Clinical Biochemistry

Table of Contents

Page created on January 6, 2020. Last updated on December 18, 2024 at 16:57

1. Purpose of laboratory test requests (screening, diagnosis, differential-diagnosis, validation, monitoring).

Why do I ask for a lab investigation?

  • What do I expect to find in the results?
  • Do I have a preliminary diagnosis for which the results will be supportive?
  • Will it affect my duties/decisions around the patient?
  • Will the lab result serve the interest of the patient?

The purpose of lab investigation:

  • Screening (healthy or not healthy)
    • Detection of subclinical disease
    • High sensitivity, low specificity
  • Lab diagnosis
    • Confirm or reject clinical diagnosis
    • High specificity, low sensitivity
  • Follow-up/monitoring
    • Monitoring progression/response to treatment
  • Evaluation of prognosis
    • Information regarding the likely outcome of the disease

2. Patient preparation before sampling. Proper method to carry out blood (venous, capillary) and urine collection. Type of tubes.

The process:

  • Preanalysis
    • Clinical question
    • Request for analyses
    • Patient sampling
    • Transit to lab
  • Analysis
  • Postanalysis
    • Collection
    • Interpretation
    • Reporting
    • Answer

Preanalytical errors (80% of all errors during the process)

  • Preparation of patient
    • Identification
    • Diet
      • Fasting affects glucose, triglycerides
    • Drugs
    • Smoking
    • Pregnancy
    • Exercise
      • Epinephrine, cortisol increase
    • Dehydration
    • Infection
      • Iron parameters wrong
      • TSH, thyroid hormones wrong
  • Sample taking
    • Posture
      • Increased in standing position: RBC, WBC, Hb, HTC, …
    • Time of sample taking
      • Cortisol highest in morning
      • TSH highest in evening
    • Strangulation
      • Max 1 min!
    • Simultaneous infusions
    • Identification of the sample
    • Patting the vein
    • Shaking the tubes too much
    • Correct tube
    • Correct order
    • Correct vessel
    • Correct filling of tube
  • Sample preparation
    • Haemolysis
      • Increased LDH, K+
      • Decreased RBC
      • Interferes with photometry, haemostasis
    • Clotting
    • Lipaemia
    • Centrifugation
    • The elapsed time from sample taking
      • Blood gas: max 30 min
    • Temperature
      • Usually room temperature
      • Store on ice: ACTH, renin, aldosterone
    • Sunlight
    • Labelling
    • Delivery

Tubes:

Order

 Tube Additive Colour Blood product Use
0. Blood culture – –

Culture

1.

 Haemostasis tube Sodium citrate Light blue Plasma Coagulation studies
2. Sedimentation tube Sodium citrate Black Plasma

ESR

3.

 Plain tube Nothing Red Serum Biochemistry, immunology, serology
4. Serum tube Gel + clot activator Yellow Serum

Biochemistry, immunology, serology

5.

 Heparin tube Lithium heparin Green Plasma Blood gas values
6. EDTA tube EDTA Purple Plasma

CBC

7.

 Glycolysis inhibitor Fluoride, oxalate Grey Plasma

Glucose, lactate

Urine sampling:

  • First morning urine
    • Unsanitary
    • Clean container
    • Duration of sample storage
  • 24-hour urine
    • Many possible errors

3. Reference values and ranges, specificity, sensitivity and predictive value of laboratory tests.

Precision – the reproducibility of an analytical result. The same investigation of the same sample should give as similar results as possible. There should be a small margin of error around the mean value.

Accuracy – how close the measured value is to the “true” value

The ideal test is both precise and accurate!

The reference range is made up of measurements made in a large, healthy population. The reference range is the mean ± 2 standard deviations. This means that the reference range includes only 95% of healthy people, 5% of healthy people will be outside the reference range!

Sensitivity – the proportion of individuals that correctly register as positive in a clinical test designed to identify a disease – how many of those who have the disease which test positive

Specificity – the proportion of individuals that correctly register as negative in a clinical test designed to identify a disease – how many of those who don’t have the disease which test negative

Positive predictive value – the proportion of individuals who test positive for a disease that actually have the disease. Influenced by disease prevalence! – how many of those that test positive actually have the disease

Negative predictive value – the proportion of individuals who test negative for a disease that actually are disease-free. Influenced by disease prevalence! – how many of those that test negative actually don’t have the disease

A highly sensitive test will have a low false negative rate, meaning that there will be very few people with the disease who receive negative test results.

A highly specific test will have a low false positive rate, meaning that there will be very few people without the disease who receive positive test results.

Receive operating characteristic (ROC) curve – plots sensitivity against specificity – compares the sensitivity and specificity of different tests.

4. Interpretation of laboratory results. Biological and analytical factors influencing the results.

Analytical results are affected by both analytical and biological variation.

Analytical variation: Random variation in analysis. Can be due to temperature in laboratory, differences in reagents, differences in pipetted volume of sample, etc.

(Intraindividual) biological variation: A test parameter fluctuates randomly around a homeostatic set point. Some fluctuations are non-random, like circadian variation in cortisol, female sex hormones in the menstrual cycle, etc.

Analytical sensitivity: The lowest detectable level of the particular analyte

Analytical specificity: The extent to which the test can identify only a specific substance, without interference from other substances

Major analytical tools:

Tools

 Description Use
Absorption photometry Measure absorbance of a sample solution

Enzymes, metabolites

Turbidimetry

 Measure loss of intensity of transmitted light through particles suspended in sample CRP, immunoglobulins, transferrin, rheumatoid factor
Nephelometry

Measure the intensity of scattered light at an angle

Densitometry

 Evaluation of electrophoresis samples
Flame photometry Electrolytes are vaporized into gaseous state, excited by flame, emits photons

Electrolytes

Fluorimetry

 Measure fluorescent light emitted by sample

Hormones, drug monitoring, tumour markers

Luminometry

Measure visible light emitted by sample

Potentiometry/ion selective electrode

 Measure electrode potential in solution pH, pO2, pCO2, electrolytes
Flow cytometry

CBC

HPLC (high performance liquid chromatography)

 HbA1c, toxicology, drug monitoring
Electrophoresis Separation of proteins in sample

Separation of serum, CSF, urine proteins

Microscopy

Blood smear, urine sediment

5. Bedside/point of care tests (POCT) and their informational value

POCT

  • Performed at or near the site of patient care
    • Bedside
  • Outside the lab
    • GP office
    • Pharmacy
    • Outpatient clinic
    • Home
  • Operators are not lab professionals
    • Nurses
    • Assistants
    • Doctors
    • Pharmacists
    • Patients

What can be measured:

  • Rapid tests
    • Pregnancy (hCG in urine)
    • Faecal occult blood
    • Procalcitonin
    • Drugs
  • Manual POCT devices
    • Glucose
    • Cholesterol
    • INR
  • Bench analyzers
    • Blood gas
    • Automatic urine dipstick evaluation

Requirements:

  • Results arrive quickly
  • Quality comparable to laboratory
  • Easy to use
  • Safe to use
  • Affordable
  • Training!
  • Regular maintenance and quality control

Advantages

  • Fast results
  • Small amounts of sample required
  • No sample storing
  • No sample packing
  • No sample transport

Disadvantages

  • Easy to fail to:
    • Perform the test properly
    • Calibrate regularly
    • Maintain regularly
    • Store reagents appropriately
    • Interpret results correctly

Methods:

  • Immunochromatography
    • Faecal occult blood test
    • Pregnancy test
    • Procalcitonin
    • Microbiology
  • Enzymatic
    • Glucose
  • Electrode
    • Blood gas

6. Disorders of water and sodium homeostasis

Parameter Sample Reference range
Sodium Serum 136 – 146 mM
Osmolality Serum 275 – 305 mOsmol/kg
Osmolality Urine 50 – 1400 mOsmol/kg

Sodium:

  • measured by flame photometry/ion-selective electrodes (potentiometry)
  • Regulated by
    • RAAS
    • ADH

