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
→ Patient variables include physical activity, diet, age, sex circardian variations, that define how all aspects of the work are done
posture, stress, obesity, smoking, and medication o Personal policies
→ Strict adherence to proper technique and site selection can minimize o Standard operating procedures (SOP)
collection variables such as hemolysis, hemoconcentration, clots, and other o Specimen collection guidelines
causes for sample rejection or erroneous results
→ Blood collection containers are color coded based on additive or preservative Quality Control
and each is suitable only for specific tests. Failure to use the proper tubes or → Emphasizes on STAT control procedure and non-STAT check procedure
filling tubes in the wrong sequence can produce erroneous results. o Linearity check
→ Blood, urine, and other body fluid constituents can change during transport o Reagent and standard check
and storage. The extent of these changes varies by analytes (Henry’s Clinical o Temperature monitor
Diagnosis)
Dwell Time
Errors in the Laboratory → Time elapsed from the initiation of the test up to the completion of analysis
Pre-Analytical Turn Around Time
→ Physiologic variable affecting blood samples (fasting, gender, race, stress, → Moment from the receiving of specimen up to release of the result
hormonal changes, and exercise → It is the total amount of time required to procure/obtain a specimen, prepare
→ Collection parameters (diet and position) the specimen, run the test, relate the result.
Analytical Errors → The supervisor should relate to the technologist the need to work with the
→ Systemic and random errors 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
Cerebrospinal Fluid or Nasal Secretion Tau Protein
- An abnormal connection between the spaces containing CSF and the → Differentiates CSF leakage from nasal secretions?
exterior, usually occurring in the region of the nose and less commonly in the → Immunofixation electrophoresis
ears is referred to as CSF fistula → Migrates slow in the beta zone next to unmodified transferring
- Drainage of CSF is called liquorrhea → Migrates together with transferring
Differentiation between CSF and Nasal secretion Findings used in the differentiation between CSF and nasal secretion
→ If liquorrhea is suspected, an immunochemical test for detection of β2-
transferrin should be conducted Investigation Cerebrospinal Fluid Nasal Secretion
o The immunoblotting assay is well suited for this purpose Β2-transferrin Detectable Not detectable
False negative result : 2 out of 88
→ Total protein and glucose determinations are unreliable for the
patients, possibly absence of
differentiation liquorrhea at the time of sampling
→ Quantitative glucose determinations does not provide unequivocal result Calcium 1.05-1.35 mmol/L 1.0-1.75 mmol/L
since glucose is detected in a large percentage of non-CSF containing nasal Cells Mainly: lymphocytes (adults) & Mainly neutrophils
secretions from healthy people as well as in patients with allergic or monocytes (children) Normally 6% eosinophils
infectious rhinitis Up to 6% neutrophils → >6% in allegic rhinitis
→ Glucose concentrations ≥ 30 mg/dL are considered to prove the presence of Fluoresecin Positive → especially suited to Negative
detection detect subclinical liquorrhea
cerebrospinal fluid
Total protein 0.2-0.5 g/dL 3-40 g/L
β2-transferrin
Glucose 46-86 mg/dL (2.7-4.8 mmol/L) Up to 10 mg/dL
- In the immunoblotting assay, CSF and serum from the patient are Always > 30 mg/dL (1.7 mmol/L) (0.6 mmol/L)
electrophoretically separated on an agarose gel plate followed by detection 60-70% of the blood glucose conc.
using specific transferring antibodies Potassium 3 mmol/L 17 mmol/L
- Serum shows a homogenous β1-transferrin band Sodium 141 mmol/L 90-148 mmol/L
- CSF shows β1 and β2-transferrin band
- Therefore, CSF is not only detectable but it can also be differentiated from Three important analytes to detect CSF
nasal or wound secretion, tear fluid or saliva → β2-transferrin
Fluorescein Detection → fluorescent detection
- Intrathecal injection of 2 mL of 5% sodium fluorescein into the lumbar region → Tau protein
and placement of three Merocel sponges into each nasal cavity for an
overnight period
- The detection of Liquorrhea is accomplished in agarose gel electrophoresis,
the detection of fluorescein labelled proteins is subsequently performed by
fluorometric scanning
- 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)
Sweat Serum or Urine
- It is a liquid secreted by the sweat glands of the skin - Serum can be differentiated from urine on the basis of comparison between
- Used for the determination of cystic fibrosis concentrations of various components
Quantities in the sweat - The following components are particularly suited : PHOSPHATE, CREATININE,
PROTEIN, GLUCOSE, and UREA
Investigation Reference Interval - The Creatinine conc. in the urine is on average 170x higher than serum
