Parasitology

METHOD PROS AND CONS (Smear Preparation Directly from Vial)

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.

Introduction:

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).

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

 
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).

ADDITIONAL INFORMATION PER PARASITOLOGY PRODUCTS