(P) Hemoglobin A1C Electrophoresis Flashcards
1
Q
- is used to monitor long-term glycemic control in individuals with diabetes, guide therapy, predict the risk of microvascular complications, and, more recently, diagnose diabetes
A
Hemoglobin A1c (HbA1c)
2
Q
An automated liquid-flow-capillary-electrophoresis method was recently developed to measure HbA1c using?
A
Capillarys 2 Flex Piercing instrument
3
Q
- SEBIA HbA1cElectrophoresis
- HbA1c Electrophoresis Test using Capillary Electrophoresis Technology
- Capillary Electrophoresis: The Next Generation of Separation Method.
A
Capillary Electrophoresis Technology
4
Q
Next Generation of Separation Method
A
Capillary Electrophoresis
5
Q
Familiarize the test available in electrophoresis
A
- Serum Protein Electrophoresis (SPE)
- Urine Protein Electrophoresis (UPE)
- Immunotyping Test (IT
- Carbohydrate Deficient Transferrin (CDT)
- Hemoglobin Electrophoresis (Hgb E)
- Hb A1c Electrophoresis (Hb A1c Elec. Test)
- CSF (cerebro spinal fluid)
- von Willebrand multimers test
6
Q
TEST AVAILABLE in ELECTROPHORESIS
- or gammopathies & pathological condition screening & confirmation (multiple myeloma, MGUS, Liver Cirrhosis, Nephrotic Syndrome, Renal Failure)
A
Serum Protein Electrophoresis (SPE)
7
Q
TEST AVAILABLE in ELECTROPHORESIS
- A complimentary test of SPE, for confirmation & screening as well.
- Check Bence Jones Protein for possible kidney damage (tubular, glomerular, or mixed)
A
Urine Protein Electrophoresis (UPE)
8
Q
TEST AVAILABLE in ELECTROPHORESIS
- In testing to recognized specifically the light and heavy chain (Immunogloculine G, A, M/ Kappa or Lambda) and to know particular treatment of the patient
A
Immunotyping test (It)
9
Q
TEST AVAILABLE in ELECTROPHORESIS
- fOR Chronic alcohol abuse
A
Carbohydrate Deficient Transferrin (CDT)
10
Q
TEST AVAILABLE in ELECTROPHORESIS
- Hemoglobinopathies (Sickle Cell, Hgb Variants) & Thalassemia’s (Alpha or Beta).
- FOR NEWBORN SCREENING
- FOR ADULT
A
Hemoglobin Electrophoresis (Hgb E)
11
Q
TEST AVAILABLE in ELECTROPHORESIS
- for precise & specific result of Hb A1c in both %, mml/mol & EAG unit can & detect Hemoglobin Variants & Thalassemia.
A
Hb A1c Electrophoresis (Hb A1c Elec. Test)
12
Q
TEST AVAILABLE in ELECTROPHORESIS
- Test application
A
CSF (cerebro spinal fluid)
13
Q
TEST AVAILABLE in ELECTROPHORESIS
- or confirmatory & specific treatment by subtyping through factor multimers.
A
von Willebrand multimers test
14
Q
- is one of the top-of-the-line tests for precise and specific results for A1c in both percentage and mm/mol.
- It could also detect the Hgb variant of thalassemia as screening while running HbA1c
A
HbA1C Elec Test
15
Q
T or F
Other tests, like CSF and vWF multimers, are done on gel electrophoresis.
A
T
16
Q
If you see this card, pls read the part about diabetes key fact
A
go na
17
Q
T or F
There is 2-card criteria used for diabetes testing
A
F (3-card criteria)
18
Q
A three-card criteria is used for diabetes testing, each way usually needs to be repeated on wwhat day to diagnose diabetes?
A
repeated on a second day
19
Q
What are the three-card criteria used for diabetes testing?
A
- A1C
- Fasting Plasma Glucose (FPG)
- Oral Glucose Tolerance Test (OGTT)
20
Q
DIABETES TESTING - DIAGNOSIS CRITERIA
HBA1C percentage which indicates diabetes
A
greater than or equal to 6.5%
21
Q
DIABETES TESTING - DIAGNOSIS CRITERIA
Normal percentage result for HBA1C
A
< 5.7%
22
Q
DIABETES TESTING - DIAGNOSIS CRITERIA
HBA1C percentage which indicates prediabetes
A
5.7 to < 6.5%
23
Q
DIABETES TESTING - DIAGNOSIS CRITERIA
Fasting Plasma Glucose (FPG) level whcih indicates diabetes
A
greater than 126 mg/dL
24
Q
DIABETES TESTING - DIAGNOSIS CRITERIA
Normal percentage result for FPG
A
< 100 mg/dL
25
# DIABETES TESTING - DIAGNOSIS CRITERIA
FPG level which indicates prediabetes?