Chloride Approx. 30 mmol/L - Protein conc. in the serum is approximately 1500x higher than in urine
Creatinine Approx. 0.5 mg/dL
Concentrations of various quantities in the serum and urine
Lysozyme 0.10 ± 0.02 mg/L
0.02 ± 0.007 mg/L
Quantity Serum Urine
a1-microglobulin In individuals with renal insufficiency : 0.10 ± 0.055 mg/L  Creatinine
(1) 0.55-1.1 mg/dL 36-130 mg/dL
→ Close correlation with the excretory renal function 49-97 umol/L 3182-11492 umol/L
b2-microglobulin 0.005 ± 0.001 mg/L Glucose 75-115 mg/dL ≤ 15 mg/dL
pH 6.3 ± 0.3 4.16-6.38 mmol/L 0.84 mmol/L
Potassium Approx. 7.5 mmol/L Phosphate 2.6-4.5 mg/dL 22-75 mg/ddL
1-2 Liters per day
Volume 0.84-1/45 mmol/L 7-24 mmol/L
172 ± 47 mL per hour  Total Protein 66-87 g/L ≤ 0.15 g/L
 (2) Urea 10-50 mg/dL 1500-2600 mg/dL
- The determination of serum trypsin for the detection of cystic fibrosis 1.75-8.3 mmol/L 250-433 mmol/L
renders the determination of electrolytes in the sweat, after pilocarpine
stimulation, dispensable to a large extent Tear Fluid
- Pilocarpine iontophoresis involves the investigation of 100 uL of sweat for its - It is collected by :
sodium and chloride concentration after stimulation with pilocarpine o Glass capillary from the lower lacrimal sac
o Cellulose sponge which is placed into the lower conjuctival sac
Pilocarpine iontophoresis Normal Abnormal - Investigations for the Characterization of Tear Fluid
Chloride (mmol/L) <50 ≥70
Sodium (mmol/L) <40 ≥50 Investigation Comment
IgE 0.9-370 kU/L (median 65 kU/L)
in the case of atopic conjunctivitis
LDH  Detectable in healthy people collected following
mechanical stimulation of the cornea and conjunctiva
 Minimal injuries of cornea result in the release of LD
 Release also occurs in herpetic keratoconjunctivitis
Lysozyme Useful in the topographic diagnosis of facial paralysis
Transferrin  Used as a marker for the detection of inflammation
 The tear/serum ratio is 0.002 in the healthy people
 0.046 in patients with verneal conjunctivitis
Total Protein Reference interval 4.6-6.9 g/L
 Values outside optimal interval may impair the motility
and/or survival of spermatozoa
Cervical Mucus
- Cervical mucus is a secretion released by pancreatic cervical glands  Non-fertile : dots and some lines start to appear
- Its consistency and quantity are subject to hormone fluctuations during  Transitional : some fern patterns start to appear
menstrual cycle  Fertile : a lot of ferning pattern
- 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 Duodenal Juice
sample can be stored in the syringe or in another container at 4 oC for up to 5 - Duodenal juice is obtained by means of a duodenal tube, in the secretin-
days pancreozymin test
- The characteristic of cervical mucus, it can be evaluated by using a score - For example : a three lume tube is used for separating collecting duodenal
system. The best scoring is 15. juice and gastric juice
- A score of > 10 indicates good cervical mucus quality, compatible with sperm - Diagnostic investigations
penetration o Duodenal juice is examined for :
- A score of <10 are indicative of unfavourable mucus conditions  Pathogens : Giardia lamblia
Investigations for the Characterization of cervical mucus  Biliary components : bilirubin and cholesterol
 Pancreatic products : fluid secretion, bicarbonate, a-
Investigations Assessment amylase, lipase, trypsin, and chymotrypsin
Volume Normally about 0.5 mL during the middle of the cycle o As part of functional diagnostic test, duodenal juices is mostly
Scores
employed in secretin-pancreozymin test for the assessment of
 0 = 0.0 mL ▪ 2 = 0.2 mL
EXOCRINE pancreatic capacity
 1 = 0.1 mL ▪ 3 = ≥0.3 mL
Consistency Scores
 0 : thick Amniotic Fluid or Urine
 1 : moderately cervical mucus - Amniotic fluid and urine often do not differ in their appearance
 2 : slightly tenacious - The following examinations can be employed in differentiation :
 3 : normal mucus (preovulatory) o Microscopy
Ferning Cervical mucus which has been air-dried on a slide : 100
o Paper test strips
fold magnification (HPO)
 0 : no crystallization o Quantitative determination of UREA and POTASSIUM
 1 : atypical ferning - Microscopy
 2 : main branch with side branches o A few drops of amniotic fluid are applied to clean slide and air-dried
 3 : complete fern like pattern (3-4 fold branching) and no cover glass is used
Elasticity The ability to stretch out a drop of mucus in the form of a o The examination is performed using 100 fold magnification (HPO)
thread is a measure of the elasticity of the cervical mucus
 A crystalline arborisation pattern confirms the presence of
 0 : <1 cm
 1 : 1-4 cm amniotic fluid
 2 : 5-8 cm  The test is positive in 96% of amniotic fluid samples
 3 : <9 cm  An arborisation pattern does not occur in the urine
pH 7.0 – 8.5 - Paper Test Strips
The pH is measured by using pH indicator paper
 o The detection of GLUCOSE and PROTEINS suggests the presence of
amniotic fluid
o Method is not very reliable since both substance may detectable by
dipstick testing in the urine as well, if diabetes mellitus and renal
disease is present

