(FINAL) CC1 LAB L3

Creatine and phosphocreatine

• Creatinine is a waste product formed from the metabolism of __________ in muscles.

• It is produced at a relatively constant daily rate, which is proportional to an individual’s muscle mass.

Muscle mass

• Creatinine is a waste product formed from the metabolism of creatine and phosphocreatine in muscles.

• It is produced at a relatively constant daily rate, which is proportional to an individual’s __________.

Glomerulus

• In the kidneys:

  • Creatinine is freely filtered by the __________

  • It is not significantly reabsorbed

  • It is slightly secreted by the renal tubules

Reabsorbed

• In the kidneys:

  • Creatinine is freely filtered by the glomerulus

  • It is not significantly __________

  • It is slightly secreted by the renal tubules

Renal tubules

• In the kidneys:

  • Creatinine is freely filtered by the glomerulus

  • It is not significantly reabsorbed

  • It is slightly secreted by the __________

Renal (kidney) function

• Because of these properties, creatinine is widely used as a marker of __________.

• Approximately 99% of creatinine is excreted in the urine, with less than 1% reabsorbed.

99, 1

• Because of these properties, creatinine is widely used as a marker of renal (kidney) function.

• Approximately ___% of creatinine is excreted in the urine, with less than ___% reabsorbed.

Kidney/s

• Since creatinine production is fairly constant and it is mainly cleared by the __________, an increase in blood creatinine levels usually indicates impaired __________ function.

GFR = V / t

Glomerular Filtration Rate (GFR)

• Definition: The specific volume of plasma filtered through the glomerular capillaries into the Bowman's capsule per unit of time.

• Mathematical Formula: 

  • __________

  • __ = Volume of Plasma filtered

  • __ = Time interval

Glomerular Filtration Rate (GFR)

• It is considered the best overall indicator of kidney function.

• It is assessed by measuring the excretion of a substance that is:

  • Freely filtered by the glomerulus

  • Minimally reabsorbed

  • Not significantly secreted by the renal tubules

10

Creatinine is the most widely used marker for estimating GFR because:

• It is produced at a relatively constant rate

• It is not bound to plasma proteins

• It is not reabsorbed by the renal tubules

• Only a small amount is secreted by the tubules (approximately ___%)

Slight tubular secretion

Although creatinine is commonly used to estimate GFR, __________ may cause a small overestimation of the actual GFR.

Creatinine clearance (CrCl)

__________

• __________ measures the amount of creatinine removed from the blood by the kidneys over a specific period of time.

• It is usually reported in mL/min.

• It provides a reasonable approximation of the glomerular filtration rate (GFR).

Glomerular Filtration Rate (GFR)

Creatinine Clearance (CrCl)

• Plasma creatinine concentration is inversely proportional to __________:

  • High plasma creatinine → Low __________

  • Low plasma creatinine → High __________

CrCl = Ucr x Vu / Pcr x t

Creatinine Clearance (CrCl)

• Formula:

  • __________

  • ___ = urine creatinine concentration

  • ___ = urine volume

  • ___ = plasma creatinine concentration

  • ___ = time of urine collection

97–137

• CrCl Reference Interval:

  • Male: _____ mL/min

  • Female: 88–128 mL/min

88–128

• CrCl Reference Interval:

  • Male: 97–137 mL/min

  • Female: _____ mL/min

Jaffe Reaction (Classical Method)

Analytical Methods for Creatinine – Chemical Methods Based

__________

• Principle: Creatinine reacts with picric acid in an alkaline solution to form an orange-red complex (creatinine picrate), which can be measured.

• Reaction:

  • Creatinine + alkaline picrate → creatinine picrate (red-orange chromogen)

• Absorbance:

  • Measured at 490–520 nm

• Advantages:

  • Simple method

  • Inexpensive

  • Can be adapted for automated analyzers

• Disadvantages:

  • Not specific for creatinine

  • Can be affected by other substances (non-creatinine chromogens), such as:

    • Glucose, Ketones, Proteins, Cephalosporins (antibiotics), Ascorbic acid (Vitamin C)

• Because of these interferences, the __________ may overestimate creatinine levels, so more specific enzymatic methods are sometimes preferred in modern laboratories.

