochromic Supernatant
- RBCs must usually remain in the CSF for
approximately 2 hours before noticeable hemolysis begins;
therefore, a xanthochromic supernatant would be the
result of blood that has been present longer than that
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CEREBROSPINAL FLUID
introduced by the traumatic tap. Additional testing for
differentiation includes microscopic examination and the
D-dimer test.
V. Cell Count
o The RBC count can be calculated by performing a
total cell count and a WBC count and subtracting the WBC
count from the total count, if necessary.
o Any cell count should be performed immediately,
because WBCs (particularly granulocytes) and RBCs
begin to lyse within 1 hour, and 40% of the leukocytes
disintegrate after 2 hours.4 Specimens that cannot be
analyzed immediately should be refrigerated.
VI. Differential Count on a CSF specimen
 The differential count should be performed on a
stained smear and not from the cells in the
counting chamber.
 To Ensure that the maximum number of cells is
available for examination, the specimen should be
concentrated before Preparing the smear.
 Methods available for specimen concentration
include Sedimentation, filtration, centrifugation,
and cytocentrifugation.
 Sedimentation and filtration are not routinely used
in the Clinical laboratory
Cytocentrifugation
 Adding albumin increases the cell yield and
decreases the Cellular distortion frequently seen
on cytocentrifuged specimens
CSF Cellular Constituents
 The cells found in normal CSF are primarily
lymphocytes and monocytes.
 Lymphocytes and monocytes Ratio in adults
(70:30)
 A high CSF WBC count of which the majority of
the cells are neutrophils is considered indicative of
bacterial meningitis.
 Moderately elevated CSF WBC count with a high
percentage of lymphocytes And monocytes
suggests meningitis of viral, tubercular, fungal, Or
parasitic origin.
Neutrophils
 Neutrophils associated with bacterial meningitis
may contain phagocytized bacteria.
 Neutrophils with pyknotic nuclei indicate
degenerating cells. They may resemble nucleated
red blood cells (NRBCs) but usually have multiple
nuclei.
 NRBCs Are seen as a result of bone marrow
contamination during the Spinal tap.
Lymphocytes and Monocytes
 Reactive lymphocytes containing increased dark
blue cytoplasm and clumped Chromatin are
frequently present during viral infections in
conjunction with normal cells.
 A moderately elevated WBC count (less than 50
WBCs/µ L) with increased normal and reactive
lymphocytes And plasma cells may indicate
multiple sclerosis or other degenerative neurologic
disorders
Eosinophils
 Increased eosinophils are seen in the CSF in
association with parasitic infections, fungal
infections (primarily Coccidioides immitis),
Macrophages
 Macrophages appear Within 2 to 4 hours after
RBCs enter the CSF
 The finding of increased macrophages indicates a
previous Hemorrhage.
 Further degradation of the phagocytized RBCs
results in the appearance of dark blue or black
iron-Containing hemosiderin granules.
 Yellow hematoidin crystals represent further
degeneration.
Nonpathologically Significant Cells
 Most frequently seen After diagnostic procedures
such as pneumoencephalography
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CEREBROSPINAL FLUID

The cells often appear in clusters and can be
distinguished from malignant cells by their uniform
appearance.
 Choroidal cells are from the epithelial lining of the
Choroid plexus. They are seen singularly and in A. CSF PROTEIN
clumps. Nucleoli are usually absent and nuclei
have a uniform appearance. i.
 Ependymal cells are from the lining of the ii.
ventricles and Neural canal. They have less iii.
defined cell membranes and are frequently seen
in clusters. Nucleoli are often present. iv.
 Spindle-shaped cells represent lining cells from
the arachnoid. They are usually seen in clusters v.
and may be seen with Systemic malignancies.
Malignant Cells of Hematologic Origin

Lymphoblasts, myeloblasts, and monoblasts in Important Key Points:
the CSF are frequently seen as a serious
complication of acute leukemias. Nucleoli are
often more prominent than in Blood smears.
 Lymphoma cells are also seen in the CSF and
indicate dissemination from the lymphoid tissue.
They resemble large and Small lymphocytes and
usually appear in clusters of large, Small, or mixed
cells based on the classification of the lymPhoma.
Nuclei may appear cleaved, and prominent
nucleoli are present.
Malignant Cells of Nonhematologic Origin Clinical Significance of Elevated Protein Values
 Metastatic carcinoma cells of nonhematologic i.
origin are priMarily from lung, breast, renal, and ii.
gastrointestinal malignanCies.
 Cells from primary CNS tumors include
astrocytomas, Retinoblastomas, and
medulloblastomas (Fig. 9–35). They Usually
appear in clusters and must be distinguished from
normal clusters of ependymal, choroid plexus,
lymphoma, and Leukemia cells.
iii.
VII. Chemistry Tests
i. CSF is formed by filtration of plasma.
ii. CSF and Plasma reference values are different.
 CSF Protein
 CSF Glucose
 CSF Lactate
 CSF Glutamine
Most frequently performed.
Normal CSF = Very small amount of Protein
Total CSF protein = 15 to 45 mg/dL (Higher in
infants & over age 40)
CSF contains protein fractions similar to those
found in serum (Varied in ratio).
Albumin makes up most of CSF protein;
prealbumin is the second most prevalent fraction
in CSF.
 Alpha Globulins - haptoglobin and ceruloplasmin
 Major Beta Globulin – transferrin
 “tau” - separate carbohydrate-deficient transferrin
fraction (in CSF, not serum)
 CSF Gamma Globulin - immunoglobulin G (IgG)
with immunoglobulin A (IgA)
 NOT FOUND IN NORMAL CSF - Immunoglobulin
M (IgM), fibrinogen, and beta lipoprotein.
Most frequently seen in pathologic conditions.
Causes of Elevated CSF Protein values:
a) Damage to the blood–brain barrier
(Meningitis & hemorrhage)
b) Immunoglobulin production within the
CNS
c) Decreased normal protein clearance from
the fluid
d) Neural tissue degeneration
Abnormally low values are present when fluid is
leaking from the CNS.
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CEREBROSPINAL FLUID
Methodology
i. The two most routinely used techniques for
measuring total CSF protein use the principles of
turbidity production or dye-binding ability.
ii. Turbidity method has been adapted to automated
instrumentation in the form of nephelometry.
ELECTROPHORESIS
TECHNIQUES
PROTEIN FRACTIONS
i. Purpose: For diagnosis of neurologic
ii.
disorders associated with abnormal CSF
protein.
ii. Protein that appears in the CSF as a result of
iii.
damage to the integrity of the blood–brain
barrier contains fractions proportional to those
in plasma, with albumin present in the highest
iv.
concentration.
iii. For igG Determination, comparisons between
serum and CSF levels of albumin and IgG
must be made.
iv. CSF/serum albumin index
a) To evaluate the integrity of the
blood–brain barrier
b) Calculated after determining the
concentration of CSF albumin in
milligrams per deciliter and the
serum concentration in grams per
deciliter.
v. Less than 9 IV = intact blood–brain barrier.
vi. The index increases relative to the amount of
damage to the barrier.
vii. CSF IgG index
a) To measure IgG synthesis within the
CNS.
b) A comparison of the CSF/serum
albumin index with the CSF/serum
IgG index, compensates for any IgG
entering the CSF via the blood–brain
barrier.
viii. Normal IgG index values vary slightly among
laboratories.
ix. Values greater than 0.70 indicate IgG
production within the CNS.
x. For measuring CSF albumin and globulin:
automated.
AND IMMUNOPHORETIC
i. Purpose: To detect oligoclonal bands, which
represent inflammation within the CNS.
Oligoclonal banding remains positive during
remission of multiple sclerosis, but disappears in
other disorders.
Location of bands: gamma region of the protein
electrophoresis (indicating immunoglobulin
production).
Such disorders (leukemia, lymphoma) and viral
infections may produce serum banding, which can
appear in the CSF as a result of blood–brain
barrier leakage or traumatic introduction of blood
into the CSF specimen.
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CEREBROSPINAL FLUID
v. Serum and CSF with HIV infection: Banding with
both systemic and neurologic involvement.
vi. Diagnosis of multiple sclerosis:
a. Presence of two or more oligoclonal
bands in the CSF that are not present in
the serum.
b. Accompanied by an increased IgG index.
vii. Other neurologic disorders (encephalitis,
neurosyphilis, Guillain-Barré syndrome) and
neoplastic disorders produce oligoclonal banding
that may not be present in the serum.
viii. CSF immunofixation electrophoresis (IFE) and
isoelectric focusing (IEF) followed by silver
staining:
a) Better resolution
b) Methods of choice to confirm whether a
fluid is actually CSF.
ix. Specimen concentration: NOT REQUIRED.
x. CSF can be identified based on the appearance
(Extra isoform “tau” only in CSF).
MYELIN BASIC PROTEIN (MBP)
i. Indicates recent destruction of the myelin sheath
that protects the axons of the neurons
(demyelination).
ii. MBP determination:
a) The course of multiple sclerosis can be
monitored.
b) Provide a valuable measure of the
effectiveness of current and future
C. CSF LACTATE
treatments.
c) Immunoassay techniques are used for
measurement.
B. CSF GLUCOSE
i. Reference value: approximately 60% to 70% that
of the plasma glucose. So if:
Plasma glucose is approximately 100
mg/dL then, CSF glucose is
approximately 65 mg/dL.
ii. Blood Glucose Test:
a) For accurate evaluation of CSF glucose.
b) Blood glucose should be drawn about 2
hours before the spinal tap to allow time
for equilibration between the blood and
fluid.
iii. CSF glucose is analyzed using the same
procedures employed for blood glucose.
iv. Specimen Consideration: IMMEDIATE processing
of test (glycolysis occurs rapidly in CSF).
v. Diagnostic Significance: to find values that are
decreased relative to plasma values.
a) Elevated CSF glucose values are always
a result of plasma elevations.
b) Low CSF glucose values can determine
the causative agents in meningitis.
vi. Decreased CSF values:
a) Primarily caused by alterations in the
mechanisms of glucose transport across
the blood–brain barrier and by increased
use of glucose by the brain cells.
b) The common tendency to associate
decreased glucose totally with its use by
microorganisms and leukocytes cannot
account for the variations in glucose
concentrations seen in different types of
meningitis and the decreased levels seen
in other disorders producing damage to
the CNS.
i. CSF Lactate levels:
a) Aid in diagnosis and management of
meningitis cases.
b) Greater than 25 mg/dL seen in bacterial,
tubercular and fungal meningitis.
 Greater than 35 mg/dL frequently
seen in bacterial meningitis.
 Lower than 25 mg/dL seen in viral
meningitis.
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CEREBROSPINAL FLUID
c) Provide more reliable information when
the initial diagnosis is difficult.
d) Sensitive method for evaluating the
effectiveness of antibiotic therapy.
e) Used to monitor severe head injuries.
ii. Tissue destruction within the CNS owing to
oxygen deprivation (hypoxia) increases CSF lactic
acid levels.
D. CSF GLUTAMINE
i. Produced from ammonia and α -ketoglutarate by
the brain cells.
ii. Normal concentration of CSF glutamine: 8-18
mg/dL.
Methods
iii. Elevated levels are associated with liver disorders.
iv. CSF Glutamine Determination: Provides an
indirect test for the presence of excess
v. ammonia in the CSF.
vi. Methods are based on the measurement of
ammonia liberated from the glutamine (CSF
Glutamine Levels).
Gram Stain
- Preferred than the direct
measurement of CSF ammonia.
- Correlates with clinical symptoms
much better than does the blood
ammonia.
vii. CSF glutamine test is frequently requested for
patients with coma of unknown origin.
VIII. Microbiology Tests
 Role of the microbiology laboratory in CSF is to
identify the causative agent in meningitis
 The microorganism must be recovered by growing
it on the appropriate culture medium for a positive
identification.
 Within 24 hours is positive in cases of bacterial
meningitis and up to 6 weeks for tubercular
meningitis.
 CSF culture is a confirmatory rather than a
diagnostic procedure.
 Gram stain
 Acid-fast stain
 India ink
 Latex agglutination tests.
 Routinely performed on CSF from all suspected
cases of meningitis and its value lies in detecting
bacterial and fungal organisms.
 All smears and cultures should be performed on
concentrated specimens
 Should be centrifuged at 1500 g for 15 minutes,
and slides and cultures should be prepared from
the sediment. Cytocentrifuge provides a highly
concentrated specimen.
 CSF Gram stain is one of the most difficult slides
to interpret because the number of organisms
present is usually small → can easily be
overlooked → resulting in a false negative report.
 False-positive reports = precipitated stain or
debris is mistaken for microorganisms

