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PRACTICALS 2 & 3 PERNICIOUS ANAEMIA This practical takes place over two sessions. Hand in the report after the second session. See the Dropbox for due dates. Background Pernicious anaemia is the end stage of chronic atrophic gastritis, an organ specific autoimmune disease affecting the stomach. The disease is characterised by gastric atrophy (wasting away of the stomach), infiltration of inflammatory cells into the stomach and production of auto-antibodies to various proteins, including intrinsic factor and the proton pump of parietal cells. Intrinsic factor is involved in the absorption of vitamin B12, which is required for the maturation of red blood cells, hence the anaemia associated with the disease. The parietal cells are found in the gastric glands, and their major function is to secrete hydrochloric acid, which is involved in the initial stages of digestion. The enzyme that is responsible for gastric acid secretion is the gastric HP+P/KP+P-ATPase, a proton pump, which consists of a 95 kDa α-subunit and a 60-90 kDa, glycosylated-β-subunit. Auto-antibodies found in the serum from patients with pernicious anaemia react with either or both of the α and β subunits of the gastric proton pump. This antibody response serves as a useful diagnostic tool for this disease. In this exercise, you will be testing antibodies from patient serum samples to determine if any of them react with the gastric proton pump antibody, and hence would suggest a diagnosis of pernicious anaemia. You will be using two different methods to determine the presence of anti-proton pump antibodies in serum samples. In Prac 2, you will separate proteins extracted from mouse stomachs by SDS-polyacrylamide gel electrophoresis, and transfer these proteins to a nitrocellulose membrane. In Prac 3, you will perform Western blotting on the gastric proteins using sera from patients who may or may not have pernicious anaemia. Also in Prac 2, you will stain a mouse stomach section with haematoxylin and eosin to become familiar with the structure and cell types within the stomach. The structure of the mouse stomach is similar to human, and human anti-proton pump antibodies cross react with the equivalent mouse protein. In Prac 3, you will perform immuno-peroxidase staining of mouse stomach sections with the same sera from patients who may or may not have pernicious anaemia. Aims To determine whether samples of patient sera contain antibodies to the gastric proton pump by Western blotting and immuno-histochemistry, and suggest which patients have pernicious anaemia.

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WESTERN BLOT PROCEDURE

SDS‐PAGE of mouse stomach proteins

Transfer proteins to nitrocellulose membrane by electrophoresis 

Stain membrane with Ponceau S to confirm protein transfer from gel to membrane. Use a pencil to mark out the lanes and label as 

per instructions. Trim edges that do not contain protein.  

De‐stain the membrane. Store in blocking buffer at 4°C until next prac.

Incubate strips of membrane with different patient sera (include positive and negative controls) 

Wash membrane strips and incubate in blocking buffer 

Incubate membrane strips in secondary antibody 

Incubate membrane strips in lumi‐light detection buffer 

Reassemble marker and test strips and visualise with Chemi‐doc system. 

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PRACTICAL 2 PERNICIOUS ANAEMIA PART 1 SDS-PAGE, Western blotting and histology Background The most widely used method for analysing protein mixtures is by SDS-polyacrylamide gel electrophoresis. Proteins are solubilised in sodium dodecyl-sulphate (SDS), an anionic detergent that binds to the hydrophobic region of proteins, causing them to unfold, and giving them an overall net negative charge. A reducing agent such as 2-β mercapto-ethanol is also included to disrupt any disulfide bonds within the proteins. When the proteins/polypeptides are electrophoresed through a polyacrylamide gel, the negatively charged proteins migrate towards the anode (positive electrode) and are separated according to their size, with smaller proteins migrating the furthest. By running a set of standard proteins of known molecular weights (see figure 1. below) along-side our samples, the size of protein/polypeptides in the mixture can be estimated. To identify specific proteins in a mix, immuno- (or Western) blotting is performed, whereby proteins separated by SDS-PAGE are electrophoretically transferred to a nitrocellulose membrane. The nitrocellulose membrane is then probed with an antibody specific for the protein of interest, and a secondary antibody conjugated to an enzyme is used to detect binding of the first antibody. The substrate for the enzyme is applied to the membrane, and the signal is detected by various means, such as colourmetric detection or chemi-luminesence.

