case study
Order Description
What is required of your case report write up.
? Introduction (background information, what testing was performed on what samples).
? Case presentation details – patient clinical history/family history and potentially impacting social factors.
? Discussion – Provide specific test results and what conclusions can be drawn from these results, there is likely to be more than one potential condition from the test results available. Indicate testing to be performed for differential diagnosis, tests to indicate prognosis where appropriate. If a firm diagnosis can be made what would be the usual course of treatment.
? Conclusion – Brief summary of the overall conclusions, future developments in the treatment of the condition where appropriate.
the supporting information file will be attached.

BLID CASE STUDY 2
Previously healthy five your old girl presented with a recent history of severe sickness, vomiting, and diarrhoea. The symptoms began with general abdominal pain followed by episodes ofdiarrhoea and vomiting without blood contamination. There were no reports of sick contacts, travel, camping, all unusual food consumption.

On the first day of symptoms, her GP diagnosed viral gastroenteritis and stool cultures were sent to a local hospital. The following day, her vomiting increased in frequency and the diarrhoea became streaked with blood and mucus. The patient was admitted to the local hospital where a complete blood count revealed a white cell count of 22,500/ml ( reference interval 5.5 – 15.5 x 1000/ml) with 89% neutrophils and 7% bands. A urinanalysis revealed positive ketones of 150mg/ml and a specific gravity of 1.030 (reference interval 1.005 – 1.030). She was transferred to a general hospital for management of neutrophilia and dehydration.

On admission, the temperature was 38.7°C, but other vital signs and physical exams were unremarkable. She was treated with intravenous fluids. Stool cultures for bacteria, shiga toxin, ova and parasites were obtained. Urinalysis showed positive protein of 300mg/dl and 4 red blood cells per high power field. On day two of admission, the patient passed cranberry coloured urine. Repeat laboratory tests were performed at this time for creatinine, blood urea, haematocrit and platelet count. She continued to demonstrate anaemia and thrombocytopenia throughout her hospital admission.

Laboratory Testing
Microbiology Testing
Preparation of a gram stain
Observation and comments on a number of pre-prepared plates
Obserbation of OF test
Preformance of oxidase test.

1. Prepare a Grams stain from the NA plate

Protocol
• Make a smear of bacteria on the slide (use the saline)
• Heat fix by passing the slide through the blue Bunsen flame
• Flood slide with crystal violet – 1min
• Flood slide with Lugols iodine – 1min
• Destain with acetone and wash with water
• Flood slide with fuschin – 30 second
• View using x100 oil immersion

2. Observe the MacConkeys and EMB agars – record the colour of the colonies and agar; interpret the results base on the Gram stain and your knowledge of these differential media

3. Observe the pre-prepared OF test and record what you can see
Remember – the media is green to begin with and changes colour to yellow if the organisms is respiring. One tube is overlayed with paraffin to make the conditions anaerobic, the other is aerobic.

4. Carry out an oxidase test

Protocol
• Break the glass vial inside the reagent tube by gently squeezing the tube
• Dispense on drop onto the filter paper disc (in the Petri dish)
• Select a colony and add it to the spot of reagent
• A purple colour is a positive result

5. Serotyping
You have a photograph of O and H serotyping
Six different serotypes have been assessed by latex agglutination
Determine the serotype from the agglutination
Results:

1- Gram stain: gram negative bacilli
2- Urineculture :MacConkey agar: lactose fermenting and round Pink mucoid colonies. – EMB :round small Greenish shiny florescent colonies. – Nutrient Agar : creamy colourless colonies
Stool culture: No growth
3- OF: colour change from green to yellow in both Aerobic and anaerobic (positive)
4- Oxidase test: Negative
5-
Haematology
Observe and report findings from the blood film
Examination of a Blood film

A blood film has been prepared from a peripheral blood sample. Examine the blood film initially under lower power magnification and then under high power magnification noting any abnormal findings.

In order to aid you in your examination of the blood film a normal blood film is also included and should be viewed initially in order for you to make appropriate observations of the patient’s blood film.

