Routine Stix testing of urine for blood, protein and sugar is obligatory in all patients suspected of having renal disease.

Blood

Haematuria may be overt, with bloody urine, or microscopic and found only on chemical testing. Currently used Stix tests for blood are very sensitive, being positive if two or more red cells are visible under the high-power field of a light microscope. Indeed, the test is too sensitive, sometimes giving positive results in normal individuals. A further disadvantage is that Stix testing cannot distinguish between blood and free haemoglobin. A positive Stix test must always be followed by microscopy of fresh urine to confirm the presence of red cells and so exclude the relatively rare conditions of haemoglobinuria or myoglobinuria. In females with a positive Stix test result for blood, it is essential to enquire whether the patient is menstruating. Bleeding may come from any site within the urinary tract:

• Overt bleeding from the urethra is suggested when blood is seen at the start of voiding and then the urine becomes clear.
• Blood diffusely present throughout the urine comes from the bladder or above.
• Blood only at the end of micturition suggests bleeding from the prostate or bladder base.

Careful urine microscopy is mandatory as the presence of red-cell casts is diagnostic of bleeding from the kidney itself, most often due to glomerulonephritis. In the absence of red-cell casts, further investigations, such as urine cytology, renal imaging and cystoscopy, are required to define the site of bleeding. Renal biopsy may be required.

Protein

Proteinuria is one of the most common signs of renal disease. Detection is primarily by Stix testing. Most reagent strips can detect a concentration of 100-200 mg/L or more in urine. They react primarily with albumin and are relatively insensitive to globulin and Bence Jones proteins.

If proteinuria is confirmed on repeated Stix testing, protein excretion in 24-hour urine collections should be measured (but see below). Healthy adults excrete up to 30 mg daily of albumin. Pyrexia, exercise and adoption of the upright posture all increase urinary protein output. Proteinuria, while occasionally benign, always requires further investigation. In some glomerular disease the protein leak is selective (e.g. minimal change) and urine electrophoresis can demonstrate the relative leaks, e.g. IgA or albumin.

Postural proteinuria.

This term is used to refer to proteinuria present on dipstick testing which becomes undetectable after a period of hours lying flat. Typically, a negative dipstick result is obtained on the first urine passed on rising in the morning, whereas subsequent specimens give a positive result. This is regarded even for insurance purposes as a benign condition.

Microalbuminuria

The term microalbuminuria is an unfortunate one since the albumin referred to is of normal molecular size and weight. Normal individuals excrete less than 20 μg of albumin per minute (30 mg in 24 hours). Dipsticks, however, detect albumin only in a concentration above 200 mg/L (300 mg per 24 hours if urine volume is normal). An increase in albumin excretion between these two levels – so-called microalbuminuria – is now known to be an early indicator of diabetic glomerular disease. It is widely used as a predictor of the development of nephropathy in diabetics and may be extended to other conditions.

Timed 24-hour urinary excretion rates provide the most precise measure of microalbuminuria. However, in clinical practice it is more convenient to test for microalbuminuria using random urine samples in which albumin concentration is related to urinary creatinine concentration. Generally an albumin : creatinine ratio of 2.5 to 20 corresponds to albuminuria of 30-300 mg daily respectively. Kits are available to test for microalbuminuria.

Glucose

Renal glycosuria is uncommon, so that a positive test for glucose always requires exclusion of diabetes mellitus.

Bacteriuria

Dipsticks are available for testing for bacteriuria based on the detection of nitrite produced from the reduction of urinary nitrate by bacteria and also for the detection of leucocyte esterase, an enzyme specific for neutrophils. Although each test on its own has limitations, a positive reaction with both tests has a high predictive value for urinary tract infection.

The overriding principle is to aim to replace what is missing.

Haemorrhage

This involves the loss of blood. The rational treatment of acute haemorrhage is therefore the infusion of a combination of red cells and a plasma substitute or (if unavailable) whole blood. (Chronic anaemia causes salt and water retention rather than volume depletion by a mechanism common to conditions with peripheral vasodilatation.)

Loss of plasma

Loss of plasma, as occurs in burns or severe peritonitis, should be treated with human plasma or a plasma substitute.

Loss of water and electrolytes

Loss of water and electrolytes, as occurs with vomiting, diarrhoea, or excessive renal losses, should be treated by replacement of the loss. If possible, this should be done with oral water and sodium salts. These are available as slow sodium (600 mg, approximately 10 mmol of each Na+ and Cl- per tablet), the usual dose of which is 6-12 tablets per day with 2-3 L of water. It is used in mild or chronic salt and water depletion, such as that associated with renal salt wasting.

