CLINICAL CHEMISTRY
Purpose of the Clinical Laboratory Test:
 To evaluate the pathophysiologic condition of a
patient
 To assist with diagnosis
 To guide or monitor therapy
 To assess risk for a disease or progression of a
disease
Quality Control (p.19)
- It is a system of ensuring accuracy and precision
in the laboratory by including quality control
reagents in every series of measurements.
 To ensure the accuracy and precision
inside the clinical chemistry section)
 What are the quality control reagents
that must be included every time there
are measurement.
- May be defined according to section.
- A process of ensuring that analytical results are
correct by testing known samples that resemble
patient samples.
 Ultimately PREVENT the reporting of
inaccurate patient test results.
THERE ARE ONLY 2 SAMPLES in CC:
1. Known samples
a) Standard samples- The most specific
analytical sample in CC.
- Only contain 1 analyte
- On the label of the vial is the
absorbance of the standard
- COLOR: Colorless/ clear
b) Control samples- measured together
with standard and unknown samples.
- To know if the patient result is with
accuracy and precision.
- Several analytes
- In control, release it. Outside
control, hold and repeat test.
- Resembles patients’ samples
- Ensures that analytical results are
correct.
- COLOR: Yellow and same
transparency of human serum
2 sources:
 Human-based control sera
 Non- infectious
 Non- hemolyzed
 Non- icteric

Bovine-based control sera
 Cow
 Goats
 Sheeps
i.
Pathologic samples (ABNORMAL)
ii.
Normal Samples
2. Unknown sample (Patient’s sample or
specimen)
UNKNOWN SOLUTION: Au x Cs
As
Cs- Concentration of the standard
Au- Absorbance of the unknown
As- Absorbance of the standard
NOTES TO REMEMBER:
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Most commonly used patients’
sample in clinical chemistry: SERUM
Only samples that are icteric:
SERUM and PLASMA
Ictericia- HYPERBILIRUBINEMIA
Icteric serum- DARK YELLOW TO
ORANGE
Parameters of quality control
1. Sensitivity- ability to measures the smallest
concentration of analytical method.
2. Specificity
3. Accuracy
4. Precision or reproducability- ability of an
analytical method to give repeated results that
agree to one another. (CLOSENESS OF
AGREEMENTS)
3 components of the analytical chemistry that must
be specific and sensitive:
1. Reagents
2. Equipment/ machine
3. Procedures
Kind of Quality Control
1. Intralab QC (Internal QC)
- Analyses of control samples together with the
patient specimens.
- Daily monitoring of accuracy and precision of
analytical methods
-
-
-
Detects random and systematic errors within a
one- week cycle.
To ensure release of current patient results
The initial control limits are established by
analyzing pool material in 20 consecutive runs
and then are reevaluated periodically.
After internal QC test, no results for a given
analyte are to be reported that has been
declared “out of control”.
The QC results are checked after each run using
a multi-rule.
2. Interlab QC (External QC)
- It is a proficiency testing program which
provides unknown samples to participating
laboratories.
- CAP proficiency program- is the gold standard
for clinical laboratory external QC testing
- To maintain long- term accuracy of the
analytical methods.
- To compare performance between laboratories
Conduct of External QC Test
Unknown samples:
-
-
Must be tested by the laboratorians who
regularly perform analysis of patient specimens
using the same reagents and equipment for
actual patient specimens.
Should be processed and treated like a patient
specimen to determine the true essence.
Interpretation of the Results of the
Proficiency Testing
Quality Control
NOTES TO REMEMBER:
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12 unknown samples (1 sample each month)
Lung center is the reference laboratory for
unknown samples in external QC in Clinical
Chemistry.
Human serum sample
Process the same time of the month and every
morning.
The releasing is every month
Only process the routine test (atleast 20 sample
requests in 24 hrs)
6 universal routine tests in CC:
 Glucose
 Cholesterol
 Triglycerides
 Urea/BUN
 Creatinine
 BUA
Ultimate goal of proficiency testing: to ensure
our clinicians that patient results are accurate
ISO 15189:2007- It has been adopted by CAP in
an effort to improve patient care through quality
laboratory practices

SPECIMEN/SAMPLE COLLECTION
Proper patient identification is the 1st step in
sample collection.
- International guidelines toand most healthcare
organizations. Requires at least 3 identifiers to
confirm patient ID
 Patient’s complete name
 Birthdate
 Hospital ID/ Patient record number
-
Venipuncture
Povidone iodine- contaminated with B.
cepacia (non fermen gram - bacilli)
2. Evacuated blood collection tubes
- Contaminated with S. marcescens and
Moraxella spp.
NOTE:


