Professional Documents
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Quality Assurance
Training Programme
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Quality Assurance Training Programme
Preface
Since the beginning of the nineties the National Public Health Laboratory has been
involved in quality assurance for medical laboratories in Nepal. Out of this a Quality
Assurance Unit has developed with the support of the Laboratory Assistance
Programme (LAP) of the International Nepal Fellowship. This now runs regular training
courses. For these courses training materials have been produced and compiled by
the Medical Technologists of LAP to form this present manual.
The main aim of this manual is to standardise commonly used laboratory techniques
while putting a special emphasis on internal quality control procedures (IQC).
We wish this manual to be a bench aid as well as a reference for the laboratory
workers in their day to day management of their work.
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Acknowledgements
We wish to thank all those who have contributed to this work.
Special thanks are due to:
• Dr Sarala Malla, Director, National Public Health Laboratory, Department of Health
Services, Teku Kathmandu
• Gabriele Mallapaty who inspired us to produce this material by providing the
format and producing the first handout on internal quality control procedures.
• The Technical Assistance Programme of the International Nepal Fellowship for the
section on maintenance and their regular input to the training of laboratory staff in
simple maintenance of equipment.
• Dot and Lines Graphics Art for their design work and drawings.
• W.H.O. Manual of Basic Techniques for a Health Laboratory (1980) from which
most of the pictures have been taken.
• All our colleagues in the National Public Health Laboratory who contributed with
valuable suggestions and comments.
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Contents
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Chapter 5 Quality Assurance in Urine 69
Sample collection 71
Sample storage and transporation 71
IQC Procedures 72
Urine deposit 73
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Chapter 1
QUALITY
ASSURANCE
PROGRAMME
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The ultimate goal of any quality assurance programme is to obtain test
results that are reliable, relevant and reproducible.
Internal Quality Control (IQC) Procedures are done during daily routine work. They
are applied to all work procedures and to every test done in the laboratory. They
provide an immediate control so that errors can be corrected immediately.
External Quality Assessment (EQA) evaluates past performance by testing
unknown samples and comparing the performance with others. It provides a forum for
improvement and correction of errors
Quality Management involves training of the laboratory staff, the use of
Standard Operating Procedures (SOPs), a standard supply of equipment and
materials, supervision in the laboratory and the organisation of the laboratory
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Quality Management
Results to NPHL
Training SOP's
LaboratoriestosamplesControl
NPHLfromadviceandFeedbac
Supply
Supervision
k
Equipment
Your laboratory
ICQ Procedures
DAILY
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Morphology
5
Example of the feeback form you will receive after sending your EQA results
National Public Health Laboratory,
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Specimen
2 ml fresh EDTA Blood or2 ml fresh Citrate Blood (not older than 24 hr. if kept at 4°C)
Equipment Requirements
• Westergren Rack
• Westergren Tubes, internal diameter 2.5 mm
• Dilution bottles to hold 2 ml (4 volumes of blood/1 volume of anticoagulant
diluent solution
• Timer (1hour)
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Special Note:
While reading the result you should also pay attention to the following: The colour
of the plasma:
• Dark yellow, indicates hepatitis
• Clear as water, indicates lack of iron
• White and turbid, indicates nephrosis, diabetes, lipaemia.
• Increased layer of white blood cells just above the red blood cells indicates
leukocytosis
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Erythrocyte Sedimentation Rate (ESR)
A-001a
Westergren Method
References
1. A handbook of Medical Laboratory TechnologyHaematology for Students and
Practitioners. Dr. Ramnik Sood M.D..Jaypee Brothers medical publishers.
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V1 = ........................
V1 = C2 x V2
C1
Example:
To make 1ml of 100mg% Glucose solution from a 500mg% stock solution:
C1 = 500mg%
V1 = ? 100 mg% x 1 ml
C2 = 100mg% V1 = = 0.2 ml
500mg%
V2 = 1 ml
Therefore measure 0.2 ml of the 500 mg% solution and make up to 1 ml with
distilled Water.
Exercise:
A. To make 1 litre of Hydrochloric acid (HCl), 0.01 mol/l from a 1 mol/l solution:
C1 =
V1 = ?
C2 = =
V2 =
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B. To make 25 ml of 350 mg% Glucose solution from a 500mg% stock solution:
C1 =
V1 = ?
C2 =
V1 = ........................ =
V2 =
Therefore measure ...............ml of the 500 mg% solution and make up to 25 ml with
distilled Water.
Universal Precautions:
• Treat all blood specimens as potentially infectious!
• Consider all equipment that has been in contact with blood specimens as potentially
infectious.
• Keep the laboratory clean. After work wipe the benches with disinfectant!
• Do not mouth pipette and wear protective coats at all times.
• Do not eat, drink or smoke in the laboratory. Cover open wounds.
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Pipetting techniques
Steps in Pipetting
1. Get a testube rack ready with as many
test tubes as needed
2. Get the solution ready
3. Take a clean glass pipette
4. Take a rubber bulb with ‚blue‘ tip
5. Blow all the air out of the bulb by pressing
very hard
6. Set the bulb – with the tip pointing into the
pipette – on top of the top end of the pipette
7. Hold the pipette upright
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2. The use of automatic pipettes
1. Preparation 2. Aspiration 3. Distribution 4. Purge 5. Home
Hold the pipette in a Immerse the pipette in the Place the pipette tip at an Wait one second, then Allow the plunger to
vertical position. Depress liquid. Allow the plunger angle against the inside depress the plunger to move up to the rest
the plunger smoothly to to move up smoothly to wall of the receiving tube. the second stop position. position.
the first stop position the rest position. Wait Depress the plunger This removes any
one second so that all the smoothly to the first stop remaining sample from
liquid has time to move position. the tip. Remove pipette
up into the tip. Keeping tip end from sidewall by
the pipette upright sliding it up the wall.
remove the pipette from
the liquid and wipe the
outside of the tip without
touching the open end.
7
1
Rest Position
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Maintenance
Good functioning laboratory equipment is an important part of quality assurance. All
our equipment has to be kept in good order, which is achieved by maintenance.
Maintenance includes
• Appropriate setting up of new equipment
• Proper daily handling
• Regular cleaning
• Routine maintenance (e.g. oiling of mechanical parts, change of bulb after x hours)
• Appropriate repairs
Remember maintenance is NOT ONLY repair.
Colorimeter
1. Light source
The light bulb gets slowly weaker usually indicated by difficulty in zeroing or instability of
the absorption signal. To prolong the life of the lamp, switch of the colorimeter after use.
2. Filter
• Check regularly that your filters are clean and not scratched or broken.
• Broken and scratched filter must be replaced.
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• Dirty filters can be cleaned with care to avoid scratches
• Always check that you are using a filter of the correct wavelength for the test method.
3. Cuvette
The quality of glass cuvettes is superior to plastic cuvettes. They can be cleaned
better and reused.
To clean off protein precipitates you can soak them in concentrated sulphuric acid/
potassium dichromate solution overnight and then rinse them with abundant
distilled water before drying.
If you are using plastic cuvettes clean them immediately after use with distillated
water or detergent solution followed by abundant distilled water. Keep them up side
down to dry. The sulphuric acid/potassium dichromate solution cannot be used, as it
would damage the plastic.
• Always check before use that cuvettes are clean and not scratched.
• Use the right kind of cuvette for your colorimeter
• Put the cuvette in the right position
Microscope
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• Staff should know how to use the microscope properly and handle it with care
• Each day before use the microscope should be cleaned and checked by
an experienced person
• The microscope should be stored safely at the end of each day
• Never place the microscope into direct sunlight.
1. Cleaning of optics
Condenser, objectives and eye pieces should be clean with a soft camel-hair brush or
a blower. Always wipe off immersion oil from the 100x objective with lens paper, soft
paper or cotton-wool. The following cleaning solution can be used for oil dried onto the
objective: Methanol 30%, ether 70%.
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Centrifuge
The Auto-pipette
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Chapter 2
Quality Assurance
in Haematology
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Sample collection
Capillary blood
Capillary blood is obtained by pricking the finger, the ear lobe or in infants the heel of the
foot. Capillary blood is used immediately and therefore does not need anticoagulants to be
added. It can be used for haemoglobin estimations, total WBC-counts, differential WBC-
counts, platelet counts, reticulocyte counts and for Malaria or Filaria films
Venous blood
Venous blood is obtained by vene-puncture. An anti-coagulant must be added to the
blood to prevent it clotting. Commonly used anti-coagulants for haematology are
EDTA (Ethylene diamine tetra-acetate), heparin and trisodium-citrate.
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Preparation of EDTA anti-coagulant bottles
Weigh out the powder and dissolve it in 100 ml of distilled water. Pour the solution into
a labelled reagent bottle.
Step three: Collect the patients blood into the bottles Add
about 2.5 ml of blood and mix gently but thoroughly.
NEVER add a pinch of EDTA powder directly to the sample bottles. - High
concentrations of EDTA lead to the RBCs shrinking and destroy the structure of the
WBCs and platelets. NEVER add the blood before the EDTA solution is completely dry
as it will dilute the blood and destroy the RBCs.
Blood films
• Blood films should be prepared within one hour of collection when EDTA blood is
used.
• Store unstained blood films in a dry place and protected from direct sunlight, dust
and flies.
• Stain blood films as soon as possible.
• Malaria and filaria positive slides should be kept in a box for future inspection by
a supervisor.
• Doubtful slides should be kept in a separate box for inspection by the supervisor.