490–520

Analytical Methods for Creatinine – Chemical Methods Based

Jaffe Reaction (Classical Method)

• Principle: Creatinine reacts with picric acid in an alkaline solution to form an orange-red complex (creatinine picrate), which can be measured.

• Reaction:

  • Creatinine + alkaline picrate → creatinine picrate (red-orange chromogen)

• Absorbance:

  • Measured at ___ nm

• Advantages:

  • Simple method

  • Inexpensive

  • Can be adapted for automated analyzers

• Disadvantages:

  • Not specific for creatinine

  • Can be affected by other substances (non-creatinine chromogens), such as:

    • Glucose, Ketones, Proteins, Cephalosporins (antibiotics), Ascorbic acid (Vitamin C)

• Because of these interferences, the Jaffe method may overestimate creatinine levels, so more specific enzymatic methods are sometimes preferred in modern laboratories.

Not specific

Analytical Methods for Creatinine – Chemical Methods Based

Jaffe Reaction (Classical Method)

• Principle: Creatinine reacts with picric acid in an alkaline solution to form an orange-red complex (creatinine picrate), which can be measured.

• Reaction:

  • Creatinine + alkaline picrate → creatinine picrate (red-orange chromogen)

• Absorbance:

  • Measured at 490–520 nm

• Advantages:

  • Simple method

  • Inexpensive

  • Can be adapted for automated analyzers

• Disadvantages:

  • __________ for creatinine

  • Can be affected by other substances (non-creatinine chromogens), such as:

    • Glucose, Ketones, Proteins, Cephalosporins (antibiotics), Ascorbic acid (Vitamin C)

• Because of these interferences, the Jaffe method may overestimate creatinine levels, so more specific enzymatic methods are sometimes preferred in modern laboratories.

Automated analyzers

Analytical Methods for Creatinine – Chemical Methods Based

Jaffe Reaction (Classical Method)

• Principle: Creatinine reacts with picric acid in an alkaline solution to form an orange-red complex (creatinine picrate), which can be measured.

• Reaction:

  • Creatinine + alkaline picrate → creatinine picrate (red-orange chromogen)

• Absorbance:

  • Measured at 490–520 nm

• Advantages:

  • Simple method

  • Inexpensive

  • Can be adapted for __________

• Disadvantages:

  • Not specific for creatinine

  • Can be affected by other substances (non-creatinine chromogens), such as:

    • Glucose, Ketones, Proteins, Cephalosporins (antibiotics), Ascorbic acid (Vitamin C)

• Because of these interferences, the Jaffe method may overestimate creatinine levels, so more specific enzymatic methods are sometimes preferred in modern laboratories.

Jaffe with Adsorbent

Analytical Methods for Creatinine – Chemical Methods Based

__________

• This modification uses adsorbent materials to remove interfering substances (non-creatinine chromogens) before measurement, improving specificity.

  • Lloyd’s reagent (sodium aluminum silicate)

  • Fuller’s earth reagent (aluminum magnesium silicate)

Lloyd’s reagent

Analytical Methods for Creatinine – Chemical Methods Based

Jaffe with Adsorbent

• This modification uses adsorbent materials to remove interfering substances (non-creatinine chromogens) before measurement, improving specificity.

  • __________ (sodium aluminum silicate)

  • Fuller’s earth reagent (aluminum magnesium silicate)

Fuller’s earth reagent

Analytical Methods for Creatinine – Chemical Methods Based

Jaffe with Adsorbent

• This modification uses adsorbent materials to remove interfering substances (non-creatinine chromogens) before measurement, improving specificity.

  • Lloyd’s reagent (sodium aluminum silicate)

  • __________ (aluminum magnesium silicate)

Kinetic Jaffe Method

Analytical Methods for Creatinine – Chemical Methods Based

__________

• Measures the rate of change in absorbance over time rather than a single endpoint reading. This helps minimize interference from non-creatinine substances.

• Interferences:

  • Positive interference (falsely increases results):

    • Cephalosporins

    • Ketoacids

  • Negative interference (falsely decreases results):

    • Bilirubin

    • Hemoglobin

• The __________ is more specific than the classical endpoint Jaffe method because it focuses on the initial reaction rate, where creatinine reacts faster than most interfering substances.