E. coli is not an enteric pathogen but an
opportunistic pathogen.
P. aeruginosa- most popular non fermentative
gram negative bacilli
Best site: Median cubital vein
Transillumination (IR light)- Hemoglobin in the blood
within the veins absorbs the light, causing the veins to
stand out as dark lines. 7 inches over the phlebotomy
site.
Note: TOURNIQUET APPLICATION
-
-
3-4 inches above site <1 minute
Blood pressure cuff, 60 mmHg
When a tourniquet is used during preliminary
vein selection, it should be released and
reapplied after 2 mins.
Reusable tourniquet SHOULD NOT BE USED bc
it has potential to transmit bacteria (MRSA)can cause organ dysfunction (Staph aureus)
DISINFECTION OF THE SITE FOR PUNCTURE
-
Traces of alcohol may cause hemolysis
Benzalkonium chloride- skin cleanser for
ethanol testing
Insert the needle bevel up at a 15-30 degree
angle
Chlorhexidine gluconate- is the recommended
skin disinfectant for BLOOD CULTURE, for
infants 2 mos and older. If less than 2 mos,
alcohol swab.
OTHER SOURCES OF CONTAMINATION CAUSING
PSEUDOBACTEREMIA:
1. IV catheters and various skin disinfectant
solutions
 Benzalkonium chloride- occasionally
contaminated with B. cepacia and
enterobacter.
ORDER OF DRAW
1. Yellow top/ Blood culture/ Sterile tube
- SPS (Sodium polyanethol sulfonate)
2. Light blue top- Citrate, for coagulation studies
3. Serum tube- with or without clot activator or
gel separator. (Orange tube- RST, Rapid serum
tube with < 5 mins)
4. Green top-Heparin, with or without gel
separator (dark green- without) (Light greenwith gel separator)
5. Lavender/pink/pearl- EDTA, blood bank. (Pink
for ABO blood typing, Cross matching) (White
pearl top for molecular test)
6. Gray top
NaF and K oxalate
- Iodoacetate and heparin
NOTE:
 Plastic tubes- di potassium EDTA (spray dried)
 Glass tubes- tri potassium EDTA (liquid form)
 Pink top- di potassium EDTA
 Only blood culture tubes, glass non additive
serum tubes, or plastic serum tubes without a
clot activator may be collected before the
anticoagulant activate.
 Date, time initals of phlebotomist must be on
the label.
 Preprinted bar code label applied after proper
patient ID and after the specimen is collected to
prevent preanalytic transcription errors
 Therapeutic drug monitoring should not be
collected in SST
- Some gels absorb certain drugs
 Use of NaF
- Fluoride- has little effect on reducing glycolysis
within the first hour of storage and may not
reach complete inhibition until 4 hours of
storage.
- for lactate sample= fluoride+ oxalate black
glycolysis
- for ethanol test
CLOT ACTIVATORS


Thrombin, silica and diatomite (celite)
Thixotropic gel
ORDER OF FILLING MICROCOLLECTION TUBES:
1. Lavender (EDTA)
2. Tubes with additives
3. Non additive tubes (serum tube)
Arterialized Capillary Blood
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Purpose: pCO2 and pH analyses
Patient: Newborn and infant
Preferred site: Earlobe
Common site: Lateral/medial heel surface
Procedure: warm the site 39-42 degree Celsius
for 3 mins.
Arterialization- warming the site
REASONS FOR RAPID SEPARATION OF BLOOD:
1.
2.
3.

