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Review Criteria for Assessment of Glycohemoglobin (Glycated or Glycosylated) Hemoglobin In Vitro Diagnostic Devices September 1991

Issued by:
Guidance Issuing Office
Office of Medical Products and Tobacco, Center for Devices and Radiological Health

This guidance was written prior to the February 27, 1997 implementation of FDA’s Good Guidance Practices, GGP’s. It does not create or confer rights for or on any person and does not operate to bind FDA or the public. An alternative approach may be used if such approach satisfies the requirements of the applicable statute, regulations, or both. This guidance will be updated in the next revision to include the standard elements of GGP’s.


This is a flexible document representing the current major concerns and 
suggestions regarding glycohemoglobin in vitro diagnostic devices.  It is 
based on 1) current basic science, 2) clinical experience and 3) previous 
submissions by manufacturers to the FDA, and 4) the Safe Medical Devices 
Act of 1990 (SMDA) and FDA regulations in the Code of Federal Regulations 
(CFR).  As advances are made in science and medicine and changes in 
implementation of Congressional legislation, these review criteria will be 
re-evaluated and revised as necessary.


This document is an adjunct to the 21 CFR Parts 800-1299.  While it is not 
to supersede the CFR, this document provides additional guidance and 
clarification on what information is necessary before the Food and Drug 
Administration (FDA) can clear a device for marketing.  The FDA can make 
more informed decisions based on a uniform data base.  We hope this will 
lead to more reliable, reproducible and standardized commercial tests.


This document discusses all generic type devices intended for use in 
clinical laboratories as in vitro diagnostic tests for the quantitative 
determination of the fraction (expressed as per cent) of glycohemoglobin 
(glycated or glycosylated) hemoglobin (GHb%) in blood samples.1




PANEL:  Hematology (81)

REVIEW REQUIRED:  Premarket Notification (510(k).


1. Clinical Indication/Significance/Intended Use

Glycohemoglobin (GHb) is being used with increasing frequency to monitor 
long-term blood glucose control and compliance in patients with diabetes 
mellitus.  The GHb test provides an index of the mean concentration of 
blood glucose during the preceding two to three months.  It complements 
more traditional measures of glucose control, such as glucose quantitation 
in urine and blood.2 

Diabetes mellitus is a metabolic disorder characterized by impairment of 
carbohydrate metabolism.  Two major types have been identified.  In type I 
insulin dependent diabetes) the pancreatic B cells fail to produce insulin, 
the protein hormone necessary for glucose oxidation to take place within 
cells of the body.  Type II or Non-Insulin-Dependent-Diabetes Mellitus 
(NIDDM) patients are of two subtypes: nonobese and obese. Nonobese Type II 
diabetics have impaired insulin production.  Type II obese NIDDM patients 
manifest ineffective insulin stimulation of target cells which is believed 
to be a post-receptor defect in target tissues. The different types of 
diabetics present with different signs and symp-toms,  but all have 
characteristic fasting hyperglycemia or elevation of glucose at 1 to 2 
hours.  The goal of treatment of diabetes mellitus has been to control 
blood glucose levels to as near normal as possible in the hope that this 
will impede, if not prevent, progression of vascular disease, e.g., stroke, 
blindness, renal failure, heart attack, circulatory insufficiency to lower 
extremities, etc.  This may be accomplished through diet, weight loss, oral 
hypoglycemic drugs or by regular injections of insulin, depending on the 
type of diabetes and individual patient differences.3,4

The term "glycohemoglobin" (GHb) refers to a series of minor hemoglobin 
components that are stable adducts formed by hemoglobin with various 
sugars.  The reaction between glucose and hemoglobin is an example of a 
nonenzymatic condensation of glucose with the free amino groups on the 
globin (protein) component of hemoglobin1.  This process is slow, 
continuous and irreversible.  The human erythrocyte is freely permeable to 
glucose.  Within each erythrocyte, GHb is formed from hemoglobin at a rate 
dependent on the ambient concentration of glucose.5  The higher the 
prevailing ambient levels of blood glucose, the higher will be the levels 
of GHb.  Per cent GHb is increased within the erythrocytes of patients with 
diabetes mellitus.6  

Tests for glycohemoglobin are unreliable for the diagnosis of diabetes 
mellitus.  Such use yields too many false negative and false positive 
results.2  It may be useful, however, to screen for those at greatest risk 
for diabetes complications.  It is not yet clear if patients diagnosed as 
having diabetes based on oral glucose tolerance test results with 
persistent normal per cent glycohemoglobin GHb%) values develop typical 
diabetic complications.  A claim for GHb tests to diagnose diabetes 
mellitus then would require premarket approval.  

