FICHA TECNICA

The subjects were asked to pass about 15- 20mls of urine (mid-stream sample) into a clean, plain universal bottle at contact. One to two-ml aliquot was removed and kept for storage at -20⁰C for subsequent analysis. The rest of the samples were analysed immediately with a Healthmate (DFI Co. Ltd., Korea) 10- parameter urinalysis multisticks strip, assessing for leukocytes, nitrites, protein, blood, specific gravity, and glucose. Other parameters not used in this study include pH, urobilinogen, bilirubin and ketones. The reaction of Multistix 10 test strips depends on colour development as an indicator of the concentration of the following test reactions:

Glucose- this is based on two enzymatic reactions- glucose-oxidase/peroxidase reaction which oxidizes the chromogen to colours ranging from green to brown.

Protein- This colorimetric method is based on colour change of certain buffered pH indicators in the presence or absence of protein. Colours range from yellow to green and blue-green and symbolized as negative, trace, 1+ to 4+. The symbol 1+ indicates 30mg/dL or 200-500mg/24hrs;

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2+ is 100mg/dL or 0.5-1.5grams/24hrs and 3+ is 300mg/dL or 2- 5grams/24hrs. 4+ indicates

>1g/dL or >5gm/24hrs.

Leukocytes - Leukocytes of the granulocyte (neutrophils) series contain esterase which catalyse the hydrolysis of the amino acid ester. The resultant reaction results in a purple colour.

Nitrite – Nitrates in the urine are converted to nitrites in the presence of Gram-negative bacteria such as Escherichia coli and Klebsiella spp.

Specific Gravity- the test reaction is based on apparent pKa changes.

Blood- this reaction is based on the liberation of oxygen from peroxide by the peroxidase-like activity of haem.

Urinary electrolytes, urea and creatinine: The aliquots of 1- 2mls of urine previously taken, were kept in cryogenic vials and stored in a freezer at -20°C for subsequent analysis of sodium, potassium, urea and creatinine, using a spectrochemical analyser.

The principles behind some of these investigations include:

Creatinine- The urine samples were diluted by a factor of 30 with distilled water before analysing for creatinine according to the manufacturer’s instructions and analysis was done using the modified Jaffe method.

Urea- Hydrolysis takes place by the enzyme urease, to ammonia and CO2 and read with a spectrophotometric reader.

Sodium and Potassium, were analysed with an ISE (Ion Selective Electrolyte) 6000 analyser by SFRI Medical Diagnostics (Bordeaux, France). The principle here uses changes in electromotive force (EMF) to determine ion concentrations. The analyser uses absorbance measurements for calculating the electrolyte values.

These analyses were conducted at the Clinical Chemistry laboratory of LUTH, Idi-Araba.

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3.8.5 Collection and analysis of blood samples

Blood samples were drawn from the antecubital vein under aseptic conditions and distributed into appropriate sample bottles. Two to three mls was put into a lithium heparin bottle and centrifuged at 5000c/min for 10 minutes. One millilitre of the serum supernatant was stored alongside the urine aliquots at a temperature of -20°C for the subsequent analysis of creatinine, urea, sodium and potassium. The rest of the samples were analysed for levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total protein, albumin, bilirubin, prothrombin time, ESR and complete blood count. Samples were also analysed for detection of Hepatitis B and anti- HCV using Diaspot rapid diagnostic kit via the immunochromatography method. The full blood count was carried out using a Beckman Coulter Counter and erythrocyte sedimentation rate (ESR) was analysed using the Westergren method.

Prothrombin time/INR was analysed with the Eurolyser, SMART 546 (Eurolyser Diagnostica, Austria). The specimen used was whole blood collected in a blue-top tube containing 3.2%

concentrated citrate. An aliquot of plasma is incubated at 37^oC with a reagent containing thromboplastin. Calcium Chloride is then added and the time required for clot formation is measured. The reference range for the laboratory was INR 0.9- 1.2.

Serum electrolytes, urea and creatinine: The serum sodium, potassium, bicarbonate, chloride, urea and creatinine were analysed with the ISE 6000 analyser (SFRI, Bordeaux, France) according to manufacturer’s specifications using the same methodology as for the urinary electrolytes.

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AST and ALT- Analysis was done using discrete reaction rate method using the Hitachi 902 automated analyser by ROCHE (F. Hoffmann-La Roche Ltd, Basel, Switzerland).

3.8.6 Radiological evaluation

ABDOMINAL ULTRASOUND SCAN

All subjects had abdominal ultrasound scans (USS) carried out on them by the investigator under the supervision of a consultant radiologist. The investigator had previously undergone a compulsory two-month rotation in the radiology department as part of the curriculum for gastroenterology in the National Postgraduate College of Nigeria, with emphasis on abdominal ultrasonography and interpretation of abdominal CT scans. Abdominal ultrasound scans were carried out using an EMP 860 B-mode Ultrasound machine equipped with a 3.5-5MHz probe.