Comparison between Amniotic Fluid and Urine

Amniotic Fluid
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
Clinical Microscopy
I. Urine Specimens
A. Specimen Evaluation
→ Before one precedes with any examination, the urine specimen must be evaluated in terms of its
acceptability
→ Considerations include
o Proper labelling
o Proper specimen for the requested examination
o Proper preservative
o Visible signs of contamination
o Whether any transportation delays may have caused significany deterioration
→ Each laboratory should have written and enforced guidelines for the acceptable or rejection of specimens
→ If a single specimen is submitted for multiple measurements, bacteriologic examination should be done first
provided that the urine has been properly collected
→ With pediatric patients and persons in acute renal failure, a notation should be made and the measurement
most pertinent to the diagnosis should be performed first
→ For quantitative measurements, timed (12-24 hour) urinary collection is preferred compared to random

B. Specimen Collection
→ In observance of standard precautions, gloves should be worn at all times when in contact with the
specimen
→ Must be collected in clean, dry, leak proof containers.
→ Disposable containers

C. Labelling
→ Three essential minimum labelling requirements include patient’s full name, and the date, and time of
collection
→ Additional information may be required by the institution such as,  as required by the institutional
protocol!

 Identification number  Time when specimen is received

 Patient’s age  Time of specimen
 Location  Possible interfering medications
 Physician’s name  Patient’s clinical information
→ Labels must be attached to the container, not to the lid and should not become detached if container is
refrigerated
o Avoid mix-ups of specimen, labels must have an adhesive that resists moisture and adheres under
refrigeration
→ A requisition form (manual or computerized) must accompany specimens delivered to the laboratory
→ The information on the form must match the information on the specimen label

D. Rejection
→ Improper labelled and collected specimen should be rejected by the laboratory and appropriate personnel
should be notified to collect a new specimen
→ Examples of unacceptable specimen
i. Unlabelled specimen
ii. Non-matching labels and requisition forms
iii. Specimens contaminated with feces or toilet paper
iv. Containers with contaminated exteriors
v. Specimens of insufficient quantity
vi. Specimens incorrectly transported
E. Specimen Handling
→ Changes may occur not only in vivo but also in vitro, thus necessitating correct handling procedures after the
specimen is collected
F. Specimen Integrity
→ Following collection, specimens should be delivered to the lab and tested within 2 hours
→ If delay is anticipated, it should be refrigerated or have an appropriate chemical preservative added
G. Specimen Preservation
→ Most routine used method of preservation is refrigeration at 2-8 oC, which decreases bacterial growth and
metabolism
o Allows specimen to return to room temperature prior in performing chemical testing by reagent
strips
→ When a specimen is to be transported, and refrigeration is impossible, chemical preservatives may be added
o The ideal preservative should be bactericidal, inhibits urease, and preserves formed elements in the
sediment.
→ Three glass collection : determine prostatic infection
→ Glucose tolerance specimens are collected to correspond with blood samples, drawn during GGT
→ 24 hour or timed specimens : quantitative chemical tests
H. Types of Urine Specimen

