Collection, Preparation and
Presentation of Samples for and
Sources of Errors in Biomedical
Analysis/Biostatistics in Clinical
Chemistry
By N.M.Hawa
Introduction
 The Chemical Pathology Laboratory processes specimens
received from both
inpatients and outpatient
departments and other medical facilities .
 Specific rejection criteria are used to determine samples
unsuitable for analysis.
 Essential function of chemical pathology is to provide
accurate and reliable data obtained from the
examination of specimens taken from patients and to
assist in the diagnosis and effective management carried
out by the clinicians.
Do you know?
 SCENE 1 :- You have to collect blood for glucose
estimation from a hemiplegic patient with the right
healthy arm running an i.v dextrose drip. Which site will
you choose ?
 SCENE 2 :- You have to transport a 24 hr urine sample for
estimation of uric acid .Which preservative will you use ?
 SCENE 3 :- You have to send samples for study of cell
morphology. Which container will you use ?
CLINICAL CHEMISTRY SAMPLES
The most commonly received sample
Blood
Urine
Cerebrospinal fluid
Stool
other body fluids such as , saliva, pericardial,
synovial, peritoneal fluid and pleural fluid etc.
SAMPLE COLLECTION
1. BLOOD
The process of collecting a blood sample is
called as phlebotomy.
-Venous
-Arterial
-Capillary
BLOOD CONT.
A) VENOUS BLOOD; Venepuncture procedure
Use : Most common sample collected is venous
sample.
Preliminary steps:• Identify :- Name ,Age, Sex, Medical Record Number,
Date of Birth, Address(most important part of sample
collection)
• Verify tests required
• Estimate amount of blood to be drawn
• Select appropriate vacutainers
BLOOD CONT.
 Location
• Vein of choice-median cubital vein in the antecubital
fossa as the vein is large and very close to skin
• Veins on back of the hand or ankle (AVOID in diabetics
and patients with poor circulation)
• Ankle
• If it is absolutely necessary to collect blood from an
arm running fluids - SHUT OFF fluids for 3 minutes and
discard first 5ml of blood
• Use a site below infusion site as retrograde blood flow
through veins doesn’t occur.
Patient position:• Patient should be comfortable :seated or supine .
• Never perform on a standing patient.
• Arm to be extended straight from wrist to shoulder.
Wear PPE e.g. gloves, gown , masks (preventing aerosol
transmission) to prevent transmission of infection from
patient
Note - do enquire about latex allergy as gloves
Needles• 19-22 gauze commonly used (larger the gauze smaller the
bore ).
• For children use 22-23 .
• If collecting a larger volume i.e 20 – 30 ml use 18 or wider
bore needle.
Avoid Venipuncture
If;
 i.v fluid running on that side
 Hemiplegia or other sensory loss
 Infections, cellulitis
 Arterio-venous fistula
 Avoid repeated aspiration from a single arm
 Sclerosed veins : repeated transfusions , chemotherapy
TRY OTHER PLACES ARM, ANKLE VEINS, FEMORAL –TAP
BLOOD SITE PREPARATION:
 Disinfect with
a) prepackaged alcohol
b) Gauze pad saturated with 70% isopropanol
c) benzalkonium chloride solution 1:750
Cleaning of puncture site must be done in a circular
motion and from the site outward.
Skin should be allowed to dry as alcohol can cause
hemolysis and interfere with results.
Timing
• The time at which a specimen is obtained is important
for constituents that have diurnal variation e.g. cortisol
and samples of therapeutic drug monitoring .
• Timing also very important for measurement of glucose
and alcohol.( medicolegal importance concentration may
change later)
Types of timed samples:• Fasting sample is best for biochemical investigations (1214 hrs or overnight fast)
• Post prandial sample is taken 2 hours after a meal
• Random sample can be taken anytime.
BLOOD CONT.
Venous occlusion
It is done to distend the veins.
Place a tourniquet 4-6inches above an intended
puncture.
A BP cuff can be used ; the pressure kept not more
than 60 mm Hg.
It should not be kept for more than one minute
Decreased pH
Increased lactate and proteins
Increased ionized calcium
Increased potassium
Pumping and taping of hand to be avoided.
BLOOD CONT.
Seal puncture site using a sterile swab
Labelling: - Label after every collection . No
batch labelling ( Put the sticker or write neatly
on tube after collecting each sample only)
Destroy and discard needle
Autoclave all disposables
BLOOD CONT.
B) ARTERIAL BLOOD:• Use :- Blood gas analysis
• Sites: Radial artery, Brachial artery, Femoral
artery
• Transport to point of testing in
Heparinized syringe, ASAP.
!!!EVERY SEC VALUES CHANGE !!!
BLOOD CONT.
