qPCR Is a Sensitive and Rapid Method for Detection of Cytomegaloviral DNA in Formalin-fixed, Paraffin-embedded Biopsy Tissue

Podsumowanie

This protocol describes qPCR detection of cytomegalovirus in formalin-fixed, paraffin-embedded biopsy tissue, which is rapid, sensitive, specific, and useful for interpreting equivocal hematoxylin and eosin or immunohistochemical staining patterns.

Streszczenie

It is crucial to identify cytomegalovirus (CMV) infection in the gastrointestinal (GI) tract of immunosuppressed patients, given their greater risk for developing severe infection. Many laboratory methods for the detection of CMV infection have been developed, including serology, viral culture, and molecular methods. Often, these methods reflect systemic involvement with CMV and do not specifically identify local tissue involvement. Therefore, detection of CMV infection in the GI tract is frequently done by traditional histology of biopsy tissue. Hematoxylin and eosin (H&E) staining in conjunction with immunohistochemistry (IHC) have remained the mainstays of examining these biopsies. H&E and IHC sometimes result in atypical (equivocal) staining patterns, making interpretation difficult. It was shown that quantitative polymerase chain reaction (qPCR) for CMV can successfully be performed on formalin-fixed, paraffin-embedded (FFPE) biopsy tissue for very high sensitivity and specificity. The goal of this protocol is to demonstrate how to perform qPCR testing for the detection of CMV in FFPE biopsy tissue in a clinical laboratory setting. This method is likely to be of great benefit for patients in cases of equivocal staining for CMV in GI biopsies.

Wprowadzenie

Interpretation of cytomegalovirus (CMV) infection in clinical specimens is critically important. CMV is a member of the betaherpesvirinae subfamily of herpesviruses. Similar to other herpesviruses, CMV has the ability to establish persistent or latent infection characterized by a lack of active viral replication¹. Reactivation of virus may occur during times of stress or other immunosuppression, leading to active viral replication characterized by virion release, which may be accompanied by disease manifestations^2-4. Gastrointestinal (GI) CMV disease symptoms frequently include abdominal pain and/or bloody stools¹.

The diagnosis of CMV gastroenteritis by histology utilizes hematoxylin and eosin (H&E) to identify CMV viral inclusions. In cases where clinical suspicion is high yet no obvious inclusions are observed, immunohistochemistry (IHC) is frequently used as an adjunct test method. However, IHC can also be hampered by rare non-classic appearing cellular staining patterns (equivocal), making interpretation difficult (Figure 2)⁵. It was sought to use DNA extracted from formalin-fixed, paraffin-embedded (FFPE) GI biopsy tissue and quantitative polymerase chain reaction (qPCR) to detect CMV in GI biopsies. This technique has been shown to be valuable in the detection of CMV in GI biopsies, and is particularly helpful in equivocal IHC staining cases^5,6. Also, qPCR has also been shown to correlate well with additional clinical CMV test data when it is available⁶. Use of qPCR in detecting CMV may lead to earlier diagnosis and treatment in cases where clinical suspicion for CMV is high, but H&E and CMV IHC are negative.

Protokół

With the approval of the Institutional Review Board, a search of the electronic laboratory database was performed to identify formalin-fixed, paraffin embedded (FFPE) blocks representing cases of cytomegalovirus (CMV) infection in gastrointestinal (GI) biopsies diagnosed by histopathology (hematoxylin and eosin (H&E) and/or immunohistochemistry (IHC)).

1. Tissue Processing from FFPE GI Biopsy Tissue

1. Collect FFPE GI biopsy tissue blocks for cytomegalovirus (CMV) quantitative polymerase chain reaction (qPCR). These blocks are stored at room temperature.
2. Pre-label 1.7 ml microfuge tubes with the block’s unique identification number.
3. Lock the microtome handwheel.
4. Seat the tissue block into the cassette clamp.
5. Using a fresh, unused blade, align the block to the knife angle on the microtome.
6. Adjust the microtome to cut 10 μm thick sections.
7. Unlock the handwheel and advance the block toward the blade.
8. Cut 5 sections of the block at 10 μm thick intervals, allowing each cut to roll into a scroll of tissue. Ensure that each scroll contains an adequate representation of the desired biopsy tissues.
9. Lock the microtome handwheel.
10. With forceps, place all 5 scrolls of tissue into the pre-labeled centrifuge tube, being careful to only manipulate the scroll by the paraffin in order to reduce potential for cross contamination. Snap the cap on the tube and place it back on the rack.
11. Remove the tissue block from the microtome.
12. Remove and discard the blade.
13. Decontaminate any surfaces or instruments that may have come in contact with the previous tissue.
14. Place a fresh, unused blade into the knife holder.
15. Repeat steps 1.4-1.14 for each additional block to be processed. The microfuge tubes containing the tissue scrolls may be stored at room temperature until the time of DNA extraction.

