Background

Lab analysis of a blood sample is used to evaluate

·       Organ function

·       Disease

·       Biochemical state

·       Mineral content

·       Drug effectiveness/Overdose

• Typical blood panel
• Blood tests in suspected infections
• Bone profile/Rheumatology
• Blood tests for bone tumors/metastases
• Patients with polytrauma /high dependency.

1.      Complete blood count

• Hemoglobin
• RBC count
• White cell count with differential
• MCV/MCH/MCHC
• Platelets 

2.      Basic metabolic panel

• Blood urea nitrogen
• Creatinine
• Electrolytes

• Earliest precursor in the marrow is the pre-erythroblast and maturation proceeds through the stages of normoblast in which there is progressive condensation of the nuclear chromatin and the development of haemoglobin in the cytoplasm.
• Disappearance of the nucleus completes the formation of the young erythrocyte (reticulocyte) which is larger than the mature form and has a faintly bluish colour with Romanowsky stains and still contains fine reticular material.
• The mature erythrocyte stains as an eosinophilic, circular biconcave disc with a diameter of 7.2 microns.
• The presence of normoblasts in blood indicates excessive or abnormal blood formation or irritation of the bone marrow.
• Excessive reticulocytes reflects increased erythropoiesis. 

Figure 1. Microscopic Features:

• Size 6-8 um, slightly smaller than a mature lymphocyte
• Lacks a nucleus
• Cytoplasm is pink-orange in color with a central pallor
• Central pallor is 1/3 the diameter

• Normal: 130-160g/L
• Amount of hemoglobin present in blood
• Involved in transport of gases (O2 and CO2)
• Low: blood loss (trauma), impaired production (B12 deficiency), increased destruction (Sickle cell anemia)
• High: polycythemia
• Important to optimize Hb prior and after surgery
• Anemia can lead to predisposition of infections, nonunion, wound healing problems, fluid overload cardiac failure.
• Restrictive red cell transfusion threshold is 70g/L depending on the clinical condition (as per NICE guidelines-May 2015)
• Haemoglobin in the normal adult consists of four paired polypeptide chains, 2 alpha chains of 141 amino acids and 2 beta chains of 146 amino acids.
• Iron is essential in the synthesis of haemoglobin and a deficiency results in over mature small red blood cells in the circulation and a microcytic anaemia.
• B12 and folate are required in the maturation of red blood cells, a deficiency state results in an increased proportion of immature blood cells in the circulation resulting in a macrocytic anaemia. 

• Normal: 4000-11000/micL
• Low: radiation, chemotherapy, AIDS, drugs like Leflunamide, sepsis
• High: infections, inflammation, trauma, surgery, and tumors.
• Low counts predispose to infections
• Differential count: Neutrophils, lymphocytes, basophils, eosinophil, monocytes.
• Granular series which includes neutrophils eosinophils and basophils and are differentiated on the basis of their staining characteristics and the earliest recognisable precursors in the marrow are myeloblasts.

• Primary white blood cell
• Mature neutrophils account for about 40-70% of the total leucocytes in the peripheral blood of a healthy adult.
• High: Acute bacterial infections, acute inflammation
• Low: radiation, chemotherapy, Indomethacin, blood cancer
• Neutrophils have a high enzyme content and are actively phagocytic to infective organisms which they digest.
• The products of autodigestion are potent stimulants of fresh neutrophil formation by the bone marrow.
• Physiological factors that increase their number in the peripheral blood include exercise, emotional stress and pregnancy.

• 15-40 % of WBC
• Lymphocytes mainly derived from stem cells in lymphoid tissue though some are found in bone marrow. The immature form is the lymphoblast which resembles a myeloblast
• Three types: NK cells, T cells and B cells
• NK cells: cell mediated cytotoxic inate immunity
• T cells: cell mediated cytotoxic adaptive immunity
• B cells: antibody driven adaptive immunity
• High: Leukemia’s, lymphomas, chronic inflammation
• Low: HIV

Types of hypersensitivity reaction:

Type I (Anaphylactic, allergy or immediate hypersensitivity)- Reagin dependent reaction mediated by IgE

Type II (Cytotoxic antibody dependent) -Mediated by humoral antibody IgG, IgM

Type III (Immune complex disease)- Caused by deposition of antigen-antibody complexes in tissues -, inflammatory reaction.

