The TOTAL- FIX® stool collection kit is a single vial system that provides a standardized method for untrained personnel to properly collect and preserve stool specimens for the detection of helminth larvae and eggs, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores. Concentrations, permanent stains, and most fecal immunoassays can be performed from a TOTAL- FIX® preserved specimen. TOTAL- FIX® is a mercury, formalin, and PVA free FIX®ative that preserves parasite morphology and helps with disposal and monitoring problems encountered by laboratories.
1. What is TOTAL- FIX® ?
TOTAL- FIX® is a FIX®ative with a proprietary formula, it is similar to UniFIX® and Zinc PVA (Z- PVA ), a commonly used fixative that has been commercially available and used in many laboratories since 1992.
2. What is PVA ?
PVA stands for polyvinyl alcohol, a plastic powder/resin that is incorporated into the fixative (Schaudinn’s or other fixatives such as Z- PVA ) and serves as an adhesive to “glue” the stool material onto the slide. PVA itself has no preservation capability.
3. Since PVA is used to “glue” the stool onto the slide prior to permanent staining, how does the material stick to the glass slide?
The TOTAL- FIX® user can add some albumin to the slide prior to preparation of the stool smear for staining. However, if the stool smear is thoroughly dry prior to staining, albumin will not be necessary.
4 How long should the slides prepared from TOTAL- FIX® be dried prior to processing for routine staining (trichrome or iron-hematoxylin)?
Spread the sample over the slide to prepare a thin smear which varies in thickness (routine preparation of slides – no changes required). Allow to dry overnight at room temperature or for several hours (minimum of 30 min; 60 min if slide is thicker) in a 37oC incubator or slide warmer (the smear will appear opaque when dry). Many labs routinely prepare their smears and then place the smears on a tray in the incubator (37ºC) for 60 min to dry. The fecal smears can be left longer in the incubator with no damage at all to the slides. Do not use a heating block; the higher temperature for most heating blocks will be detrimental to any organisms (protozoa, coccidia, microsporidia) present.
5. What should Giardia lamblia and other protozoa look like when stained from normal patient clinical specimens (no lag time between specimen passage and fixation)?
Organism morphology should appear no different from organisms preserved in Z- PVA , with good overall morphology for trophozoites, cysts (routine trichrome or iron-hematoxylin), coccidian oocysts (modified acid-fast stains), and microsporidial spores (modified Trichrome).
ALGORITHM FOR THE USE OF TOTAL-FIX® (UNIVERSAL FIXATIVE)
MEDICAL CHEMICAL CORPORATION, (www.Med-Chem.com)
(1). SPECIMEN VIAL CONTAINING TOTAL- FIX AND FECAL SPECIMAN
(Ratio of 1/3 stool, 2/3 fixative – MIX WELL )
(Allow to fix 30 min before processing)
(2). USE THE FLUID AT THE TOP OF THE VIAL TO RUN THE IMMUNOASSAYS
(Some kit directions recommend shaking the vial. After shaking, allow particulate matter to settle, and then use the fluid at the top for the immunoassays). Use centrifuged specimen for DFA and special stains (coccidia, microsporidia) (Step 7)
(3). TUBE OR CONCENTRATION DEVICE
(Add approximately 1-2 mL fixative/specimen mix to the tube or device)
(Mix well prior to pouring into the tube or device)
(DO NOT ADD ANY RINSE FLUIDS: Saline, water, formalin)
(Rinse fluids will prevent routine permanent staining.)