Figure 1. Precision Plus Protein™ Kaleidoscope™ Standards #161-0375

Materials

Precast-Tris Glycine-polyacrylamide gel Gel running buffer (25 mM Tris, pH 8.3, 0.192 M Glycine, 0.1% SDS) Gel apparatus Power supply (Caution: electrocution risk, mop up spills) Mouse stomach protein (pre-diluted to 2 µg per µL) 5 x SDS sample buffer (0.315 M Tris, pH 6.8, 25% Glycerol, 10% SDS, 5% 2-beta mercaptoethanol, 0.025% Bromophenol blue) (Caution: irritant and health hazard, wear safety glasses and gloves) Protein standard (Biorad Precision Plus Protein™ Kaleidoscope™ Standards #161-0375) Set of auto-pipettes and tips

7  Sterile microfuge tubes Gel loading tips Biorad Turbo-blot Transfer apparatus Rapid Transfer buffer (prepared fresh from Biorad 5x concentrate) Nitrocellulose membrane Ultra thick filter paper 0.1% Ponceau in 1% acetic acid (Caution: corrosive, wear safety glasses and gloves) 0.1 M NaOH (Caution: corrosive, wear safety glasses and gloves) TBS (Tris buffered saline) TBST (Tris buffered saline with Tween) Blocking solution (TBS containing 5% skim milk powder) Sample trays Plastic gel wedge Metal gel cassette opener Pencils Gel roller tool Heat sealer Heat sealing bags Methods Electrophoresis

1. The precast polyacrylamide gel has been placed into the electrophoresis tank, and both reservoirs have been filled with Tris-glycine running buffer. (Caution: electrocution risk, mop up spills)

2. To prepare the stomach protein sample add 50 µL of 5 x SDS-sample buffer to the tube containing 200 µL of mouse stomach protein) (Caution: irritant and health hazard, wear safety glasses and gloves).

3. Critical step: Mix well by pipetting up and down until the solution looks homogeneous. Avoid introducing bubbles to your sample as you mix by controlling the way you aspirate the solution into the tip (your demonstrator will show you how to do this).

4. Seal the tube and hold at room temperature until required.

5. Your demonstrator will load the MW marker into the first well.

6. Load 25 µL aliquots of prepared protein into each of the sample wells.

7. Connect the apparatus to the power supply and run the gel at 200 V for 40 min-1 h or until

the yellow band on the marker reaches the bottom of the gel. Critical Step: Check the gel every 10 mins to monitor the buffer level in the inner chamber. It should remain higher than the short gel plate (Caution: electrocution risk, mop up spills).

Transfer

1. Turn off the power supply and remove the gel from the apparatus noting the position of the protein marker on the gel. Cool the gel cassette under running water.

2. With the help of your demonstrator, open the cassette by prying the plates apart with the

cassette opening tool where the arrows indicate. Use the plastic gel wedge to cut the lanes off the gel. Cut the corner off the top of the gel in the lane with the marker so that you can recognize it.

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3. To remove the gel, place the plate containing the gel into a tray containing a just enough transfer buffer to immerse the plate. You may need to gently detach the edges of the gel with the plastic gel wedge. Gently rock the container to loosen the gel. Once the gel is detached, remove the gel plate and set the gel aside.

4. To transfer the proteins to a nitrocellulose membrane, Critical Step set up the following on the base of the Turbo-blot transfer cassette in the order following order (see diagram below). Each layer must be soaked in transfer buffer prior to being placed on the apparatus. Ensure there are no air bubbles between the layers by smoothing with the roller provided (wear gloves!!).

a) 1 piece of ultra-thick filter paper (bottom of stack) b) nitrocellulose membrane (clearly labeled, label facing up) c) gel (align the gel to the correct orientation with the cut corner near the M label of the

nitrocellulose) d) 1 piece of ultra-thick filter paper (top of stack)

Your transfer stack should look like the diagram below.

5. Place the lid on the Turbo-blot cassette and turn to the lock position. Insert the cassette into the TurboBlot transfer apparatus. Set the transfer at a constant current of 25V for 7 mins. Start the transfer.

6. Once the transfer has finished, turn off the power supply, disassemble the transfer set-up and place the nitrocellulose membrane into a container with 50 mL 0.1% Ponceau stain. Place on the rocker for 1 min.

7. To visualize the lanes and confirm the transfer of your proteins, rinse the membrane with

deionised water until protein bands can be seen. Take note whether the bands appear smeary and degraded or crisp and straight.