Types Purpose Considerations Additional Info

Routine screening to
Random detect obvious
abnormalities
 Ideal screening specimen
First morning  Avoid false negative on
(most concentrated) pregnancy test
 Orthostatic protein
Fasting Diabetic screening or
(second morning) monitoring
Diabetic monitoring with
2-hour postprandial
insulin therapy
Catheterized Bacterial culture
Mid-stream clean Bacterial culture
catch Routine urinalysis
Cytology
Suprapubic
Bladder urine for bacterial
aspiration
culture
Pediatric specimen Routine or culture
Drug specimen
collection Drug testing
(timed specimen)
May also produce erroneous result
caused by dietary intake or Ease of collection
physical activities prior to Convenient for patients
collection
Patient instructed to collect
Assurance detection of
specimen immediately upon
chemicals and formed
arising and deliver to the
elements
laboratory within 2 hours
Specimen will not contain any
metabolites from the food Second voided specimen
ingested prior to beginning of after a period of fasting
fasting period
Patient instructed to void shortly
Result compared with
before consuming a routine meal
fasting specimen and
and to collect a specimen 2 hours
blood glucose test
after eating
Specimen collected under sterile conditions by passing a
catheter through urethra into the bladder
When cleansing is done, patient
Provide specimen that is
voids first into the toilet, then
less contaminated by
collect an adequate amount of
epithelial cells and
urine in the sterile container and
bacteria
finish voiding into the toilet
Provides sample for bacterial culture that is completely free of
extraneous contamination (can culture mycobacteria)
 Soft clear plastic bags with adhesive to attach to the
genitalia area of both boys and girls
 Care must be taken not to touch the inside the bag when
applying it
Chain of custody (COC) is the process that can provide
documentation of proper sample identification from the time of
collection to the receipt of lab results
I. Urine Cytology
i. Obtain a first morning specimen
ii. Submit the specimen to the lab within 30 minutes of collection
iii. Avoid touching the insides of the container and/or the lid in order to maintain sterility

J. 24 Hour Urine Specimens

→ The collection container should be kept cool throughout the collection period
→ It may be placed on the refrigerator or in a pan with ice during collection
Procedure
1. Upon arising in the morning, patient voids to empty his bladder completely and discards the specimen
Note the exact time and print it on the container label.
2. Collect all urine voided for 24 hours after the time in the container provided. All urine passed during the 24 hour
time period (day and night) must be saved. Urine passed during bowel movements must be also collected.
Be careful not to contaminate urine specimen with feces
3. Refrigerate the collected urine between all voiding or keep in a cool place
4. At exactly the same time the following morning, void completely again (first time after awakening) an add this
sample to the collection container. This completes the 24 hour collection
5. Be sure to label specimen with patient’s name, date, and time the collection began and ended
6. Upon arrival of the specimen to the laboratory, the entire 24 hour specimen is thoroughly mixed and the
volume is accurately measured and recorded
7. An aliquot is saved for testing and additional or repeat testing. Discard remaining urine
II. Clinical Chemistry
A. Carbohydrates
i. Whole blood, serum, pkasma, CSF, serous fluids and urine can be used as samples
ii. Standard clinical specimen is venous plasma glucose
iii. Fasting blood sugar should be obtained after 10 hours of fasting (at least 8 hours)
iv. A serum specimen is appropriate for glucose analysis if serum is separated from the cells within 30 minutes
(Bishop) or within 1 hour (Henry’s)
v. Glucose is metabolized at room temperature at the rate of 7mg/dL/hour
vi. At 4oC, glucose decrease by approx. 2mg/dL/hour
vii. 2 mg (NaF) /mL (whole blood) prevents glycolysis for 48 hours
viii. Fluoride binds to Mg which causes inhibition of enolase
ix. CSF glucose concentration is approximately 60-70% that of the plasma concentration
x. NO to hemolysis