Hyponatraemia

  • Etiology
    • Excessive renal salt loss
      • Addison disease
      • Kidney disease
      • Diuretics
    • Excessive ADH secretion
      • SIADH
    • Water overload
      • Heart failure
      • Cirrhosis
      • Kidney disease
  • Symptoms
    • Vomiting
    • Weakness
    • Confusion
    • Altered mental status
    • (Coma)

Hypernatraemia

  • Etiology
    • Excessive water loss
      • Diarrhoea
    • Poor fluid intake
    • Diabetes insipidus
    • Hyperaldosteronism
  • Symptoms
    • Increased thirst
    • Tremor
    • Altered mental status
    • Seizures
    • (Coma)

7. Disorders of potassium balance

Parameter Sample Reference range
Potassium Serum 3,8 – 5,2 mM

Potassium

  • Regulate cardiac contraction and rhythm
  • Regulate muscle contraction
  • Regulation
    • RAAS

Hypokalaemia

  • Etiology
    • Intracellular shift
      • Alkalosis
      • Insulin therapy
    • Extrarenal loss
      • Diarrhoea
      • Vomiting
    • Renal loss
      • Renal tubular acidosis I, II
      • Potassium-losing diuretics
    • Hyperaldosteronism
    • Hypercortisolism
  • Symptoms
    • Weakness
    • Constipation
    • Fatigue
    • Arrhythmia
  • ECG
    • U waves
    • ST depression
    • Flat T waves (no pot, no tea!)

Hyperkalaemia

  • Etiology
    • Extracellular shift
      • Acidosis
      • Insulin deficiency
    • Renal failure
      • Acute
      • Chronic
      • Potassium-sparing diuretics
      • Renal tubular acidosis IV
    • Adrenal failure
      • Hypoaldosteronism
    • Cell lysis
      • Myolysis
      • Tumor lysis
      • Haemolysis
  • Symptoms
    • Weakness
    • Diarrhoea
    • Fatigue
    • Arrhythmia
  • ECG
    • Peaked T waves
    • Flat P waves
    • Broad QRS

8. Laboratory findings in metabolic type changes of the acid/base balance

Parameter Sample Reference range
pH Heparin anticoagulated blood 7,35 – 7,45
pCO2 Same 35 – 45 mmHg
pO2 Same 75 – 100 mmHg
Standard HCO3– Same 20 – 26 mM
K+ Serum 3,8 – 5,2 mM
Anion gap 10 – 16 mM
  • Arterial blood gas!
  • Anion gap
    • Measured cations – measured anions = unmeasured anions
    • (Na+ + K+) – (Cl– + HCO3–)

Metabolic acidosis

  • Increased production of H+ or acids
  • Increased ingestion of H+ or acids
  • Loss of HCO3–
  • Etiology
    • High anion gap metabolic acidosis
      • Lactic acidosis
        • Tissue hypoxia
        • Metformin
      • Ketoacidosis
      • Uraemia
      • Exogenous acids
        • Methanol
        • Ethylene glycol
    • Normal anion gap metabolic acidosis
      • Renal tubular acidosis
      • Loss of HCO3–
        • Diarrhoea
  • Labs
    • pH decreased
    • Bicarbonate decreased
    • pCO2 decreased (compensation)
    • K+ increased (H+ into cell, K+ out)
    • Free Ca2+ increased (more albumin binds H+, less albumin binds Ca2+)
  • Symptoms
    • Depression of consciousness
    • Arrhythmia
    • Death at pH < 6,8

Metabolic alkalosis

  • Loss of H+
  • Increased production of HCO3–
  • Increased ingestion of HCO3–
  • Etiology
    • Chloride-responsive metabolic alkalosis
      • Vomiting
      • Nasogastric suction
      • Diuretics
    • Chloride-resistant metabolic alkalosis
      • Hyperaldosteronism
      • Cushing syndrome
      • Ingestion of HCO3–
  • Labs
    • pH increased
    • Bicarbonate increased
    • pCO2 increased (compensation)
    • K+ decreased
    • Free Ca2+ decreased
  • Symptoms
    • Depression of consciousness
    • Tetany
    • Laryngospasm
    • Death at pH > 8,0

9. Laboratory findings in respiratory type changes of the acid/base balance

Parameter Sample Reference range
pH Heparin anticoagulated blood 7,35 – 7,45
pCO2 Same 35 – 45 mmHg
pO2 Same 75 – 100 mmHg
Standard HCO3– Same 20 – 26 mM
K+ Serum 3,8 – 5,2 mM

Respiratory acidosis

  • Alveolar hypoventilation
  • Etiology
    • COPD exacerbation
    • Bronchial asthma
    • Respiratory muscle weakness
    • CNS depression
  • Labs
    • pH decreased
    • pCO2 increased
    • Bicarbonate increased (compensation)
    • K+ increased
    • Free Ca2+ increased
  • Symptoms
    • Depression of consciousness

Respiratory alkalosis

  • Alveolar hyperventilation
  • Etiology
    • Panic attacks
    • Anxiety
    • High altitude (hypoxia)
    • Drug intoxication
    • Mechanical ventilation
  • Labs
    • pH increased
    • pCO2 decreased
    • Bicarbonate decreased (compensation)
    • K+ decreased
    • Free Ca2+ decreased
  • Symptoms
    • Depression of consciousness
    • Tetany
    • Laryngospasm

10. Laboratory approaches for the detection of disorders in calcium, magnesium and phosphate homeostasis

Parameter Sample Reference range
Calcium Serum 2,1 – 2,6 mM
Magnesium Serum 0,7 – 1,0 mM
Phosphate Serum 1 – 1,5 mM

Calcium

  • Roles
    • Comprises bone
    • 1 kg in adults
      • 99% in bone
    • Muscle contraction
    • Neurotransmission
    • Membrane transport
    • Enzymes
    • Coagulation
  • 45% free
  • 45% protein bound
  • 10% anion bound
  • Regulation
    • Kidney
    • PTH
      • Decreased calcium stimulates PTH
      • Increased phosphate stimulates PTH
      • Increases Ca2+ reabsorption in tubule
      • Increases phosphate excretion in tubule
      • Increases vitamin D production
      • Increases bone resorption
    • Vitamin D
      • Increases Ca2+ reabsorption in tubule
      • Increases Ca2+ reabsorption in GI tract
      • Decrease phosphate excretion in tubule
      • Increase bone mineralization
    • (Calcitonin)
      • Opposite of PTH
      • Tumor marker
  • Hypocalcaemia
    • Hypoparathyroidism
    • Malabsorption of calcium
    • Vitamin D deficiency
    • Renal failure
    • Pancreatitis (saponification)
  • Hypercalcaemia
    • Hyperparathyroidism
      • Primary – due to tumor
      • Secondary – due to low calcium level (CKD)
      • Tertiary – due to chronic secondary hyperPTH -> hyperplasia of PTH gland
    • Excess vitamin D
    • Tumours
      • Osteolytic metastases
      • Multiple myeloma
  • Related lab parameters
    • Kidney function
    • ALP
    • Bone specific ALP
    • Acid base
    • Total protein
    • PTH
    • Vitamin D
    • Urine
      • Phosphate excretion
      • Type 1 collagen fragments

Magnesium

  • Roles
    • Enzyme cofactor
    • Calcium homeostasis
      • Magnesium is a calcium antagonist, prevents intracellular accumulation of calcium
  • Hypomagnesaemia
    • Malnutrition
    • Laxative abuse
    • Hyperparathyroidism
  • Hypermagnesaemia
    • Renal failure
    • Rhabdomyolysis

Chloride

  • Roles
    • Maintains osmolality, electric neutrality, blood volume
  • Hypochloraemia
    • Vomiting
    • Diuretics
    • Metabolic alkalosis
  • Hyperchloraemia
    • (Normal anion gap) Metabolic acidosis (RTA, diarrhoea)
    • Dehydration
    • Excessive IV saline