C) CAPILLARY BLOOD:- Skin is punctured
a small volume of blood is collected .
with a lancet and
• Uses:a) Limited sample volume e.g. paediatric ward.
b) Repeated venepunctures have caused severe vein damage
c) Burns or bandaged areas- veins are not available for
venipuncture
d) Blood collected on special filter papers for neonatal
screening and molecular genetics testing
• Sites:tip of a finger
An ear-lobe
Heel or big toe of infants
BLOOD CONT.
Steps: Clean site
 Puncture with a sharp stab but not deeper than 2.5cm
to avoid contact with bone
 Use different site for each puncture to prevent
infection
 Don’t press or massage as it causes tissue debris and
tissue fluid comes out
 First drop of blood is wiped off and the subsequent
drops are collected in appropriate collection tubes
 Fill rapidly as site will clot
PATIENT PREPARATION
Preparation of Patient for OGTT: The Patient is instructed to have good
 Carbohydrate diet for 3 days prior to the test.
 Diet containing about 30-50gm of carbohydrate should
be taken on the evening prior to the test.
 Patient should avoid oral hypoglycemics for at least 2
days prior.
 No Strenuous exercise on previous day.
 Patient should be on 12 hr fast 8PM to 8 AM next
morning. NO TEA –COFFEE – BISCUITS etc .
 No smoking during the test
PATIENT PREPARATION CONT.
Preparation of Patient for LIPID PROFILE : The Patient is instructed to have normal diet for 3 days
prior to the test.
 No extra butter/ghee or other oils in food. Avoid meat
and eggs day before testing.
 Patient should avoid oral cholesterol lowering
medicines (eg.STATINS)for at least 2 days prior.
 No Strenuous exercise on previous day.
 Patient should be on 12 hr fast 8PM to 8 AM next
morning.
 No smoking during the test .
Containers and preservatives for blood
URINE COLLECTION
Type of specimen: Random – obtained at anytime of the day
 Early morning sample – A clean, early morning fasting sample - most
preferred for detection of abnormal constituents like proteins, or
compounds like HCG.
 Double voided sample ; GTT. Its excreted during a timed period after a
complete emptying of bladder
 Mid-Stream sample- For bacteriological examinations, the first 10 ml
of voided urine is discarded then rest is collected. External genitalia
must be cleaned properly for such collection.
 Catheterisation of the bladder through the urethra for urine collection
i.e., in a comatose or confused patient. This procedure risks
introducing infection and traumatizing the urethra and bladder, thus
producing iatrogenic infection or hematuria.
• 24-HOUR SAMPLE - Obtained by collecting urine excreted in 24
hours. E.g. 8am of one day to 8am of next day discarding early
morning sample of first day. (Volume - 1200 to 1500ml.)
URINE PRESERVATIVES:• Substances added to urine to reduce bacterial action or
chemical decomposition or to solubilize constituents that might
precipitate out of solution.
• Commonly used are : HCl -6mol/L -30ml per 24 hour collection (pH below 3)
Na2CO3 ,NaOH is used to preserve specimens for Uric acid,
urobilinogen and porphyrins. (pH – 8- 9). Uric acid will form
clump like precipitate with HCl.
Others; - Thymol, Acetic acid, Na2CO3 ,HNO3, Boric acid.
OTHER BODY FLUIDS
1) CSF
Uses Meningitis, CVA, demyelinating diseases like
multiple sclerosis, meningeal involvement in
malignant disease
Procedure – Lumbar Puncture
 Contraindications of lumbar puncture – A very
high intra-cranial pressure in infants and children
can cause instant death if LP is carried out as CSF
gushes out too fast and brain stem herniation
occurs
2. Ascitic fluid
• Uses – Ascites of any cause – cirrhosis,
peritonitis etc
• Procedure -Abdominal tap at most dependent
portion at maximum dullness
3. Amniotic fluid
• Uses – prenatal diagnosis of congenital
disorders, assessment of fetal maturity & Rh
iso immunization
• Procedure -Amniocentesis
4. Effusions-Ascites, pleural OR pericardial:
Fresh fluid specimen should be transported
promptly to the lab.
• If it cannot be immediately delivered,
preserve in heparin to prevent clotting, and
place in refrigerator until it can be delivered.
• Female genital tract – Monolayer Paps:
Obtain an adequate sample.
• Procedure; Rinse the spatula after use in
preservative solution . Tighten the cap on the
vial and label it with the patient’s name before
placing it in a transport bag.
• Use ; HPV, GC and CT testing.
FAECES
• Uses–
1) Feacal fat – obstructive jaundice and
malabsorption
2) Parasites – microscopy
3) Heme and occult blood – for GI bleeding
4) Feacal nitrogen- malabsorption
• Care to be taken to prevent contamination with urine
during collection.
• Usually fresh specimens to be used to avoid need for
preservation.
BIOCHEMICAL ERRORS IN CHE.
