Specimen Considerations

Key Points Quality Laboratory Process

→ Errors and variables in the pre-analysis stage can affect test results → Includes analytical process and general policies, practices, and procedures that define how
→ Patient variables include physical activity, diet, age, sex circadian variations, posture, stress, all aspects of the work are done
obesity, smoking, and medication o Personal policies
→ Strict adherence to proper technique and site selection can minimize collection variables o Standard operating procedures (SOP)
such as hemolysis, hemoconcentration, clots, and other causes for sample rejection or o Specimen collection guidelines
erroneous results
→ Blood collection containers are color coded based on additive or preservative and each is Quality Control
suitable only for specific tests. Failure to use the proper tubes or filling tubes in the wrong → Emphasizes on STAT control procedure and non-STAT check procedure
sequence can produce erroneous results. o Linearity check
→ Blood, urine, and other body fluid constituents can change during transport and storage. o Reagent and standard check
The extent of these changes varies by analytes (Henry’s Clinical Diagnosis) o Temperature monitor

Errors in the Laboratory Dwell Time

Pre-Analytical → Time elapsed from the initiation of the test up to the completion of analysis
→ Physiologic variable affecting blood samples (fasting, gender, race, stress, hormonal Turn Around Time
changes, and exercise) → Moment from the receiving of specimen up to release of the result
→ Collection parameters (diet and position) → It is the total amount of time required to procure/obtain a specimen, prepare the specimen,
Analytical Errors run the test, relate the result.
→ Systemic and random errors → The supervisor should relate to the technologist the need to work with the stated limits
→ Performed during testing Throughput
Post-Analytical Errors → Number of test that an instrument can perform
→ Delayed reporting
→ Errors within the reports (miscoding) Ten Common Errors in Specimen Collection
→ Misunderstanding the information 1. Misidentification of the patient
2. Mislabelling of the specimen
Total Quality Management 3. Short draws or wrong anticoagulant blood ratio (affects the protime)
→ Is considered a quality system implemented to ensure quality 4. Mixing problems or clots (affects CBC parameter)
→ Process is also referred as : 5. Wrong tubes or anticoagulant
o Total Quality Control 6. Hemolysis or lipemia
o Total Quality Leadership 7. Hemoconcentration from prolonged tourniquet time (cause hemolysis)
o Continuous Quality Management 8. Exposure to light/extreme temperatures (bilirubin)
o Quality Management Science 9. Improperly timed specimens or delayed delivery to the laboratory
o Industrial Quality Management 10. Processing errors
a. Incomplete centrifugation (affects hematocrit)
b. Incorrect log-in
c. Improper storage
Clinical Microscopy → Labels must be attached to the container, not to the lid and should not become
I. Urine Specimens detached if container is refrigerated
A. Specimen Evaluation → Avoid mix-ups of specimen, labels must have an adhesive that resists moisture
→ Before one precedes with any examination, the urine specimen must be and adheres under refrigeration
evaluated in terms of its acceptability → A requisition form (manual or computerized) must accompany specimens
→ Considerations include delivered to the laboratory
o Proper labelling → The information on the form must match the information on the specimen label
o Proper specimen for the requested examination D. Rejection
o Proper preservative → Improper labelled and collected specimen should be rejected by the laboratory
o Visible signs of contamination and appropriate personnel should be notified to collect a new specimen
o Whether any transportation delays may have caused significant → Examples of unacceptable specimen
deterioration i. Unlabelled specimen
→ Each laboratory should have written and enforced guidelines for the acceptable ii. Non-matching labels and requisition forms
or rejection of specimens iii. Specimens contaminated with feces or toilet paper
→ If a single specimen is submitted for multiple measurements, bacteriologic iv. Containers with contaminated exteriors
examination should be done first provided that the urine has been properly v. Specimens of insufficient quantity
collected vi. Specimens incorrectly transported
→ With pediatric patients and persons in acute renal failure, a notation should be E. Specimen Handling