Frequently Encountered Organisms:

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CEREBROSPINAL FLUID
 Streptococcus pneumoniae (gram-positive cocci)
 Haemophilus influenzae (pleomorphic gram-
negative rods)
 Escherichia coli (gram-negative rods
 Neisseria meningitidis (gram-negative cocci)
Newborns:
 Gram-positive cocci Streptococcus agalactiae
 Gram-positive rods Listeria monocytogenes
Acid-fast or fluorescent antibody stains
 Not routinely performed on specimens unless
tubercular meningitis is suspected. A positive
report to culture mycobacteria from this smear is
extremely valuable.
 Possible cases of fungal meningitis are Gram
stained and India ink preparation performed to
detect the presence of thick encapsulated
Cryptococcus neoformans.
 Cryptococcal meningitis is now commonly
encountered in the clinical laboratory and
Cryptococcus is seen more by gram stain.
 C. Neoformans- a Latex agglutination tests to
detect the presence of antigen in serum and CSF
provide a more sensitive method than the India ink
preparation.
 Rheumatoid factor - most common cause of
false- positive reactions
 Pretreatment techniques- incubation of
dithiothreitol or pronase and boiling with
ethylenediaminetetra-acetic acid.
 Immunoassay enzyme technique- produce
fewer false-positive results.
 Lateral flow assay (LAF)- a rapid method for
detecting C. neoformans.
 ELISA- a rapid means for detecting identifying
microorganisms in CSF.
 LAF Procedure= reagent strip coated with
monoclonal antibodies that react with the
cryptococcal polysaccharide capsule.
Detected by Test kits:
 Streptococcus group B
 influenzae Type B
 S. pneumoniae
 meningitidis A, B, C, Y, W135
 Mycobacterium tuberculosis
 Coccidioides immitis
 E. coli K1 antigens.
Bacterial Antigen test (BAT)
 Not appear to be as sensitive to N. meningitidis
 Used in combination with results from the
hematology and clinical chemistry laboratories for
diagnosing meningitis
Naegleria fowleri
 Opportunistic parasite found in ponds, small lakes,
and in chlorinated swimming pools.
 Enters the nasal passages and migrates along the
olfactory nerves to invade the brain.
 Motile trophozoites: microscopically by examining
a wet preparation of CSF.
 Nonmotile trophozoites: seen on cytocentrifuged
stained smears by ↑WBCs and no bacteria.
IX. Serologic Testing
 Performed to detect the presence of neurosyphilis
 Use of penicillin in the early stages of syphilis has
reduced the number of neurosyphilis cases.
 Number of requests for syphilis is low
 Detecting the antibodies in syphilis remains a
necessary diagnostic procedure.
Venereal Disease Research Laboratories (VDRL)
 Used diagnose neurosyphilis (CDC)
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CEREBROSPINAL FLUID
 Not as sensitive as the fluorescent treponemal
antibody-absorption (FTA-ABS) test for syphilis

Rapid Plasma Regain (RPR) test

 Not recommended.
 It is less sensitive than the VDRL.

FTA-ABS
 Care must be taken to prevent contamination with
blood.
 Remains positive in the serum of treated cases of
syphilis.

Reference:
Strasinger, S.K, Di Lorenzo, M.S (2014). Urinalysis and
Body Fluids (6th ed.) Philadelphia: F.A Daviss
Company

Group 3 Members
BMLS 3A
Nicomedez, Nashren A.
Hormillada, Hanyan T.
Gabao, Monica L.
Valdez, Hilborj Reigm Gullon V.
Tuban, Rose Anne
Pongase, Deive A.
Labarda, Dwight E.
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SEMEN
PHYSIOLOGY
-Semen is composed of four fractions that are contributed
by the testes, epididymis, seminal vesicles, prostate
gland, and bulbourethral gland
 Each fraction differs in its composition, and the
mixing of all four fractions during ejaculation is
essential for the production of a normal semen
specimen
SEMEN COMPOSITION
Spermatozoa 5%
Seminal Fluid 60% - 70%
Prostate Fluid 20%- 30 %
Bulbourethral glans 5%
-Testes are paired glands in the scrotum that contain the
seminiferous tubules for the secretion of sperm.
-The external location of the scrotum contributes to a lower
scrotum temperature that is optimal for sperm
development.
-Germ cells for the production of spermatozoa are located
in the epithelial cells of the seminiferous tubules.
 Specialized Sertoli cells provide support and
nutrients for the germ cells as they undergo
mitosis and meiosis (spermatogenesis).
 When spermatogenesis is complete, the immature
sperm (nonmotile) enter the epididymis.
-In the epididymis, the sperm mature and develop flagella.
The entire process takes approximately 90 days. The
sperm remain stored in the epididymis until ejaculation, at
which time they are propelled through the ductus deferens
(vas deferens) to the ejaculatory ducts.
-The ejaculatory ducts receive both the sperm from the
ductus deferens and fluid from the seminal vesicles.
-The seminal vesicles produce most of the fluid present
in semen (60% to 70%), and this fluid is the transport
medium for the sperm.
-The fluid contains a high concentration of
 fructose and
 flavin
-Spermatozoa metabolize the fructose for the energy
needed for the flagella to propel them through the female
reproductive tract.
-In the absence of fructose, sperm do not display motility
in the semen analysis.
 Flavin is responsible for the gray appearance of
semen. Various proteins secreted by the seminal
vesicles are involved in the coagulation of the
ejaculate.
-The muscular prostate gland, located just below the
bladder, surrounds the upper urethra and aids in propelling
the sperm through the urethra by contractions during
ejaculation.
-Approximately 20% to 30% of the semen volume is acidic
fluid produced by the prostate gland.
 The milky acidic fluid contains high concentrations
of acid phosphatase, citric acid, zinc, and
proteolytic enzymes responsible for both the
coagulation and liquefaction of the semen
following ejaculation.
-The bulbourethral glands, located below the prostate,
contribute about 5% of the fluid volume in the form of :
 thick, alkaline mucus that helps to neutralize
acidity from the prostate secretions and the
vagina.
-It is important for semen to be alkaline to neutralize the
vaginal acidity present as a result of normal bacterial
vaginal flora. Without this neutralization, sperm motility
would be diminished.
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SEMEN
SPECIMEN COLLECTION
-most of the sperm are contained in the first portion of the
ejaculate, making complete collection essential for
accurate testing of both fertility and post vasectomy
specimens.
 first portion of the ejaculate is missing, the sperm
count will be decreased, the pH falsely
increased, and the specimen will not liquefy.
 last portion of ejaculate is missing, the semen
volume is decreased, the sperm count is falsely
increased, the pH is falsely decreased, and the
specimen will not clot.
-Specimens are collected following a period of sexual
abstinence of at least 2 days to not more than 7 days.
Specimens collected following prolonged abstinence tend
to have higher volumes and decreased motility. When
performing fertility testing, the World Health Organization
(WHO) recommends that
 two or three samples be collected not less than 7
days or more than 3 weeks apart, with two
abnormal samples considered significant.
-laboratory should provide the patient with warm sterile
glass or plastic containers.
-specimen is collected in a room provided by the
laboratory. However, if this is not appropriate, the
specimen should be kept at room temperature and
delivered to the laboratory within 1 hour of collection.
-Laboratory personnel must record the
 patient’s name and birth date, the period of sexual
abstinence, the completeness of the sample,
difficulties with collection, and the times of
specimen collection and specimen receipt.
-Specimens awaiting analysis should be kept at 37°C.
Specimens should be collected by masturbation. If this is
not possible, only non- lubricant containing rubber or
polyurethane condoms should be used. Ordinary
condoms are not acceptable because they contain
spermicides.
SPECIMEN HANDLING
-All semen specimens are potential reservoirs for HIV and
hepatitis viruses, and standard precautions must be
observed at all times during analysis.
 Specimens are discarded as biohazardous
waste. Sterile materials and techniques must be
used when semen culture is to be performed or
when the specimen is to be processed for
bioassay, intrauterine insemination (IUI), or in vitro
fertilization (IVF).
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.
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SEMEN

