Category: Technical Brief

Background Information

Trichomonas is a common cause of vaginitis and the most common nonviral sexually transmitted disease worldwide.¹ Studies show that T. vaginalis is an important cause of premature rupture of membranes, premature delivery, pelvic inflammatory disease, urethritis, and chronic prostatitis.

Diagnosis of Trichomonas infection proves challenging in that the traditional wet mount method relies on detecting motility of the parasite, which is often lost during delays in transport or refrigeration. Wet mount preparations are less than optimal when the transport time exceeds six hours.² Optimal transport time is one hour or less. Alternative methods for diagnosis of Trichomonas infection include EIA methods, in-office physician-performed microscopy, culture, and molecular methods. Molecular diagnostic methods have cost-to-patient charges that are significantly higher than other methods. Wet mount microscopy has a reported sensitivity of 58% versus culture.³

The OSOM® Trichomonas Rapid Test is an FDA-approved, CLIA-waived test that offers a simple, inexpensive option with improved sensitivity for diagnosis of Trichomonas infection as compared to wet prep. The test is intended for qualitative detection of Trichomonas vaginalis antigen from vaginal swabs or from the saline solution prepared when making wet mounts from vaginal swabs.

Clinical Indications

The OSOM^® Trichomonas Rapid Test is intended for the qualitative detection of Trichomonas vaginalis antigen from genital swabs. The test is intended for use in patients with symptoms of vaginosis or suspected exposure to the organism.

Interpretation

If Trichomonas is present in the sample, it will form a complex with the primary anti-Trichomonas antibody conjugated to blue-colored particles. The complex will then be bound by a second anti-Trichomonas antibody coated on the nitrocellulose membrane. The appearance of a visible blue test line along with the red control line (internal control) indicates a positive result.

Limitations

The OSOM^® Trichomonas Rapid test has not been approved for urine samples; the test has only been validated for qualitative detection of T. vaginalis antigen from vaginal swabs. Molecular methodology is recommended for urine specimens.

A negative result may be obtained if the specimen is inadequate or if the antigen concentration is below the sensitivity of the test.

Samples contaminated with preparations containing iodine or by the immediate prior use of vaginal lubricants are not recommended.

The test does not differentiate between viable and non-viable organisms, nor does it differentiate between acute infection and carrier states.

Staphylococcus aureus in specimens at concentrations higher than 1 X 108 cfu/ml may interfere with the test results in negative samples. These concentrations are higher than would be expected to be present in normal patient samples.

The OSOM^® Trichomonas Rapid Test is reported to detect as little as 2500 organisms/ml per manufacturer’s package insert.

Methodology

The OSOM^® Trichomonas Rapid Test is an immunochromatographic test that utilizes capillary flow dipstick technology. The test requires solubilization of the Trichomonas proteins from the swab by mixing the sample in the sample buffer. A sample test strip is then added allowing the subsequent migration of the sample along the membrane surface of the dipstick.

References

1. Campbell L, Woods V, et al. Evaluation of the OSOM Trichomonas Rapid Test versus Wet Preparation Examination for Detection of Trichomonas vaginalis Vaginitis in Specimens from Women with a Low Prevalence of Infection. Journal of Clinical Microbiology. 2008; 3467-3469.

2. Huppert JS, Batteiger BE, et al. Use of an Immunochromatographic Assay for rapid Detection of Trichomonas vaginalis in Vaginal Specimens. Journal of Clinical Microbiology. 2005; 684-687.

3. Kingston MA, Bansal D, Carlin EM. Shelf life of Trichomonas vaginalis, International Journal of STD & AIDS. 2003;14:28-29.

4. OSOM® Trichomonas Rapid Test product information and package insert, Sekisui Diagnostics, Framingham, MA, 2011.

Mixing Study, Incubated Activated Partial Thromboplastin Time (APTT)

Technical Brief:

Mixing Study, Incubated Activated Partial Thromboplastin Time (APTT)

Test Name

PTT Incubated Mixing Study (PTTIM)

CPT Codes

85730
85610
85732
85670
85520
85390

Methodology

Clot Detection

Turnaround Time

1 – 3 days

Specimen Requirements

Volume:
4 mL

Minimum Volume:
1.5 mL

Specimen Type:
Plasma

Collection Container:
Light Blue Sodium Citrate Coagulation Tube

Transport Temperature:
Frozen

3.2% sodium citrate is the preferred anticoagulant recommended by CLSI.

Specimen Collection & Handling

The presence of heparin, fondaparinux, dabigatran, or a direct thrombin inhibitor in the specimen may interfere with test results.

Discontinue coumadin therapy for 14 days prior to collection.

Discontinue direct thrombin inhibitors and heparin 2 days prior to collection.

Stability

Ambient:
7 days

Citrated plasma for ADAMSTS13 Antibody remains stable at room temperature and refrigerated for 7 days, but no tests for ADAMSTS13 Activity or Inhibitor can be performed on such specimens.

Reference Range

See Interpretation

PT Screen:
8.4-13.0 seconds

APTT Screen:
< 33.2 seconds

APTT Immediate Mix:
33.2 seconds

APTT Incubated Mix:
< 36.0 seconds

Thrombin Time:
< 18.6 seconds

Heparin Assay:
< 0.1 U/mL

Additional Information

Background

Background Information

The activated partial thromboplastin time (APTT) is one of the most commonly used tests to investigate bleeding patients, monitor anticoagulant therapy, and screen patients before surgery. The APTT measures the integrity of the intrinsic and common pathways of the coagulation cascade. The prothrombin time (PT), another common screening test, measures the integrity of the extrinsic and common coagulation pathway.

The APTT is measured as the number of seconds for the patient’s plasma to form a fibrin clot after the addition of an intrinsic pathway activator, phospholipid, and calcium. A prolonged APTT can be caused by a coagulation factor deficiency or the presence of an inhibitor. The mixing study, incubated APTT, is used to investigate the cause of a prolonged APTT result. The mixing study is performed by measuring the APTT in the patient’s plasma, then mixing an equal volume of the patient’s plasma and normal pooled plasma (NPP), and repeating the APTT tests immediately and after one-hour incubation.

The components of the panel include PT screen, APTT screen, APTT Immediate Mix, and APTT Incubated Mix, as well as a thrombin time and heparin anti-Xa assay if needed.

The principle of the mixing study can be summarized as:

1. If the prolonged APTT screen is due to a factor deficiency, mixing with an equal volume of NPP (which has approximately 100% of all coagulation factors) will replace the patient’s deficient factor. This result in an APTT immediate mix is shortened or corrected into the reference range.

2. If the prolonged APTT screen is due to the presence of an inhibitor, mixing with an equal volume of NPP will not shorten or correct the prolongation of APTT in repeated tests. The reason is the inhibitor in the patient’s plasma is present in excess and binds to coagulation factors or protein/phospholipid complexes in both the patient’s plasma and NPP.

Correction of the APTT in the mixing study suggests a coagulation factor deficiency in either the intrinsic pathway (factors VIII, IX, XI and XII, high-molecular-weight kininogen [HMWK] or prekallikrein [PK]), or in the common pathway (also prolonged PT) such as factor II, V, and X. Deficiency of factors VIII, IX, and XI will present with bleeding; however, deficiency of factor XII or prekallikrein will not increase bleeding risk, but may increase thrombotic risk. Further testing, such as clotting factor assays, is necessary to diagnose a specific factor deficiency. See Figure 1 for the diagnostic algorithm used in the laboratory.

There are three different types of inhibitors:

1. Inhibitors directly against specific factors, such as factor VIII or factor V inhibitors.

2. Anticoagulants such as heparins, fondaparinux, dabigatran, and other direct thrombin inhibitors.

3. Non-specific inhibitors, such as lupus anticoagulants.

Some inhibitors will demonstrate a delayed-type inhibitor pattern, with time and/or temperature dependence. In cases with a delayed-type inhibitor, the APTT Immediate Mix will correct to within the reference range; however, the APTT Incubated Mix will be prolonged.

Although rare, the presence of a factor inhibitor, such as a factor VIII inhibitor, will increase the risk of life-threatening bleeding. The presence of a factor inhibitor can be confirmed by a Bethesda assay for that factor. The presence of heparins, fondaparinux, dabigatran, or other direct thrombin inhibitors can cause prolongation of both the APTT Immediate Mix and APTT Incubated Mix. Careful clinical and medication history, and additional thrombin time with heparin assay (anti-Xa inhibition assay) can exclude the presence of anticoagulants.

The presence of lupus anticoagulants, which are antibodies against protein-phospholipid complexes, will increase the risk of thromboembolism. The presence of low-level, nonspecific inhibitors in the patient’s plasma may demonstrate a prolonged APTT Incubated Mix similar to a delayed-type inhibitor. If the clinical history suggests a lupus anticoagulant, further testing, including phospholipid based screening tests, phospholipid dependency assays, and exclusion of the presence of inhibitors, in addition to mixing study, incubated APTT, is necessary (refer to Figure 1 for lupus anticoagulant).

The adequate performance of the mixing test and accurate interpretation is important because the presence of a specific factor inhibitor, non-specific inhibitors, such as lupus anticoagulant, anticoagulants, or factor deficiency, have different clinical manifestations and require different clinical management.