made and the measurement most pertinent to the diagnosis should be → Changes may occur not only in vivo but also in vitro, thus necessitating correct
performed first handling procedures after the specimen is collected
→ For quantitative measurements, timed (12-24 hour) urinary collection is preferred F. Specimen Integrity
compared to random → Following collection, specimens should be delivered to the lab and tested within
B. Specimen Collection 2 hours
→ In observance of standard precautions, gloves should be worn at all times when → If delay is anticipated, it should be refrigerated or have an appropriate chemical
in contact with the specimen preservative added
→ Must be collected in clean, dry, leak proof containers. G. Specimen Preservation
→ Disposable containers → Most routine used method of preservation is refrigeration at 2-8 oC, which
C. Labelling decreases bacterial growth and metabolism
→ Three essential minimum labelling requirements include patient’s full name, and o Allows specimen to return to room temperature prior in performing
the date, and time of collection chemical testing by reagent strips
→ Additional information may be required by the institution such as,  as required → When a specimen is to be transported, and refrigeration is impossible, chemical
by the institutional protocol! preservatives may be added
o The ideal preservative should be bactericidal, inhibits urease, and
 Identification number  Time when specimen is received preserves formed elements in the sediment.
 Patient’s age  Time of specimen → Three glass collection : determine prostatic infection
 Location  Possible interfering medications → Glucose tolerance specimens are collected to correspond with blood samples,
 Physician’s name  Patient’s clinical information drawn during GGT
→ 24 hour or timed specimens : quantitative chemical tests
 collection
documentation of proper sample identification from the time
(timed
of collection to the receipt of lab results
specimen)
H. Types of Urine Specimen
I. Urine Cytology
Types Purpose Considerations Additional Info i. Obtain a first morning specimen
May also produce ii. Submit the specimen to the lab within 30 minutes of collection
Routine screening to erroneous result caused iii. Avoid touching the insides of the container and/or the lid in order to maintain
Ease of collection
Random detect obvious by dietary intake or sterility
Convenient for patients
abnormalities physical activities prior
to collection J. 24 Hour Urine Specimens
 Ideal screening → The collection container should be kept cool throughout the collection period
specimen Patient instructed to → It may be placed on the refrigerator or in a pan with ice during collection
First morning  Avoid false collect specimen Procedure
Assurance detection of chemicals
(most negative on immediately upon arising
and formed elements 1. Upon arising in the morning, patient voids to empty his bladder completely and
concentrated) pregnancy test and deliver to the
 Orthostatic laboratory within 2 hours discards the specimen
protein Note the exact time and print it on the container label.
Specimen will not 2. Collect all urine voided for 24 hours after the time in the container provided. All
Fasting contain any metabolites urine passed during the 24 hour time period (day and night) must be saved. Urine
Diabetic screening Second voided specimen after a
(second from the food ingested
or monitoring period of fasting passed during bowel movements must be also collected.
morning) prior to beginning of
fasting period Be careful not to contaminate urine specimen with feces
Patient instructed to 3. Refrigerate the collected urine between all voiding or keep in a cool place
void shortly before 4. At exactly the same time the following morning, void completely again (first time
2-hour Diabetic monitoring consuming a routine Result compared with fasting after awakening) and add this sample to the collection container. This completes
postprandial with insulin therapy meal and to collect a specimen and blood glucose test
the 24 hour collection
specimen 2 hours after
eating 5. Be sure to label specimen with patient’s name, date, and time the collection began
Specimen collected under sterile conditions by passing a and ended
Catheterized Bacterial culture
catheter through urethra into the bladder 6. Upon arrival of the specimen to the laboratory, the entire 24 hour specimen is
When cleansing is done, thoroughly mixed and the volume is accurately measured and recorded
patient voids first into 7. An aliquot is saved for testing and additional or repeat testing. Discard remaining
the toilet, then collect an Provide specimen that is less