11. Clinical biochemistry of osteoporosis. Laboratory tests to assess joint and bone disorders.

  • bone
    • 65% minerals (hydroxyapatite)
    • 35% matrix
      • Collagen
      • Osteoblasts
      • Osteoclasts
  • Markers
    • High biological and analytical variability
      • Diurnal variation
      • Seasonal variation
      • Food intake
      • Exercise
    • Made in same lab, collected at same time
  • Markers of bone formation
    • Total alkaline phosphatase
    • Bone specific alkaline phosphatase
    • Osteocalcin – increased in high bone turnover
    • Procollagen propeptides
      • PICP – procollagen type I C-terminal
      • PINP – procollagen type I N-terminal
  • Markers of bone resorption
    • Urine (corrected for creatinine)
      • Deoxypyridinoline (cross-linking collagen cleavage products)
      • N-terminal cross links (NTX) – N-telopeptide of type I collagen
      • C-terminal cross links (CTX) – C-telopeptide of type I collagen
    • Serum
      • TRACP5b – tartate resistant acid phosphatase
    • Serum and urine
      • beta-crosslaps

12. Laboratory analysis of plasma proteins

Parameter Sample Reference range
Total protein Serum 66 – 83 g/L
Total protein Urine < 0,1 g/L
Albumin Serum 35 – 53 g/L
  • Total protein
    • Biuret test (copper forms purple complex with proteins in alkaline solution)
    • Colour intensity proportional to protein concentration – photometry
  • Albumin
    • Albumin forms green complex with BCG at pH 4,1
    • Photometry
  • Specific proteins
    • Measurement based on immunogenicity – use of monoclonal or polyclonal antibodies
    • Immunoassay – biochemical test measuring concentration of protein by using antibody
      • Enzyme immunoassay (EIA)
      • Enzyme-linked immunosorbent assay (ELISA)
      • Radioimmunoassay (RIA)
      • Fluorescence
    • Proteins
      • Microalbumin (high sensitivity albumin measurement)
      • Hormones
      • Tumour markers
      • Drugs
      • B12
      • Folic acid
      • Transferrin
      • Ferritin
      • EPO
      • HCG
      • Troponin
      • Procalcitonin
  • Separation techniques
    • Chromatography
    • Electrophoresis – sample added to gel -> electric field applied -> molecules separated by electric charge -> stained to make charged molecules visible
      • Qualitative, semi-quantitative!
      • Serum, not plasma (fibrinogen forms a band)
      • Serum electrophoresis – monoclonal paraproteins
      • CSF electrophoresis – multiple sclerosis
      • Haemoglobin electrophoresis – haemoglobinopathies
      • Lipid electrophoresis – dyslipidaemias
      • Urine electrophoresis – Bence-Jones protein
  • Serum electrophoresis
    • Prealbumin=transthyretin
    • Albumin
    • Alpha-1 band
      • alpha1-antitrypsin
      • alpha1-acid glycoprotein (AGP)
      • alpha1-microglobulin
    • Alpha-2 band
      • Haptoglobin
      • alpha2-macroglobulin
      • Caeruloplasmin
    • Beta band
      • Transferrin
      • Beta2-microglobulin
      • Complement factors
    • Between beta and gamma
      • CRP
    • Gamma band
      • Immunoglobulins

13. Laboratory analysis of plasma enzymes

  • Enzymes
    • Measurement based on enzyme activity
    • Enzyme + substrate ->
      • coloured product -> photometry
      • or a secondary reaction which forms a coloured product -> photometry
      • or there is a change in NADH -> photometry with UV light
    • 1 unit = 1 µmol substrate per minute
    • U/L = µmol/min/L
  • Most frequently measured enzymes
    • ASAT
    • ALAT
    • GGT
    • ALP
    • LDH
    • CK
    • Amylase
    • Lipase

14. Disturbances of non-immunoglobulin plasma proteins

  • Prealbumin
    • Binds thyroxin (T3) and vitamin A
    • Marker of nutritional status
    • Negative acute phase protein
  • Albumin
    • Transports steroids, fatty acids, unconjugated bilirubin, drugs, calcium
    • Bisalbuminaemia – harmless
    • Negative acute phase protein
  • Alpha1-antitrypsin
    • Deficiency – cirrhosis, emphysema
  • Alpha1-microglobulin
    • Increased in urine in tubular dysfunction
  • beta2-microglobulin
    • Increased in urine in tubular dysfunction
    • Increased in serum in lymphoma
  • Caeruloplasmin
    • Reduced in Wilson disease
  • CRP
  • Ferritin
    • Iron status
  • Haptoglobin
    • Reduced in haemolysis
  • Thyroid-binding globulin
    • Disturbed in thyroid disease
  • Sex hormone binding globulin
  • Transferrin
    • Iron status

15. Disorders of immunoglobulins and paraproteins

  • Monoclonal gammopathies
    • Multiple myeloma
      • Total protein increased
      • IgG, IgA or IgM increase OR free kappa/lambda light chain increase
      • Serum electrophoresis: Peak in gamma-band (M-peak)
      • Urine protein electrophoresis: Bence jones proteins
      • ESR increased
      • Calcium increased
      • Kidney function decreased
    • MGUS
    • Waldenström macroglobulinaemia
    • Light chain amyloidosis
  • Polyclonal gammopathies
    • Infection
    • Inflammation

16. Assessment of acute phase proteins, diagnosis and monitoring of sepsis

Positive acute phase proteins

  • CRP
  • Ferritin
  • Fibrinogen
  • Alpha1-antitrypsin
  • Serum amyloid A
  • Haptoglobin
  • Procalcitonin
  • IL-6

Negative acute phase proteins

  • Albumin
  • Transferrin
  • Antithrombin

Sepsis

  • Leukocytosis
  • Thrombocytopaenia
  • Procalcitonin increase
  • CRP increase
    • Less sensitive than procalcitonin
  • Serum lactate increase

17. Laboratory findings in inflammatory disorders

Parameter Sample Reference range
CRP Serum < 5 mg/L
ESR (Westergren) Blood + citrate (black tube) 1 – 15 mm/h
  • CRP
    • Positive acute phase protein
    • Ring shaped pentameric protein
    • Activates classical pathway of component
    • Opsonizes
    • Increase begins within 6 – 12 hours, peaks at 24 – 48 h
    • Measured by immunoturbidimetric assay
    • Elevated in
      • Bacterial infection
      • Fungal infection
      • Necrosis
        • Acute pancreatitis
        • MI
        • Tumor
      • Autoimmune
        • RA
        • Vasculitis
        • Crohn’s disease
      • Trauma
  • High sensitivity CRP
    • Cardiovascular disease risk assessment
  • Procalcitonin
    • Precursor of calcitonin
    • Increase begins 3 – 4 hours, peaks at 24 hours
    • Elevated in
      • Systemic bacterial infection
      • Shock
      • Multiple organ failure
      • Fungal infection
    • Measured by electrochemiluminescence immunoassay (expensive)
  • ESR
    • Black tube
    • Manual or automatic
    • Less sensitive and specific
    • Elevated in inflammation and necrosis
    • Depends on albumin/globulin ratio, RBCs, fibrinogen

18. Laboratory diagnosis of malignant hematologic disorders; complete blood count and flow cytometry

Parameter Sample Reference range
WBCs 4,0 – 10,0 G/L
RBCs 4,5 – 6,0 T/L
Hb 137 – 175 g/L
Hct 40,1 – 51 %
Thrombocytes 140 – 440 G/L
  • CBC
    • WBCs
      • Neutrophil
      • Lymphocytes
      • Monocytes
      • Eosinophils
      • Basophils
    • RBCs
      • RBC count
      • Hb
      • Hct
        • RBC x MCV
      • MCV
        • Measured directly
      • MCH
        • Hb / RBC
      • MCHC
        • Hb / Htc
      • RDW
        • Price-Jones curve
      • Reticulocytes
    • PLT
      • PLT count
      • MPV
  • Flow cytometry (EDTA tube (purple))
    • Hydrodynamic focusing method -> one cell flow
    • Cell measured and identified by
      • Impedance
        • Number of RBC and PLT
        • Volume of RBC and PLT
      • Optical measurement
        • Forward scatter – cell size
        • Side scattered laser – granularity
      • Fluorescence – stains DNA, RNA
        • WBC, nucleated RBC, reticulocytes
  • Malignant haematological disorders
    • Blood smear
    • CBC
    • Bone marrow biopsy/aspirate
    • Chronic disorders
      • < 20% blasts in bm
      • Myelodysplastic syndrome
        • Dysplastic cells in blood
    • Acute disorders
      • > 20% blasts in blood and/or bm
      • LDH increase
      • Cytopaenias
    • Multiple myeloma
      • Total protein increase
      • Immunoglobulin or light chain increase
      • M peak on serum electrophoresis
      • Bence Jones protein in urine
      • Anaemia
      • Hypercalcaemia
      • Extremely high ESR