100 to < 126 mg/dL
26
# DIABETES TESTING - DIAGNOSIS CRITERIA
OGTT level which indicates diabetes?
greater than 200 mg/dl
27
# DIABETES TESTING - DIAGNOSIS CRITERIA
Normal OGTT level?
< 140 mg/dL
28
# DIABETES TESTING - DIAGNOSIS CRITERIA
OGTT level which indicates prediabetes?
140 to < 200 mg/dL
29
# DIABETES TESTING - DIAGNOSIS CRITERIA
Which criteria is shown based on value of normal, prediabetes, and diabetic
Normal: < 140 mg/dL
Prediabetic: 140 to < 200 mg/dL
Diabetic: > 200 mg/dL
Oral Glucose Tolerance Test (OGTT)
30
# DIABETES TESTING - DIAGNOSIS CRITERIA
Which criteria is shown based on value of normal, prediabetes, and diabetic
Normal result: < 5.7%
Prediabetes: 5.7 to < 6.5%
Diabetic: >6.5%
A1c
31
# DIABETES TESTING - DIAGNOSIS CRITERIA
Which criteria is shown based on value of normal, prediabetes, and diabetic
Normal: < 100 mg/dL
Prediabetes: 100 to < 126 mg/dL
Diabetic: > 126 mg/dL
Fasting Plasma Glucose (FPG)
32
Currently, diabetes is being diagnosed and followed up through what specific criteria?
through A1C
| it’s been used for monitoring diabetes
33
T or F
In the last decade, A1C has only been used for monitoring diabetes
F (has also been used for monitoring and **diagnostic test**)
34
What are the two important bodies used to standardized A1C tests?
* National Glycohemoglobin Standardization Program (NGSP)
* International Federaiton of Clinical Chemistry (IFCC)
35
# BODIES FOR STANDARDIZING A1C TESTS
* Started standardization in 1996
National Glycohemoglobin Standardization Program (NGSP)
36
# BODIES FOR STANDARDIZING A1C TESTS
* Developed the gold standard (reference system) for HbA1c (IFCC-RM) in 2004
International Federaiton of Clincal Chemistry (IFCC)
37
# BODIES FOR STANDARDIZING A1C TESTS
Master's equation?
NGSP = 0.09148x x IFCC) + 2.15
38
# MEASURING A1C
Formula setup by IFCC for measuring Hb A1C
% Hb A1c = Hb A1c / (Hb A1c + Hb A0)
39
# MEASURING A1C
* This assay should be performed using a certified method (NGSP, DCCT, IFCC).
HbA1c assay
40
T or F
Factors influencing blood glucose levels, such as hemoglobinopathies, should be ignored when interpreting the Hb A1c value
F (Factors influencing blood glucose levels, such as **hemoglobinopathies, should be considered** when interpreting the Hb A1c value)
41
2 current methods for A1C testing?
* Separation method
* Chemical method
42
# 2 METHODS FOR A1C TESTING
* deals with the differences in charges of the molecule of Hemoglobin or the protein
* utilized in HPLC Ion Exchange, Capillary Electrophoresis, and Affinity Chromatography for point-of-care testing
Separation method
43
# 2 METHODS FOR A1C TESTING
* utilized in Immunoassays (IA) and enzymatic assays
* IA is for the chemistry machines.
* Enzymatic Assays or the ELISA or the machine that is using antibody-antigen technology.
Chemical method
44
# 2 METHODS FOR A1C TESTING
* chemical method used for the chemistry machines
| 2 concepts under chemical method
Immunoassay
45
# 2 METHODS FOR A1C TESTING
* chemical method using antibody-antigen technology
| 2 concepts under chemical method
Enzymatic Assays or the ELISA
| ELIZAAAAA
## Footnote
I put myself back in the narrative
46
Familiarize some of the concepts under separation method
* HPLC Ion Exchange
* Capillary Electrophoresis
* Affinity Chromatography
| for point-of-care testing
47
* Gold standard for HbA1c measurement
* Basis for being the gold standard is DCCT study
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
(HPLC)
48
# HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
* A study which is the basis why HPLC is the gold standard
* The first clinical study, which lasted from 1983 to 1993, demonstrated the central role of A1c in the management of diabetic patients.
* The machine used during this trial was an HPLC method.
