Repeat Lumbar Puncture at 24 Versus 48 Hours After Traumatic Lumbar Puncture in Neonates
Condition: Neonate; Meningitis; Spinal Puncture
MeSH terms: Meningitis; Wounds and Injuries
Study type: Interventional

Summary: 
      Visibly traumatic as well as microtraumatic lumbar punctures (LP) are very common in the
      neonatal period. The presence of blood makes it difficult to interpret cerebro-spinal fluid
      (CSF) findings. Clinicians often perform a repeat LP in the hope that some of the red blood
      cells would have cleared by then, allowing a better interpretation of the CSF findings. There
      is no published information whether a repeat LP provides any added information to the
      original traumatic LP, and if so what is the best time to repeat an LP after a traumatic LP.
      In this randomised controlled trial (RCT), we plan to randomly allocate neonates following a
      visibly traumatic LP to either undergo a repeat LP at 24 hours or 48 hours later to determine
      which LP gives more accurate results.
    

Detailed description: 
      STUDY DESIGN An open-label Randomised Control Trial, with stratification and blocking. STUDY
      SETTING The Newborn unit and Neonatal Unit of Pediatric Emergency of Post graduate Institute
      of medical education and research (PGIMER).

      The former is a level 3 Neonatal Intensive Care Unit (NICU). STUDY PERIOD Mentioned elsewhere
      ELIGIBILITY CRITERIA Mentioned elsewhere CONSENT A parent information sheet was provided.
      Informed written consent was obtained from the parents of the patient following which
      subjects were included in the study. The consent included information about the procedure and
      study related to it. It also included the risk factors associated with and after the
      procedure. All subjects were included after obtaining wilful consent from respective parents.

      PROCEDURE Post enrolment uncorrected White blood cell (WBC), corrected WBC count, red blood
      cell (RBC) count, Glucose concentration, Protein concentration, CSF Procalcitonin
      concentration, Gram stain and culture and sensitivity of microorganism was performed.

      Post-LP the subject was randomized and repeat LP was performed either after 24 hours or 48
      hours, and uncorrected WBC, corrected WBC count, RBC count, Glucose concentration, Protein
      concentration, Gram stain, and Culture and sensitivity of microorganism was studied.

      BASELINE PARAMETERS Gestation, Birth weight, Sex, Postnatal age, Current weight, Indication
      for initial lumbar puncture in the neonate, Seniority level of the person doing the initial
      lumbar puncture, Platelet count when available before initial lumbar puncture, The
      coagulation study report, if available, before initial lumbar puncture and repeat lumbar
      puncture, Whole blood glucose measured by glucometer before the initial LP.

      Parameters measured in the initial lumbar puncture

      o Uncorrected and corrected WBC count: Cells were counted using two methods. One was in
      Neubauer chamber within 30 minutes of performing an LP. 60 μL of CSF was split in 2 Eppendorf
      tubes, labeled A and B. 27 μL of CSF in the first Eppendorf tube i.e labeled A was diluted
      with Methylene Blue using lab standard micropipettes and was used to charge the chamber. Cell
      count of both WBC and RBC were performed as standard procedure.

      Another 27 μL CSF from Eppendorf tube labeled B was diluted using 3 μL Turk's fluid with
      composed of Gentian Violet and Glacial Acetic. The Turk's fluid essentially lyses the RBCs.
      Following which one side of the chamber is charged and WBCs are calculated in the light grey
      squares (n). The WBC count per microlitre = n x 25/9. For RBC count - 3 μL of Methylene Blue
      was added to 27 μL of CSF to make 30 μL. One side of the chamber was charged and RBCs were
      counted on the dark grey squares (n). The total RBCs counted per microliter = n x 500/9. As
      mentioned WBCs and RBCs were also counted in automated machines available in the Main
      Hematology lab as well as the emergency lab. The fully automated machine analyzer is the
      Sysmex XN-1000(Sysmex-Kobe, Japan). The XN series analyzer has research mode which uses
      fluorescence flow cytometry and impedance measurements to count the number of WBCs and RBCs
      in CSF. To measure RBCs, both the optical(for lower concentration ranges) and impedance (for
      higher concentration) techniques were used. In the WBC Differential Fluorescence channel, the
      total WBC and differential WBC counts are based on flow cytometry measurements (e.g side
      scatter, forward scatter, and DNA/RNA fluorescence). Before analysis using the XN hsA
      research mode, the sample didn't require any preparation.

      Glucose concentration- Glucose concentration in CSF was estimated using a colorimetry-based
      method after enzymatic oxidation in the presence of glucose oxidase but didn't break down the
      protein. The hydrogen peroxide produced during the reaction reacts with phenol and
      4-aminophenazone to form a red-violet quiononimine dye which acts as an indicator. The
      intensity of the color that is produced is directly proportional to the glucose concentration
      and is measured at a wavelength of 505 nm. The machine model is Adiva 1800 from Siemens.

      Protein concentration- This is also a quantitative determination by a photometric color test
      performed in a Beckman Coulter analyzer. Pyrogallol red is combined with molybdate to form a
      red complex with maximum absorption at a wavelength of 470 nm. The assay is based on the
      shift absorption that occurs when the pyrogallol red-molybdate complex binds basic amino acid
      groups of protein molecules. A blue purple complex is formed with a maximum absorbance at a
      wavelength of 600 nm. The absorbance of this complex is directly proportional to the protein
      concentration in the sample.

