Elisa

1. Diagnostic Challenges in Body
Fluid Analysis
Dr. mehrdad vanaki
DCLS / EMBA
Trainer & consultant of QMS in medical labs

2. Reference
 Urinalysis and body fluids / Susan King Strasinger,
Marjorie Schaub Di Lorenzo.Seventh edition , [2021]
 TIETZ TEXTBOOK OF LABORATORY MEDICINE, SEVENTH
EDITION 2022
 Guideline NCCLS / C49 Analysis of Body Fluids in
Clinical Chemistry; Approved Guideline second
edition 2018

3. ‫مایعات‬ ‫بندی‬ ‫طبقه‬
‫بدن‬
3
‫سلولی‬ ‫خارج‬ ‫مایعات‬
(
1/3
TBW
)
‫سلولی‬ ‫داخل‬ ‫مایعات‬
(
2/3
TBW
)
‫پالسما‬ Interstiti
al fluid
Transcellular
fluid
CSF
‫سروزی‬ ‫مایعات‬
‫صفاق‬ ‫یا‬ ‫ئن‬ِ‫و‬‫پریت‬ ‫مایع‬
‫ی‬
‫پلور‬ ‫مایع‬
‫یا‬ ‫پریکارد‬ ‫مایع‬
‫قلب‬ ‫آبشامه‬
‫سینوویال‬ ‫مایع‬
‫امنیوتیک‬ ‫مایع‬

4. Introduction
 Recognition of diagnostic challenges is a prerequisite
for Standardization and harmonization of Body fluids
Analysis* in a medical laboratory.
 This study explains the challenges regarding the pre-
analytic, analytic, and post analytical variables in body
fluid analysis, and finally, provides guidance on how
deviations might affect final interpretation of test results
and change clinical decisions.
 Body fluids Include CSF, Serous Body Fluid
(Pleural, peritoneal, pericardial), Synovial fluids, etc.

5. pre-analytic challenges
in body fluid analysis
 1- Different approach of medical laboratories
for acceptance and rejection criteria for body
fluid samples, and Concession for acceptance of
invasive body fluid

6. pre-analytic challenges
in body fluid analysis
 2- insufficient knowledge of clinical physician
and nurses about body fluid specimen collection
procedures,such as :
1- distribution in proper anti-Coagulant tubes,
2-proper keeping body fluid samples in clinical wards
3- proper transportation and sample delivery to labs

7. Preanalytical Variables
 In some body fluids, the proper order
of draw is important (the incidence of cellular
contamination from tube to tube is reduced).
 Hemolyzed and clotted specimens are not recommended
 However, circumstances may arise when
it is not possible to acquire another
specimen from a patient.
 These exceptions to standard practice
must be clearly defined in the site’s
Standard Operating Procedures.

8. Specimen Collection
Cerebrospinal Fluid, CSF
 No anticoagulant is necessary,
since spinal fluid does not clot except
occasionally if the puncture is traumatic.

9. Specimen Collection and handling
CSF is collected by lumbar puncture between third, fourth, fifth
lumbar vertebrae. It requires certain precautions and careful technique
to prevent the introduction of infection or the damaging of neural tissue.
CSF usually collected in three sterile tubes
•Label 1 / Tube 1 – used for chemical and serologic test
( tubes are frozen)
•Label 2 / Tube 2 – used for microbiology lab
( room temp.)
•Label 3 / Tube 3 – used for hematology (cell count)
( refrigerated)

10. Specimen Collection
Cerebrospinal Fluid, CSF

11. Specimen collection
 Before antibiotic therapy
Number specimens:
 Collected 3 or more sterile tube
 Ideal :3-4ml (minimum : 1ml)
for TB : 10ml

12. Specimen Transport
Delay of several hours in processing specimen :
Incubating the CSF ( with screw-caps loosened)
at 35° in a 5% co2 ,may improve bacterial survival
If culture is not possible:
Inoculated aseptically into T-I medium with a
syringe & held overnight at 35°

13. Trans-Isolate (T-I) medium

14. Specimen processing CSF for culture and smear
If less than 1 ml of CSF ; should not be centrifuged
directly for culture & gram stain
If >1 ml ,must be centrifuged (1200 xg ,10-15 min)
Supernatant ;for antigen detection
Sediment well mix & prepare for staining & culture
Rapid screening for antigen is not recommend
Unopened sample prefer for PCR