To prevent glycolysis
2 mg NaF/ mL of blood- prevents enolase
To prevent shift of electrolytes
To prevent hemolysis
Increased conc. of
LD (erythrocytic) (most abundant enzyme), ACP,
transaminases
 K+ (most abundant ion), Mg+, P+. Fe +
 Total protein, albumin
LIPEMIA
DISCARD TEST TUBE
-
-
Is normally needed only when the sample is
drawn from a winged set or intravenous
catheter.
Clear top or cover
CAPILLARY PUNCTURE (SKIN PUNCTURE)
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1.75 mm- length of lancet
<2.0/<2.5 mm- incision depth
-
Elevated conc of lipids in plasma
It occurs when TAG exceeds 400 mg/dL
Lipemic plasma and serum- MILKY
It inhibits AMS, urate, urea, CK, bilirubin, Total
bilirubin
Requires chilling or “cold incubation”
-
Blood pH and gases, ammonia, hormones (PTH,
catecholamines, gastrin)
Lactate, renin any pyruvate
BIOTIN INTERFERENCES
-
Can create analytic interference in biotin-based
immunoassays
Increased levels of biotin in plasma leads to:
False elevations: T3 and FT4, Vit D conc
-
Biotiin ibterferes with immunoassays that use
stratavidin and biotinylated AB
-
It inhibits or enhances competitive binding
assay
( Free biotin in the px serum binds to the
streptavidin and block the binding of the
capture Ab to the beads resulting
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INTERFERENCES IN MOLECULAR DIAGNOSTICS
-
Laboratory manipulations of nucleic acids are
susceptible to interferences at various stages,
including specimens collection and processing.
RNA is labile in blood or tissues
-
Thus these spec must be stored by rapid
freezing by liquid nitrogen esp when extraction
is delayed.
CSF (Preparing for lumbar puncture)
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If possible, 3 tubes (1 ml)
Microbio, Chemistry and hema?
Protein and glucose
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CARBOHYDRATES
Hormones that promote hypoglycemia:
1. Insulin- hormone that lowers blood glucose
- Glycogenesis- process to lower blood glucose
by insulin.
Hormones that promote hyperglycemia:
1.
2.
3.
4.
5.
6.
7.
Glucagon- major promoter of glycogenolysis (1)
Cortisol and corticosteroids- gluconeogenesis
Catecholamines- glycogenolysis (2)
Growth hormone- Somatotrophic
T3 and T4 Thyroid- Glycogenolysis (3)
ACTH Adenocorticotropic hormone
anterior pituitary gland
2nd promoter of gluconeogenesis
Somatostatin
DIABETES MELLITUS
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Is a group of metabolic disorder characterized
by HYPERGLYCEMIA resulting from defects in
insulin secretion, insulin receptors or both
FBS: ≥ 126 mg/dL
A. GLUCOSURIA
- Plasma glucose level exceeds 180 mg/dL (9.99
mmol/L) with normal renal function
-
B. FULMINANT TYPE 1 DIABETES
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It is recommended that adults ages 45 and
older be screened for DM every 3 years, but
screening should be performed earlier and
more frequently if the individual is at high risk
PREFERRED TEST: Fasting plasma glucose or
HbA1C (monitoring glucose control)
SCREENING TEST: FBS
Early indicator of glomerular dysfunction
associated w/ type 1 DM.
Routine test for DM patients
(+) microalbuminuria= 2 out of 3 specimens
collected w/in a 6-month period are abnormal
Method: Random-spot albumin-creatinine
ratio
Reference value: 0-29 microliter/mg creatinine
Creatinine confirmatory test – Creatinine test
Microalbuminuria: 30-300 microliter/mg
creatinine
Clinical albuminuria: <300 microliter/mg
creatinine
C. GESTATIONAL DIABETES MELLITUS
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Formerly known as Idiopathic Type 1 DM or
type 1b.
Strongly inherited and associated with the
absence of beta cell autoantibodies
(-) beta cell autoantibodies
(+) rapid and complete beta cell destruction
MICROALBUMINURIA TEST
-
RECOMMENDATION:
FBS NORMAL RANGE: 70-99 mg/dL
Impaired ability to metabolize carbohydrate
usually caused by a deficiency of insulin or
insulin resistance
Placental hormones- promotes insulin
resistance that contributes to GDM
It occurs in pregnancy and disappears after
delivery
Diabetic women who become pregnant are
NOT included in this category
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SCREENING TEST: 2-hour OGTT (24-28 weeks of
gestation)
Method:
 One step OGTT- 75g glucose load (most
commonly used)
 Two step OGTT- 50g GLT; 100g follow-up load
GLUCOSE METHODOLOGIES:
-