• For transportation each slide should be wrapped in a piece of paper and kept in a
box to avoid breakage.
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EDTA anti-Coagulated Venous blood
When EDTA anti-coagulated blood cannot be tested within 1 to 2 hours it must be
refrigerated at 4-8oC to prevent cellular changes affecting test results. Manual or
automated blood cell counts, reticulocyte counts, and PCV change little in EDTA blood
at 4-8oC when stored for up to 24 hours. Haemoglobin concentration is stable for 2 to
3 days at 4-8oC providing there is no haemolysis.
Blood
• Where running water is available, pour the blood into a sink that is connected to a
soak pit. Where running water is not available pour blood into a bucket that contains
a 10% lysol solution, soak over night and dispose off with solid waste (bury).
• 1% Virex solution can also be used. Soak for half an hour and dispose off with
solid waste.
Glass slides
• Soak used glass slides in 5% lysol solution at least over night. Clean and rinse next
day.
IQC Procedures
Westergren ESR
• Ensure that the correct dilution of blood and tri-sodium citrate solution is used
• Store the trisodium citrate solution in the refrigerator
• The trisodium citrate solution should not be turbid
• Avoid air-bubbles in the Westergren tube
• Place the Westergren tube in a vertical position
• Temperatures above 23°C increase the speed of the ESR. Therefore keep the ESR
rack in the coolest place of the lab and out of direct sun light.
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• If the haemoglobin concentration is very low inform the doctor or health-care
worker immediately.
Haemoglobin Colorimetric method
A new calibration graph must be prepared whenever the colorimeter, cuvette type or
the test method is changed.
• Always allow the colorimeter to warm up before measuring the test samples.
• Ensure that the correct filter (540nm wavelength—yellow/green filter) is in place.
• To test for accuracy use a control sample of known value.
• Control samples are either commercially available control samples or samples
of known (true) value measured by a very reliable laboratory.
• Repeat the test on a single specimen to control for reproducibility (precision).
• A new stock of Drabkin’s solution must be checked against the old solution with
samples of known value before it is used for patients
• Drabkin’s solution should be clear and pale yellow in colour. If it is turbid or loses its
colour it must be discarded.
• Handle Drabkin’s solution with care. It is very poisonous. Do not mouth pipette.
Leave the blood mixed with Drabkin’s solution for 10 minutes before reading the
optical density so that the haemoglobin can convert to cyanmethaemoglobin.
• Be careful with cuvettes with frosted sides. The clear side must face the light path.
• Be careful to avoid air bubbles in the cuvette.
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Chapter 3
Quality Assurance
in Biochemistry
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Sample Collection
Blood for sugar estimation is normally requested on a fasting specimen. You should be
familiar with the following terms:
Fasting specimen
• This means no food or drink except water has been taken since the night before
and the blood sample is collected in the morning before any drink or food is taken.
Random
• This means the specimen has been collected at any time of the day, irrespective
of food intake.
The normal range of blood sugar differs in serum & whole blood, whole blood levels
are about 15% higher.
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Sample Storage and Transportation:
• Carry out the biochemistry test as soon as possible after separation of the serum
from the blood clot.
• Not all analytes are stable, which means their concentration will reduce over time.
• Generally, stability is prolonged if the serum is kept in the refrigerator.
• Some analytes such as bilirubin are also affected by light.
IQC Procedures:
General
• Do not examine the specimen when the blood is haemolysed.
• Do not examine the specimen when the time of the sample collection is not clear.
• Label the specimen with patient name and lab number to avoid confusion.
• Take great care when pipetting samples and reagents.
• Care for your colorimeter. Cover it with a protective cover when not in use.
• Handle filters with great care. Do not touch the filter with fingers, always hold from
the side.
• Prepare standard curves whenever you use new reagents or equipment.
• If possible use commercially available control sera to control accuracy.
• Do repeated tests or redo tests from the day before to check your precision.
• Be careful when repeating and redoing tests as some analytes are not stable.
• The serum for sugar has to be separated from the blood clot within one hour after
the collection and the test should be done within two hours after collection as the
concentration of glucose decreases over time due to glycolysis.
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Quality assurance in the analytical stage
Reliable test results depend on the detection and correction of errors at an early
stage. Good laboratory practice and quality control helps keep errors of imprecision
and inaccuracy low.
1kg
Mr Poudyal regularly checks his equipment and closely supervises his shop assistant
so the bags always contained the same amount of salt and as close as possible to the
exact weight. They were accurate and precise.
Mr Shrestha and his assistant were careless and imprecise when filling the bags, which
did not contain the correct weight but varied from day to day. They were neither
accurate nor precise.
Mr Yadav’s assistant was careful in filling the bags, but Mr Yadav never checked his scales,
which were inaccurate and weighing too little. They were inaccurate but precise.
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When to check for accuracy?
Always check when changing reagent, instrument and methodology, as a routine once
a week or as appropriate and when there is doubt about the result.
Errors of accuracy are also known as errors of bias, they are consistent and
systematic. They can be caused by incorrect calibration, due to inaccurate standard
and pipette, incubation at the wrong temperature, reading the test at the wrong
wavelength and the use of an incorrect calibration factor. The use of poor quality
reagents and standards also leads to errors of accuracy.
Errors of precision are also known as errors of scatter, they are irregular and random.
They can be caused by inadequate mixing of the sample and reagent, inconsistent
pipetting, incubation at variable temperature and dirt or air bubbles in the colorimeter.
Incorrect storage and handling of samples and dirty glassware will also lead to errors
of precision.
Accuracy is best checked with commercially available quality control sera and
through the External Quality Assurance Programme control sera. At least two levels
of control sera should be used, one normal and one pathological.
Errors of precision can be avoided by internal quality control procedures and
quality management.
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Expected result of Blood Glucose 120 mg%
Example One
• Repeated Results of the Blood Glucose show the following values:
101, 103, 99, 101, 102 mg %
140 mg %
120 mg %
100 mg %
Example Two
• Repeated Results of the Blood Glucose show the following values:
122, 119, 121, 120, 123 mg %
140 mg %
120 mg %
100 mg %
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Establishing Performance Standard
Mean
Describes the midpoint of a population or in other words gives the calculated average
of a set of values.
I.e.: If 10 tests have been performed, the 10 results are added and this sum is divided
by 10.
No.
1 .............................................. 107 mg%
2 .............................................. 118 mg%
3 .............................................. 102 mg%
4 .............................................. 114 mg%
5 .............................................. 110 mg%
6 .............................................. 108 mg%
7 .............................................. 117 mg%
8 .............................................. 109 mg%
9 .............................................. 112 mg%
10............................................ 113 mg%
Total 1110 mg%
Before any test method is used for patients we must first make sure that the method
is reliable and that it can be performed within acceptable limits of variation.
Once a test has been introduced it must be controlled routinely.
To assess reliability first determine the
• Standard Deviation (SD) then the
• Optimal Conditions of Variance.
1. Standard Deviation
• In statistical terms the distribution or scatter of values around the mean can
be expressed as standard deviation.
• A range of 2 standard deviations (± 2 SD) is generally considered as the limit for
a control value to be acceptable.
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Usually
• 68% of results will fall within ±1SD
• 95% of results will fall within ±2SD
• 99.7% of results will fall within ±3SD
• To establish the SD perform 20 measurements on the same control serum.
• List the values for each of the 20 estimations as shown in the example below.
• Calculate the mean of these 20 values
• For each result work out the difference in value from the mean and record it
in column 2
• Multiply each difference value by itself (to give the squared difference value).
Enter these values in column 3
• Add up the values in the third column to obtain the sum of the squared differences.
Test no. 1 2 3
Test results Diffrence from the mean Squared differences
1 72 74-72= 2 22 4
2 74 74-74= 0 02 0
3 75 74-75= -1 -12 1
4 74 74-74= 0 0
02
5 72 74-72= 2 4
22
6 78 74-78= -4 -42 16
7 74 74-74= 0 02 0
8 72 74-72= 2 22 4
9 73 74-73= 1 12 1
10 74 74-74= 0 02 0
11 75 74-75= -1 -12 1
12 76 74-76= -2 -22 4
13 73 74-73= 1 12 1
14 74 74-74= 0 02 0
15 72 74-72= 2 22 4
16 74 74-74= 0 02 0
17 72 74-72= 2 4
22
18 77 74-77= -3 -32 9
19 74 74-74= 0 02 0
20 75 74-75= -1 -12 1
Mean 74 Sum = 54
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• Calculate the Standard Deviation from the formula :
SD = sum of the squared differences
n-1 n = number of results.
E.g.: SD = 54 = 1.69
19 n = 20
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The above OCV values are suggested OCV maximum % that you may use to access
the analytical methods you are using. Different books and Quality Control web sites
may give slightly different values. These are considered suitable for a district laboratory.
OCV (Optimal Conditions Variance) can be obtained only in ideal conditions, the same
technician carefully performing 20 measurement of the same control serum, at a given
time.
The RCV (Routine Conditions Variance) is obtained in routine conditions when different
technicians are performing the same test at different times. The acceptable value may
be up to twice the OCV.
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Month: October
Analyte: Urea Low
45
44.5
44
43.5
43
42.5
42
41.5
41
40.5
40
39.5
39
38.5
38
37.5
37
36.5
36
35.5
35
34.5
34
33.5
33
32.5
32
31.5
31
Mean
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30.5
30
29.5
-2 sd
TrainingAssuranceQuali
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28.5 +2sd
28
27.5
27
26.5
26
25.5
25
24.5
24
23.5
23
22.5
22
21.5
21
20.5
20
19.5
19
ty
18.5
18
17.5
17
16.5
Progra
16
mme
15.5
15 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32
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Interpretation of Results
• Control Serum value within + 2 SD limits is a good sign and it can be assumed that
the patient results are reliable and can be reported with confidence.