Examine the blood film with a scan of the slide using the low-power objective. This step is necessary to assess the overall quality of the blood film, including abnormal distribution of RBCs, suggesting the presence of rouleaux or autoagglutination and/or the presence of a disproportionate number of large nucleated cells, such as monocytes or neutrophils. In addition, the low power magnification examination allows for the rapid detection of large abnormal cells such as blasts, reactive lymphocytes, and parasites.

Using high power magnification and oil immersion find an area of the blood film inwhich the RBCs are evenly distributed and barely touching one another (two or three cellsmay overlap). Scan 8 to 10 fields in this area note the RBC, WBC and platelet morphology and estimate the numbers in relation to the normal blood film.

Evaluation of the RBC morphology is an important aspect of the smear evaluation andis used in conjunction with the RBC indices to describe cells as normal or abnormal in size,shape, and color.

Make a note of your observations.
Biochemistry and Haematology laboratory Results

Laboratory Test
Result
Blood Urea Nitrogen
9.3 mmol/L
Haemoglobin
82g/L
Haematocrit
30.7%
Platelet Count
28 x 109/L
White Blood Cell count 22 x 109/L
Haptoglobulins
Absent
Ketones
150mg/ml
Direct Antiglobulin Test Negative
Urine Analysis:
Haematuria
Proteinuria
4 RBCs per low power field
300mg/dL

Cell Type Cell size (µm) Nucleus Chromatin Cytoplasm Granules Adult Reference range
Peripheral Blood (%) Cell x 109/L
Segmented neutrophil (Seg) polymorphonuclear neutrophil (Poly PMN) 10 – 15 2 – 5 lobes connected by thin filaments without visible chromatin Coarsely clumped Pale ink, cream coloured or colourless 1o: Rare
2o: Abundant 50 – 70 2.3 – 8.1
Band Neutrophil 10 – 15 Constricted but chromatin must be visible within the thinnest part Coarsely clumped Pale blue to pink 1o: Few
2o: Abundant 0 -5 0.0 – 0.6
Lymphocyte 7 – 18 Round to oval; may be slightly indented occasional nucleoli Condensed to deeply condensed Scant to moderate; sky blue ± Few azurophilic 20 – 40 0.8 – 4.8
Monocyte 12 – 20 Variable; may be round, horseshoe or kidney shaped, often has folds producing brain like convolutions Moderately clumped; lacy Blue-gray may have pseudopods; vacuoles may be absent or numerous Many fine granules frequently giving the appearance of ground glass 3 – 11 0.5 – 1.4
Eosinophil 12 – 17 2- 3 lobes connected by thin filaments without visible chromatin Coarsely clumped Cream to pink; may have irregular borders 1o: Rare
2o: Abundant red to orange, round 0 – 5 0.0 – 0.4
Basophil 10 – 14 Usually two lobes connected by thin filaments without visible chromatin Coarsely clumped Lavender to colourless 1o: Rare
2o: Lavender to dark purple, variable in number with uneven distribution; may obscure nucleus or was out during staining. 0 – 1 0.0 – 0.1

Results:

Abnormal RBC morphology and number
• Schistocytes – fragmented part of a red cell
• Spherocytes – lack central pallor and may appear smaller
• Reticulocytes – Immature red cells, larger, contain RNA
• Echinocytes – RBCs covered in spicules
Reduction or absence of platelets
May or may not present with leukocytosis
Determine Prothrombin Time

Vinyl gloves and goggles to be worn for all parts of practical.
METHOD TO DETERMINE PROTHROMBIN TIME (PT)

Materials:
1. 12 x 75 test tubes
2. Oxford pipettes 200 and 100 µl
3. Pipette tips
4. Water bath 37?C
5. Test tube rack
6. Stop watch
7. Calcium chloride/tissue thromboplastin (PT reagent)
8. Controls, Level I and III (normal and abnormal)
9. Patient plasma specimens
Principle:

The PT measures functional activity of the extrinsic and common pathways. The PT evaluates patients suspected of having an inherited or acquired deficiency in these pathways. The procedure uses a tissue thromboplastin reagent with calcium chloride (CaCl) to provide a one step procedure for evaluating plasma clotting.
This test was devised on the assumption that when an optimal amount of calcium and an excess of thromboplastin are added to decalcified plasma, the rate of coagulation depends on the concentration of prothrombin in the plasma. The prothrombin time is therefore the time required for the plasma to clot after an excess of thromboplastin and an optimal concentration of calcium have been added.

