Clinical Chemistry 1

CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

MIDTERM LESSON 1: ➢ B. Measure of Spread

METHOD EVALUATION AND QUALITY MANAGEMENT ✓ Range - Largest value in the data minus the
smallest value
OUTLINE ✓ Standard Deviation ((SD, s or σ) - Represents the
I. Introduction “average” distance from the mean; Degree of
II. Descriptive Statistics precision of a measurement
III. Method Evaluation ✓ Coefficient of variation (CV) - Allows comparing
IV. Quality Control SD with different units
V. Reference Interval Studies
VI. Diagnostic Efficiency

INTRODUCTION
QUALITY MANAGEMENT
A. Method Evaluation
➢ Used to verify
the
acceptability of
new methods.
B. Quality Control
➢ Insurance of a
method to
remain valid
over time.

DESCRIPTIVE STATISTICS ➢ C. Measure of Shape

➢ Used for monitoring test performance and QC. ✓ Gaussian (Normal) distribution “bell curve”
✓ 68.3% is between X±1SD, 95.4% is between
X±2SD, 99.7% is between X±3SD

➢ Summarize the data generated by the laboratory METHOD SELECTION AND EVALUATION

➢ Sources of Analytic Variability

✓ Operator technique
✓ Instrument differences
✓ Test accessories
✓ Contamination A. METHOD SELECTION
✓ Environmental conditions (temperature, humidity)
✓ Reagents
✓ Power Surges
✓ Matrix effects (hemolysis, lipemia, etc.)

➢ A. Measure of Center
✓ Mean - Average
✓ Median - Middle of the data after the data have been
ranked
✓ Mode - The most frequent occurring value in the
dataset

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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

2. Constant error: The

magnitude of error is
constant and not
dependent on analyte
concentration.
B. Can be determined by recovery study, interference
study and comparison of methods
Recovery - Ability of a test to measure a known
amount of analyte (e.g. Ca2+) added to a matrix (e.g.
CSF)
a. Recovery studies - detects proportional error

1. Analytic Sensitivity - Ability of a method to detect

small quantities of an analyte
2. Analytic Specificity - Ability of a method to detect
only the analyte it is designed to determine
3. Specificity - Ability of a method to measure only the
analyte of interest
4. AMR (Analytic Measurement Range) - Linear or
dynamic range; Range of analyte concentrations that
can be directly measured without dilution or
concentration Interference - Effect of an interferent (e.g. Hb) on the
5. CRR (Clinically Reportable Range) - Range of accuracy of detection of an analyte (e.g. troponin);
analyte that a method can quantitatively report; detects constant error
Allows for dilution and concentration used to extend Common interference
AMR o Hemolysis
6. LoD (Limit of Detection) - Lowest amount of o Lipemia
analyte accurately detected by a method. o Bilirubin
o Anticoagulant
B. METHOD EVALUATION o Preservatives
1. Determination Imprecision
o Dispersion of repeated
measurements about the
mean due to analytic
(random) error.
o Random error varies from
sample to sample.
Determined by Repeated Analysis Study - detects
random error that affects reproducibility
a. Repeated Analysis study (Precision study)
o 2 x 2 x 10 study
– 2 controls are run twice a day for 10 days
– E.g. glucose in hyperglycemic (150 C. Comparison-of-Methods Studies
mg/dL) and normal range (90 mg/dL) o Detects
– Estimate long terms changes occurring Systemic error
over o The test
method is
compared with
a reference
method (gold
standard)
o 40 -100
specimens were run everyday over 8 -20 days
o A plot of the test-method data (y-axis) versus the
comparative method (x-axis) is generated.

2. Determination of Inaccuracy
A. Difference between a measured
value and its true value due to
systemic error (proportional or
constant)
Systemic error: always in one
direction
1. Proportional error: The
magnitude of error is
dependent on analyte
concentration.