REFERENCE VALUES FOR SEMEN ANALYSIS LIQUEFACTION

VOLUME 2- 5 mL
VISCOSITY Pours in droplets -A fresh semen specimen is clotted and should liquefy
pH 7.2- 8.0 within 30 to 60 minutes after collection. Therefore,
SPERM >20 million/mL recording the time of collection is essential for evaluating
CONCENTRATION semen liquefaction.
SPERM COUNT >40 million/ejaculate  Failure of liquefaction to occur within 60 minutes
MOTILITY >50% within 1h may be caused by a deficiency in prostatic
QUALITY >2.0 or a, b, c (sperm grading) enzymes and should be reported.
MORPHOLOGY >14% normal forms ( strict  Analysis of the specimen cannot begin until
criteria) liquefaction has occurred.
>30% normal forms (routing -If after 2 hours the specimen has not liquified, an equal
criteria) volume of physiologic Dulbecco’s phosphate-buffered
ROUND CELLS <1.0 million/ mL saline or proteolytic enzymes such as alpha- chymotrypsin
or bromelain may be added to induce liquefaction and
APPERANCE allow the rest of the analysis to be performed.
 These treatments may affect
-Normal semen has a gray-white color, appears o biochemical tests
translucent, and has a characteristic musty odor. o sperm motility
o and sperm morphology, so their use must
 When the sperm concentration is very low, the be documented.
specimen may appear almost clear. -The dilution of semen with bromelain must be accounted
 Increased white turbidity indicates the presence of for when calculating sperm concentration.
white blood cells (WBCs) and infection within the  Jelly-like granules (gelatinous bodies) may be
reproductive tract. present in liquefied semen specimens and have
no clinical significance.
MICROSCOPIC EXAMINATION  Mucus strands, if present, may interfere with
semen analysis.
 WBCs must be differentiated from immature sperm REMEMBER: Gently mixing the sample container during
(spermatids). The leukocyte esterase reagent strip test liquefaction can help produce a homogeneous sample.
may be useful to screen for the presence of WBCs.
 Varying amounts of red coloration are associated with
the presence of red blood cells (RBCs) and are
abnormal.
 Yellow coloration may be caused by urine
contamination, specimen collection following
prolonged abstinence, and medications. Urine is
toxic to sperm, thereby affecting evaluation of motility.
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SEMEN
PROCEDURE
Semen Dilution With Physiologic Saline
Prepare an equal volume of diluent and semen (1 part
diluent and 1 part semen) using Dulbecco’s phosphate-
buffered saline. Repeated pipetting of the prepared
dilution will induce liquefaction.
Preparation of Dulbecco’s Phosphate-Buffered
Saline
1. Using a 1-L volumetric flask, add the following:
a. 750 ml of purified water
b. 0.20 g of potassium chloride (KCL)
c. 0.20gofpotassiumdihydrogenphosphate(KH2PO4)
d. 0.10 g of magnesium chloride hexahydrate
(MgCl2.6H2O)
e. 8.0 g of sodium chloride (NaCl)
f. 2.16 g of disodium hydrogen phosphate heptahydrate
(Na2HPO4.7H2O)
g. 1.00 g of D-glucose
2. In a 10-mL volumetric flask, dissolve 0.132 g of
calcium chloride dehydrate (CaCl2.2H2O) in 10 mL of
purified water.
3. To prevent precipitation, add calcium chloride
dehydrated solution to the 1-L flask slowly, stirring
continuously.
4. Adjust the pH to 7.4 with 1 mol/L sodium hydroxide
(NaOH).
5. Make up to 1000 mL with purified water.
PROCEDURE
Digestion With Bromelain
1.Prepare 10 IU/mL bromelain in Dulbecco’s phosphate-
buffered saline.
a. Into a 100-mL volumetric flask, add 1000 IU of
bromelain.
b. Add 60 mL Dulbecco’s phosphate-buffered saline.
c. Mix to dissolve. It takes about 15 to 20 minutes. Bring
volume to calibration mark using buffered saline.
2. Dilute one part semen 1:2 with the 10 IU/mL
bromelain (1 part semen + 1 part bromelain solution).
3. Stir with a pipette tip.
4. Incubate at 37°C for 10 minutes.
5. Mix the sample well before analysis.
VOLUME
-Normal semen volume ranges between 2 and 5 mL.
 It can be measured by pouring the specimen into
a clean graduated cylinder calibrated in 0.1-mL
increments.
o Increased volume may be seen following periods
of extended abstinence.
o Decreased volume is more frequently associated
with infertility and may indicate improper
functioning of one of the semen producing organs,
primarily the seminal vesicles. Incomplete
specimen collection must also be considered.
VISCOSITY
-Specimen viscosity refers to the consistency of the fluid
and may be related to specimen liquefaction.
 Incompletely liquefied specimens are clumped
and highly viscous.
-The normal semen specimen should be easily drawn into
a pipette and form small discrete droplets that do not
appear clumped or stringy when falling by gravity from the
pipette.
 Droplets that form threads longer than 2 cm are
considered highly viscous and are recorded as
abnormal.
o Ratings of 0 (watery) to 4 (gel-like) can be
assigned to the viscosity report.
-Viscosity can also be reported as low, normal, or high.
 Increased viscosity and incomplete liquefaction
impede testing for sperm motility, sperm
concentration, anti-sperm antibody detection, and
measurement of biochemical markers
pH
-The pH of semen indicates the balance between the pH
values from the acidic prostatic secretion and the alkaline
seminal vesicles secretion.
 The pH should be measured within 1 hour of
ejaculation due to the loss of CO2 that occurs.
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SEMEN
 The normal pH of semen is alkaline with a range
of 7.2 to 8.0 .
o Increased pH indicates infection within the
reproductive tract
o A decreased pH may be associated with
increased prostatic fluid, ejaculatory duct
obstruction, or poorly developed seminal
vesicles.
-Semen for pH testing can be applied to the pH pad of a
urinalysis reagent strip and the color compared with the
manufacturer’s chart. Dedicated pH testing paper also can
be used.
SPERM CONCENTRATION AND SPERM COUNT
-Various factors can affect sperm concentration, such as
the days of sexual abstinence before the collection,
infection, or stress. Therefore, more than one semen
specimen should be evaluated for infertility studies.
-Reference values for sperm concentration are commonly
listed as
 greater than 20 to 250 million sperm per milliliter.
 concentrations between 10 and 20 million per
milliliter are considered border line.
-The total sperm count for the ejaculate can be calculated
by multiplying the sperm concentration by the specimen
volume.
 Total sperm counts greater than 40 million per
ejaculate are considered normal (20 million per
milliliter 2 mL).
MOSTCOMMONLY USE DILUTION
-is 1:20 prepared using a mechanical (positive-
displacement) pipette.
-Dilution of the semen is essential because it immobilizes
the sperm before counting.
 The traditional diluting fluid contains sodium
bicarbonate and formalin, which immobilize and
preserve the cells however, good results can also
be achieved using saline and distilled water.
NEUBAUER HEMOCYTOMER
-sperm are usually counted in the four corner and center
squares of the large center square, similar to a manual
RBC count.
-Both sides of the hemocytometer are loaded and allowed
to settle for 3 to 5 minutes then they are counted, and the
counts should agree within 10%. An average of the two
counts is used in the calculation.
 If the counts do not agree, both the dilution and
the counts are repeated.
 Counts are performed using either phase or
bright-field microscopy. The addition of stain,
such as crystal violet, to the diluting fluid aids in
visualization when using bright-field microscopy.