Cephalosporins, Ketoacids

Analytical Methods for Creatinine – Chemical Methods Based

Kinetic Jaffe Method

• Measures the rate of change in absorbance over time rather than a single endpoint reading. This helps minimize interference from non-creatinine substances.

• Interferences:

  • Positive interference (falsely increases results):

    • __________

    • __________

  • Negative interference (falsely decreases results):

    • Bilirubin

    • Hemoglobin

• The kinetic Jaffe method is more specific than the classical endpoint Jaffe method because it focuses on the initial reaction rate, where creatinine reacts faster than most interfering substances.

Bilirubin, Hemoglobin

Analytical Methods for Creatinine – Chemical Methods Based

Kinetic Jaffe Method

• Measures the rate of change in absorbance over time rather than a single endpoint reading. This helps minimize interference from non-creatinine substances.

• Interferences:

  • Positive interference (falsely increases results):

    • Cephalosporins

    • Ketoacids

  • Negative interference (falsely decreases results):

    • __________

    • __________

• The kinetic Jaffe method is more specific than the classical endpoint Jaffe method because it focuses on the initial reaction rate, where creatinine reacts faster than most interfering substances.

Creatininase – CK

Analytical Methods for Creatinine – Enzymatic Methods (More Specific)

__________ Method

• Creatinine is measured through a series of enzymatic reactions that ultimately produce a measurable change (usually involving NADH consumption).

• Reaction:

1. Creatinine + H₂O —(__________)→ Creatine

2. Creatine + ATP —(__________)→ Creatine phosphate + ADP

3. ADP + PEP —(pyruvate kinase)→ ATP + Pyruvate

4. Pyruvate + NADH + H⁺ —(lactate dehydrogenase, LDH)→ Lactate + NAD⁺

• Measurement:

  • The decrease in NADH is measured spectrophotometrically

  • The change is directly proportional to the creatinine concentration

• Requirement:

  • Requires a relatively large volume of pre-incubated sample

• __________ Method is highly specific because each step is catalyzed by specific enzymes, minimizing interference from other substances.

Creatininase, creatine kinase, pyruvate kinase, lactate dehydrogenase

Analytical Methods for Creatinine – Enzymatic Methods (More Specific)

Creatininase – CK Method

• Creatinine is measured through a series of enzymatic reactions that ultimately produce a measurable change (usually involving NADH consumption).

• Reaction:

1. Creatinine + H₂O —(__________)→ Creatine

2. Creatine + ATP —(__________)→ Creatine phosphate + ADP

3. ADP + PEP —(__________)→ ATP + Pyruvate

4. Pyruvate + NADH + H⁺ —(__________)→ Lactate + NAD⁺

• Measurement:

  • The decrease in NADH is measured spectrophotometrically

  • The change is directly proportional to the creatinine concentration

• Requirement:

  • Requires a relatively large volume of pre-incubated sample

• Creatininase – CK Method is highly specific because each step is catalyzed by specific enzymes, minimizing interference from other substances.

Pre-incubated sample

Analytical Methods for Creatinine – Enzymatic Methods (More Specific)

Creatininase – CK Method

• Creatinine is measured through a series of enzymatic reactions that ultimately produce a measurable change (usually involving NADH consumption).

• Reaction:

1. Creatinine + H₂O —(creatininase)→ Creatine

2. Creatine + ATP —(creatine kinase, CK)→ Creatine phosphate + ADP

3. ADP + PEP —(pyruvate kinase)→ ATP + Pyruvate

4. Pyruvate + NADH + H⁺ —(lactate dehydrogenase, LDH)→ Lactate + NAD⁺

• Measurement:

  • The decrease in NADH is measured spectrophotometrically

  • The change is directly proportional to the creatinine concentration

• Requirement:

  • Requires a relatively large volume of __________

• Creatininase – CK Method is highly specific because each step is catalyzed by specific enzymes, minimizing interference from other substances.

Creatininase–H₂O₂

Analytical Methods for Creatinine – Enzymatic Methods (More Specific)

__________ Method

• Creatinine is converted through a series of enzymatic reactions, ultimately producing hydrogen peroxide (H₂O₂), which is then used to form a colored compound that can be measured.