• Control Serum value outside + 2 SD is unacceptable and the patient results must not be
reported. Take a fresh control serum and repeat with a few of the patient samples.
- If the repeat CS is within +2SD and the repeat tests agree with those of the
first testing all the patients’ results can be reported.
- If the repeat CS is within +2SD but the repeat test results do not agree with those
of the first testing all the patients’ tests should be repeated.
- If the CS is still not acceptable do not report patient results. Check for errors -e.g.
reagent deterioration, incorrect preparation of reagents, faulty equipment, or
wrong filter in the colorimeter. Correct the error and repeat the batch with CS.
• Serum Control Values moving towards the TAKE ACTION ZONE report patient
results, but a drift upwards or downwards is a warning that the test is
becoming unreliable and the cause must be investigated.
analyte: month:
I II III IV V
2SD
mean
2SD
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31
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Control serum
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Chapter 4
Quality Assurance
in Bacteriology
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Sample Collection
If the pathogens are to be isolated successfully, the type of specimen, method of
collection, the time and method of dispatch to laboratory must be appropriate.
Things to be remembered
A. Use of aseptic collection technique
B. Correct type of specimen: Eg, MSU, Endocervical swabs, Sputum
etc. C. Time of Collection
D. Containers/swabs for specimen collection
E. Proper labeling of specimen
Sputum for TB
As a rule three sputum samples are collected:
1. When the patient comes to the health centre.
2. The following morning at home
3. At the clinic during the second visit at the health centre.
• It is best to collect the first sputum in the morning
• Before collecting the sputum, the patient may drink a glass of hot water.
• The patient should stand if possible and take a few very deep breaths filling the lungs.
• The lungs should be empty in one breath coughing as hard and deeply as possible
• The patient should spit what he/she brings up into the clean collection container.
• Write the name and patient number on the container to avoid confusion.
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• Clearly mark from which site each smear is taken
• Take a fold of skin between your index finger and thumb and squeeze tightly
to prevent the blood flow and make a cut without releasing the pressure
• Collect the tissue fluid
• If there is lots of blood do not collect the sample but make a new cut again
squeezing the skin tightly
Pus
• Collection by experienced medical staff
• Possible pathogens:
Staphylococcus aureus Pseudomonas aeruginosa
Streptococcus pygoens Proteus spp.
Other streptococci Klebsiella
Clostridium tetani E. coli
Clostridium perfringens Bacteroids spp. etc.
• Pus from an abscess is best collected at the time it is incised and drained, or after
it has ruptured itself.
• When collecting pus from abscesses, wounds or other sites, special care should be
taken to avoid contaminating the specimen with commensal organisms from the skin.
• A specimen from a wound should be collected before antiseptic dressing is
applied/ before antimicrobial therapy is started.
Urine
• Patients should be instructed about proper urine collection technique (MSU).
• Procedure for Mid Stream Urine Collection
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• Possible pathogens from urine:
Note: It is advisable to screen all urine samples sent for culture with a screening
method for urinary tract infection (UTI). The screening method avoids unnecessary
culture of uninfected samples and saves time and money. (see SOP-C002)
Female
Spread the legs apart and with your left hand spread the labia apart. Keep holding
the labia apart during the entire collection process.
Male
Pull back the foreskin hold it and back during the entire collection period.
Male and female
Pass a small amount of urine but do not collect it. Move the collection container under
the stream of urine and collect some into the container. Do not collect the last few
drops of urine. Close the lid of the container and take the urine sample immediately to
the laboratory for examination.
Blood
Blood for culture must be collected under comletely sterile conditions to
avoid contamination.
• Always collect blood for culture before antibiotic treatment has been started
• If possible collect the blood at the time when the patient’s temperature is rising
• Remove the blood culture bottles from the fridge and allow them to reach
room temperature.
• If the culture broth appears turbid do not use for blood culture
• Possible pathogens:
Salmonella typhi, paratyphi E. coli
Streptococcus viridans Klebsiella
Streptococcus pneumoniae Proteus
Staphylococcus aureus Haemophilus influenzae etc.
• Bacteraemia/septicaemia
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Method of Collection:
Must be collected asceptically
• Make the patient sit comfortably.
• Locate the suitable vein in the arm.
• Cleanse thoroughly the skin over the vein with 70% ethanol
• Do not touch the skin after disinfection.
• Using a sterile syringe, draw 5 ml of blood and immediately dispense into blood
culture bottle containing 50 ml culture broth, (for children, 3 ml into 30 ml of broth).
• Recap the bottle, label the specimen and dispatch to microbiology laboratory.
Sputum for TB
• Smears are prepared and heat-fixed when the sputum is received at the laboratory.
• Usually slides are prepared and heat-fixed before sending them to a
reference laboratory. Sputum is only sent when culture is required.
• For transportation wrap each individual slide in a piece of paper or use a slide box.
• Store slides in a box away from flies, dust and direct sunlight.
Waste Disposal
• Used sputum containers, slides and wooden applicators must be collected in a
waste container that is covered with a lid.
• Dig a deep pit of 1 metre and throw the waste into the pit. Cover the pit with soil
when it is about half full.
• All the waste material must be burnt.
• Do not leave the waste in the open uncovered.
• Do not throw the waste into a river.
• Do not reuse slides of TB and leprosy smears.
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Sterilisation
Sterilisation is the killing of all living micro-organisms including bacterial spores.
Methods of sterilisation
1. Steam under pressure (autoclave, steam steriliser or pressure cooker).
• Sterilisation is achieved through pressure and temperature.
• Recommended conditions are 121°C for 15 minutes at a pressure of 15 psi.
• It is used for sterilising culture media and instruments.
2. Incineration
• Incineration is burning of material. It is the most effective method of
destroying infected disposable material.
• Waste material for awaiting incineration should be kept covered and well protected
to prevent access by people, animals and insects.
3. Flaming
• Fire kills all living organisms.
• This is use to disinfect reusable metal or glass objects, like wire-loops, glass slides
and the necks of blood culture bottles.
Disinfection
Disinfection is the killing or removal of pathogenic (causing disease) micro-
organisms. Methods of Disinfection
1. Chemicals
The chemicals most commonly used for disinfection are Phenols,
Aldehydes, Alcohols and Halogens.
2. Boiling in water
Boiling in water for 20 – 30 minutes can be used. At altitudes above 2000 feet 30
minutes is recommended.
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Dilution Use
Phenols • 5% solution of Lysol to Glassware / Floor /Benches
disinfect glassware and
other reusable items.
• 10% solution of Lysol to Body fluids.
disinfect body fluids.
Aldehydes • 10% Formalin Preservation of stool
• Not commonly use for parasites.
disinfection of instruments
as it is highly irritating to
skin, eyes and lungs.
Alcohols • 70% alcohol Skin disinfection
• Methylated spirit (no need Smear fixation
to dilute)
Halogens • 0.5% Hypochlorite solution Hand disinfection
(Bleach)
• 0.5% Iodine solution Glassware Skin disinfection
• 1% Virex Glassware, bench top
IQC Procedures
Gram stain
• Follow proper sample collection procedures.
• Do not overheat the slide when heat-fixing the smear.
• Filter the crystal violet before use.
• Discard Lugol’s iodine when the colour has faded.
• Decolourise carefully. Just few seconds are requesred then wash off with water.
• Stain known samples of Gram-negative and Gram-positive bacteria once a week
and whenever new stains have been prepared.
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Gram Stain
Clinical Significance
1. Gram stain results can have a dramatic effect on patient care.
2. Hospitalisation may be required when the Gram stain results indicate bacteria
are present in a normally sterile body fluid.
3. The initial choice of antibiotic therapy is guided by Gram stain results.
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Staining Procedure
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Theory
Gram-positive and Gram-negative bacteria stain differently because of the structure
of their cell walls.
Microscopy
Examine the slide under oil immersion and record the presence of any host
cells, bacteria, or yeast.
Reporting Results
Use systematic, descriptive terminology to report Gram stain results.
It is NOT possible to determine the species of bacteria from Gram stain results
alone! Definitive identification requires culture and biochemical testing
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Morphology
Cells
1. Polymorphonuclear Neutrophils
A polymorphonuclear neutrophil (PMN) is
distinguished from other white blood cells by its
segmented nucleus.PMN’s are attracted to the site
of infection in response to bacterial and host
inflammatory products.PMN’s are short-lived,
phagocytic, and usually appear in the acute stages
of bacterial infection.
2. Lymphocytes
Lymphocytes are smaller than PMN’s. The nucleus
is round and has a thin border of
cytoplasm.Lymphocytes are uncommon on direct
Gram stains except from cerebrospinal fluid.A
smaller number of lymphocytes are present in CSF
from healthy individuals. A large number of
lymphocytes usually indicates viral meningitis.
3. Macrophages
Macrophages are slightly larger than PMN’s. The
nucleus is large, non-segmented and slightly
indented.Macrophages are polymorphic; they may
resemble large, atypical lymphocytes.Macrophages
are widely distributed in tissues throughout the
body.Macrophages are long-lived, phagocytic, and
are more prevalent in chronic infections.