Clinical Indications

The mixing test, incubated APTT, is indicated when the cause of a prolonged APTT result needs to be investigated.

Interpretation

APTT Screen:
Prolonged

APTT, Immediate Mix:
Normal

APTT, Incubated Mix:
Normal

If the APTT Screen is prolonged with a normal APTT Immediate Mix and APTT Incubated mix, this indicates a factor deficiency in the intrinsic or final common pathway.

If the PT is normal, this suggests an intrinsic pathway deficiency (VIII, IX, XI, XII, PK, HMWK).

If the PT is prolonged, this suggests a common pathway deficiency (fibrinogen, II, V, X).

APTT Screen:
Prolonged

APTT, Immediate Mix:
Normal

APTT, Incubated Mix:
Abnormal

If the APTT Screen is prolonged, with a normal APTT Immediate Mix, but an abnormal APTT Incubated Mix, this indicates the presence of a delayed inhibitor such as specific factor inhibitors, most commonly factor VIII inhibitor, and small numbers of lupus anticoagulant.

APTT Screen:
Prolonged

APTT, Immediate Mix:
Abnormal

APTT, Incubated Mix:
Abnormal

If the APTT Screen is prolonged, with an abnormal APTT Immediate Mix and abnormal APTT Incubated Mix, this favors a non-specific inhibitor, such as a lupus anticoagulant, and anticoagulants such as heparin, fondaparinux, dabigatran, or other direct thrombin inhibitors.

Methodology

The PT Screen is performed using Innovin® (Dade Behring, Inc.) reagent and STAR Evolution® Analyzer (Diagnostica Stago, Inc.). The PT Screen is included to localize abnormalities to common, intrinsic, and extrinsic pathways.

The APTT Screen is performed using the PTT-Automate reagent and STAR Evolution® analyzer (both Diagnostica Stago, Inc).

The mixing studies are performed by mixing the patient’s plasma with an equal volume of the NPP (Cryocheck; Precision Biologic, Inc).

For the APTT Immediate Mix, the APTT is performed immediately after mixing the plasmas. For the APTT Incubated Mix, the APTT is performed after one-hour incubation at 37ºC. The NPP serves as a negative control; two levels of positive control are performed; lupus positive plasma (Precision Biologic, Inc) and weak lupus positive plasma (Precision Biologic, Inc).

The thrombin time (Diagnostica Stago, Inc) will be measured in specimens with prolonged APTT. If the TT is prolonged, a heparin assay (anti-Xa inhibition assay; Rotachrom Heparin kit, Diagnostica Stago, Inc.) by a chromogenic assay will be performed to distinguish a heparin effect from a direct thrombin inhibitor.

Legionella pneumophila by Real-Time PCR

Technical Brief:

Legionella pneumophila by Real-Time Polymerase Chain Reaction (PCR)

Test Name

Legionella pneumophila PCR (LEGPCR)

CPT Codes

87541

Methodology

Polymerase Chain Reaction (PCR)

Turnaround Time

3 days

Specimen Requirements

Specimen Type:
Bronchoalveolar lavage (BAL)

Volume:
3 mL

Minimum Volume:
2 mL

Collection Container:
Sterile specimen container

Transport Temperature:
Ambient

Alternative Specimen

Specimen Type:
Sputum, induced
Aspirate, tracheal

Volume:
1 mL

Collection Container:
Sterile specimen container

Transport Temperature:
Ambient

If aliquoting is necessary, sterile tubes must be used.

Stability

Ambient:
24 hours

Refrigerated:
7 days

Frozen:
30 days

Reference Range

No Legionella pneumophila detected

Additional Information

Background

Background Information

Legionella pneumophila is the most common pathogenic species of the 42-recognized Legionella species and is associated with significant mortality in elderly patients and those with severe underlying disease. Diagnostic delay also may result in increased mortality. Target sequences within the genes that encode the 5S and 16S ribosomal subunits, as well as the MIP (macrophage infectivity potentiator) gene, in conjunction with real-time PCR, are useful for the detection of the Legionella genus and, specifically, the species L. pneumophila.

The MIP gene, which encodes a 24-kDa protein virulence factor that facilitates the entry of legionellae into amoebae and macrophages, has sufficient sequence variability between the Legionella species to allow for the specific detection of L. pneumophila by real-time PCR. This assay has 100% specificity and sensitivity.

Clinical Information

Multiple laboratory methods should be employed to ensure the diagnosis of Legionnaire’s disease (LD), a bacterial pneumonia caused by L. pneumophila (90% of cases) or other Legionella species. Molecular methods are more sensitive than culture for the diagnosis of LD.

The PCR assay performed at Cleveland Clinic will not detect disease caused by Legionella species other than L. pneumophila.

Culture for Legionella species from respiratory sites is a sensitive (~80-90%) method for diagnosing severe, untreated disease, but insensitive (~20%) for the diagnosis of mild disease. Specimens from non-respiratory sites should not be submitted for Legionella culture unless there is a high index of clinical suspicion to support the request. Urine antigen assays for L. pneumophila serogroup 1 will be positive in ~90-95% of patients with severe disease due to the Pontiac monoclonal subtype of serogroup 1, but positive in only 50% of these patients with mild disease. The urine antigen assay is unreliable for the diagnosis of severe LD caused by L. pneumophila other than serogroup 1 or a different Legionella species (detects less than 5-40% of cases).

Interpretation

PCR results are reported qualitatively as positive or negative for Legionella pneumophila.

Limitations

This assay does not contain an internal amplification control; instead, the false-negative rate, which to date has been 0%, is monitored according to the College of American Pathologists’ guidelines.

This assay only detects L. pneumophila, as Legionella species other than L. pneumophila infrequently cause legionellosis. The implementation of this test was intended to replace the less-sensitive direct immunofluorescence assay, and it is intended to be used in conjunction with culture.

Methodology

The LightCycler FastStart DNA Master Hybridization Probe Kit (Roche Diagnostics, Indianapolis, Ind.) is used in conjunction with species-specific Legionella pneumophila primers and probes. The PCR is performed on the LightCycler system (Roche).

Qualitative positive and negative results are determined based on analysis of the amplification curves and post-amplification melt curve.

Hepatitis C Virus Genotyping

Technical Brief

Hepatitis C Virus Genotyping

Test Name

Hepatitis C Genotype (HEPGEN)

CPT Codes

87902

Methodology

Reverse Transcription/Polymerase Chain Reaction (RT/PCR)

Turnaround Time

5 – 7 days

Specimen Requirements

Type:
Plasma

Volume:
3 mL

Minimum Volume:
1 mL

Tube/Container:
White BD Hemogard™ K₂EDTA Plasma Preparation Tube

Transport Temperature:
Refrigerated

Separate plasma from whole blood by centrifugation within 6 hours of collection.

Do not pour-off.

Alternative Specimen

Type:
Plasma

Volume:
3 mL

Minimum Volume:
1 mL

Tube/Container:
Lavender BD Hemogard™ K₂EDTA Tube

Transport Temperature:
Refrigerated

Separate plasma from whole blood by centrifugation within 6 hours of collection. Transfer plasma to a sterile, polypropylene screw-cap tube.

Alternative Specimen

Type:
Serum

Volume:
3 mL

Minimum Volume:
1 mL

Tube/Container:
Gold BD Hemogard™ Serum Separation Tubes (SST)™

Transport Temperature:
Refrigerated

Centrifuge within 6 hours of collection.

Do not pour-off.

Stability:

Plasma

Ambient:
3 days

Refrigerated:
3 days

Frozen:
60 days

Stability:

Whole blood

Ambient:
15-30°C for up to 6 hours prior to centrifugation

Refrigerated:
2-8°C for up to 6 hours prior to centrifugation

Frozen:
Unacceptable

Reference Ranges

Capable of discriminating among HCV subtypes 1a, 1b, 2a-2c, 3a-3c, 4a-4h, 5a, and 6a

Additional Information

Background

Background Information

Chronic infection with the hepatitis C virus (HCV) remains one of the world’s most important clinical and public health problems. It has been estimated that approximately 3% of the world’s population is infected with HCV, which represents nearly 170 million people worldwide.^1,4 In the United States, up to 3.9 million people (1.8% of the population) are currently living with HCV, of which as many as 2.7 million suffer from chronic infection.^2,3 In the Western world, chronic damage from hepatitis C is the primary cause for end-stage liver disease that requires liver transplantation.

Clinical Significance

The 1989 discovery of the hepatitis C virus was a major development. Previously, it was clear that a major cause of acute hepatitis after a blood transfusion was related to neither hepatitis A nor to hepatitis B. This resulted in the early name for this disease: non-A, non-B hepatitis. It is now known that HCV is the cause for most of the non-A, non-B hepatitis cases.

HCV is a single-stranded RNA virus that has approximately 9400 nucleotides that demonstrate significant genetic variation. HCV replicates in the liver and is detectable in serum during acute and chronic infection. The RNA polymerase lacks the proofreading functions of DNA polymerase and introduces random nucleotide errors. This results in a relatively high rate of spontaneous nucleotide substitutions. As a consequence, HCV is a highly-heterogeneous virus with at least six known major genotypes and more than 80 subtypes identified worldwide.⁵ Genotypes are classified based on differences in the amino acid sequence of specific proteins.

