Nov 21, 2022
Lumbar Puncture
- Clemens Scherzer1,2,
- Bradley Hyman3,2,
- Charles Jennings1,2
- 1Brigham and Women's Hospital;
- 2Harvard Medical School;
- 3Massachusetts General Hospital
Protocol Citation: Clemens Scherzer, Bradley Hyman, Charles Jennings 2022. Lumbar Puncture. protocols.io https://dx.doi.org/10.17504/protocols.io.q26g78x11lwz/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 18, 2021
Last Modified: May 31, 2024
Protocol Integer ID: 47407
Keywords: lumbar, puncture, ASAPCRN
Abstract
This protocol explains the Standard Operating Protocol for performing a Lumbar Puncture.
Guidelines
FREEZER STORAGE
Freezers are divided into 4 shelves, with 6 racks per shelf, and 24 boxes that can be held in each shelf. In total, 576 boxes, approximately 2,160 sample sets, can be stored in one -80°C freezer. The first three shelves are designated by visit number: Shelves A1-6 (top shelf) house samples from enrollment visits, shelves B1-6 (2nd shelf) house samples from the 1st year follow-up, and shelves C1-6 (3rd shelf) house samples from the 2nd year follow-up. Shelves D1-6 contain packed red blood cell tubes (PRBC), DNA, and RNA, extracted from blood as described in the protocols above. CSF is designated between two freezers in selected racks. Freezer storage and transactions of samples are recorded in the Freezerworks Inventory software.
Materials
MATERIALS:
Safety Lumbar Puncture Kit (SIMS mfg., 5858-A34420*163041)
Kit includes:
- Three Sponge Sticks
- Three 2 X 2 Gauze pads
- Fenestrated Paper Drape
- Lidocaine Hydrochloride USP, 1%, 5 ml ampule
- Needle-pro® device with 22g X 1 ½ in needle
- Point-Lok® device
- Plastic syringe (5 ml Luer Lock) Needle-pro® device and 25g X 5/8 in needle
- Filter straw, 5µM
- 4 way Stopcock, No Caps, Luer Slip
- Manometer
- Flexible extension unit
- Needle stick pad
- Specimen vials for cell count, protein and glucose
- Adhesive Bandage (Band-Aid)
- Povidone-Iodine solution
- Quincke Spinal Needle (20G x 3 ½ in.)
Additional materials:
- 22g. Sprotte Atraumatic Spinal Needles and Introducers PAJ-021151-30C
- 24g. Sprotte Atraumatic Spinal Needles and Introducers PAJ-121151-30A
- 13 mL transfer tubes with clear caps
- 50 mL Falcon tube
- Styrofoam transfer cooler
- Dry ice
The following “stock” items will also be used:
- Sterile gloves in correct size for person performing the LP (one plus extras for backup)
- 2 Blue pads (plus extras for backup)
- Bottle of Betadine solution (not Betadine scrub)
- Individually wrapped alcohol wipes
- Sterile 25g, 1 1/2"needle for deep infiltration of lidocaine (one, plus extras for backup)
- Sterile 3 cc syringe with 20g needle attached (in case more lidocaine is needed). Attached needle is used for drawing up lidocaine, but NOT for injecting it.
- Bottle of lidocaine (in case more lidocaine is needed)
- Sterile 4 by 4 gauze pads (extras)
- Extra adhesive bandages (Band-Aids)
- Clean washcloths and towels
Safety warnings
Please refer to Safety Data Sheets (SDS) for health and environmental hazards. Gain all required consent and experimental approvals before beginning any procedures.
Before start
*Optimum time delay between withdrawal and freezing should be within 1-2 hours per European consensus.
Ref:
CITATION
Lumbar Puncture
Lumbar Puncture
Label the collection tubes with the sample ID as appropriate.
Place aliquot tubes on dry ice prior to procedure so they are pre-cooled.
Perform lumbar puncture using the atraumatic technique, inserting the needle with the bevel in parallel to the dura fibers. If bloody, discard the first 0.5 mL to clear any blood then collect. Record time of lumbar puncture.
Collect 1 mL CSF into one 14 mL polypropylene tube at Room temperature for routine diagnostic labs.
Collect 20 mL CSF into two 10 mL syringes at Room temperature . Then, transfer all 20 ml into one 50 ml Falcon tube, mix gently by inverting 3-4 times.
Place 0.5 mL CSF in one transfer tube for routine cell count, and 0.5 mL CSF in another transfer tube for glucose and total protein. Send at Room temperature to core lab with completed requisition form.
Note
Send to local clinical lab for basic CSF analyses - must be analyzed within 4 hours of collection.
- Cell count (500 µl)
- Total protein and Glucose (500 µl)
Spin at 400 x g, Room temperature, 00:10:00 right after CSF is collected. Record time of spinning.
Time of Spinning:
Pipet 0.5 mL CSF into the remainder (34-36 aliquots) 1.5 mL pre-cooled Fisherbrand silconized polypropylene transfer tubes.
Freeze aliquots immediately on dry ice and transfer to Scherzer lab.
Identify samples with obvious blood contamination.
Scan specimens and place in -80°C freezer within 2 hours. Record time of freezing.
Time of Freezing:
Time of LP collection, spinning and freezer time will be recorded and entered into freezerworks.
Specimens will be scanned, frozen, and stored in two separate freezers at the Scherzer lab.
Citations
Teunissen CE, Petzold A, Bennett JL, Berven FS, Brundin L, Comabella M, Franciotta D, Frederiksen JL, Fleming JO, Furlan R, Hintzen RQ, Hughes SG, Johnson MH, Krasulova E, Kuhle J, Magnone MC, Rajda C, Rejdak K, Schmidt HK, van Pesch V, Waubant E, Wolf C, Giovannoni G, Hemmer B, Tumani H, Deisenhammer F. A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking.
https://doi.org/10.1212/WNL.0b013e3181c47cc2