VOLUME NO.27

Towards Excellent Customer Service • Utas Maju Sdn Bhd Newsletter

THE VALUEOF PROTEINELECTROPHORESIS

OUREDITORIAL

BOARD

Asalamualaikum w.b.t and have a great day!

How’s your 2016 so far? We hope to hear that life is smooth and

productive. We would like to thank everyone for your continuous

support and encouragement especially to the strategic partner and

team of UMSB.

In this issue, we highlight the values and importance of Protein

Electrophoresis. This article is from International Myeloma

Foundation the first organization dedicated solely to myeloma, and

is the largest currently. The IMF provides programs and services

to aid the research, diagnosis, treatment, and management of

myeloma. The IMF ensures that no one brave the myeloma battle

alone.

This year we have another chance to share the continuous issue

of method verification on “How to perform in the biochemistry

laboratory” by En Sairi Satari. On this part, writer will share on total

precision. As for the poster, we share the result from evaluation

study of SPE & IF with Hospital Ampang.

On the closing portion we share our memory of 2015 and beginning

of a challenging 2016 with our strategic partner and our glorious

team of UMSB!

Zamzani SyaifulEditor • Brisk News

ADVISOREn. Adnan Md Noh

En. Ahmad Khusyairi Zubir

CHIEF EDITORZamzani Syaiful

ASSISTANT EDITOR & MARKETINGAtiqah Zulme

CONTRIBUTING EDITORAmen Fadzly Rahmat Ahmad

Nurmurhasuani Murad

MEDIAAhmad Athar Mohd Yusoff

DESIGNERPlum Rose & Thorn (PRT) Advertising

CONTENTS3 The Value of Protein Electrophoresis

5 Method Verification: How to perform in the biochemistry laboratory

6 SPE

8 Event Highlights

FROM THE

EDITOR’SDESK

“Hope won’t be cut from the soul that has not expired” - Turkish proverb

Thank you for a wonderful 2015 & Happy New Year 2016

BRISK NEWS • VOLUME NO. 27 • 3

WHAT IS PROTEIN ELECTROPHORESIS?

Protein Electrophoresis is a laboratory analysis based on separation of protein under electric current. To run this test,

laboratories may use a solid medium (such as agarose gel) a very thin silica tubes, called capillaries, filled with liquid. When

sample containing a mixture of different proteins is applied on a gel or into a capillary, different proteins of the mixture

will be separated according to their electrical charge. When searching for a monoclonal protein, laboratories will analyze

serum and urine by protein electrophoresis, which is the only test that can confirm the monoclonality unequivocally.

SERUM PROTEIN ELECTROPHORESIS

Serum contains a variety of different proteins that will be separated by electrophoresis into five or six fractions (according

to the method used by the laboratory). These fractions (also referred to as “zones” or “regions”) are called Albumin, Alpha

1, Alpha 2, Beta (which can be separated into Beta 1 and Beta 2), and Gamma. Polyclonal (normal) immunoglobulins are

found mostly in the Gamma zone. Hence, they are known as gamma globulins. The diverse, normal immunoglobulins

present in serum differ slightly from each other in their structure and electrical charge. For that reason, when they

are subjected to electrophoresis, they form a large zone, which is spread out and symmetrical, without any visible

deformation.

Monoclonal proteins are produced by one clone of plasma cells,

and thus all the molecules are identical and have the same electrical

charge. That is why on electrophoresis a monoclonal protein will

migrate as a narrow spike (this spike appears most often in the

gamma zone, but sometimes can be present in Beta 2 or Beta 1,

or even the Alpha 2 zone, although the latter is very rare). Serum

electrophoresis can be used to search for a monoclonal protein as

well as to monitor the amount of monoclonal protein.

The kidney acts as a filter, eliminating only a few molecules and

leaving most of the proteins in the bloodstream. Although some

small proteins do pass through the kidney filter, they are later

resorbed and recycled into amino acids. Thus, normally urine

contains only traces of proteins. When monoclonal protein is

present in serum, often the excess of free light chains will be found in the urine as Bence Jones protein (it will look like

a narrow spike, usually in the Gamma or Beta zone). Urine electrophoresis is used to search for Bence Jones protein

and to monitor its concentration. It can also help to assess kidney damage (which is a common complication of multiple

myeloma).