Mid-stream Bacterial culture urine
adequate amount of contaminated by epithelial cells
clean catch Routine urinalysis
urine in the sterile and bacteria
container and finish
voiding into the toilet
Cytology
Suprapubic Provides sample for bacterial culture that is completely free of
Bladder urine for
aspiration extraneous contamination (can culture mycobacteria)
bacterial culture
 Soft clear plastic bags with adhesive to attach to the
Pediatric genitalia area of both boys and girls
Routine or culture
specimen  Care must be taken not to touch the inside the bag when
applying it
Drug specimen Drug testing Chain of custody (COC) is the process that can provide
 D. Acid-Base Balance
II. Clinical Chemistry
i. Anticoagulant : sodium heparin (100 units/mL) [excess heparin cause false
A. Carbohydrates
decrease of pH]
i. Whole blood, serum, pkasma, CSF, serous fluids and urine can be used as samples
ii. Must use anaerobic collections for pH and blood gas studies
ii. Standard clinical specimen is venous plasma glucose
iii. STAT
iii. Fasting blood sugar should be obtained after 10 hours of fasting (at least 8 hours)
iv. Must be placed in iced water or ice bath(ice cubes can cause hemolysis)
iv. A serum specimen is appropriate for glucose analysis if serum is separated from
v. Seal specimen with rubber stopper
the cells within 30 minutes (Bishop) or within 1 hour (Henry’s)
vi. Don’t use vacutainer tubes
v. Glucose is metabolized at room temperature at the rate of 7mg/dL/hour
vi. At 4oC, glucose decrease by approx. 2mg/dL/hour pH pO2 pCO2
vii. 2 mg (NaF) /mL (whole blood) prevents glycolysis for 48 hours Blood is exposed to air Increase Increase Decrease
viii. Fluoride binds to Mg which causes inhibition of enolase Increase
Sealed specimen was left Decrease
ix. CSF glucose concentration is approximately 60-70% that of the plasma → due to waste
at room temp. for a long Decrease → due to
concentration products of
period of time glycolysis
x. NO to hemolysis glycolysis
Bubbles in the syringe Increase Increase Decrease
B. Glycosylated Hemoglobin
→ For every 1% increase of HbA1C – 35 mg/dL is added to plasma glucose E. Collection Variables
i. Hemolysis
C. Oral Glucose Tolerance Test (Guidelines)  If present when the serum or plasma layer is pink
1. Patient is asked to consume a normal to high carbohydrate intake (150g/day) for  Hemolysis can falsely INCREASE blood constituents such as
3 days prior to the test o Potassium, Magnesium, Iron, LDH, Phosphorus, Ammonium and
2. Patient is asked to fast overnight and to avoid excessive physical activity Total protein
Patent should fast at least 8-10 hours but not greater than 16 hours ii. Hemoconcentration or Hemodilution
3. OGT testing should be performed on the morning to prevent hormonal diurnal  To avoid problems with hemoconcentration and hemodilution, the patient
effect on glucose should be seated in a supine position for 15-20 minutes before the blood is
4. Patient should be ambulatory drawn
5. Patient should refrain from exercise, eating, or drinking (except water) and  Extended application of the tourniquet can cause hemoconcentration,
smoking which increases the concentration of analytes and cellular components
6. Fasting blood glucose is measured before giving the glucose load ; a fasting iii. Proper Anticoagulation
glucose higher than 140 mg/dL, the test must be terminated ; fasting glucose  When blood collection tubes that contain various anticoagulants or
must be less than of 140 mg/dL additives are used
7. Glucose load should be given to the patient  It is important to follow the proper order of draw and to thoroughly mix
8. Glucose load for an adult : 75 g an anticoagulated tube of blood after it has been filled
Children receive 1.75 g per kg of body weight, maximum of 75 g  Failure to mix a tube containing an anticoagulant will result in failure to
anticoagulate the entire blood specimen, and small clots may be formed.
9. Blood glucose is taken every 30 minutes for 2 hours
  Can cause erroneous cell counts  Improper blood collection tube specimen
 If a clot is present, it may interfere with automated analyzers.  Short draws, wrong volume  Contaminated specimen or
 It is very important that the proper anticoagulant be used for the test leaking container
ordered
iv. Icteric or Lipemic Serum F. Common Test Status Determination (Action Importance : STAT  Timed  Fasting  Routine)
 Lipemia occurs when serum triglyceride levels exceed 4.6 mmol/L or 400
mg/dL
STATUS MEANING COLLECTION CONDITIONS PRIORITY
 Artifactually induced values in some laboratory determinations result