8. Critical step With the help of your demonstrator, label your nitrocellulose membrane as shown below with a pencil. Do not allow your membrane to dry out. It is important to mark out the lanes while stained with ponceau S (Caution: corrosive, wear safety glasses and gloves) so you can cut strips of protein for antibody testing accurately in the next session.

 M       +ve   –ve     P1    P2    P3    P4      P5    P6

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8. Rinse the membrane briefly in 50 mL 0.1 M NaOH on the rocker until the stain disappears.

(Caution: corrosive, wear safety glasses and gloves)

9. Rinse the membrane briefly on the rocker in 50 mL TBS.

10. To block non-specific binding of proteins, add 50 mL blocking solution (TBS containing 5% skim milk powder) to the membrane.

11. Heat-seal (Caution: Hot, risk of burns) the membrane in a plastic pouch with enough

blocking buffer to keep it moist and label with your names and prac session. Remove as many air bubbles as possible. Your membrane will be stored in this buffer at 4oC until the next session.

NB Please do not use symbols such as asterisks or stars to label tubes or samples. This is creates confusion if you are sick or late.

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Morphology of the stomach In order to become familiar with the structural features of the stomach, you will stain a formalin-fixed, paraffin-embedded (FFPE) section of a mouse stomach with haematoxylin and eosin. Materials Slide containing section of mouse stomach (one per pair) Histoclear Ethanol Haematoxylin Acid alcohol Scott's tap water Eosin DPX mounting media Coverslips Methods (use gloves for all procedures) 1. Incubate the slide in histoclear for 2 min then in ethanol for 2 min (Caution: flammable, wear

safety glasses and gloves, avoid ignition sources). 2. Rehydrate in tap water for 30 s. 3. Incubate the slide in haematoxylin for 2 min.

4. Rinse in tap water for 30 s by dipping up and down. Blot the end of the slide on paper towel. 5. Dip into 1% acid alcohol for 3 s (Caution: corrosive and flammable, wear safety glasses and

gloves, avoid ignition sources) 6. Rinse immediately in water for 30 s. 7. Incubate slide in Scott's tap water for 30 s. 8. Rinse in water for 30 s. 9. Place slide in eosin Y for 4 min. Blot excess stain off onto paper towel.

10. Place slide in 90% ethanol for 30 s. Dip up and down a few times. Drain excess liquid onto

blotter (Caution: flammable, wear safety glasses and gloves, avoid ignition sources)

11. Place slide in 100% ethanol for 30 s. Dip up and down a few times. Drain excess liquid onto blotter (Caution: flammable, wear safety glasses and gloves, avoid ignition sources)

12. Incubate the slide in 100% ethanol for 2 min then allow to air dry. 13. Your demonstrator will place a drop of Organolimoline mounting medium on the section, cover

with a cover slip (Caution: flammable, wear safety glasses and gloves, avoid ignition sources)

14. Examine the slide under the microscope. Read the description of stomach morphology on the next page then draw 2 diagrams of the section, noting the following features:

11  Diagram 1 should show the layers at low power (40x magnification) showing the following features of the stomach wall

Gastric mucosa (purple staining) Muscularis mucosal layers (pink staining) Submucosa (Pink/Red staining) Blood vessels (if present) Gastric pits

Diagram 2 should show cells of the gastric gland (100-400x magnification)

a gastric pit mucous secreting cells parietal cells (fried egg appearance) chief cells Blood vessels (if present)

15. Ensure your diagrams have titles, magnification, all labels and no shading. 16. Get your demonstrator to clearly initial and date the drawings before you leave. Scans or

photos of the diagrams can be used as figures in your report. Add a figure legend to ensure each diagram can be interpreted independently from the text.

Figure 2 Morphology of the stomach and small intestine. Source: Color Atlas of Histology, 5th Ed. Gartner and Hiatt

12   The wall of the stomach is divided into distinct layers (Figure 2). The innermost layer facing the lumen is the gastric mucosa, containing the gastric glands. A thin layer of muscle separates the mucosa from the sub-mucosa, which contains connective tissues, blood vessels and nerves. There are outer layers of muscle and connective tissue. The gastric glands consist of a number of different cell types. The top regions of the glands closest to the lumen contain small mucous cells, which secrete mucous to protect the mucosa from the acidic environment. The middle and lower regions of the glands contain parietal cells, which are large pink staining cells that contain the proton pump and secrete acid. The base of the glands contain zymogenic, or chief cells, which stain a dark pink/purple colour. These cells secrete pepsinogen, which is involved in protein breakdown. Refer to recommended journal article by Toh, et al. 1997 for information on the structure of the mouse stomach.

