MED3008 Clinical Chemistry and Immunochemistry -Protein Electrophoresis Lab

Report
Poon Wing Lam 21002405

Introduction:
Serum contains loads of individual proteins with their specific functions, anomalies in the
distribution and concentration of the serum proteins can imply different physiological and
pathological conditions. Serum protein electrophoresis is a screening technique used in
laboratories to separate and quantify serum protein into major fractions/ bands including
albumin, alpha 1, alpha 2, beta and gamma globulins based on their charge to mass ratio. The
objective of this report is to make presumptive diagnosis for patients through utilizing their
serum protein electrophoresis results.

Material and Methods:

25 mL of diluted buffer was poured into the inner section of the gel chamber. Then, control 1
(normal), control 2 (abnormal), and patient sample 1, 2, 3 were diluted 1 in 4 using Tris
Barbital Buffer. After that, 3.0uL of the 5 samples were applied to each slit of the agarose gel
and waited for 4 minutes. Electrophoresis of the sample gel were carried out at 120 volts for
20 minutes. The gel was then fixed by methanol and stained by the staining set. At last, the
gel was scanned and evaluated using the Bio-Rad ChemiDoc Imaging System.

Result:
Control 1: lane 6 Range % Observation
Total Protein= 57 g/L / /
Albumin=59.5%× 57≈ 33.9 g/L 48.3-61.1 /
Alpha1=2.7%× 57≈ 1.5 g/L 1.9-5.4 /
Alpha2=8.1% × 57≈ 4.6 g/L 6.9-15.7 /
Beta=11.6% × 57≈ 6.6 g/L 12.6-19.0 Low
Gamma=18.1%× 57≈ 10.3 g/L 10.8-17.0 High
A/G ratio
=33.915/(57-33.915)
≈ 1.47
Control 2: lane 7 Reference range Observation
Total Protein= 72 g/L / /
Albumin=49.9%× 72≈ 35.9 g/L 45.5-54.1 /
Alpha1=2.1%× 72≈ 1.5 g/L 2.1-4.9 /
Alpha2=6.7%× 72≈ 4.8 g/L 9.5-14.3 Low
Beta=8.3% × 72≈ 6.0 g/L 9.1-12.7 Low
Gamma=33.1%× 72≈ 23.8 g/L 20.1-27.7 High
A/G ratio
=35.928/(72-35.928)
≈ 0.996
Case 1: lane 8 Reference range Observation
Total Protein= 118 g/L 60-82 g/L High
Albumin=39.2%× 118≈ 46.3 53-65% Low
g/L
Alpha1=0.4%× 118≈ 0.5 g/L 2.5-5% Low
Alpha2=3.8% × 118≈ 4.5 g/L 7-13% Low
Beta=7.7% × 118≈ 9.1 g/L 8-14% Low
 Gamma=48.8%× 118≈ 57.6 g/L 12-22% High
A/G ratio
=46.256/(118-46.256)
≈ 0.645

Case 2: lane 9 Reference range Observation

Total Protein= 98g/L 60-82 g/L High
Albumin=52.9%× 98≈ 51.8 g/L 53-65% Slightly lower
Alpha1=3.7%× 98≈ 3.6 g/L 2.5-5% /
Alpha2=12.1% × 98≈ 11.9 g/L 7-13% /
Beta=6.7% × 98≈ 6.6 g/L 8-14% Low
Gamma=24.5%× 98≈ 24.0 g/L 12-22% High
A/G ratio
=51.842/(98-51.842)
≈ 1.12
Case 3: lane 10 Reference range Observation
Total Protein= 58g/L 60-82 g/L Slightly lower
Albumin=64.1%× 58≈ 37.2 g/L 53-65% /
Alpha1=2.6%× 58≈ 1.5 g/L 2.5-5% /
Alpha2=7.6% × 58≈ 4.4 g/L 7-13% /
Beta=9.6% × 58≈ 5.6 g/L 8-14% /
Gamma=16.1%× 58≈ 9.3 g/L 12-22% /
A/G ratio
=37.178/(58-37.178)
≈ 1.79

Discussion:
For control 1, it is a normal control sample that is known to have normal levels and
distributions of the serum proteins. It can be used to ensure that the serum electrophoresis
used in the experiment can correctly separate all 5 protein fractions into identifiable bands
with all 5 concentrations falling within the expected range %. This helps ensuring the
reliability of the result which the test can accurately identify normal patient samples’ serum
proteins patterns. From the test result located at lane 6, all 5 bandings, albumin, alpha 1,
alpha 2, beta and gamma globulins were clearly seen and separated with normal patterns in
the gel photo. The Albumin to Globulin ratio (A/G ratio) measures if the body is producing
too much or too little of either protein. The A/G ratio of control 1 is 1.47 which lies within
the normal range 1.1-2.5. When it comes to the percentages of each protein fractions, the 3
fractions, albumin (59.5%), alpha 1 (2.7%) and alpha 2 (8.1%) all lies within the control’s
range percentage. Yet, the percentage for the beta fraction is 11.6% which is lower than the
given range percentage 12.6-19.0%, while the percentage for the gamma fraction is 18.1%
which is slightly higher than the given range 10.8-17.0%. It might be due to technical errors
during the electrophoresis process or poor staining skills.