Recent understanding of the natural history of chronic hepatitis C has greatly expanded, and more effective therapeutic strategies have been developed. Several studies have shown a strong correlation between HCV genotype and a patient’s response to various treatments. Determining genotypes is necessary because some hepatitis C viruses with certain genetic variations are harder to treat successfully and usually require a different treatment approach; others are much easier to treat and respond well to shorter treatment schedules.

For many years, the standard of care for patients with chronic hepatitis C infection was peginterferon (PegIFN) alfa and ribavirin (RBV) taken for 24 to 48 weeks, depending on the genotype; however, less than 50% of patients responded to this therapy. In May 2011, the FDA approved the use of two new protease inhibitors – Incevik (telaprevir) and Victrelis (boceprevir) – for the treatment of hepatitis genotype 1, the most common form of hepatitis in North America. The drugs, used in conjunction with the standard interferon and ribavirin therapies, represent the first major therapeutic advance in the treatment of hepatitis C in more than a decade. The new protease inhibitors block the replication of an enzyme that is crucial for the hepatitis C virus to reproduce. In patients with hepatitis genotype 1, the use of the new protease inhibitor combined with the traditional antiviral treatment eliminated the virus in 70 to 80% of all cases. Patients with the other types of hepatitis C continue to be treated with peginterferon and ribavirin, which is successful in 80% or more in those with genotypes 2 and 3 infections.^6-8

Clinical Indications

Pre-treatment analysis of hepatitis C genotype is used to determine the duration of therapy and predict therapeutic response.

Interpretation

The predominant HCV genotypes in the United States are 1a, 1b, 2a, 2b, and 3a; the other subtypes (4, 5, 6) are more prominent in other parts of the world.⁹

Limitations

This assay is based on the sequence variability of the 5’UTR, allowing differentiation between HCV genotypes 1 to 6. Subtyping may occasionally be limited.

Methodology

The gold standard for genotyping is determining the nucleotide sequence of an HCV isolate. Currently, this method is not practical for the clinical diagnostic laboratory, and a less labor-intensive technique, such as Line Probe Assay (LiPA), is employed.

The LiPA method uses genotypic-specific oligonucleotide probes immobilized onto membrane strips. The end products obtained from RT-PCR of the 5’UTR region of the clinical isolate are then hybridized onto the membrane containing the immobilized-oligonucleotides. A purple-brown line develops where sequence homology occurs between the biotinylated PCR products and the probe. Hybridization of 5’UTR amplification products with genotype-specific probes is capable of discriminating among HCV subtypes 1a, 1b, 2a-2c, 3a-3c, 4a-4h, 5a, and 6a.¹⁰

References

1. Organization WH. Hepatitis C surveillance and control. Available at: http://www.who.int/=csr/disease/hepatitis/whocdscsrlyo2003/en/index4.html#incidence.

2. Armstrong GL, Wasley A, Simard EP, McQuillan GM, Kuhnert WL, Alter MJ. The prevalence of hepatitis C virus infection in the United States, 1999 through 2002. Ann Intern Med. 2006;144:705-14.

3. Centers for Disease Control. www.cdc.gov/hepatitis/PDFs/disease_burden.pdf.

4. WHO Hepatitis C fact sheet. 2011.

5. Davis GL. Hepatitis C Virus genotypes and quasispecies. Am J Med. 1999;107(6B):21S-25S.

6. Poordad, F, et al. (March 2011). “Boceprevir for Untreated Chronic HCV Genotype 1 Infection”. N Engl J Med. 364(13):1195 206. doi:10.1056/NEJMoa1010494. PMID 21449783.

7. Bacon, B, et al. (March 2011). “Boceprevir for Previously Treated Chronic HCV Genotype 1 Infection”. N Engl J Med. 364(13):1207-17. doi:10.1056/NEJMoa1009482. PMC 3153125. PMID 21449784.

8. McHutchison JG, Manns MP, Muir AJ, et al. (April 2010). “Telaprevir for previously treated chronic HCV infection”. N Engl J Med. 362 (14): 1292–303. doi:10.1056/NEJM oa0908014. PMID 20375406.

9. Mahaney K, Tdeschi, V, Maertens G, et al. Genotypic analysis of hepatitis C virus in American patients. Hepatology; 1994; 20:1405-1411.

10. Stuyver L, Wyseur A, Van Amhem W et al. Second generation line probe assay for hepatitis C virus genotyping. J Clin Microbiol. 1996; 24:2259-2266.

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine, Endocervical, Vaginal, and Urethral Specimens

Technical Brief

Detection of Chlamydia trachomatis and Neisseria gonorrhoeae in Urine, Endocervical, Vaginal, and Urethral Specimens

Test Name

GC/Chlamydia Amplification, Genital, Rectal and Oral Specimens (GCCT)

GC Amplification, Genital, Rectal and Oral Specimens (GC)

Chlamydia Amplification, Genital, Rectal and Oral Specimens (CT)

CPT Codes

GCCT

87491
87591

87591

87491

Methodology

Target amplification nucleic acid probe, qualitative

Turnaround Time

1 – 4 days

Specimen Requirements

Type:
Swab

Source:
Vaginal, urethral, endocervical, rectal, throat

Specimen Container:
Aptima^® Unisex Swab Specimen Collection Kit

Transport Temperature:
Ambient

Alternative Specimen

Type:
Cervical

Specimen Container:
ThinPrep^® Pap Test

Transport Temperature:
Ambient

Cytyc PreservCyt Solution (ThinPrep) is not recommended unless performed in conjunction with a ThinPrep^® PAP test.

Prior to cytology testing, and within 30 days of collection, transfer a 1 mL aliquot into an Aptima^® Specimen Transfer Tube. Note: the specimen must have been stored at 2 – 30°C.

Stability:

Aptima^® Swab

Ambient:
60 days

Refrigerated:
60 days

Frozen:
1 year

Stability:

ThinPrep^® Solution in Aptima^® Transport Media

Ambient:
14 days

Refrigerated:
30 days

Frozen:
1 year

Stability:

ThinPrep^® Solution, unprocessed

Ambient:
30 days

Refrigerated:
30 days

Frozen:
Unacceptable

Additional Information

Background

Background Information

Sexually transmitted diseases (STDs) continue to be a major cause of deteriorating reproductive health throughout the world. Chlamydia trachomatis and Neisseria gonorrhoeae remain as two of the most common causes of STDs in the United States.¹ C. trachomatis infections have comprised the largest proportion of all STDs reported to the CDC since 1994, with a reported 1,244,180 cases in 2009 for a rate of 409.2/100,000 population. This was a 2.8% increase in rate from that reported in 2008.^1,2 The increase in reported chlamydial infections during the last 20 years reflects the expansion of chlamydia screening activities and the use of increasingly sensitive assays for the detection of C. trachomatis. The CDC recommends annual chlamydia screening of all sexually active women younger than 25 years of age.³

In 2009, there were 301,174 cases of N. gonorrhoeae infections reported for a rate of 99.1/100,000 population. This rate was a 10.5% decrease since 2008. The national gonorrhea rate declined by 74% between 1975 and 1997 following the implementation of a national gonorrhea control program in the mid-1970s. However, since 1997, these rates have reached a plateau and are not continuing to decline.^1,2 Infections due to both C. trachomatis and N. gonorrhoeae are a major cause of pelvic inflammatory disease (PID) in the U.S. and both have been shown to facilitate the transmission of HIV as well.

Rapid and sensitive methods for the laboratory diagnosis of these two agents have been developed, making it reasonable to test for both simultaneously when the diagnosis of an STD is being considered.² The estimate of mixed infections with both agents can be as high as 40%, making it important to consider ordering both agents when sending material off to the laboratory for testing. Nucleic acid amplification tests (NAAT) are recommended for detection of reproductive tract infections caused by C. trachomatis and N. gonorrhoeae infections in men and women with and without symptoms. NAAT should be used for diagnosing both C. trachomatis and N. gonorrhoeae in women with cervicitis; testing can be performed on vaginal, cervical, or urine samples. In men with urethritis, NAAT testing of urine or urethral swabs is recommended.³

Clinical Indications

Both C. trachomatis and N. gonorrhoeae cause urethritis in the male and cervicitis in the female. A significant number of cases, however, remain asymptomatic in both males and females. In addition, both can cause epididymitis and rectal infections in the male, and PID in the female.

Neonates, who contract chlamydial infection during birth, can develop inclusion conjunctivitis and/or pneumoniae; pregnant women can infect their newborns, causing ophthalmia neonatorum; gonorrheal infections can produce joint infections, pharyngitis, and disseminated disease.

Cleveland Clinic Laboratories offers a target amplification nucleic acid probe (APTIMA, Gen-Probe, Inc, San Diego, CA) for the laboratory diagnosis of C. trachomatis and N. gonorrhoeae from urethral and urine specimens from males suspected of these infections, and from cervical, vaginal, and urine samples from females. Numerous articles have been published demonstrating the excellent performance of NAAT testing for the diagnosis of both of these STD agents.^4-8

Methodology

The laboratory diagnosis of Neisseria gonorrhoeae can include culture of urethral or cervical specimens, gram stain of the urethral secretions in symptomatic males, detection by specific nucleic acid gene probes, and amplification of N. gonorrhoeae nucleic acids. Amplification of N. gonorrhoeae nucleic acids has been shown to be a very sensitive and specific method of detection.^4,5 The sensitivity is equivalent to culture, but it is not fraught by the problem of organism fragility that can easily occur with delays in specimen transport.

