Investigations


Haematological

CBE

Haemoglobin (11.5 - 16.5g/dl)
RBC (4.0 - 5.5 x1012/l)
Reticulocytes (less than 1%)
PCV (35 - 47%)
MCV (81 - 99fl)
Platelets (150 - 450 x109/l)
White Cell Count (4.0 - 11.0 x109/l)
Neutrophils (2.2 - 7.7 x109/l) ~ 60 - 70%
Lymphocytes (1 - 3.5 x 109/l) ~ 25 - 35%
Monocytes (1.6 - 1.8 x 108/l) ~ 4 - 8%
Eosinophils (less than 0.45 x 109/l) ~ 1 - 4%

Red Blood Cells

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.

Haemoglobin is the oxygen transport system of blood and 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.

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.

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.

White Blood Cells

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.

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. Mature neutrophils account for about 70% of the total leucocytes in the peripheral blood of a healthy adult. Physiological factors which increase their number in the peripheral blood include exercise, emotional stress and pregnancy.

Immature granulocytes represented by metamyelocytes are found when the production of leucocytes is being stimulated by sever pyogenic infections. Appearance of myeloblasts reflects a serious disturbance of marrow function such as leukaemia.

Eosinophils are also phagocytic and their granules contain histamine and are concerned in processes involving foreign proteins such as hypersensitivity reactions.

Basophils are non phagocytic and have granules containing heparin and histamine.

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. Lymphocytes are mainly concerned with the formation and transport of antibodies. 'T' lymphocytes are concerned with cell mediated immunity and 'B' lymphocytes with humoral antibody synthesis.

Types of hypersensitivity reaction:

Type I
Anaphylactic or immediate hypersensitivity, reagin dependent reaction mediated by IgE
Type II
Cytotoxic antibody (eg autoimmune haemolytic anaemia) 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 (T cell) usually viral infections but also TB, brucellosis, pertussis and syphilis
Type V
Stimulating antibody where the antibody complex ® stimulation of certain cell receptor sites (eg thyroid stimulating antibodies)
Type VI
Antibody dependant cell mediated cytotoxicity which may be important in autoimmune disease and tumour rejection.
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. Mature cells have a lobulated nucleus and cloudy blue cytoplasm, are actively motile and phagocytic.

Platelets

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.
Bleeding tends to occur when the platelet count falls below 40 x109/l

ESR (Westergren)

Defined as the distance in millimetres that a column of erythrocytes fall 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

Most commonly used in orthopaedic surgery to diagnose and then monitor the progression of or recovery from infection

The interval between the onset of symptoms and the return of the sedimentation rate to normal may be 3 - 4 months (the CRP will fluctuate more rapidly indicating response to treatment)

ESR will increase after an acute fracture but returns to normal after about one month

After THR the ESR will often remain slightly elevated for one year or so but if more than 40mm more than one year after THR suggests infection until proven otherwise.

Technique

Blood is mixed with sodium citrate in a glass tube and allowed to settle over one hour. Normal value is 6 - 16mm per hour

Women tend to have a higher baseline ESR than men

Higher values occur in elderly patients, during pregnancy, and when taking certain medications such as heparin and oral contraceptives

Conditions that can lower the sedimentation rate include the administration of steroidal and non steroidal anti-inflammatories, the presence of sickle cell disease and haemolytic anaemia, haemoglobinopathy hereditary spherocytosis, polycythemia, hypofibrinogenaemia, deficiency of pyruvate kinase, etc

C Reactive Protein

Precipitation of this protein with the C polysaccharide of the pneumococcal cell wall
Increases in 6/24 and decreases within 2/7 after treatment

Microbiological

Gram Stain

Distinguish bacteria as positive or negative on the basis of whether or not they decolourise with acetone, alcohol or aniline after staining with a para rosaline dye (methyl violet) and subsequent treatment with iodine
Gram positive resist decolourisation and contain no mucopeptides in their wall, their walls are thicker and stronger

Culture on chocolate agar plates ® gonococcus

Ziehl Neelsen stain

Diagnosis positive in 80 - 90% of cases of TB
A strongly acting dye with a mordant (eg carbol fuchsin) is required with the application of heat to facilitate impregnation which is followed by decolourisation and application of a counter stain of malachite green or methylene blue.