• Reaction:

1. Creatinine + H₂O —(creatininase)→ Creatine

2. Creatine + H₂O —(creatinase)→ Urea + Sarcosine

3. Sarcosine + H₂O + O₂ —(sarcosine oxidase)→ Glycine + Formaldehyde + H₂O₂

4. H₂O₂ + hydrogen donor + 4-aminoantipyrine —(peroxidase)→ Quinoneimine (colored compound)

• Measurement:

  • The intensity of the colored quinoneimine is measured

  • Color intensity is directly proportional to creatinine concentration

• __________ Method is highly specific and widely used because it minimizes interference and provides more accurate results compared to the Jaffe method.

• Advantages: No interference from acetoacetate or cephalosporins

• Interference: Positive interference: lidocaine

Creatininase, creatinase, sarcosine oxidase, peroxidase

Analytical Methods for Creatinine – Enzymatic Methods (More Specific)

Creatininase–H₂O₂ Method

• Creatinine is converted through a series of enzymatic reactions, ultimately producing hydrogen peroxide (H₂O₂), which is then used to form a colored compound that can be measured.

• Reaction:

1. Creatinine + H₂O —(__________)→ Creatine

2. Creatine + H₂O —(__________)→ Urea + Sarcosine

3. Sarcosine + H₂O + O₂ —(__________)→ Glycine + Formaldehyde + H₂O₂

4. H₂O₂ + hydrogen donor + 4-aminoantipyrine —(__________)→ Quinoneimine (colored compound)

• Measurement:

  • The intensity of the colored quinoneimine is measured

  • Color intensity is directly proportional to creatinine concentration

• Creatininase–H₂O₂ Method is highly specific and widely used because it minimizes interference and provides more accurate results compared to the Jaffe method.

• Advantages: No interference from acetoacetate or cephalosporins

• Interference: Positive interference: lidocaine

Acetoacetate or cephalosporins

Analytical Methods for Creatinine – Enzymatic Methods (More Specific)

Creatininase–H₂O₂ Method

• Creatinine is converted through a series of enzymatic reactions, ultimately producing hydrogen peroxide (H₂O₂), which is then used to form a colored compound that can be measured.

• Reaction:

1. Creatinine + H₂O —(creatininase)→ Creatine

2. Creatine + H₂O —(creatinase)→ Urea + Sarcosine

3. Sarcosine + H₂O + O₂ —(sarcosine oxidase)→ Glycine + Formaldehyde + H₂O₂

4. H₂O₂ + hydrogen donor + 4-aminoantipyrine —(peroxidase)→ Quinoneimine (colored compound)

• Measurement:

  • The intensity of the colored quinoneimine is measured

  • Color intensity is directly proportional to creatinine concentration

• Creatininase–H₂O₂ Method is highly specific and widely used because it minimizes interference and provides more accurate results compared to the Jaffe method.

• Advantages: No interference from __________

• Interference: Positive interference: lidocaine

Lidocaine

Analytical Methods for Creatinine – Enzymatic Methods (More Specific)

Creatininase–H₂O₂ Method

• Creatinine is converted through a series of enzymatic reactions, ultimately producing hydrogen peroxide (H₂O₂), which is then used to form a colored compound that can be measured.

• Reaction:

1. Creatinine + H₂O —(creatininase)→ Creatine

2. Creatine + H₂O —(creatinase)→ Urea + Sarcosine

3. Sarcosine + H₂O + O₂ —(sarcosine oxidase)→ Glycine + Formaldehyde + H₂O₂

4. H₂O₂ + hydrogen donor + 4-aminoantipyrine —(peroxidase)→ Quinoneimine (colored compound)

• Measurement:

  • The intensity of the colored quinoneimine is measured

  • Color intensity is directly proportional to creatinine concentration

• Creatininase–H₂O₂ Method is highly specific and widely used because it minimizes interference and provides more accurate results compared to the Jaffe method.