Although culture or the use of a nucleic acids probe can be employed for the detection of C. trachomatis, nucleic acid amplification is the most sensitive method, with studies indicating that it may be up to 40% more sensitive than culture. The same assay that detects Chlamydia trachomatis nucleic acids is also used by Cleveland Clinic Laboratories to detect Neisseria gonorrhoeae nucleic acids, thus providing a convenient approach to dual detection.

Collection & Transport

Specimen Collection & Transport

Acceptable specimens include urethral, endocervical, and vaginal swabs, as well as urine. A vaginal swab is optimal for screening asymptomatic females, while a first-catch urine is optimal for screening asymptomatic men.

Specimen collection/transport using Aptima^® devices is preferred.

Urethral, Endocervical Specimens

The Aptima^® Unisex Swab Specimen Collection Kit for urethral or endocervical specimens contains a white cleaning swab to be used for removing excess mucus. The blue swab must be used for collection of specimens that are submitted for testing.

For urethral specimens:
- Patients should not urinate within 1 hour prior to specimen collection.
- Insert the blue shaft swab 2 to 4 cm into the urethra.
- Gently rotate swab clockwise for 2 to 3 seconds and withdraw carefully.

For endocervical specimens:
- Remove excess mucus using cleaning swab and then insert blue shaft swab into the endocervical canal.
- Rotate swab for 10-30 seconds in the endocervical canal to ensure adequate sampling and withdraw carefully (avoid contact with vaginal mucosa).

Place swab into the transport tube and carefully break at the scoreline. Use care to avoid splashing contents. Discard top portion of swab shaft and recap transport tube tightly.

Maintain the specimen at 2ºC to 30ºC.

Vaginal Specimens

The Aptima^® Multitest Swab Transport Media Kit is optimal for testing asymptomatic women.

For vaginal specimens:
- Hold the swab with forefinger and thumb covering the scoreline (do not hold the shaft below the scoreline).
- Carefully insert the swab about 2 inches into the vagina and gently rotate the swab for 10-30 seconds.
- Make sure the swab touches the walls of the vagina so that moisture is absorbed by the swab, then withdraw the swab without touching the skin.
- While holding the swab in the same hand, unscrew the cap from the tube, being careful not to spill contents of the tube.

Immediately place the swab in the transport tube and carefully break swab shaft at score line against side of the tube. Use care to avoid splashing contents. Discard top portion of swab shaft and recap transport tube tightly.

Maintain the specimen at 2ºC to 30ºC.

Urine Specimens

The Aptima^® Urine Specimen Collection Kit is used for the collection and transport of male or female urine specimens for chlamydia and/or gonorrhea testing.

For urine specimens:
- Patients should not urinate within one hour of collection.
- Collect the first catch urine (approximately 20-30 ml of initial urine stream; collecting larger volumes of urine will reduce test sensitivity).
- Within 24 hours of collection, transfer 2 mL of urine into the Aptima^® urine transport tube using the disposable pipette provided in the collection kit. The correct volume of urine has been added when the fluid level is between the black lines on the transport tube label.

Maintain the specimen at 2ºC to 30ºC.

ThinPrep^® Pap Test Specimens

Alternatively, if a ThinPrep^® vial is being used for a Liquid Cytology PAP Test, the same sample can be submitted for detection of C. trachomatis and N. gonorrhoeae as well.⁹

The assay can only be performed on ThinPrep^® vials if 1 mL of Cytyc PreservCyt Solution is transferred to an Aptima^® Specimen Transfer Tube before the specimen is processed in Cytology for a PAP test.

Maintain the specimen at 2ºC to 30ºC.

Interpretation

Amplification is performed Monday through Friday. Internal controls are run with each specimen in order to detect any inhibitors in the sample.

Results will be reported as “positive for C. trachomatis and/or N. gonorrhoeae by amplification” when the relative light unit (RLU) result is above our positive cut-off value.

Within a narrow range of RLU results, as determined by the assay manufacturer, an “equivocal result” will be reported with a request that a repeat specimen be submitted.

If the internal control indicates inhibition, and the result is negative for C. trachomatis and/or N. gonorrhoeae, the report will be: “Inhibition detected; N. gonorrhoeae, and/or C. trachomatis, if present, would not be detectable. Please send an additional specimen.”

All results for N. gonorrhoeae and/or C. trachomatis that are lower than the laboratory’s derived positive cut-off, but within the instrument derived positive results, will be confirmed with a repeat amplification assay before reports are released. This is done to avoid any problems with false-positive results that might occur with low positive results.¹⁰

Limitations

There is currently no FDA clearance for use of amplification assays on specimens outside of the genitourinary tract. Culture is recommended for testing specimens from the throat, eye, or rectal area. However, a laboratory can validate the use of NAAT for rectal and pharyngeal specimens. In addition, for specimens obtained from infants and children, or if the information from the laboratory is to be used for legal purposes, culture is the preferred method.⁷

Since NAAT is more sensitive, it may be run in conjunction with culture for purposes of treatment decision-making. Although “test-of-cure” samples are not recommended from patients in whom the diagnosis has previously been made within the last 4-6 weeks, if required, a culture is the preferred request.

If a culture is needed for any of these purposes, the collection swab for Neisseria gonorrhoeae needs to be placed into a culturette and NOT the Aptima^® transport tube, and a specific request for culture should be made.

References

1. Centers for Disease Control and Prevention. Sexually Transmitted Disease Surveillance, 2009. Atlanta, GA: U.S. Department of Health and Human Services; November 2010. Printed copies and the on-line version of this report can be obtained at the following website: http://www.cdc.gov/std/pubs.

2. http://www.cdc.gov/std/stats09/chlamydia.htm

3. Centers for Disease Control and Prevention. Sexually transmitted Diseases treatment Guidelines. MMWR. 2010;59(RR#2):1-110.

4. Chernesky M, Martin DH, Hook EW et al. Ability of Aptima CT and Aptima GC assays to detect Chlamydia trachomatis and Neisseria gonorrhoeae in male urine and urethral swabs. J Clin Microbiol. 2005;43:127-31.

5. Gaydos CA, Quinn TC, Willis D, Weissfeld A, Hook EW, Martin DH, Ferrero DV, Schachter J. Performance of the APTIMA Combo 2 assay for the multiplex detection of Chlamydia trachomatis and Neisseria gonorrhoeae in female urine and endocervical swab specimens. J Clin Microbiol. 2003;41:304-309.

6. Fang J, Husman C, Dasilva L et al. Evaluation of self collected vaginal swab, first void urine, and endocervical swabs for the detection of C. trachomatis and N. gonorrhoeae in adolescent females. J Pediatr Adolesc Gynecol. 2008;21:355-60.

7. Blake DR, Maldeis N, Barnes MR et al. Cost-effectiveness of screening strategies for C. trachomatis using cervical swabs, urine, and self-obtained vaginal swabs in a sexually transmitted disease clinic setting. Sex Transm Dis. 2008;35:649-55.

8. Schachter J, Chernesky MA, Willis DE, et al. Vaginal swabs are the specimens of choice when screening for C. trachomatis and N. gonorrhoeae: results from a multicenter evaluation of the Aptima assays for both infections. Sex Transm Dis. 2005;32:725-8.

9. Chernesky M, Jang D, Smieja M et al. Validation of the Aptima Combo 2 assay for detection of C. trachomatis and N. gonorrhoeae in Sure-Path liquid-based pap test samples taken with different collection devices. Sex Transm Dis. 2009;36:581-2.

10. Farrell, DJ. Evaluation of AMPLICOR Neisseria gonorrhoeae PCR using cppB nested PCR and 16S rRNA PCR. J Clin Microbiology. 1999;37:386-90.

Antiphospholipid Antibody Testing (Lupus Anticoagulant Testing)

Technical Brief:

Antiphospholipid Antibody Testing (Lupus Anticoagulant Testing)

Test Name

Lupus Anticoagulant Diagnostic Interpretive Panel (LUPUSP)

CPT Codes

85670
86147 (x3)
85613 (x2)
85597
85610
85390
85730 (x3)
86146 (x2)
85732 (x3)
85520

Methodology

Refer to individual components

Turnaround Time

3 – 5 days

Specimen Requirements

Volume:
1 mL

Minimum Volume:
0.2 mL

Specimen Type:
Serum

Collection Container:
Gold BD Hemogard™ Serum Separation Tubes (SST)™

Transport Temperature:
Frozen

Indicate each tube as serum or plasma.

Volume:
5 mL

Minimum Volume:
2.5 mL

Specimen Type:
Plasma

Collection Container:
Light Blue VACUETTE® Sodium Citrate Coagulation Tube

Transport Temperature:
Frozen

Indicate each tube as serum or plasma.

3.2% sodium citrate is the preferred anticoagulant recommended by CLSI.

Stability

Ambient:
Unacceptable

Refrigerated:
Unacceptable

Frozen:
2 months

Specimen Collection & Handling

Collection of blood by routine venipuncture in a 3.5 mL light blue top tube containing 9:1 ratio of blood to 3.2% trisodium citrate anticoagulant.

Patient Preparation

Discontinue heparin therapy for 2 days prior to collection.