Compensated polarised light microscopy

With polarising filters and a first order red compensator monosodium urate crystals are 3-20mm in length needle shaped and strongly negatively birefringent (yellow in colour when the crystal axis is parallel to the compensator)
Calcium pyrophosphate crystals are short, rhomboidal and weakly positively birefringent (blue when the crystal axis is parallel to the compensator)

Radiological

Plain Radiographs

Introduced in 1895
The initial investigative procedure in almost every situation of bony pathology
Radiographs are insensitive to the earliest changes of bone pathology since about 30% local decalcification is required before a lesion is visible on the plain radiograph.

Ultrasound

May be diagnostic or therapeutic (employs different frequency ® different penetration and generation of heat)
No harmful effects have been identified

Physics

Involves the generation of sound waves with a frequency higher than detectable by the human ear (more than 20,000 Kz)

Sound waves are generated using a reverse piezo-electric effect when a signal is applied to a crystal and causes it to deform

The frequency at which the crystal vibrates is dependent on both the nature of the electrical stimulus applied and the characteristics of the crystal

The crystal is a transducer which transforms electrical stimulus into vibration and then acts as a receiver transforming reflected vibration into an electrical signal (about 1/1000 acts as a stimulus and 999/1000 as a receiver)

As the vibration travels from one substance into another with a different density some sound energy is reflected and recorded, the bigger the change in sound transmission the greater the reflection (sound transmission is best in liquid and worst in air)

Tissue penetration is better with lower frequencies but spatial resolution is better with higher frequencies

'A' Mode
Initial mode and not used clinically
Like a fish finder or depth gauge to measure the depth of the eye globe
'B' Mode
(Brightness Modulation) where the return signal is shown by a variable brightness on a screen
Using digital technology can be displayed in real time on a screen and grey scale gives better definition of tissue planes

Therapeutic

Utilises 1MHz
The effect is obtained through the generation of heat by vibration of tissues and stimulation of circulation
The thermal effect is not uniform as tissues with higher protein content are heated the most therefore heating is more marked in joint capsules, ligaments and muscles than fat
High thermal absorption coefficient of bone can result in excessive heating of bone surface and a periosteal reaction

Myelography

Contrast Media

Omnipaque (iohexol) is a triiodinated non-ionic water soluble radiographic contrast and is indicated for lumbar, thoracic, cervical and total columnar myelography and in computerised tomography of the CNS in adults.

Contraindications

Known sensitivity to iohexol
Should not be administered to patients with thyrotoxicosis, anuria or decompensated cardiac insufficiency
Intrathecal administration of corticosteroids with omnipaque is contraindicated

Precautions

Previous tolerance to iodine-containing contrast media and for a history of allergy

At least 48 hours should ordinarily be allowed before repeat examination

Patients should be well hydrated prior to the study and maintain a normal diet up to two hours prior to the study

Should discontinue neuroleptic drugs at least 48 hours prior to the study

During the procedure keep the patients head elevated above the highest level of the spine and do not lower the head of the bed more than 15o in order to move contrast cranially

Inject contrast slowly over 1 - 2 minutes, avoid intracranial entry of contrast and after the procedure raise the head of bed 15o - 45o and maintain patient in a semi sitting position for the first few hours

Recommend to maintain close supervision of the patient for at least 12 hours post myelogram and after 8 hours the patient may be lowered to the horizontal position for a further 16 hours and encourage oral fluids throughout

Dosage

Recommended dose is 180mg/ml for examination in the lumbar region and if more extensive examination is required higher concentrations are needed (240 - 300mg/ml) to compensate for dilution of the contrast with CSF
Maximal dose of 3g iodine or a concentration of 300mg/ml should not be exceeded

Adverse reactions

Headache in ~ 25%
Nausea in ~ 6%
Vomiting in ~ 3%

These reactions generally occur 3 - 8 hours after the study and almost all occur within 24 hours of the study.

Symptoms are usually mild or moderate in degree, lasting for a few hours and usually disappearing within 24 hours. Rarely symptoms may persist for days and be severe

Back ache, pain in the arms, neck stiffness, weakness, numbness and paraesthesia or sciatic type pain have occurred less frequently often in the form of transient exacerbation of pre-existing symptomatology

Bone Scanning

Ideally use a radionuclide which emits gamma rays which are not accompanied by alphe or beta particle emission which result in a greater radiation dose to the tissues

Isotopes in common use are Tc99m MDP (methylene diphosphonate) and Tc99m EHDP (ethyl hydroxy-diphosphonate)

Tc99 is the degradation product of Molybdenum99

Tc99 is bound in the generator to aluminium hydroxide and the technitium is washed off with saline to form pertechnate (TcO4) which can be bound to diphosphonate for bone scanning (generators received weekly from Lucas Heights)

Diphosphonates are the most commonly used ligands and are phosphate-carbon-phosphate compounds which are rapidly cleared from the blood producing high bone to soft tissue ratios and marked accumulation within areas of bony pathology within 2 - 3 hours of injection.

