Kamis, 25 Agustus 2011

BRONCHO-ALVEOLAR LAVAGE (BAL) - CMV SURVEILLANCE

Figure6 I. Introduction

Broncho-alveolar lavage specimens (BAL) for CMV surveillance collected on day 35 post-bone marrow transplant patients or routine screening of solid organ transplant patients are examined for CMV only.  http://bahankuliahkesehatan.blogspot.com/ 

 

 

II. Collection and Transport

BAL fluid should be collected into a clean, sterile container. If a delay in transport or processing is anticipated, keep the specimen at 4 oC.

 

III. Procedure

A. Processing of Specimen:

a) Approximately 2-3 mL of centrifuged sediment should be received from the specimen receiving planting area.

b) Transfer 2 mL of sediment to a sterile freezer vial containing 4 drops (0.2 mL) gentamicin (1 mg/mL) and 2 drops (0.1 mL) of fungizone (250 mg/mL) to a final concentration of 100 mg/mL and 10mg/mL respectively.

c) Allow to stand at room temperature for 10 minutes

d) Refer to Appendix II for Shell Vial inoculation.

 

B. Direct Examination:

Not done.

 

C. Isolation and Identification:

Method

Cell Linesa

Incubation at 36oC

Stainb used

Shell Vial

MRC-5

2 days

CMV-IE

aMRC-5 = Human fibroblast cells

bCMV-IE= Monoclonal antibody IFA stain for CMV immediate early antigen

Refer to Appendix II for Shell Vial staining and interpretation.

D. Interpretation and Processing of Cultures:

a) At 48 hours observe shell vial coverslips using the inverted microscope:

i) If <50% or no CPE - Perform DFA for CMV immediate early antigen

(See Appendix V).

ii) If >50% CPE - Prepare a cytospin slide of scraped cells and then perform DFA for CMV immediate early antigen.

iii) If positive, freeze shell vial supernate.

 

IV. Reporting Results

Shell Vial: Negative report: “Negative for Cytomegalovirus.”

Positive report*: “POSITIVE for Cytomegalovirus.”

 

V. Reference

1. Gleaves, Curt A. et al. Cumitech 15A “Lab Diagnosis of Viral Infections”.

American Society for Microbiology, August 1994.

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