The normal values for the prothrombin time range from 10.0 to 13.0 seconds. An elevated prothrombin time may indicate the presence of vitamin K deficiency, DIC, liver disease, presence of FSP’s or a deficiency in one or clotting factors. In addition, inhibitors can cause prolonged PT’s.

Procedure:
1. Mix well prior to pipetting thromboplastin reagent at any step in this procedure.
2. Using a volumetric pipette, add 1mls of the tissue thromboplastin-calcium chloride reagent (PT reagent) into a 12 x 75 mm test tube and place in a 37oC waterbath for 10 minutes to equilibriate to 37oC temperature.
3. Pipette 100 µLof normal control into 2 test tubes.
4. Allow at least one 1 minute for the normal control to reach 37oC.
5. Pipette 200µLof PT reagent into one of the tubes containing the control. Start the stop watch simultaneously.
6. Mix the tube and leave in the heat block for a minimum of 7-8 seconds. Then remove, wipe the exterior, tilt back and forth gently until a visible clot is formed. As the clot forms, the mixture will gelatinize and may turn cloudy.
7. Stop the stop watch immediately when the clot begins to form and record the time in seconds. Record you results to 1 decimal place.
8. Repeat the procedure for the second run of normal control. Record the time.
9. The results from run 1 and run 2 should be within ±1 second from each other, average the two results and report with appropriate units. For manual PT, results should match within 1.0 second (if result is less than 20 seconds). Results over 20 seconds should match within 2.0 seconds.
10. Repeat steps 3-10 for the abnormal control, including the same criteria for accepting the results.
11. Repeat steps 3 – 10 for the patient plasma sample.
Results:
PT = 13 seconds – Normal

Biochemistry
Determine plasma creatinine level.

METHOD FOR CREATININE CLEARANCE

1 Prepare a suitable series of working standards (6) and the stock standard of 1.0mmol/l suggested range 0-1000µmol/l suggested values 0, 50, 100, 400, 800, 1000µmol/l (Note units). Prepare 1ml of each standard and you will use the whole 1ml in the method
2
1.0mmol/l creatinine std (ml) Water (ml) Creatinine conc. µmol/l
0 1.0
0.05 0.95
0.1 0.9
0.4 0.6
0.8 0.2
1.0 0.0

3 Label up a test tube for the patient plasma sample (P1). You will now have a total of 7 tubes.

4 Add 1ml of plasma to the labelled patient tube.

5 Add 3.5ml of distilled water to all 7 tubes.

6 Mix well and add 0.5ml or 500µl of sodium tungstate reagent to all tubes. Mix well and add 1ml of sulphuric acid to all tubes

7 You will find that the patient’s sample has precipitated out the proteins. Centrifuge this tube at 3000rpm for 5 minutes.Carefully return to rack

8 Label up a second series of 10 tubes as before,0, 200, 400, 600, 800, 1000, P1

9 Transfer 3ml from of each of the standard tubes into the fresh tubes and also 3ml of supernatant from the patient’s plasma sample to fresh tubes.

10 Add 1ml of picric acid to all tubes and mix well.

11 Add 1 ml sodium hydroxide to all the tubes and mix well.

12 Stand tubes at room temperature for 15minutes for the colour to develop and read the absorbance at 500nm.

13 Plot a standard curve and extrapolate the patient result.
Result:
• Plasma creatinine : 97.2µmol/l
•
Determine plasma bilirubin level

METHOD FOR PLASMA BILIRUBIN

DETERMINATION OF TOTAL PLASMA BILIRUBIN

PRINCIPLES
Treatment of bilirubin with a diazotised sulphanilic acid reagent results in the production of an azobilirubin with an absorption peak at 600nm.
Free (unconjugated) bilirubin reacts only slowly in aqueous solution and the reaction carried out in such conditions provides a measure of the immediately reacting conjugating bilirubin only.
Inclusion of a solubilising agent such as caffeine-sodium benzoate permits free bilirubin to react with similar rapidity and the reaction carried out under these conditions therefore gives a measure of the total bilirubin present in the sample.
The reaction is allowed to proceed for 10minutes at room temperature after which it is stopped by the addition of ascorbic acid. Alkaline tartrate reagent is then added to convert the red azobilirubin to a blue-green form which improves the sensitivity of the assay.
Please note that for this practical only we will be adding patient/standard samples to our blank tubes. This is unusual. We are doing it so that we can “blank out” the yellow colour of the sample due to the bilirubin. We only want to measure the colour developed when bilirubin reacts with the dye and not the yellow “icterus”

REAGENTS
Note you will need to make up some of these reagents marked with # immediately before use. The sodium nitrite will be prepared for you.