Sodium bicarbonate (500 mg, 6 mmol each of Na+ and HCO3- per tablet) is used in doses of 6-12 tablets per day with 2-3 L of water. This is used in milder chronic sodium depletion with acidosis (e.g. chronic renal failure, post-obstructive renal failure, renal tubular acidosis). Sodium bicarbonate is less effective than sodium chloride in causing positive sodium balance.

Oral rehydration solutions are described in Box 2.9. Intravenous fluids may sometimes be required (Table 12.6). Rapid infusion (e.g. 1000 mL per hour or even faster) is necessary if there is hypotension and evidence of impaired organ perfusion (e.g. oliguria, confusion); in these situations, plasma expanders (colloids) are often used in the first instance to restore an adequate circulating volume. Repeated clinical assessments are vital in this situation, usually complemented by frequent measurements of central venous pressure. Severe hypovolaemia induces venoconstriction, which maintains venous return; over-rapid correction does not give time for this to reverse, resulting in signs of circulatory overload (e.g. pulmonary oedema) even if a total body ECF deficit remains. In less severe ECF depletion (such as in a patient with postural hypotension complicating acute tubular necrosis), the fluid should be replaced at a rate of 1000 mL every 4-6 hours, again with repeated clinical assessment. If all that is required is avoidance of fluid depletion during surgery, 1-2 L may be given over 24 hours, remembering that surgery is a stimulus to sodium and water retention and that over-replacement may be as dangerous as under-replacement. Regular monitoring by fluid balance charts, bodyweight and plasma biochemistry is mandatory.

Loss of water alone

This causes extracellular volume depletion only in severe cases, because the loss is spread evenly among all the compartments of body water. In the rare situations where there is a true deficiency of water alone, as in diabetes insipidus or in a patient who is unable to drink (after surgery, for instance), the correct treatment is to give water.

If intravenous treatment is required, water is given as 5% dextrose, because pure water would lead to osmotic lysis of blood cells.

Table 12-6. Intravenous fluids in general use for fluid and electrolyte disturbances

	Na+ (mmol/L)	K+ (mmol/L)	HCO3- (mmol/L)	Cl- (mmol/L)	Indication (see footnote)
Normal plasma values	142	4.5	26	103
Sodium chloride 0.9%	150	-	-	150	1
Sodium chloride 0.18% + glucose 4%	30	-	-	30	2
Glucose 5% + potassium chloride 0.3%	-	40	-	40	3
Sodium bicarbonate 1.26%	150	-	150	-	4

1. Volume expansion in hypovolaemic patients. Rarely to maintain fluid balance when there are large losses of sodium. The sodium (150 mmol/L) is higher than in plasma and hypernatraemia can result. It is often necessary to add KCl 20-40 mmol/L. 2. Maintenance of fluid balance in normovolaemic, normonatraemic patients. 3. To replace water. Can be given with or without potassium chloride. May be alternated with 0.9% saline as an alternative to (2). 4. For volume expansion in hypovolaemic, acidotic patients alternating with (1). Occasionally for maintenance of fluid balance combined with (2) in salt-wasting, acidotic patients.

Peripheral blood

A low haemoglobin should always be considered in relation to:

• the white blood cell (WBC) count
• the platelet count
• the reticulocyte count (as this indicates marrow activity)
• the blood film, as abnormal red cell morphology may indicate the diagnosis.

Where two populations of red cells are seen, the blood film is said to be dimorphic. This may, for example, be seen in patients with ‘double deficiencies’ (e.g. combined iron and folate deficiency in coeliac disease, or following treatment of anaemic patients with the appropriate haematinic).

Bone marrow

Examination of the bone marrow is performed to further investigate abnormalities found in the peripheral blood (Practical box 8.1). Aspiration provides a film which can be examined by microscopy for the morphology of the developing haemopoietic cells. The trephine provides a core of bone which is processed as a histological specimen and allows an overall view of the bone marrow architecture, cellularity and presence/absence of abnormal infiltrates.

The following are assessed:

• cellularity of the marrow
• type of erythropoiesis (e.g. normoblastic or megaloblastic)
• cellularity of the various cell lines
• infiltration of the marrow
• iron stores.

Special tests may be performed: cytogenetic, immunological, cytochemical markers, biochemical analyses (e.g. deoxyuridine suppression test), microbiological culture.