If the plasma glucose level measured 1 hour after the
load is:
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≥ 130 mg/ dL (7.2 mmol/L)
135 mg/dL (7.5 mmol/L)
140 mg/dL (7.8 mmol/L)
- Step 2: Proceed to 100-g OGTT using fasting sample
D. OTHER TYPES OF DIABETES MELLITUS (OTODM)
1. DM due to pancreatic disorder
- AKA type 3C DM- pancreatogenic DM
- Chronic pancreatitis, malignancy (pancreatic
cancer), or maybe caused by pancreatectomy
2. DM related to endocrine disorders
3. DM caused by genetic syndromes
4. DM associated w/ exocrine disorders- Cystic Fibrosis
5. DM due to viral infections
6. Drug- induced or chemically-induced DM
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Glucose in whole blood is 10-15% lower than in
serum or plasma bc red cells consume glucose.
Venous plasma is recommended for diagnosis of
DM
Serum is separated from the cells within 30
mins to prevent glycolysis (7 mg/dL/hour
decrease at room temp.) (2 mg/dL/ hour
decrease at 4 degree Celsius)
NaF with citrate is preferred over oxalate
AACC and ADA recommended for blood glucose test:
-
4. Dextrostics (cellular strip)
- Glucose oxidase method embedded
- Is important for establishing correct insulin
amount for next dose
The use of citrate buffer which is considered a
rapid inhibitor.
Individual with a capillary glucose of ≥140 mg/dL
(7.8 mmol/L)- should be rescreened with a FPG.
The standard clinical laboratory analysis for glucose is
performed
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On plasma or serum deried from a phlebotomy
specimen
INTERSTITIAL GLUCOSE MEASURING DEVICE
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For the diagnosis of diabetes:
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Only plasma and never serum
Laboratory plasma glucose measurements are
recommended.
ENZYMATIC METHODS
Note: Dubowsky reaction- Glacial acetic acid reagent
1.
-
Glucose Oxidase ( Colorimetric)
Only measures the beta D glucose
Not only in blood
Routine method
2. Hexokinase Method
- The most specific method for measurement of
glucose
- Reference method for glucose
- G6PD enzyme- most specific reagent use for
glucose
- Avoids hemolysis
1.
2.
3.
4.
3. Glucose Dehydrogenase Method
- Provides results in close agreement with
hexokinase procedures (GDH method)
 Isotope dilution gas chromatography
For continuous monitoring of glucose levels in
persons with DM
It utilizes electrochemical process to
automatically measure glucose levels in the
interstitial fluid of dermis
Only supplemental and cannot replace
conventional home blood glucose monitoring
SAMPLES FOR ANALYSIS
Random Blood Sugar
- Emergency purposes (lost of consciousness,
insulin shock)
- If RBS ≥160 mg/dL (8.9 mmol):
FPG should be performed
Fasting Blood Sugar/ FPG
- It is the best indication of overall glucose
homeostasis
- Measures the relationship of Insulin and
glucagon by glycogenesis.
2- Hour Post Prandial Blood Sugar
- Evaluates hyperglycemia and hypoglycemia
- 2 hrs after eating; If standard, just once
- Modified: 75g glucose
- If 2 blood draws, 2HPPT + FBS
- Anytime of the day
- ≥140 mg/dL= hyperglycemia
Glucose Tolerance Test
- Multiple blood sugar test
- Aids in the diagnosis of DM and GDM
- Confirmatory test for impaired fasting glucose
- >126 mg/dL= DO NOT proceed with the test
- REQUIREMENTS FOR OGTT:
 Ambulatory
 Unrestricted diet of 150 g/day for 3
days prior to testing
 Unlimited physical activity
 Avoid smoking and alcohol
 8-14 hrs fasting
 Glucose load (OGTT)
a. 75g- Standard load (WHO)
b.
c. 100g- Pregnant (2 step)
d. 1.75 g- glucose/ kg BW PEDIATRIC
PATIENT 1 y/o-19y/o)
NOTE: Max of 75 g
 Intravenous GTT
- Use for DM patients with GI disorders
- Requirement: FBS is required
- Glucose load: 0.5 g glucose x kbw (given within
3 mins)
- 2nd blood collection: after 5 mins of IV glucose
PROCEDURE:
1. Extract FBS (0 hr sample)
2. Give glucose load (5 mins)
3. Collect 1st hr, 2nd hr and 3rd hr blood samples
respectively.
5. Glycosylated Hemoglobin (HbA1C)
-
Glycated hemoglobin
Largest sub-fraction of normal Hgb A in
both DM and non DM
Red cell is the sample
With FBS (directly proportional)
1st step is to use lysing reagent but never
use hemolyzed samples.
DIAGNOSTIC SIGNIFICANCE:
-
A reliable method of monitoring long-term
glucose control or treatment of DM.
2-4 months average glucose
2 factors (2-4 patients) average plasma
glucose
SAMPLE FOR TESTING:

EDTA whole blood
- Episodic or chronic hemolysis to low HbA1C
- Method: Immunoassay, chromatography
- Interpretation: High risk (Pre-DM): 5.7%6.4%; DM: ≥???
DIAGNOSIS OF PRE-DIABETES AND DM:
1. NORMAL (NON- DM)
 FBS (Screening)= 70- <mg/dL (<5.6 mmol/L)
- Children= 60-100 mg/dL
 2nd-hr OGTT (Confirmatory) = <140 mg/dL
2. DIABETES MELLITUS
3. FBS= 1-125 mg/dL (impaired glucose tolerance)
 Confirmatory test- 2nd hour OGTT= 140-199
mg/dL
<140mg/dL
140-199 mg/dL
>200
NON- DM
IMPAIRED GLUCOSE
TOLERANCE
DM
INTERFERENCES/ VARIATIONS:
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
False increased HbA1c: HbF, carbamylated Hb
False Decreased HbA1c: (+) Hgb C and S
Vitamin C and E
NOTES:

2 hours after a glucose load, blood glucose should
return to the fasting level
 Symptoms of DM such as polyuria and polyphagia (are
observable if the plasma glucose concentration reach
≥200 mg/ dL
 Lab result: (+) glucosuria but normoglycemia
- Interpretation: renal tubular dysfunction
 Defective gene causing renal glucosuria- SLC5A2
 Formal OGTTs are NOT generally recommended for
routine clinical use in the diagnosis of DM EXCEPT:
 Diagnosis of cystic fibrosis- related diabetes (Type
3bDM)
- Annual screening with a 2 hr 75g (1.75g/kg) OGTT is
recommended beginning at least by age 10 years
NOTES:

Effects of Hyper glycemia to HbA1cassociated with decrease in erythrocyte
survival

6. Fructosamine
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Mostly composed of glycosylated or glycated
albumin (plasma protein ketoamine)- AKA;
remaining portions are globulins and
lipoproteins
Monitors short- term glucose control (2-3
weeks)
Alternate test of HbA1c
Use for monitoring:
a. Chronic hemolutic anemia
b. Hgb variants (HbS or HbC)
c. Shortened RBC lifespan
Low albumin (<20 g/dL)= Low fructosamine
Methods: Chromatography, enzymatic and
immunoassay
Reference values: 205-285 umol/L
Iron deficiency anemia: Higher HbA1c and
higher fructosamine,
< 50 mg/dL – Impairment of cerebral
function starts
Test for Hypoglycemia- Whipple’s triad
a. Symptoms consistent w/ hypoglycemia
b. Decrease in plasma glucose
c. Relief symptoms
DIAGNOSIS
-
Should NOT be made unless a patient meets the
criteria of Whipple’s triad w typical symptoms
alleviated by glucose administration.
Tolbutamide Tolerance Test:
-
Determines fasting hypoglycemia
Blood Samples: drawn at 2 mins to 2 hours
interval, 6 SPECIMENS, to measure glucose and
insulin.
Mixed-meal Tolerance Test
1,5 anhydroglucitol (1,5-AG)
-
Measures very short glycemic control in DM
It reflects to 1 to 2 weeks post prandial glycemia
Predicts rapid changes in glycemia than A1C and
fructosamine
- INTERPRETATION:
 Normal: 10-31 ug/mL
 Recent hyperglycemia: < 10 ug/mL (non
compliance, suggest for FBS)
Ketone Test
-
It is recommended when plasma glucose
reached 300 mg/dL
Nitroprusside test: Acetatoacetate
Nitroprusside + Glycine: Acetoacetate+ Acetone
Increase in Acetone= Defect in CHO metabolism
Hypoglycemia
-
Imbalance between glucose utilization and
production
INTERPRETATION:
 65-70 mg/dL = Glucagon and glycemic
hormones are released into the
circulation
 50-55 mg/dL- Observable symptoms of
hypoglycemia appear
 ≤ 50 mg/dL – Considered low value and
abnormal for infants
-
Determines reactive hypoglycemia
By measuring the response of insulin to a
cocktail meal
Post prandial blood sample: 15, 30, 45, 60, 90
and 120 minutes with a baseline fasting plasma
glucose.
Notes:
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Alimentary (reactive) hypoglycemia occurs
usually within 4 hours after eating a meal.
Hypoglycemia should be considered if the
fasting serum glucose is <50 mg/dL
Strongly suggest hypoglycemia: If a series of
random fasting sera is <60 mg/ dL
GLYCOGEN STORAGE DISEASE
-
An inherited deficiencies of enzyme that control
the synthesis or breakdown of glycogen
Most common GSD: Von Gierke Disease (can
cause hepatomegaly)
IV galactose Tolerance Test- test for type 1 GSD
(decrease in glucose levels)
LIPIDS