If tests are abnormal, the following tests may be ordered and billed:

Factor II (FIIC)
Factor V (FVC)
Factor X (FXC)
Factor VIII (FVIIIC)
von Willebrand Factor Antigen (VWF)
Ristocetin Co-factor (RISCOF)
Factor IX Assay (FIXC)
Factor XI Assay (FXIC)
Factor XII Assay (FXIIC)
Heparin Xa Inhibition (HEPASY)
Fibrinogen (FIBCT)
Bethesda Assay (BETHDA)

Reference Range

Refer to Table 1

Additional Information

Background

Background Information

Antiphospholipid syndrome (APS) is the most common cause of acquired thrombophilia, and the presence of antiphospholipid antibody (APA) is associated with significant morbidity and mortality across diverse patient populations. Both primary and secondary forms of APAs exist, the difference being whether they arise spontaneously or in association with another condition. These antibodies — also known as lupus anticoagulants due to their prevalence in patients with systemic lupus erythematosus — are extremely heterogeneous and are directed against a wide variety of anionic phospholipids, including cardiolipin, ß2 glycoprotein 1 (B2GP1), cell-membrane phosphatidylserine, and many others. Paradoxically, APAs prolong clot-based assays in vitro while predisposing to thrombosis in vivo. In fact, approximately 30% of APA patients will experience thrombosis. A panel of assays is necessary to detect APAs, as no single test presently available is sufficient.

Diagnosis of antiphospholipid syndrome is made by clinicopathologic evaluation. In addition to clinical criteria, such as vascular thrombosis or pregnancy morbidity, repeated laboratory testing of APA is required for the diagnosis because of transient low-level increase of APA in many clinical conditions including infection. The laboratory criteria include positive testing for one of the following on 2 or more occasions, at least 12 weeks apart: 1. lupus anticoagulant; 2. anticardiolipin antibodies (IgG or IgM) in medium or high titer; 3. B2GP1 antibodies (IgG or IgM).

Lupus Anticoagulant (LA) Testing:

Based upon consensus criteria from the International Society for Thrombosis and Haemostasis (ISTH), confirmation of a LA requires that the following criteria are met:

Performing two or more phospholipid-dependent clotting tests and demonstrating prolongation of at least one test (i.e. aPTT or dilute Russell Viper Venom Test (dRVVT))
Evidence for inhibitory activity shown by the effect of patient plasma on normal pooled plasma. (i.e. positive mixing study)
Demonstration of phospholipid-dependence of the inhibitor on a confirmatory test shown by shortening of the clotting time with the addition of more phospholipid
Exclusion of a co-existing specific factor inhibitor, particularly factor VIII or an anticoagulant drug such as heparin or direct thrombin inhibitor (DTI)

Anticardiolipin Antibody (ACA) IgG, IgM or IgA, and B2GP1 Antibody IgG or IgM Testing:

ACAs recognize a complex of cardiolipin, a naturally found phospholipid, bound to a protein called B2GP1. Complexes of anionic phospholipids and endogenous plasma proteins provide more than one epitope recognized by natural autoantibodies.

An enzyme-linked immunosorbent assay (ELISA) is performed for APA testing. Because the antigen target of ACAs is B2GP1 bound to cardiolipin, B2GP1 antibodies are considered to be more specific than ACA assays.

Clinical Indications

Suspicion for APS in patients with an elevated aPTT, unexplained thrombocytopenia, or a history of arterial and venous thrombosis and/or obstetric complications.

Interpretation

Lupus Anticoagulant (LA)
Tests for LA are interpreted as positive, indeterminate or negative. A narrative interpretation is issued for each patient panel:

Positive:
The panel of tests meets all four diagnostic criteria. If one screening test, one mixing test, and one confirmatory test are positive and there is no evidence for a factor inhibitor or anticoagulant drug effect, the diagnostic criteria for LA are fulfilled.

Indeterminate:
Fewer than four diagnostic criteria are met. If clinical suspicion exists, the patient should be retested at a later date.

Negative:
None of the four diagnostic criteria is met.

Anticardiolipin Antibodies and B2GP1 Antibodies
Tests for ACA and B2GP1 are interpreted as positive, equivocal, or negative. The reference range of each test in the diagnostic panel is shown in Table 1.

Methodology

Laboratory testing for LA consists of a panel of assays (at least two assays on different principles in each criterion) specifically performed together to maximize diagnostic potential.

Test Category

Tests Performed

Screening Tests:

aPTT, aPTT Screen, dRVVT Screen, Hexagonal PL Screen

Four screening tests are performed: the standard laboratory automated aPTT, a more APA-sensitive manual aPTT screen reagent (which contains a different phospholipid composition), the dilute Russell’s viper venom test (dRVVT), a clot-based assay that uses snake venom to activate Factor X directly, and the hexagonal PL screen, which uses a very dilute aPTT reagent to increase sensitivity to phospholipids.

Mixing Studies:

Mixing Study aPTT (immediate and delayed), dRVVT Mix

Patient plasma and normal control plasma are mixed 1:1, and an aPTT and dRVVT test is performed on the mixed sample.

In the presence of an inhibitor in the patient’s plasma, the normal plasma also is affected, and the clotting time will not correct into the normal range. However, if the initial prolonged clotting time was due to a factor deficiency in the patient’s plasma, the normal plasma corrects this deficiency and the resultant clotting time will be normal.

The aPTT mixing study includes a one-hour incubation step to check for more slow-acting specific factor inhibitors

PL Confirmatory Tests:

dRVVT Confirm Ratio, Hexagonal PL Confirm, Platelet Neutralization

Several tests are used to confirm the phospholipid-dependence of an inhibitor:

• The dRVVT confirm ratio is performed by adding PL to plasma and repeating the dRVVT assay. The ratio is calculated by the dRVVT screen/dRVVT confirm.

• The hexagonal phase phospholipid test (STAclot) confirm is performed by adding hexagonal PL to plasma and repeating the hexagonal PL screen. The Delta is calculated by the hexagonal PL screen — the hexagonal PL confirm.

• The platelet neutralization procedure (PNP) uses phospholipid-containing platelet membranes to neutralize the aPTT-prolonging effects of an LA. A PNP test is positive when the prolonged aPTT is shortened by the addition of platelet lysate.

Exclusion Assays:

The presence of other inhibitors must be excluded to confirm the presence of an APA. These include drugs (heparin, DTIs) and specific factor inhibitors (factor VIII is the most common). Tests for each of these are included in the panel, as required per the LA algorithm.

Specific antibodies against cardiolipin and B2GP1 are measured by solid-phase ELISA assay.

Abbreviations:

APTT: Activated partial thromboplastin time
dRVVT: Dilute Russell’s viper venom test

FVIII: Factor VIII
LA: Lupus anticoagulant

PL: Phospholipid
PNP: Platelet neutralization procedure

TT: Thrombin time

Limitations

LAs are heterogeneous in terms of antigenic recognition, and aPTT reagents are variable in terms of phospholipid composition. Thus, variability in detection of LAs may exist between individual reagents, between different panel tests, and/or between laboratories.

Consequently, a normal aPTT cannot definitively exclude the presence of an LA; therefore, if clinical suspicion is high, the full panel may be performed.

Both ACA and B2GP1 APA assays are recommended as using one B2GP1 antibody assay can miss some cases of APA.

- Table 1

Table 1: Reference Ranges of Each Test in the Lupus Anticoagulant Diagnostic Interpretive Panel

Test

Reference Range

aPTT

23.0 – 32.4 sec

PT/INR

8.4 – 13.0 sec / 0.8 – 1.2 sec

<18.6 sec

Mixing Study, Incubated aPTT

Negative

Hexagonal Phase PL Test

Screen:
48.9 – 70.2 sec

Delta:
< 9.0

DRVVT

Screen:
32.7 – 46.7 sec

1:1 Mix:
32.7 – 46.7 sec

Confirm Ratio:
< 1.21

PNP

Negative

ACA IgA

Negative:
< 12 APL

Equivocal:
12 – 40 APL

High Positive:
> 40 APL

ACA IgG

Negative:
< 10 GPL

Equivocal:
10 – 40 GPL

High Positive:
> 40 GPL

ACA IgM

Negative:
< 12 MPL

Equivocal:
12 – 40 MPL

High Positive:
> 40 MPL

B2GP1 Autoabs

IgG:
< 20 Units

IgM:
< 20 Units

Heparin Assay/Factor Xa Inhibition

< 0.10 IU/mL

References

1. Pengo V, Tripodi A, Reber G, et al. Update of the guidelines for lupus anticoagulant detection. J. Thromb Haemost. 2009: 7:1737.

2. Kottke-Marchant K. An Algorithmic Approach to Hemostasis Testing. CAP Press (2008).

3. Miyakis S, Lockshin MD, Atsumi T et al. International consensus statement on an update of the classification criteria for definite antiphospholipid antibody syndrome (APS). J. Thromb Haemost. 2006; 4:295.

4. Moffat KA, Ledford-Kraemer MR, Plumhoff EA et al. Are laboratories following published recommendations for lupus anticoagulant testing? An international evaluation of practices. Thromb Haemost. 2009:101:178.

5. Hoppensteadt D, Walenga J. The relationship between the antiphospholipid syndrome and heparin-induced thrombocytopenia. Hematol Oncol Clin N Am. 2008:22:1.