19. Laboratory diagnostic approaches in anaemias.

  • Anaemia definition: RBC/Haemoglobin/Haematocrit below normal
  • Normochromic normocytic anaemia
    • Increased reticulocytes – bone marrow works hard
      • Bleeding
      • Haemolysis
    • Decreased reticulocytes – bone marrow doesn’t work
      • Aplastic anaemia
      • Leukaemia
      • Myelodysplasia
      • Myelofibrosis
      • Chronic kidney disease (EPO deficiency)
  • Hypochromic microcytic anaemia
      • Iron deficiency
      • Anaemia of chronic disease
  • Hyperchromic macrocytic anaemia
    • Megaloblastic anaemia
      • B12 deficiency
        • Malabsorption
        • Pernicious anaemia
          • Autoantibodies against parietal cells/intrinsic factor -> no B12 absorption -> megaloblastic anaemia
      • Folate deficiency
        • Malabsorption
        • Pregnancy
    • No B12 or folate deficiency
      • Aplastic anaemia
      • Myelodysplasia
  • Haemolytic anaemia
    • LDH increased
    • K+ increased
    • Unconjugated bilirubin increased
    • (Free) Haptoglobin decreased
    • Reticulocytes increased
    • Causes
      • Autoimmune haemolytic anaemia
      • Haemoglobinopathies
      • DIC
      • Artificial heart valve

20. Haemoglobinopathies: disorders of the porphyrin metabolism.

  • Haemoglobinopathies
    • Sickle cell
      • Haemoglobin electrophoresis
        • Separates types of haemoglobin
      • Sickle cell on blood smear
    • Thalassaemia
      • Haemoglobin electrophoresis
      • DNA analysis
      • Target cells, teardrop cells on blood smear
  • Porphyrias
    • Deficient enzyme in haeme biosynthesis -> precursor accumulation
    • Porphyrins and porphyrin precursors in blood and (24 hour) urine
      • Makes urine tea-coloured or purple, especially when exposed to sunlight
      • Detection
        • direct photometry
        • Photometry after Ehrlich reaction

21. Laboratory findings in the disorders of iron metabolism.

  • Serum iron
    • Diurnal variation
    • 30% Biological variation
    • Influenced by inflammation, diet
    • Detected by coupled enzymatic reaction which form coloured complexes -> photometry
  • Transferrin
    • Binds 2 Fe3+ molecules, transports in plasma
    • Measured by immunoturbidimetry
    • Elevated when iron storages are empty
    • Transferrin saturation – how much of transferrin is saturated
  • Ferritin
    • Stores iron in liver, etc
    • Binds 4500 iron atoms
    • Small amounts leak into circulation
    • Decreased in iron deficiency
    • Measured by electrochemiluminescent immunoassay
  • Soluble transferrin receptor
    • Binds transferrin, transports it into cells
    • Elevated in iron deficiency
  • Reticulocytes
    • Low: Iron deficiency anaemia
    • If iron studies show low iron stores but reticulocytes are normal -> latent iron deficiency
    • If iron studies show normal iron stores but reticulocytes are low -> functional iron deficiency (anaemia of chronic disease)
Disorder Serum iron Transferrin Ferritin Soluble transferrin receptor Reticulocytes
Iron deficiency anaemia ↓ ↑ ↓ ↑ ↓
Haemoglobinopathy Normal Normal Normal Normal Normal
Anaemia of chronic disease ↓ Normal/↓ Normal/↑ Normal ↓
Haemochromatosis ↑ ↓ ↑ ↓ Normal

22. Blood coagulation: cellular components (platelets, endothelial cells) and their laboratory analysis.

  • Vasculopathies – endothelial dysfunction
    • Ehlers-Danlos
    • Marfan
    • Bleeding time prolonged
  • Thrombocytopathies – plt dysfunction
    • Platelet count and volume
      • Measured by impedance or light scatter with flow cytometry
    • Thrombocytopaenia
      • Heparin induced (HIT)
        • Immunoassay tests for antibodies (type 2)
    • Thrombocytosis
      • Inflammation
      • Iron deficiency
    • Thrombocythaemia
      • Myeloproliferative disease
    • Thrombocyte dysfunction
      • von Willebrand disease
      • Bernard-Soulier syndrome
      • Glanzmann thrombasthenia
    • Platelet aggregometry
      • Photometric measurement
      • Pro-aggregation factors are added (ADP, arachidonic acid, ristocetin)
        • Sample clears up -> transmittance increases
        • Disease examined depends on the factor added
          • Arachidonic acid – monitor aspirin
          • ADP – monitor clopidogrel, etc
          • Ristocetin – monitor von Willebrand disease, Bernard-Soulier

23. Blood coagulation: functional tests (PT, APTT, TT, fibrinogen, D-dimer).

Parameter Sample Reference range
Prothrombin time Citrated blood 80 – 120% of normal
INR Citrated blood 0,9 – 1,15
  • Prothrombin time
    • Thromboplastin reagent – recombinant tissue factor + synthetic phospholipids
    • Time from addition of reagent to clot formation -> optical detection
    • Reflects extrinsic + common pathway
      • VII, X, V, II, I
    • Monitor oral anticoagulant therapy (warfarin)
  • INR
    • Thromboplastin reagent is variable -> standardization is needed
    • International sensitivity index: sensitivity of reagent compared to WHO reference preparation
    • Recommended range: 2,0 – 3,0
    • Artificial heart valve: 2,5 – 3,5 (+ aspirin)
  • aPTT – activated partial thromboplastin time
    • Partial thromboplastin reagent – synthetic phospholipids + silica (no tissue factor)
    • Time from addition of reagent to clot formation -> optical detection
    • Reflects intrinsic + common pathway
      • XII, XI, IX, VIII, X, V, II, I
    • Monitor unfractionated heparin
    • Recommended range: 1,5 – 2,5 x healthy control
  • Thrombin time
    • Time for fibrin polymers to form after adding bovine thrombin
    • Reflects common pathway
    • Use to reflect disorders of fibrin or monitor fibrinolytic therapy
  • Fibrinogen
    • Bovine thrombin added in excess -> thrombin converts fibrinogen to fibrin
    • The time it takes to clot is compared to a calibration curve to determine the concentration of fibrinogen in the plasma
    • Decreased
      • Liver disease
      • DIC
    • Increased
      • Acute phase reaction
  • D-dimer
    • D-dimer is a fibrin degradation product
    • Measured with turbidometry and immunoassay
    • High negative predictive value
      • Negative D-dimer rules out DVT, PE
    • Not specific
      • Positive D-dimer does not mean the patient had thrombus

24. Laboratory monitoring of anti-coagulant therapies.

  • Antiplatelet therapy
    • Aspirin – platelet aggregometry with arachidonic acid
    • Clopidogrel etc – platelet aggregometry with ADP
  • Coumarins
    • INR – 2,0 – 3,0
  • Heparin
    • aPTT – 1,5 – 2,5x healthy control
  • LMWH
    • Anti-factor Xa assay
  • DOAC
    • No specific tests widely available