Table 23-2. Investigations used in skin disorders

Test	Use	Clinical example
Skin swabs	Bacterial culture	Impetigo
Blister fluid	Electron microscopy and viral culture	Herpes simplex
Skin scrapes	Fungal culture	Tinea pedis
	Microscopy	Scabies
Nail sampling	Fungal culture	Onychomycosis
Wood’s light	Fungal fluorescence	Scalp ringworm Erythrasma
Blood tests	Serology	Streptococcal cellulitis
	Autoantibodies	Discoid lupus erythematosus
	HLA typing	Dermatitis herpetiformis
	DNA analysis	Epidermolysis bullosa
Skin biopsy	Histology	General diagnosis
	Immunohistochemistry	Cutaneous lymphoma
	Immunofluorescence	Immunobullous disease
	Culture	Mycobacteria/fungi
Patch tests	Allergic contact eczema	Hand eczema
Urine	Dipstick (glucose)	Diabetes mellitus
	Cytology (red cells)	Vasculitis
Dermatoscopy (direct microscopy of skin)	Assessment of pigmented lesions	Malignancy

Haemoglobin function

The biconcave shape of red cells provides a large surface area for the uptake and release of oxygen and carbon dioxide. Haemoglobin becomes saturated with oxygen in the pulmonary capillaries where the partial pressure of oxygen is high and Hb has a high affinity for oxygen. Oxygen is released in the tissues where the partial pressure of oxygen is low and Hb has a low affinity for oxygen.

In adult haemoglobin (Hb A), a haem group is bound to each of the four globin chains; the haem group has a porphyrin ring with a ferrous atom which can reversibly bind one oxygen molecule. The haemoglobin molecule exists in two conformations, R and T. The T (taut) conformation of deoxyhaemoglobin is characterized by the globin units being held tightly together by electrostatic bonds. These bonds are broken when oxygen binds to haemoglobin, resulting in the R (relaxed) conformation in which the remaining oxygen-binding sites are more exposed and have a much higher affinity for oxygen than in the T conformation. The binding of one oxygen molecule to deoxyhaemoglobin increases the oxygen affinity of the remaining binding sites – this property is known as ‘cooperativity’ and is the reason for the sigmoid shape of the oxygen dissociation curve. Haemoglobin is, therefore, an example of an allosteric protein. The binding of oxygen can be influenced by secondary effectors – hydrogen ions, carbon dioxide and red-cell 2,3-bisphosphoglycerate (2,3-BPG, formerly called 2,3-diphosphoglycerate (2,3-DPG)). Hydrogen ions and carbon dioxide added to blood cause a reduction in the oxygen-binding affinity of haemoglobin (the Bohr effect). Oxygenation of haemoglobin reduces its affinity for carbon dioxide (the Haldane effect). These effects help the exchange of carbon dioxide and oxygen in the tissues.

Oxygenated and deoxygenated haemoglobin molecule. The haemoglobin molecule is predominantly stabilized by α-β chain bonds rather than α-α and β-β chain bonds. The structure of the molecule changes during O2 uptake and release. When O2 is released, the β chains rotate on the α1β2 and α2β1 contacts, allowing the entry of 2,3-BPG which causes a lower affinity of haemoglobin for O2 and improved delivery of O2 to the tissues.

Red cell metabolism produces 2,3-BPG from glycolysis. 2,3-BPG accumulates because it is sequestered by binding to deoxyhaemoglobin. The binding of 2,3-BPG stabilizes the T conformation and reduces its affinity for oxygen. The P50 is the partial pressure of oxygen at which the haemoglobin is 50% saturated with oxygen. P50 increases with 2,3-BPG concentrations, which increase when oxygen availability is reduced in conditions such as hypoxia or anaemia. P50 also rises with increasing body temperature, which may be beneficial during prolonged exercise. Haemoglobin regulates oxygen transport as shown in the oxyhaemoglobin dissociation curve. When the primary limitation to oxygen transport is in the periphery, e.g. heavy exercise, anaemia, the P50 is increased to enhance oxygen unloading. When the primary limitation is in the lungs, e.g. lung disease, high altitude exposure, the P50 is reduced to enhance oxygen loading.

Haemoglobin synthesis

Haemoglobin performs the main functions of red cells – carrying O2 to the tissues and returning CO2 from the tissues to the lungs. Each normal adult Hb molecule (Hb A) has a molecular weight of 68 000 and consists of two α and two β globin polypeptide chains (α2β2) which have 141 and 146 amino acids respectively. HbA comprises about 97% of the Hb in adults. Two other types, Hb A2 (α2δ2) and Hb F (α2γ2), are found in adults in small amounts (1.5-3.2% and < 1%, respectively).

Haemoglobin synthesis occurs in the mitochondria of the developing red cell. The major rate-limiting step is the conversion of glycine and succinic acid to δ-aminolaevulinic acid (ALA) by ALA synthase. Vitamin B6 is a coenzyme for this reaction, which is inhibited by haem and stimulated by erythropoietin. Two molecules of δ-ALA condense to form a pyrrole ring (porphobilinogen). These rings are then grouped in fours to produce protoporphyrins. Finally, iron is inserted to form haem. Haem is then inserted into the globin chains to form Hb.