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Epithelial Cells
Sperm Cells
Bacteria
Gram-positive Cocci
The Gram-positive coccus is a spherical bacterium.
Gram-positive cocci may appear in four groupings.
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Gram-positive Rods
The Gram-positive rod or bacillus is a rectangular shaped bacterium.
Rods are variable in length, width, and staining characteristics.
There are four clinically significant shapes of Gram-positive rods....
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Gram-negative Cocci
Gram-negative cocci may appear in two clinically significant groupings...
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Gram-negative Rods
The Gram-negative rod or bacillus is a rectangular shaped bacterium.
Rods are variable in length, width, and staining characteristics.
There are five clinically significant shapes of Gram-negative rods....
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Yeasts
Budding Yeasts
Yeasts commonly appear as “budding yeasts.”
Note: do not confuse single yeasts with sperm cells
Pseudohyphae
Yeasts may also appear as pseudohyphae which
are elongated projections from yeasts.
Pseudohyphae have tapered ends.
Artifacts
Cellular Debris
Direct Gram stains often show a variety of
background debris which must be distinguished
from host cells, fungi, or poorly staining
bacteria.The debris can include the following:
mucus strands, ruptured or disintegrating cells, and
protein precipitate.
Decolorization
Overdecolorization
Gram-positive organisms may appear partially or completely Gram-negative if they
have been overdecolorized.Overdecolorization may be due to poor staining technique.
The timing of the acetone-alcohol step is critical.Overdecolorization may occur if the
Organisms are old or dead. This is due to damage to the organism’s cell wall.
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Underdecolorization
Epithelial cells, WBC’s, mucus, and cellular debris should stain Gram-negative.Thick
areas of a Gram stained smear are more likely to be undercolorized.
Antibiotic Effects
The presence of antibiotics may alter the size, shape, and arrangement of some
species of bacteria.
For example, the bacteria may appear swollen or elongated.
Specimen Sites
Cerebrospinal Fluid
Normal
Cerebrospinal fluid is normally sterile.
Rarely lymphocytes may be present.
PMN’s are not normally present.
Infection
Any organism is considered significant in CSF.
Usually only a single morphotype of bacterium or yeast is present.
The presence of PMN’s and/or mononuclear cells usually indicates infection. PMN’s
predominate in bacterial infection.
Note: Gram negative bacteria can be very difficult to see on a CSF gram stain, it is
therefore compulsory to do a simple Methylene blue stain first to detect the presence
of any bacteria and then to do a Gram stain.
Blood
Normal
Blood is usually not directly Gram stained. Rather, it is cultured in a blood culture bottle,
and the fluid from a positive culture is Gram stained.
Blood is normally sterile. Depending on the blood culture media used, white blood cells
can be seen in a Gram stain from a normal blood culture.
Septicemia
Any organism is considered significant in blood. Interpretation of positive blood cultures
is complicated by the possibility of contamination during specimen collection.
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Contamination
Contamination during collection may complicate interpretation of blood culture results.
Skin flora (eg. Staphylococci, micrococci, corynebacteria) can be pathogenic in certain
circumstances and therefore cannot be automatically disregarded as contaminants.
Sputum
Contaminated Sputum
Gram-stained sputum specimens should be examined under low power to determine
if the specimen has been contaminated with saliva.
A contaminated sputum specimen is characterized by abundant squamous
epithelial cells and very few PMN’s.
Identification of bacteria from contaminated specimens will provide misleading
results. Acceptable Sputum
An acceptable sputum specimen should contain very few squamous epithelial cells.
Acceptable sputum specimens are more likely to provide accurate culture results.
Only acceptable sputum specimens should be cultured.
Pneumonia
The lower respiratory tract is normally sterile.
The presence of ciliated columnar epithelial cells helps to confirm that the specimen
is from the lower respiratory tract.
Large number of PMN’s and bacteria are usually present in sputum specimens
from patients with bacterial pneumonia.
Urine
Normal
Urine is normally sterile.
Rare epithelial cells may be present.
PMN’s are normally present in urine specimens.
Infection
PMN’s are common in the urine of patients with urinary tract infections.
The presence of a single bacterium per oil immersion (100x objective) field
indicates infection with approximately 100 000 organisms per millilitre of urine.
The absence of PMN’s and bacteria does not rule out a urinary tract infection.
Contamination
Urine is easily contaminated with bacteria from the periurethral area.
Contaminated urine contains squamous epithelial cells and a mixture of Gram-
positive organisms.
A few Gram-negative rods may be present in a contaminated urine specimen.
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Genital
Normal vagina
Squamous epithelial cells are commonly seen on normal vaginal smears.
Lactobacilli are the predominant normal flora present in the vagina.
Occasional PMN”s are considered normal.
Vaginosis
“Clue cells” are squamous epithelial cells with large numbers of adherent coccobacilli.
“Clue cells” are associated with bacterial vaginosis. Gram-variable
coccobacilli are a predominant organism in bacterial vaginosis. Urethritis
(male)
Intracellular Gram-negative diplococci from a male urethral specimen suggest
a diagnosis of gonorrhea.
PMN”s are commonly present on a urethral discharge smear.
Trichomonas vaginalis
Typical Morphology:
oval-shaped, can resemble a white blood cell or a small epithelial cell.
Wound
Not Infected
If the wound specimen is from a non-sterile site, normal flora an be present.
If the wound specimen is from a normally sterile site, no bacteria should present.
Epithelial cells may be present.
Infected
Large numbers of PMN’s are usually present in wound infections.
A wide variety of bacterial species can cause wound infections.
Wound infections caused by a mixture of bacteria are common.
Eye
Normal
The conjunctiva of the eye is normally colonized with non-pathogenic corynebacteia and
staphylococci from the skin.
PMN’s are rarely seen under normal conditions.
Infection
The presence of PMN’s indicate an infection.
Usually only a single morphotype of bacteria is present.
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Joint Fluid
Normal
Synovial fluid is normally sterile.
PMN’s are not normally present.
Cellular debris may be present.
Infection
Any organism is considered significant in synovial fluid.
Usually only a single morphotype of bacteria is present.
The presence of PMN’s usually indicates infection.
Stool
Normal
In some circumstances, Gram stains of stool specimens may be useful; however,
they are not commonly performed.
Normal stool specimens contain abundant Gram-positive and Gram-negative organisms.
PMN’s are not usually present in normal stool specimens.
Infection
The presence of PMN’s in stool usually indicates that an invasive pathogen is present.
Reduction of the normal faecal flora is associated with diarrhoea.
It is difficult to distinguish pathogens from normal faecal flora on Gram
stains. Bacterial stool pathogens are usually Gram-negative rods
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Chapter 5
Quality Assurance
in Urine
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Sample Collection
Biochemical tests
• For chemical tests such as sugar, protein or bile pigment the method of collection is
less important. Any random urine sample can be used. It is always best to request
for a mid-stream urine sample.
• The concentration of analytes will be highest in the first morning urine.
• Tell the patient to collect about 20 ml of urine into a clean, dry container.
• Label the container immediately with the patient’s name and the laboratory number.
• Examine as soon as possible
Urinary deposit
• For urinary deposit you must instruct the patient to collect a clean urine sample or
a so-called mid-stream urine sample (MSU) See separate sheet!
Urine culture
• For urine culture you need a mid-stream urine sample collected into a
sterile container.
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IQC Procedures
• Always note the colour of the urine and report unusual colours. For example, urine
can show a red colour when containing blood, or brownish green colour in patients
with hepatitis, or pale almost white colour in patients with diabetes, or milky white
colour when urine contains lymph gland fluid (chyluria – search for microfilaria).
• The urinary deposit must be examined within one hour of collection as bacteria
will multiply, cells become unclear & crystals increase.
• Examine urine for culture within 30 minutes after collection.
• When preparing new reagents, test the reagent with a known positive urine sample
or prepare your own positive control.
• Always refer to a picture atlas if you find structures that you do not know very well.
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Urine Deposits
Examination of urine is most important in patient care in that it aids diagnosis, assists
in monitoring disease and treatment and helps provide valuable information on the
patient’s well being.
The kidney has a prime role in maintaining normal healthy life, therefore many
early changes owing to disease may be reflected in the urine well before they
become clinically obvious.
Urine examination is also essential for the diagnosis of Urinary Tract Infection.
Principle
Routine urine analysis consists of physical, chemical and microscopic analysis.
Normal urine is almost clear and contains very few microscopic elements. In
some diseases the appearance of urine, chemical content and content of
microscopic elements changes. These changes are examined and reported.
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CELLS
Normal urine may contain a variety of cellular elements in small numbers, but
an increased number of any types can have diagnostic significance. The cells
must therefore be identified and quantified.
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Epithelial Cells
Epithelial cells may be seen in small numbers in
normal urine. The presence of increased numbers
of transitional or renal cells may indicate disease. To
differentiate the cells we look at the size, shape,
appearance and nuclear to cytoplasmic ratio. They
are usually reported as number per high power (40
x objective) field.
Squamous epithelial cells
Large, flat, irregularly shaped cells with small nuclei.
Large number of squamous epithelial cells may
indicate improper specimen collection. In female
patient it is usually of vaginal contamination.
Transitional cells:
Round, oval or pears shaped cells usually have a
centrally located nucleus. They come from the
renal pelvis to the terminal urethra, they have
ability to absorb large amounts of water and
therefore may appear swollen.
Renal epithelial cells:
The shape can vary depending on the location of
origin within the kidney. Renal cells may be very
difficult to distinguish from small transitional cells.