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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

QUALITY CONTROL b. 13S Rule: If 1 control observation exceeds

A. Introduction mean ±3s, reject the run for probable random
1. Check the reproducibility of a method by error.
including control specimens in the run
a. Equipment out of calibration
b. Reagent may be deteriorating
c. Technologist may have made an error
2. Done by Running QC Materials (Controls)
a. Should be the same as the specimens to be
tested
b. Should span the clinically important range of the
analyte (cutoff values) i.e. Glucose assays c. 22S Rule: If 2 consecutive control values are
• In reference range (80 mg/dL) greater than mean ±2s, reject the run for
• Hypoglycemia (50 mg/dL) probable systemic error. x ± 2SD (95%) = 1:20
• Hyperglycemia (150 mg/dL)
3. Types of Controls
a. Lyophilized (dehydrated powder)
• Reconstituted with its diluents.
b. Stabilized frozen controls
• Needs to be evaluated for stability.
B. Quality Control Charts
Levey-Jennings Control Chart - Used to detect errors d. R4S Rule: If the difference between the 2
(inaccuracy and imprecision) over time controls is ≥4s, reject the run for probable
a. Random errors random error.
1. Due to variations in technique
b. Systemic errors
• Poorly made (contaminated) standards &
reagents
• Instrumentation problems (function &
calibration)
• Poorly written procedure
c. Control values should be within statistical limits
(expresses as the mean ‡ SD) e. 41S Rule: If 4 consecutive values exceed the
same mean ±1s limit, reject the run for probable
C. Operation of QC System systemic error.
1. Shift and Trend
Shift - More than six consecutive values fall in one
side of the mean

f. 10x Rule: If 10 consecutive control values fall

on 1 side of mean, reject the run for probable
systemic error
Trend - More than six consecutive values steadily
increase or decrease.

o Once a rule has been violated, reject the analytic run, take
action to remedy errors
2. Multi-rule Procedure o Find the cause of error, take corrective action, reanalyze
a. 12S Rule: b. 12S Rule: If 1 control observation control and patient data
exceeds mean ±2s, A warning rule. x ± 2SD
(95%) = 1:20

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 Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

REFERENCE INTERVALS DIAGNOSTIC EFFICIENCY

A. DEFINITION
1. Referred as reference ranges.
o A pair of medical decision points that span the limits of
results expected for a given condition.
2. Normal Range
o Range of results between medical decision levels that
correspond to ±2SD of results from a healthy patient.
3. Confidence Interval
o Range of values that include a specific probability, usually
90% or 95%
4. Medical Decision level
o Value for an analyte that represents the boundary between
different therapeutic approaches.
5. Therapeutic Range
o Reference interval applied to a therapeutic drug.
6. Establishing a reference interval
o Done when there is no existing assay for an analyte or
methodology in the laboratory
o May require from 120 to ≈700 study individuals
7. Verifying a reference interval (transference)
o Confirm the validity of an existing reference interval for an
analyte using the same type of analytic system
o Can require 20 study individuals

B. CATEGORIES
1. DIAGNOSIS OF A DISEASE OR CONDITION
A. DEFINITIONS
1. Analytic Sensitivity - Refers to the lower limit of detection
for a given analyte
2. Clinical sensitivity - proportion of individuals with that
disease who test positively with the test 22 Stat Fax 4500
Sample & reagent mixture
3. True positive - patient w/ a condition who are classified by
a test to have the condition
4. False negative - patient with a condition who are classified
by a test as not having the condition

2. MONITORING OF A PHYSIOLOGIC CONDITION B. MEASURES OF DIAGNOSTIC EFFICIENCY

1. Diagnostic sensitivity - Ability of a test to detect a given
disease or condition

2. Diagnostic specificity - Ability of a test to detect the absence

of a given disease or condition

3. Positive Predictive Value - Refers to the probability of an

individual having the disease if the result is outside the
reference range
3. THERAPEUTIC MANAGEMENT

4. Negative Predictive Value - Refers to the probability that a

patient does not have a disease if the result is within the
reference range

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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

PATHWAYS OF GLUCOSE METABOLISM

GLYCOLYSIS

MIDTERM LESSON 2:
CARBOHYDRATES

CONTENTS
1. Introduction
2. Pathways of Glucose Metabolism
3. Regulation of Glucose Metabolism
4. Disease states in Glucose Metabolism
a. Hyperglycemia
b. Hypoglycemia
5. Diagnosis of patients with Glucose Metabolic Alterations