-Sperm concentration is usually performed using the

Neubauer counting chamber.
 The sperm are counted in the same manner as
cells in the cerebrospinal fluid cell count, that is,
by diluting the specimen and counting the cells in
the Neubauer chamber. The amount of the
dilution and the number of squares counted vary
among laboratories.
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SEMEN
-Fully developed sperm should be counted
 Immature sperm and WBCs, often referred to as
“round” cells, must not be included. However,
their presence can be significant, and they may
need to be identified and counted separately.
 Stain included in the diluting fluid aids in
differentiating between immature sperm cells
(spermatids) and leukocytes, and they can be
counted in the same manner as mature sperm.
-A count greater than 1 million leukocytes per milliliter is
associated with
 inflammation or infection of the reproductive
organs that can lead to infertility.
-The presence of more than 1 million spermatids per
milliliter indicates disruption of spermatogenesis.
 This may be caused by viral infections, exposure
to toxic chemicals, and genetic disorders.
CALCULATING SPERM CONCENTRATION AND
SPERM COUNT
Calculation of sperm concentration depends on the dilution
used and the size and number of squares counted.
-When using the 1:20 dilution and counting the five
squares (RBCs) in the large center square as described
previously, the number of sperm can be multiplied by
1,000,000 (add 6 zeros) to equal the sperm concentration
per milliliter.
 unlike blood cell counts, the sperm concentration
is reported in millions per milliliter rather than
microliters.
-Sperm concentration can also be calculated using the
basic formula for cell counts.
 Because this formula provides the number of cells
per microliter, the figure must then be multiplied by
1000 to calculate the number of sperm per
milliliter.
 The total sperm count is calculated by multiplying
the number of sperm per milliliter by the specimen
volume.
SPERM MOTILITY
-Sperm motility should be assessed using a well-mixed,
liquefied semen specimen within 1 hour of specimen
collection.
-To provide continuity in reporting, laboratories should
place a consistent amount of semen on a slide under the
same size cover slip, such as 10 μL under a 22 22 mm
cover slip using a calibrated positive-displacement pipette,
and allow it to settle for 1 minute.
 This procedure should be done in duplicate for
accuracy.
-The percentage of sperm showing actual forward
movement can then be estimated after evaluating
approximately 20 high-power fields.
 An alternate procedure is to examine 200 sperm
per slide and count the percentages of the
different motile categories using a manual cell
counter.
-Motility is evaluated by both speed and direction.
Grading can be done using
o scale of 0 to 4, with 4 indicating rapid, straight-line
movement
o 0 indicating no movement.
o A minimum motility of 50% with a rating of 2.0
after 1 hour is considered normal.
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SEMEN
-The WHO uses a rating scale of a, b, c, d. Interpretation Sperm concentration and morphology are also
states that within 1 hour, 50% or more of the sperm should included in the analysis.
be motile in categories a, b, and c, or 25% or more should -Currently, CASA instrumentation is found primarily in
show progressive motility (a and b). laboratories that specialize in andrology and perform a
high volume of semen analysis.
SPERM MOTILITY GRADING
GRADE WHO CRITERIA SPERM MOTILITY SPERM MORPHOLOGY
ACTION
4.0 a Rapid, straight line -Sperm morphology is evaluated with respect to the
motility structure of the head, neckpiece, midpiece, and tail.
3.0 b Slower speed, some Abnormalities in head morphology are associated with
lateral movement poor ovum penetration, whereas neckpiece, midpiece, and
2.0 b Slow forward tail abnormalities affect motility.
progression,
noticeable lateral  The normal sperm has
movement an oval-shaped head
1.0 C No forward approximately 5 μm long and
progression 3 μm wide and a long,
0 d No movement flagellar tail approximately 45
μm long.
-The WHO Laboratory Manual for the Examination and
Processing of Human Semen (2010) currently
recommends a simpler system for grading motility that
does not include speed because of the difficulty in
standardized reporting. -Critical to ovum penetration is the enzyme containing
MOTILITY IS GRADED acrosomal cap located at the tip of the head.
o progressive motility (PM)  The acrosomal cap should encompass
o nonprogressive motility (NP) approximately half of the head and cover
o and immotility (IM). approximately two thirds of the sperm nucleus.
-Motility must be specified as total motility (PM and NP) or  The neckpiece attaches the head to the tail and
progressive motility (PM) . the midpiece. The midpiece is approximately 7.0
ALTERNATIVE SPERM MOTILITY GRADING μm long and is the thickest part of the tail because
CRITERIA it is surrounded by a mitochondrial sheath that
Progressive motility (PM) Sperm moving linearly or produces the energy required by the tail for
in a large circle motility.
Non-progressive motility Sperm moving with and EVALUATED
(NP) absence of progression
Immotility (IM) No movement -Sperm morphology is evaluated from a thinly smeared,
stained slide under oil immersion.
-instrumentation capable of performing computer-assisted  Smears are made by placing approximately 10 μL
semen analysis (CASA) has been developed of semen near the frosted end of a clean
 CASA provides objective determination of both microscope slide.
sperm velocity and trajectory (direction of motion).  Place a second slide with a clean, smooth edge
in front of the semen drop at a 45° angle and
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SEMEN
draw the slide back to the edge of the drop of REMEMBER: Sperm motility can be evaluated at
semen, allowing the semen to spread across the room temperature or 37°C. When assessing motility at
end. 37°C, the sample should be incubated at this
STAINING PERFORMED USING: temperature and the preparation made with
o Wright’s prewarmed slides and cover slips.
o Giemsa
o Shorr, or Papanicolaou stain and is a matter of CALCULATING ROUND CELLS
laboratory preference.
-Air-dried slides are stable for 24 hours. At least 200 𝑁𝑥𝑆
sperm should be evaluated and the percentage of 𝐶=
100
abnormal sperm reported. This method can be used when counting cannot be
performed during the hemocytometer count and to verify
ABNROMALITIES IN SPERM MORPHOLOGY counts performed by hemocytometer.
-Routinely identified abnormalities in
o head structure include double heads, giant and ADDITIONAL TESTING
amorphous heads, pinheads, tapered heads, and -The most common are tests for sperm vitality, seminal
constricted heads fluid fructose level, sperm agglutinins, and microbial
-Abnormal sperm tails are frequently infection.
o doubled, coiled, or bent. ADDITIONAL TESTING FOR ABNORMAL SEMEN
-An abnormally long neckpiece may cause the sperm head ANALYSIS
to bend backward and interfere with motility. ABNORMAL POSSIBLE TEST
RESULT ABNORMALITY
ADDITIONAL PARAMETERS IN EVALUATING SPERM Decreased Vitality -Eosin-
MORPHOLOGY motility with nigrosine stain
o measuring head normal count
o neck Decreased Lack of seminal -Fructose level
o and tail size count vesicle support
o measuring acrosome size and evaluating for the medium
presence of vacuoles. Decreased Male antisperm -Mixed
-Inclusion of these parameters is referred to as Kruger’s motility with antibodies agglutination
strict criteria. clumping reaction and
 Strict criteria evaluation requires the use of a stage immunobead
micrometer or morphometry. At present, tests
evaluation of sperm morphology using strict -Sperm
criteria is not routinely performed in the clinical agglutination
laboratory but is recommended by the WHO. with male serum
Normal analysis Female -Sperm
-Normal values for sperm morphology depend on the with continued antisperm agglutination
evaluation method used and vary from greater than infertility antibodies with female
30% normal forms when using routine criteria to serum
greater than 14% normal forms when using strict
criteria.
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SEMEN
SPERM VITALITY
-Decreased sperm vitality may be suspected when a
specimen has a normal sperm concentration with markedly
decreased motility.
 Sperm vitality should be assessed within 1 hour
of ejaculation.
-Vitality is evaluated by mixing the specimen with an eosin
nigrosin stain, preparing a smear, and counting the
number of dead cells in 100 sperm using a bright- field or
phase-contrast microscope.
o Living cells are not infiltrated by the dye and
remain bluish white, whereas
o Dead cells stain red against the purple
background
SEMINAL FLUID FRUCTOSE
-Low sperm concentration may be caused by lack of the
support medium produced in the seminal vesicles which
can be indicated by a low to absent fructose level in the
semen.
 Low fructose levels are caused by abnormalities
of the seminal vesicles, bilateral congenital
absence of the vas deferens, obstruction of the
ejaculatory duct, partial retrograde ejaculation,
and androgen deficiency.
 Specimens can be screened for the presence of
fructose using the resorcinol test that produces
an orange color when fructose is present
PROCEDURE:
SEMINAL FRUCTOSE SCREENING TEST
1. Prepare reagent (50 mg resorcinol in
33 mL concentrated HCl diluted to 100
mL with water).
2. Mix 1 mL of semen with 9 mL of
reagent.
3. Boil.
4. Observe for orange-red color.
-A normal quantitative level of fructose is equal to or
greater than 13 μmol per ejaculate.
o This can be determined using spectrophotometric
methods. Specimens for fructose levels should be
tested within 2 hours of collection or frozen to
prevent fructolysis.
ANTISPERM ANTIBODES
Can be present in both men and women
-They may be detected in semen, cervical mucosa, or
serum, and are considered a possible cause of infertility. It
is not unusual for both partners to demonstrate antibodies,
although male antisperm antibodies are more frequently
encountered.
-The presence of antibodies in a male subject can be
suspected when clumps of sperm are observed during a
routine semen analysis.
 Sperm-agglutinating antibodies cause sperm to
stick to each other in a head-to-head, head-to-tail,
or tail- to-tail pattern.
 The agglutination is graded as “few,” “moderate,”
or “many” on microscopic examination.
-The presence of antisperm antibodies in a female subject
results in a normal semen analysis accompanied by
continued infertility.
 The presence of antisperm antibodies in women may
be demonstrated by mixing the semen with the female
cervical mucosa or serum and observing for
agglutination.
FREQUENTLY USED TEST TO DETECT THE
PRESENCE OF BODY COATED SPERM
1. Mixed agglutination reaction (MAR) test
-The MAR test is a screening procedure used primarily
to detect the presence of immunoglobulin G (IgG)
antibodies.
2. Immunobead test
-The immunobead test is a more specific procedure in
that it can be used to detect the presence of IgG, IgM,
and IgA antibodies and demonstrates what area of the
sperm (head, neckpiece, midpiece, or tail) the
autoantibodies are affecting.
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SEMEN
MICROBIAL AND CHEMICAL TESTING SPERM FUNCTION TEST
-Routine aerobic and anaerobic cultures and tests for
 Chlamydia trachomatis, Mycoplasma hominis, TEST DESCRIPTION
and Ureaplasma urealyticum are most frequently Hamster egg penetration Sperm are incubated with
performed. species nonspecific hamster
eggs and penetration is
-Additional chemical testing performed on semen may observed microscopically
include determining the levels of Cervical mucus penetration Observation of sperm’s ability
 neutral α-glucosidase, free L-carnitine, to penetrate partner’s
glycerophosphocholine, zinc, citric acid, glutamyl midcycle cervical mucus
transpeptidase, and prostatic acid phosphatase. Hypo-osmotic swelling Sperm exposed to low-
sodium concentrations are
 Motile sperm can be detected for up to 24 hours after evaluated for mem- brane
integrity and sperm viability
intercourse, whereas nonmotile sperm can persist for In vitro acrosome reaction Evaluation of the acrosome to
3 days. As the sperm die off, only the heads remain produce enzymes essential
and may be present for 7 days after intercourse. for ovum penetration