CALR Mutation Detection

Technical Brief

CALR Mutation Detection

Test Name

CALR (Calreticulin) Exon 9 Mutation, Blood (CALR)

CALR (Calreticulin) Exon 9 Mutation Analysis, Marrow (CALRM)

CPT Codes

81219
G0452

Methodology

Polymerase Chain Reaction with fragment length analysis by capillary electrophoresis

Turnaround Time

7 days

Specimen Requirements

Type:
Aspirate, bone marrow

Volume:
2 mL

Minimum Volume:
1 mL

Specimen Container:
Lavender BD Hemogard™ K₂EDTA Tube

or:

Type:
Formalin-fixed paraffin-embedded tissue/bone marrow clot

Volume:
1 block

Transport Temperature:
Ambient

or:

Type:
Blood, whole

Volume:
4 mL

Minimum Volume:
2 mL

Specimen Container:
Lavender BD Hemogard™ K₂EDTA Tube

Stability

Ambient:
48 hours

Refrigerated:
7 days

Frozen:
Unacceptable

Formalin-fixed paraffin-embedded tissue/bone marrow clot:
Indefinitely

Additional Information

Background

Background Information

The BCR/ABL1-negative myeloproliferative neoplasms (MPN) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF).¹ The JAK2 V617F point mutation occurs in >95% of cases of PV and approximately 50-60% of cases of ET and PMF. In ET and PMF lacking the JAK2 V617F mutation, approximately 10-20% contain a mutation in MPL exon 10 while 60-80% of cases have a mutation in CALR.^2,3

The identification of a JAK2, MPL, or CALR mutation is diagnostically useful to separate MPN from a reactive leukocytosis that may mimic a myeloid neoplasm. Cases of ET and PMF with mutations of JAK2, MPL, or CALR may also show prognostic differences.^4–7

Approximately 80% of CALR mutations can be classified as either type 1 (a 52-bp deletion) or type 2 (a 5-bp insertion). The remaining mutations represent other, variably sized insertions and deletions. All CALR mutations (type 1, type 2 or other) create a frameshift with the production of an altered C-terminus of the calreticulin protein.

Cleveland Clinic Laboratories has developed, validated and implemented a sensitive PCR assay for the detection of CALR mutations in peripheral blood, bone marrow or formalin-fixed, paraffin-embedded tissues.

Clinical Indications

CALR mutation testing is useful in the workup of suspected myeloproliferative neoplasms, especially those that are negative for JAK2 V617F.

Interpretation

Normal results are reported as “CALR mutation not detected.”

Positive results are reported as “CALR mutation detected,” and an interpretation is provided that includes a description of the mutation (type 1, type 2, or other).

Methodology

Genomic DNA is extracted from the sample and CALR exon 9 is amplified by PCR. Fragment length analysis is performed to assess for insertion/deletion mutations.

Limitations

This assay has a sensitivity of 5% mutant alleles. This assay detects only insertion/deletion mutations in CALR exon 9, and a negative result does not exclude the possibility of a myeloproliferative neoplasm.

References

1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Lyon: IARC Press; 2008.

2. Nangalia J, Massie CE, Baxter EJ, et al. Somatic CALR mutations in myeloproliferative neoplasms with nonmutated JAK2. N Engl J Med. 2013;369:2391-405.

3. Klampfl T, Gisslinger H, Harutyunyan AS, et al. Somatic mutations of calreticulin in myeloproliferative neoplasms. N Engl J Med. 2013;369:2379-90.

4. Tefferi A, Lasho TL, Finke CM, et al. CALR vs JAK2 vs MPL-mutated or triple-negative myelofibrosis: clinical, cytogenetic and molecular comparisons. Leukemia. 2014;28:1472-7.

5. Tefferi A, Wassie EA, Lasho TL, et al. Calreticulin mutations and long-term survival in essential thrombocythemia. Leukemia. 2014;28:2300-3.

6. Rumi E, Pietra D, Pascutto C, et al. Clinical effect of driver mutations of JAK2, CALR, or MPL in primary myelofibrosis. Blood. 2014;124:1062-9.

7. Rumi E, Pietra D, Ferretti V, et al. JAK2 or CALR mutation status defines subtypes of essential thrombocythemia with substantially different clinical course and outcomes. Blood. 2013;123:1544-1551.

MYD88 L265P Mutation Detection

Technical Brief

MYD88 L265P Mutation Detection

Test Name

MYD88 L265P Mutation Analysis (MYD88)

CPT Codes

81305
G0452

Methodology

Allele-specific Polymerase Chain Reaction (PCR)

Real-Time PCR

Turnaround Time

7 days

Specimen Requirements

Type:
Blood, whole

Volume:
4 mL

Minimum Volume:
1 mL

Specimen Container:
Lavender BD Hemogard™ K₂EDTA Tube

Transport Temperature:
Refrigerated

Type:
Bone marrow

Volume:
2 mL

Minimum Volume:
0.5 mL

Specimen Container:
Lavender BD Hemogard™ K₂EDTA Tube

Transport Temperature:
Refrigerated

Type:
Paraffin block, formalin-fixed
Bone marrow clot

Volume:
1 block

Transport Temperature:
Ambient

Stability

Blood, Bone Marrow

Ambient:
24 hours

Refrigerated:
5 days

Frozen:
Unacceptable

Stability

FFPE, Bone Marrow Clot

Ambient:
Indefinitely

Refrigerated:
Indefinitely

Frozen:
Indefinitely

Reference Range

MYD88 L265P mutation not detected.

Additional Information

Background

Background Information

Lymphoplasmacytic lymphoma (LPL) is a small B-cell neoplasm with plasmacytic differentiation that typically involves the bone marrow, but may also involve spleen and lymph nodes. In most cases, LPL is associated with an IgM paraprotein (Waldenstrom’s macroglobulinemia).^1,2 Distinguishing LPL from other small B-cell neoplasms that may show plasmacytic differentiation, especially marginal zone lymphomas, is often challenging.

Recently, the MYD88 L265P mutation has been identified in >90% of cases of LPL.³ This mutation may also be found in diffuse large B-cell lymphomas, especially those with a nongerminal center phenotype, but the mutation is only rarely found in other small B-cell neoplasms. The detection of an MYD88 L265P mutation can, therefore, assist in establishing the diagnosis of LPL.^3-5

Cleveland Clinic Laboratories has developed, validated and implemented a sensitive PCR assay for the detection of MYD88 L265P in peripheral blood, bone marrow, or formalin-fixed, paraffin-embedded tissues.

Clinical Indications

MYD88 L265P mutation testing is useful in the evaluation of small B-cell neoplasms, especially those with plasmacytic differentiation.

Interpretation

Normal Results:

“MYD88 L265P mutation not detected.”

Positive Results:

“MYD88 L265P mutation detected.” An interpretation is also provided.

Methodology

DNA is extracted from the sample. Real-time PCR is performed using primers specific for the L265P mutation and a reference primer set for a non-mutated portion of the MYD88 gene.

Limitations

This assay has a sensitivity of 0.5% mutant alleles.

This assay detects only the L265P point mutation, and a negative result does not exclude a diagnosis of lymphoplasmacytic lymphoma.

References

1. Swerdlow S.H., Berger F., Pileri S.A., et al. Lymphoplasmacytic lymphoma. In: WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC: Lyon 2008. Swerdlow SH, Campo E. Harris EL, et al (Eds). Pp 194-195.

2. Treon SP, Hunter ZR, Castillo JJ, et al. Waldenström macroglobulinemia. Hematol Oncol Clin North Am. 2014 Oct;28(5):945-70.

3. Treon SP, Xu L, Yang G, et al. MYD88 L265P somatic mutation in Waldenström’s macroglobulinemia. N Engl J Med. 2012 Aug 30;367(9):826-33.

4. Hamadeh F, MacNamara SP, Aguilera NS, et al. MYD88 L265P mutation analysis helps define nodal lymphoplasmacytic lymphoma. Mod Pathol. 2014 Sep 12. [Epub ahead of print]

5. Ondrejka SL, Lin JJ, Warden DW, et al. MYD88 L265P somatic mutation: its usefulness in the differential diagnosis of bone marrow involvement by B-cell lymphoproliferative disorders. Am J Clin Pathol. 2013 Sep;140(3):387-94.

ADAMTS13 Evaluation for Thrombotic Thrombocytopenic Purpura (TTP)

Technical Brief:

ADAMTS13 Evaluation for Thrombotic Thrombocytopenic Purpura (TTP)

Test Name

ADAMTS13 Activity Assay (ADM13A)

ADAMTS13 Inhibitor Assay (ADM12I)

ADAMTS13 Antibody Test (ABADM)

CPT Codes

ADAMTS13 Activity Assay

85397
85390

ADAMTS13 Inhibitor Assay

85335
85390

ADAMTS13 Antibody Test

83520
85390

Methodology

Enzyme Immunoassay (EIA)

Turnaround Time

2 – 4 days

Specimen Requirements

Volume:
2 mL

Specimen Type:
Plasma

Collection Container:
Light Blue VACUETTE® Sodium Citrate Coagulation Tube

Transport Temperature:
Frozen

3.2% sodium citrate is the preferred anticoagulant recommended by CLSI.