2. DNA Extraction from Scrolls of FFPE GI Biopsy Tissue

NOTE: This protocol is adapted from the package insert of the FFPE tissue DNA extraction kit(CAUTION: refer to Material Safety Data Sheet (MSDS)).

1. Add 1 ml xylene (CAUTION: Refer to MSDS) to each sample microfuge tube. Close the lid and vortex vigorously for 10 sec.
2. Proceed with the DNA extraction as specifically outlined in the manufacturer’s product insert with the following modifications.
3. First add 6 μl of the internal control (IC) to each microfuge tube.
   1. Following the ATL/proteinase K lysis incubation at 56 °C for 1 hr, incubate at 99 °C for 30 min, rather than 90 °C for 1 hr.
   2. During the final DNA elution step, carefully open the lid of the DNA purification column and apply 50 μl distilled DNAse/RNAse-free water at the center of the membrane, rather than buffer ATE.
      NOTE: Refer to the package insert for the appropriate storage and preparation of reagents. DNA should be extracted from all 5 FFPE scrolls. Perform all centrifugation steps at room temperature (15-25 °C).

3. qPCR for CMV

1. Polymerase Chain Reaction (PCR) Procedure
   1. Generate an Excel PCR worksheet for the CMV assay run. This Excel worksheet calculates the total amount of each reagent needed to make up the master mix with Mg. It multiplies the amount of each reagent needed per sample (listed below) times the number of samples and controls plus one (to compensate for loss due to pipetting).
      1. Include the following reagents in each PCR reaction in the amount indicated:
         Reagent                                      Amount per Sample
         CMV LC master mix vial               12.5 μl
         Mg Solution yellow vial                 2.5 μl
         Total volume                                15.0 μl
   2. Include in every assay, run a water or no target control (NTC), negative control, a low positive control, a high positive control, and either QS3 or QS4, and all patient samples. The capillaries should be set up in this order.
   3. Obtain the cooling block specific for the real-time PCR instrument that is stored at 4 °C. Place the appropriate capillary tubes in the cooling block. Do not touch the surface of the capillary tubes. Always use gloves when handling the capillaries.
   4. Set up all polymerase chain reactions in a clean hood not contaminated with amplification products.
      1. Remove the correct number of master vials from the PCR kit to test all patient samples, plus 5 controls. Let the master vials, Mg solution, and PCR grade water thaw in the cooling block. Each master vial contains enough reagents for 12 tests.
      2. Gently vortex the master vials and quick spin them in the centrifuge at the maximum speed.
      3. Combine the contents of each master vial into one vial, and gently vortex again.
      4. Combine the master mix and Mg solution in the amounts indicated on the PCR worksheet into a single sterile microfuge tube.
      5. Mix gently and aliquot 15 μl of master mix with Mg into each capillary tube, except the capillary in position 5 which is for the quantitation standard (QS).
   5. Move the cooling block containing the capillary tubes and master mix with Mg from the clean hood to a processing hood.
   6. Add 2 μl of internal control (IC) to the remaining 30 μl of master mix with Mg. Mix gently and aliquot 15 μl into the capillary in position 5 (position for the QS).
   7. Pipette 10 μl of water, control DNA, or sample DNA into the appropriate capillary tube. Cap the capillary tubes using the capping tool.
   8. Take cooling block/samples to the real-time PCR instrument.
2. Program the real-time PCR Instrument (see Table 1)
3. Amplification and Detection performed on the real-time PCR instrument
   1. Log onto the computer
   2. Double click on the instrument icon and log into the software.
   3. Turn on the real-time PCR instrument
   4. Click on “CMV/EBV” from the front screen.
   5. Follow the prompts of the macro wizard.
   6. Select the box to run a self-test when prompted by the software. A self-test must be performed once each day the instrument is in use.
   7. Name the run.
   8. Adjust the sample count located in the upper left corner of the page to the sum of controls and patients included in the run and enter the identification number of each patient sample.
   9. Click on the “Abs Quant” tab.
   10. Under “Sample type”, assign the appropriate sample type for the standard.
   11. Type in the expected concentrations for the quantitation standard (QS).
   12. Remove the capillary tubes from the cooler block and place them into the corresponding position in the carousel (cooling block sample #1 should be placed in position #1 on the carousel, etc.).
   13. Place the carousel into the instrument’s centrifuge and press start.
   14. Open centrifuge and remove the carousel.
   15. Place the carousel into the real-time PCR instrument. Close the lid.
   16. Press “Start Run”.
   17. The real-time PCR instrument will check each position for a sample. Do not walk away from the instrument until this check has been completed, especially if one of the capillaries is not in the carousel. The instrument will note this and ask if the run is to be continued. Answer yes before leaving.
   18. Once the run is complete, close the report that was generated.
   19. Click on “Absolute Quantitation”.
   20. Click on the arrow next to Color Compensation.
   21. Click “Select Color Compensation” from the drop-down box.
   22. Choose the most current color compensation file to apply to the run.
   23. Click OK when the box pops up.
   24. Click on the arrow by Standard Curve.
   25. Select “use external” from the drop-down menu.
   26. Select the most current external standard curve file, then click OK.
   27. Look at each curve under the Absolute Quantification menu to ensure that curves are present and not flat line, if a concentration is assigned. The Absolute Quantification for the 705/Back 530 is automatically analyzed.
4. Quality control
   1. Generate a new standard curve whenever a new lot number of reagents is received, or every six months, whichever is more frequent.
   2. Add 10 μl of each standard to the master mix with Mg. Consider the standards included with the kit as previously extracted DNA.
   3. Add 1 μl of internal control for each standard to the master mix with Mg used in generating the standard curve in order to assure accurate quantitation. Do not add IC to the master mix with Mg for the No Template Control (NTC).
   4. Prepare a NTC and three replicates of Quantitation Standards QS 4, 3, 2, 1, and a 1:10 dilution of QS4 for the run to generate the standard curve, totaling in 16 data points.
   5. Select “replicate of” in the drop down menu for repeated standards
   6. Choose channel 530/back none and turn on color compensation when analyzing the run,
   7. Under “Standard Curve (In Run)”, select “save as external” and name the curve. Enter the comment “applied as standard curve” when saving.
   8. Graph the results. The resulting linearity coefficient must be ≥0.81.
   9. For daily runs, include one copy of QS3 or QS4 as a control in the run. The current external standard curve may be imported into the run during the analysis phase.
   10. Run Controls: no DNA control, negative control, low, and high positive controls. Expected viral load is determined and adjusted for each vendor and lot number.
   11. Add an IC to each sample and control during the DNA extraction.
   12. If any of the controls fall out of range, record the unacceptable results and refer the assay for troubleshooting.
5. Frequency of controls
   1. Include in every run: a NTC, negative, low positive, high positive, and standard control.