25. Current analytical protocol to detect acute myocardial infarction; international recommendations.

  • Ideal marker
    • High concentration in myocardium
    • Found only in myocardium
    • Released early in injury
    • Release proportional to injury
    • Cheap to test
  • Myoglobin
    • Rapid release within 2 hours
    • Not cardiac specific
    • Very short half-life
    • Not widely used
    • Can rule out AMI in early stages
  • Creatine kinase-MB
    • CK-MB more specific to myocardium than total CK
    • Good to evaluate reinfarction (because of short half-life)
    • Not cardiac specific – also found in skeletal muscle
    • Low sensitivity during early AMI or late AMI
  • Troponin
    • Two isotypes: I and T – similar
      • Cardiac and skeletal muscle subtypes
    • cTnT and cTnI – best sensitivity and specificity for AMI
    • Elevated up to 2 weeks after AMI
    • Bad at detecting small re-infarctions
    • Positive when above the 99th percentile of the upper reference limit
    • Not 100% specific – must be evaluated with ECG, clinical picture – also elevated in
      • myocarditis
      • Decompensated heart failure
      • PE
      • Arrhythmia
      • Renal failure
      • Sepsis
  • Ischaemia-modified albumin
    • New ischaemia marker
    • Rises within minutes of ischaemia
    • Less specific than troponin
  • Fourth universal definition of MI:
    • Acute myocardial infarction is: clinical evidence of acute myocardial ischaemia and with detection of a rise and/or fall of cardiac troponin

26. Laboratory monitoring the therapy of myocardial infarction. Laboratory approaches in chronic heart failure (BNP, proBNP, electrolytes).

  • Monitoring therapy of AMI
    • Decrease in troponin after treatment -> treatment successful
    • High troponin -> Worse prognosis
    • Immediately following reperfusion, troponin may temporarily increase
  • Chronic heart failure
    • BNP – brain natriuretic peptide
      • Produced in ventricles – production is proportional to volume and pressure overload
      • Very specific for congestive heart failure
      • Higher levels -> worse prognosis
      • High levels -> may cause hyponatraemia
      • Normal value increases with age
    • NT-proBNP – precursor of BNP
      • Increases more than BNP
    • Hyponatraemia

27. Laboratory diagnostics of the striated muscle diseases.

  • Degenerative striated muscle disorders
    • Duchenne muscle dystrophy
      • Elevated total creatine kinase
      • Creatine in urine
    • Rhabdomyolysis
      • Elevated total creatine kinase (very high levels, 5x upper limit)
      • Elevated myoglobin
      • Elevated LDH, K+, phosphate
      • Myoglobinuria – orange or brown
      • Renal impairment parameters
        • Urea
        • Electrolytes

28. Diagnostic criteria of diabetes mellitus (WHO criteria). Laboratory monitoring of patients with diabetic history

Parameter Sample Reference range
(Fasting) glucose Glycolysis inhibited plasma 4 – 7,0 mM
Microalbumin Urine < 30 mg/L
Total protein Urine < 0,1 g/L
HbA1c Serum < 5,7 %
  • Preanalytical errors in carbohydrate testing
    • Correct tube (should contain sodium fluoride to inhibit glycolysis + heparin)
    • Correct time
    • Correct patient preparation (diet, fasting)
  • Diagnostics of diabetes mellitus
    • Diagnosis (either one of these)
      • HbA1c > 6,5%
      • Fasting plasma glucose > 7,0 mM
      • OGTT > 11,1 mM
      • Symptoms of hyperglycaemia + random plasma glucose > 11,1 mM
    • Fasting plasma glucose
      • Normal – < 6,1 mM
      • Impaired fasting glucose – 6,1 – 7,0 mM
      • Diabetes mellitus – > 7,0 mM
    • Random plasma glucose
      • Diabetes mellitus – > 11,1 mM
    • Oral glucose tolerance test
      • Fasting -> Glucose intake (75g) -> blood test after 2 hours
      • Normal < 7,8 mM
      • Impaired glucose tolerance – 7,8 – 11,1 mM
      • Diabetes mellitus – > 11,1 mM
    • HbA1c
      • Corresponds to average glucose of 120 days (lifespan of RBC)
      • HbA1c is the result of an Amadori reaction between Hb and glucose
      • Can be measured with POCT
      • Higher HbA1c -> higher risk of complications
      • Measured as percent of total Hb or as mmol HbA1c/mol Hb
      • Normal – < 5,7% = 39 mmol/mol
      • Prediabetes – 5,7 – 6,4% = 39 – 46 mmol/mol
      • Diabetes mellitus – > 6,4% = 46 mmol/mol
  • Fructosamine
    • = reflects total serum glycated proteins
    • Corresponds to average glucose of 30 days
    • Strongly affected by albumin metabolism and thyroid dysfunction
    • Much less used than HbA1c
  • Glycated albumin
    • = reflects serum glycated albumin
    • Corresponds to average glucose of 30 days
    • Strongly affected by albumin metabolism and thyroid dysfunction
    • Much less used than HbA1c
  • Monitoring of diabetes mellitus
    • Kidney function
      • Creatinine
      • Electrolytes
    • Blood lipids
    • Eye function
    • Blood pressure
    • HbA1c
    • Glucose
    • Urine
      • Protein
      • Glucose
      • Microalbuminuria
        • 30 – 300 mg/day
        • Early sign of diabetic nephropathy
      • Ketones

29. Clinical biochemistry of hypoglycaemia and hyperglycaemia

Parameter Sample Reference range
(Fasting) glucose Glycolysis inhibited plasma 4 – 7,0 mM
  • Hypoglycaemia
    • Glucose < 4,0 mM
    • Counter-regulatory response (glucagon, catecholamines) -> cognitive disturbance -> coma (< 1,0 mM)
    • Often occurs in diabetics
    • Tremor
    • Moist skin
  • Hyperglycaemia
    • Diabetic ketoacidosis (DKA)
      • Hyperglycaemia
      • Electrolyte abnormalities
        • Low intracellular K+
      • Decreased bicarbonate
      • Ketones in plasma and urine
      • Metabolic acidosis
      • Dehydration
      • Kussmaul breathing
    • Hyperosmotic hyperglycaemic syndrome (HHS)
      • Hyperglycaemia (higher than DKA)
      • Hyperosmolarity
      • No ketones
      • No acidosis
      • Dehydration
      • No Kussmaul breathing

30. Lipids and lipoproteins in the blood plasma

Parameter Sample Reference range
Cholesterol Serum 4,0 – 5,6 mM
LDL Serum 0,0 – 3,4 mM
HDL Serum 0,9 – 1,7 mM
  • Analysis of lipids
    • Enzymatic assay
      • Cholesterol
      • HDL, LDL
      • FFA
      • Triglycerides
    • Immunoassay
      • ApoA1
      • ApoB
      • Lp(a)
    • Lipoprotein electrophoresis
    • Ultracentrifugation – sorted after density
  • Cholesterol
    • Component of cell membrane
    • Precursor of steroid hormones and bile acids
  • Triglycerides
    • Energy store
    • Risk factor for CVD, acute pancreatitis
  • HDL
    • Contains mostly protein and phospholipid
    • Transports cholesterol back to liver from peripheral tissues
    • Negative risk factor for CVD
  • LDL
    • Contains mostly cholesterol
    • Transports cholesterol from liver to peripheral tissues
    • Risk factor for CVD
    • Small, dense LDL
      • Greater atherogenic potential than other LDL subtypes
      • Less affinity to LDL receptor
      • Higher endothelial permeability
  • VLDL
    • Contains mostly triglycerides
    • Transports triglycerides from liver to peripheral tissues
  • Lipoprotein(a)
    • LDL-like particle
    • Contains mostly cholesterol
    • Increased level -> increased risk for CVD, atherosclerosis
    • Independent predictor of CVD
    • Recommended to be tested in people with moderate or high risk for CVD
  • Chylomicron
    • Contains mostly triglycerides
    • Transports dietary triglycerides to peripheral tissues and liver
  • Nonfasting lipid panel
    • Measures HDL and total cholesterol
    • Screening every 5 years from around 40 y old, from 20y if high risk
  • Fasting lipid panel
    • Measures HDL, total cholesterol, triglycerides, LDL
    • LDL can be calculated: LDL = TC – HDL – (TG/5)
  • Hereditary dyslipidaemias
    • Familial hypercholesterolaemia
      • Mutated LDL receptor
      • High risk for AS and xanthoma
    • Familial combined hyperlipidaemia
      • Decreased LDL receptor
    • Familial hypertriglyceridaemia
      • Can cause pancreatitis
  • Metabolic syndrome
    • Definition: 3 or more of the following:
      • Insulin resistance
      • Hypertension
      • Hypertriglyceridaemia
      • Low HDL
      • Abdominal obesity