15.  The major Analytical challenge is the lack of competent staff
with sufficient practical knowledge and awareness about
unconformities related to body fluids, e.g. weakness of staff
in differential diagnosis of microorganisms with artifact
elements in gram stain in CSF analysis
 weakness of Education department of medical labs for
planning effective training course,
 obvious deviation between the results of manual and
automated methods, especially in field of cytology of body
fluid analysis are among the main challenges
Analytic challenges
in body fluid analysis

16. CSF turbidity
 WBC >200/μL
 RBC > 400/μL
 RBC >6000/μl bloody CSF.
 Protein>150mg/dl
 Bacteria fungi radiographic substance
16

18. Clues Useful in Differentiating Traumatic Tap
from CNS Hemorrhage
 Traumatic tap demonstrates
 maximum amount of blood in first
sample tube with progressive decrease
in subsequent sample tubes
 the sample from a CNS hemorrhage
demonstrates blood evenly mixed in all
collection tubes
 After CSF sample centrifugation,
 the supernatant from a traumatic tap is
clear
 The supernatant is
“xanthochromic” from a
hemorrhage
 A very bloody tap may demonstrate blood
clots in the CSF sample, while clots are not
usually associated with CNS hemorrhage.
Graphic accessed URL http://www.webmm.ahrq.gov/media/cases/images/case69_fig2.jpg,

19. Analytical challenge for cytology CSF
 Before starting the hematological analysis of the spinal fluid, we
should mix the sample well into the tube, then put a drop between
the slide and the slide check with a 40 lens if the number of white
cells was more than 50, use a diluent if it was less. I can. Let's start
the cell count directly with a homocytometer without preparing a
dilution.
Cell counter should never be used for spinal fluid count.
Routine spinal fluid cell count is a white blood cell count.

20. Microscopic Examinations
 Cell counts
 Total
 Leukocyte
 RBC
 Differential
 Cytology
http://www.neuropat.dote.hu/jpeg/liquor/kmcarc1.jpg

21. Analytical challenge for cytology CSF
 Routine spinal fluid cell count is a white blood cell count.
 Red blood cell count is less important due to traumatic tap.
However, it should be noted that in traumatic tap due to the
entry of leukocytes and proteins into the spinal fluid, the
count should be corrected.
 White and red spinal fluid counts should be done within
one hour after sampling so that there is no false reduction
due to cell lysis. It should be noted that after two hours,
40% of leukocytes are lysed.
 The white blood cell count of normal spinal fluid is less
than 5 white cells per microliter, which is higher in infants
and most of the cells are mononuclear.

22.  Counting with automation system is associated with high
and false counting and is not recommended.
 Spinal fluid transparency is sometimes up to 400 cells per
microliter, so microscopic counting is required regardless of
spinal fluid transparency.
 Manual counting method with modified neobar slide is the
proposed and selected method in spinal fluid cell counting.
 White cell counting formula in microliters ;
 Number of whites in ten squares White count X index
Dilution X index Volume counted (10 * 0.1 equivalent to 1)
Analytical challenge for cytology CSF

23. If the spinal fluid sample is bloody, a number of
white blood cells are added to the spinal fluid.
In this case, with the following formula, we calculate
the number of white blood cells added to the spinal fluid
and subtract the total white count in the spinal fluid.
It should be noted that in order to correct the white
matter count of spinal fluid, we have access to the
simultaneous count of white and red blood cells of the
patient.
Excess white blood cells in spinal fluid = red
blood cells in spinal fluid multiplied by white
blood cells in blood / red blood cells in blood
Correction of spinal fluid white blood cells
in bloody CSF samples

24. general formula for white or total count
in a variety of clinical specimens
(blood / spinal fluid / serous fluids / sperm, etc.)
 White blood cell count formula in microliters of body fluid:
 index of dilution (1) X index of counted volume (1) X
Number of whites in ten squares White and red counts
 index Counted volume = 10 (number of squares counted) multiplied by
0.1 (volume of each white square) = equivalent to 1
 Volume of each square white count = length multiplied by width
multiplied by height . Cubic millimeters 0.1 = 1 x 1 x0.1
 index of dilution If a sample of spinal fluid without dilution is counted
on a homocytometer, it is equal to 1 and is equal to 10 for dilution of one
tenth and equal to twenty for dilution of one twentieth.