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PERNICIOUS ANAEMIA PART 2 Western Blotting (continued) and calculation refresher

Materials Nitrocellulose membrane from the previous session Positive control serum (from a patient with pernicious anaemia) Negative control serum (from a normal person) Serum samples from patients (P1-6) Sheep anti-human Ig-HRP conjugate (diluted 1:2000) TBS/Tween (Tris buffered saline, 20 mM Tris, pH 7.5, 137 mM NaCl containing 0.05% Tween-20) Lumi-Light reagents (Roche) Scissors Containers for washing Parafilm Forceps

Methods

1. Remove your membrane from the storage pouch carefully

2. Slice the remaining membrane into 6-8 strips depending on how many patient sera there are to test (+ve, -ve, P1, P2, P3, P4, P5 and P6).

3. Each pair will be allocated two serum samples to test, either the positive control, negative

control or patient samples. The serum samples have already been diluted appropriately.

4. Place the strips onto a piece of parafilm and pipette 100 µL of the appropriate sera onto the strip. Cover lightly with a second piece of parafilm to prevent the strip from drying out. Do not press down. The entire strip needs to be covered in a thin layer of serum and there should be no air bubbles.

5. Incubate for 1 h at room temperature.

6. Wash the strips 3 x 10 min in TBST with rocking.

7. Place each strip onto a fresh piece of parafilm and pipette 100 µL of the secondary antibody,

sheep anti-human Ig-HRPO (already diluted 1:2000), evenly onto each cover with parafilm as before and incubate for 45 mins.

8. Wash the strips 3 x 5 min in TBS with rocking.

9. Leave the strips at the end of the bench in TBS buffer.

10. When the whole class is ready the technical staff will take the strips to the Chemidoc.

11. Just before analysis the strips will be incubated in 4 mL of the freshly prepared Lumi-Light

substrate for 5 mins.

12. The strips, plus the marker, will then be aligned on a piece of parafilm.

13. The membrane will be placed into a Chemidoc apparatus so that the light emitted from the substrate can be detected and the results recorded. An image of the membrane will be placed on CloudDeakin.

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14. When your results are posted on CloudDeakin you will need to have 2 images. One image of the marker and one of the blot. These will be the same size.

15. You will need to crop the two images to create the figure for analysis. An example of how to do this can be found on CloudDeakin.

Immuno-peroxidase staining of mouse stomach sections

Background Immuno-histochemistry is a method used to either detect the presence of a specific antigen in a tissue section, or to detect specific antibodies within a serum sample. In this exercise, we are testing patient sera for the presence of anti-proton pump antibodies by incubating the sera on sections of mouse stomach, which contain the proton pump antigen. Once any unbound serum is washed away, the section is incubated with a secondary antibody specific for human Ig molecules. The secondary antibody is conjugated to an enzyme, horseradish peroxidase, which will convert the substrate to a coloured product in the areas of the tissue section to which the primary antibodies have bound. Each pair is to perform the experiment on two slides using the same serum samples they tested in the Western blotting.

Materials Slides containing section of mouse stomach (two per pair) Histoclear Ethanol (Caution: flammable, wear safety glasses and gloves, avoid ignition sources) PBS 0.3% H2O2 in PBS 1% BSA in PBS Positive control serum (from a patient with pernicious anaemia) Negative control serum (from a normal person) Serum samples from patients #1-4 Sheep anti-human Ig HRPO conjugate (diluted 1:300) DAB substrate (Demonstrator to prepare fresh) Haematoxylin Organolimolene mounting media (Caution: flammable, wear safety glasses and gloves, avoid ignition sources) Coverslips Timers

Methods Note: Due to time constraints, steps 1-5 have been performed for you. Start at step 9. Do not let the sections dry out at any stage as this will result in high background staining. Note that a water-resistant circle has been drawn around the section to keep the liquid reagents confined to the small area containing the tissue. 1. Incubate the slide in histoclear for 2 min.

15  2. Incubate the slide in ethanol for 2 min. (Caution: flammable, wear safety glasses and gloves,

avoid ignition sources) 3. Rinse in tap water for 30 s.