For control 2, it is an abnormal control sample that is known to have abnormal levels and
distribution of serum proteins. It can be used to ensure that the serum electrophoresis used in
the experiment can correctly separate protein fractions into identifiable bands with abnormal
protein patterns like the presence of M-bands or missing bands. This helps ensuring the
reliability of the result which the test can accurately identify patient samples’ abnormal serum
proteins patterns. From the test result located at lane 7, all 5 bandings, albumin, alpha 1,
alpha 2, beta and gamma globulins were clearly observed and separated in the gel photo with
a distinct M-band shown at the gamma region showing the abnormal serum protein patterns.
The A/G ratio of control 2 is 0.996 which is lower than the reference this might indicate an
overproduction of globulin or under production of albumin. The percentages of the 2
fractions albumin (49.9%) and alpha 1 (2.1%) lies within the control’s range percentage.
While alpha 2 (6.7%) and beta fractions (8.3%) are lower than the range percentage 9.5-
14.3% and 9.1-12.7% respectively while gamma fractions (33.1%) is higher than the range
percentage 20.1-27.7%. Similar as control 1, it might be due to technical errors in poor
staining skills. Also, it might be due to the degradation of the control sample when it was
poorly handled and stored.

For case 1, all 5 fractions can be seen clearly in lane 8 matches the bands in the normal
control located in lane 6. Yet, an obvious M-band distinct from lane 6 was observed at the
gamma region in lane 8. From the lane 8’s lane and band analysis in the image report, a sharp
M-spike can also be seen in the gamma region. The presence of M-spike often indicates an
abnormal proliferation of a single type of plasma cell known as monoclonal protein. The total
protein of the sample in case 1 is 118g/L which is much higher than the reference range 60-
82 g/L indicating the presence of hyperproteinemia, an increase in total protein could be due
to conditions like dehydration, chronic inflammation and multiple myeloma. Besides, through
investigating the band percentages of case 1, all 4 fractions, including albumin (39.2%), alpha
1 (0.4%), alpha 2 (3.8%) and beta (7.7%), their band percentages are lower than the reference
range. As for the gamma fraction, its band percentage is 48.8% which is significantly higher
than the reference range 12-22% which is often seen in chronic inflammation, multiple
myeloma and autoimmune disorder. The A/G ratio of sample 1 is 0.645 which is less than the
reference range 1.1-2.5, with the low albumin showed in the band percentage, it probably
indicates overproduction of globulins usually occurs in multiple myeloma or autoimmune
disease. With the above results, the presumptive diagnosis would be multiple myeloma,
serum immunofixation electrophoresis would be suggested as further test to qualify the
monoclonal protein. It can be used to identify immunoglobulin type of the M-band in serum
protein electrophoresis, which could be both light and heavy chain or just either one of them.

For case 2, all 5 fractions can be seen clearly in lane 9, matches the bands in the normal
control located in lane 6. However, an extra M-band was observed at the gamma region in
lane 9. From the lane 9’s lane and band analysis in the image report, a M-spike can also be
seen in the gamma region indicating the presence of M-protein. The total protein of the
sample in case 2 is 98g/L which is higher than the reference range 60-82 g/L indicating the
presence of hyperproteinemia, an increase in total protein could be due to conditions like
dehydration, chronic inflammation and multiple myeloma. For the band percentages in case
2, the 2 alpha fractions including alpha 1 (3.7%) and alpha 2 (12.1%) lies within the reference
range. The band percentage of albumin (52.9%) is slightly lower than the reference range 53-
65%, while beta (6.7%) is also lower than the reference range 8-14%. For the gamma
fraction, its band percentage is 24.5% which is higher than the reference range 12-22% which
is often seen in chronic inflammation, multiple myeloma and autoimmune disorder. From the
fraction distribution percentages, the elevated total protein level is probably due to the
increased percentage of the gamma globulin. The A/G ratio of sample 2 is 1.12 which is
normal and lies within the reference range 1.1-2.5. With the above results, the presumptive
diagnosis would be multiple myeloma though the A/G ratio appeared to be normal. Possible
reasons might be because the relative increase in the gamma region is just slightly higher than
the reference range which might not be large enough to alter the A/G ratio significantly, so
the ratio lies within the normal range. Besides, the increase in globulin portion by the gamma
globulin might be compensated by the decrease in beta globulins so the A/G ratio remains
normal. For further test, serum immunofixation electrophoresis would be suggested to qualify
the monoclonal protein. It can be used to identify immunoglobulin type of the M-band in
serum protein electrophoresis, which could be both light and heavy chains or just either one
of them.

For case 3, all 5 fractions can be seen clearly in lane 10 with all the bandings matched with
the bands in the normal control lane 6 with no extra banding nor M-band found. The total
protein of the sample in case 3 is 58g/L which is just slightly lower than the reference range
60-82 g/L, this could be due to malnutrition. For the band percentages in case 3, all 5 bands
albumin (64.1%), alpha 1 (2.6%), alpha 2 (7.6%), beta (9.6%) and gamma (16.1%) fall within
the reference range showing the patient’s serum protein distribution is normal. For the A/G
ratio, the A/G ratio of sample 3 is 1.79 which lies within the normal range 1.1-2.5. With the
above results, the presumptive diagnosis for case 3 would be healthy with slight protein
malnutrition suspected. Nutritional assessment may be a further test to assess the nutrition
status of the patient for further investigation.

For the errors encountered in the experiment, some scratches were accidentally made on the
gel and were observed after staining, so the gel should be handled more carefully next time to
avoid scratches that may affect the result of the band % during the scanning process.

Conclusion:
Presumptive diagnosis are successfully made through utilizing the serum protein
electrophoresis result. With case 1 and case 2 patients were both suspected with multiple
myeloma while case 3 patient was suspected to have mild protein malnutrition.