Caffeine/Benzoate 75g Sodium benzoate dissolved in 800ml of water at 60°C, 50g hydrated sodium acetate, 1g EDTA made up to 1 litre.

Alkaline tartrate 100g Sodium Hydroxide and 250g Sodium Potassium Tartrate dissolved in water made up to 1 litre.

Ascorbic acid # 200mg in 5ml water

Sulphanilic acid 5g sulphanilic acid in 15ml concentrated hydrochloric acid
made up to 1 litre.

Sodium nitrite Prepare immediately before use – 500mg in 100ml water.

Diazo reagent # Prepare immediately before use – 10ml sulphanilic acid and 0.25ml Sodium Nitrite solution.

METHOD

Set up a blank tube and 2 sample tubes for the patient sample P1, P2 (perform patient test in duplicate) and also for the bilirubin standard (6 tubes in total) and treat as shown below:

REAGENT TUBES BLANK
TEST (std, P1,P2)µl (stdBl, P1Bl, P2Bl)µl

Water 400 400
Serum 100 100
Ascorbic acid – 50
Diazo reagent 250 250
Caffeine/Benzoate 1000 –

STAND THE TUBES AT ROOM TEMPERATURE FOR 10 MINUTES THE ADD:-
Ascorbic acid 50 –
Caffeine/Benzoate – 1000
Alkaline tartrate 750 750

Read the absorption of each at 600nm. Tabulate your results

N.B. there has to be a blank tube for each test because the yellow colour (icterus) of the samples interferes with the developed substrate colour. We add sample to the blank tubes to get the yellow colour and then we stop the reaction occurring in the blank tubes. If this colour was not blanked out when we zero on the blank tube then this colour would give a false result.

Finally to calculate the Total Bilirubin results use the following equation

Patient result = Absorbance patient x standard value µmol/l
Absorbance standard

Health and Safety Information / Personal Protective Equipment (PPE)

Reagent Hazard CLP Label Control Measure
Ascorbic acid 200mg Not Classed as hazardous
0.5% sulphanilic acid warning
H315 Causes skin irritation.
H317 May cause an allergic skin reaction.
H319 Causes serious eye irritation Whilst this is a dilute solution it may still cause eye irritation so it best to avoid contact with skin, eyes and clothing

Vinyl gloves and goggles
0.5% Sodium Nitrite
Danger
H272 – May intensify fire; oxidizer
H301 – Toxic if swallowed
H319 – Causes serious eye irritation
H400 – Very toxic to aquatic life Whilst this is a dilute solution it may still cause eye irritation so it best to avoid contact with skin, eyes and clothing

Vinyl gloves and goggles
Caffeine/benzoate solution
Caffeine50g/l Warning
H302 Harmful if swallowed. Vinyl gloves and goggles
Sodium Benzoate
75g/l Warning
H319 – Causes serious eye irritation Whilst this is a dilute solution it may still cause eye irritation so it best to avoid contact with skin, eyes and clothing

Vinyl gloves and goggles
Ethylene Diamine tetraacetic acid (EDTA)
1g/l Warning
H319 – Causes serious eye irritation
H332 – Harmful if inhaled Vinyl gloves and goggles
Sodium Acetate 125g/l Not classed as hazardous
Alkaline tartrate solution contains
Sodium hydroxide
100g/l Danger H290 – May be corrosive to metals
H314 – Causes severe skin burns and eye damage
Sodium Potassium tartrate
250g/l Not classed as hazardous

Based on available data, the classification criteria are not met
Bilirubin Standard
Autonorm Not classed as hazardous
Patient sample 1
350 µmol/l (autonorm) Not classed as hazardous
Patient sample 1
35µmol/l (autonorm) Not classed as hazardous

Result:
• Total bilirubin : 50µmol/L

case study
Order Description
What is required of your case report write up.
? Introduction (background information, what testing was performed on what samples).
? Case presentation details – patient clinical history/family history and potentially impacting social factors.
? Discussion – Provide specific test results and what conclusions can be drawn from these results, there is likely to be more than one potential condition from the test results available. Indicate testing to be performed for differential diagnosis, tests to indicate prognosis where appropriate. If a firm diagnosis can be made what would be the usual course of treatment.
? Conclusion – Brief summary of the overall conclusions, future developments in the treatment of the condition where appropriate.
the supporting information file will be attached.