4. Transfer the slide to a Sequenza Coverplate™ clip and insert into the Sequenza rack (Fig. 3)

5. To remove endogenous peroxidase activity, add 200 µL of 0.3% H2O2 in PBS and incubate

for 30 min at room temperature. 6. Wash the slide in PBS for 2 min

7. Add 200 µL of 1% BSA (bovine serum albumin) in PBS (Phosphate buffered saline) for 30

min to block non-specific binding of proteins. Do not wash slide after this step. 8. Add 200 µL of the patient’s serum sample to the section by pipetting into the top of the

Sequenza clip into the space between the slide and the coverplate as shown in Fig. 3 and incubate overnight at 4°C.

Coverplate™ Sequenza Rack™

Addition of reagents to the Coverplate™ system

Figure 3. Thermo Shandon Coverplate™ System

9. Wash slides 3 x 2 min in with 200 µL of PBS. 10. Add 200 µL of the sheep anti-human Ig HRPO conjugate (diluted 1:300) to the section and

incubate for 45 min.

16  11. Wash slides 3 x 2 min in PBS, then rinse briefly in water. 12. Add 200 µL of DAB substrate to the section and incubate for 5-10 min. 13. Rinse slides in 3 x 1 min with 200 µL of PBS.

14. Get your demonstrator to remove the slide from the Sequenza rack.

15. Dip into haematoxylin for 3 s. 16. Wash slides by dipping in water for 30 s. Agitate gently to remove excess stain. 17. Incubate the slide in ethanol for 2 mins. 18. Ask your demonstrator to place a small drop of Organolimolene mounting medium on the

section for you.

19. Cover the section with a cover slip.

20. Clean excess mounting medium from the slide then examine under the microscope.

21. Determine whether or not the parietal cells have been stained.

22. Prepare a diagram of a stomach section that has a positive antibody reaction. Label as per the previous prac and point out where the DAB staining occurs. Get your demonstrator to sign and date your diagram.

23. Collaborate with your group members to get the results for all patients. Make sure you do not leave without these results. Summarise the results in a table. Determine whether any results may be unreliable because of errors so you can account for any false positives or negatives obtained by other pairs in your discussion.

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Pernicious anaemia practical report Before you begin:

Read the article entitled “Pernicious anaemia” (1997) by Toh, B.H., van Driel, I.R. and Gleeson, P.A., New England Journal of Medicine, 337, p1441-1448

Obtain an entire set of results for all of the serum samples used for the Western blotting and

immuno-peroxidase staining from the other students at your bench. Summarise the results in a table.

Determine which of the patients can be diagnosed with pernicious anaemia.

Avoid the temptation to cut and paste anything from the manual, journal articles or web pages. It is really obvious and easily detected by anti-plagiarism software. See the document “Harvard 2” on CloudDeakin for tips on referencing, paraphrasing and avoiding plagiarism. Your Prac reports should include the following:

Title and author: A title should reflect the report content. Below the title include your name and practical partner’s name and practical session

A short paragraph that describes the Aim/Aims of the Prac An Introduction that gives a background to the experiments. What is pernicious anaemia?

Provide some background to the experimental procedure and what you hope to achieve. You can make reference to published articles here. This should be half to one page in length.

A Methods section that summarises the main steps you followed with reference to the detail in the Prac manual. Do not include materials lists. You can use sub-headings for different techniques. Write in paragraph form, not dot-points. It should not look anything like the manual. The manual is full of unnecessary details. Make sure your methods reflect modifications made to procedures. Don’t copy straight from the manual. If you used 200µL instead of 100µL of serum your methods should state the correct figure.

The Results section should systematically describe your results in detail, with reference to Figures and Tables that display your data. All data presented in Figures or Tables, including diagrams, should also be commented on in the text of the Results section. Provide Figure Legends to accompany your Figures and a heading for Tables. You can use subheadings in this section if you like.

In the Discussion you should discuss the significance of your findings. Explain what your Results mean and how they contribute to understanding pernicious anaemia. Mention if you achieved your Aims and if there were any factors that adversely contributed to the experiment. If you had technical problems and you did not obtain good results explain what occurred, give a scientific explanation for the failed result. Suggest how the problem might be remedied. Could you improve the experiment? Provide references to support your argument.