They are slightly larger than leukocytes and
contain a large, usually eccentric, round nucleus.
Increased numbers of renal cells may indicate
tubular necrosis.
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CASTS
• Casts are structures formed in the distal tubules and collecting ducts. The cells of
these parts of the renal system form a simple protein. In certain conditions this
protein forms fibrils which become attached to the tubule walls. If urinary flow is
decreased, more protein accumulates around the fibrils forming casts.
• If cells are present they may be incorporated into the cast matrix.
• Like cells, casts need to be quantified. They are quantified by number per low
power field (10 x objective).
Hyaline Casts
Hyaline casts are composed primarily of simple
proteins.
This kind of cast may be very difficult to see.
Lowering the light level, focusing up and down will
make it easier to see hyaline casts clearly.
Hyaline casts can be seen following fever, stress,
exercise or postural changes in normal individuals.
They are not indicative of any particular renal
disorder but increased numbers may be seen in all
diseases of the kidney.
Granular casts
Granular casts may be the result of degeneration of
a cellular cast or the results of direct aggregation of
serum protein granules into the matrix of protein.
They may be present in normal persons after
strenuous exercise. They may be found in a wide
variety of renal diseases, often associated with
the presence of cellular casts.
Waxy casts
Waxy casts represent the last stage in the
degeneration of hyaline, granular or cellular casts.
They are highly refractile and more easily seen than
hyaline casts. They have usually smooth blunt ends
and sharply defined edges.
Waxy casts are always accompanied by
a positive biochemical test for protein.
They usually indicate end stage renal disease.
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Fatty Casts
Fatty casts may contain lipid droplets or oval fat
bodies. They may be seen together with free fat
droplets in the urine and a strongly positive
biochemical test for protein. They are
frequently associated with nephrotic syndrome.
Renal Casts
They are hyaline casts with renal cells incorporated in
the matrix. They may occasionally be seen in normal
person due to normal fall off of dead tubular cells. The
presence of more than an occasional renal cast per
low power field is indicative of tubular injury.
RBC Casts
RBC casts contain red blood cells in the cast matrix
and are usually red or reddish brown in colour.
They are the most diagnostic of all elements in
urinary sediment and their presence is always
pathological. They indicate acute disorder of the
glomerulus. RBC casts indicate that other free
RBCs in the sediment came from the kidney.
You must see a sharp red cell outline in at least
part of the cast to identify it as a red blood cell
cast. Usually accompanied by positive blood and
protein tests.
WBC Casts
WBC casts contain white blood cells in the cast
matrix. You must see a sharp cell outline in at
least part of the cast to identify it as WBC cast. They
are associated with inflammation or infection within
the nephron.
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CRYSTALS
• Most crystals, which appear in the urine, are normal and are of little
clinical significance.
• There are a few abnormal crystals, which the microscopist must be able
to differentiate and identify.
• Normal crystals may be found at either acid or alkaline pH. All abnormal crystals
and most drug crystals are found in urine with an acid pH.
Normal crystals
Uric acid
pH of urine : acid
Soluble in : alkali and heat
Insoluble in : hydrochloric acid and acetic acid
• Urine acid may be found in multiple forms
• Uric acid is highly birefringent
Hippuric acid
pH of urine: acid but may also be found in neutral
or alkaline urine.
Soluble in : alkali
Insoluble in : acetic acid
• Hippuric acid crystals are colourless six-sided
prisms, needles or plates.
• They are uncommon and sometimes
confused with triple phosphate crystals.
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Triple Phosphate
pH of urine: alkaline
Soluble in : acetic acid
Insoluble in: sodium hydroxide and ammonium
hydroxide.
• The typical appearance of triple phosphate
crystals is a coffin-lid form (1) but they may have
a fern leaf (2) appearance if freshly formed.
Calcium Carbonate
pH of urine: alkaline
Soluble in : effervesces with hydrochloric or acetic
acid
Insoluble in: alkali
• Calcium carbonate crystals are seen as
granules or dumbbells.
Calcium Phosphate
pH of urine: alkaline
Soluble in : dilute acetic acid
Insoluble in: alkali
• Calcium phosphate crystals are large flat-shaped
or wedge-shaped prisms.
Ammonium biurate
pH of urine: alkaline
Soluble in : acetic acid
Insoluble in: ammonium hydroxide
• Ammonium biurate crystals are yellowish-brown
in colour with a characteristic round with thorny
projection shape.
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Abnormal Crystals
Leucine
pH of urine : acid
Soluble in : sodium hydroxide, hot water
Insoluble in : hydrochloric acid
• Leucine crystals are round with concentric
striations, yellow-brown, and oily looking.
• They may be seen in a wide variety of
liver disorders.
• Leucine crystals are often seen with
tyrosine crystals.
Tyrosine
pH of urine : acid
Soluble in : hydrochloric acid and sodium
hydroxide
Insoluble in : alcohol, acetic acid
• Tyrosine crystals will appear as colourless to
yellow-brown single needles or clumped or
rosettes.
• They may be seen in a wide variety of liver
disorders or in the genetic condition tyrosinemia.
• Tyrosine crystals often appear with leucine
crystals.
Cystine Crystals
pH of urine : acid
Soluble in : hydrochloric acid, sodium hydroxide
and ammonium hydroxide
Insoluble in : acetic acid
• Cystine crystals are thin, colourless, hexagonal
plates and may appear wrinkled when dissolving.
• Cystine crystals are found in an
inherited condition known as cystinuria.
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Bilirubin Crystals
pH of urine: acid
Soluble in : acetic acid, hydrochloridric acid,
sodium hydroxide and acetone
Insoluble in : alcohol
• Bilirubin crystals are typically yellow-brown
needles or sometimes granules.
• They should be accompanied by a positive
biochemical test for bilirubin.
• Bilirubin crystals are found in a wide variety
of hepatic disorders.
• Bilirubin may stain the entire sediment yellow.
Cholesterol crystals
pH of urine : acid
Soluble in : chloroform or ether
Insoluble in : dilute acids / alkalis
• Cholesterol crystals appear as regular or
irregular flat plates.
• Increased urinary protein, increased
serum cholesterol, and decreased serum
albumin usually accompany them.
• Cholesterol crystals are associated with nephrotic
syndrome.
Sulfonamide Crystals
pH of urine : acid
Soluble in : acetone and alkali
Insoluble in : acetic acid
• Sulfonamide crystals may be seen in a wide
variety of shapes.
• They should be confirmed with biochemical test
before reporting.
• Sulfonamide crystals are seen in the urine as
a result of drug therapy.
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Yeast
• Yeast is a common cause of urinary tract infection
in immunocompromised or diabetic patients.
• The usual organism is Candida albicans.
• Candida albicans may be seen as budding or
non-budding or may show pseudohyphae.
• Non-budding forms may be confused with red
blood cells. These may be differentiated by using
dilute acetic acid which will lyse the red blood
cells but leave the yeast intact.
Parasite
• Trichomonas vaginalis is common parasite seen
in urine. It is most frequently seen in females
but may also be seen in males.
• Trichomanas may be confused with white blood
cells.
• Flagella motility is necessary for positive
identification.
Sperm
• May be seen in urine from males or females.
• Sperm is not usually considred a clinically
significant finding.
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Bacteria
• The presence of bacteria in a fresh, clean catch
urine may indicate a urinary tract infection,
especially if white blood cells are also present.
• The most common bacteria causes of urinary
tract infection also react to cause a positive
nitrate biochemical test.
• Large amounts of bacteria without white
blood cells present may indicate poor
specimen handling.
Fibers
• Fibers may be present as contaminants
from clothing or faecal material.
• Fibers may be confused with hyaline or waxy
casts. Look carefully at the end of the fiber.
Starch
• Starch crystals may frequently be seen in
the urinary sediment as a contaminant from
powdered gloves.
• Starch crystals frequently have a characteristic
greenish appearance and a t-shaped notch in
the centre.
• Small starch crystals may be confused with
fat droplets.
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Chapter 6
Quality Assurance
in Parasitology
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STOOL PARASITES
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Stool examination flow-chart
Macroscopic examination
Note the colour and consistency
Look for adult worms and segments of tapeworms
Formed or soft stools
Liquid, watery or bloodstained stools
Examine within 24 hours! Examine within 1 hour of collection
Keep in cool place!
Microscopic examination
Search for helminths eggs or larvae and Search for helminths eggs or larvae,
protozoa cysts protozoa, Amoebic trophozoites and
Cyclospora
Report
Write the name of the intestinal parasite found and the grade of infection (few, some,
many).Report the presence of red blood cells and faecal leucocytes.
Intestinal Parasites
• Follow proper collection procedures to ensure accurate diagnosis, e.g.
amoebic trophozoites begin to degenerate within 1/2 hours after collection.
• Cysts, flagellates and eggs also undergo changes especially if the stool is left at
high temperatures.
• Label the specimen properly with patient name and lab number to avoid confusion.
• Only accept fresh specimens and refuse specimens contaminated with dirt or urine.
• If you cannot examine specimens immediately, leave them in a cool place
not exposed to sunlight.
• Always examine watery and blood-stained specimens first.
• Store Lugol’s iodine in brown bottles. Prepare Lugol’s iodine fresh every two weeks.
• Select portions of the stool that are coated with blood or mucus when preparing
the smears.
• Keep prepared slides in a wet chamber to prevent them from drying.
• Do not touch the stool or the smear with your bare fingers. Stool may
contain infectious material. (Health hazard)
• If you are in doubt about structures that resemble eggs or cysts refer to the
pictures and charts.