Immediately collect, test and report result
when triglyceride levels are elevated (turbidity) on the basis of STAT Immediately (Statim) Alert lab staff when delivered First
absorbance of light of various lipid particles ER stats have priority over other stats
 Inhibition of assays : amylase, bilirubin, creatine kinase, total protein, Collect at a specific Collect as close as possible requested time
Timed Record actual time collected Second
urates, and urea may be observed time
o To correct for artifactual absorbance readings, “blanking Second or Third
procedures and dual-wavelength methods may be used ASAP As soon as possible Follow hospital protocol for type of test depending on
test
o Blanking procedures : the blank contains serum but lacks a crucial
No food or drink
element to complete the assay Fasting Verify patient has fasted Fourth
except water
o A blanking process may not be effective in some cases of Nothing by mouth Do not give patient water or food
turbidityh, and ultracentrifugation may be necessary to clear the NPO Refer requests to nurse/physician N/A
(nulla per os)
serum or plasma of chylomicrons Pre-op Before an operation Collect before patient goes to surgery Same as ASAP
v. Order of Draw Post-op After an operation Collect when patient is out of surgery Same as ASAP
1. Blood culture tubes (yellow) Relating to establish Collect in timely manner but no urgency
Routine None
2. Coagulation sodium citrate tube (blue stopper) procedure involved
3. Serum tubes with or without activator or gel separator(red) Specimen Collection and Handling
4. Heparin tubes with or without gel (green)  Proper patient identification is the first step in sample collection
5. Ehylenediamine tetra acetic acid tubes (lavender) Patient Identification Procedure
1. Conscious inpatients or Hospitalize patients
6. Glycolytic inhibitors (gray)
a. Verbally ask their full names
vi. Note For Venipuncture b. Verify the name using identification bracelets which includes :
 Routine gauge needle : 19, 20, 21
 Length of the needle : 1.0-1..5 inch → First and last name → Room or bed number
 Angle : 15o → Hospital number → Physician’s name
2. Sleeping patients
 Two attempts allowed
a. Identify in same manner as conscious in-patients
 Tourniquet placement : 2-3 inches or 7.5-10 cm above
b. Must be awakened before blood collection
vii. Reasons Specimen Rejection
3. Unconsious, Mentally incompetent patients
 Hemolysis or lipemia  Improper transport conditions a. Identigy by asking the attending nurse or relative
 Clots present in an (ice for blood gases) b. ID bracelet
anticoagulated specimen  Discrepancies between 4. Infants and Children
 Non-fasting specimen when requisition and specimen label a. A nurse or a relative may identify the patient
test requires fasting  Unlabelled or mislabelled b. By means of ID bracelet
 5. Outpatient/Ambulatory iii. Cause of thick smear
a. Verbally ask their full names ; address or birth date and countercheck with driver’s  Large blood drop
license or ID card with photo  High angle
b. If the patient has ID card or bracelet, same manner as with hospitalized patient  Fast speed in spreading
III. Hematology iv. Need of buffy coat smear
A. Coagulation Testing  When patient’s WBC count <1 x 109/L
i. Non-traumatic venipuncture  Demonstration of LE cell
 Hemolyzed sample should not be used for platelet aggregation studies v. Thick blood smear : presence of blood parasites
 Because RBC’s contain ADP
ii. Order of draw C. Staining
 Sodium citrate (3.8 or 3.2% - is recommended)  Romanowsky stain : contains methylene blue (or Azure B) and eosin
 Calcium is necessary for both coagulation studies and platelet  Fixative : methanol
aggregation studies  For best result : blood smears should be stained at 2-3 hours of
 Coagulation samples should NOT be drawn first when using evacuated specimen collection
tubes because of contamination with tissue thromboplastin (activates  pH 6.8 for blood and bone marrow staining
coagulation) as the needle pierces the skin  pH 7.2 for malarial parasites
 If only test ordered is coagulation study, draw small amount in a tube that D. Staining Problems
is discarded i. Excessively Blue stains
iii. Use of plastic or silicone-coated glass tubes → Thick films
 Glass activates factor XII → Prolonged staining time
 Platelets will adhere to glass → Inadequate washing
iv. Ratio of blood to anticoagulant (9:1) → Too high of alkalinity of stain or diluents tends to cause basophilia
v. Specimen processing ii. Excessively Pink Stains
 Recommendations include processing within → Insufficient staining