In your Prac write-up, include the answers to the following questions in whichever section you think appropriate eg: Introduction, Materials and Methods, Discussion etc:

18  Questions a. What is the expected size of the protein being detected by the sera from pernicious

anaemia patients? b. Which subunit of the proton pump does this correspond to?

c. Describe another method that could be used to detect and quantitate the levels of anti-

proton pump antibodies in patient sera? d. The secondary antibody that was used in the Western and immuno-histochemistry was

a sheep anti-human Ig-HRP conjugate. What is the antigen that this antibody is specific for? What animal was this antibody raised in? What is the enzyme that is conjugated to the antibody?

Refer to published papers (primary literature) as well as secondary literature in your Prac report

and use Endnote to reference your write up Obtain electronic images of your mouse stomach diagrams from your phone or the Library

scanner Ensure all figures and tables are labelled properly. Can they be interpreted without reading the

text? Use legends where necessary. Submit your report to SLE346 Dropbox. Word limit 2000-2500

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PERNICIOUS ANAEMIA EXAMPLE RUBRIC – (Please check on CloudDeakin for

the final version of the rubric as it will be modified since this manual was published).

Criteria Missing Unsatisfactory Fair Good Excellent

Title, Author and

Introduction to the Prac

0-0.9 points

Missing title, author and/or Introduction

1 -1.9point

Missing either title, author or Introduction

2-2.9points

Title and Introduction missing, but Introduction not complete

3-3.9 points

Title included. Introduction included and

fairly comprehensive, referencing fair

4 points

Title included and

Introduction very well

articulated, referencing

goodAims of the

Prac 0-0.24 points

Missing or incorrect

0.25-0.4 points

Inappropriate, incomplete or

unclear

0.5-0.9points

Stated but not very clear, or

copied verbatim from

the manual

1-1.4 point

Stated and reasonably

clear

1.5 points

Stated and articulated very

well

Materials and

Methods

0-0.4 points

Missing or incorrect

0.5-0.9 points

Incorrect format for the materials and

methods section eg lists.

1-1.9 point

Either insufficient or

too much detail present.

Poor presentation

2-2.9 points

Generally sufficient and

accurate detail present. Good presentation

3 points

Excellent detail present.

Accurate. Exemplary

presentation.

Results text 0-1.9 points

Missing or incorrect

information provided

2-2.9 points

Incomplete description of

results or results contain material that should be in discussion

3-3.9 points

Basic description of results with reference to figures and tables in the

text

4-4.9 points

Good description of results with

reference to all figures and tables in the

text

5 points

Clear and accurate

description of results with

reference to all figures and

tablesFigures

(including diagrams) and Tables

0-1.4 points

Missing, Poorly presented/

mostly incomplete

1.5-2.9 points

Some figures and tables

missing AND/OR legends

missing or incomplete

3-3.9 points

All Figures and Tables including

Figure legends and

titles. Presentation

average.

4-4.9 points

Clear and mostly

consistent presentation of

Figures and Tables and

fairly comprehensive Figure legends

and titles

5 points

Clear, well presented, consistent style for

Figures and Tables and

comprehensive Figure legends

and titles

Discussion 0 -1.4 points

Missing or incorrect. Does

not interpret results.

1.5-2.9 points

Discussion includes partial

or oversimplified

interpretation of Results

3-3.9 points

Discusses the significance of most results

with acceptable detail and complexity

4-4.9 points

Good interpretation and mostly

clear/correct discussion of significance of

Results

5 points

Correct interpretation

and clear discussion of significance of Results to a

high scientific level.

20  

Questions 0-1.4 points

Missing, incorrect or

incomprehensible

1-1.9 point

Less than half the questions

answered correctly.

Insufficient detail.

2-2.9 points

Half the questions answered

correctly with good scientific understanding

3-3.9 points

Most or all of the questions

answered correctly,

concisely and to a high scientific standard

4 points

All of the questions answered correctly, clearly,

concisely and to a high scientific standard

Reference List (any common

style used in Medical

Science is acceptable)

0-0.4 points

Missing in-text citation and

reference list

0.5 -0.9 points

Either in-text citations or

reference list is missing

1-1.9 points

Some references but poor

formatting

2-2.9 points

References complete, with

variable formatting

3 points

References complete with

consistent formatting

Overall Score

Inadequate 0 or more

Unsatisfactory

8 or more

Fair

16.5 or more

Good

23 or more

Excellent

30 or more