(10 min at 500 Xg)
(5). POUR OFF MOST OF EXCESS FIXATIVE
(6). MIX SEDIMENT WITH REMAINING FIXATIVE
(7). PREPARE SLIDES FOR PERMANENT STAINS and/or DFA; STAIN SLIDES
(Allow slides to dry for ~30-45 min at 37ºC)
(Slides can be placed on trays and put in the incubator)
(Room temperature drying requires ~60 min)
(8). ADD RINSE FLUID TO REMAINING STOOL SEDIMENT (FROM STEP 5)
(Mix sediment and rinse fluid well before centrifugation)
PROCEED WITH ROUTINE CONCENTRATION PROCEDURE
(Single rinse including Ethyl Acetate step is recommended)
(DO NOT ADD Ethyl Acetate if the specimen contains a lot of mucus)
(9). RING DEBRIS LAYER, POUR OFF EXCESS FLUID
MIX AND EXAMINE SEDIMENT AS CONCENTRATION SEDIMENT SLIDE
Entire 22 x 22mm coverslip: low power (10X objective)
1/3 – 1/2 coverslip: high dry power (40X objective)
*ALTERNATE METHOD FOR SMEAR PREPARATION DIRECTLY FROM VIAL
Allow stool to settle to the bottom of the vial.
Using a pipette (without creating bubbles), remove fecal material from the bottom of the vial. Smear this fecal material onto the glass slide for subsequent permanent staining (after the slides are allowed to dry for ~30-45 min at 37ºC or ~60 min at room temperature). Allow stool to settle to the bottom of the vial.
METHOD PROS AND CONS (Smear Preparation Directly from Vial)
|Eliminates one centrifugation step from the algorithm – however, this centrifugation step (Step 4) ensures sufficient stool and concentrates the stool prior to smear preparation.||If the specimen is thin/runny/very little stool, there is a chance the slide may not contain sufficient stool material for staining and/or organism identification.|
|If bubbles are created with the pipette, the fecal material will have to settle out again before taking the sample.|
NOTE: In order to ensure maximum organism recovery and the best overall results, the algorithm should be used as originally written and recommended. However, with practice, sufficient fecal material can be taken from the vial and used to prepare smears for permanent staining.
Total-Fix patent number 8,338,130.
TOTAL-FIX® VALIDATION STUDIES FOR THE ROUTINE LABORATORY
The TOTAL-FIX® (Universal Fixative, Medical Chemical Corporation, Torrance, CA) stool collection kit is a single vial system that provides a standardized method for untrained personnel to properly collect and preserve stool specimens for the detection of helminth larvae and eggs, protozoan trophozoites and cysts, coccidian oocysts, and microsporidian spores. Concentrations, permanent stains, and most fecal immunoassays can be performed from a TOTAL-FIX® preserved specimen. TOTAL-FIX® is a mercury, formalin, and PVA free fixative that preserves parasite morphology and helps with disposal and monitoring problems encountered by laboratories. TOTAL-FIX® is a fixative with a proprietary formula and is similar to Unifix and Zinc PVA (Z-PVA), a commonly used fixative that has been commercially available and used in many laboratories since 1992. PVA stands for polyvinyl alcohol, a plastic powder/resin that has been incorporated into the fixative (Schaudinn’s or other fixatives such as Z-PVA) to serve as an adhesive to “glue” the stool material onto the slide. PVA itself has no preservation capability and is inert. However, if the TOTAL-FIX® stool smear is thoroughly dry prior to staining, PVA will not be necessary and can be eliminated.
The approach to validation of TOTAL-FIX® is based on the user’s current fecal fixative. Various fecal fixatives and the recommended validation approach are indicated below. See also Table 1.
QUALITY CONTROL SPECIMEN USED FOR VALIDATION STUDIES
Fixatives for fecal specimens are checked for quality control by the manufacturer before sale, generally with the use of living protozoa. If you prepare your own fixatives, the following approach can be used for quality control. The specimen used for quality control presented below is designed to be used with fixatives from which permanent stained smears will be prepared (Universal Fixatives, Schaudinn’s fluid, fixatives with/without PVA, fixatives with a mercury base, fixatives with a zinc base, SAF, or MIF). The same quality control specimen can also be used in a concentration; the white blood cells (WBCs) can be seen in the concentrate sediment (sedimentation concentration) or in the surface film (flotation concentration).
1. Add approximately 2 g (size of 2 medium-large grapes) of soft, fresh fecal specimen (normal stool, containing no parasites) to a vial of the fixative(s) being tested. MIX WELL.
