WS/T 632-2018Detection of Babesia spp. - Blood smear microscopy examination method (English PDF)

4.1 Optical microscope (100× oil mirror, 10× eyepiece)

4.2 Blood smear staining rack

4.3 Blood smear drying rack

4.4 Slide box

4.5 Dyeing tray and dyeing tank

4.6 Counter

5.1 Gibbs staining solution (Giemsa)

5.2 PH7.2 Phosphate Buffer (PBS)

5.3 Standard Slides (Clean Slides, or Adhesive Slides with Positive Charges on the Surface)

5.4 Methanol (analytical grade)

5.5 Cedar oil or special immersion oil (refractive index >= 1.5)

5.6 Xylene (analytical grade)

5.7 Glycerin

6.1 Production of blood smear

6.1.1 Blood collection site and blood collection method

Blood is taken from the tip (the fingertip or the earlobe). Use a 75% alcohol cotton ball to disinfect the blood collection area. After the alcohol is evaporated, use your thumb and forefinger to tighten.

Pinch the top of the earlobe or the fingertip of the ring finger, and hold the disposable blood collection needle in the other hand to quickly penetrate the skin and gently squeeze the bleeding. Babies can be from the big toe or the heel is stabbed to take blood.

6.1.2 Thick film production

Take a sterilized slide (or adhesive slide) as a pusher, use a corner of the pusher to take 4µL ~ 5 µL of blood to make the blood and the other 1 the flat glass slide (or adhesive slide) is placed in the center-right position and rotates from the inside to the outside. Draw 2 to 4 turns and coat it to a diameter of 0.8 cm.

Round thick thick film of 1 cm size (see Appendix A).

6.1.3 Thin film production

Dry the cotton ball to remove the blood stain from the lower corner of the pusher. Use the middle of the same pusher to take about 1 µL~1.5µL of blood. Place blood on the left side of the thick film with a margin of about 0.3 cm, the edge of the pusher is placed against the slide, and the upper and lower sides are gently moved to spread the blood along the edge of the pusher to a width of 2 cm.

The slides are kept at an angle of 25° to 35°, and the push piece is pushed forward from right to left to push the tongue into a thin blood film (see Appendix A).

6.1.4 Number

After the blood smear is prepared, place it horizontally, and after drying thoroughly, mark the number and date on the frosted glass on one side of the slide or on the side near the thick film.

period.

6.2 Immobilization and hemolysis

6.2.1 Thin film fixation

Place the thin film at a 45° angle at one end, draw the methanol solution with a disposable pipette, and evenly apply it on the surface of the thin blood film to avoid touching the thick blood.

membrane.

6.2.2 Thick blood film hemolysis

A few drops of distilled water were added to the dried thick blood film to completely cover the blood film, and the blood was dissolved for several minutes. When the blood film was light gray, the hemolyzed blood was decanted.

6.3 Gill's stain

6.3.1 Preparation of Gill's dye solution

The commonly used Gill's dye liquor contains 2%, 3%, and 10% three concentrations, which are buffered by Gill's dye solution and pH 7.2 phosphate.

The liquid (PBS) is prepared in proportion, and can also be purchased in proportion to commercialized Gill's dye solution plus experimental pure water (see Appendix B).

6.3.2 Gibbs dyeing

See Appendix B for dyeing methods.

6.4 Microscopy

Add 1 drop of cedar oil or special immersion oil to the stained blood smear, using 100× (times) oil mirror, 10×(times) eyepiece optical microscopy Mirror inspection. The order of the blood film is observed as the thin blood film starts from the tip of the tail and the thick blood film starts from the upper or lower end (see Appendix A).

6.5 Result determination

6.5.1 Babesia test positive

Babesia was found to be positive in the blood membrane (see Appendix C).

6.5.2 Babesia insect infection rate count

6.5.2.1 Babesia infection rate counting method for thin blood film

The thin blood membrane was examined under a microscope, and the total number of infected red blood cells and red blood cells in each field was more than 2,000. Calculate the bar by the following formula Beetle insect infection rate.