o 4 hours for APTT at 4 oC → Prolonged washing time
o 24 hours for PT → Too high of acidity of the stain or buffer
vi. Temperature : testing must be performed at 37 oC
iii. Other Staining problems
 Labile Factors V and VII will breakdown at temperatures above 37 oC
→ Inadequately stained red cells, nuclei, or eosinophilic granules may be due
 Factors Vii and XI will be activated at cold temperatures
to under-staining or excessive washing
vii. Lipemic smaples may cause problems with coagulation and aggregation studies
o Prolonging the staining or reducing the washing may solve the
 may obscure changes in optical density
problem
B. Blood Smear Preparation
→ Precipitate on the film may be due to
i. Blood drop : 2-3 mm
o Unclean slides
 0.25 inch away from the slide end
o Especially failure to hold the slide horizontally during initial
 0.5 inch from the other end
ii. Cause of thin smear washing
 Small blood drop o Inadequate filtration of the stain
 Low angle o Permitting dust to settle on the slide or smear
 Slow speed in spreading
IV. Histopatholgy  Actual tissue sample or fluid aspirate is always superior to a swab specimen for
 Fix specimen first the recovery of pathogenic organisms
 Complete labelling (proper patient identification, and type of specimen)  Use required collection and transport materials to preserve specimen integrity
 Use the appropriate solutions, reagents and stains for tissue processing
V. IS/BB
 Pro-zone : excess antibody C. Specimen collection
 Post-zone : excess antigen  Specimens should be obtained from site of infection with minimal contamination
from adjacent tissue and organ secretion
 Physiologic variations  All specimens should be collected in a sterile container (except stool) and
 Cyclic variations : changes in analyte concentration occur at different times during labelled with
the day, week or month 1. Name 3. Source of specimen
 Diurinal variations : variations according to sleeping and waking times (iron and 2. Hospital ID number 4. Time of collection
cortisol)  Communicate clear orders and source of information
 Expedite the transport of specimens to the laboratory and do not allow them to
→ ACP → Insulin sit in collection areas
→ ACTH → Iron
→ Aldosterone → Thyroxine D. Specimen storage, labelling and requisition
→ Cortisol → prolactin  Specimen suspected of containing anaerobic bacteria should never be stored in
→ Growth hormone the refrigerator
 Circardian variations : occurs during 24 hour period  Urine, stool, viral specimens, sputa, swabs, and foreign device such as catheters
 Circaannual variation : occurs twice a year, related to seasonal changes in climate should be stored at 4oC
and diet  Serum for serologic studies
 Elevated in the summer, decrease in the winter o May be frozen for up to 1 week at -20 oC
 Tissues or specimen for long term storage
VI. Microbiology o Should be frozen at -70 oC
A. General concepts for specimen collection, handling and transport  The specimen or test requisition is an order form that is sent to the laboratory
 Collect specimens from site of infection before initiating therapy along with the specimen
 Collect adequate volume of sample for testing required  (requisition may be in the form of paper or electronically sent)
 For all cultures, tissue, fluid, or aspirate material is always superior to a swab
specimen E. Specimen storage
 Some specimens that will not be transported or processed immediately can be
B. Specimen volume maintained by being stored under certain conditions
 Volume should be adequate  Important : best storage environment for each type
 If swab is not used : polyester-tipped swab on plastic shaft is acceptable
 Swabs are not optimal for detection of anaerobes, mycobacteria, or fungi and Refrigerate Room temperature
they should not be used when these organisms are suspected :
o Cotton tip swabs are toxic for N. gonorrheae
o Wooden shafts are toxic to Chlamydia trachomatis
 i. Catheter tips (IV) a. Abscess, Lesion, Wound → Anaerobes : usually can’t grow in presence of oxygen and must use the
ii. CSF for virus b. CSF for bacteria atmosphere of anaerobe jars, bags or chambers is composed of :
iii. Ear : outer c. Ear : inner  5-10% hydrogen  80-90% nitrogen
iv. Feces for C. Difficile toxin d. Feces (preserved)  5-10% carbon dioxide  0% oxygen
(up to 3 days) e. Genital → Capnophiles : requires concentration of (5-10% carbon dioxide) and (15%
(>3 days stored at -70oC) f. Nasal, N/P, throat oxygen)
v. Sputum g. Tissue → Microaerophiles : grows under reduced oxygen (5-10%) and increased carbon
vi. Urine (unpreserved h. Urine (preserved) dioxide (8-10%)