Type IV (Delayed cell mediated hypersensitivity )- Memory t cells activate macrophages.

Type V (Autoimmune diseases)- Receptor mediated like Graves disease, myasthenia Gravis.

• 2.3% of WBC
• Combat parasites
• Associated with allergy/asthma
• High: Parasitic infections, Rheumatoid arthritis, Hodgkin’s disease

• 0.4% of WBC
• Seen in certain allergic response

• 5-6 % of WBC
• Monocytes are formed from stem cells in the spleen and lymphoid tissue and to a lesser extent bone marrow and the immature cell is a monoblast which are similar to myeloblasts and lymphoblasts.
• Part of innate immune system
• Involved in phagocytosis, antigen presentation and cytokine production
• Monocytes are formed from stem cells in the spleen and lymphoid tissue and to a lesser extent bone marrow and the immature cell is a monoblast which are similar to myeloblasts and lymphoblasts.
• High: Chronic inflammation, stress, sarcoidosis

• Range 140,000 - 390,000 (mm³)
• Platelets are small (2 - 4 micron) hyaline non-nucleated bodies with blue or purple granules.
• Derived from megacaryocytes which are very large cells containing multilobulated nuclei and granular cytoplasm.
• Contribute to hemostasis
• Activation of clotting cascade
• Bleeding tends to occur when the platelet count falls below 40 x10⁹/l
• Low: DIC, ITP, Heparin induced thrombocytopenia, Gauchers disease, aplastic anemia
• High: Acute blood loss, chronic inflammation/infection.

• Blood urea nitrogen
• Creatinine
• Electrolytes

•  Normal: 6-20mg/dL
• Produced in urea cycle from digestion of protein
• Raised: high protein diet, renal failure, hypervolemia, heart failure, catabolic states, GI bleeding, and fever.
• Low: Anabolic states, severe liver disease

• Normal: 0.6-1.2 mg/dL
• Breakdown of creatine phosphate in muscle
• Excreted chiefly by Kidneys
• Raised: Renal failure, hypervolemia, rhabdomyolysis.
• Low: Late stage muscle loss

• Normal: 135-145 mEq/L
• Involved in salt and water balance
• Maintain normal blood pressure, normal function of nerves and muscles.

• Hyponatremia:

-Renal failure, heart failure, liver cirrhosis, etc.

-Headache, confusion, memory loss, seizures, brain herniation

-Gradual drop even to low levels may be tolerated (eg old patients prior to surgery).

- Rapid correction can lead to central pontine myelinosis 

• Hypernatremia:

-Usually caused by deficit of water.

-Diabetes insipidus, diarrhea, intake of hypertonic fluids.

-Lethargy, weakness, irritability, edema.

- Treat by water replacement

-Rapid correction can lead to cerebral edema, brain damage and death

• Normal: 3.5- 5.0 mEq/L
• Crucial for heart function, smooth and skeletal muscle contraction.

• Hypokalemia:

- Caused by diarrhea, laxatives, diuretics, diabetic ketoacidosis or inadequate intake.

-Asymptomatic or muscle cramps, pain, palpitations, psychosis, delirium.

-Managed by treating cause and K supplementation

• Hyperkalemia:

-Caused by renal failure, NSAIDS, rhabdomyolysis, massive blood transfusion, massive hemolysis, and excessive intake.

- Palpitations, muscle weakness, arrhythmias, sudden cardiac death.

- Raised level can be due to hemolysis, hence repeat levels if needed

-Perform urgent ECG if risk of cardiac arrhythmias

- Treat by eliminating cause, insulin with glucose and in severe cases hemodialysis.