• Preserve the stool in 10% formalin for examination by a visiting expert or for referral
of the specimen if in doubt as to what it contains.
• Always examine the slide systematically.
BLOOD PARASITES
Sample Collection
Malaria parasites & Microfilariae
Capillary blood is usually used and the following preparations are examined:
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Direct wet preparation
• Screening for Microfilariae
Note: You can prepare a thick and a thin film on one slide together.
Ram 23
Thick Film n n
Thin Film
Sample Collection
Leishmania
It is difficult to find Leishmania amastigotes in capillary blood.
It is best to examine the layer of white cells (buffy coat) after centrifugation of EDTA
anti-coagulated blood.
Buffy coat
• EDTA blood is used for buffy coat examination.
Collection time
The number of certain parasites in the blood depends on the time of collection.
Malaria
• Highest number of parasites is found during fever attacks and before the start
of treatment
Microfilariae
• Take the specimens at night between 10 p.m. – 2 a.m. (W.bancrofti and
Brugia malayi)
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IQC Procedures
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Medical Parasitology studies human illnesses caused directly or indirectly by parasites.
What is a parasite ?
A parasite is an organism that lives in or on a living
organism of a different species and obtains its
food from it.
Host ?
The host is the organism from which the parasite
takes is food. The definitive host carries the adult
stage of the parasite. The intermediate host
carries the larval stage of the parasite.
Vector ?
The vector carries the parasite from one host to
another, it can sometimes also be an
intermediate host.
Classification of parasites
By habitat
• Ectoparasite
• Endoparasite
By phylum
• Protozoa (which can be categorized by the
way they move.)
• Helminthes with the 3 following groups:
- Nematodes (round worms)
- Cestodes (tape worms)
- Trematodes (flukes)
What samples?
• Stool
• Urine
• Sputum
• Blood
• Cerebrospinal fluid (CSF)
• Other secretions or tissues
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How do you collect samples ?
Generally speaking, samples for parasitology do not need to be collected in a sterile
way. However if the same sample has to be examined for bacteria, it must be
collected under sterile conditions. The sample should always be sent first for
bacteriological examination before parasitological processing.
Stool
Use the right container
• Waxed cardboard box
• Tin with a lid·Plastic box
• Glass container
Things not to do
• Never leave stool specimens exposed to the air
in containers without lids.
• Never set aside stool specimens for examination
at the end of the morning (i.e. 2-3 hours later)·
Never accept stools mixed with urine.
• Never place the stool specimen container on
the examination request form.
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3. Gently pull the tape away from the slide and loop it
over the end of the spoon handle.
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Urine
In parasitology urine samples are collected to
check for filariasis or schistosomiasis.
If there are a lot of RBCs without WBCs or other
signs of infection in any urine deposit, you
should look for schistosomiasis.
• The patient should be asked to do some physical
exercises before collecting the urine.
• Collect the final urine passed.
Sputum
In rare cases sputum may reveal
amoebic abscesses of the lung.
• Collect sputum as for AFB.
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Blood
Blood is collected for research of malaria, filaria
or leishmania.
Make a thick and a thin film. (EDTA anticoagulated
blood can be used, however it is better to use fresh
blood collected from the finger prick.)
For details on the method, see the course
on Malaria.
Other
CSF and other secretions and tissues are generally collected by the doctor.
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Principle:
The stool are mixed with saturated solution of sodium chloride (increasing the
specific gravity). The eggs are lighter in weight and float to the surface where they
can be collected.
Materials:
• 10ml penicillin vials
• wooden applicator
• coverslip
• ethanol
• petri dish
• saturated saline solution
Reagent preparation:
• Saturated sodium chloride solution
Sodium chloride....................................................................... 125 g
Distilled water ........................................................................ 500 ml
Dissolve the sodium chloride by heating the mixture to boiling point. Leave standing
to cool. Check that some of the salt remains undissolved. If it has all dissolved add
an extra 50 g. Filter and store.
Method:
1. Prepare grease-free coverslips by cleaning them
with 95% ethanol. Air dry completely.
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• Recommended for:
- all eggs
- larvae
- cysts of protozoa
• NOT suitable for:
- motile forms of amoebae
- flagellates
Materials:
• Electric centrifuge
• 15 ml conical centrifuge tubes with caps
• Funnel
• Gauze
• Graduated cylinder
• Wooden sticks
• Formaldehyde solution (10%)
• Pure ether
• Lugol iodine solution
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Reagent preparation:
Method:
1. Take 1 cm2 of stool. Crush and mix it in 10ml
of 10% formaldehyde solution.
2. Stir the mixture well and let it stand for 5 minutes.
4. Add 3 ml ether.
WARNING: Ether is highly flammable. Make
sure there is no open flame in the laboratory.
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Materials:
• Microscope slides
• Coverslips, 20 mm x 20 mm
• Wooden applicators or wire loops
• Sodium chloride solution
• Lugol iodine solution, diluted 1:5
Reagent Preparation
• Sodium chloride solution (8.5 g/L)
Sodium chloride........................................................................ 8.5 g
Distilled water ...................................................................... 1000 ml
Mix sodium chloride until fully dissolved.
• Lugol iodine solution
Iodine ........................................................................................... 1g
Potassium iodide (KI) .................................................................. 2g
Distilled water ........................................................................ 100 ml
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Method
1. Take a slide and put :
• 1 drop of sodium chloride on the left half
• 1 drop of the iodine solution on the right half
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A. Characteristics Of Eggs
Eggs laid by parasitic worms and found in stools
are identified by:
• size
• shape
• shell
• content
and occasionally by
• colour
• external features
For example: the egg of Schistosoma mansoni
Size : 150 mm
Shape : oval
Shell : external shell, thin and turgid;
internal shell, thin, membranous
and less distinct
Content : 1 ciliated embryo
Colour : pale yellow
External feature: 1 lateral spine
B. Size of Eggs
The size can be estimated by comparison with
that of a red blood cell, which measures 7.5-8 mm
1 micrometre (1 mm) = 1/1000 of a mm
The size in mm given in this manual is that of
the long side of the egg.
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DUODENALE
MOUTH INTESTINE RUDIMENDOD GENITAL ORGAN
ESOPHAGUS
STRONGLYLOIDES
EMBRY STERCORALIS
WITH POLAR GLOBULES
ONATED
HETERODERA MARIONI
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Hook worm
1. Ancylostoma duodenale
Size : 50-60 µm
Shape : oval
Shell : very thin, appears as a black line
Colour : the inside cells are pale grey
Contents : varies as the egg matures
Type A (fresh stool): 4,8 or 16 granular cells
Type B (stool a few hours old): uniform mass
of many small grey granular cells
Type C (stool 12-48 hours old): the egg is filled with
a small larva folded around itself
Thread worm
1. Strongyloides stercoralis
The larvae are highly motile in the stools.
Size : 200-300 µm, 15 µm thick
Tail : tapered
Mouth : short, 4 µm
Genital part : long, ~22 µm
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Round worm
1. Ascaris lumbricoides
There are four types of Ascaris eggs:
Type A : Fertilised eggs with double shell
Size : about 60 -70 µm
Shape : oval or sometimes round
Shell : There are two distinct shells
• External shell - rough, brown, and covered
with little lumps
• Internal shell - smooth, thick, colourless
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Pin worm
1. Enterobius vermicularis/Oxyuris
Size : 50-60 µm
Shape : asymmetrical oval which is flattened
on one side and rounded on the
other
Shell : smooth and thin, but a double line is
visible
Contents :
Type A : small, granular, oval mass
Type B : small folded larva
Colour : transparent, colourless
Whip worm
1. Trichuris trichiura
Size : 50 µm
Shape : large lemon-shaped
Shell : very thick, smooth with two layers
Colour : brown shell, yellow contents
Other features : transparent plug at each pole
Contents : uniform granular mass
IMPORTANT : Specify whether there are many or
few whip worm eggs present.
Schistosomes
1. Schistosoma haematobium
Eggs are found in urine, and occasionally in stools.
Size : 120-150 µm
Shape : oval; poles are different
Spine : terminal
Shell : smooth, very thin
Colour : grey or pale yellow
Contents : well-formed, ciliated embryo,
surrounded by an internal
membrane
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2. Schistosoma mansoni
Size : 150 µm
Shape : oval; one pole more round than the
other
Spine : lateral spine
Shell : smooth, very thin
Colour : pale yellow
Contents : ciliated embryo, surrounded by a
membrane
3. Schistosoma japonicum
Size : 70-80 µm
Shape : oval, almost round
Colour : transparent or pale yellow
Spine : difficult to see, lateral and very small
Contents : ciliated embryo
Tape worms
1. Taenia saginata (beef tape worm)/ Taenia
solium (pork tape worm)
The eggs of these two species are almost identical.
Size : 30-40 µm
Shape : round
Shell : very thick, lined, and smooth
Colour : yellowish brown shell, light yellowish
grey contents
Contents : granular mass surrounded by a
membrane containing 3 pairs
of hooklets
External sac: sometimes the egg is enclosed in
a transparent sac
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Flukes
1. Fasciola hepatica (Giant liver fluke)
Size : 130-150 µm
Shape : oval with rounded poles
Shell : smooth and fine, with double line
Colour : yellow to dark brown
Other features : operculum may be seen at one
pole; thickening at the other pole
Contents : a large mass of cells that are
not clearly defined. Cells
appear granulated.