False results could suggest incorrect long term blood glucose control. When 
used with the more immediate testing of glucose in blood and urine, 
however, false GHb% test results can be evaluated before disastrous 
measures are taken, because there could be short term low or high glucose 
levels in blood and/or urine with high or low GHb% levels (long term 

Specimen types:

List all specimen types/matrix(ices) claimed by the manufacturer for use in 
the test.  Matrix is defined as the milieu containing the analyte in the 
patient sample submitted for analysis (hereinafter "specimen type" includes 
considerations for matrix or milieu)15.  It can be whole blood, packed 
erythrocytes and/or lysed washed packed red cells, etc.


Discuss the principles of the device methodology and whether it is 
well-established or new and unproven.


FDA requests different types and amounts of data and statistical analyses 
in applications to market in-vitro diagnostic devices.  The amount and type 
of data requested depends on: 1) the test analyte, 2)  the intended use 
(which determines whether the application is a 510(k), an original 
Premarket Approval application (PMA), 3) whether the test is quantitative 
or qualitative, 4) whether the data design is independent or paired, and 5) 
certain claims made by the manufacturer.

The performance of the device can be established by comparison of the 
device to a predicate device (any legally marketed device).  The National 
Committee for Clinical Laboratory Standards (NCCLS) is an example of a 
source for reference methods.  Prove all claims for substantial equivalence 
and specific parameters for using the device.  Include data to support use 
of the test with all claimed specimen types/matrices.

A. Analytical/Laboratory/In Vitro Studies.

1. Statistical 

Describe statistical methods, assumptions used, statistical analyses and 
corresponding computer outputs and references well enough so a 
knowledgeable reader with access to the original data can verify the 
reported results.

Provide data and statistical analyses determined with the device to support 
performance parameters specific to and important for operating the device, 
e.g., reproducibility.

Discuss the statistical methods for the type of data submitted, e.g., 
quantitative continuous data, qualitative discrete data, etc.  The 
distribution of data (normal vs. Non-normal type of data (paired vs. 

Use references for study design and statistical methods that are from
standard texts and/or refereed biomedical journals. 

Test data:

2. Performance Characteristics

Establish the linearity of the test in relation to the hemoglobin
concentration of the sample.  Using varying concentrations of hemoglobin,
document the range of hemoglobin in which accurate GHb% results are

In the package insert state the hemoglobin levels which may cause the test
to become over- or under-loaded and give aberrant results.  Give
instructions in the package insert for the action to be taken when samples
are above or below linearity ranges. 

3. Specificity/Cross-Reactivity/Interference Studies

If the manufacturer makes a claim for restricted assay specificity, e.g.,
it is claimed that an immunologically-based test, electrophoresis or
cation exchange methodology detects HbA1c rather than HbA1 only, the
specificity of that test for the claimed restricted species should be

For Tests Employing Cation Exchange, HPLC and Electrophoresis
Methodologies, present chromatograms from gas-liquid and liquid
chromatography or electrophoregrams from electrophoretic techniques so
that users can see the efficiency of the separation of the claimed
glycohemoglobin peak from other peaks and observe the resolution from
interfering substances in the matrix.  Express migration by Rf (or
retention times for columns).  State temperature conditions.  Describe in
detail the solvents or carriers and their order of use.17  Include similar
types of information for electrophoresis as for chromatography as

Present chromatograms or electrophoregrams of samples containing
Hemoglobin F to determine whether of not there is interference.

Present chromatograms or electerophoregrams of samples from patients with
various hemoglobinopathies (such as HbS, HbG, HbH, Hb Wayne, HbC,
thalassemia, etc., to determine whether or not there is interference. 
Describe the exact source of columns and packing materials, and their
handling.  Give the solvent sources and solvent compositions, and describe
the history of experience with each column.  