• Normal: 96-106 mEq/L
• Involved in acid-base and fluid balance
• Transmits nerve impulses

• Hypochloremia:

- Caused by Addison’s disease, dehydration, metabolic alkalosis, vomiting 

• Hyperchloremia:

-Caused by renal failure, diarrhea, and metabolic acidosis.

• Normal: 22-26 mEq/L
• Involved in acid-base balance

• Low:

- Caused by metabolic acidosis, renal failure, aspirin overdose, etc.

• High:

-Caused by metabolic alkalosis, COPD, Cushing’s disease.

1.      WBC with differential

2.      ESR

3.      CRP

4.      Blood culture.

• Defined as the rate at which red cells sediment in one hour
• This is determined by the aggregation of erythrocytes into rouleaux which in turn is determined by the characteristics of the erythrocytes, the viscosity of the plasma and the electrostatic forces of the surrounding macromolecules
• Erythrocytes usually repel each other due to a negative charge of the carboxyl group of N-acetylneuraminic acid located on the surface of the cell.
• Absorbing force is provided by plasma macromolecules (acute phase reactants) many of which are positively charged and stick to the RBCs which then -> aggregates which have an increased combined weight relative to their surface area and settle to the bottom through the plasma
• There is an increase in the concentration of acute phase reactants in situations of infection, malignancy or inflammatory conditions such as collagen vascular disease etc
• Normal: Men – 3mm,Women- 7mm, children -3-13mm
• Women tend to have a higher baseline ESR than men
• Non-specific inflammatory marker.
• Septic arthritis:
• 1 of the 4 Kocher’s criteria (others WBC, fever, NWB)
• Rises after 2 days of infection, comes back to normal after 4 weeks
• Prosthetic joint infection: ESR takes 90 days to come back to normal after surgery, if still raised suspect infection. If > 40mm one year after THA surgery suggests infection until proven otherwise
• Osteomyelitis: Rises rapidly in 3-5 days but declines too slowly to guide treatment. May be normal in 10 % patients.

• Acute phase protein produced by the liver.
• Normal: < 10
• Non-specific inflammatory marker.
• Septic arthritis:
• CRP most helpful marker
• Rises few hours after infection, normalizes within a week of treatment
• Used to assess efficacy of treatment
• > 20 is strongest independent risk factor for infection
• < 20 with weight bearing virtually rules out septic arthritis.
• Prosthetic joint infection: CRP takes 21 days to come back to normal after surgery, if still raised suspect infection.
• Osteomyelitis: Rises rapidly in 6 hours and most sensitive to monitor treatment response.

• Three separate specimens from different sites and at different times.
• Ideally before starting antibiotics
• Aerobic, anaerobic and sometimes mycobacterial culture performed
• Bottles incubated for 5 days
• 30-50 % positive cultures in osteomyelitis and septic arthritis.
• Helps identify organism. (Gram staining and culture)

• Cytokine released by tissue monocytes and fibroblasts in response to infections
• Detected earlier than CRP in patients with infections
• Expensive and limited utility due to short half-life.
• Highest correlation in prosthetic joint infections.

Indicated for investigations of calcium and bone disease

• Calcium
• Phosphate
• Albumin
• Alkaline phosphate
• Rheumatoid factor
• PTH
• Vit D

• Normal: 2.2-2.6 Mmol/L (Total)
• 1.3-1.5          Mmol /L (Ionized)

• Always check serum albumin as only ionized calcium that is not bound to albumin has a biological effect.
• Calcium functions in clotting, muscle contraction, nerve function, as a neurotransmitter and as a supporting material in bones (Calcium phosphate)

• Hypocalcaemia:

-   Caused by hypoparathyroidism, vitamin D deficiency, renal osteodystrophy, and massive blood transfusion.

-   Presents as paresthesias, cramps, tetany, convulsions and positive Chvostek’s / Trousseau’s sign

-   Treat with Vit D and calcium.