31. Laboratory tests that predict hepatic disorders.

Parameter Sample Reference range
Total bilirubin Serum 5 – 20 µM
Albumin Serum 35 – 53 g/L
Total protein Serum 66 – 83 g/L
AST Serum < 44 U/L
ALT Serum < 50 U/L
Gamma-GT Serum < 70 U/L
Alkaline phosphatase Serum 40 – 130 U/L
  • Parameters of hepatocellular damage
    • Alanine aminotransferase (ALT) = GPT
      • L for liver specific
      • P for perfect for liver diseases
      • Specific to hepatocytes
    • Aspartate aminotransferase (AST) = GOT
      • Not specific to hepatocytes (muscle, heart, RBCs)
      • In mitochondria – only elevated in severe damage, where mitochondria are affected
    • AST/ALT ratio
      • Healthy: ratio < 1
      • Viral hepatitis, NASH: Elevated but ratio < 1
      • Alcoholic hepatitis: Elevated and ratio > 2
    • LDH
      • Nonspecific but often elevated
  • Parameters of cholestasis
    • Gamma-glutamyl transpeptidase (GGT)
    • Alkaline phosphatase (ALP)
      • Various form found in various tissues (bone, liver, kidney, etc.)
        • If no bone disease or pregnancy -> specific for cholestasis
      • Slight elevation (1 – 2x normal): Parenchymal liver disorders
      • High elevation (3 – 10x normal): extrahepatic or intrahepatic biliary obstruction
    • Direct bilirubin
    • Indirect bilirubin
  • Parameters of hepatic synthesis
    • Albumin
    • Alpha-foetoprotein (AFP)
    • alpha2-macroglobulin
    • haptoglobin
    • apolipoprotein A1
  • Acute hepatitis B
    • HBsAg
    • HBsAb – IgM

32. Laboratory diagnostics of alcoholic liver damage. Laboratory tests to assess liver fibrosis

  • Alcohol liver damage
    • Alcoholic fatty liver
      • AST > ALT (AST/ALT > 2)
      • Carbohydrate-deficient transferrin
        • Most specific marker of heavy alcohol use (independent of liver disease)
    • Alcoholic hepatitis
      • AST > ALT (AST/ALT > 2)
      • Mixed hyperbilirubinaemia
      • Decreased albumin
      • Increased prothrombin time (decreased clotting factors)
  • Liver fibrosis
    • Mixed hyperbilirubinaemia
    • AST, ALT increased
    • GGT increased
    • Albumin decreased
    • Increased prothrombin time (decreased clotting factors)
    • Thrombocytopaenia (hypersplenism)
    • B12/folate deficiency
    • FibroTest
      • Uses six biomarkers to assess degree of liver damage
        • alpha2-macroglobulin
        • haptoglobin
        • apolipoprotein A1
        • Bilirubin
        • GGT
        • Age
        • Gender
      • Very specific and sensitive

33. Disorders of bilirubin metabolism

Parameter Sample Reference range
Total bilirubin Serum 5 – 20 µM
  • Measurement of bilirubin – Diazo method or enzymatic
  • Prehepatic hyperbilirubinaemia
    • Haemolysis
    • Unconjugated
  • Intrahepatic hyperbilirubinaemia
    • Hepatitis
    • Mixed
  • Posthepatic hyperbilirubinaemia
    • Cholestasis
    • Conjugated
  • Crigler-Najjar syndrome & Gilbert syndrome
    • Unconjugated

34. Laboratory diagnosis of acute pancreatitis

Parameter Sample Reference range
Amylase Serum 28 – 100 U/L
  • Elevated pancreatic enzymes
    • Lipase (highly elevated -> very specific)
    • Amylase (not specific)
      • Serum and urinary
      • Other causes of elevation: Salivary gland inflammation, peptic ulcer, cancer, kidney failure …
    • Not proportional to severity or prognosis!
  • Additional
    • Hypocalcaemia (saponification)
    • Leukocytosis
    • CRP, procalcitonin increase
    • Hct
      • Increased (haemoconcentration) – third spacing of fluid
      • Decreased – haemorrhagic pancreatitis
  • To determine etiology
    • Triglyceride levels – hypertriglyceridaemia cause
    • Calcium levels – hypercalcaemia cause
    • ALP, bilirubin – obstructive cause

35. Laboratory diagnosis of chronic pancreatitis

  • Elevated pancreatic enzymes (if acute inflammation and not end-stage disease)
    • Lipase
    • Amylase
      • Serum and urine
  • Pancreas function test
    • Glucose tolerance
    • Oral administration of fluorescein dilaurate (metabolization requires pancreatic enzyme)
    • 13C-triolein breath test – radioactive triglyceride is ingested – content of exhaled CO2 is measured
    • Faecal fat content measurement (lipase deficiency)
    • Faecal elastase activity

36. Laboratory diagnosis and monitoring of malignant and inflammatory bowel diseases

  • IBD
    • Systemic inflammatory markers
      • CRP
      • ESR
    • More specific markers
      • Anti-saccharomyces cerevisiae antibodies (ASCA)
      • Perinuclear antineutrophil cytoplasmic antibodies (P-ANCA)
      • Calprotectin (stool)
        • Negative predictive value
        • Detect flareups
        • Monitor treatment response
  • Coeliac disease
    • Anti-tTG antibody
      • Tissue transglutaminase
    • Anti-endomysial antibody (EMA)
    • Anti-deamidated gliadin peptide (anti-DGP)
  • Tests for malabsorption
    • General
      • Serum proteins
      • Anaemia
      • Iron studies
      • Prothrombin time
    • Functional tests
      • Xylose test – carbohydrate malabsorption
      • Hydrogen breath test – test for lactose, fructose malabsorption or bacterial overgrowth
      • Stool tests for nutrients
  • Malignancies
    • No specific tumor marker
    • General tests
      • Inflammatory markers: CRP, ESR, leucocytosis
      • Tissue damage: LDH, K+
      • Blood loss: Anaemia
    • Tumor markers
      • CEA
      • CA 19-9

37. Laboratory diagnosis of acute renal diseases.

Parameter Sample Reference range
Creatinine Serum 62 – 106 µM
Urea Serum 2 – 9 mM
Sodium Serum 136 – 146 mM
Potassium Serum 3,8 – 5,2 mM
Osmolality Urine 50 – 1400 mOsmol/kg
  • Creatinine
    • Daily muscle turnover
    • Serum level depends on muscle mass, diet, exercise
    • Freely filtered, little tubular excretion
    • Increased in acute renal failure
    • eGFR
      • Creatinine clearance = GFR
      • Based on age, gender, serum creatinine
      • Interference with drugs, diet, age, comorbidity, BMI
  • Urea
    • Increased in acute renal failure
  • Oliguria (< 400 mL/day)
    • Anuria in postrenal failure
  • Fluid, electrolyte imbalances
    • Hyperkalaemia
    • Metabolic acidosis
  • Cystatin-C
    • Precise GFR estimation
    • When precise GFR is needed or there are interferences with eGFR
  • Tubule function parameters
    • Proximal tubule
      • Proteinuria
      • Glucosuria
      • Urine sodium concentration
    • Distal tubule
      • Urine osmolality
Test Prerenal failure Acute renal failure
Urea Highly elevated Moderately elevated
Creatinine Less elevated Moderately elevated
Urea:creatinine > 20:1 < 20:1
Proteinuria No Yes
Urinary sodium concentration Low High
Urine osmolality High Low

One student in the comments told us that knowing the formula “Clearance = urine concentration x urine flow rate / plasma concentration” or “Clearance = UxV/P” was, according to their examiner, important to know for the exam.