SPECIFIC METHOD SEMEN ANALYSIS QUALITY CONTRIL

-is the detection of seminal glycoprotein p30 (prostatic
specific antigen [PSA]), which is present even in the
absence of sperm. Further information can often be
obtained by performing ABO blood grouping and DNA
analysis on the specimen.
REFERENCE SEMEN CHEMICAL VALUES
Neutral α-glucosidase >20 mU/ejaculate
Zinc >2.4 μmol/ejaculate
Citric acid >52 μmol/ejaculate
Acid phosphatase >200 Unites/ ejaculate
POSTVASECTOMY SEMEN ANALAYSIS
-is a much less involved procedure when compared with
infertility analysis because the only concern is the
presence or absence of spermatozoa.
 Specimens are routinely tested at monthly
intervals, beginning at 2 months postvasectomy
and continuing until two consecutive monthly
specimens show no spermatozoa.
 Recommended testing includes examining a wet
preparation using phase microscopy for the
presence of motile and nonmotile sperm. A
negative wet preparation is followed by specimen
centrifugation for 10 minutes and examination of
the sediment.
 The analysis is rated as a high complexity test
under the Clinical Laboratory Improvement
Amendments, and testing personnel standards
must be observed.
 The standardized procedures developed by the
WHO have provided a basis for laboratory testing
and reporting.
 The use of CASA has aided in reducing the
subjectivity of the analysis. However, even
computerized analysis has been shown to vary
among operators.
 Laboratories can now participate in proficiency
testing programs offered by the College of
American Pathologists and the American
Association of Bioanalysts that include sperm
concentration, vitality, and morphology.
 Commercial quality control materials and training
aids are available and should be incorporated into
laboratory protocols.
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SEMEN
REFERENCE:
Strasinger, S.K, Di Lorenzo, M.S (2014). Urinalysis and
Body Fluids (6th ed.) Philadelphia: F.A Daviss Company

MEMBERS: BMLS 3A
o Barbarona, Shaira Mae
o Echavez, Cyrelle Margarette
o Camellon, Rich John Mark
o Jaime, Jaira
o Langilao, Norjanna
o Lasaca, Christine
o Plasabas, Rochellane
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SYNOVIAL FLUID
PHYSIOLOGY Beneficial test most frequently performed
- often referred to as “joint fluid,” is a viscous on synovial fluid:
liquid found in the cavities of the movable - WBC count
joints (diarthroses) or synovial joints - differential test
- the bones in the synovial joints are lined - Gram stain
with smooth articular cartilage and separated - culture
by a cavity containing the synovial fluid. - crystal examination
- the joint is enclosed in a fibrous joint
capsule lined by the synovial membrane

Synovial membrane
- contains specialized cells called
synoviocytes

Smooth articular cartilage and synovial

fluid
- reduce friction between the bones during
joint movement

Synovial Fluid provides: Variety of Conditions associated with

- lubricants in the joints arthritis:
-provides nutrients to the articular cartilage - infection
and lessens the shock of joint compression - inflammation
- metabolic disorder
Synovial Fluid is formed: - trauma
- as an ultrafiltrate of plasma across the - physical stress
synovial membrane - advance age
- filtration is nonselective except for the
exclusion of high-molecular-weight proteins
- most of the chemical constituents, although
seldom of clinical significance, have
concentrations similar to plasma values

Synoviocytes
- cell of the synovial fluid
- secrete a mucopolysaccharide containing
hyaluronic acid and a small amount of
protein

Large Hyaluronate molecules

- contribute the noticeable viscosity to the
synovial fluid

Arthritis
- damage to the articular membranes
produces pain and stiffness in the joints
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SYNOVIAL FLUID
Consideration for amount of fluid
collected or present:
- size of the joint
- extent of fluid build up in the joint

Normal Synovial Fluid

- does not clot (fluid from a diseased joint
may contain fibrinogen and will clot)
- fluid is often collected in a syringe that has
been moistened with heparin

NOTE:
- Powdered anticoagulants should not be
used because they may produce artifacts
that interfere with crystal analysis
- The nonanticoagulated tube for other tests
must be centrifuged and separated to
prevent cellular elements from interfering
with chemical and serologic analyses
-All testing should be done as soon as
possible to prevent cellular lysis and possible
changes in crystals.

COLOR AND CLARITY

A report of the gross appearance is an
essential part of the synovial fluid analysis.

Normal synovial fluid

- appears colorless to pale yellow.

“synovial”
SPECIMEN COLLECTION AND HANDLING - comes from the latin word for egg, ovum.
Normal viscous synovial fluid
Arthrocentesis - resembles egg white
- synovial fluid is collected by needle
aspiration
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SYNOVIAL FLUID
 The color becomes a deeper yellow in essential for the proper joints lubrication
the presence of noninflammatory and
inflammatory effusions and may have Arthritis
a greenish tinge with bacterial - affects both the production of hyaluronate
infection. and its ability to polymerize, thus decreasing
the fluid viscosity
NOTE:
- As with cerebrospinal fluid, in synovial fluid
the presence of blood from a hemorrhagic Method in measuring the viscosity of
arthritis must be distinguished from blood Synovial fluid
from a traumatic aspiration. - observe the fluid’s ability to form a string
from the tip of a syringe
How?
-by observing the uneven distribution of  A string measuring 4 to 6 cm is
blood or even a single blood streak in the considered normal
specimens obtained from a traumatic Hyaluronate polymerization
aspiration - can be measured using a Ropes, or mucin
clot test.
Turbidity
- is frequently associated with the presence  When added to a solution of 2% to 5%
of WBCs. acetic acid, normal synovial fluid forms a
Also produces turbidity: solid clot surrounded by clear fluid.
- synovial cell debris and fibrin  As the ability of the hyaluronate to
polymerize decreases, the clot becomes
 Presence of Crystals less firm, and the surrounding fluid
- fluid may appear milky increases in turbidity.
 Noninflammatory: The mucin clot test is reported in terms
- Clear, yellow fluid of:
 Inflammatory Immunologic origin: -  good (solid clot)
Cloudy, yellow fluid  fair (soft clot)
 low (friable clot)
 Crystal-induced origin:
 poor (no clot)
- Cloudy or milky fluid
The mucin clot test is not routinely performed,
 Septic:
- Cloudy, yellow-green fluid because all forms of arthritis decrease
viscosity and little diagnostic information is
 Hemmorhagic: obtained.
- Cloudy, red fluid
VISCOSITY Formation of a mucin clot after adding acetic
- Synovial fluid viscosity comes from acid can be used to identify a questionable
polymerization of the hyaluronic acid and is fluid as synovial fluid.
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SYNOVIAL FLUID
CELL COUNTS
Total leukocytes count
- most frequently performed cell count on
synovial fluid.
RBC count
- seldom requested
Cell counts
- should be perform as soon as possible.
Very viscous fluid
- may need to be pretreated by adding one
drop of 0.05% hyaluronidase in phosphate
buffer per milliliter of fluid and incubating at
37°C for 5 minutes.
Manual counts
-Mixed specimens using the Neubauer
counting chamber. Clear fluids can usually
be counted undiluted, but dilutions are
necessary when fluids are turbid or bloody.
Dilutions
- traditional WBC diluting fluid cannot be
used because it contains acetic acid that
causes the formation of mucin clots.
 If necessary to lyse the RBCs,
hypotonic saline (0.3%) or saline that
contains saponin is a suitable diluent.
 Methylene blue added to the normal
saline stains the WBC nuclei, permitting
separation of the RBCs and WBCs
during counts performed on mixed
specimens.
Recommended technique
 line a petri dish with moist paper and
place hemocytometer on two small
sticks to elevate it above the moist
paper.
 Fill and count both sides of the
hemocytometer for compatibility.
Acceptable ranges are determined by
the laboratory.
Counting
 For counts less than 200 WBCs/L,
count all 9 large squares.
 For counts greater than 200 WBCs /L in
the above count,
 count the 4 corner squares.
 For counts greater than 200 WBCs /L in
the above count,
 Count the 5 small squares used for a
RBC count.
( Automated cell counters- can be used)
-Incubating the fluid with hyaluronidase
decreases specimen viscosity-
Analyzing scatter grams
- Can aid in detecting tissue cells and debris.
Properly controlled automated counts
provide higher precision than manual counts.
WBC Counts
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SYNOVIAL FLUID
<200 cells/L - Normal and may reach
100,000 cells/L or higher in severe
infections.
Considerable overlap of elevated
leukocyte counts between septic and
inflammatory forms of arthritis.
DIFFERENTIAL COUNTS
 Should performed cytocentrifuged
preparations or on thinly smeared slides.
Fluid- should incubated with hyaluronidase
prior to slide preparation.
Primary cells seen in Synovial Fluid
 Mononuclear cells, including monocytes,
macrophages, and synovial tissue cells.
 Neutrophils should account for < 25% of
the differential count and lymphocytes <
15%.
 Increased neutrophils indicate a septic
condition, whereas an elevated cell
count with a predominance of
lymphocytes suggests a nonseptic
inflammation.
Lipid droplets
- present after crush injuries.
Hemosiderin granules
- pigmented villonodular synovitis.
CRYSTAL IDENTIFICATION
- Microscopic examination of synovial
fluid for the presence of crystals is
an important diagnostic test in
evaluating arthritis
Causes of crystal formation
 metabolic disorders
 decreased renal excretion that produce
elevated blood levels of crystallizing
chemicals
 degeneration of cartilage and bone
 injection of medications
(corticosteroids)
Types of Crystals
1. Monosodium urate (uric acid) (MSU)
- found in cases of gout
- routinely seen as needle-shaped crystals
Caused by:
 impaired metabolism of purines
 increased consumption of
high-purine-content foods, alcohol, and
fructose
 chemotherapy treatment of leukemias
 decreased renal excretion of uric acid
are the most frequent causes of gout.
Location:
- may be extracellular or located within the
cytoplasm of neutrophils.
- they are frequently seen sticking through
the cytoplasm of the cell
2. Calcium pyrophosphate dihydrate
(CPPD)
- rhomboid-shaped or square but may
appear as short rods.
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SYNOVIAL FLUID
- seen with pseudogout
- Pseudogout is most often associated with
degenerative arthritis, producing cartilage
calcification and endocrine disorders that
produce elevated serum calcium levels.
3. Cholesterol crystals
- associated with chronic
inflammation
4. Corticosteroids
- after injections
5. Calcium oxalate crystals
- in renal dialysis patients
6. hydroxyapatite (basic calcium
phosphate)
- associated with calcified cartilage
degeneration
Artifacts
 talcum powder
 starch from gloves
 precipitated anticoagulants
 Dust
 scratches on slides and cover slips.
NOTE:
Slides and cover slips should be examined
and if necessary cleaned again before use.
Slide Preparation
- Ideally, crystal examination should
be performed soon after fluid
collection to ensure that crystals are
not affected by changes in
temperature and pH
- Both MSU and CPPD crystals are
reported as being located
extracellularly and intracellularly
(within neutrophils); therefore, fluid
must be examined before WBC
disintegration.
1. Fluid is examined as an unstained
wet preparation.
2. One drop of fluid is placed on a pre
cleaned glass slide and cover
slipped.
3. The slide can be initially examined
under low and high power using a
regular light microscope
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SYNOVIAL FLUID
4. Crystals may be observed in
Wright’s-stained smears; however,
this should not replace the wet prep
examination and the use of polarized
and red-compensated polarized light
for identification.
Crystal Polarization
 Positive identification is made using
first-order red-compensated
polarized light
 Use betamethasone acetate
corticosteroid as control slide to
polarize the properties of MSU –
highly birefringent
 If the crystal run parallel to the long
axis aligned with slow vibration
produces a yellow color – negative
birefringence
If the crystal run perpendicular to the long
axis aligned with slow vibration produces a
blue color – positive birefringence
CHEMISTRY TESTS
 Glucose determination – frequently
requested
 Normal synovial fluid glucose values
are based on the blood glucose level
 Samples are obtained after 8 hours
of fasting
 Normal synovial fluid glucose – 10
mg/dL
 Other tests: Total protein, uric acid
determination
MICROBIOLOGIC TEST
Infection
- may occur as a secondary complication of
inflammation
Caused by:
 trauma or through dissemination of a
systemic infection
Gram Stains and Cultures
- two of the most important tests
performed on synovial fluid
- must be performed on all specimens
Bacterial Infections
- most frequently seen
- fungal, tubercular, and viral infections
also can occur.
- if suspected, use special culturing
procedures
Routine Bacterial Cultures
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SYNOVIAL FLUID
- use enrichment medium- chocolate agar
because in addition to Staphylococcus
and Streptococcus
- Haemophilus species and Neisseria
gonorrhoeae
(common fastidious organisms that
infect synovial fluid)

