Test ID: CDCOM
Celiac Disease Comprehensive Cascade

Evaluating patients suspected of having celiac disease, including patients with compatible symptoms, patients with atypical symptoms, and individuals at increased risk (family history, previous diagnosis with associated disease). It should be noted that HLA typing is not required to establish a diagnosis of celiac disease. Consider ordering CDSP/89199 Celiac Disease Serology Cascade if HLA typing is not desired or has been previously performed.

If IgA is age-specified normal, then tissue transglutaminase (tTG) IgA will be performed at an additional charge.

If tTG IgA is equivocal, then endomysial antibodies IgA and deamidated gliadin antibody IgA will be performed at an additional charge.

If IgA is > or =1.0 mg/dL but lower than age-specified normal, then tTG IgA, tTG IgG, deamidated gliadin IgA, and deamidated gliadin IgG will be performed at an additional charge.

If IgA is below detection (<1.0 mg/dL), then tTG IgG and deamidated gliadin IgG will be performed at an additional charge.

The following algorithms are available in Special Instructions:

-Celiac Disease Comprehensive Cascade

-Celiac Disease Comprehensive Cascade for Patients on a Gluten-Free Diet

-Celiac Disease Diagnostic Testing Algorithm

-Celiac Disease Routine Treatment Monitoring Algorithm

-Celiac Disease Serology Cascade

• Celiac Disease Diagnostic Testing Algorithm
• Celiac Disease Comprehensive Cascade
• Celiac Disease Comprehensive Cascade for Patients on a Gluten-Free Diet
• Celiac Disease Routine Treatment Monitoring Algorithm
• Celiac Disease Serology Cascade

IGA/8157: Nephelometry

CELI2/31928: Polymerase Chain Reaction (PCR)/Sequence-Specific Oligonucleotide Probe (SSO)

TTGA/82587, TTGG/83660, DAGL/89029, DGGL/89030: Enzyme-Linked Immunosorbent Assay (ELISA)

EMA/9360: Indirect Immunofluorescence Assay (IFA)

(PCR is utilized pursuant to a license agreement with Roche Molecular Systems, Inc.)

Blood and serum are required.

Specimen Type: Blood

Container/Tube: Yellow top (ACD [solution B])

Specimen Volume: 6 mL

Collection Instructions: Do not transfer blood to other containers.

Specimen Type: Serum

Container/Tube:

Preferred: Red top

Acceptable: Serum gel

Specimen Volume: 2 mL

Celiac disease (gluten-sensitive enteropathy, celiac sprue) results from an immune-mediated inflammatory process following ingestion of wheat, rye, or barley proteins that occurs in genetically susceptible individuals.(1) The inflammation in celiac disease occurs primarily in the mucosa of the small intestine, which leads to villous atrophy.(1) Common clinical manifestations related to gastrointestinal inflammation include abdominal pain, malabsorption, diarrhea, and constipation.(2) Clinical symptoms of celiac disease are not restricted to the gastrointestinal tract. Other common manifestations of celiac disease include failure to grow (delayed puberty and short stature), iron deficiency, recurrent fetal loss, osteoporosis, chronic fatigue, recurrent aphthous stomatitis (canker sores), dental enamel hypoplasia, and dermatitis herpetiformis.(3) Patients with celiac disease may also present with neuropsychiatric manifestations including ataxia and peripheral neuropathy, and are at increased risk for development of non-Hodgkin lymphoma.(1,2) The disease is also associated with other clinical disorders including thyroiditis, type I diabetes mellitus, Down syndrome, and IgA deficiency.(1,3)

Celiac disease tends to occur in families; individuals with family members who have celiac disease are at increased risk of developing the disease. Genetic susceptibility is related to specific HLA markers. More than 97% of individuals with celiac disease in the United States have DQ2 and/or DQ8 HLA markers compared to approximately 40% of the general population.(3)