25. Cell Counts
 Reference range leukocytes “normal” adult
CSF
0-5 cells/mL
 mononuclears
 RBC count is of limited use, but can be used
to correct CSF leukocyte counts* & CSF
protein values of a traumatic tap CSF.
W* = WBCf - WBCb X RBCf
RBCb
 RBC count is calculated by subtracting the
WBC count from the total cell count.
http://www.tpub.com/content/medical/14295/img/14295_279_1.jpg
This space is one
square mm. On h
e hemacytometer its
depth is 1/10 of
a
mm. For content
of this space,
multiply by 10 to
get content of
one cubic mm
Each of these small
squares is 1/25 mm square (50)
One WBC added for every 700 RBC
8mg/dl protein for every 10.000 RBC

26. Cell Counts
Manual counts
 0 RBC/µL should be seen in normal CSF specimens, regardless
of patient’s age.
 WBC RIs
a) Adult = up to 5 mononuclear WBC /µL
b) Newborn = up to 30 mononuclear WBC /µL
c) Children (1 - 4 yrs) = up to 20 mononuclear WBC /µL
d) Children (5 yrs - puberty) = up to 10 mononuclear
WBC/µL

27. Normal CSF Differential Cell Count

28. Prominent Cells in CSF and Clinical Significance

29. Glucose CSF
 The spinal fluid glucose test method is measured similar to blood sugar, and because
the correct reference value for spinal fluid glucose is 60% blood sugar, a patient's blood
sample should be taken half an hour before the spinal fluid test and the amount of spinal
fluid sugar relative to blood sugar To be measured
 Example: The patient's blood sugar is 500 mg / dl and the spinal fluid glucose is
110 mg / dL (the normal range of spinal fluid glucose for this patient is 60% of
blood sugar, ie about 300 mg / dL Therefore, spinal fluid glucose level 110 shows
a significant decrease in spinal fluid glucose and has a high diagnostic value and
the possibility of bacterial or fungal meningitis can be raised)
 Note: Spinal fluid sugar should be done no later than 0.5 to one hour after collecting
the spinal fluid sample, and too much time is associated with false reduction results,
which is not interpretable for the doctor,
 so emergency spinal fluid glucose is necessary (especially in the sample Blood
vessels that drop in blood sugar very quickly)

31. Increasing IgG INDEX in CSF
 In neurosyphilis
 Guillain-Barré syndrome
 Multiple sclerosis (diagnosed in MS with 90% sensitivity)
 Vascular collagen diseases
31

35. Gram stain CSF challenge
 Immediately and without wasting time, the spinal fluid sample sent to the microbe section
should be centrifuged for 15 minutes at 2000 rpm.
 Note: Working on the spinal fluid specimen in the microbial section has priority over all other
work in the emergency department
 2- Transfer the supernatant to another tube with Pasteur pipette (usable for serology or
biochemistry of spinal fluid) and shake the sediment of the tube.
 3- Put 1 to 2 drops of sediment on two clean slides without spreading the drops (for direct smear
and gram stain) and use the rest of the sediment for culture in chocolate and blood media
(containing sheep blood) and liquid brain broth environment. And transfer the plates to a jar of
carbon dioxide or candle jar. In addition to culturing on a plate and chocolate, a drop of sediment should be
transferred to the liquid broth environment to allow re-passage of the sample.
 To reduce false positives in the report of spinal fluid hot smear, smears should be prepared on
a new smear without contamination (without finger contact).
 Positive and negative gram control slides should also be used
along with gram staining of CSF to ensure the correct performance and quality
of the gram stain used.