SEROLOGIC TEST

- diagnosis of joint disorders

- most of these tests are performed on serum
and synovial fluid analysis
-serves as confirmatory measure in cases
that are difficult to diagnose

Autoimmune Diseases Rheumatoid

Arthritis and Systemic Lupus
Erythematosus
- very serious joint inflammation
- diagnosed in the serology laboratory by
demonstrating the presence of their
particular autoantibodies in the
patient’s serum.
-same antibodies can also be
demonstrated in synovial fluid, if
necessary.

Arthritis
- frequent complication of Lyme disease

Thus,
Demonstrating antibodies to the causative
agent Borrelia burgdorferi in the patient’s
serum can confirm the cause of the arthritis.

REFERENCES:
Strasinger, S.K, Di Lorenzo, M.S (2014).
Urinalysis and Body Fluids (6th ed.)
Philadelphia: F.A Daviss Company
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SEROUS FLUID
 The closed cavities of the body:
o pleural
o pericardial, and;
o peritoneal cavities
They are lined by the two membranes referred
to as serous membranes.
 Serous membranes
I. Parietal membrane – lines the cavity
wall
II. Visceral membrane – covers the
organ with cavity
 Serous fluid – the fluid between the
membranes that provides lubrication between
the parietal and visceral membranes.
 Lubrication –to prevent the friction between
the two membranes that occurs as a result of
movement of the enclosed organs.
FORMATION
 Are formed as ultrafiltrates of plasma
 Production and reabsorption are subject to
hydrostatic pressure and colloidal
pressure (oncotic pressure).
 Colloidal pressure from serum proteins is the
same in the capillaries on both sides of the
membrane.
 Hydrostatic pressure in the parietal and
visceral capillaries causes fluid to enter
between the membranes.
 Filtration of the plasma ultrafiltrate results in
increased oncotic pressure in the capillaries
that favors reabsorption of fluid back into the
capillaries.
 Effusion – increase in fluid between
membranes due to the disruption of the
mechanisms of serous fluid formation and
reabsorption
- 
- Congestive heart failure
- Hypoproteinemia
- Inflammation and infection
- Tumors
SPECIMEN COLLECTION AND HANDLING
 Thoracentesis – pleural
 Pericardiocentesis – pericardial
 Paracentesis – peritoneal
 Abundant fluid - >100 mL
 EDTA tube – cell count and differential
 Sterile heparinized or sodium polyanethol
sulfonate (SPS) evacuated tubes –
microbiology and cytology
 Centrifugation –performed for better
recovery of microorganisms and abnormal
cells.
 Chemistry tests - run on clotted specimens in
plain or heparin tubes.
 pH- maintained anaerobically in ice
TRANSUDATES AND EXUDATES
Transudates
- Effusions due to systemic disorder that
disrupts the balance in the regulation of fluid
filtration and reabsorption
- Hydrostatic pressure (congestive heart failure)
& Hypoproteinemia (nephrotic syndrome)
Exudates
 Produced by the conditions that directly
involve the membranes or particular cavity
including infections and malignancies
Tests
 Appearance
 Total protein
Lactic dehydrogenase
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SEROUS FLUID
 Cell counts  Turbidity – presence of WBC’s and
 Spontaneous clotting indicated bacterial infection, tuberculosis,
 Fluid blood ratios for protein and lactic or an immunologic disorder
dehydrogenase – most reliable  Blood – hemothorax, membrane damage,
or a traumatic aspiration
GENERAL LABORATORY PROCEDURES
 Milky pleural fluid – chylous material
Test on serous fluid: (high concentration of triglycerides) or
pseudochylous material (high
- Evaluation of the appearance concentration of cholesterol)
- Differentiation between transudate and b) Hematology Tests
exudate.
 Differential cell count - most
 WBC counts greater than 1000/L and RBC diagnostically significant hematology test
counts greater than 100,000/L indicate an
 Macrophages – 64% to 80%
exudate.
 Lymphocytes – 18% to 30% (noticeably
 Neubauer counting chamber – used for
present in both transudates and exudates)
manual counting of serous fluid cell counts
 Neutrophils - 1% to 2% (increased in
 Differential cell counts - routinely performed
effusions resulting from pancreatitis and
on serous fluids, preferably on Wright’s-
pulmonary infarction)
stained, cytocentrifuged specimens or on
 Increased eosinophil levels (>10%) may
slides prepared from the sediment of
be associated with trauma resulting in the
centrifuged specimens.
presence of air or blood (pneumothorax
and hemothorax) in the pleural cavity.
1. PLEURAL FLUID
 Mesothelial cells -pleomorphic; they
 Obtained from the pleural cavity, located
resemble lymphocytes, plasma cells, and
between the parietal pleural membrane
malignant cells.
 Procedures:
- single small or large round cells with
I. Pleural fluid cholesterol and fluid:
abundant blue cytoplasm and round
serum cholesterol ratio - A pleural
nuclei with uniform dark purple
fluid cholesterol >60 mg/dL or a
cytoplasm and may be referred to as
pleural fluid: serum cholesterol ratio
“normal” mesothelial cells
>0.3 provides reliable information that
- “reactive” mesothelial cells may appear
the fluid is an exudate.
in clusters; have varying amounts of
II. Pleural fluid: serum total bilirubin
cytoplasm, eccentric nuclei, and
ratio - ratio of 0.6 or more also
prominent nucleoli; and be multinucleated,
indicates the presence of an exudate.
thus more closely resembling malignant
a) Appearance
cells
 Clear and pale yellow – normal and
transudate pleural fluids
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: AUBF
SEROUS FLUID
c) Chemistry tests
 most common chemical tests performed
on pleural fluid are glucose, pH,
adenosine deaminase (ADA), and
amylase
 Triglyceride levels - measured to confirm
the presence of a chylous effusion
 Glucose – decreased levels are seen
with tuberculosis, rheumatoid
inflammation, and purulent infections.
- less than 60 mg/dL are
considered decreased
 Pleural fluid pH- lower than 7.2 may
indicate the need for chest-tube drainage
- 0.30 degrees lower than the
blood pH is considered significant
- A pH value as low as 6.0
indicates an esophageal rupture that
is allowing the influx of gastric fluid.
 ADA levels higher than 40 U/L are highly
indicative of tuberculosis.
d) Microbiologic and serologic tests
 Associated with pleural effusions:
o Staphylococcus aureus,
Enterobacteriaceae, anaerobes,
and Mycobacterium tuberculosis.
 Serologic testing - differentiate effusions
of immunologic origin from
noninflammatory processes
 Antinuclear antibody (ANA) and
rheumatoid factor (RF)- are the most
frequently performed.
 Tumor markers carcinoembryonic antigen:
I. CA 125 – metastatic uterine
cancer
II. CA 15.3 and CA 549 – breast
cancer
III. CYFRA 21-1 – lung cancer
2. PERICARDIAL FLUID
 10 to 50 mL
 primarily the result of changes in the
membrane permeability due to infection
(pericarditis), malignancy, and trauma-
producing exudates.
 Primary cause of transudates: uremia,
hypothyroidism, and autoimmune
disorders
 An effusion is suspected when cardiac
compression (tamponade) is noted during
the physician’s examination
a.) Appearance
 Clear and pale yellow - normal and
transudate pericardial fluid
 Turbid- infection and malignancy
 Blood streaked – malignant effusions
 Milky fluids - chylous and
pseudochylous effusions
b.) Laboratory tests
 Cytologic examination of pericardial
exudates for the presence of
malignant cells is an important part of
the fluid analysis.
 Bacterial cultures and Gram stains -
endocarditis is suspected
 Acid-fast stains and chemical tests for
adenosine deaminase are often
requested on pericardial effusions.
3. PERITONEAL FLUID
 Ascites - Accumulation of fluid between
the peritoneal membranes
 Ascitic fluid – fluid
 Cirrhosis – frequent cause of ascitic
transudates
 Peritonitis - a result of intestinal
perforation or a ruptured appendix
 Malignancy - most frequent causes of
exudative fluids
 DAVAO DOCTORS COLLEGE
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SEROUS FLUID
 Normal saline - introduced into the
peritoneal cavity as a lavage to detect
abdominal injuries that have not yet
resulted in fluid accumulation
 Peritoneal lavage - sensitive test to
detect intra-abdominal bleeding in blunt
trauma cases
 RBC counts greater than 100,000/mL are
indicative of blunt trauma injuries
a.) Transduates versus Exudates
 serum-ascites albumin gradient
(SAAG) - Recommended over the
fluid:serum total protein and LD ratios
to detect transudates of hepatic origin
 A difference (gradient) of 1.1 or
greater suggests a transudate
effusion of hepatic origin, and lower
gradients are associated with
exudative effusions
b.) Appearance
 Clear and pale yellow - normal
peritoneal fluid
 Exudates are turbid with bacterial or
fungal infections.
 Green or dark-brown - presence of
bile
 Blood-streaked fluid - seen after
trauma and with tuberculosis,
intestinal disorders, and malignancy.
 Chylous or pseudochylous material -
present with trauma or lymphatic
vessels blockage.
c.) Laboratory tests
 Normal WBC counts- <350 cells/µL
 Absolute neutrophil count- to
distinguish bacterial peritonitis and
cirrhosis.
o >250 cells/uL or >50% of total
WBC- indicates infection
 Lymphocytes- predominant cell in
tuberculosis.
 Cellular examination
o Examination of ascitic exudates is
important for the detection of tumors
of primary and metastatic origin.
o Malignancies are most frequently of
gastrointestinal prostate, or ovarian
origin.
o Cells present in ascitic fluid
includes:
 leukocytes,
 abundant mesothelial cells,
and
 macrophages, including
lipophages
o Microorganisms including
 bacteria,
 yeast, and
 Toxoplasma gondii (may also
be present )
 Chemical testing
o Chemical examination of ascitic
fluid consists primarily of;
o Glucose- decreased below
serum levels in bacterial and
tubercular peritonitis and
malignancy
o Amylase- determined on ascitic
fluid to ascertain cases of
pancreatitis, and it may be
elevated in patients with
gastrointestinal perforations
o Alkaline phosphatase- when
elevated is also highly diagnostic
of intestinal perforation.
 DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: AUBF