BLID CASE STUDY 2
Previously healthy five your old girl presented with a recent history of severe sickness, vomiting, and diarrhoea. The symptoms began with general abdominal pain followed by episodes ofdiarrhoea and vomiting without blood contamination. There were no reports of sick contacts, travel, camping, all unusual food consumption.

On the first day of symptoms, her GP diagnosed viral gastroenteritis and stool cultures were sent to a local hospital. The following day, her vomiting increased in frequency and the diarrhoea became streaked with blood and mucus. The patient was admitted to the local hospital where a complete blood count revealed a white cell count of 22,500/ml ( reference interval 5.5 – 15.5 x 1000/ml) with 89% neutrophils and 7% bands. A urinanalysis revealed positive ketones of 150mg/ml and a specific gravity of 1.030 (reference interval 1.005 – 1.030). She was transferred to a general hospital for management of neutrophilia and dehydration.

On admission, the temperature was 38.7°C, but other vital signs and physical exams were unremarkable. She was treated with intravenous fluids. Stool cultures for bacteria, shiga toxin, ova and parasites were obtained. Urinalysis showed positive protein of 300mg/dl and 4 red blood cells per high power field. On day two of admission, the patient passed cranberry coloured urine. Repeat laboratory tests were performed at this time for creatinine, blood urea, haematocrit and platelet count. She continued to demonstrate anaemia and thrombocytopenia throughout her hospital admission.

Laboratory Testing
Microbiology Testing
Preparation of a gram stain
Observation and comments on a number of pre-prepared plates
Obserbation of OF test
Preformance of oxidase test.

1. Prepare a Grams stain from the NA plate

Protocol
• Make a smear of bacteria on the slide (use the saline)
• Heat fix by passing the slide through the blue Bunsen flame
• Flood slide with crystal violet – 1min
• Flood slide with Lugols iodine – 1min
• Destain with acetone and wash with water
• Flood slide with fuschin – 30 second
• View using x100 oil immersion

2. Observe the MacConkeys and EMB agars – record the colour of the colonies and agar; interpret the results base on the Gram stain and your knowledge of these differential media

3. Observe the pre-prepared OF test and record what you can see
Remember – the media is green to begin with and changes colour to yellow if the organisms is respiring. One tube is overlayed with paraffin to make the conditions anaerobic, the other is aerobic.

4. Carry out an oxidase test

Protocol
• Break the glass vial inside the reagent tube by gently squeezing the tube
• Dispense on drop onto the filter paper disc (in the Petri dish)
• Select a colony and add it to the spot of reagent
• A purple colour is a positive result

5. Serotyping
You have a photograph of O and H serotyping
Six different serotypes have been assessed by latex agglutination
Determine the serotype from the agglutination
Results:

1- Gram stain: gram negative bacilli
2- Urineculture :MacConkey agar: lactose fermenting and round Pink mucoid colonies. – EMB :round small Greenish shiny florescent colonies. – Nutrient Agar : creamy colourless colonies
Stool culture: No growth
3- OF: colour change from green to yellow in both Aerobic and anaerobic (positive)
4- Oxidase test: Negative
5-
Haematology
Observe and report findings from the blood film
Examination of a Blood film

A blood film has been prepared from a peripheral blood sample. Examine the blood film initially under lower power magnification and then under high power magnification noting any abnormal findings.

In order to aid you in your examination of the blood film a normal blood film is also included and should be viewed initially in order for you to make appropriate observations of the patient’s blood film.