A definitive diagnosis of celiac disease requires a jejunal biopsy demonstrating villous atrophy.(1-3) Given the invasive nature and cost of the biopsy, serologic and genetic laboratory tests may be used to identify individuals with a high probability of having celiac disease. Subsequently, those individuals with positive laboratory results should be referred for small intestinal biopsy, thereby decreasing the number of unnecessary invasive procedures (see Celiac Disease Comprehensive Cascade testing algorithm in Special Instructions). An individual suspected of having celiac disease may be HLA typed to determine if the individual has the susceptibility alleles DQ2 and/or DQ8.(4) In terms of serology, celiac disease is associated with a variety of autoantibodies, including endomysial (EMA), tissue transglutaminase (tTG), and deamidated gliadin antibodies.(4) Although the IgA isotype of these antibodies usually predominates in celiac disease, individuals may also produce IgG isotypes, particularly if the individual is IgA deficient.(2) The most sensitive and specific serologic tests are tTG and deamidated gliadin antibodies.

The treatment for celiac disease is maintenance of a gluten-free diet.(1-3) In most patients who adhere to this diet, levels of associated autoantibodies decline and villous atrophy improves (see Celiac Disease Routine Treatment Monitoring Algorithm in Special Instructions).This is typically accompanied by an improvement in clinical symptoms.

For your convenience, we recommend utilizing cascade testing for celiac disease. Cascade testing ensures that testing proceeds in an algorithmic fashion. The following cascades are available; select the appropriate 1 for your specific patient situation. Algorithms for the cascade tests are available in Special Instructions.

-CDCOM/89201 Celiac Disease Comprehensive Cascade: complete testing including HLA DQ typing and serology

-CDSP/89199 Celiac Disease Serology Cascade: complete serology testing excluding HLA DQ

-CDGF/89200 Celiac Disease Comprehensive Cascade for Patients on a Gluten-Free Diet: for patients already adhering to a gluten-free diet.

To order individual tests, see Celiac Disease Diagnostic Testing Algorithm in Special Instructions.

IMMUNOGLOBULIN A (IgA)

0-<5 months: 7-37 mg/dL

5-<9 months: 16-50 mg/dL

9-<15 months: 27-66 mg/dL

15-<24 months: 36-79 mg/dL

2-<4 years: 27-246 mg/dL

4-<7 years: 29-256 mg/dL

7-<10 years: 34-274 mg/dL

10-<13 years: 42-295 mg/dL

13-<16 years: 52-319 mg/dL

16-<18 years: 60-337 mg/dL

> or =18 years: 61-356 mg/dL

HLA-DQ TYPING

Presence of DQ2 or DQ8 alleles associated with celiac disease

Immunoglobulin A (IgA):

Total IgA levels below the age-specific reference range suggest either a selective IgA deficiency or a more generalized immunodeficiency. For individuals with a low IgA level, additional clinical and laboratory evaluation is recommended. Some individuals may have a partial IgA deficiency in which the IgA levels are detectable but fall below the age-adjusted reference range. For these individuals both IgA and IgG isotypes for tTG and deamidated gliadin antibodies are recommended for the evaluation of celiac disease; IgA-tTG, IgG-tTG, IgA-deamidated gliadin, and IgG-deamidated gliadin antibody assays are performed in this cascade. For individuals who have selective IgA deficiency with undetectable levels of IgA, only IgG-tTG and IgG-deamidated gliadin antibody assays are performed.