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Size : 100-200 µm
Shape : oval or rectangular with rounded
corners
Colour : yellow
Contents : transparent with no granulations;
lines may or may not be present
3. Soaps
Size : 20-100 µm
Shape : round, oval, or irregular
Colour : brownish yellow or colourless
Contents : lines around the edge, pointing
inwards; nothing in the centre
4. Air bubbles
Size : variable, can be any size
Shape : perfectly round
False shell : a circular ring, very shiny (several
rings in the case of oil bubbles)
Contents : none
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5. Plant hairs
Size : very variable, 50-300 µm
Shape : rigid, often curved; clear cut at
one end and tapered at the other
Colour : pale yellow
Contents : a narrow, empty central tube
between two transparent
shiny layers
WARNING : Do not mistake plant hairs for
Strongyloides larvae
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Examination
1. Examine a chain of segments to identify the
arrangement of the pores along the side of
the segments.
2. Examine a single segment gently
flattened between two slides.
3. Hold the slide against the light to count the
number of uterine branches with the naked eye.
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s
h
t
n
o
m
2
n
a
m
n
larvae penetrate mucosa, enter
i
e l
c
y lymphatics and venules, to right
c
n heart and lungs break out into
o
i
t
Alveoli, mount twice, ascend
a
r
u
respiratory tree, descend
oesophagus to mature in the
t
a
M
intestine.
Adult life span1 0
-
1
2
m
n
h
s t
a
rs
e
Eosinophilia
y
-
s
n
th
o
m
le
b
ia
V
.
s
k
e
e
w
2
-
1
in
re
a
tu
OVA
ADULTS
150 - 200 X 2 - 4 mm.
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The Hookworms
Ancylostoma duodenale Necator americanus
Pneumonitis
Old world
New world
Occult
blood s
y
a
d
5 Eosinophilia
3
Bursa n Anaemia Bursa
a
Dorsal ray, shallow cleft, tips m Dorsal ray, deep cleft, bifid tips
tridigitate i n
Ovum f
m
r
Life size
8 - 11 x 0.45 mm. 10 - 13 x 0.6 mm.
250 µm -700 µm
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Strongyloides stercoralis
Rhabditiform larva
250 x 20 µm
Filariform larva
600 x 20 µm
Larvae mature in
duodenum (or
bronchus ) Fertilised enters mucosa,
lays eggs which hatch to
rhabditiform larvae, these
then make their ways to bowel
lumen
s
y
a
d
Eosinophilia
7
1
n
a
m
i
n
e
g
a
t
s
n Rhabditiform larvae
o
Enter circulation i
t
a
and via heart, lungs, r
Fil
Survive weeks
Fil in soil
Under unfavourable New host Same host Same host
Rhab
F skin Bowel wall
environment
i
l
conditions meta-
marphose to infective h 12 - 24 h h
a
R
R
filariform larvae a b
Free living b
12 - 24 h
mou
lts
Direct cycle Direct cycle Autoinfection Hyperinfection
Indirect cycle
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Maturation
in man
3 months
Contaminated
soil, food, etc.
Mainly caecum
Ovum
50 - 22 µm
Life size
35 - 50 mm.
30 - 45 mm.
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Life cycle
5 - 10 metres
Strobila
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Life cycle
Ovum
31 - 43 µm
Human cystricercosis Infection with adult
AUTO - INFECTION
Cysticercus is liberated,
scolex evaginates, attaches
itself to mucosa of small
intestine. Develops to adult.
Maturation time 3 months.
Life span up to 25 years.
Development of cysticercus
(Cysticercus cellulosae - 5 x 8 - 10mm.)
2 - 8 metres.
4 suckers - 2 rows of
large and small hooks
25 - 30 7 - 12 uterine
branches on
Section of human brain showing each side.
viable larva of T. solium
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Dwarf Tapeworms
Hymenolepis nana (Vampirolepts nana)
Adult
4 Suckers 20 - 30 hooks
Autoinfection Ova ingested in
in children contaminated food
via hands etc. 40 X 0.5 - 0.9 mm.
Life cycle
200 segments
No intermediate host required
(RODENTS) Segment
45 X 35 µm
Liberated embryo penetrates villus
and becomes cysticercoid in 4 days.
Cysticercoid re-enters lumen, attaches
itself to mucosa nd develops into
adult worm in 10 - 12 days
70 X 50 µ
NO polar
filament
(hexacanth
embryo)
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Cytoplasm
(ecto and endoplasm)
Nucleus
Pseudopodium (used for movement)
Ectoplasm
Endoplasm
Vaculoes
Contents:
a) red blood cells
b) bacteria, yeast cells, debris
Nuclear membrane
Flagellum
Undulating membrane
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Cytoplasm
Nuclei
Nuclear membrane
(chromatin)
Regular, thin Irregular,
chromatin rough chromatin
Karyosome
Small, central Large, off-center
Karyosome karyosome
Chromatoid body
(shiny)
Rounded Sharp
chromatoid body chromatoid body
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Amoebae
1. Entamoeba histolytica (dysentery amoeba)
This amoeba can cause dysentery or intestinal abscesses. It is the only
intestinal amoeba that causes disease in man. E. histolytica can be found in
liquid or diarrhoeal stool.
TROPHOZOITE
• Size :12-35 µm (about as long as 3 to 4 red blood
cells)
• Shape :when moving, irregular and changing;
when not moving, round
• Motility :moves in one direction
• Cytoplasm :
- Ectoplasm = transparent
- Endoplasm = greyish and granular, may have
yellowish-green areas that contain vacuoles
• Nucleus : not seen unless stained with iodine;
nucleus has a single circular
membrane with a black dot in the
centre (karyosome)
• Vacuoles : contains red blood cells
CYST
• Size : 12-15 µm
• Shape : round
• Nuclei : 1-4 nuclei
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2. Entamoeba coli
TROPHOZOITE
• Size : 20-40 µm
• Shape : oval or long oval, irregularly-shaped
• Motility : often not moving, or moving very
slowly; pseudopodia go in all
directions
• Cytoplasm : ectoplasm and endoplasm look the
same; both are granular
• Inclusion bodies : numerous and varied (bacteria,
yeast cell, debris), but there are
never any red blood cells
• Nucleus : visible without staining;
membrane is irregular and looks like
a beaded necklace, the karyosome
is large and off-centred
CYST
• Size : 12-20 µm
• Shape : round or slightly oval, sometimes
irregular
• Nuclei : 1-8 nuclei
- membrane : irregular, thick in parts like a
beaded necklace
- karyosome : large, not clearly seen, off-centred
• Cytoplasm : (iodine) yellow, brighter than in E.
histolytica
• Chromatid bodies : sharp, knife-shaped or
needle-shaped; not found in all cysts
• Vacuole: sometimes a large vacuole is seen
in between two nuclei
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Comparison of Entamoeba histolytica and Entamoeba coli
TROPHOZOITES E. histolytica E. coli
Motion in one direction in all directions
Motility motile not usually motile
Ectoplasm transparent; different from little or no difference
endoplasm between the endoplasm
and ectoplasm
Inclusion bodies red blood cells bacteria; yeast cells;
debris; NO red blood cells
Nucleus (fresh state) invisible visible
Nucleus (iodine) regular membrane; small, irregular membrane;
dense, central karyosome large, off-centred
karyosome
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Differences Between Amoebic (Entamoeba histolytica)
and Bacterial (Shigella dysenteriae) Dysentery by Stool
Examination
Amoebic dysentery Bacterial dysentery
Naked eye
pH reaction Acidic alkaline
smell extremely strong usually does not smell
stool always present maybe absent; blood and
mucus only
mucus small amount large amount
Microscopic
Bacteria (1) Numerous may be few
Pus cells (2) few, cells intact very numerous, cells
damaged
Red blood cells (3) often in rolled formation scattered
(rouleaux)
Large macrophages (4) None may be numerous; may
have ingested red cells
(do NOT mistake for
amoebae)
Charcot-Leyden crystals(5) may be present absent
E. histolytica (6) Present absent
Amoebic Dysentery Bacterial Dysentery
6 1 2 1
2 3 4
5 6 3
4
2
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Flagellates
1. Giardia lamblia
TROPHOZOITE
• Size : 10-18 µm
• Shape :
- Front view : tear drop-shaped; numerous
pairs of flagella
- Side view : spoon-shaped
• Motility : moves in one direction, sometimes
turning in a loop; movement is only seen in
fresh liquid stools
• Contents : two large, oval nuclei; slightly visible
CYST
• Size : 8-12 µm
• Shape : oval
• Shell : thick shell with a double wall
• Nuclei : 2-4 oval nuclei
- membrane : very fine
- karyosome : small, central, slightly coloured
• Cytoplasm : clear, shiny when unstained; pale
yellowish-green (iodine)
• Fibril : shiny, looks like hair; can be folded in half
or S-shaped
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2. Trichomonas hominis
T. hominis is the most resistant flagellate, it can
be found as a trophozoite even in old stools.
TROPHOZOITE
• Size : 10-15 µm
• Shape : oval with two pointed poles; usually four
flagella
• Motility : turns in all directions with vibrations
• Undulating membrane : on one side only;
moves in a fast wave-like manner
• Nucleus : one nucleus, difficult to see
3. Trichomonas vaginalis
IMPORTANT: T. vaginalis is found only in urine or
genital discharge specimens.