36. Bacteria report in gram stain CSF
by classification of cocci and coccobacilli or diplococci
 Observation of gram-negative intracellular and extracellular diplococci of
brown seed = possibility of meningococcal meningitis
 Observation of gram-positive cocobacilli = possibility of listeria
meningitis
 Observation of gram-positive cocci = possibility of bacterial meningitis
caused by group B streptococcus (Agalactia) or Staphylococcus aureus
 Observation of gram-negative bacilli = possibility of meningitis due
to Escherichia coli and other Enterobacteriaceae
 Observation of gram-negative coccobacilli = possibility of
meningitis due to Haemophilus influenzae

37. Post -analytic challenges
in body fluid analysis
The main challenge in post-analytical phase
is lack of standard reference interval for
types of body fluids that may cause
misinterpretation of physician.

38. Important point for
critical and double emergency gram stain CSF reporting
 The spinal fluid sample is one of the vital and emergency
samples of the laboratory and its analysis should be
completed within 1 to 2 hours after collection and the
observed cases (whether positive or negative) should be
reported in writing by phone to the doctor or nurse.
 Be responsible for reporting and the reporting time of the
reporter and the reporter and the recipient of the report and
the text of the report should be recorded in the telephone
reporting table.

39. Synovial Fluid

40. Synovial Fluid
 Collection
- Arthrocentesis

41. Synovial Fluid
- Composition and formation
 Secreted by cells of synovial membrane
 Very viscous, clear ultrafiltrate of plasma
- Contains:
 Hyaluronicacid
 Mucopolysaccharides
 Limited amount of plasma protein
 Glucose & uric acid levels equivalent to plasma

42. Specimen Collection
Synovial Fluid
 The amount of fluid depends on the size of the
joint and effusion.
 A 3- to 5-mL sample is ideal for laboratory
analysis.
 Specimens should not be rejected because of
small volumes, since even a drop may provide
definitive diagnosis in crystalline joint
disease and only small volumes are needed for
cell count and differential.
 lithium heparin and EDTA can be used or not?

43. Specimen Collection
Synovial Fluid

44. Synovial Fluid Lab. Testing
 Physical properties
- Viscosity
- Screening –‘String Test’ drop from pipette
- Evaluates viscosity
- Normal = @ 5+ cm long before breaking
- Rope’s test for mucin clot, Mucin Clot Test
- Measures degree of hyaluronate polymerization
The test is qualitative and involves the addition of
2% acetic acid to synovial fluid.
- Good / normal = tight ropey mass
- Poor = appears friable or fails to form

45. String Test
Mucin Clot Test

46. 46
Specimen Collection, Cont’d

48. Pathologic Classification of
Synovial Fluids

49. Quantitative Assessment
Manual Counting, Procedure
 Mix the specimen well by rotation on an automated mixer:
maximum of 2 to 5 min. (excessive rocking may damage
cells) or hand mix by inverting the tube 10 to 15 times.
 The exception is synovial fluid, which must be mixed for 5 to
10 min due to the viscosity of the fluid.
 If the fluid is in a conical tube, flick the bottom of the conical
tube several times
 The more turbid the sample, the greater the mixing process
impacts cell count accuracy.

50. Specimen Dilutions
 Different diluents,
- Isotonic saline: white and red cell
- acetic acid or hypotonic saline may be used to
lyse red cells for white cell dilutions.
 Acetic acid should not be used as a diluent for
synovial fluid manual nucleated cell counts, since
mucin will coagulate.
 If manual nucleated cell counts are performed on
synovial fluid samples, erythrocytes can be lysed,
with preservation of nucleated cells, by using a
hypotonic saline solution (0.3%).

51. MSU

53. Mono-sodium urate: Intracellular
Intracellular crystals
indicate acute flare

54. CPPD

55. CPPD

56. ‫در‬ ‫موجود‬ ‫ترکیبات‬ ‫مرجع‬ ‫دامنه‬
SF
56

57. serous fluid
plural fluid
peritoneal fluid
pericardial fluid
57

58. 58

59. Serous Body Fluids
Schematic representation of the three body cavities

60. Specimen Collection
and Handling
An ethylenediaminetetraacetic acid (EDTA) tube is used
for cell counts and the differential.
Sterile heparinized or sodium polyanethol sulfonate (SPS)
evacuated tubes are used for microbiology and cytology.
For better recovery of microorganisms and abnormal
cells, concentration of large amounts of fluid is
performed by centrifugation.
Chemistry tests can be run on clotted specimens in plain
tubes or in heparin tubes.
Specimens for pH must be maintained anaerobically in
ice

61. Serous Body Fluids,
Composition & Formation
 Effusion
- an increase in the serous fluid due to
some disruption in production &/ re-
absorption processes.
- Classification of cause of an effusion is
aided by determining if the fluid is a
“transudate” or an “exudate”.