      Procalcitonin concentration - CSF procalcitonin was determined using a two-step sandwich
      electrochemiluminescence immunoassay using biotin-labeled procalcitonin specific antibody and
      a monoclonal procalcitonin specific antibody labeled with a Ruthenium complex (Roche
      Diagnostics, Germany). 200 μL of the CSF sample was incubated at 37 degrees C with the Biotin
      and Ruthenium labeled anti- procalcitonin antibodies. In the second incubation,
      streptavidin-coated anti microparticles were added which binds the complex via the
      biotin-streptavidin interaction. The reaction mixture is aspirated into a measuring cell
      where microparticles are magnetically captured onto the surface of the electrode. Unbound
      substances are removed by washing and upon application of a voltage to the electrode,
      chemiluminescence would be induced which is measured by a photomultiplier. The measuring
      range of the assay is 2 ng/dl - 10000ng/dl with a functional sensitivity of 6ng/dl and
      analytical sensitivity of 2 ng/dl.

      Gram stain of traumatic lumbar puncture Culture and sensitivity of traumatic lumbar puncture
      As per unit policy, CSF cell count was performed within 30 minutes of LP in the microscope in
      the NICU side lab by personnel trained in CSF cell count. This was done to avoid degeneration
      of WBCs and RBCs and drop in WBC count due to delay in the analysis.

      RANDOMISATION SEQUENCE Eligible subjects were enrolled within less than 24 hours after the
      initial LP. A block randomization sequence was generated from the website
      www.randomization.com. Permuted, even-numbered, randomly varying block sizes with a 1:1
      allocation ratio were used in this study.

      ETHICS COMMITTEE APPROVAL The study was approved by The Institutional Ethics Committee of
      PGIMER, Chandigarh (INT/IEC/2019/000557) POST-RANDOMIZATION EXCLUSIONS Mentioned elsewhere
      CONCEALMENT OF ALLOCATION The random allocation sequence was concealed using serially
      numbered opaque sealed envelopes which contained slips of a paper, mentioning a group of
      randomization. After enrolment, the name of the patient was written on the envelope. Only
      after writing the name, the envelope was torn and the allocated intervention inside the
      envelope was implemented subsequently.

      INTERVENTION GROUPS Subjects were randomly allocated to 2 groups. One group consisted of
      patients where a repeat LP was done 24 hours after the initial traumatic LP. The 2nd group
      consisted of patients where LP was repeated at 48 hours after the initial traumatic LP.

      BLINDING It was not possible to blind doctors and nurses working in the NICU. However,
      Microbiology, Hematology and Biochemistry lab personnel were blinded to the group of
      allocation.

      OUTCOMES MEASURED IN REPEAT LP:

      Blood glucose before doing LP, Whether CSF sample obtained, CSF sample: whether frank blood,
      visibly blood-tinged, micro-traumatic, or nontraumatic, CSF RBC count, CSF WBC count
      (uncorrected and corrected), CSF glucose, CSF protein, CSF Gram stain, culture, and
      sensitivity. Similar to the first LP, CSF cell count was performed within 30 minutes of LP in
      the microscope in the NICU side lab by personnel trained in CSF cell count.

      REFERENCE STANDARD Definite meningitis CSF culture and/or gram stain positive Probable
      meningitis CSF procalcitonin (PCT) positive. To define CSF PCT positive, we took a cut-off of
      33 ng/dL or more. This was based upon the study by Reshi et al(18). The study was conducted
      in the Neonatal Intensive care unit of the Department of Neonatology and Pediatrics at SKIMS,
      Srinagar, over a period of 2 years (January 2014 to December 2015). The study was a
      prospective study that included infants 28 days old, who qualified LP as a part of sepsis
      workup. They defined traumatic lumbar puncture as a CSF sample with ⩾500 red blood cells/mm3,
      and leukocyte count adjustments were done at an average ratio of 500 red blood cells to 1
      white blood cells. The diagnosis of bacterial meningitis was made in neonates with sepsis
      with either positive CSF and/or blood culture or positive gram staining. They excluded
      infants who were more than 28 days old or neonates who had received antibiotics prior to
      hospital admission, neonates who had undergone recent brain surgery if another focus of
      infection was present in addition to meningitis or Viral meningitis (herpes simplex virus).
      Post exclusion criteria the study was concluded on 168 neonates. They observed, at a value of
      0.33ng/ml, CSF PCT has a sensitivity of 92%, a specificity of 87%, a positive predictive
      value (PPV) of 85.2%, and a negative predictive value (NPV) of 93%. We in our study didn't
      include other CSF conventional parameters for probable meningitis, such as CSF WBC, CSF
      Sugar, and CSF Protein for the definition of probable meningitis because we intended to study
      these tests as index tests, and hence they could not also be part of the reference standard.

      COMPARISON OF OUTCOME MEASURES Mentioned elsewhere
    

Intervention name(s): 24 hours; 48 hours
Intervention type(s): Other; Other
Intervention description(s): Performing a lumbar puncture at 24 hours after a traumatic lumbar puncture; Performing a lumbar puncture at 48 hours after a traumatic lumbar puncture
Allocation: Randomized
Intervention model: Parallel Assignment
Masking: Single (Outcomes Assessor)
Primary purpose: Diagnostic
Keywords: neonate; meningitis; lumbar puncture; traumatic