2. Obtain a fresh, anticoagulated blood specimen (lavender top, EDTA, 7 ml specimen), centrifuge, and obtain a buffy coat sample (try and find a specimen with a high WBC count).
3. Add the buffy coat specimen to the fixative/stool mixture (TOTAL-FIX®/stool) and the second fixative to be tested (your current fecal collection vial). MIX WELL.
4. Allow 30 min for fixation, and then prepare several fecal smears (as you normally would). Allow the smears to dry thoroughly (60 min) at room temperature or 30 min in an incubator (approximately 35°C). Do NOT use a heat block, and do not make the smears too thick.
5. Stain the slides by the normal staining procedure (trichrome, iron-hematoxylin).
6. After staining, if the WBCs appear well fixed and display typical morphology and color, one can assume that any intestinal protozoa placed in the same lot number of preservative would also be well fixed, provided that the fecal sample was fresh and fixed within the recommended time limits.
7. This bulk quality control specimen can be concentrated as for a normal patient specimen. If the fixative is performing correctly, the WBCs will be visible in the concentration sediment or surface film (depending on the method used).
8. Record all quality control results. If the WBC morphology does not confirm good fixation, describe the results and indicate what corrective action procedures were used (repeated the test, prepared new fixative).
TABLE FOR VALIDATION STUDY (PERMANENT STAINED FECAL SMEAR)
|Step 1||Prepare Quality Control specimen as indicated above, using three different stool specimens and three different buffy coat layers||3 different specimens (stools, EDTA)||You will have 3 vials of TOTAL-FIX® QC and 3 vials of your regular fixative QC|
|Step 2||Prepare and stain 2 slides from each of the six vials
6 slides from TOTAL-FIX®
6 slides from your regular fixative
|6 slides from the TOTAL-FIX® vials and 6 from your regular fixative QC vial||You will have a total of 12 slides for routine staining (trichrome or iron-hematoxylin)|
|Step 3||Examine stained smears and record results (descriptions of WBCs); nuclear and cytoplasmic colors; lobed nuclei, any granules if present (eosinophils, or basophils), and overall morphology||12 smears total||Have 2 different people examine the slides for comparison|
Note: If your regular fixative is Unifix or Zinc PVA (Z-PVA) (Medical Chemical Corporation), the proprietary formulations are essentially the same and removal of the inert PVA plastic powder has no impact on organism morphology. In this case, validation would be at the discretion of the user, but should not be required. No organism differences are seen in fecal concentrations (larvae, eggs, oocysts).
If anything, the removal of PVA will provide a clearer fecal smear with less background haze, thus making the microscope examination easier than before.
REMEMBER, THE FECAL SMEARS MUST BE THOROUGHLY DRY PRIOR TO STAINING.
A. Room temperature – 60 min or overnight
B. Incubator (standard bacteriology incubator): 30-60+ min; drying beyond 60 min will not be detrimental to the smears
TABLE FOR VALIDATION STUDY (PERMANENT STAINED FECAL SMEAR)
|Step 1||Save positive fecal specimens (Giardia and/or Cryptosporidium) for fecal immunoassay(s) (EIA, FA, or rapid cartridge formats)||3 different specimens||You will have 3 vials of your regular fixative that are positive|
|Step 2||Obtain positive TOTAL-FIX® fecal specimens (Giardia and/or Cryptosporidium) for fecal immunoassay(s) (EIA, FA, or rapid cartridge formats)||3 different specimens||You will have 3 vials of TOTAL-FIX® fixative that are positive|
|Step 3||Perform duplicate runs using your regular fecal immunoassay format from the 3 vials (current fixative – known positives) and the 3 vials (TOTAL-FIX® – known positives)||Record results for the duplicate runs on the 6 positive vials: total of 12 IA results||Have 2 different people perform the testing (one per run of 6 tests, the other per run of the 6 duplicates)|
TOTAL-FIX® FIXATIVE AND COMPATIBILITY WITH VARIOUS IMMUNOASSAYS
The TOTAL-FIX® (Patent Pending) fixative is manufactured by Medical Chemical Corporation and is a single vial system for the preservation of fecal specimens. TOTAL-FIX®-preserved fecal specimens can be used for fecal concentrations, permanent stained smears (trichrome or iron-hematoxylin; special stains for coccidia and microsporidia), and certain fecal immunoassays for Cryptosporidium spp. and Giardia lamblia. TOTAL-FIX® (2807-05, 100 vials/case) is available from Medical Chemical Corporation (www.Med-Chem.com).