62. Transudate vs. Exudate
 Transudate
 result of a systemic disorder that disrupts the balance of
fluid production / fluid re-absorption.
Examples:
 Pleural transudate–congestive heart failure;
 Pericardial transudate–nephrotic syndrome,
metastatic cancer

63. Transudate vs. Exudate
 Exudates
 term to classify the effusion that is a result of a problem with the
membranes themselves.
 Produced by conditions that directly involve the membranes of the
particular cavity, ex. infections, inflammation, and malignancies
 Thought of as an inflammatory process
 Exudate examples:
 Pleural exudate–carcinoma, pneumonia, trauma
 Pericardial exudate–infection, cardiovascular
disease (CV) trauma, cancer

64. ‫الیتز‬ ‫معیارهای‬
‫اگزودا‬ ‫تران‬
‫ز‬
‫ودا‬ ‫آزمایشگاهی‬ ‫تست‬
‫خونی‬ ‫و‬ ‫کدر‬ ‫شفاف‬
‫کمرنگ‬ ‫زرد‬ ‫ظاهری‬ ‫نظر‬ ‫از‬
‫از‬ ‫بیش‬
3
‫درصد‬ ‫گرم‬ 3
‫کمتر‬ ‫یا‬ ‫درصد‬ ‫گرم‬ ‫تام‬ ‫پروتئین‬
‫از‬ ‫بیش‬
0.5 ‫از‬ ‫کمتر‬
0.5 ‫به‬ ‫مایع‬ ‫پروتئین‬ ‫نسبت‬
‫پروتئین‬
‫سرم‬
‫از‬ ‫بیش‬
0.6 ‫از‬ ‫کمتر‬
0.6 ‫نسبت‬
LD
‫به‬ ‫مایع‬
LD
‫سرم‬
‫از‬ ‫بیش‬
ULN
‫سرم‬
×
0.67 ‫از‬ ‫کمتر‬
ULN
‫سرم‬
×
0.67 LD
‫مایع‬
‫بیش‬
‫از‬
1.015 ‫کمتر‬
‫از‬
1.015 ‫مخصوص‬ ‫وزن‬
64

66. Serous Fluids
 Serous fluids for the cytology laboratory should be
sent as soon as possible.
 If storage is necessary, the specimen should be
refrigerated at 4 °C without a fixative.
 Serous fluids have a high protein content, cellular
detail with Papanicolaou (PAP), H & E, or other stains
will be adequately preserved with refrigeration for
several days.

67. Fecal Leukocytes
 Microscopic screening is performed as a preliminary test to determine whether diarrhea is being
caused by invasive bacterial pathogens, including Salmonella, Shigella, Campylobacter, Yersinia, and
enteroinvasive E. coli .
 Bacteria that cause diarrhea by toxin production, such as Staphylococcus aureus and
Vibrio spp.; viruses; and parasites usually do not cause the appearance of fecal
leukocytes
 Specimens can be examined as wet preparations stained with methylene blue or as
dried smears stained with Wright’s or Gram stain
 In an examination of preparations under high power, as few as three neutrophils per
high-power field can be indicative of an invasive condition
 Methylene Blue Stain for Fecal Leukocytes
 1. Place mucus or a drop of liquid stool on a slide.
 2. Add two drops of Löffler methylene blue.
 3. Mix with a wooden applicator stick.
 4. Allow to stand for 2 to 3 minutes.
 5. Examine for neutrophils under high power.