SERUM AND URINE IMMUNOFIXATION Once a narrow spike of protein is detected by protein electrophoresis, the presence of monoclonal protein may be

suspected. It is necessary then to confirm its presence and to determine its type by identifying which types of heavy chains

and light chains are involved in its structure. Knowing the type of M-protein is important in establishing a diagnosis and in

monitoring the patient. To do this, another method of electrophoresis, called immunofixation (or IFE, for ImmunoFixation

Electrophoresis), will be used. Immunofixation methods are more sensitive to the presence of faint monoclonal proteins

and may detect them even if electrophoresis does not show any visible abnormality. But immunofixation does not

determine how much M-protein is present. Therefore, both methods are used together: electrophoresis to detect the

monoclonal protein and to quantify it, and immunofixation to identify its type.

Albumin

gamma

alpha-1 alpha-2 beta

Fig 1. Representation of an abnormal Serum Protein Electrophoresis result, with myeloma cells producing the

M-protein, creating an M-spike in the gamma zone.

THE VALUE OF PROTEIN ELECTROPHORESISArticle by: MISS NAZIRAH AHMAD HASSAN • Application Specialist for Utas Maju Sdn Bhd

4 • BRISK NEWS • VOLUME NO.27

Article by: MISS NAZIRAH AHMAD HASSAN • Application Specialist for Utas Maju Sdn Bhd

WHY IS PROTEIN ELECTROPHORESIS IMPORTANT? Why have booklets about protein electrophoresis? The main reason is that production of a single, monoclonal protein

(M-protein) is a characteristic feature of multiple myeloma. The test used to measure the amount of monoclonal protein

in the blood or urine is protein electrophoresis. This M-protein is manufactured (synthesized) by malignant plasma cells

(or myeloma cells). The amount of protein produced and released into the serum (the liquid part of the blood that is

left after the blood cells are removed), and sometimes into the

urine, reflects the amount of myeloma present in the body at

any given time. This protein, which is released into the serum or

urine, is called a serum or urine tumor marker. Only a very few

cancers have this type of a marker which, in this case, makes

it possible to assess the myeloma burden at the time of initial

diagnosis and track the amount of myeloma throughout the

course of the disease. One can look at response to treatment,

depth of remission, and, if necessary, the patient’s relapse using

exact numbers, which is a unique advantage. For example, we

can determine if a response is: Partial (PR)=50% improvement;

very good partial (VGPR)=90% improvement; or complete

(CR)=no protein detected. We can also identify a ≥25% increase

in protein level, which we call relapse.

MEASUREMENT OF M-PROTEIN WITH ELECTROPHORESIS

If the M-protein, which is characteristic of myeloma, is present in serum, the electrophoresis test for it is called Serum

Protein ElectroPhoresis (SPEP). Likewise, when monoclonal protein is found in urine, the test for it is called Urine Protein

ElectroPhoresis (UPEP).

To measure the amount of the monoclonal or M-protein one needs two pieces of information:

• What is the total amount of protein in the serum or urine?

• What percentage of the total is the M-protein?

The crucial information comes from SPEP and/or UPEP. By calculating the size of the

“spike”, which depicts the amount of monoclonal protein (done by measuring the area

between the top of the spike and the baseline of the graph), one gets the percentage

of the total protein that represents the M-protein. For example:

• Spike is 60% of total protein

• Total protein=12 grams/deciliter (g/dL)

• Spike level=7.2 g/dL (60% of 12)

Both the total protein and percentage of monoclonal protein change over time. With

response to treatment, the spike can drop to 40%, and total protein to 9 g/dL, for

example. This gives a spike level of 3.6 g/dL, which is a 50% drop in M-protein, or a

partial response. This type of serial measurement and assessment is key to all myeloma

disease monitoring. This is why SPEP and UPEP are so important. An additional test,

called immunofixation, is used to determine the type of M-protein. This is important at

the time of baseline diagnosis and at the point of maximum response with treatment.

If there is a complete response (CR), the immunofixation test is completely negative*;

that is, no monoclonal protein can be identified.

Albumin

alpha-1 alpha-2 beta gamma

Fig 2. Representation of a normal Serum Protein Electrophoresis result.