38. Laboratory diagnosis and monitoring of chronic renal diseases

Staging based on GFR

Stage GFR (mL/min)
G1 > 90
G2 60 – 90
G3a 45 – 60
G3b 30 – 45
G4 15 – 30
G5 < 15

Staging based on albuminuria

Stage Albumin excretion rate (mg/day)
A1 < 30
A2 (microalbuminuria) 30 – 300
A3 (macroalbuminuria) > 300
  • POTE staging
    • Stage 1 – Normal urea and creatinine
    • Stage 2 – Increased urea and creatinine
    • Stage 3 – Increased urea and creatinine, electrolyte imbalance
      • Symptoms of uraemia
      • Hyperkalaemia
      • Hyperphosphataemia
      • Hypocalcaemia
        • Can’t produce vit D
      • Metabolic acidosis
      • PTH increased
      • Bleeding time increased
      • Anaemia of chronic renal disease

39. Laboratory findings in proteinuria and haematuria

  • Nephrotic syndrome
    • Definition
      • Proteinuria > 3,5g/day
      • Oedema
      • Hypoalbuminaemia
      • Hyperlipidaemia
    • Loss of ATIII – hypercoagulability
    • Hypogammaglobulinaemia
  • Nephritic syndrome
    • Definition
      • Haematuria
      • RBC casts in urine
      • Proteinuria (less than in nephrotic syndrome)
      • Hypertension
  • Haematuria
    • Nephritic syndrome
    • UTI
    • RCC, urothelial cancer
    • Urolithiasis
    • Myoglobinuria
  • Proteinuria
    • Glomerular proteinuria
      • Medium-sized proteins, like albumin
      • If severe, large proteins: IgG
    • Tubular proteinuria
      • Beta-2-microglobulin in urine
    • Overflow proteinuria
      • Multiple myeloma
      • Haemolysis
      • Myolysis

40. Laboratory assessment of increased serum uric acid levels

  • Uric acid
    • Catabolites of purines
    • Triggers gout
    • Can trigger uric acid nephropathy
      • Deposition of crystals in kidney
    • Elevation
      • Lesch-Nyhan syndrome
      • Tumor lysis syndrome
      • Increased dietary intake
      • Alcohol
      • Renal failure

41. Laboratory tests of the cerebrospinal fluid and other body fluids.

Parameter Sample Reference range
Microalbumin Urine < 30 mg/L
Osmolality Urine 50 – 1400 mOsmol/kg
Total protein Urine < 0,1 g/L
  • CSF
    • Compared to plasma
      • Less protein
      • Different electrolyte composition
      • Very few cells
    • CNS infection or blood in CNS changes composition of CSF
    • Sampled from lumbar puncture
    • Biochemical analysis within 2 hours! Cooling!
    • Biochemical tests
      • Glucose
      • Total protein
      • Albumin
      • IgG
      • Lactate
      • Cells
      • Electrophoresis

CSF in different disorders

Parameter Healthy Bacterial meningitis Viral meningitis Multiple sclerosis
Colour Clear Cloudy Clear Clear
RBCs Very few Normal Normal Normal
WBCs Very few Increased (polymorphs) Increased (lymphocytes) Increased (lymphocytes)
Lactate Normal Increased Normal Normal
Glucose > 60% of serum Decreased Normal Normal
Protein < 0,45 g/L Increased Normal Normal
Microbiology Sterile Culture positive Sterile/virus detectable Sterile
Oligoclonal bands Negative Negative Negative Positive

Urine

  • Sample taking
    • First morning urine
    • Midstream urine
    • Analysis within 2 hours
  • Gross appearance
    • Cloudy – UTI
    • Odour – ketonuria
  • Urine output
    • 800 – 1800 mL/day
    • Oliguria < 400 mL/day
    • Anuria < 100 mL/day
  • Microscopy
    • Urine sediment analysis
    • Cells
      • RBCs
      • WBCs
      • Epithelial cells
    • Casts
    • Crystals
    • Bacteria
  • Biochemistry
    • Dipstick
      • Test strips have coloured reagent blocks – when binding to specific structures in the urine the colour changes
        • Oxidation reactions
          • Glucose
      • Specific gravity/density
      • pH
      • Leukocytes
        • UTI
        • Inflammation
      • Haemoglobin/blood
        • UTI
        • Glomerulonephritis
      • Nitrite
        • UTI
      • Ketones
      • Bilirubin
        • Conjugated bilirubin
      • Protein
      • Glucose
    • Special investigations
      • Protein
        • Nephrotic syndrome
        • Micro/macroalbuminuria
        • Tubular failure
          • alpha and beta globulins
      • Electrophoresis
        • Bence Jones protein
      • Porphyrins
      • Toxicology
      • Urinary catecholamines and metabolites
        • Phaeochromocytoma
      • Serotonin metabolite
        • Carcinoid tumors

42. Pre-analytical considerations of the hormone tests.

  • Measured by serum in plain tube (red) or gel tube (yellow)
  • Small peptide hormones
    • Unstable, transported quickly and on ice
    • TRH
    • Renin
  • Amino acid derivatives
    • T3, T4
      • Only free fraction is measured
      • Protein binding influences measurement
    • Adrenalin
  • Steroids
    • Cortisol
    • Aldosterone
    • Sex hormones
    • Both fractions are measured
      • Binding protein should also be measured
  • Biological variation
    • GH – pulsatile release
    • Cortisol – influences by stress, diurnal rhythm
    • Should be measured in the morning
    • Log-linear relationship – small change in TRH can cause significant change in TSH
  • Analytical variations
    • Measured by immunoassays
    • Immunoassay interference
      • Heterophilic antibodies – low affinity antibodies
      • Anti-animal antibodies
      • Autoantibodies
      • Antibodies from treatment
      • Rheumatoid factor
    • Matrix effect
      • High level of molecules in plasma -> molecules can cover epitopes of analyte -> underestimate level of analyte
    • High-dose hook effect
      • More analyte than capture antibody -> analyte binds to detection antibody without being captured -> detection antibodies depleted -> fewer captured analytes can be labelled -> false negative result

43. Clinical biochemistry of the hypothalamus, hypophysis

  • Dynamic function test
    • Administer insulin -> measure cortisol, GH
    • Administer GnRH -> measure FSH, LH
    • Administer TRH -> measure TSH
  • GH excess
    • Administer oral glucose -> GH still high (usually decreases)
  • GH deficiency
    • Low levels of IGF-1
    • Administer GHRH -> GH still low (usually increases)
  • Hyperprolactinaemia
    • Serum prolactin
    • Administer dopamine agonist -> prolactin still high (usually decreases)
  • Diabetes insipidus
    • Water deprivation -> urine osmolality unchanged (usually increases)
    • Administer desmopressin -> urine osmolality increases (normalizes)

44. Laboratory assessment of the thyroid function

  • Hypothyroidism
    • Elevated TSH – more specific than thyroid hormones!
    • Low fT3, fT4
    • (TPO antibody) – if autoimmune
    • LDL increased
    • cholesterol increased
  • Hyperthyroidism
    • Decreased TSH
    • High fT3, fT4 (can be normal if subclinical)
    • Anti-TSHR – Graves disease
  • Drugs which affect thyroid function
    • Lithium – decreases thyroid hormone release
    • Amiodarone – hypo or hyperthyroidism
    • SSRIs – increase TSH
  • Pregnancy
    • hCG stimulates TSH-R -> hyperthyroidism
  • Thyroid cancer
    • Calcitonin – tumor marker
    • Thyroglobulin – follow-up after thyroidectomy