68. lactoferrin latex agglutination test
for detecting fecal leukocytes
 A lactoferrin latex agglutination test is available
for detecting fecal leukocytes and remains
sensitive in refrigerated and frozen specimens.
The presence of lactoferrin, a component of
granulocyte secondary granules, indicates an
invasive bacterial pathogen

69. Interpretation fecal fat
 Normal specimens may contain fewer than 100 small droplets, less than
4 μm in diameter, per high-power field.
 The same number of droplets measuring 1 to 8 μm is considered slightly
increased,
 whereas 100 droplets measuring 6 to 75 μm is considered
increased and commonly seen in steatorrhea.
 An increased amount of total fat on the second slide with normal fat content on
the first slide is an indication of malabsorption, whereas maldigestion is
indicated by increased neutral fat on the first slide

70. Split Fat Stain
 1. Mix emulsified stool with one drop of 36% acetic acid.
 2. Add two drops of saturated Sudan III.
 3. Mix and apply the cover slip.
 4. Heat gently almost to boiling.
 5. Examine under high power.
 6. Count and measure the orange droplets per highpower
field.

71. Guaiac-Based Fecal Occult Blood Tests
 fecal occult blood testing (FOBT) is necessary. Annual testing for occult blood has a high positive
predictive value for detecting colorectal cancer in the early stages and is recommended by the American
Cancer Society, particularly for people over 50 years of age.
 Guaiac-Based Fecal Occult Blood Tests
 The screening test used most frequently for fecal blood is the guaiac-based test for occult blood (gFOBT) based
on detecting the pseudoperoxidase activity of hemoglobin
 Hemoglobin + H2O2 + (colorless) guaiac psudo peroxidase oxidized guaiac (blue color) + H2O

72.  1. Open the front flap of the guaiac-impregnated filter paper slide.
 2. Using an applicator stick, take a sample from the center of the stool specimen.
 3. Apply a thin smear of stool on the filter paper inside the box marked “A” on the slide.
 4. Using the same applicator stick, take a second sample from a different part of the center of
the stool.
 5. Apply a thin smear of the second sample inside the box marked “B.”
 6. Close the cover of the filter paper slide.
 7. Allow the stool samples to soak into the filter paper slide for 3 to 5 minutes.
 8. Open the back of the slide.
 9. Add two drops of hydrogen peroxidase developer to the boxes marked “A” and “B.”
 10. Read results within 60 seconds. Any trace of blue on or at the edge of the smear is positive
for occult blood.
 11. Add 1 drop of hydrogen peroxidase developer between the positive and negative internal
control.
 12. Read quality control results within 10 seconds. The positive control will appear blue, and no
color will be present in the negative control.
Guaiac-Based Fecal Occult Blood Tests

73. Neutral Fat Stain
 1. Homogenize one part stool with two parts water.
 2. Mix emulsified stool with one drop of 95% ethyl
alcohol on the slide.
 3. Add two drops of saturated Sudan III in 95% ethanol.
 4. Mix and apply the cover slip.
 5. Examine under high power.
 6. Count the orange droplets per high-power field.

74.  False-Positive
 Aspirin and anti-inflammatory medications
 Red meat
 Horseradish
 Raw broccoli, cauliflower, radishes, turnips
 Melons
 Menstrual and hemorrhoid contamination
 False-Negative
 Vitamin C >250 mg/d
 Iron supplements containing vitamin C
 Failure to wait the specified time after sample is applied
 to add the developer reagent

75. Immunochemical Fecal Occult Blood Test
 The immunochemical fecal occult blood test (iFOBT) is
specific for the globin portion of human hemoglobin and uses
 polyclonal antihuman hemoglobin antibodies. Because this method
is specific for human blood in feces, it does not require dietary or
drug restrictions.
 iFOBT is more sensitive to lower GI bleeding that could be an
indicator of colon cancer or other GI disease and can be used for
patients who are taking aspirin and other anti-inflammatory
medications. The iFOBT tests do not detect bleeding from other sources,
such as a bleeding ulcer, thus decreasing the chance for false-positive
reactions. Hemoglobin from upper GI bleeding is degraded by
bacterial and digestive enzymes before reaching the large intestine
and is mmunochemically nonreactive.