The ultrasound scan is a safe diagnostic tool with a sensitivity of up to 72% and specificity of up to 98% for the diagnosis of liver cirrhosis. ⁽²⁸⁾ Subjects were required to fast for at least 6 hours before the USS. Following a description of the procedure, the patient was asked to lie on an examination couch, exposing his abdomen and upper trunk up to the nipple line. A special lubricating gel was applied to the skin and the transducer to prevent friction and help transmit the sound waves. The transducer sends high-frequency sound waves through the body which echo as they encounter a dense object, such as the liver or bone. The echoes are then reflected back into the computer where images are generated from these echoes. The transducer was placed at the midline initially and then moved around to get images and measurements of the liver, kidneys and the spleen. It also assessed the gallbladder, hepatic and portal vessels; the presence of ascites, varices and intrahepatic masses.

59 3.8.7 Liver biopsy

A liver biopsy was carried out in only two patients as part of the routine investigations for evaluating unexplained deranged liver enzymes. The pre-procedure requirements included:

 The patient’s informed consent

 Haematocrit of at least 30%

 INR <1.3 and/or Prothrombin time difference between control and test of less than 4 seconds

 Platelet count of at least 100,000 cells/mm³

 Absence of massive ascites, dilated intrahepatic bile ducts, cyst or haemangioma determined on abdominal ultrasound examination.

 Absence of cardiac failure, respiratory failure, pneumothorax, severe ribcage skin infection or pleural effusion.

 2 units of fresh whole blood made available.

The required materials included: 5- 10mls of 1% plain xylocaine injection, 20mls of 0.9%

normal saline infusion, two 10cc syringes, two 23G needles, two 21G needles, methylated spirit or povidone iodine, a sterile dressing pack, one disposable Menghini needle, sterile cotton wool/

gauze and plaster.

The rest of the procedure is discussed in appendix VI.

3.8.8. Working definitions.

1. Liver cirrhosis was diagnosed in the presence of a combination of (1)- peripheral stigmata of chronic liver disease (leukonychia, palmar erythema, finger clubbing, spider naevi etc.) with or without ascites; (2), laboratory features such as low platelet counts (<100 x 10⁹/L), APRI score

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≥2; (3), radiological features such as hepatic nodularity, heterogeneous parenchymal echoes, hypertrophic caudate lobe, splenomegaly, ascites and intra-abdominal varices; as well as (4), histologically where possible.^{(2, 12)}

2. APRI score – Aspartate aminotransferase-to-Platelet Ratio Index. This is calculated thus- APRI= AST level

ULN x 100

Platelet count(109/L) ULN, AST upper limit of normal.

3. Risk factors for liver cirrhosis: The risk factors for liver cirrhosis were determined by a positive screening test for Hepatitis B or C or history suggestive of significant alcohol ingestion.

4. Significant alcohol ingestion is taken as consumption of 30grams of alcohol/day for > 10 years. ⁽¹²⁴⁾

5. Renal dysfunction refers to both functional and organic causes of abnormal renal function in liver cirrhosis. ⁽²⁶⁾ In this study, these include AKI, hyponatraemia and HRS.

6. Acute kidney injury – The conventional criteria for diagnosis of AKI in cirrhosis comprises a percentage increase in SCr of ≥ 50% from baseline to a final value of SCr >

1.5mg/dL.⁽¹⁷⁾ In this current study, AKI has been defined using an extrapolation of the conventional criteria, where ≥ 50% from baseline ≥1.5mg/dL, assumes that baseline value is 1mg/dL. Similar studies by Carvalho, Shahid and Schekpe have also defined AKI using SCr ≥1.5mg/dL. (16, 97, 98)

7. CKD – subjects with CKD were excluded.

8. Severity of renal failure - if an increase by ≥50% gives a value >1.5mg/dl in patients with liver cirrhosis, it may be assumed in this study that the baseline is 1mg/dl. Thus, the

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subjects with SCr values ≥1.5mg/dl can be classified using the AKIN criteria into stages as shown below:

Stage 1: ↑SCr to ≥ 1.5-2-fold from baseline Stage 2: ↑ SCr to ≥ 2-3-fold from baseline Stage 3: ↑SCr of >3-fold

Thus Stage 1 = 1.5- 1.9mg/dl; Stage 2 = 2- 2.9mg/dl and Stage 3 ≥ 3mg/dl.