About IMF The International Myeloma Foundation (IMF) was founded in 1990 by Brian and Susie Novis shortly

after Brian’s myeloma diagnosis at the age of 33. It was Brian’s dream that future patients would have easy access

to medical information and emotional support throughout their battle with myeloma. He established the IMF with the

3 goals of treatment, education, and research. He sought to provide a broad spectrum of services for patients, their

families, friends, and health care providers. Although Brian died 4 years after his initial diagnosis, his dream didn’t.

Today, the IMF reaches out to an international membership of more than 195,000. The IMF was the first organization

dedicated solely to myeloma, and today it remains the largest. The IMF provides programs and services to aid in the

research, diagnosis, treatment, and management of myeloma. The IMF ensures that no one must brave the myeloma

battle alone. We care for patients today, while working toward tomorrow’s cure.

BRISK NEWS • VOLUME NO. 27 • 5

METHOD VERIFICATIONHow To Perform In The Biochemistry Laboratory: Part 2

Preliminary Study: Total Precision

Example 1: Albumin, there are two levels of QC

The calculation for this preliminary study is only for level 2 QC. The mean level 2 is 38 g/l, CV within run is 0.5% and Total

CV is 1.1% (from insert kit). In real situation, we must perform precision study for level 1 and level 2. The data triplicate

for five days with the mean and variance as below:

Day Run 1 Run 2 Run 3 Mean Variance

1 37.7 37.5 37.8 37.73 0.0633

2 37.3 37.5 37.7 37.50 0.0400

3 38.4 37.6 38.1 38.03 0.1633

4 38 37.3 38.2 37.83 0.2233

5 38.1 37.8 38 37.97 0.0233

Note: formula for mean and variance

Mean = (∑x)/ n , n = number of observation or number of run

Variance, α2 = ∑(Xi - Mean )2 / (n-1)

Overall mean and variance Overall mean = (37.73 + 37.50+38.03+37.83+37.97)/5 = 37.81 g/l

Overall variance = (0.0633+0.0400+0.1633+0.2233+0.0233)/5 = 0.1027

Total PrecisionThe total precision is calculated from adjustable overall variance and variance between day.

Adjustable overall variance = (N-1)/N x overall variance, N = number of replicate

= 2/3 x 0.1027

= 0.068

Variance between day = 0.044 (the data in the table below)

Mean day 1 Mean day 2 Mean day 3 Mean day 4 Mean day 5 Overall mean Variance

37.73 37.50 38.03 37.83 37.97 37.81 0.044

Note:

Variance = ∑(Xi – Overall mean )2 / (n-1), Xi = Mean day1.......day 5,

n = number of day.

Total Variance = Adjustable overall variance + variance between day

= 0.068 + 0.044

= 0.112

Total SD = √Total variance

= √ 0.112

= 0.3347

Total Precision/Total CV = (Total SD/Overall mean) x 100

= (0.3347/37.81) x 100

= 0.9% Total CV analytical

Total CV analytical < manufacturer claim (1.1%), so we accept manufacturer claim.

Example 2: Creatinine. There are two levels total of QC

The calculation for this preliminary study is only for level 1 QC. The mean for level 1 QC is 117 umol/l, CV within run is

0.5% and Total CV is 1% (from insert kit). In real situation, we must perform precision study for level 1 and level 2.

6 • BRISK NEWS • VOLUME NO.27

Preliminary study: Within RunDay Run 1 Run 2 Run 3 Mean Variance

1 117.5 118.4 118.2 118.03 0.2233

2 120 120.6 120.1 120.23 0.1033

3 116.2 118.1 117.6 117.30 0.9700

4 117.7 118 118.4 118.03 0.1233

5 120 119.2 119.4 119.53 0.1733

2. Overall mean= 118.63 umol/l Overall variance (α12= 0.3187) 3. SD = 0.56454. CV = 0.48%

Preliminary study: Total PrecisionBased on the preliminary study: within run table (the above), the table for daily mean, overal mean and variance is below.