T1/2 is 6 hours for the Tc99 but the biological T1/2 is less due to renal excretion

Abnormality can be indicated by an increase in the blood supply to a region as indicated by rapid sequential images after injection of the isotope indicating arterial blood flow (Phase 1). Phase 2 follows immediately and is designed to image the static blood pool and relative vascularity of a lesion. Phase 3, delayed images are taken after 2 - 3 hours and indicate increased bone turnover.

Arterial phase scans may be of use in the assessment of RSD and arterio-venous malformations etc.

Occasionally the patient may be re-scaned at 24 hours, particularly for pelvic pathology in an attemp to reduce activity in the overlying bladder.

Gallium has similar phases but takes longer for cellular uptake therefore scan initially at 6 hours and then at 48 hours (taken up by cells with a high transferrin content, particularly WBCs, tumour cells particularly lymphoma and lung)

T1/2 of Gallium is 72 hours

Indium111 labelled WBC scan performed by taking 80 - 90ml of venous blood ® separate the leucocytes, label the leucocytes in vitro with In111 by incubating the cells in In111 oxide for 10 - 20 minutes at room temperature and replace WBCs in the patient ® scan 18 - 24 hours later (the whole process takes ~ 3 hours to label the cells)

Can be coupled with computer tomography (SPECT; Single Photon Emission Computer Tomography)

Indications

  1. Infection results in increased uptake in both the blood pool and delayed scans, and usually evident within the first 24 hours of the disease
    The use of Gallium or Indium labelled WBCs may improve the accuracy of the study but greater delay is required to enable incorporation of isotope into pathological area and a little more radiation is administered.
    Sensitivity of 98% in children and older infants but only 32% in neonates, in adults ~ 92% accurate
    Cellulitis results in increased activity in the blood pool scans
    Septic arthritis results in a diffusely increased activity in all the structures of the involved joint
  2. Avascular bone results in a signal void in the delayed scan and absence of activity in the blood pool may be evident if a large area is involved
    Diagnostic accuracy of bone scan in Perthes disease is reported to be 98% (Sutherland et al 1980)
    In traumatic AVN the presence of avascularity is not a reliable prognostic sign of subsequent osteonecrosis and should not in itself be used to indicate the need for joint replacement
  3. Bone trauma and the presence of stress fractures of compression fractures of cancellous bone
    Useful to identify multiple injuries in NIA
    May indicate the degree of healing activity at a fracture and indicate the likelihood of progression to union (no or little activity indicates a non-union or pseudarthrosis)
    Can also be used to assess the activity of growth plates and likelihood of further growth
  4. Tumours both benign and malignant may be located on bone scan
    Benign conditions associated with a normal scan include bone island, fibrous cortical defect, non-ossifying fibroma, simple bone cyst, single or multiple exostoses and osteoma
    Other lesions may have moderate increase in activity such as ABC, enchondroma, osteochondroma and EG (up to 50% may not show increased activity)
    Benign conditions resulting in a hot scan include, osteoid osteoma, osteoblastoma, chondroblastoma, fibrous dysplasia and Pagets disease
    Malignant primary bone tumours show variable increased activity, those with osteogenic potential show increased activity, fibrous or connective tissue tumours may show decreased delayed activity but increased activity in a blood pool or arterial phase scan
    Good for identifying skip lesions or distant metastases and useful to indicate the effectiveness of treatment or progression of disease
    Myeloma produces little osteoblastic activity, Histiocytosis X and neuroblastoma deposits may not be identified on bone scan and lymphoma and as with leukaemia may show increased normal or decreased activity
    Ewings gives a picture of even and intense uptake
  5. Can be used to indicate the activity of heterotopic new bone formation and when activity has reduced to that of surrounding normal bone surgical resection may be indicated
  6. Used in Total Joint Replacement to indicate the presence of infection around a prosthesis or if the components are loose (NB a component will sow increased activity for up to 12 months following its insertion)
  7. Metabolic bone disease such as Pagets where 30% of patients will have normal X-Rays with typical bone scan features of the disease.
    Also increased uptake in osteomalacia and may be increased in hyperparathyroidism and secondary hyperparathyroidism ® super scan
  8. Arthritidies are also evident on the scan but it is generally not a useful study in these patients in terms of modifying treatment but can help to localise the painful a degenerate patello-femoral joint in anterior knee pain
May be used to identify sacroiliitis in ankylosing spondylitis etc
In general misinterpretation of scans will be avoided if the scan is interpreted with regard to the clinical background and plain radiographs