Examine the blood film with a scan of the slide using the low-power objective. This step is necessary to assess the overall quality of the blood film, including abnormal distribution of RBCs, suggesting the presence of rouleaux or autoagglutination and/or the presence of a disproportionate number of large nucleated cells, such as monocytes or neutrophils. In addition, the low power magnification examination allows for the rapid detection of large abnormal cells such as blasts, reactive lymphocytes, and parasites.

Using high power magnification and oil immersion find an area of the blood film inwhich the RBCs are evenly distributed and barely touching one another (two or three cellsmay overlap). Scan 8 to 10 fields in this area note the RBC, WBC and platelet morphology and estimate the numbers in relation to the normal blood film.

Evaluation of the RBC morphology is an important aspect of the smear evaluation andis used in conjunction with the RBC indices to describe cells as normal or abnormal in size,shape, and color.

Make a note of your observations.
Biochemistry and Haematology laboratory Results

Laboratory Test
Result
Blood Urea Nitrogen
9.3 mmol/L
Haemoglobin
82g/L
Haematocrit
30.7%
Platelet Count
28 x 109/L
White Blood Cell count 22 x 109/L
Haptoglobulins
Absent
Ketones
150mg/ml
Direct Antiglobulin Test Negative
Urine Analysis:
Haematuria
Proteinuria
4 RBCs per low power field
300mg/dL

Cell Type Cell size (µm) Nucleus Chromatin Cytoplasm Granules Adult Reference range
Peripheral Blood (%) Cell x 109/L
Segmented neutrophil (Seg) polymorphonuclear neutrophil (Poly PMN) 10 – 15 2 – 5 lobes connected by thin filaments without visible chromatin Coarsely clumped Pale ink, cream coloured or colourless 1o: Rare
2o: Abundant 50 – 70 2.3 – 8.1
Band Neutrophil 10 – 15 Constricted but chromatin must be visible within the thinnest part Coarsely clumped Pale blue to pink 1o: Few
2o: Abundant 0 -5 0.0 – 0.6
Lymphocyte 7 – 18 Round to oval; may be slightly indented occasional nucleoli Condensed to deeply condensed Scant to moderate; sky blue ± Few azurophilic 20 – 40 0.8 – 4.8
Monocyte 12 – 20 Variable; may be round, horseshoe or kidney shaped, often has folds producing brain like convolutions Moderately clumped; lacy Blue-gray may have pseudopods; vacuoles may be absent or numerous Many fine granules frequently giving the appearance of ground glass 3 – 11 0.5 – 1.4
Eosinophil 12 – 17 2- 3 lobes connected by thin filaments without visible chromatin Coarsely clumped Cream to pink; may have irregular borders 1o: Rare
2o: Abundant red to orange, round 0 – 5 0.0 – 0.4
Basophil 10 – 14 Usually two lobes connected by thin filaments without visible chromatin Coarsely clumped Lavender to colourless 1o: Rare
2o: Lavender to dark purple, variable in number with uneven distribution; may obscure nucleus or was out during staining. 0 – 1 0.0 – 0.1

Results:

Abnormal RBC morphology and number
• Schistocytes – fragmented part of a red cell
• Spherocytes – lack central pallor and may appear smaller
• Reticulocytes – Immature red cells, larger, contain RNA
• Echinocytes – RBCs covered in spicules
Reduction or absence of platelets
May or may not present with leukocytosis
Determine Prothrombin Time

Vinyl gloves and goggles to be worn for all parts of practical.
METHOD TO DETERMINE PROTHROMBIN TIME (PT)

Materials:
1. 12 x 75 test tubes
2. Oxford pipettes 200 and 100 µl
3. Pipette tips
4. Water bath 37?C
5. Test tube rack
6. Stop watch
7. Calcium chloride/tissue thromboplastin (PT reagent)
8. Controls, Level I and III (normal and abnormal)
9. Patient plasma specimens
Principle:

The PT measures functional activity of the extrinsic and common pathways. The PT evaluates patients suspected of having an inherited or acquired deficiency in these pathways. The procedure uses a tissue thromboplastin reagent with calcium chloride (CaCl) to provide a one step procedure for evaluating plasma clotting.
This test was devised on the assumption that when an optimal amount of calcium and an excess of thromboplastin are added to decalcified plasma, the rate of coagulation depends on the concentration of prothrombin in the plasma. The prothrombin time is therefore the time required for the plasma to clot after an excess of thromboplastin and an optimal concentration of calcium have been added.