SEROUS FLUID
 Microbiology tests
o Gram stains and bacterial
cultures- are perfromed when
bacterial peritonitis is suspected.
o Acid-fast stains, adenosine
deaminase, and cultures - for
tuberculosis
 Serologic tests
o Presence of CA 125 antigen with
a negative CEA suggests the
source is from the ovaries,
fallopian tubes, or
endometrium.

REFERENCE:
Strasinger, S.K, Di Lorenzo, M.S (2014). Urinalysis
and
Body Fluids (6th ed.) Philadelphia: F.A Daviss
Company
  After of the first trimester- fetal urine is the major
DAVAO DOCTORS COLLEGE
MEDICAL LABORATORY SCIENCE DEPARTMENT
STUDENT NOTES: AUBF

AMNIOTIC FLUID

PHYSIOLOGY
FUNCTION
 Present in the amnion
 Amnion- a membranous sac that surrounds the fetus
 Metabolically active
 Involved in the exchanges of water and
 Chemicals between the fluid, the fetus, and the
maternal circulation; and produces peptides, growth
factors, and cytokines.
 Primary functions of the amniotic fluid are to provide a
protective cushion for the fetus;
 Allow fetal movement, stabilize
 The temperature to protect the fetus from extreme
temperature changes;
 And permit proper lung development.
VOLUME
 Increases in quantity throughout pregnancy;
 Reaching a peak of approximately 800 to 1200 mL
during the third trimester;
 Polyhydramnios- an amniotic fluid volume greater
than 1200 Ml. Failure of the fetus to begin swallowing
results in excessive.
 Accumulation of amniotic fluid. (may be secondarily
associated with fetal structural anomalies, cardiac
arrhythmias, congenital infections, or chromosomal
abnormalities).
 Oligohydramnios- an amniotic fluid volume less than
800 mL. Increased fetal swallowing, urinary tract
deformities, and membrane leakage are possible
causes of decreased amniotic fluid. (may be
associated with congenital malformations, premature
rupture of amniotic membranes, and umbilical cord
compression, resulting in decelerated heart rate and
fetal death).
 First trimester- approximately 35 mL of amniotic fluid
is derived.
 Third to half of pregnancy- secretes a volume of
lung liquid necessary to expand the lungs with
growth.
 Lung surfactants: lecithin, sphingomyelin, and
phosphatidyl glycerol.
 contributor to the amniotic fluid amniotic fluid volume.
CHEMICAL COMPOSITION
 Has a composition similar to that of the maternal
plasma and contains a small amount of sloughed fetal
cells from the skin, digestive system, and urinary
tract.
 Bilirubin, Lipids, Enzymes, Electrolytes, Urea,
Creatinine, Uric Acid, Proteins, Hormones
 The chemical composition of the amniotic fluid
changes when fetal urine production begins.
 Increased concentrations- creatinine, urea, and uric
acid;
 Decreased concentrations- glucose and protein
concentrations.
 Electrolytes, enzymes, hormones, and metabolic end
products also vary but are of little clinical significance.
Measurement of amniotic fluid creatinine
 Prior to 36 weeks gestation: level ranges between
1.5 and 2.0 mg/dL. It then rises above 2.0 mg/dL,
providing a means of determining fetal age greater
than 36 weeks.
Differentiating Maternal Urine From Amniotic Fluid
Aids in the Differentiation
1. Chemical analysis of creatinine, urea, glucose, and
protein.
2. Creatinine does not exceed 3.5 mg/dL;
3. Urea does not exceed 30 mg/dL
 4. Measurement of glucose and protein by a reagent
strip is a less reliable indicator.
5. Fern test
 Used to evaluate premature rupture of the
membranes
 “Fern-like” crystals is seen in a positive screen
for amniotic fluid
SPECIMEN COLLECTION
INDICATIONS FOR AMNIOCENTESIS
 Amniocentesis is recommended for neural tube
defects when screening blood test.
 Fetal body measurements – taken with
ultrasonography, estimate the gestational age of
fetus.
COLLECTION
 Fetal epithelial cells in amniotic fluid indicate the
genetic material of the fetus and the biochemical
substances that the fetus has produced.
 Cells can be separated from the fluid,
cultured, and examined for chromosome
abnormalities by karyotyping, fluorescence
in situ hybridization (FISH), fluorescent
mapping spectral karyotyping (SKY), and
DNA testing
 Biochemical substances produced by the
fetus can be analyzed by thin-layer
chromatography
 Amniocentesis – is a safe procedure, particularly
when performed after the 14th week of gestation
 16 weeks’ gestation - Fluid for chromosome analysis
is usually collected.
 End of the Second Trimester - Tests for intrauterine
growth retardation are performed.
 Third Trimester - Tests for fetal distress and maturity
are performed.
INDICATION FOR PERFORMING AMNIOCENTESIS
Amniocentesis may be indicated at 15 to 18 weeks’
gestation for the following conditions to determine
early treatment or intervention:
 Mother’s age of 35 or older at delivery
 Family history of chromosome abnormalities, such
as
 trisomy 21 (Down syndrome)
 Parents carry an abnormal chromosome
rearrangement
 Earlier pregnancy or child with birth defect
 Parent is a carrier of a metabolic disorder
 Family history of genetic diseases such a sickle
cell disease, Tay-Sachs disease, hemophilia,
muscular dystrophy, sickle cell anemia, Huntington
chorea, and cystic fibrosis
 Elevated maternal serum alpha-fetoprotein
 Abnormal triple marker screening test
 Previous child with a neural tube disorder such as
spina
 bifida, or ventral wall defects (gastroschisis)
 Three or more miscarriages
Amniocentesis is indicated later in the pregnancy (20 to
42 weeks) to evaluate:
 Fetal lung maturity
 Fetal distress
 HDN caused by Rh blood type incompatibility
 Infection
 Amniotic fluid is obtained by needle aspiration into
the amniotic sac, a procedure called amniocentesis.
 The procedure most frequently performed is a
transabdominal amniocentesis.
 Vaginal amniocentesis may also be performed;
however, this method carries a greater risk of
infection.
 A maximum of 30 mL of amniotic fluid is collected in
sterile syringes.
 The first 2 or 3 mL collected can be contaminated.
 Specimens should be transferred to sterile plastic
containers.
 Fluid for bilirubin analysis in cases of hemolytic
disease of the newborn (HDN) must be protected
from light at all times.
SPECIMEN HANDLING AND PROCESSING
 Fluid for Fetal lung maturity (FLM) test should be
placed in ice for delivery to the laboratory and kept
refrigerated.
 Specimen for bilirubin testing must be immediately
protected from light by placing the specimen in
amber-colored tubes.
 Wrap the collection tube in foil or black plastic cover.
 Specimen for cytogenetic or microbial studies must
be processed aseptically and maintain at room
temperature (37 °C)
 All fluid for chemical testing should be separated from
cellular element and debris as soon as possible using
centrifugation or filtration.
 Time and speed of centrifugation depends in the test
and lab protocol.
 COLOR AND APPEARANCE  Amniotic fluid bilirubin is measured by
 Normal amniotic fluid is colorless and may exhibit spectrophotometric analysis using serial dilutions.
slight to moderate turbidity from cellular debris.  Bilirubin from RBC destruction appears in the
 The presence of bilirubin gives the fluid a yellow color amniotic fluid.
and is indicative of red blood cell destruction resulting  The amount of unconjugated bilirubin present
from HDN. correlates with the amount of RBC destruction.
 MECONIUM  Spectrophotometric analysis of fluid optical density
 usually defined as a newborn’s first bowel (OD) between 365 and 550 nm is plotted on
movement, is formed in the intestine from fetal semilogarithmic graph paper.
intestinal secretions and swallowed amniotic  In normal fluid, the OD is highest at 365 nm and
fluid. decreases linearly to 550 nm, illustrated by a straight
 dark green, mucus-like material line.
 Bilirubin causes OD rise at its maximum absorbance
Amniotic Fluid Color level of 450 nm; difference between theoretic baseline
Color Significance and 450 nm peak is the ΔA450 represent amniotic
Colorless Normal fluid bilirubin concentration.
Blood-streaked Traumatic tap,  Difference is plotted on a Liley graph to determine the
abdominal trauma, severity of the hemolytic disease.
intra-amniotic
hemorrhage
Yellow Hemolytic disease of
the newborn (bilirubin)
Dark Green Meconium
Dark Red-Green Fecal death