NOTE: Acceptable limits for controls. No peaks should be observed for the no template (NTC) and negative controls. Peaks should be present in the 530 channel and expected viral loads are determined and adjusted for each vendor and lot number for the low and high positive controls.

Wyniki

A total of 228 tissue blocks were tested by quantitative polymerase chain reaction (qPCR), which was comprised of 91 cytomegalovirus (CMV) positive cases based on histology and positive immunohistochemistry (IHC), 18 with equivocal CMV IHC, and 79 negative controls. As illustrated in Figure 2, CMV positive cases would have demonstrated typical CMV viral inclusions (A) and/or positive IHC staining (B). Equivocal cases would have demonstrated rare, non-classic appearing st...

Dyskusje

Many laboratory methods are available for the diagnosis of cytomegalovirus (CMV) infection, including serology, viral culture, molecular viremia assays, histology of biopsy material, and, as recently demonstrated, molecular assays of formalin-fixed, paraffin-embedded (FFPE) biopsy tissue^5,6. Serology, once a mainstay of diagnosis, only reliably identifies exposure and does not correlate well with acute CMV infection⁷. Viral culture, both conventional and early antigen shell vial, is hampered by leng...

Materiały

Name	Company	Catalog Number	Comments
1.7 mL microfuge tubes	Costar	3620
serrated forceps	Electron Microscopy Services	62086-1S	Micro Forceps MF-1
Tabletop centrifuges (microfuge)	Eppendorf	5424
Xylene	Fisher	Fisher Scientific: X3P	1 gallon
Ethanol	Fisher	Fisher Scientific: ET108	96-100%
microtome	Leica	RM2255
QIAamp DNA FFPE Tissue Kit	Qiagen	56404
artus CMV LightCycler PCR ASR	Qiagen	4500025
CMV LC PCR Supplement Kit	Qiagen	1031873
LightCycler centrifuge	Roche	75005087
LightCycler Cooling Block	Roche	10800058001
LightCycler Capping Tool	Roche	3357317
Color Compensation Kit	Roche	2158850
LightCycler 20 μL Capillaries	Roche	1 909 339
blades	Sakura	Fisher Scientific: 4689	Accu-Edge low profile