F. Specimen priority I. Criteria for specimen rejection

i. Any specimen received in formalin
Leve Description Specimens ii. 24 hour sputum collections
l iii. Specimens placed in container with leaks
1 Critical or invasive Amniotic fluid, Pericardial fluid iv. Out-date or dried out specimens
Blood, brain, CSF & Heart valves v. Specimens contaminated with barium, chemical dyes, or oily chemicals
2 Unpreserved Body fluids (not listed for level 1) vi. Specimens from tip of Foley catheters
Bone, wound drainage vii. Duplicate specimen (except blood) received in 24 hour period
Feces, sputum, and tissue viii. Info on the label doesn’t match the info on the request slip
3 Quantitation required Catheter tip ix. Quantity Not Sufficient (QNS)
Urine x. One swab for multiple specimens
4 Preserved Feces in preservative
Urine in preservative
J. Specimens should be rejected for anaerobic culture
Swabs in holding medium
i. Gastric washings
→ (aerobic and non-aerobic)
ii. Urine other than suprapubic aspirate
iii. Stool (except for recovery of clostridium difficile)
G. Specimen preparation
iv. Oropharyngeal specimens except deep tissue samples obtained during a surgical
i. Many specimens require some form of initial treatment before inoculation onto
procedure
primary plating media. Such procedures include:
v. Sputum
 Homogenization (grinding) of tissue)
vi. Swabs for ileostomy or coloscopy sites
 Concentration (centrifugation or filtration) of large volumes of body fluids
vii. Superficial skin specimens
Decontamination of specimens for mycobacteria or legionellae
→ For fungal exams (KOH preparation)

H. Incubation conditions
i. 28oC for fungi
ii. 35-37oC for most bacteria, virus and mycobacteria
iii. A number of different environmental conditions exist
→ Aerobes : grows in ambient air, which contains (21% oxygen and 0.03% carbon
dioxide)
 - The fluorescein detection is considered to be a sensitive test for the detection of liquorrhea
Glucose
→ In order to rule out false positive results due to possible blood-admixture, a determination
of hemoglobin should be conducted (using test strips)