PATHOLOGY
Pre-Analytical Errors (46 – 68.2%)
• Insufficient sample
• Sample condition
• Sample handling
• Incorrect Identification
• Incorrect sample
BIOCHEMICAL ERRORS CONT.
 Analytical errors
• (7 – 13.3 %)
• Test System Not Calibrated
• Results reported when control results out of range
• Improper measurements of specimens and/or reagents
• Reagents prepared incorrectly
• Reagents stored inappropriately or used after expiration
date
• Instrument maintenance not dance
• Dilution and pipetting err
• Inaccuracy
Invalidates reference ranges and cut-off points
May lead to inappropriate therapy or failure to treat
May lead to misdiagnosis
May lead to failure to diagnose
• Imprecision Issues
Poor reproducibility invalidates patient monitoring using
laboratory results
• Insensitivity
 A change in assay sensitivity leads to issue of detection
 Affects robustness of results near the detection limit
• Linearity Issues
 A clear understanding of the linearity of a method is
essential to ensure that grossly inaccurate results are not
reported where there are high concentrations or activities
of analytes
Post-Analytical Error
•
•
•
•
•
18.5 – 47 %
Transcription errors in reporting
Report sent to the wrong location
Report illegible
Report not sent
Impact of Errors
•
•
•
•
•
Inadequate or improper patient care
Inconvenience to patient
Misdiagnosis
Harm to patient
Death
How to avoid such!!!
Quality assurance
• Quality assurance has two components;
- Internal Quality Control (IQC) ; set of procedures
undertaken by the HC professionals in their day to day
activities to ensure release of reliable biomedical
analysis results.
- External Quality Assessment (EQA; Tool for assessing
implementation of an IQC programme and is aimed to
improve performance.
Characteristics
 Done by an independent agency for comparisons.
This assessment in contrast to IQC is retrospective
and periodic.
Essential elements of a quality
assurance programme
Commitment
 Facilities and Resources
Technical Competence
Technical Procedures; Accurate technical
procedures are necessary to provide quality
results. Three groups of procedures are as follows.
1. The pre-analytical procedures or variables
2. The analytical procedures or variables
3. The post- analytical procedures or variables
Problem solving mechanism to provide the link
between the identification of a problem and the
implementation of a solution to that problem.
The basic quality control
statistics
Biostatics Measures
 Measures of central tendency; Variable usually has a point
(center) around which the observed values lie. The three most
commonly used averages are:
 The arithmetic mean: The mean (X) is the sum of the control
observations (x1, x2, ... xi) divided by the number (n) of
observations
• The Median; It is the middle observation in a series of
observation after arranging them in an ascending or descending
manner.
-The rank of median for is (n + 1)/2 if the number of observation
is odd and n/2 if the number is even
• The Mode; The most frequent occurring value in the data is the
mode
Biostatics Measures cont.
• Measures of Dispersion; The measure of
dispersion describes the degree of variations
or scatter or dispersion of the data around its
central values:
– Range - R
– Variance -V
– Standard Deviation - SD
– Coefficient of Variation -CV
Range
• is the difference between the largest and
smallest values and is the simplest measure of
variation.
• Disadva. ; it is based only on two of the
observations and gives no idea of how the
other observations are arranged between
these two.
• Also, it tends to be large when the size of the
sample increases
Variance
• Average of differences between the mean and each
observation in the data, reduce each value from the
mean and then sum these differences and divide it by
the number of observation.
Variance V = Σ (mean – x) / n
• The value of this equation will be equal to zero because
the differences between each value and the mean will
have negative
• To overcome this zero, square the difference between
the mean and each value so the sign will be always
positive
V = Σ (mean – x)2 / n - 1
Standard Deviation (SD)
• It is the commonly used measure of dispersion
around the mean
• Calculation:
– X ± 1SD includes 68% observations
– X ± 2SD includes 95% observations
– The higher the SD, the more the observation varies
(deviates) from the mean.
SD = √ Σ (mean – x)2 / n - 1
68-95-99.7 Rule
• For any normal curve with
mean mu and standard
deviation sigma:
• 68 percent of the observations
fall within one standard
deviation sigma of the mean.
• 95 percent of observation fall
within 2 standard deviations.
• 99.7 percent of observations
fall within 3 standard deviations
of the mean.
Coefficient of
variation
• The coefficient of variation expresses the SD
as a percentage of the sample mean.
C. V = SD / mean * 100
• C.V demonstrates the relative size of the
variability in the data.
How Are These Values Used?
• Mean and SD are calculations that assess the accuracy
and precision of the analysis statistically.
• Errors of accuracy may be assessed by examining
changes in the measured concentration of the control
over time and comparing these concentration values
to mean and SD ranges of the control.
• By contrast, an imprecision problem will be
demonstrated by an increase in the SD and %CV of
results of the control concentration over time.
THANK YOU!!!!!!!!!!