Data should be provided to demonstrate that the specimen does not exhibit
test interference if it is hemolyzed, lipemic (affinity chromatography
only) or bilirubinemic at specified levels24.  Any of these conditions
found to interfere with the test should be declared as interfering
conditions in the package insert.

It is already documented that electrophoresis tests are unaffected by
hypertriglyceridemia25 and that tests employing cation exchange methodology
are affected by severe lipemia26 and bilirubinemia27.  HPLC is also 
by bilirubinemia27.  If these limitations are declared and referenced in
the respective package insert, the respective data does not have to be

Labile GHb is an acutely generated, non-enzymatic, reversibly linked
glucose intermediary product present in blood after a heavy meal which can
cause a falsely elevated GHb% test.  
Submit data to support a claim that the presence of labile GHb does not
cause a statistically significant elevation of the true GHb% value. 
Several test protocols are acceptable for this purpose.

Show that normal and diabetic specimens incubated for 3 to 4 hours at 37oC
with at least 55 Mm/L or 1400mg/Dl of glucose28,29 give the same GHb% 
as the original untreated specimen. 

If the test cannot meet this stringent, non-physiological test, it will be
acceptable to show that for 21 samples in the high GHb range there is not
a clinical statistically significant difference between GHb% values
obtained with the new test vs. GHb% values obtained using an accepted
(published) labile removal method analyzed by paired student's t test, and
that the "labile-removed" value is lower than that with no labile removal,
i.e., labile GHb was present in the sample.  For example, take 21 samples
with HbA1c values between 10 and 15%.  Divide each sample into 3 aliquots:
a) aliquot #1 - no treatment, b) aliquot #2 remove labile by the method
being tested, c) aliquot #3 - remove labile by incubation of washed
erythrocytes in saline at 37oC for 5 hours or by another accepted
(published) method.  Run aliquots #1, #2 and #3 in the same assay.  
Aliquot #2 and #3 should give the same results and be lower than #1 (or
equal to #1 if the assay itself is not affected by labile GHb).

If an interference is not examined, then add a statement to the
Limitations Section of the package insert that the device has not been
tested for cross-reactivity or interference of one or more substances.

Analysis of Variance of Reproducibility17,19,20,22,23

The National Committee for Clinical Laboratory Standards (NCCLS)
recommends23 an analysis of variance experiment testing two clinically
significant levels near medical decision limits (subnormal, normal, or
elevated) of an analyte, in this case normal and elevated.  Use controls
simulating patient samples or actual patient specimens 2 times in the same
run and in two different runs each day for 20 days.  This permits separate
estimation of between-day, between-run and within-day standard deviations
(SD), as well as within-run and total SDs.  Acceptable alternatives that
include only one run per day are also discussed in the cited document.23 
The three important assumptions (homogeneity of error variances,
additivity, and normality) of analysis of variance should be used to
demonstrate the validity of the above results.

Calculate total, between- and within-day, and between- and within-run
means and coefficients of variation of imprecision for each set of values.

5. Comparison Studies.

Compare the device to a FDA-cleared device.  In addition the device may be 
compared to a reference method.  GHb A1c by HPLC or an affinity 
chromatography method is recommended for use as a reference method14.  HPLC 
products are commercially available.  HPLC shows excellent assay precision 
and permits rapid separation of HbA1c from the other minor components.3  
The A1c peak suffers less variability from sample storage does the HbA1 
peak.4 Affinity chromatography has far fewer interferences including sample 
degradation problems than do any of the ion-exchange or electrophoresis 
methods.  The reference method chosen should be used for all comparisons 
throughout the study.  