• Size : 15 µm
• Shape : round; 4 flagella on one end
• Motility : motile in fresh urine, turning motion
• Undulating membrane : on one side only
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Cilates
1. Balantidium coli
B. coli is a very common parasite in pigs. It rarely
causes disease in humans. When in humans, it
is associated with pig farming.
TROPHOZOITE
• Size : very large; 50 µm (similar size to
Ascaris egg)
• Shape : oval; transparent
• Cilia : covered with very small cilia which move
very quickly
• Motility : moves very rapidly in stools; moves in
definite direction; sometimes turning in circles
• Nucleus : large kidney-shaped nucleus next to
a small round nucleus
• Mouth : has a “dent” on one side that is used
for feeding
CYST
• Size : 50-70 µm
• Shape : round
• Shell : thin, double wall
• Nuclei : one large kidney-shaped nucleus, one
small nucleus like a thick dot
• Cytoplasm : granular, greenish, filled with
inclusion bodies
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Plasmodium vivax
• The first symptoms usually occurs from 7-10 days after a mosquito bite.
• Sometimes, symptoms such as headache, sensitivity to light, muscle aches, loss
of appetite, nausea, and vomiting may occur before parasites can be detected in
the bloodstream.
• Sometimes parasites can be found before symptoms occur.
• During the first few days, the patient may not exhibit regular fever cycles but may
have a low grade fever. Once established, fevers recur in a cycle of 48-hours.
• Relapses may occur after weeks, months, or up to 5 or more years.
• P. vivax affects only the reticulocytes (immatue RBCs), therefore the presence
of parasites is usually limited to a maximum of 2% of the available RBC’s.
• Enlargement of the spleen occurs during the first few weeks of the infection, and
the spleen will progress from being soft and palpable to hard.
• A decreased Total Leucocyte Count is usually present, but sometimes an increased
TLC is present during episodes of fever.
• Serum potassium may be increased as a result of RBC lysis.
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Plasmodium falciparum
• Invades all ages of RBC’s and the proportion of infected cells may be between
30 and 40%.
• Onset of an attack occurs from 8-12 days after infection and is preceded by 3-4 days
of vague symptoms such as pains, headache, fatigue, loss of appetite, or nausea.
• The onset is characterised by fever, a more severe headache, and nausea
and vomiting.
• If a fever cycle is established it is less than 48hrs.
• Severe or fatal complications of P.falciparum malaria can occur at any time during
the infection and are related to the plugging of vessels in the internal organs by the
multiplying merozoites (see Fig. 1)
• Disseminated Intravascular Coagulation – vascular endothelial damage from
endotoxins and bound blood cells containing parasites may lead to clot formation
in small vessels.
• Cerebral malaria is most often seen in P. flaciparum malaria. If the onset is
gradual, the patient may become disorientated or violent or may develop severe
headaches and pass into coma.
• Extreme fevers, 107oF (41.7oC) or higher may develop. Without vigorous therapy,
the patient usually dies.
• Remittent fever involves the liver with symptoms including abdominal pain,
nausea, and severe and persistent vomiting containing evidence of bile and fresh
blood. There may be severe diarrhea or dysentery with resulting dehydration. The
liver is large and tender, the skin becomes yellowish, and the urine contains bile.
• Diarrhea or dysentery without liver involvement or yellowing of the skin may be seen.
• Blackwater fever, which is often fatal, can occur in patients with a history of
previous malarial attacks. Sudden, intravascular haemolysis results in a dramatic
colour change in the urine.
• Acute renal failure may also occur.
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• Always prepare blood smears before antimalarial drugs have been given.
• Stain thin and thick films with Giemsa stain. However, Wright’s or Wright-Giemsa
stain can also be used.
• For thin films, 200 – 300 oil immersion fields should be examined before the
blood film is considered negative. NOTE: The presence of a small number of
malaria parasites in the blood is easily missed.
• Additional blood specimens should be examined over a 36 hr time frame, since
one set of negative films will not rule out malaria
• Note the following information which will be very useful in the diagnosis of malaria:
- Has malaria been diagnosed in the patient before? If so, what species
was identified.
- What medication (prophylaxis or other) has the patient received, how often
and when was the last dose?
- When was the blood specimen drawn and was the patient symptomatic at
the time? (E.g. with fever).
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Malaria : Laboratory diagnosis - Preparation of thin films
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Preparation of thin and thick films on a single slide
During the malaria season, there are a large number of patients needing to be
screened for malaria. During this period it is possible to make the thin and thick films
on a single slide as shown below:
Place the ingredients in a bottle containing glass beads and shake. Shake the
bottle 3 times a day for 4 consecutive days. Filter.
• Measuring cylinders (10, 100 ml)
• Methanol in drop bottle
• Buffered water
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Procedure
1. Mix 1.5 ml Giemsa stock solution in 50 ml of
buffered water (3% solution).
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Identification of malaria parasites
Plasmodium falciparum
YOUNG TROPHOZOITE (ring form)
• Frequently found - even in light infection
• Multiple ring forms
• Double chromatin dot
• ring form lying on red cell membrane surface
• Cytoplasm : small thin pale blue ring
• Chromatin : 1-2 small red dots
MATURE TROPHOZOITE
• Not usually seen
SCHIZONT
• Not usually seen
GAMETOCYTE
• Fairly frequently found in severe infections
• Shape : like a banana
• Colour : reddish-blue (male) or dense blue
(female)
• Nucleus : reddish-pink
• Pigment : a few blue-black granules in the
centre of the cytoplasm or scattered
RED CELLS
• Normal in size
PARASITE DENSITY
• Often high percentage of cells infected
with parasites (30-40%)
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Plasmodium vivax
YOUNG TROPHOZOITE (ring form)
• Frequently found - even in light infections
• Ring forms are larger than in P. falciparum
• May resemble a perfectly shaped ring
• Cytoplasm: irregular blue ring, quite thick
• Chromatin: 1 rather large dot
MATURE TROPHOZOITE
•· Sometimes seen
• Cytoplasm : large, blue, irregular
(sometimes divided into 2,3, or 4)
• Chromatin : 1 red dot
SCHIZONT
• Fairly frequently found in severe
infection· Merozoites 12-24
GAMETOCYTE
• Fairly frequently found in severe infections
• Female : oval or rounded, dense blue; dense red
triangular nucleus often at one end; many
orange pigment particles in cytoplasm
• Male : rounded, pale blue; round central pale
red nucleus; some orange pigment particles in
cytoplasm
RED CELLS
• Enlarged in size
• Schuffner’s dots
PARASITE DENSITY
• Up to 2% of cells can be infected
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Plasmodium malariae
YOUNG TROPHOZOITE (ring form)
• Frequently found - even in light infections
• Bird’s eye ring form may be present
• Cytoplasm : thick, dense, blue ring with
some granules of black pigment
• Chromatin : 1 large red dot
MATURE TROPHOZOITE
• Cytoplasm
1. round, compact, dark blue, with many
black pigment particles;
2. in band form
• Chromatin : a round dot or red band
SCHIZONT
• Fairly frequently found in severe infections
• Merozoites 6-12
GAMETOCYTE
• Fairly frequently found in severe infections
• Shape : large, oval or rounded
• Colour : dense blue (female) or pale blue (male)
• Nucleus : 1 round red spot (chromatin) near
one edge
• Pigment : large black granules in cytoplasm
RED CELLS
• Normal size
PARASITE DENSITY
• Rarely more than 1% can be infected.
Therefore, it can be easily missed!
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Plasmodium falciparum
TROPHOZOITES
SCHIZONTS
GAMETOCYTES
Thin film Thick film
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Plasmodium vivax
TROPHOZOITES
SCHIZONTS
GAMETOCYTES
Thin film Thick film
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Plasmodium malariae
TROPHOZOITES
SCHIZONTS
GAMETOCYTES
Thin film Thick film
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Plasmodium ovale
TROPHOZOITES
SCHIZONTS
GAMETOCYTES
Thin film Thick film
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P. falciparum
Diagnostic points
1. Red Cells are not enlarged.
2. Rings appear fine and delicate and there may be several in one cell.
3. Some rings may have two chromatin dots.
4. Presence of marginal or applique forms.
5. It is unusual to see developing forms in peripheral blood films.
6. Gametocytes have a characteristic crescent shape appearance.
However, they do not usually appear in the blood for the first four weeks of infection.
7. Maurer’s dots may be present.
P. vivax
Diagnostic points
1. Red cells containing parasites are usually enlarged.
2. Schuffner’s dots are frequently present in the red cells as shown above.
3. The mature ring forms tend to be large and coarse.
4. Developing forms are frequently present.
P. malariae
Diagnostic points
1. Ring forms may have a squarish appearance.
2. Band forms are a characteristic of this species.
3. Mature schizonts may have a typical daisy head appearance with up to
ten merozoites.
4. Red cells are not enlarged.
5. Chromatin dot may be on the inner surface of the ring.
P. ovale
Diagnostic points
1. Only found in Africa.
2. Red cells enlarged.
3. Comet forms common (top right)
4. Rings large and coarse.
5. Schuffner’s dots, when present, may be prominent.
6. Mature schizonts similar to those of P. malariae but larger and coarser.
(Division of Laboratory Medicine at Royal Perth Hospital, Malaria an On-line Resource)
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Chapter 7
Quality
Assurance in CSF
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Sample collection
IQC PROCEDURES
• Always consider CSF as urgent and report the results as soon as possible
• Handle the sample carefully as CSF collection is difficult and quite often cannot be
repeated
• Physical inspection (colour, turbidity) should be performed immediately after
puncture at the patient’s side.
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