Specimen Collection & Handling

The preferred blood specimen is collected by routine venipuncture in 1.8 mL light blue top tube containing a 9:1 ratio of blood to 3.2% sodium citrate anticoagulant.

Citrated plasma with an appropriate ratio of anticoagulant (3.2% sodium citrate) is acceptable.

The presence of heparin, fondaparinux, dabigatran or another direct thrombin inhibitor in the specimen may interfere with test results.

Stability

Ambient:
7 days

Citrated plasma for ADAMSTS13 Antibody remains stable at room temperature and refrigerated for 7 days, but no tests for ADAMSTS13 Activity or Inhibitor can be performed on such specimens.

Reference Range

See Interpretation

Additional Information

Background

Background Information

Many studies on the pathophysiology of thrombotic thrombocytopenic purpura (TTP), a rare life-threatening disease characterized by microangiopathic hemolytic anemia, thrombocytopenia and multi-organ failure, have been published over the last two decades. The most significant finding was the identification of ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif, member 13) that is involved in the regulation of the size of von Willebrand factor (VWF), a plasma protein responsible for regulating the interaction of platelets with von Willebrand factor (VWF) and physiologic proteolytic cleavage of ultra-large (UL) VWF multimers at the Tyr(1605)-Met(1606) bond in the A2 domain of VWF.

Reduced or absent ADAMTS13 activity can retain UL VWF that can trigger intravascular platelet aggregation and microthrombi causing clinical symptoms or signs of thrombotic thrombocytopenic purpura (TTP). Measurement of ADAMTS13 activity, its inhibitor, and antibody (in some cases) is crucial in the diagnosis of TTP, potentially fatal thrombotic microangiopathy (TMA) syndrome and further differentiation of congenital (Upshaw-Schulman syndrome) versus acquired (e.g. autoimmune-related disorder) etiology.

TTP has an estimated incidence of four to six cases per million, and affects women more often, particularly pregnantor postpartum women (estimated incidence of one per 25,000 pregnancies) and African-Americans. TTP is primarily diagnosed clinically, and its correct diagnosis is often very difficult. TTP is characterized by microangiopathic hemolytic anemia including numerous schistocytes in the peripheral blood smear, thrombocytopenia, neurologic symptoms, fever, renal dysfunction, variable organ damage and ischemia, and deficient ADAMTS13 activity, usually less than 30%. Approximately two-thirds of patients with a clinical diagnosis of idiopathic TTP will have less than 10% ADAMTS13 activity.

Two forms of ADAMTS13 deficiency, an acquired and a congenital form, are recognized; both will eventually result in microvascular thrombosis and TTP. Acquired TTP is more common than the congenital form, and may be considered to be primary or idiopathic (the most frequent type) or associated with distinctive clinical conditions (secondary TTP). The majority of acquired, idiopathic TTP patients with severe ADAMTS13 deficiency are related to circulating anti-ADAMTS13 autoantibodies (inhibitors) that can neutralize ADAMTS13 activity. ADAMTS13 inhibitor is observed in 44-93% of patients with severely deficient ADAMTS13 activity according to literatures. 10-15% of TTP patients with severe ADAMTS13 deficiency have lacked neutralizing antibodies (non-inhibitors). These patients have non-neutralizing IgG or IgM antibodies and ADAMTS13 deficiency may be related to increased antibody-mediated clearance or yet unknown other mechanisms. However, both types, inhibitor and non-inhibitor, may be simultaneously present in some TTP patients.

Congenital TTP (Upshaw-Shulman syndrome) is a rare inheritable disease with an autosomal recessive pattern, and caused by compound heterozygous or homozygous genetic mutations within the ADAMTS13 gene producing non-functional ADAMTS13 protein. Half of these patients will present acute TTP within their first years of life (early-onset), and the remaining half will remain asymptomatic until adulthood, usually 20-40 years of age (late-onset). These patients will have severely deficient ADAMTS13 activity with high risk for recurrent episodes of TTP often being triggered by events such as pregnancy or heavy alcohol intake. These patients usually do not develop autoantibodies to ADAMTS13.

Quantitative measurement of the ADAMTS13 activity, inhibitor and autoantibody will help to confirm clinical diagnosis of TTP and be useful to distinguish patients with TTP from other thrombocytopenic conditions such as hemolytic uremic syndrome (HUS), immune thrombocytopenic purpura (ITP) or heparin-induced thrombocytopenia (HIT). Severely decreased ADAMTS13 activity (less than 5-10%) is considered as a relatively specific laboratory finding for the clinical diagnosis of TTP. ADAMTS13 inhibitor assay can detect most of TTP patients with neutralizing autoantibodies. ADAMTS13 autoantibody assay can detect some additional TTP patients with non-neutralizing autoantibodies (non-inhibitor). Figure 1 shows the diagnostic algorithm of ADAMTS13 evaluation for TTP using ADAMTS13 activity, inhibitor and autoantibody assays as a panel.

Early detection and initiation of therapeutic plasma exchange is critical for better survival of patients and can save approximately 70% of TTP patients. Current therapy is based on support and plasmapheresis to remove both circulating antibodies against ADAMTS13 and UL VWF multimers, and replace ADAMTS13 via fresh frozen plasma. In addition to the diagnosis of TTP by ADAMTS13 assay using the specimen collected prior to any therapy, ADAMTS13 assay can be useful for treatment selection or monitoring because of short turnaround time. TTP patients with ADAMTS13 autoantibodies can consider immunosuppressive drugs in addition to plasma exchange.Approximately 20-25% of TTP patients will experience relapse. Persistence of severe deficiency in ADAMTS13 activity or an inhibitor suggests a high risk of relapse in symptomatic TTP. Persistency of autoantibodies during clinical remission or high titers of autoantibodies also suggests an increased risk of clinical relapse.

Figure 1: Diagnostic Algorithm of ADAMTS13 Evaluation for Thrombotic Thrombocytopenic Purpura

Abbreviations:

IU: Inhibitor Unit

TTP: Thrombotic Thrombocytopenic Purpura

Clinical Indications

The ADAMTS13 activity (ADM13A), inhibitor (ADM13I), and autoantibody (ABADM) assays are useful for the diagnosis of the congenital or acquired form of TTP.

Interpretation

Diagnosis of TTP is difficult, due to the rarity of the disease and the poor specificity of clinical and laboratory signs and symptoms. Decreased ADAMTS13 activity (less than 68%) can be observed in idiopathic (autoimmune-related) TTP, TMA syndrome, congenital ADAMTS13 deficiency (Upshaw Schulman syndrome) and secondary to other clinical conditions such as HUS, ITP, solid organ or bone marrow transplantation, sepsis, DIC, HIV infection, inflammation, bloody diarrhea, liver disease, pregnancy, malignancy, or certain drug effects (e.g., clopidogrel, cyclosporine, mitomycin C, ticlopidine, tacrolimus, etc.).

1. Normal ADAMTS13 activity (>=68%) and negative ADAMTS13 inhibitor (<=0.4 IU):
No laboratory evidence of TTP

2. Mildly decreased ADAMTS13 activity (30-67%) and negative ADAMTS13 inhibitor (<=0.4 IU):
Unlikely idiopathic TTP by laboratory findings and suggestive of TTP secondary to other clinical conditions. However, if there is a strong clinical suspicion of idiopathic TTP, autoantibody assay can be performed

3. Mildly decreased ADAMTS13 activity (30-67%) and positive ADAMTS13 inhibitor (>0.4 IU):
Diagnostic of idiopathic TTP or prior therapy effect in TTP patients

4. Decreased ADAMTS13 activity (<30%) and positive ADAMTS13 inhibitor (>0.4 IU):
Diagnostic of idiopathic TTP

5. Decreased ADAMTS13 activity (<30%) and negative ADAMTS13 inhibitor (<=0.4 IU):
Further evaluation of ADAMTS13 autoantibody assay

a) Positive ADAMTS13 autoantibody (>18 U/mL):
Diagnostic of idiopathic TTP

b) Negative ADAMTS13 autoantibody (<=18 U/mL):
Suggest ADAMTS13 sequencing to rule out congenital TTP with positive ADAMTS13 gene mutation

Methodology

ADAMTS13 activity is measured by change of fluorescence energy transfer (FRET) technology with recombinant VWF86 substrate (American Diagnostica Inc/Sekisui, Stamford, CT) in citrated plasma. The basic principle of the method is that proteolytic cleavage of the VWF86-ALEXA FRET substrate between the Tyr-Met residues by ADAMTS13 uncouples the ALEXA fluorochromes resulting in an increase in fluorescence.

ADAMTS13 inhibitor is measured by using a mixing study. After the patient’s plasma is mixed with normal pooled plasma (1:1) and incubated for 1 hour at 37°C, the residual ADAMTS13 activity of the mixture is measured using FRET technology. ADAMTS13 inhibitor level (Bethesda Unit) is calculated. One inhibitor unit is considered as the concentration of inhibitor that can reduce ADAMTS13 activity by 50%.

ADAMTS13 autoantibody is measured by sandwich enzyme immunoassay modified from Technozym ADAMTS13 INH kit (Technoclone Inc, Vienna, Austria). After binding with pre-coated recombinant human ADAMTS13, anti-ADAMTS13 IgG and conjugate, resulting color is measured photometrically. The color intensity is proportional to the concentration of ADAMTS13 IgG antibodies.