Compare results obtained using packed red cells (if claimed) and/or whole 
blood samples free from interfering substances from 40 to 100 persons 
covering the whole assay range (from normal to clinically relevant high 
levels of GHb%) to results obtained with another test already on the market 
or to a reference method.17,18

Analyze the data using linear regression methods18. (The X axis is the 
independent variable or comparison test.  The Y axis is the dependent 
variable or new test.)19,20  Linear-regression analysis is often most 
useful for estimating the differences or errors between two analytical 
methods, because the errors can be calculated at any medically important 
concentration within the range studied; furthermore, the slope and 
intercept may give some indication of the type of systematic error, which 
may aid in reducing the analytical errors.  Because the reliability of the 
estimates of slope and intercept can be affected by nonlinearity in the 
data set, outliers, a narrow range of data, and variability of the 
comparison method, samples preferably should cover the complete range of 
concentrations that might be encountered.21  The slope, intercept, and 
their estimated standard errors, correlation coefficient, the standard 
error of the estimate, the assay range, and nature and size of samples 
tested should be reported in the Performance Characteristics section of the 
package insert. 

B. Clinical Data/Reference Ranges.

The device results may correlate well using linear regression (slope close 
to 1.0 and intercept close to zero)17,18 with a method that has a published 
reference range for healthy individuals.  If the device results correlate 
well, 40-60 subjects are sufficient to confirm agreement.30,31  

If the device results do not correlate well, establish a reference range 
with samples from 120 to 200 normal persons characterized by age, sex, 
geographic location, any symptoms of disease and any other factors that 
would influence the values obtained, e.g., pregnancy.31

It is suggested that the statistics used to characterize the population and 
the confidence limits used be stated in the package insert.  The 
populations studied should be characterized according to age and disease 
status.  The number of persons tested should be stated.  Investigate all 
sample type(s) claimed in the intended use unless other data proves that 
there is no difference between them.  A range of analyte values for samples 
from specified patient groups, e.g., diabetics, may also be provided. 


Do not make unproven claims for clinical significance in the package

The following are additional details for labeling.

A. The Intended Use Statement [ 809.10(b)(2)]

Whether it is for use in clinical laboratories, doctors' offices, or over-
the-counter (OTC). (The Limitations section should include any specific
training required for test performance or use.)

Whether it is for screening, monitoring, confirmation or exclusion and/or
to aid in the diagnosis as an adjunct to other procedures.

Clinical significance, if it can be stated in a few words.  (If the
clinical significance statement is lengthy or complicated, create a
separate heading entitled "Clinical Significance".)

A typical intended use statements:

"ABC's *** test is a laboratory test intended for the quantitative
determination of percent glycated hemoglobin in whole blood by
[methodology] using the ABC automated system (if applicable) to monitor
long term blood glucose control in individuals with diabetes mellitus."

Conditions for Use

Describe any special applications of the device or specific 
contraindications or indications for use not addressed in the Intended Use 
Statement, e.g., "Despite serious consideration the measurement of 
glycohemoglobin has not been shown to be reliable in the diagnosis of 
diabetes mellitus."2,5  For example, one study shows that even though a 
cutoff of three standard deviations above the mean of a "normal" population 
has a specificity of 99% for diabetes, the sensitivity is only 48%49.  

B. Summary and Explanation of the Test [ 809.10(b)(3)]

It is suggested that this section should discuss the following merits and
limitations of this test:

1. For Affinity Chromatographic Methods Only;
This method detects all glycohemoglobins, not just HbA132,

This method is not affected by the presence of abnormal hemoglobins.

This method is unaffected by carbamlyated hemoglobins in uremic patients. 
In uremic patients there is condensation of urea-derived cyanate with the
N-terminal amino groups on the beta chains of HbA.  This urea bound
hemoglobin elutes with HbA1 on cation-exchange columns producing falsely
elevated results33,34.

2. Labile Glycohemoglobin (All methods, if data so demonstrates.)  

This method is unaffected by the presence of "labile" glycosylated

C. Specimen collection and preparation for analysis [ 809.10(b)(7)]

Data or literature references should be provided in the 510(k) to back all
statements made.

Include a description of: 

The type of specimen to be collected, e.g., whole blood, packed cells,
washed packed cells, and acceptable anticoagulants. 

Acceptable anticoagulants or other additives, preservatives, etc., to
maintain specimen.

Collection Precautions:

Special conditions for patient preparation, e.g., fasting, timing of
collection, intervals of collection, etc.

For routine clinical use, testing every 3 to 4 months is generally
sufficient.  In certain clinical situations such as diabetic pregnancy, or
after a major change in therapy, it may be useful to obtain GHb% values in
2 to 4 week intervals5.