The normal values for the prothrombin time range from 10.0 to 13.0 seconds. An elevated prothrombin time may indicate the presence of vitamin K deficiency, DIC, liver disease, presence of FSP’s or a deficiency in one or clotting factors. In addition, inhibitors can cause prolonged PT’s.

Procedure:
1. Mix well prior to pipetting thromboplastin reagent at any step in this procedure.
2. Using a volumetric pipette, add 1mls of the tissue thromboplastin-calcium chloride reagent (PT reagent) into a 12 x 75 mm test tube and place in a 37oC waterbath for 10 minutes to equilibriate to 37oC temperature.
3. Pipette 100 µLof normal control into 2 test tubes.
4. Allow at least one 1 minute for the normal control to reach 37oC.
5. Pipette 200µLof PT reagent into one of the tubes containing the control. Start the stop watch simultaneously.
6. Mix the tube and leave in the heat block for a minimum of 7-8 seconds. Then remove, wipe the exterior, tilt back and forth gently until a visible clot is formed. As the clot forms, the mixture will gelatinize and may turn cloudy.
7. Stop the stop watch immediately when the clot begins to form and record the time in seconds. Record you results to 1 decimal place.
8. Repeat the procedure for the second run of normal control. Record the time.
9. The results from run 1 and run 2 should be within ±1 second from each other, average the two results and report with appropriate units. For manual PT, results should match within 1.0 second (if result is less than 20 seconds). Results over 20 seconds should match within 2.0 seconds.
10. Repeat steps 3-10 for the abnormal control, including the same criteria for accepting the results.
11. Repeat steps 3 – 10 for the patient plasma sample.
Results:
PT = 13 seconds – Normal

Biochemistry
Determine plasma creatinine level.

METHOD FOR CREATININE CLEARANCE

1 Prepare a suitable series of working standards (6) and the stock standard of 1.0mmol/l suggested range 0-1000µmol/l suggested values 0, 50, 100, 400, 800, 1000µmol/l (Note units). Prepare 1ml of each standard and you will use the whole 1ml in the method
2
1.0mmol/l creatinine std (ml) Water (ml) Creatinine conc. µmol/l
0 1.0
0.05 0.95
0.1 0.9
0.4 0.6
0.8 0.2
1.0 0.0

3 Label up a test tube for the patient plasma sample (P1). You will now have a total of 7 tubes.

4 Add 1ml of plasma to the labelled patient tube.

5 Add 3.5ml of distilled water to all 7 tubes.

6 Mix well and add 0.5ml or 500µl of sodium tungstate reagent to all tubes. Mix well and add 1ml of sulphuric acid to all tubes

7 You will find that the patient’s sample has precipitated out the proteins. Centrifuge this tube at 3000rpm for 5 minutes.Carefully return to rack

8 Label up a second series of 10 tubes as before,0, 200, 400, 600, 800, 1000, P1

9 Transfer 3ml from of each of the standard tubes into the fresh tubes and also 3ml of supernatant from the patient’s plasma sample to fresh tubes.

10 Add 1ml of picric acid to all tubes and mix well.

11 Add 1 ml sodium hydroxide to all the tubes and mix well.

12 Stand tubes at room temperature for 15minutes for the colour to develop and read the absorbance at 500nm.

13 Plot a standard curve and extrapolate the patient result.
Result:
• Plasma creatinine : 97.2µmol/l
•
Determine plasma bilirubin level

METHOD FOR PLASMA BILIRUBIN

DETERMINATION OF TOTAL PLASMA BILIRUBIN

PRINCIPLES
Treatment of bilirubin with a diazotised sulphanilic acid reagent results in the production of an azobilirubin with an absorption peak at 600nm.
Free (unconjugated) bilirubin reacts only slowly in aqueous solution and the reaction carried out in such conditions provides a measure of the immediately reacting conjugating bilirubin only.
Inclusion of a solubilising agent such as caffeine-sodium benzoate permits free bilirubin to react with similar rapidity and the reaction carried out under these conditions therefore gives a measure of the total bilirubin present in the sample.
The reaction is allowed to proceed for 10minutes at room temperature after which it is stopped by the addition of ascorbic acid. Alkaline tartrate reagent is then added to convert the red azobilirubin to a blue-green form which improves the sensitivity of the assay.
Please note that for this practical only we will be adding patient/standard samples to our blank tubes. This is unusual. We are doing it so that we can “blank out” the yellow colour of the sample due to the bilirubin. We only want to measure the colour developed when bilirubin reacts with the dye and not the yellow “icterus”

REAGENTS
Note you will need to make up some of these reagents marked with # immediately before use. The sodium nitrite will be prepared for you.