Limitations

ADAMTS13 activity by FRET-based assay can be interfered by high levels of endogenous VWF, hyperlipemia, elevated plasma hemoglobin level (>2 g/dL; potent inhibitor of ADAMTS13), hyperbilirubinemia (>15 mg/dL) or other proteases that may cleave ADAMTS13. In addition, recent plasma exchange or transfusion can potentially mask the diagnosis of TTP because of false normalization of ADAMTS13 activity. ADAMTS13 autoantibody assay, usually measuring IgG by enzyme immunoassay, is sensitive but less specific than functional inhibitor assay, and can be detected in other immune-mediated disorders such as systemic lupus erythematosis, antiphospholipid syndrome or patients with high titer of IgG, and some healthy individuals (10-15%).

Hypercoagulability / Thrombophilia Testing

Technical Brief:

Hypercoagulability / Thrombophilia Testing

Test Name

Hypercoagulation Diagnostic Interpretive Panel (HYPER)

Please complete a Hemostasis & Thrombosis Evaluation: Clinical History Form and include with the specimens.

CPT Codes

86147 (x3)
83090
85610
85730 (x2)
85300
85384
85306
85303
85240
85307
86140
85732
81240
85390

Methodology

Refer to individual components

Turnaround Time

3 – 5 days

Specimen Requirements

Specimen Type:
Serum

Volume:
2 mL

Minimum Volume:
1 mL

Collection Container:
Gold BD Hemogard™ Serum Separation Tubes (SST)™

Transport Temperature:
Frozen

Indicate each tube as serum or plasma.

Specimen Type:
Whole blood

Volume:
4 mL

Minimum Volume:
2 mL

Collection Container:
Lavender BD Hemogard™ K₂EDTA Tube

Transport Temperature:
Frozen

Specimen Type:
Plasma

Volume:
6 mL

Minimum Volume:
3 mL (citrated plasma)

Collection Container:
Light Blue Sodium Citrate Coagulation Tube

Transport Temperature:
Frozen

Indicate each tube as serum or plasma.

3.2% sodium citrate is the preferred anticoagulant recommended by CLSI.

Stability

Ambient:
4 hours

Refrigerated:
Unacceptable

Frozen:
14 days at -20°C
6 months at -70°C

Specimen Collection & Handling

Collection of blood by routine venipuncture in a 3.5 mL light blue top tube containing 9:1 ratio of blood to 3.2% trisodium citrate anticoagulant.

Patient Preparation

Discontinue coumadin therapy for 7 days, heparin therapy for 2 days, and thrombolytic therapy for 7 days prior to the test, if possible.

If tests are abnormal, the following tests may be ordered and billed:

Dilute Russell Viper Venom Time (DRVVT)
Platelet Neutralization (PLTNEU)
Factor V Leiden (FVLEI)
Thrombin Time (TT)
Reptilase (REPTM)
Fibrinogen Antigen (FIBRAG)
Protein C Immunologic (PRCAG)
Protein S Immunologic (PROTSI)
Heparin Xa Inhibition (HEPASY)

Reference Range

Refer to individual components.

Additional Information

Background

Background Information

Venous thromboembolism (VTE) is a major health issue, with more than 300,000 first-lifetime cases per year and around 1 million deaths annually in the United States alone. Thrombophilia (or hypercoagulability), although not a disease itself, is a major contributing factor in the development of VTE. Thrombophilia is the propensity to develop thromboses due to an acquired or inherited defect in the coagulation system. The predominant clinical manifestation of thrombophilia is venous thromboembolism.

Anti-phospholipid antibody syndrome (APS) is the most common cause of acquired thrombophilia. Additional causes include acquired or inherited deficiency of anticoagulant or procoagulant factors (e.g., protein C, protein S, antithrombin or fibrinogen), acquired or inherited elevation in procoagulant factors, such as factor VIII or homocysteine (>95th percentile). Inherited genetic mutations, including Factor V Leiden [FVL] and prothrombin gene, also predispose to thrombosis.

Not all abnormalities are associated with thrombophilia. For example, thrombophilic risk factors include advancing age (>50), major surgery, trauma, immobilization, malignancy, pregnancy, prior to oral contraceptive or hormonal replacement therapy, and chemotherapy. As with many disease-modifying risk factors, thrombophilic risk factors are synergistic — a combination magnifies the risk for thrombosis.

Acquired fibrinogen deficiency can occur in liver disease, disseminated intravascular coagulation (DIC), or hyperfibrinolysis. Acquired protein C or protein S deficiency can be associated with liver disease, anticoagulant therapy (warfarin), acute thrombosis, infections, DIC, postoperatively, uremia, or chemotherapy. Acquired antithrombin deficiency can be associated with DIC, liver disease, heparin therapy, acute thrombosis, nephrotic syndrome, or L-asparaginase therapy.

Currently, there is no single laboratory test that can identify all hypercoagulable defects; therefore, a combination of laboratory analyses is needed to accurately identify thrombophilic patients. Many of these tests are affected by other — often concurrent — clinical conditions so that the correct interpretation of these specialized laboratory test results can be complicated, and always require clinical correlation.

Clinical Indications

Patients with a personal or family history of unexplained or recurrent thrombosis and/or pregnancy complications.

May potentially be of benefit for screening patients who will be placed at increased risk of thrombosis.

Interpretation

This panel of tests is not simply reported as positive or negative; a narrative interpretation is issued for each patient panel.

Each test is reported separately, taking into account the patient’s clinical context.

Each positive test result increases the relative risk of thrombophilia independently of the other test results.

Limitations

Results from a hypercoagulability workup are difficult to interpret in the setting of acute thrombosis or anticoagulant medication therapy; thus, testing should be performed approximately 30 days after VTE or discontinuation of medication including warfarin, heparin, direct thrombin inhibitors (DTIs), and fibrinolytic agents.

Other clinical conditions (e.g. pregnancy, inflammatory states, liver disease, etc.) may affect certain assay results as well. The test requestor should provide appropriate clinical information in regards to these conditions to assist the laboratory in making the best possible interpretation of results. Alternatively, thrombophilic testing may be delayed until these clinical conditions have subsided.

Rarer thrombophilic mutations do exist for which testing is not currently performed. In this case, a patient may have an apparently-negative thrombophilic workup while still exhibiting a thrombotic phenotype. Clinical judgment is necessary to guide the therapy of these patients.

Methodology

Laboratory testing for thrombophilia consists of a panel of assays specifically performed together to maximize diagnostic potential.

Key:

Initial Core Panel Laboratory Testing

Reflex Testing, depending on Core Panel results

Thrombophilia Risk Factors

Abbreviations:

APTT: Activated partial thromboplastin time
CAC: Circulating anticoagulant assay (mixing study)

DRVVT: Dilute Russell’s viper venom test
PNP: Platelet neutralization procedure

PT: Prothrombin time
SNP: Single nucleotide polymorphism

Functional Testing

Anti-Phospholipid Antibody

APA, lupus anticoagulant

Automated and manual aPTT, and the hexagonal phase phospholipid dependence assay.

Protein S

A turbidometric clot-based assay.

If a deficiency is suggested, an antigen level can be measured for confirmation.

Protein C & Antithrombin

Chromogenic substrate assays in which the normal ability to cleave substrate molecules causes a color change.

If a deficiency is suggested, an antigen level can be measured for confirmation.

Activated Protein C Resistance

APC; a surrogate for the FVL mutation

An aPTT-based assay using the ratio of APTTs with and without additional APC.

If the ratio is decreased (<2), molecular testing is used as confirmation of FVL mutation.

Homocysteine Levels

Chemiluminescence immunoassay.

While the methylenetetrahydrofolate reductase (MTHFR) gene mutation may be confirmed by molecular methods, this usually is considered unnecessary.

Fibrinogen

Clauss variation of the thrombin time assay (clot-based).

Factor VIII

Clot-based assay.

C-Reactive Protein

Levels assist in determining whether Factor VIII and fibrinogen are elevated as part of an acute phase response.

Antigenic Testing

Specific Antibodies Against Cardiolipin

By ELISA assay.

If positive, antibodies against ß2 glycoprotein 1 are measured.

Protein S (Free & Total) Antigenic Testing

Testing may be performed to confirm and/or subtype a deficiency detected by a decrease in protein S functional activity.

Protein C Antigen Level

May be measured to confirm and/or subtype a deficiency detected by a decrease in protein C functional activity.

Antithrombin Antigen Level

May be measured to confirm and/or subtype a deficiency detected by a decrease in antithrombin functional activity.

Genetic/Molecular Testing

Prothrombin Gene (G20210A)

A single nucleotide polymorphism (SNP) in a regulatory region of the prothrombin gene (G20210A) accounts for most cases of elevated prothrombin.

This SNP is assayed by fluorescence melt-curve analysis.

Factor V Leiden

FVL may be confirmed (after a decreased APC-R result) by fluorescence melt-curve analysis.

References

1. Colman RW et al. Hemostasis and Thrombosis: Basic Principles and Clinical Practice, 5th Ed. Lippincott Williams and Wilkins (2006).

2. Heit J. Thrombophilia: Common Questions on Laboratory Assessment and Management. Hematology. 2007;127-35.

3. Kottke-Marchant K. An Algorithmic Approach to Hemostasis Testing. CAP Press (2008).