Babesia insect infection rate = Total number of red blood cells Infected red blood cells × 100%.

6.5.2.2 Abdominal blood staining of Babesia

If the rate of infection is less than 0.1%, the whole thick blood film is examined under the microscope, and the number of Babesia and red blood cells of the whole thick blood film are counted.

total. The rate of insect infection of Babesia was calculated by the following formula.

The infection rate of Babesia = × 100% (the number of red blood cells is 5 million/µL for men and 4.5 million for women) /µL blood calculation).

Appendix A

(normative appendix) Operation diagram

Figure A.1 Schematic diagram of the manufacturing process of thick and thin blood film blood smear

Figure A.2 Schematic diagram of the production of thick blood film blood smear

Figure A.3 Schematic diagram of a thick blood film smear

Appendix B

(normative appendix) Reagent preparation and blood smear staining and preservation method B.1 Gill's staining solution preparation Put 5.0 g of Gill powder in a mortar, add a small amount of glycerin and grind it thoroughly, then add side grinding until 250 mL of glycerin is added.

Pour into a 500 mL stoppered dark glass bottle. Add a small amount of methanol to the mortar, wash off the remaining part, pour into the bottle, add methanol again, wash Then pour into the bottle and wash the glycerin in the mortar in 250 mL of methanol. Keep it out of the air and keep it out of direct sunlight. Plug the stopper tightly to avoid evaporation and oxidation caused by high humidity. Shake the solution vigorously for 5 min every day, and use it after 3 days. The longer the storage time, the better the dyeing effect. According to the day Often, use a dry pipette to draw a small amount of dye solution into a closed vial (approximately 25 mL). Never add water to the stock solution.

B.2 Preparation of pH 7.2 Phosphate Buffer (PBS) Weigh 8.0 g NaCl, 0.2 g KCl, 0.24 g KH2PO4 (or 1.44 g Na2HPO4) and 1.8 g K2HPO4, dissolved in in 800 ml of distilled water, adjust the pH of the solution to 7.2 with HCl, and finally dilute to 1 L with distilled water. Store in a refrigerator at 4 ° C for later use.

B.3 Gill's dyeing working fluid preparation B.3.1 Preparation of 2% Gills dyeing solution Add 2 mL of Gill's stain solution to 98 mL of pH 7.2 phosphate buffer and mix, or add 98 mL of experimental water to the solution.

mL of commercial Gill's stained stock solution and mix well.

B.3.2 Preparation of 3% Gill's dye solution Add 3 mL of Gill's stain solution to 97 mL of pH 7.2 phosphate buffer and mix, or add 97 mL of experimental water to the solution.

mL of the product Gill's dye solution and mix.

B.3.3 Preparation of 10% Gie's dyeing solution Add 10 mL of Gill's stain solution to 90 mL of pH 7.2 phosphate buffer and mix, or add 90 mL of experimental water.

10 mL of the commercial Gill's solution is mixed and mixed.

B.3.4 Precautions

Do not shake the dyed stock bottle when drawing the Gill's stain solution. Gill's dyeing working fluid should be used now, do not use unused Gills the dyeing working solution is poured back into the original liquid bottle.

B.4 Single blood smear staining Single blood smear Gibbs staining is commonly used for the detection of Babesia microscopic examination in patients with clinical Babesia. Dyeing with 3% Gill dye Min or longer. The stained blood smear can be stored for a long time. Can also be dyed with 10% Gill dye for 8 min to 10 min, but the stained blood smear is not suitable for long-term storage.

B.4.1 3% Gill dye staining B.4.1.1. After the methanol-fixed thin blood film smear is sufficiently dried, the blood film is placed horizontally upward in the dyeing tray.

B, 4.1.2. Use a pipette to draw about 3 mL of the newly prepared 3% Gill dye solution, and add it to the thick and thin blood film until the dye solution evenly covers the blood.

Membrane, does not overflow.

B.4.1.3. Static staining for about 30 min.