TESTS FOR FETAL DISTRESS

 Oldest routinely performed laboratory test on amniotic
fluid evaluates the severity of the fetal anemia
produced by Hemolysis Disease of the Newborn
(HDN). LILEY GRAPH
 Plots ΔA450 against gestational age.
Hemolysis Disease of the Newborn (HDN)
 Consists of three zones based on hemolytic severity.
 Also called “Erythroblastosis fetalis”
 Zone I: mildly affected fetus.
 Most commonly Rh-negative mothers
 Zone II: requires careful monitoring.
 Other red blood cell (RBC) antigens can also produce
 Zone III: severely affected fetus, may require
HDN
induction of labor or intrauterine exchange
 Fetal cells with antigens enter maternal circulation
transfusion.
and cause production of maternal antibodies
 Maternal antibodies cross the placenta and destroy
fetal cells with the corresponding antigen
NEURAL TUBE DEFECTS
 One of the most common birth defects in the United
States. It can be detected by maternal serum alpha-
fetoprotein (MSAFP) blood test, high-resolution
ultrasound, and amniocentesis
 Alpha-fetoprotein (AFP) produced by the fetal liver
prior to 18 weeks’ gestation.
 Increased levels in maternal blood or amniotic fluid
indicate possible anencephaly or spinal bifida.
 Increased levels are found when skin fails to close
over neural tissue.
 Measure maternal blood first, then amniotic fluid.
TEST OF FETAL DISTRESS (DISADVANTAGES)
 Normal values based on weeks of gestation
(maximum AFP 12 to 15 weeks)
 Report multiples of the median (MoM)
 Median is laboratory’s reference level for a given
week of gestation.
 More than two times the median (MOM) is abnormal.
 Follow with fluid amniotic acetylcholinesterase
(AChE): more specific for neural disorders.
- Do not perform on a bloody specimen
TESTS FOR FETAL MATURITY
FETAL LUNG MATURITY
 Respiratory distress syndrome (RDS)
 most frequent complication of early delivery
 seventh most common cause of morbidity
and mortality in the premature infant
 caused by an insufficiency of lung surfactant
production and structural immaturity of the
fetal lungs
LABORATORY TESTS:
 Amount of surfactant in fetal lungs can be estimated
by measuring the amount of surfactants in amniotic
fluid.
LECITHIN-SPHINGOMYELIN RATIO
 A test of fetal amniotic fluid to assess for fetal lung
immaturity
 Lecithin is the primary component of the
surfactants that make up the alveolar lining and
account for alveolar stability
 Prior to 35 weeks’ gestation, the L/S ratio is usually
less than 1.6 because large amounts of lecithin are
not being produced at this time.
 The L/S ratio will rise to 2.0 or higher as the lecithin
production increases to prevent alveolar collapse.
 When the L/S ratio reaches 2.0, a preterm delivery is
usually considered to be a relatively safe procedure.
 Falsely elevated results are encountered in fluid
contaminated with blood or meconium because both
these substances contain lecithin and sphingomyelin.
 Quantitative measurement of lecithin and
sphingomyelin is performed using thin-layer
chromatography (TLC).
PHOSPHATIDYL GLYCEROL
 Presence of another lung surface lipid and also
essential for adequate lung maturity and can be
detected after 35 weeks’ gestation
 The production of PG normally parallels with lecithin,
but its production is delayed in cases of maternal
diabetes.
 In this circumstance, respiratory distress occurs in the
presence of an L/S ratio of 2.0. Therefore, a thin-layer
chromatography lung profile must include lecithin,
sphingomyelin, and PG to provide an accurate
measurement of FLM.
FOAM STABILITY INDEX
 Measure the individual lung-surface lipid
concentrations, a mechanical screening test, called
the “foam” or “shake” test, was used to determine
their presence.
PROCEDURE FOR FOAM SHAKE TEST
1. Mix equal parts of amniotic fluid with 95% ethanol.
2. Vigorously shake for 15 seconds.
3. Allow to sit undisturbed for 15 minutes.
4. Observe for the presence of a continuous line of
bubbles around the outside edge.
The presence of bubbles indicates that a sufficient amount of
phospholipid is available to reduce the surface tension of the
fluid even in the presence of alcohol, an antifoaming agent.
PROCEDURE FOR FOAM STABILITY INDEX
1. Add 0.5 mL of amniotic fluid to tubes containing
increasing amounts of 95% ethanol ranging from
0.42 to 0.55 mL in 0.01-mL increments.
2. Vigorously shake for 15 seconds.
3. Allow to sit undisturbed for 15 minutes.
4. Observe for the presence of a continuous line of
bubbles around the outside edge.
5. Values ≥ 47 indicate fetal lung maturity.
The Foam Stability Index has shown good correlation with the PROCEDURE:
L/S ratio and tests for phosphatidyl glycerol. The test cannot 1. Mix the AF by inverting capped container 5 times.
be used with contaminated amniotic fluid because blood and 2. Transfer sample to clear test tube for visual
meconium also reduce surface tension, yielding a falsely inspection.
3. Visually inspect the sample for obvious mucus, WB,
mature index result.
or meconium.
4. Cap tube and mix by gentle inversion or placing it to
LAMELLAR BODIES
tube rocker for 2 minutes.
 Surfactant (90% phospholipid and 10% proteins) - 5. Flush platelet channel; analyze instrument’s diluents
buffer until a background count deemed acceptable
layered storage granules - Lamellar bodies.
by laboratory is obtained in 2 consecutive analyses.
 Secreted by Type II pneumocytes of fetal lung at 24 6. Process specimen through cell counter and record
weeks of gestation. platelet channel as LBC.
 Absorbed into alveolar spaces to provide surfactant.
 Enter amniotic fluid at about 26 weeks of gestation
50,000 to 200,000/ul by the end of trimester.
REFERENCE:
 Fetal lung matures = ^lamellar body production =
Strasinger, S.K, Di Lorenzo, M.S (2014). Urinalysis and Body
^amniotic fluid phospholipid = L/S ratio.
Fluids (6th ed.) Philadelphia F.A Daviss Company
 # of lamellar bodies (Amniotic fluid) = # of
phospholipid (Fetal lung)
MEMBERS:
 Lamellar bodies increase the OD of the amniotic fluid.
 Arcenas, Naomi
 Specimens centrifuged at 2000g for 10 minutes and
 Mabalot, Rianne Ymanuel
examined at 650nm.
 Monocillo, Mae Anne
 OD of 0.150 correlate well with L/S ratio of >/= 2.0
 Paican, Rosa
and presence of phosphatidyl glycerol.
 Siso, Nadine
 Vidoy, Kimberly
Lamellar Body Count
 Lamellar body diameter ranges from 1.7-7.3Fl, or 1-
5um.
 Automated Hematology analyzers (Optical or
impedance method).
Advantages:
1. Rapid turnover time
2. Low reagent cost
3. Wide availability
4. Low degree of technical difficulty
5. Low volume of amniotic fluid required
6. Excellent clinical performance
 AF containing WB, meconium, and mucus should not
be used.
 Specimens may be stored at 2 degree Celsius to 8
degree Celsius.
 Results reported in units of lamellar bodies/ml.
 Consensus protocol for noncentrifuged samples
<15,000/ul as immature. Results in between are
considered indeterminate and further testing is
recommended.