B. Glycosylated Hemoglobin
→ For every 1% increase of HbA1C – 35 mg/dL is added to plasma glucose

C. Oral Glucose Tolerance Test (Guidelines)

1. Patient is asked to consume a normal to high carbohydrate intake (150g/day) for 3 days prior to the test
2. Patient is asked to fast overnight and to avoid excessive physical activity
Patent should fast at least 8-10 hours but not greater than 16 hours
3. OGT testing should be performed on the morning to prevent hormonal diurnal effect on glucose
4. Patient should be ambulatory
5. Patient should refrain from exercise, eating, or drinking (except water) and smoking
6. Fasting blood glucose is measured before giving the glucose load ; a fasting glucose higher than 140 mg/dL,
the test must be terminated ; fasting glucose must be less than of 140 mg/dL
7. Glucose load should be given to the patient
8. Glucose load for an adult : 75 g
Children receive 1.75 g per kg of body weight, maximum of 75 g
9. Blood glucose is taken every 30 minutes for 2 hours

D. Acid-Base Balance
i. Anticoagulant : sodium heparin (100 units/mL) [excess heparin cause false decrease of pH]
ii. Must use anaerobic collections for pH and blood gas studies
iii. STAT
iv. Must be placed in iced water or ice bath(ice cubes can cause hemolysis)
v. Seal specimen with rubber stopper
vi. Don’t use vacutainer tubes

pH pO2 pCO2
Blood is exposed to air Increase Increase Decrease
Sealed specimen was left at Decrease Increase
room temp. for a long period of Decrease → due to → due to waste
time glycolysis products of glycolysis
Bubbles in the syringe Increase Increase Decrease
E. Collection Variables
i. Hemolysis
 If present when the serum or plasma layer is pink
 Hemolysis can falsely INCREASE blood constituents such as
o Potassium, Magnesium, Iron, LDH, Phosphorus, Ammonium and Total protein
ii. Hemoconcentration or Hemodilution
 To avoid problems with hemoconcentration and hemodilution, the patient should be seated in a
supine position for 15-20 minutes before the blood is drawn
 Extended application of the tourniquet can cause hemoconcentration, which increases the
concentration of analytes and cellular components
iii. Proper Anticoagulation
 When blood collection tubes that contain various anticoagulants or additives are used
 It is important to follow the proper order of draw and to thoroughly mix an anticoagulated tube of
blood after it has been filled
 Failure to mix a tube containing an anticoagulant will result in failure to anticoagulate the entire blood
specimen, and small clots may be formed.
 Can cause erroneous cell counts
 If a clot is present, it may interfere with automated analyzers.
 It is very important that the proper anticoagulant be used for the test ordered
iv. Icteric or Lipemic Serum
 Lipemia occurs when serum triglyceride levels exceed 4.6 mmol/L or 400 mg/dL
 Artifactually induced values in some laboratory determinations result when triglyceride levels are
elevated (turbidity) on the basis of absorbance of light of various lipid particles
 Inhibition of assays : amylase, bilirubin, creatine kinase, total protein, urates, and urea may be
observed
o To correct for artifactual absorbance readings, “blanking procedures and dual-wavelength
methods may be used
o Blanking procedures : the blank contains serum but lacks a crucial element to complete the
assay
o A blanking process may not be effective in some cases of turbidityh, and ultracentrifugation
may be necessary to clear the serum or plasma of chylomicrons
v. Order of Draw
1. Blood culture tubes (yellow)
2. Coagulation sodium citrate tube (blue stopper)
3. Serum tubes with or without activator or gel separator(red)
4. Heparin tubes with or without gel (green)
5. Ehylenediamine tetra acetic acid tubes (lavender)
6. Glycolytic inhibitors (gray)
vi. Note For Venipuncture
 Routine gauge needle : 19, 20, 21
 Length of the needle : 1.0-1..5 inch
 Angle : 15o
 Two attempts allowed
 Tourniquet placement : 2-3 inches or 7.5-10 cm above
vii. Reasons Specimen Rejection

 Hemolysis or lipemia  Improper transport conditions (ice for blood

 Clots present in an anticoagulated specimen gases)
 Non-fasting specimen when test requires  Discrepancies between requisition and
 fasting specimen label
 Improper blood collection tube  Unlabelled or mislabelled specimen
 Short draws, wrong volume  Contaminated specimen or leaking container