PARA-TECT Giardia EIA
|Medical Chemical Corp.|
|PARA-TECT Giardia EIA||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|PARA-TECT Cryptosporidium EIA||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|PARA-TECT Giardia/Cryptosporidium DFA||Compatible with TOTAL-FIX®||Perform test on concentrate sediment (specimen rinsed with saline or formalin)|
|SIMPLE-READ Giardia (rapid)||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|Xpect Cryptosporidium RAPID||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|Xpect Giardia RAPID||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|Xpect Giardia/Cryptosporidium RAPID||Compatible with TOTAL-FIX®||Perform test on concentrate sediment (specimen rinsed with saline or formalin)|
|ProSpect (EIA) Cryptosporidium||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|ProSpect (EIA) Giardia||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|ImmunoCard STAT Crypto/Giardia RAPID||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|MERIFLUOR Cryptosporidium/Giardia DFA||Compatible with TOTAL-FIX®||Perform test on concentrate sediment (specimen rinsed with saline or formalin)|
|Cryptosporidium II EIA||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
|Giardia II EIA||Compatible with TOTAL-FIX®||Use clear liquid from the top of the vial (antigen in solution)|
ADDITIONAL INFORMATION PER PARASITOLOGY PRODUCTS
|ProSpecT Entamoeba histolytica EIA||Fresh, frozen, or Cary Blair||Suspect this is still not compatible with TOTAL FIX®, as well as with other fixatives|
|Entamoeba histolytica II EIA||Fresh or frozen specimens||Suspect this is still not compatible with TOTAL FIX®, as well as with other fixatives|
|Test for the Entamoeba histolytica/E. dispar group no longer available|
|Triage Rapid Cartridge||Fresh or frozen specimens||Giardia, Cryptosporidium, Entamoeba histolytica/E. dispar|
COMMENTS REGARDING PREPARATION OF FECAL SMEARS FOR PERMANENT STAINING [TRICHROME, IRON-HEMATOXYLIN, MODIFIED ACID-FAST (COCCIDIA), MODIFIED TRICHROME (MICROSPORIDIA)] FROM TOTAL-FIX®
In order to increase the sensitivity of the permanent stained smear, the fecal smears can be prepared from concentrated stool sediment. HOWEVER, if the concentrated sediment has been rinsed with saline and/or formalin, one may need to use albumin to “glue” the material onto the slide prior to staining. If the fixative used contains PVA (plastic powder used to glue the stool onto the slide), then albumin may not be necessary. HOWEVER, AGAIN, if rinses have been performed using saline/formalin, then the PVA has been rinsed out.
Some of the fixatives that do not contain PVA (single vial systems that can be used for the O&P, as well as the fecal immunoassays, including TOTAL-FIX®), will also stick to the slides if the specimen is centrifuged and the slides prepared from the first centrifugation sediment (no rinse fluid used). Then, the full concentration with rinses can be performed. Also, these slides can be dried for two hours in the incubator or overnight at room temp. If prepared and dried using these recommendations, the stool will not fall off the slides during staining (even without PVA or albumin). Each laboratory can try the albumin vs no albumin approach to see if their method requires extra “glue” for adherence of the fecal material to the slide (similar to the approach used for sodium acetate-acetic acid-formalin (SAF). TOTAL FIX® does not contain PVA, so specimens collected in this fixative can also be used for fecal immunoassay testing (see table above).
Remember, specimens collected in this fixative can also be used for fecal immunoassay testing (see table above).
*If the vial is shaken prior to testing, allow the large fecal debris to settle out before taking the specimen for immunoassay testing.