INTRODUCTION
o Primary energy
source for brain,
RBC & retinal
cells
o Stored primarily
as liver and
muscle glycogen
o Contain C, H & O
[Cx(H20)y] w/
C=O & -OH groups
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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

GLUCONEOGENESIS

REGULATION OF GLUCOSE METABOLISM

➢ Insulin
➢ Glucagon
➢ Epinephrine
➢ Cortisol
➢ Growth hormone
➢ Thyroxine
➢ Somatostatin

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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

TYPE 1 (IDDM)
PATHOGENESIS
I. β-Cell destruction
II. Absolute insulin deficiency
III. Autoantibodies
• Islet cell
• Insulin
• Glutamic acid
EPIDEMIOLOGY
I. 10-20% of all cases
II. Childhood & adolescence
CHARACTERISTICS
I. Abrupt
II. Insulin dependence, ketosis tendency (acetoacetate,
acetone, β- hydroxybutyrate)
III. Symptoms:
o Polydipsia
o Polyphagia
o Polyuria
o Microvascular problems
– nephropathy
– neuropathy

TYPE 2 (NIDDM)
PATHOGENESIS
I. Relative insulin deficiency
II. Insulin resistance
DISEASE STATES IN GLUCOSE METABOLISM III. Insulin is present (hyperinsulinemia)
A. HYPERGLYCEMIA IV. Attenuated glucagon secretion
➢ Increase in plasma glucose V. ↑ w/ age, obesity & lack of exercise
EPIDEMIOLOGY
I. 90% of all cases
II. Adult onset
CHARACTERISTICS
I. Non-Insulin dependent
II. Ketosis tendency is seldom (↓ lipolysis)
III. Greater tendency to develop nonketotic hyperosmolar
states

GESTATIONAL DIABETES

DIABETES MELLITUS
o Metabolic disease characterized by hyperglycemia
▪ Type 1 (IDDM)
▪ Type 2 (NIDDM) PATHOGENESIS
▪ Gestational I. Due to metabolic and hormonal changes
II. During pregnancy
III. Glucose intolerance
IV. Risk of respiratory distress,

LABORATORY FINDINGS
A. ↑ glucose in plasma and urine
B. ↑ urine specific gravity
C. ↑ serum and urine osmolality
D. Ketonemia & ketonuria
E. ↓ blood and urine pH (acidosis)

CATEGORIES OF FPG
Normal fasting glucose <100 (<5.6)
Impaired fasting glucose BG 100-125 mg/dL
Provisional diabetes diagnosis ≥126 (≥7.0)
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 Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