Caffeine/Benzoate 75g Sodium benzoate dissolved in 800ml of water at 60°C, 50g hydrated sodium acetate, 1g EDTA made up to 1 litre.

Alkaline tartrate 100g Sodium Hydroxide and 250g Sodium Potassium Tartrate dissolved in water made up to 1 litre.

Ascorbic acid # 200mg in 5ml water

Sulphanilic acid 5g sulphanilic acid in 15ml concentrated hydrochloric acid
made up to 1 litre.

Sodium nitrite Prepare immediately before use – 500mg in 100ml water.

Diazo reagent # Prepare immediately before use – 10ml sulphanilic acid and 0.25ml Sodium Nitrite solution.

METHOD

Set up a blank tube and 2 sample tubes for the patient sample P1, P2 (perform patient test in duplicate) and also for the bilirubin standard (6 tubes in total) and treat as shown below:

REAGENT TUBES BLANK
TEST (std, P1,P2)µl (stdBl, P1Bl, P2Bl)µl

Water 400 400
Serum 100 100
Ascorbic acid – 50
Diazo reagent 250 250
Caffeine/Benzoate 1000 –

STAND THE TUBES AT ROOM TEMPERATURE FOR 10 MINUTES THE ADD:-
Ascorbic acid 50 –
Caffeine/Benzoate – 1000
Alkaline tartrate 750 750

Read the absorption of each at 600nm. Tabulate your results

N.B. there has to be a blank tube for each test because the yellow colour (icterus) of the samples interferes with the developed substrate colour. We add sample to the blank tubes to get the yellow colour and then we stop the reaction occurring in the blank tubes. If this colour was not blanked out when we zero on the blank tube then this colour would give a false result.

Finally to calculate the Total Bilirubin results use the following equation

Patient result = Absorbance patient x standard value µmol/l
Absorbance standard

Health and Safety Information / Personal Protective Equipment (PPE)

Reagent Hazard CLP Label Control Measure
Ascorbic acid 200mg Not Classed as hazardous
0.5% sulphanilic acid warning
H315 Causes skin irritation.
H317 May cause an allergic skin reaction.
H319 Causes serious eye irritation Whilst this is a dilute solution it may still cause eye irritation so it best to avoid contact with skin, eyes and clothing

Vinyl gloves and goggles
0.5% Sodium Nitrite
Danger
H272 – May intensify fire; oxidizer
H301 – Toxic if swallowed
H319 – Causes serious eye irritation
H400 – Very toxic to aquatic life Whilst this is a dilute solution it may still cause eye irritation so it best to avoid contact with skin, eyes and clothing

Vinyl gloves and goggles
Caffeine/benzoate solution
Caffeine50g/l Warning
H302 Harmful if swallowed. Vinyl gloves and goggles
Sodium Benzoate
75g/l Warning
H319 – Causes serious eye irritation Whilst this is a dilute solution it may still cause eye irritation so it best to avoid contact with skin, eyes and clothing

Vinyl gloves and goggles
Ethylene Diamine tetraacetic acid (EDTA)
1g/l Warning
H319 – Causes serious eye irritation
H332 – Harmful if inhaled Vinyl gloves and goggles
Sodium Acetate 125g/l Not classed as hazardous
Alkaline tartrate solution contains
Sodium hydroxide
100g/l Danger H290 – May be corrosive to metals
H314 – Causes severe skin burns and eye damage
Sodium Potassium tartrate
250g/l Not classed as hazardous

Based on available data, the classification criteria are not met
Bilirubin Standard
Autonorm Not classed as hazardous
Patient sample 1
350 µmol/l (autonorm) Not classed as hazardous
Patient sample 1
35µmol/l (autonorm) Not classed as hazardous

Result:
• Total bilirubin : 50µmol/L