Labelled WCC scans

May also be useful (Indium labelling of WCC separated from 50ml of autogenous blood, reinjected and the scan performed ~ 48 hours later

MRI

Non invasive method of imaging by measuring the emitted energy from certain atomic nuclei in a magnetic field when stimulated by radiofrequency energy
Avoids ionising radiation and can image in any plane
There are no known biologic hazards at the currently used field strengths
Patients with claustrophobic tendencies do not tolerate the examination ® 2 - 3% refuse to continue with the examination
Examination takes more time than a CT scan and depending on the area and type of scan required takes from 15 to 45 minutes

Physics

Nucleus with unpaired protons or neutrons have angular momentum, spin which leads to a current loop with north and south poles and in the absence of an external magnetic field the vectors of theses moments are randomly orientated.

Once exposed to a magnetic field they align within the field in one of two positions either parallel (low energy state) or anti-parallel (high energy state)

In addition to spinning the nuclei precess or wobble a few degrees off axis, the frequency of precession is known as the resonance frequency and is proportional to the strength of the magnetic field applied

It is the hydrogen nucleus, which accounts for 2/3 of the atoms in the body and is a single proton that is used for clinical magnetic resonance imaging.

If radiofrequency energy is applied at the proper frequency (resonant frequency) the protons can be caused to realign from their original orientation within the magnetic field. When the radiofrequency is turned off the protons return to their original position and a small amount of energy is given off, which is detected by the receiving coil.

The intensity of the voltage (spin density) is related to the concentration of nuclei in the site being imaged

The time it takes for the voltage to decay or relaxation to occur is expressed by the T1 and T2 relaxation times

T1 relaxation (longitudinal or spin lattice) is the time taken for 63% of longitudinal magnetisation to recover following a RF pulse.

T1 weighted images are good for identifying the anatomy with good tissue plane differentiation and water has a low signal intensity, fat has a high signal intensity

T2 relaxation (transverse or spin-spin) is due to randomly varying inhomogenicities in the magnetic field created by adjacent nuclei and equals the time necessary to reduce transverse magnetisation to 37% of original value following the RF pulse

T2 weighted images assist in distinguishing normal and abnormal soft tissues and water appears white with high signal intensity

By varying the conditions under which the radiofrequency pulses are applied can emphasise the spin density resulting in a T1 or T2 weighted image

TR is the repetition time between radiofrequency pulses and the echo time. The longer the delay the more the image depends on proton density, and the shorter the delay the more T1 weighted the image will become

TE is the time between application of the RF pulse and the recording time

Application of the radiofrequency results in the protons flipping 90 - 180o in the magnetic field and it is the characteristics of the tissue that governs the rate of regaining alignment in the axis of the magnetic field. Application of a second radiofrequency pulse when the orientation of the protons is out of phase gives further information about the characteristics of the tissue.

Blood flow results in a signal void as do substances with a low proton density

Gadolinium-diethylenetriamine penta-acetic acid (Gadolinium DPTA) contain paramagnetic moieties which create local magnetic fields leading to quicker relaxation of adjacent tissues and its effect is seen indirectly by its effect on adjacent tissues (shortens the T1 relaxation time of vascular tissue)

Surface coils are small RF receivers wrapped around or placed on body parts which result in better signal to noise ratios

Contra-indications

Pacemakers ® convert to the fixed or asynchronous mode when exposed to a magnetic field of greater than 10 gauss
Aneurisms clips may toggle and potentially result in vascular mishap

Indications

Shown to be more accurate than discography in identifying disc degeneration (lowered water content) but does not indicate if the disc is painful
Metallic appliances create a local signal void but show considerably less image degradation than with CT and the artefacts are greater with higher field strengths
In orthopaedics good for soft tissue pathology and including ligament and meniscal pathology

Best test for vascularity of bone with changes evident with avascularity of the marrow well before other investigations will alter.