Categories of OGTT & RPG: mg/dL (mmol/L) ENZYMATIC

Normal glucose tolerance <140 (<7.8) a. Glucose oxidase (Saifer
Impaired glucose tolerance 140-199 (7.8-11.1) Gernstenfield)
Provisional diabetes diagnosis ≥200 (≥11.1) ➢ Β-D-glucose + O2 +
H2O – glucose
DIAGNOSTIC CRITERIA FOR DM oxidase → gluconic
1. Random plasma glucose: acid + H2O2
≥200 mg/dL, + symptoms of DM ➢ H2O2 + reduced chromogen – peroxidase → oxidized
2. Fasting plasma glucose (FPG): chromogen + H2O
≥126 mg/dL a. Couple reaction is known as Trinder’s reaction
3. 2-h plasma glucose: False ↓ results due to ↑ uric acid, bilirubin &
≥200 mg/dL during an OGTT ascorbic acid
4. HbA1C: b. O2 consumption electrode (polarographic
≥6.5% glucose) – measure O2 depletion
b. Hexokinase (reference method)
DIAGNOSTIC CRITERIA FOR GESTATIONAL DIABETES ➢ Glucose + ATP – hexokinase
1. Fasting plasma glucose (FPG) → glucose 6-PO4 + ADP
≥92 mg/dL ➢ G6-PO4 + NADP+ – G-6-PD
→ NADPH + H++ 6-
DISEASE STATES IN GLUCOSE METABOLISM phosphogluconate
B. HYPOGLYCEMIA a. False ↓ results due to gross hemolysis and ↑
CAUSES OF HYPOGLYCEMIA bilirubin
1. Insulinoma
2. Islet hyperplasia 2-HOUR POSTPRANDIAL TEST
3. Factitial hypoglycemia (insulin/sulfonylurea) ➢ Glucose load (75 g soln’) is administered. Blood glucose is
4. Severe exercise determined in the 2nd hr.
5. Ketotic hypoglycemia
6. Von Gierke Disease (↓ G-6-P, inh. glycogenolysis) ORAL GLUCOSE TOLERANCE TEST
➢ FBS is taken. Glucose load is
DIAGNOSIS OF PATIENTS WITH GLUCOSE METABOLIC administered. Blood glucose is
ALTERATIONS determined in 30 min, 1st, 2nd &
3rd hrs.
CONSIDERATIONS ➢ Ambulatory and in a normal
1. WB glucose conc. is 11% lower than plasma carbohydrate intake for 3 days
2. Obtain Serum/plasma within 1 hr Fasting for 8 - 10 hours
3. Obtain FPG in the morning after 8-10 hrs. fasting (not >16
hours) HbA1C MEASUREMENT
➢ Index for long term plasma glucose control (2-3 months
METHODS
period)
1. Fasting Blood Glucose
➢ Based on charged differences between HbA1C and Non-
2. 2-Hr Post Prandial Sugar
HbA1C
3. OGTT
➢ Specimen requirement is EDTA WB sample.
4. HbA1C
N.V → 4.0% to 6.0%
5. Ketone
6. Microalbuminuria
NON-ENZYMATIC
1. Nelson Somogyi
2. Hagedorn Jensen
3. Ortho-toluidine (Dubowski)
ENZYMATIC
1. Glucose oxidase (Saifer Gernstenfield)
2. Hexokinase (Reference method)
HbA1C MEASUREMENT BASED ON:
NON-ENZYMATIC A. Structural differences
1. Nelson Somogyi 1. Immunoassays
➢ Copper reduction 2. Affinity
method Chromatography
➢ Glucose + B. Charge differences
arsenomolybdic acid 1. Cation-exchange
→ arsenomolybdenum Chromatography
blue 2. Electrophoresis
2. Hagedorn Jensen 3. Isoelectric
➢ Ferric reduction method (inverse colorimetry) focusing
➢ Glucose + ferricyanide (yellow) → Ferrocyanide 4. HPLC
(colorless)
3. Ortho-toluidine (Dubowski) KETONE
➢ Condensation of carbo. w/ aromatic amines to Schiff o Produced by the liver through metabolism of stored lipids
bases o 3 KETONE BODIES
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 Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

– Acetone (2%)
– Acetoacetic acid(20%)
– 3-β-hydroxybutyric acid (78%)
o Ketonemia accumulation of ketones in blood
o Ketonuria accumulation of ketones in urine

KETONE MEASUREMENT
1. Gerhardt’s Test
Acetoacetic acid + Ferric chloride → Red color
2. Nitroprusside (Acetest tablet & Reagent Strip)
Acetoacetic acid + Na nitroprusside – alkaline pH →Purple
color
3. Enzymatic
Acetoacetic acid (pH 7) + NADH + H+ + ← β HBD → NAD+β

MICROALBUMINURIA
o Early stage diabetic renal nephropathy
o Persistent albuminuria (30-299 mg/24 hr)
o Albumin-creatinine ratio (30-300 µg/mg)

MIDTERM LESSON 3:
LIPIDS AND LIPOPROTEIN

CONTENTS
1. Lipid Chemistry
2. Lipoprotein Structure
3. Lipoprotein Physiology & Metabolism
4. Lipid Disorders
5. Lipid and Lipoprotein Analyses

LIPID CHEMISTRY

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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

LIPOPROTEIN STRUCTURE

LIPOPROTEIN PHYSIOLOGY & METABOLISM

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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

LIPID AND LIPOPROTEIN ANALYSIS

1. Cholesterol
2. Triglyceride
3. Lipoprotein
i. General Mtds.
ii. HDL Mtds.
iii. LDL Mtds
iv. Apolipoprotein Mtds.
LIPID DISORDERS

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Clinical Chemistry 1
CCHM321 LECTURE
Our Lady of Fatima University – Nueva Ecija BSMLS – 3rd Year – YA-3
College of Medical Laboratory Science Ms. Gladys Anne Marie Limson, RMT

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