Mean day 1

Mean day 2

Mean day 3

Mean day 4

Mean day 5

Overall mean

Variance o22

118.03 120.23 117.30 118.03 119.53 118.63 1.4669

Variance between day (α22) = 1.4669Total Variance = Adjustable overall variance + variance between day = (2/3x 0.3187) + 1.4669 = 0.2125 +1.4669 = 1.6794Total SD = √Total variance = √ 1.6794 = 1.3Total Precision/total CV = (Total SD/Overall mean) x 100 = 1.3/118.63) x 100 = 1.1% Total CV analytical

Total CV analytical > manufacturer claim (total CV analytical is 1.1% and total CV manufacturer claim is 1%), so we proceed verification of total precision.

Verification Total Precision The calculation of verification total precision, we defined the total standard deviation verification (TSDV) from Total CV manufacturer, overall mean, percentage point (chi-square distribution) and degree of freedom are as the formula below:

TSDV = TCV x OM x √C √ TTCV = Total CV manufacturerOM = Overall Mean data analyticalC = Percentage point (☐2 (0.05/L, T), L = Total level of QCT = Degree of freedom T = {( n-1) x (α12 / n) + (n x α22)} 2 [(n-1)/D] x (α12)2 + { n2 x (α22)2 } ( D-1 )

α12 = Overall Variance within run α22 = Variance between dayD = number of dayn = number of replicate

If total standard deviation analytical (TSD) < Total standard deviation verification (TSDV), we accept total CV manufacture claim. If total standard deviation analytical (TSD) > Total standard deviation verification (TSDV), the total CV manufacture claim is unacceptable and manufacturer must perform the troubleshooting and precision check will be performed again until acceptable to the manufacturer claim.

The data for creatinine is calculation as below:Overall mean (OM) = 118.63 umol/lOverall variance within run (α12) = 0.3187Variance between day (α22) = 1.4669Total SD analytical = 1.3n = 3D = 5L = 2

T = {(3-1)x(0.3187)/3) + (3x1.4669)}2

[(3-1)/5 ]x (0.3187)2 + 32x (1.4669)2

(5-1)T = ( 0.2125 + 4.4)2

0.041+ 4.842T = 21.28 4.883T = 4.3580

C = ☐2 (0.05/L, T)C = ☐2 (0.025, 4.3580)C = 11.14

TSDV = TCV x OM x √C √ TTSDV = (1/100) x 118.63 x√11.14 √4.3580 = 3.8583 2.0876 = 1.848 Total SD verification

Total SD analytical = 1.3 and Total SD verification = 1.848, meaning, Total SD analytical < total SD verification, so we accept total CV manufacturer.

Article by: MR. SAIRI SATARISenior Biochemist for Penang Hospital

CV within run manufacturer claim is 0.5%

Within run manufacturer claim is acceptable

}

8 • BRISK NEWS • VOLUME NO.27

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EVENTS Compilations by:Atiqah Binti ZulmeMarketing Executive for UMSB

International Pathology Day 2015 at IKNFor the fi rst time, on 30th November 2015, National Cancer Institute together with “Persatuan Perubatan Pascasiswazah Hospital Kuala Lumpur” has conducted an International Pathology Day 2015 at national level. The event was launched by the Minister of Health, Y.Bhg. Datuk Seri Dr Subramaniam. The participants of the event were among Head of Pathology Department, Pathologist, Science Offi cer, and JTMP from all over Malaysia. Utas Maju take part as one of the strategic partner which Highlight the Pre-Laboratory Solutions “Robo as a Phlebotomy Automated System”.

The Prophet SAW when digging a trench in the battle of the Trench:“... Constantinople (now Istanbul) will fall into the hands of the Muslim army. Its KING is the best KING; his Army is the best Army ...”(Narrated by Imam Ahmad)Hadith the Prophet was realized almost 800 years later by Sultan Muhammad Al-Fateh, the Caliph of the Ottoman Empire 7th with 150,000 troops. This is the starting of the Great conquest during the era of Islam Empire. Utas Maju get a meaningful chances to explore the land full with historical stories, various personalities, diff erent cultures, food and art in every corner of the place.

Protein Electrophoresis Workshop at Hospital AmpangTuesday, 27 October 2015 - Workshop is mainly focus on the use of protein analysis in clinician aspect and interpretation for the basic analysis such as provide knowledge and skill in diagnosing and monitoring of protein disorders using gel electrophoresis. 30 participants had joined this workshop including Pathologist and science offi cer from Peninsular Malaysia and Brunei.