F. Common Test Status Determination (Action Importance : STAT  Timed  Fasting  Routine)

STATUS MEANING COLLECTION CONDITIONS PRIORITY

Immediately collect, test and report result
STAT Immediately (Statim) Alert lab staff when delivered First
ER stats have priority over other stats
Collect as close as possible requested time
Timed Collect at a specific time Second
Record actual time collected
Second or Third
ASAP As soon as possible Follow hospital protocol for type of test
depending on test
No food or drink
Fasting Verify patient has fasted Fourth
except water
Nothing by mouth Do not give patient water or food
NPO N/A
(nulla per os) Refer requests to nurse/physician
Pre-op Before an operation Collect before patient goes to surgery Same as ASAP
Post-op After an operation Collect when patient is out of surgery Same as ASAP
Relating to establish Collect in timely manner but no urgency
Routine None
procedure involved

Specimen Collection and Handling

 Proper patient identification is the first step in sample collection
Patient Identification Procedure
1. Conscious inpatients or Hospitalize patients
a. Verbally ask their full names
b. Verify the name using identification bracelets which includes :
→ First and last name
→ Hospital number
→ Room or bed number
→ Physician’s name
2. Sleeping patients
a. Identify in same manner as conscious in-patients
b. Must be awakened before blood collection
3. Unconsious, Mentally incompetent patients
a. Identigy by asking the attending nurse or relative
b. ID bracelet
4. Infants and Children
a. A nurse or a relative may identify the patient
b. By means of ID bracelet
5. Outpatient/Ambulatory
a. Verbally ask their full names ; address or birth date and countercheck with driver’s license or ID card with
photo
b. If the patient has ID card or bracelet, same manner as with hospitalized patient
III. Hematology
A. Coagulation Testing
i. Non-traumatic venipuncture
 Hemolyzed sample should not be used for platelet aggregation studies
 Because RBC’s contain ADP
ii. Order of draw
 Sodium citrate (3.8 or 3.2% - is recommended)
 Calcium is necessary for both coagulation studies and platelet aggregation studies
 Coagulation samples should NOT be drawn first when using evacuated tubes because of
contamination with tissue thromboplastin (activates coagulation) as the needle pierces the skin
 If only test ordered is coagulation study, draw small amount in a tube that is discarded
iii. Use of plastic or silicone-coated glass tubes
 Glass activates factor XII
 Platelets will adhere to glass
iv. Ratio of blood to anticoagulant (9:1)
v. Specimen processing
 Recommendations include processing within
o 4 hours for APTT at 4 oC
o 24 hours for PT
vi. Temperature : testing must be performed at 37 oC
 Labile Factors V and VII will breakdown at temperatures above 37 oC
 Factors Vii and XI will be activated at cold temperatures
vii. Lipemic smaples may cause problems with coagulation and aggregation studies
 may obscure changes in optical density

B. Blood Smear Preparation

i. Blood drop : 2-3 mm
 0.25 inch away from the slide end
 0.5 inch from the other end
ii. Cause of thin smear
 Small blood drop
 Low angle
 Slow speed in spreading
iii. Cause of thick smear
 Large blood drop
 High angle
 Fast speed in spreading
iv. Need of buffy coat smear
 When patient’s WBC count <1 x 109/L
 Demonstration of LE cell
v. Thick blood smear : presence of blood parasites

C. Staining
 Romanowsky stain : contains methylene blue (or Azure B) and eosin
 Fixative : methanol
 For best result : blood smears should be stained at 2-3 hours of specimen collection
 pH 6.8 for blood and bone marrow staining
 pH 7.2 for malarial parasites
 D. Staining Problems
i. Excessively Blue stains
→ Thick films
→ Prolonged staining time
→ Inadequate washing
→ Too high of alkalinity of stain or diluents tends to cause basophilia
ii. Excessively Pink Stains
→ Insufficient staining
→ Prolonged washing time
→ Too high of acidity of the stain or buffer
iii. Other Staining problems
→ Inadequately stained red cells, nuclei, or eosinophilic granules may be due to under-staining or
excessive washing
o Prolonging the staining or reducing the washing may solve the problem
→ Precipitate on the film may be due to
o Unclean slides
o Especially failure to hold the slide horizontally during initial washing
o Inadequate filtration of the stain
o Permitting dust to settle on the slide or smear

IV. Histopatholgy
 Fix specimen first
 Complete labelling (proper patient identification, and type of specimen)
 Use the appropriate solutions, reagents and stains for tissue processing

V. IS/BB
 Pro-zone : excess antibody
 Post-zone : excess antigen

 Physiologic variations
 Cyclic variations : changes in analyte concentration occur at different times during the day, week or month
 Diurinal variations : variations according to sleeping and waking times (iron and cortisol)