45. Clinical biochemistry of the disorders of adrenal medulla/cortex

  • Adrenal cortex
    • Hypofunction
      • Hyponatraemia
      • Hyperkalaemia
      • Metabolic acidosis
      • Addison disease
        • Low cortisol
        • ACTH high
        • ACTH stimulation -> cortisol remains low (usually rises)
      • Secondary/tertiary adrenal insufficiency
        • Low cortisol
        • ACTH low
        • ACTH stimulation -> cortisol increases
      • Congenital adrenal hyperplasia
        • High levels of 17-hydroxyprogesterone
    • Hyperfunction
      • Hypernatraemia
      • Hypokalaemia
      • Metabolic alkalosis
      • Cushing syndrome
        • 24 hour urine cortisol high
        • Low-dose dexamethasone suppression test -> cortisol remains high (usually decreases)
        • Cushing disease
          • High-dose dexamethasone -> cortisol decreases
        • Ectopic ACTH production
          • High-dose dexamethasone -> no change in cortisol
  • Adrenal medulla
    • Phaeochromocytoma
      • Metanephrines and catecholamines in 24 hour urine
      • Genetic testing

46. Clinical biochemistry of the disorders of the human reproductive system

  • Anti-Mullerian hormone
    • Assesses woman’s ovarian reserve
  • PCOS
    • High testosterone
    • High LH
    • FSH, oestradiol normal
  • Prenatal lab tests
    • HCG – increased in Downs
    • AFP – decreased in Downs
    • Oestriol – decreased in Downs
    • Evaluate chance for genetic conditions

47. The most important non-specific laboratory tests that suggest the presence of malignant diseases.

  • Multiple myeloma
    • Extremely high ESR
  • GI cancer
    • Anaemia
  • Leukaemias
    • WBC increase
    • Anaemia
    • Thrombocytopaenia
    • Blasts in bm or blood
    • Abnormal differentiation
  • Bone tumors
    • ALP increase
  • Lung cancer
    • LDH increase
  • Bile duct cancer
    • Bilirubin increase
  • Neuroendocrine tumors
    • Hormones
  • During chemo
    • Bone marrow depression
    • LDH increase
    • Uric acid increase

48. Tumor markers and their informational value in the clinical laboratory practice

  • Tumor markers
    • A molecule originating from tumors which can be quantitatively analysed by non-invasive methods
    • Often produced by tumor cells, but can be produced by neighbouring cells
    • Synthesized in normal cells too -> reference range
    • Not for screening – low sensitivity and specificity
    • Not for diagnosis
    • For prognosis, follow up and check for relapse
    • To laboratory ASAP
  • PSA – prostate cc
    • Not to be taken after rectal examination (artificially increases value)
    • Free PSA can be measured (percentage of how much is bound to protein) – the lower the free % the higher the risk for cancer
  • Calcitonin – Thyroid cancer
  • AFP
    • HCC, testicular cc
  • CEA – carcinoembryonic antigen
    • CRC
    • Breast cancer
    • Elevated in smokers!
  • CA 19-9
    • Hepatobiliary cc
    • Pancreatic cc
  • MCA – Breast cc
  • CA 125 – Ovarian cancer
  • hCG
    • Germ cell tumors
    • Trophoblastic tumors
  • Paraneoplastic syndrome
    • Clinical feature caused by tumors but not by mass effect or metastasis
    • General: Cachexia, thrombosis
    • Thymoma – myasthenia gravis
    • Cushing syndrome – SCLC, Pancreas
    • SIADH – SCLC
    • Hypercalcaemia – SCC, RCC

49. Molecular biology applications in the practice of clinical laboratories

  • Benefit
    • Diagnosis
    • Prognosis
    • Therapy response
  • Monogenic disorders
    • Sickle cell anaemia
    • Thalassaemia
  • Molecular biology methods
    • High sensitivity and specificity
    • Expensive
    • Real-time PCR
      • Visualization – fluorescent dyes, hybridization
    • DNA sequencing
      • Next generation sequencing
      • Whole exome sequencing
  • Microbiological assays
    • HepC
    • HepB
    • CMV
    • EBV
    • HSV
    • Gonorrhoea
  • Inherited conditions
    • Factor V Leiden
    • Prothrombin mutation
    • Gilbert syndrome (UGT)
    • Congenital metabolic disorders
    • NF1
    • CFTR
    • Neurodegenerative diseases
  • Pharmacogenomics
    • Polymorphisms in Pgp
    • CYP2C19 – clopidogrel
    • CYP2C9, VKORC1 – warfarin
    • TPMT – azathioprine

50. Therapeutic drug monitoring (TDM)

  • Methods
    • High performance liquid chromatography
    • Immunoassay
  • Indications
    • Non-linear kinetics
    • When there is poor compliance
    • If the therapeutic effect can’t be assessed otherwise
    • If the therapeutic index is low
      • Warfarin
      • Aminoglycosides
      • Antipsychotics
      • Digoxin
      • Lithium
    • When there are signs of overdose
    • To determine pharmacokinetic interactions

51. Toxicology tests in the clinical laboratory

  • Methods
    • HPLC
      • Can detect many drugs at the same time
    • Immunoassay
    • Alcohol -> enzymatic reaction + UV detection, gas chromatography
    • Blood, urine
  • Qualitative, sometimes quantitative
  • Drugs
    • Benzos
    • Barbs
    • Illegal drugs
    • Alcohol
  • Indications
    • Suspected poisoning
    • Unexplainable sickness

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Clinical Radiology

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Basics of blood transfusion

Parent page:
Fourth year

23 thoughts on “Clinical Biochemistry”

  1. Hi, could you maybe add the formula for clearance of kidney? because I was asked on my exam last year and I failed because of that:(
    Thank you

    1. Do you know which formula he meant? The Cockcroft-Gault formula, the formula based on serum and urine creatinine, or something else?

      1. I think he meant this one> CL=U*V/P
        CL> clearance, U>urine concentration of a substance, V>flow rate, P>plasma concentration of a substance

        1. I understand. Was that the only question you answered wrong in your exam when you failed?

          1. It was a long time ago but I think I answered some other things wrong too and I failed after not being able to write this formula. apparently this formula was really important to him and he had told his class that this is necessary to know for the exam. then for the next exam I just read your notes once again plus this formula and got a 5 haha(I had looked through a bit of the lectures for my A chance too but your notes were the main help)
            Thank you 🙂

  3. Hello,

    I had my exam recently and was asked about the other glycated product that is used for diabetes monitoring. glycated albumin measured by the “Fructose amine test”. Maybe its worth adding it to topic 29 🙂

    Thank you for all your work!

  4. Hey for topic 26 and cardiac necroenzyme levels you wrote that if they decrease its good and if they increase that is worse prognosis; this is true after some time
    but what they mentioned in the lecture is that immediately after thrombolytic therapy, reperfusion will cause an immediate strong increase in troponin and myoglobin showing successful reperfusion which will ofcrs decrease over time!

  5. hello greek.doctor
    in topic 43: for Hyperprolactinaemia, shouldn’t it be dopamine agonist administration (bromocriptine) or we use dopamine antagonist for Hyperprolactinaemia?
    thanks alot.

  6. Hey greek.doctor

    What do you recommend for “evaluating laboratory results “ part of the exam ?

    1. I wouldn’t worry about that. First of all, few people are asked to evaluate a lab result on the exam (mostly those who are close to failing or want a better grade). Besides, you’ll be more than capable to evaluate lab results after having studied the topics.

  7. For question 21. you wrote that for iron deficiency anemia the reticulocyte is low but in the table you have the arrow pointing up, I think this is a mistake. Also, for anemia in chronic disease you have the arrow pointing up but I believe reticulocyte count is also low here.

    1. Hey

      Yeah, pre-diabetes refers to having either impaired fasting glucose, impaired glucose tolerance or HbA1c 5,7 – 6,4%

  9. Hello , thanks for these notes
    I want to ask where did you collect the info that the lectures doesn’t cover? I’m confused where to study from
    Thanks in advance and good luck , you really deserve all the best ❤️

    1. The extra info isn’t just from one source, it’s mostly from random pages on the internet I think, and amboss. I tried using the book they recommend but it sucks balls.

      Thank you too, you also deserve the best <3

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