Tau Protein
→ Differentiates CSF leakage from nasal secretions?
→ Immunofixation electrophoresis
→ Migrates slow in the beta zone next to unmodified transferring
→ Migrates together with transferring
Cerebrospinal Fluid or Nasal Secretion Findings used in the differentiation between CSF and nasal secretion
- An abnormal connection between the spaces containing CSF and the exterior, usually
occurring in the region of the nose and less commonly in the ears is referred to as CSF Investigation Cerebrospinal Fluid Nasal Secretion
fistula
- Drainage of CSF is called liquorrhea Detectable
False negative result : 2 out of 88
Differentiation between CSF and Nasal secretion Β2-transferrin Not detectable
patients, possibly absence of liquorrhea
→ If liquorrhea is suspected, an immunochemical test for detection of β2-transferrin should be at the time of sampling
conducted
o The immunoblotting assay is well suited for this purpose Calcium 1.05-1.35 mmol/L 1.0-1.75 mmol/L
→ Total protein and glucose determinations are unreliable for the differentiation
→ Quantitative glucose determinations does not provide unequivocal result since glucose is Mainly: lymphocytes (adults) & Mainly neutrophils
Cells monocytes (children) Normally 6% eosinophils
detected in a large percentage of non-CSF containing nasal secretions from healthy people
Up to 6% neutrophils → >6% in allegic rhinitis
as well as in patients with allergic or infectious rhinitis
→ Glucose concentrations ≥ 30 mg/dL are considered to prove the presence of cerebrospinal Fluoresecin Positive → especially suited to detect
Negative
fluid detection subclinical liquorrhea
β2-transferrin Total protein 0.2-0.5 g/dL 3-40 g/L
- In the immunoblotting assay, CSF and serum from the patient are electrophoretically
separated on an agarose gel plate followed by detection using specific transferring 46-86 mg/dL (2.7-4.8 mmol/L)
Up to 10 mg/dL
antibodies Glucose Always > 30 mg/dL (1.7 mmol/L)
(0.6 mmol/L)
60-70% of the blood glucose conc.
- Serum shows a homogenous β1-transferrin band
- CSF shows β1 and β2-transferrin band Potassium 3 mmol/L 17 mmol/L
- Therefore, CSF is not only detectable but it can also be differentiated from nasal or wound
secretion, tear fluid or saliva Sodium 141 mmol/L 90-148 mmol/L
Fluorescein Detection
- Intrathecal injection of 2 mL of 5% sodium fluorescein into the lumbar region and placement Three important analytes to detect CSF
of three Merocel sponges into each nasal cavity for an overnight period → β2-transferrin
- The detection of Liquorrhea is accomplished in agarose gel electrophoresis, the detection → fluorescent detection
of fluorescein labelled proteins is subsequently performed by fluorometric scanning → Tau protein
  Pancreatic products : fluid secretion, bicarbonate, a-amylase, lipase,
trypsin, and chymotrypsin
o As part of functional diagnostic test, duodenal juices is mostly employed in
secretin-pancreozymin test for the assessment of EXOCRINE pancreatic capacity

Tear Fluid
- It is collected by : Sweat
o Glass capillary from the lower lacrimal sac - It is a liquid secreted by the sweat glands of the skin
o Cellulose sponge which is placed into the lower conjuctival sac - Used for the determination of cystic fibrosis
- Investigations for the Characterization of Tear Fluid Quantities in the sweat

Investigation Comment Investigation Reference Interval

IgE 0.9-370 kU/L (median 65 kU/L) Chloride Approx. 30 mmol/L
Creatinine Approx. 0.5 mg/dL
in the case of atopic conjunctivitis
Lysozyme 0.10 ± 0.02 mg/L
LDH  Detectable in healthy people collected following mechanical
0.02 ± 0.007 mg/L
stimulation of the cornea and conjunctiva
In individuals with renal insufficiency : 0.10 ± 0.055 mg/L
 Minimal injuries of cornea result in the release of LD a1-microglobulin
→ Close correlation with the excretory renal
 Release also occurs in herpetic keratoconjunctivitis
function
Lysozyme Useful in the topographic diagnosis of facial paralysis
b2-microglobulin 0.005 ± 0.001 mg/L
Transferrin  Used as a marker for the detection of inflammation pH 6.3 ± 0.3
 The tear/serum ratio is 0.002 in the healthy people Potassium Approx. 7.5 mmol/L
 0.046 in patients with verneal conjunctivitis 1-2 Liters per day
Volume
Total Protein Reference interval 4.6-6.9 g/L 172 ± 47 mL per hour

Duodenal Juice - The determination of serum trypsin for the detection of cystic fibrosis renders the
- Duodenal juice is obtained by means of a duodenal tube, in the secretin-pancreozymin test determination of electrolytes in the sweat, after pilocarpine stimulation, dispensable to a
- For example : a three line tube is used for separating collecting duodenal juice and gastric large extent
juice - Pilocarpine iontophoresis involves the investigation of 100 uL of sweat for its sodium and
- Diagnostic investigations chloride concentration after stimulation with pilocarpine
o Duodenal juice is examined for :
 Pathogens : Giardia lamblia
Pilocarpine Normal Abnormal
 Biliary components : bilirubin and cholesterol
iontophoresis
 Chloride (mmol/L) <50 ≥70
Sodium (mmol/L) <40 ≥50