HLA-DQ Typing:

Approximately 90% to 95% of patients with celiac disease have the HLA DQ2 allele; most of the remaining patients with celiac disease have the HLA DQ8 allele. Individuals who do not carry either of these alleles are unlikely to have celiac disease. However, individuals with these alleles may not, during their lifetime, develop celiac disease. Therefore, the presence of DQ2 or DQ8 does not conclusively establish a diagnosis of celiac disease. Individuals with DQ2 and/or DQ8 alleles, in the context of positive serology and compatible clinical symptoms, should be referred for small intestinal biopsy. HLA typing may be especially helpful for those patients who have begun to follow a gluten-free diet prior to a confirmed diagnosis of celiac disease.(4)

Tissue Transglutaminase (tTG) Antibody, IgA/IgG:

Individuals positive for tTG antibodies of the IgA isotype likely have celiac disease and small intestinal biopsy is recommended. For individuals with selective IgA deficiency, testing for tTG antibodies of the IgG isotype is performed. In these individuals, a positive IgG-tTG antibody result suggests a diagnosis of celiac disease. However, just as with the IgA-tTG antibody, a biopsy should be performed to confirm the diagnosis. Negative tTG IgA and/or IgG antibody serology does not exclude a diagnosis of celiac disease, as antibody levels decrease over time in patients who have been following a gluten-free diet.

Gliadin (Deamidated) Antibody, IgA/IgG:

Positivity for deamidated gliadin antibodies of the IgA isotype is suggestive of celiac disease, and small intestinal biopsy is recommended. For individuals with selective IgA deficiency, testing for deamidated gliadin antibodies of the IgG isotype is performed. In these individuals, a positive IgG-deamidated gliadin antibody result suggests a diagnosis of celiac disease. However, just as with the IgA-deamidated gliadin antibody, a biopsy should be performed to confirm the diagnosis. Negative deamidated gliadin IgA and/or IgG antibody serology does not exclude a diagnosis of celiac disease, as antibody levels decrease over time in patients who have been following a gluten-free diet.

Endomysial (EMA) Antibody, IgA:

Positivity for EMA antibodies of the IgA isotype is suggestive of celiac disease, and small intestinal biopsy is recommended. For individuals with selective IgA deficiency, evaluation of EMA antibodies is not indicated. Negative EMA antibody serology does not exclude a diagnosis of celiac disease, as antibody levels decrease over time in patients who have been following a gluten-free diet.

This cascade should not be solely relied upon to establish a diagnosis of celiac disease. It should be used to identify patients who have an increased probability of having celiac disease and for whom a small intestinal biopsy is recommended.

This cascade should not be used in patients who have previously been or are currently being treated with a gluten-free diet. For these individuals, CDGF/89200 Celiac Disease Comprehensive Cascade for Patients on a Gluten-Free Diet should be ordered.

This cascade should not be used in patients for whom HLA DQ2/DQ8 typing has already been performed. For individuals who are positive for either DQ2 and/or DQ8, CDSP/89199 Celiac Disease Serology Cascade should be ordered to assess the levels of autoantibodies associated with celiac disease. For individuals who are negative for DQ2 and DQ8, no further testing is necessary as a diagnosis of celiac disease is unlikely.

See individual unit codes

1. Green PHR, Cellier C: Medical progress: Celiac disease. N EngL J Med 2007;357:1731-1743 

2. Green PHR, Jabri J: Celiac disease. Ann Rev Med 2006;57:207-221 

3. Harrison MS, Wehbi M, Obideen K: Celiac disease: More common than you think. Cleve Clinic J Med 2007;74:209-215 

4. Update on celiac disease: New standards and new tests. Mayo Communique 2008

Immunoglobulin A (IgA):

Total IgA levels are measured by immunonephelometry. Rabbit antisera that specifically recognizes human IgA is added to the patient sample. Immune complexes form between the human IgA and the rabbit immunoglobulins; the immune complexes scatter light that is passed through the sample. The intensity of the scattered light is related to the amount of human IgA in the sample. The concentration of IgA in the sample is calculated from a multipoint calibration curve.(Behring Nephelometer II Operations Instruction Manual. Dade Behring, Inc. Newark, DE 19714)

HLA-DQ Typing:

LABType applies Luminex technology to the reverse SSO DNA typing method. First, target DNA is PCR-amplified using a group-specific primer. The PCR product is biotinylated, which allows it to be detected using R-Phycoerythrin-conjugated streptavidin. The PCR product is denatured and allowed to rehybridize to complementary DNA probes conjugated to fluorescently coded microspheres. A flow analyzer, identifies the fluorescent intensity of phycoerythrin on each microsphere. The HLA Class II allele or allele groups of the sample is determined by the positive and negative bead ID's using a computer software program. The assignment of the HLA typing is based on the reaction pattern compared to patterns associated with published HLA gene sequences.(Package insert: One Lambda, LABType SSO Typing)

Tissue Transglutaminase (tTG) Antibody, IgA/IgG:

IgA and IgG antibodies to tTG are detected by enzyme-linked immunosorbent assay (ELISA). Recombinant human tTG antigen expressed in Escherichia coli is adsorbed to wells of a microtiter plate under conditions that preserve the native state of the antigen. Diluted patient sera are added to the microtiter plate wells under conditions that allow binding of the antibodies to the tTG antigen. Unbound sample constituents are washed away and horseradish peroxidase (HRP)-labeled antihuman IgA or IgG antibody conjugate is added to each well. After a second incubation, unbound HRP-label is removed and bound conjugate is detected by adding tetramethylbenzidine (TMB) chromogenic substrate. After a final incubation, colored product is measured spectrophotometrically; the absorbance of the patient sample is compared to the positive calibrator. The absorbance is directly proportional to the level of IgA or IgG antibodies to tTG, which is expressed in arbitrary units.(QUANTA Lite R h-tTG IgA and IgG. Inova Diagnostics, Inc. San Diego, CA, 92131)

Gliadin (Deamidated) Antibody, IgA/IgG:

IgA and IgG antibodies to deamidated gliadin peptides are detected by enzyme-linked immunosorbent assay (ELISA). Purified peptides are adsorbed to wells of a microtiter plate under conditions that preserve the native state of the antigen. Diluted patient sera are added to the microtiter plate wells under conditions that allow binding of the antibodies to the deamidated gliadin peptides. Unbound sample constituents are washed away and HRP-labeled antihuman IgA or IgG antibody conjugate is added to each well. After a second incubation, unbound HRP-label is removed and bound conjugate is detected by adding tetramethylbenzidine (TMB) chromogenic substrate. After a final incubation, colored product is measured spectrophotometrically; the absorbance of the patient sample is compared to the positive calibrator. The absorbance is directly proportional to the level of IgA or IgG antibodies to deamidated gliadin peptides, which is expressed in arbitrary units.(QUANTA Lite Gliadin IgA II and IgG II. INOVA Diagnostics, Inc. San Diego, CA, 92131)

Endomysial (EMA) Antibody, IgA:

IgA endomysial antibodies are detected by indirect immunofluorescence assay (IFA). Frozen sections of rhesus monkey esophagus substrate are overlaid with dilutions of patient sera and incubated. After washing, the slides are overlaid with fluorescein-conjugated IgA antisera and incubated. After a final washing, the slides are analyzed through fluorescence microscopy.(Chorzelski TP, Beutner EH, Sulet J, et al: IgA anti-endomysium antibody: A new immunological marker of dermatitis herpetiformis and coeliac disease. Br J Dermatol 1984;111:395-402)

IgA: Monday through Saturday; 3 p.m.

HLA-DQ Typing: Monday through Friday; 7:30 a.m.-5:00 p.m.

tTG IgA: Monday through Saturday;  p.m.

Endomysial antibodies: Monday through Friday; 7 a.m.-5 p.m.

Gliadin IgA: Monday through Saturday; 3 p.m.

tTG IgG: Monday through Saturday;  p.m.

Gliadin IgG: Monday through Saturday; 3 p.m.

82784-IgA

86816-HLA-DQ typing

83516-Deamidated gliadin IgA (if appropriate)

83516-Deamidated gliadin IgG (if appropriate)

83516-tTG IgA (if appropriate)

83516-tTG IgG (if appropriate)

86256-Endomysial antibodies (if appropriate)