Electrophoresis Methodology

A statement that hypertriglyceridemia does not interfere electrophoresis

For Tests Employing Cation Exchange Methodology, severely lipemic samples
may show elevated results26. If a sample appears lipemic, it is recommended
that washed packed red cells be used as the sample.

For Tests Employing Cation-Exchange or HPLC Test Methodology, it has been
reported that samples with elevated levels of bilirubin may exhibit
falsely elevated levels of glycohemoglobin27.

State in the package insert in range of oC (low to high), collection,
transport, handling and storage conditions to maintain stability of the
specimen.  Do not use terms such as "room temperature" without
qualification.  Provide data or appropriate literature references in the
510(k) to back any claims made.  

Due to instability of the samples for Cation-Exchange and Electrophoresis
methodologies consistent storage conditions and time intervals between
samples should be recommended.  For example, all samples should be
prepared into hemolysates on the same day of collection and tested on the
morning of the fourth day after collection.

A statement that each laboratory must develop and evaluate sample handling
procedures based on their specific situation should be made.  The
laboratory should be responsible for educating clinicians concerning the
large analytical errors that can be introduced by inappropriate sample

[Storage conditions may contribute significantly to both cation-exchange
and electrophoresis assay imprecision6

The temperature variation for some cation exchange minicolumns may be 1%
HbA1 per 1oC46.  This temperature problem must be addressed in any one of
several ways, for example:

However, reports indicate that a temperature vs. GHb concentration
conversion chart47 may not provide accurate results6.  Such charts do not
compensate for temperature fluctuations during assay11.

Use three level calibrators to compensate for temperature variations and
recommendations for methods of strict temperature control.

Various substances other than sugars can form adducts with hemoglobin, 
thereby altering its charge characteristics.  Falsely elevated results can 
occur if these adducts comigrate with GHb.  Examples include individuals 
with opiate addiction44, lead poisoning, uremia, and alcoholism,5 as well 
as those receiving large doses of aspirin (acetylated hemoglobin).45 
Clinically the most important of these interfering adducts occurs in 
uremia.5  This increase correlates with the BUN (blood urea nitrogen) and 
appears to result at least in part from the carbamylation of hemoglobin by 
urea-derived cyanate (carbamylated hemoglobin).33  

D. Quality Control [ 809.10(b)(8)(vi)]

Describe specimens or commercially available products that should be used
for positive and negative control including recommended levels of analyte,
if materials are not provided in the kit.

Recommendations for frequency and placement of quality control samples 
within run and from run to run.  Directions for interpretation of the 
results of quality control samples (satisfactory limits of performance). 
Conclude with a statement similar to the following: "If controls do not 
behave as stated (above), test results are invalid."
E. Limitations of the Procedure [ 809.10(b)(10)]

The following are examples of the types of limitation statements for GHb%
that are suggested for inclusion:

1. Limitation applying to all GHb% test methods

This test is unreliable for the diagnosis of diabetes mellitus2,5.  There
are too many false positive and/or false negative results, depending upon
where the test cutoff is set.  For example, one study shows that even
though a cutoff of three standard deviations above the mean of a "normal"
population has a specificity of 99% for diabetes, the sensitivity is only

This test is not useful in judging day-to-day glucose control, and should
not be used to replace daily home testing of urine and blood glucose5.
As with any other laboratory procedure, a large discrepancy between
clinical impression and test results usually warrants investigation.  Some
of the following test limitations should be considered.

Any cause of shortened red cell survival will reduce exposure of red cells
to glucose with a consequent decrease in %Ghb values, e.g., hemolytic
anemia or other hemolytic diseases, pregnancy, recent significant blood
loss, etc.  Per cent Ghb results are not reliable in any patients with
chronic blood loss and consequent variable erythrocyte 
2. All tests except those with data demonstrating lack of interference by 
labile Ghb. [If the manufacturer claims that the methodology of the test 
removes or is unaffected by labile glycohemoglobin, data should be 
presented in the 510(k) to back the claims.]  Falsely elevated %Ghb test 
results can be caused by "labile Ghb", an acutely generated, reversible, 
non-enzymatically linked glucose intermediary product present after a heavy 
meal.  The higher the prevailing ambient levels of blood glucose, the 
higher will be the potential for falsely elevated results caused by labile 
Ghb.  The package insert should advise users that labile Ghb may be removed 
by any one of the following methods, depending on the test methodology 

1) Incubation of a twenty-fold sample dilution overnight in isotonic
saline at room temperature39.