Amniotic Fluid or Urine

- Amniotic fluid and urine often do not differ in their appearance Serum or Urine
- The following examinations can be employed in differentiation : - Serum can be differentiated from urine on the basis of comparison between concentrations
o Microscopy of various components
o Paper test strips - The following components are particularly suited : PHOSPHATE, CREATININE, PROTEIN,
o Quantitative determination of UREA and POTASSIUM GLUCOSE, and UREA
- Microscopy - The Creatinine conc. in the urine is on average 170x higher than serum
o A few drops of amniotic fluid are applied to clean slide and air-dried and no cover - Protein conc. in the serum is approximately 1500x higher than in urine
glass is used Concentrations of various quantities in the serum and urine
o The examination is performed using 100 fold magnification (HPO)
 A crystalline arborisation pattern confirms the presence of amniotic fluid Quantity Serum Urine
 The test is positive in 96% of amniotic fluid samples  (1) 0.55-1.1 mg/dL 36-130 mg/dL
 An arborisation pattern does not occur in the urine Creatinine 49-97 umol/L 3182-11492 umol/L
Glucose 75-115 mg/dL ≤ 15 mg/dL
- Paper Test Strips
4.16-6.38 mmol/L 0.84 mmol/L
o The detection of GLUCOSE and PROTEINS suggests the presence of amniotic fluid
Phosphate 2.6-4.5 mg/dL 22-75 mg/ddL
o Method is not very reliable since both substance may detectable by dipstick
0.84-1/45 mmol/L 7-24 mmol/L
testing in the urine as well, if diabetes mellitus and renal disease is present
 Total
Comparison between Amniotic Fluid and Urine 66-87 g/L ≤ 0.15 g/L
Protein
 (2) Urea 10-50 mg/dL 1500-2600 mg/dL
Amniotic Fluid
1.75-8.3 mmol/L 250-433 mmol/L
Examination Up to the 20th After the 20th Urine
week of GA week of GA

Urea 12-50 mg/dL 10-120 mg/dL 926-2103 mg/dL

2.0-8.3 mmol/L 1.7-20 mmol/L 154-350 mmol/L

Potassium 3.3-4.6 mmol/L 2.9-5.6 mmol/L 48 mmol/L

 Elasticity The ability to stretch out a drop of mucus in the form of a thread is a
measure of the elasticity of the cervical mucus
 0 : <1 cm
 1 : 1-4 cm
 2 : 5-8 cm
 3 : <9 cm
pH 7.0 – 8.5
The pH is measured by using pH indicator paper
Values outside optimal interval may impair the motility and/or
survival of spermatozoa

 Non-fertile : dots and some lines start to appear

 Transitional : some fern patterns start to appear
Cervical Mucus  Fertile : a lot of ferning pattern
- Cervical mucus is a secretion released by pancreatic cervical glands
- Its consistency and quantity are subject to hormone fluctuations during menstrual cycle
- It is collected by using, for example, a needless syringe on the 12 th – 15th day.
- If the investigations of the secretions are not immediately assessed, the sample can be
stored in the syringe or in another container at 4oC for up to 5 days
- The characteristic of cervical mucus, it can be evaluated by using a score system. The best
scoring is 15.
- A score of > 10 indicates good cervical mucus quality, compatible with sperm penetration
- A score of <10 are indicative of unfavourable mucus conditions
Investigations for the Characterization of cervical mucus

Investigations Assessment
Volume Normally about 0.5 mL during the middle of the cycle
Scores
 0 = 0.0 mL ▪ 2 = 0.2 mL
 1 = 0.1 mL ▪ 3 = ≥0.3 mL
Consistency Scores
 0 : thick
 1 : moderately cervical mucus
 2 : slightly tenacious
 3 : normal mucus (preovulatory)
Ferning Cervical mucus which has been air-dried on a slide : 100 fold
magnification (HPO)
 0 : no crystallization
 1 : atypical ferning
 2 : main branch with side branches
 3 : complete fern like pattern (3-4 fold branching)