76. APT Test (Fetal Hemoglobin)
 Grossly bloody stools and vomitus are seen sometimes in neonates as the
result of swallowing maternal blood during delivery.
 Should it be necessary to distinguish between the presence of fetal
blood or maternal blood in an infant’s stool or vomitus, the APT test
may be requested
 The material to be tested is emulsified in water to release hemoglobin (Hb) and, after
centrifugation, 1% sodium hydroxide is added to the pink hemoglobin-containing
supernatant. In the presence of alkali-resistant fetal hemoglobin, the solution remains
pink (HbF), whereas denaturation of the maternal hemoglobin (HbA) produces a
yellow-brown supernatant
 after standing for 2 minutes. The APT test distinguishes between not only
HbA and HbF but also between maternal hemoglobins AS, CS, and SS and HbF.
The presence of maternal thalassemia major would produce erroneous results due to
the high concentration of HbF. Stool specimens should be tested when fresh. They
may appear bloody but should not be black

77. APT Test
 1. Emulsify specimen in water.
 2. Centrifuge.
 3. Divide pink supernatant into two tubes.
 4. Add 1% sodium hydroxide to one tube.
 5. Wait 2 minutes.
 6. Compare color with that in the control tube.
 7. Prepare controls using cord blood and adult blood

79. Vaginal secretions specimen collection
 A speculum moistened with warm water is used to visualize the vaginal
fornices.
 Lubricants may contain antibacterial agents and must not be used.
 The specimen is collected by swabbing the vaginal walls and vaginal
pool to collect epithelial cells along with the vaginal secretions using one
or more sterile,polyester-tipped swabs on a plastic shaft or
swabs specifically designated by the manufacturer.3
 Cotton swabs should not be used because cotton is toxic to Neisseria
gonorrhoeae, the wood in a wooden shaft may be toxic to Chlamydia
trachomatis, and calcium alginate can inactivate herpes simplex virus (HSV) for
viral cultures.4
 places the swab in a tube containing 0.5 to 1.0 mL of sterile physiological
salineThe swab should be twirled in the saline vigorously to dislodge
particulates from the swab

80.  An alternative method of specimen preparation is to dilute a sample
of vaginal discharge in one to two drops of normal saline solution
directly on a microscope slide.
 Then a second sample is placed in 10% KOH solution in the same
manner. Cover slips are placed over both slides for microscopic
examination
 Specimens must be kept at room temperature to preserve the
motility of Trichomonas vaginalis and the recovery of N.
gonorrhoeae,
 whereas specimens for C. trachomatis and HSV must be refrigerated
to prevent overgrowth of normal flora.
 Specimens for T. vaginalis should be examined within
2 hours of collection
Vaginal secretions specimen collection

81. Normal Findings in Vaginal Secretions
 Appearance : White, flocculent discharge
 pH :3.8–4.2
 Amine (whiff) test : Negative
 WBCs :Rare to 2+
 Lactobacilli :Predominant
 Clue cells : Absent
 Other cells : Absent (except RBCs during menses)
 Other organisms : Other lactobacilli subgroups / occasional yeast

82.  Keep specimens for suspected T. vaginalis at
room temperature and examine within 2 hours
of collection to visualize movement of the
flagella or undulating membrane on a wet prep.
 When not moving, Trichomonas may resemble a
white blood cell (WBC), transitional, or renal
tubular epithelial (RTE) cell.

83. Clue Cells
 Clue cells are an abnormal variation of the squamous epithelial
cell and are distinguished by coccobacillus bacteria attached in
clusters on the cell surface, spreading past the edges of the cell
and making the border appear indistinct or stippled.
 Bacteria should cover at least 75% of the epithelial cell. This
gives the cell a granular, irregular appearance sometimes
described as “shaggy.”
Clue cells are diagnostic of BV caused by G. vaginalis
 The presence of clue cells also can be found in urine sediment
and should be confirmed by the procedures already described.

86. Amine (Whiff) Test
 1. Apply one drop of the saline vaginal fluid suspension to the surface of a clean
glass slide.
 2. Add one drop of 10% KOH directly to the vaginal sample.
 3. Holding the slide in one hand, gently fan above the surface of the slide with the
other hand and assess for the presence of a fishy amine odor.
4. Report as positive or negative.
 Positive: The presence of a fishy odor after adding KOH.
 Negative: The absence of a fishy odor after adding KOH

87. Conclusion
The main prerequisite for
standardization and harmonization of
body fluid analysis is recognition of
unconformities in this field.