DCCT Study: A study in the US from Diabetes Control and Complications Trial (DCCT)
49
# HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY
* was used as the reference anchor for the DCCT trial and was adopted by the NGSP in the US.
* It is a floor-type machine
Bio-Rad Biorex 70 HPLC
50
* Which important body realized that Biorex 70 HPLC was NOT specific, as the **HbA1c fraction was co-eluting with other substances that had the same elution** behavior as HbA1c but were not HbA1c by definition.
International Federation of Clinical Chemistry (IFCC)
51
Why did IFCC realize that Biorex 70 HPLC
was NOT specific?
| HbA1c fraction was?
HbA1c fraction was **co-eluting** with other substances hat had the same elution behavior as HbA1c but were not HbA1c by definition
## Footnote
other hemoglobin variants or interferences could affect the A1c itself during HbA1c runnin
52
T or F
Biorex 70 HPLC can cause overestimatiob (false elevated) or underestimate (false decreased) of A1c result
T
53
# COMPARISON OF NATIONAL REFERENCE METHOD
Match the following reference method
1. NGSP
2.: Mono-S
3. JDS/JDCC
a. US
b. SWEDEN
C. JAPAN
D. KOREA
## Footnote
In different countries, they have their standardization
1. US: NGSP
2. Sweden: Mono-S
3. Japan: JDS/JDCC
54
Comparison of each of the National Reference Method was
a. Specific
b. Arbitrarily chosen
c. DIfferent numbers
d. Obsolete
e. all of the above
f. a, b, c
g. c, d
h. b, c, d
h. b, c, d
| a is wrong since national refrence methods are not specific
55
T or F
There is a need to achieve a more uniform standardization of HbA1c Measurement.
T
56
In what year did the IFCC develop a new reference method to overcome HPLC's lack of specificity and standardized HbA1c testing worldwide
2002
57
Two (2) new international reference methods that specifically measure the concentration of A1c developed by IFCC
* LC/MS: Liquid Chromatography Mass Spectrometry
* LC/CE: Liquid chromatography capillary electrophoresis
58
Arrange the LC/MS process in chronological order
A. Detected by mass spectrophotometry
B. Digest the Glu-C protein
C. Separated by liquid chromatography
B, C, A
59
# LC/MS process
How much hemoglobin (Hb) is used in the LC/MS process?
| (1) Digest the Glu-C protein
1 mg Hb
60
# LC/MS process
What are the conditions for the Glu-C digestion in the LC/MS process?
pH 4.3, 37°C, 18 hours
61
# LC/MS process
What chromatography column is used in the LC/MS process?
| (2) Separated by liquid chromatography
Zorbax SB-CN, 2.1 x 150 mm
62
# LC/MS process
What are the temperature and flow rate settings for liquid chromatography?
| (2) Separated by liquid chromatography
50oC, 300 ul/min
63
# LC/MS process
What ion is detected in mass spectrometry for hemoglobin analysis?
| (3) Detected by mass spectrometry
[M + 2H]+2
64
Arrange the LC/CE process in chronological order
A. Glu-C is digested
B. Separated via liquid chromatography
C. Collect and concentrate
D. Separate via capillary electrophoresis
A, B, C, D
65
# EFFECTS OF IFCC STANDARDIZATION OF HBA1c MEASUREMENT
T or F
Because of standardization, there was eventually harmonization of result in 2010-present
T (TYL)
66
# EFFECTS OF IFCC STANDARDIZATION OF HBA1c MEASUREMENT
* one of the expert heads of the IFCC, for the American Diabetes Association (ADA), European Association of Standardization (EADF), International Diabetes Federation (IDF) Working Group of the HbA1c Assay recommends
David Sacks
67
* The current IFCC reference method
LC/MC or the LC/CE technology
68
* What is the new solution/breakthrough for HbA1c analysis
* This technology will give a very precise and accurate result without any analytical and biological interference.
* Screening for **hemoglobinopathy and thalassemia** at the same time we run A1c
SEBIA CAPILLARY ELECTROPHORESIS
69
Which three SEBIA machine array are used in the Philippines?
* Minicap
* Capillarys 2 FlexPiercings
* Capillarys 3.
70
# SEBIA MACHINE ARRAY
* Could run 8-10 samples/hour
* 5-6mins per sample
* A very nice platform that can run different electrophoresis tests
MINICAP FLEX PIERCING
71
Identify if CAPILLARYS 2 FLEX PIERCING, CAPILLARYS 3, CAPILLARYS TERA MC, CAPILLARYS 3 TERA MC:
* for 78 samples/day
CAPILLARYS 2 FLEX PIERCING
72