• Advantages: No interference from acetoacetate or cephalosporins

• Interference: Positive interference: __________

Isotope Dilution Mass Spectrometry

__________ (Reference Method)

• Detection of characteristic fragments following ionization; quantification using an isotopically labeled compound

• Highly specific; accepted reference method

24-hour urine collection

Clinical Significance – Disease Correlations / Clinical Uses

• Creatinine measurement is used to:

  • Assess renal filtration function

  • Determine the adequacy of kidney function

  • Evaluate the severity of kidney damage

  • Monitor the progression of kidney disease

  • Check completeness of __________ (via urine creatinine)

Acute kidney injury (AKI), Chronic kidney disease (CKD)

Clinical Significance – Increased Creatinine can indicate:

• Impaired renal function:

  • __________

  • __________

• Prerenal azotemia (decreased blood flow to kidneys):

  • Shock

  • Dehydration

• Postrenal causes (urinary obstruction):

  • Kidney stones

  • Tumors

  • Prostatic hypertrophy

• Other contributing factors:

  • Muscle disease

  • Gigantism

  • High meat intake

Prostatic hypertrophy, gigantism, high meat intake

Clinical Significance – Increased Creatinine can indicate:

• Impaired renal function:

  • Acute kidney injury (AKI)

  • Chronic kidney disease (CKD)

• Prerenal azotemia (decreased blood flow to kidneys):

  • Shock

  • Dehydration

• Postrenal causes (urinary obstruction):

  • Kidney stones

  • Tumors

  • __________

• Other contributing factors:

  • Muscle disease

  • __________

  • __________

Reduced muscle mass, pregnancy

Clinical Significance – Decreased Creatinine may be due to:

• __________:

  • Cachexia

  • Malnutrition

  • Muscular dystrophy

• __________ (due to increased renal clearance)

Hemolysis, lipemia, and icterus

Specimen Collection and Handling

• Proper specimen handling is important to avoid inaccurate creatinine results

• __________ can interfere with laboratory measurements

Heparin or EDTA

Specimen Collection and Handling

• Specimen Types:

  • Serum

  • Plasma

  • Urine

• Anticoagulants:

  • __________ is acceptable

Fasting

Specimen Collection and Handling

• Patient and Specimen Preparation:

  • __________ is NOT required

  • Urine Handling:

    • Refrigerate urine after collection

    • If storage is longer than 4 days, freeze the specimen

Refrigerate, freeze

Specimen Collection and Handling

• Patient and Specimen Preparation:

  • Fasting is NOT required

  • Urine Handling:

    • __________ urine after collection

    • If storage is longer than 4 days, __________ the specimen

Plasma creatinine

Estimated GFR (eGFR) and Laboratory Reporting

• Sensitivity Issue: __________ alone is not sensitive enough to detect mild renal dysfunction.

• Reporting Standards: Labs are encouraged to report an estimated GFR (eGFR) whenever serum creatinine is ordered to improve early detection of kidney disease.

• Calibration: To ensure accuracy, the National Kidney and Disease Education Program (NKDEP) recommends assays be traceable to an IDMS (Isotope Dilution Mass Spectrometry) method.

Estimated GFR (eGFR)

__________ and Laboratory Reporting

• Sensitivity Issue: Plasma creatinine alone is not sensitive enough to detect mild renal dysfunction.

• Reporting Standards: Labs are encouraged to report an __________ whenever serum creatinine is ordered to improve early detection of kidney disease.

• Calibration: To ensure accuracy, the National Kidney and Disease Education Program (NKDEP) recommends assays be traceable to an IDMS (Isotope Dilution Mass Spectrometry) method.

IDMS (Isotope Dilution Mass Spectrometry)

Estimated GFR (eGFR) and Laboratory Reporting

• Sensitivity Issue: Plasma creatinine alone is not sensitive enough to detect mild renal dysfunction.

• Reporting Standards: Labs are encouraged to report an estimated GFR (eGFR) whenever serum creatinine is ordered to improve early detection of kidney disease.

• Calibration: To ensure accuracy, the National Kidney and Disease Education Program (NKDEP) recommends assays be traceable to an __________ method.

eGFR (mL/min/1.73m^2) = 175 x (SCr)^-1.154 x (age)^0.203 x (0.742 if female) x (1.210 if black)

The MDRD Equation (Abbreviated) – Historically used for individuals aged 17–70, utilizing four variables: serum creatinine (SCr), age, sex, and race.

• Formula (IDMS-traceable):

  • __________

• Normalization: Results are normalized to a standard body surface area of 1.73 m².

eGFR (mL/min/1.73m^2) = 0.413 x height (cm) / SCr

The Bedside Schwartz Equation (Pediatric):

• Used for patients under 18 years old.

• Formula: __________

• Improvement: This modified version corrects for the 20%–40% overestimation found in the original Schwartz equation.