9. AKI was classified into prerenal and acute tubular necrosis (ATN) based on the relevant clinical features, the fractional excretion of sodium and/or urine specific gravity. Where subjects had multifactorial causes (>1 cause), the main classification was based on the predominant clinical picture with a supportive laboratory test. Thus, subjects must fulfill at least two out of three criteria listed above.

(i). Pre-renal AKI - defined using relevant clinical features (history of fluid losses- vomiting, diarrhoea, upper gastrointestinal bleeding; along with signs of volume depletion- dehydration, low mean arterial pressure (MAP) <80mmHg) and laboratory parameters. The laboratory parameters include a urine specific gravity (SpG) ≥1.020 and/or Fractional excretion of sodium- FENa <1%. ^{(26, 125)} i.e.

- FENa% = 100 x (UNa x SCr)/ (SNa x UCr). UNa= Urinary sodium; SCr= serum creatinine; SNa=serum sodium; UCr= urinary creatinine

(ii). Acute tubular necrosis - diagnosis of acute tubular necrosis was made in the presence of a history of use of nephrotoxic agents, infection and prolonged volume depletion, along with urine specific gravity (SpG) < 1.020 and/or FENa > 2%.

(iii). Post-renal AKI – post-renal failure was to be confirmed by imaging, demonstrating hydronephrosis in the setting of oliguria/ anuria.

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10. Hyponatraemia – in these subjects is defined as serum sodium <130mEq/L. ^{(87, 102)}

11. HRS – defined by the International Club of Ascites (ICA-AKI) in the presence of the following-

(i). Absence of parenchymal kidney disease as evidenced by proteinuria (urine protein >500 mg/day) and/or haematuria (red blood cells -RBC) count >50 cells per high power field),

(ii). Renal failure (Serum creatinine ≥1.5mg/dL) (iii). Advanced cirrhosis + ascites

(iv). Absence of shock.

(v). No use of nephrotoxic drugs over the preceding seven days;

(vi). No improvement of serum creatinine (decrease to a level <1.5 mg/dl) after two days with diuretic withdrawal and volume expansion by means of human albumin infusion at 1 g/kg body weight/day up to a maximum of 100 grams/day. ^{(17, 93)}

For the purpose of this study, HRS has been modified, defined and diagnosed in the presence of ascites, low MAP (<80mmHg); urine sodium < 10mEq/L; serum creatinine ≥1.5mg/dL and hyponatraemia (serum sodium ≤ 130mEq/L)

12. Acidosis – serum bicarbonate less than 22mEq/L (reference range 22-30mEq/L). ⁽¹¹⁵⁾ 13. Serum Anion gap- difference between the serum cations and anions. i.e. (Na⁺ + K⁺) –

(HCO3‾ + CL‾). Normal anion gap is 3-11mEq/L ⁽¹⁰⁰⁾

14. Low MAP- The value for low mean arterial pressure (MAP) was taken as MAP <80mmHg

(76, 106, 116) where MAP =𝐷𝑃 + ¹₃ (𝑆𝑃 − 𝐷𝑃). (DP = diastolic pressure, SP = systolic pressure).

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15. Infection - Laboratory findings of leucocytosis (WBC > 7.8 x 10⁹/L) with neutrophilia and/or constitutional symptoms e.g. fever, dysuria, cough, or positive peritoneal stretch test, support the presence of an infection.^{(126, 127)}

16. Electrolyte derangements- abnormal ranges of the following electrolytes⁽¹¹⁵⁾:

i. Sodium- serum sodium <130mEq/L(hyponatraemia), >145mEq/L(hypernatraemia) ^{(83, 87)} ii. Potassium- serum potassium <3.5mEq/L(hypokalaemia), >5.5mEq/L(hyperkalaemia) iii. Bicarbonate- serum bicarbonate <22mEq/L(acidosis), >30mEq/L(alkalosis).

iv. Chloride- serum chloride <95mEq/L(hypochloraemia), >105mEq/L(hyperchloraemia).

17. ISCO - 08

The International Labour Organization (ILO) have an international standard classification of occupations (ISCO - 8), which was used to classify the subjects based on their occupation. The nature of a person’s job may predispose him to certain risk factors for liver cirrhosis e.g. many sailors and soldiers are known to take a lot of alcohol. Group 0 represents members of the armed forces, 1 represents managers and 9 represents elementary workers. The rest of the classification is as shown in Appendix VII.

3.8.9 Funding/cost of the study.

Expenses were incurred in the performance of all the urine investigations, serum electrolytes, urea and creatinine. In addition, the cost of the FBC, ESR, liver enzymes, serum albumin and PT/INR was also borne by the researcher. Other expenses were incurred in the procurement of stationery.