→ ACP → Insulin
→ ACTH → Iron
→ Aldosterone → Thyroxine
→ Cortisol → prolactin
→ Growth hormone
 Circardian variations : occurs during 24 hour period
 Circaannual variation : occurs twice a year, related to seasonal changes in climate and diet
 Elevated in the summer, decrease in the winter
VI. Microbiology
A. General concepts for specimen collection, handling and transport
 Collect specimens from site of infection before initiating therapy
 Collect adequate volume of sample for testing required
 For all cultures, tissue, fluid, or aspirate material is always superior to a swab specimen

B. Specimen volume
 Volume should be adequate
 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 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
 Actual tissue sample or fluid aspirate is always superior to a swab specimen for the recovery of pathogenic
organisms
 Use required collection and transport materials to preserve specimen integrity

C. Specimen collection
 Specimens should be obtained from site of infection with minimal contamination from adjacent tissue and
organ secretion
 All specimens should be collected in a sterile container (except stool) and labelled with
1. Name 3. Source of specimen
2. Hospital ID number 4. Time of collection
 Communicate clear orders and source of information
 Expedite the transport of specimens to the laboratory and do not allow them to sit in collection areas

D. Specimen storage, labelling and requisition

 Specimen suspected of containing anaerobic bacteria should never be stored in the refrigerator
 Urine, stool, viral specimens, sputa, swabs, and foreign device such as catheters should be stored at 4 oC
 Serum for serologic studies
o May be frozen for up to 1 week at -20 oC
 Tissues or specimen for long term storage
o Should be frozen at -70 oC
 The specimen or test requisition is an order form that is sent to the laboratory along with the specimen
 (requisition may be in the form of paper or electronically sent)

E. Specimen storage
 Some specimens that will not be transported or processed immediately can be maintained by being stored
under certain conditions
 Important : best storage environment for each type

Refrigerate Room temperature

 i. Catheter tips (IV) a. Abscess, Lesion, Wound
ii. CSF for virus b. CSF for bacteria
iii. Ear : outer c. Ear : inner
iv. Feces for C. Difficile toxin d. Feces (preserved)
(up to 3 days) e. Genital
(>3 days stored at -70oC) f. Nasal, N/P, throat
v. Sputum g. Tissue
vi. Urine (unpreserved h. Urine (preserved)

F. Specimen priority

Leve Description Specimens

l
1 Critical or invasive Amniotic fluid, Pericardial fluid
Blood, brain, CSF & Heart valves
2 Unpreserved Body fluids (not listed for level 1)
Bone, wound drainage
Feces, sputum, and tissue
3 Quantitation required Catheter tip
Urine
4 Preserved Feces in preservative
Urine in preservative
Swabs in holding medium
→ (aerobic and non-aerobic)
G. Specimen preparation
i. Many specimens require some form of initial treatment before inoculation onto primary plating media. Such
procedures include:
 Homogenization (grinding) of tissue)
 Concentration (centrifugation or filtration) of large volumes of body fluids
Decontamination of specimens for mycobacteria or legionellae
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)
→ Anaerobes : usually can’t grow in presence of oxygen and must use the atmosphere of anaerobe jars, bags
or chambers is composed of :
 5-10% hydrogen  80-90% nitrogen
 5-10% carbon dioxide  0% oxygen
→ Capnophiles : requires concentration of (5-10% carbon dioxide) and (15% oxygen)
→ Microaerophiles : grows under reduced oxygen (5-10%) and increased carbon dioxide (8-10%)
I. Criteria for specimen rejection
i. Any specimen received in formalin
ii. 24 hour sputum collections
iii. Specimens placed in container with leaks
iv. Out-date or dried out specimens
v. Specimens contaminated with barium, chemical dyes, or oily chemicals
vi. Specimens from tip of Foley catheters
vii. Duplicate specimen (except blood) received in 24 hour period
viii. Info on the label doesn’t match the info on the request slip
 ix. Quantity Not Sufficient (QNS)
x. One swab for multiple specimens
J. Specimens should be rejected for anaerobic culture
i. Gastric washings
ii. Urine other than suprapubic aspirate
iii. Stool (except for recovery of clostridium difficile)
iv. Oropharyngeal specimens except deep tissue samples obtained during a surgical procedure
v. Sputum
vi. Swabs for ileostomy or coloscopy sites
vii. Superficial skin specimens
→ For fungal exams (KOH preparation)