• Hypercalcemia:

-Caused by hyperparathyroidism, Vit D excess, malignancy, myeloma and sarcoidosis.

- Bone pains, renal stones, constipation, psychic moans.

-Treat cause, iv fluids, loop diuretics and Bisphosphonates.

·     Normal: 0.87-1.45 Mmol/L

·     Inversely affects level of calcium in blood.

·     High: Renal disease, Vit D excess, hypoparathyroidism.

·     Low: Hyperparathyroidism, starvation, high calcium levels.

·     Normal: 44-147 IU/L

·     Produced primarily by liver and bone

·     In bone, produced by osteoblasts

·     High: Osteoblastic conditions like Paget’s disease, Rickets, hyperthyroidism, osteosarcoma, healing fractures, and osteoporosis treatment.

·     Low: Hypothyroidism, malnutrition

• Autoantibody
• Part of disease criteria for rheumatoid arthritis
• Positive in many other conditions like Hep B, SLE, tuberculosis, leukemia, etc.
• Many patients with rheumatoid disease begin seronegative but seroconversion occurs later.
• Helps in both diagnosis and prognosis70 % sensitive and 78 % specific for rheumatoid arthritis.

• Regulates calcium and phosphate
• Converts inactive to active Vit D
• Low: produce hypocalcaemia
• High: Primary, secondary or tertiary.

·       Increases Ca and PO4

·       Low: Rickets, osteomalacia

1. Basic blood panel including LFT’s
2. Infective parameters like ESR, CRP (differential diagnosis)
3. Bone profile including Vit D and PTH
4. Tumor markers- PSA, Myeloma screen, LDH (Ewing’s), etc.

• CBC
• Total protein including albumin
• LFT
• Bone profile (Ca. PO4, ALP)
• Serum/urine electrophoresis

• Electric current separates serum proteins into 5 fractions depending on size and electric charge- albumin, alpha1 globulins, alpha 2 globulins, beta globulins and gamma globulins
•  Abnormal pattern is seen in myeloma with large spike in globulin region- M spike 

Figure 7. Typical normal pattern for serum protein electrophoresis. 

Figure 8. Abnormal serum protein electrophoresis pattern in a patient with multiple myeloma. Note the large spike in the gamma region

1. ABG
2. Serum Lactate
3. Complete blood panel including RFT, LFT
4. Coagulation screen

Normal: pH: 7.35-7.45

              PO2: >10 KPa

              PCO2: 4-7.6 KPa

              HCO3: 22-26 mmol/L

How to read ABG?

• Look at the pH first: <7.35 is acidosis, > 7.45 is alkalosis
• CO2 is an acid while HCO3 is base
• CO2 represents respiratory component while HCO3 represents metabolic component
• Raised CO2 in other words is respiratory acidosis. If general pH acidic, then primary respiratory acidosis. If general pH alkalosis, then compensatory respiratory acidosis (for primary metabolic alkalosis)
• Lowered CO2 in other words respiratory alkalosis. If general pH alkalosis, then primary respiratory alkalosis. If general pH acidic, then compensatory respiratory alkalosis (for primary metabolic acidosis)
• Raised HCO3 in other words metabolic alkalosis. If general pH alkalosis, then primary metabolic alkalosis. If general pH acidic, then compensatory metabolic alkalosis (for primary respiratory acidosis)
• Lowered HCO3 in other words metabolic acidosis. If general pH acidic, then primary metabolic acidosis. If general pH alkalosis, then compensatory metabolic acidosis (for primary respiratory alkalosis)

Normal: Arterial :0. 5-1.6mmol/L

             Venous: 0.5-2.2 mmol/L

·       Measures hypoxia and lactic acidosis

·       Marker for sepsis and trauma

·       One of the endpoints in Damage Control orthopedics (Lactate< 2mmol/L, consider definitive surgery)

·       High levels associated with increased mortality for sepsis