2) Dialysis overnight at 4oC vs. two changes of volumes of buffer
containing 4.59 g  NaH2PO4.H2O, 1.18 g  Na2HPO4 and 0.65 g KCN per liter 

3) Incubation at 37oC for 5 hours in 0.9% saline41.

4) Incubation for 30 minutes at 38oC in 30 mM semicarbazide and 12 mM
aniline, pH 5.  Note: These chemicals are toxic28.

5) Lysis of erythrocytes for 15 minutes in 50 volumes of 0.05M potassium
biphthalate lysing buffer, Ph 5 at 37oC.  Care must be taken to avoid
denaturation of hemoglobin29,42.

3. For all cation exchange (including HPLC), and electrophoresis

Note that elevated levels of Hemoglobin F (HbF) usually found in infants
and some pregnant women result in falsely elevated results because HbF
comigrates with HbA1c43.

Patients with various hemoglobinopathies (such as HbS, HbG, HbH, Hb Wayne,
HbC, thalassemia, etc.) may have incorrect results depending on charge
characteristics of these variants6.

4. For assays quantifying HbA1 only, note that in most of the following
situations, including uremia, the HbA1a and b fraction is more affected
than the HbA1c fraction. Thus assays that quantify HbA1c specifically show
only slight alterations (rarely greater than 1 GHb%) from these

F.Interpretation of Results/Expected Results [ 809.10(b)(11)]

Actions to be taken if a samples' test result is above or below linearity
ranges according to hemoglobin concentration, e.g., dilution procedures.

Ranges for healthy persons

Ranges for defined disease groups.

Reference ranges from the scientific literature

Alternatively, reference range studies may also be reported from
literature references, especially if the new test was used by the authors,
or if the performance characteristics demonstrate results equivalent to
those obtained with a test used by the author, e.g., regression equation
slope = 1.0, r = approximately 0.95.  Otherwise all expected values should
be determined with the submitted product.

In normal healthy individuals,  GHb comprises approximately 4 to 8% of the
total hemoglobin.  This level may be doubled in the diabetic patient. 
Because glucose binding to hemoglobin occurs slowly and depends on the
circulating level of blood glucose, the GHb% level represents a time-
averaged blood glucose level.  There is a time lag of approximately two to
four weeks before GHb% reflects changes in the blood glucose level.

Known insulin-dependent diabetic patients usually have elevated GHb%
levels48. Percent GHb quantitations on these patients may be in the 9-17%
range depending on the degree of hyperglycemia.  Diabetic patients in good
control may have GHb% values in the normal range.  To date, there is no
specific GHb% value that is accepted as indicating "good" or "poor"
control.  When using GHb% to monitor the diabetic patient, results must be
interpreted individually; that is, the patient should be monitored against
him- or herself and values compared to the normal range for that
particular method.

For Electrophoresis and High Performance Liquid Chromatography Test
Methodology.  Provide Graphics

Provide examples of electrophoregrams from common hemoglobinopathies,
e.g., HbS and C.  Provide a detailed discussion with examples of how to
calculate GHb% in the presence of genetically abnormal hemoglobins. 

G. Performance Characteristics [  809.10(b)(12)]

Provide the statistical results of the comparison study.  Scattergrams are
recommended but optional.

Report the mean, standard deviation and/or per cent coefficient of
variation for the various levels of %GHb in the within-run, within-day,
between-day and total assay precision studies.  Mean values should be
arranged from lowest to highest (or vice-versa) to illustrate recognizable

Summarize studies performed to establish assay linearity depending on
hemoglobin concentration, and state conclusions. 


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HFK-440 NChace/chron  2/24/91 Version 9/27/91

This document was still considered current as of July 1997.
It will be reviewed again in July 1998.

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