B.4.1.4. Move the staining tray to the flushing tank and rinse with the slow running water along the upper edge of the blood smear for 1 min.

B.4.1.5. Insert the stained blood smear into the blood sample drying rack with the blood film facing down and dry.

B.4.2 10% Gill's dye fast dyeing method B.4.2.1. After the methanol-fixed thin film smear is sufficiently dried, the blood film is placed horizontally on the staining plate.

B.4.2.2. Pipette a freshly prepared 10% Gill's dye solution to about 3 mL, and add it to the thick and thin blood film until the dye solution evenly covers the blood film.

Do not overflow.

B.4.2.3. Static staining for 8 min ~ 10 min.

B.4.2.4. Move the staining tray to the flushing tank and rinse with slow running water for 1 min along the upper edge of the blood smear.

B.4.2.5. Insert the stained blood smear into the blood sample drying rack with the blood film facing down and dry.

B.5 Batch blood smear staining Batch blood smear staining is commonly used for Babesia microscopic examination in population epidemiological investigations, generally using 2% Gill dye solution for batch Blood smear staining.

B.5.1. Insert the blood film of each blood smear into the dyeing cylinder in one direction, or insert the blood film of each pair of blood smears outward into the dyeing cylinder.

B.5.2. Pour the newly prepared 2% Gill dye solution into the thick, thin blood film.

B.5.3. Static staining for more than 30 min.

B.5.4. Injecting tap water into the dyeing tank to overflow, remove the dross on the surface of the dyeing liquid, pour out the residual dyeing liquid in the dyeing tank, and add it.

Water, rinse slowly 2 to 3 times.

B.5.5. Remove the blood smear and insert the blood film face down into the blood sample drying rack and let it dry.

B.6 Blood smear preservation Absorb the cedar oil on the surface of the blood film of the blood test smear with absorbent paper, add 2 drops to 3 drops of xylene on the blood film, and then absorb it with absorbent paper.

dry. Place the blood smear in the slide box, away from light, dry and cool, for review. Note. special immersion oil does not need to be cleaned with xylene.

Drain directly with absorbent paper.

Appendix C

(informative appendix) morphology C.1 Form Babesia has a variety of morphology when parasitized in red blood cells. Common worm morphology is ring, round, rod, point, pear, and Ami Ba shape and so on. The typical form is pear-shaped, often with multiple parasites in one red blood cell, mostly in one to four worms, forming a triple the body or quadruplet type, that is, the Maltese cross, and can be a worm body of different developmental stages. After Gy staining, the cytoplasm is blue and the nucleus is purple color.

Figure C.1 Bone worm in the thin blood film

Figure C.2 Differences in Babesia in thin blood membranes (from Mørch K, Holmaas G, Frolander, et al. Severe human Babesia

Divergens infection in Norway.Int J Infect Dis, 2015, 33.37-38)

Figure C.3 Duncan Babesia in a thin blood film

Figure C.4 Orion Babesia in a thin blood film (from Sun Y, Li SG, Jiang JF, et al. Babesia venatorum Infection)

In Child, China.Emerg Infect Dis, 2014, 20(5). 896-897) references [1] Liu Enyong, Zhao Junlong. Babesiasis (first edition) [M]. Wuhan. Hubei People's Publishing House, 2001, 21-30.

[2] Wu Guanling. Human Parasitology (4th Edition) [M]. Beijing. People's Medical Publishing House, 2013, 233-237.

[3] WS/T 569 Plasmodium detection blood smear microscopy.

[4] Zhou X, Xia S, Huang JL, et al. Human babesiosis, an emerging tick-borne disease in the People's Republic of China [J]. Parasites [5] Mørch K, Holmaas G, Frolander, et al. Severe human Babesia divergens infection in Norway [J]. Int J Infect Dis, 2015, 33.37-38.

[6] Sun Y, Li SG, Jiang JF, et al. Babesia venatorum Infection in Child, China [J]. Emerg Infect Dis, 2014, 20(5). 896-897.

[7] WS/T 564 Babesia diagnosis.