Method for the analysis of cerebrospinal fluid (CSF) samples

Abstract

The present invention provides a flow cytometry method for the detection of target cells in a cerebrospinal fluid (CSF) sample with blood contamination.

Description

[0001] Method for the analysis of cerebrospinal fluid (CSF) samples

[0002] Cerebrospinal fluid (CSF) analysis plays a critical role in diagnosing and monitoring neurological conditions and assessing drug effects targeting cells within the central nervous system (CNS). However, the likelihood of blood contamination during CSF collection procedure is high due to chances of a traumatic Lumbar Puncture (LP). Traumatic LP happens when the needle used to perform the collection procedure causes accidental bleeding in the subarachnoid space. Due to a very high concentration of white blood cells (WBCs) in the blood compared to CSF, this poses a significant challenge to accurate assessment of cells originating in CSF. Given that CSF cells are typically very low in numbers, minute traces of blood in the sample can disproportionately skew CSF cell counts, complicating data interpretation. Furthermore, this could be exacerbated by differences in the relative abundance of cell subsets in blood vs CSF. This issue becomes particularly critical in drug development and therapeutic response monitoring where precise quantification of drug effects on CSF-resident cells is essential. For drugs targeting CNS cells, it is imperative to accurately distinguish between changes in CSF cell populations and contributions from contaminating peripheral blood. Failure to do so may result in incorrect conclusions regarding drug efficacy or safety, ultimately compromising clinical decisions.

[0003] Several approaches to detect blood contamination in CSF have been explored in the past as described below:

[0004] Spectrophotometry for hemoglobin detection: In the past, spectrophotometry has been used to detect hemoglobin derivatives in CSF, which indicates blood contamination (Ann Clin Bio- chem 1987; 24: 189 - 197.

[0005] Biomarker ratios for assessing contamination: Often albumin quotient (CSF / Serum) or immunoglobulin assessments can demystify blood contamination (Front Neurol. 2019 Jun 12: 10:584).

[0006] Such approaches only elucidate whether there is blood contamination but not necessarily allowing correction for it.

[0007] On a cellular level, correction for blood contamination has been explored and one of the approaches is by manually counting the red blood cells (RBCs) in the CSF under a microscope. An elevated RBC count indicates blood contamination. Formulas or correction factors, such as the White Blood Cell (WBC) to RBC ratio in blood, are subsequently used to estimate and correct for the number of WBCs originating from the blood in the CSF sample (BMC Pediatr. 2022 Aug 16;22:488).

[0008] However, this approach involves a manual process subjected to high variability and the risk of misidentification or miscount of the target cell type. Furthermore, it does not concretely allow correction for different WBC subsets originating from blood.

[0009] Flow cytometry has been long used to analyze CSF cellular fraction and also for certain diagnoses in CSF, mainly hematological CNS malignancies. Theoretically, a flow cytometry based im-munophenotyping approach could be used to distinguish blood-derived cells from CSF cells if unique blood specific markers existed. However, so far no such markers have been described. In addition to that, there are other intricate issues with the CSF sample such as; limited volume, low cell density, low cell stability and lack of consensus protocols for processing, antibody panels and gating strategies (Nature scientific reports 2024; 14:2463).

[0010] Therefore, there is a need for real time correction for cells originating from blood in CSF due to traumatic lumbar puncture.In a first aspect, the present invention provides a flow cytometry method for the determination of cross contaminating target cells in a CSF sample which stem from a blood contamination in the CSF sample, the method comprising:

[0011] detecting red blood cells (RBC) in the cerebrospinal fluid (CSF) sample by a gating step based on the red blood cell scatter parameters (in the blue laser), and

[0012] calculating the number of cross contaminating target cells in the CSF sample stemming from the blood contamination based on the result of step a).

[0013] In a second aspect, the present invention provides a flow cytometry method for the detection of target cells in a cerebrospinal fluid (CSF) sample with blood contamination comprising:

[0014] detecting red blood cells (RBC) in the cerebrospinal fluid (CSF) sample by a gating step based on the red blood cell scatter parameters (in the blue laser), and

[0015] calculating the number of cross contaminating target cells in the CSF sample stemming from the blood contamination based on the result of step a).

[0016] In an embodiment of the present invention, the method further comprises the step of correcting the flow cytometry primary results of the target cells in the CSF sample by subtracting the number of target cells calculated in step b).

[0017] In an embodiment of the present invention, the CSF sample is a sample of a human individual. In an embodiment of the present invention, the calculation in step b) is based on average statistics including the average ratio of RBC : target cell ratio.

[0018] In an embodiment of the present invention, the calculation in step b) is based on the RBC : target cell ratio calculated from a blood sample taken from the human individual as the CSF sample.

[0019] In an embodiment of the present invention, the calculation in step b) involves a flow cytometric assessment of a paired blood sample using target cell lineage specific markers such as CD3 (for T-cells, CD 19 for B-cells) to get specific target cell sub-population cell counts, allowing for target cell subset-specific correction.

[0020] In an embodiment of the present invention, the target cell is a white blood cell, preferably a CD20+ B lymphocyte.

[0021] In an embodiment of the present invention, the blood sample is taken within 0 - 14 days before the CSF sample, preferably at the same day as the CSF sample.

[0022] The invention provides a flow cytometry based approach to identify and potentially correct for blood-contaminating cells in CSF. Unlike the conventional approach of counting RBCs in CSF samples manually, this approach provides advantage in terms of real time assessment of total RBCs in samples and differential assessment at sub-population level in blood and CSF (paired collection). This provides an added advantage because of the fact that the cellular composition and relative frequencies of different WBC subpopulations is very different in blood compared to CSF.

[0023] Short description of the figures:

[0024] Fig. 1 : Intact RBCs are identified in an initial gating step based on their scatter parameters as well as their scatter parameters in the blue laser line.

[0025] FSC-A stands for Forward Scatter-Area. It is a parameter used to measure the size and volume of a cell or particle as it passes through the laser beam. FSC-H stands for Forward Scatter-Height.

[0026] SSC-A stands for Side Scatter-Area. This parameter is used to provide information about the internal complexity or granularity of a cell. SSC-B-A refers to a specific fluorescence channel.Fig. 2: Comparison of expected vs calculated RBC counts at each level of spike-in (range of contamination of O.OOlpl of blood to lOpl of blood in 10ml mock CSF).

[0027] Fig. 3: Stability of RBC over time in Transfix tubes. RBC events and corresponding blood volumes were determined from day 0 to day 3 post-fixation to assess stability of RBC in TransFix tubes over time. TransFix samples were stored at 4°C for the period of stability assessment.

[0028] Fig. 4: Stability of lymphocyte subsets and monocytes in TransFix tubes was established using fresh CSF samples from patients (diagnostic suspects) for up to 3 days.

[0029] Example 1 :

[0030] Blood was collected from healthy donors. Whole blood cell count was performed using a Sys-mex hematological analyzer (in 2 out of 4 experiments) to get red blood cells (RBC), lymphocyte and monocyte counts in blood. Blood was split into two parts. Primary peripheral blood mononuclear cells (PBMCs) were harvested from one part by density gradient centrifugation and spiked into PBS+2%FBS at cell density of 5000-10000 cells per ml to achieve mock cerebrospinal fluid (CSF) at physiological cell densities. From the part-2 whole blood fraction, different volumes were spiked into mock 10ml CSF (lOOpl of spike-in volume from serial dilutions of 1:10-1:100.000, resulting in corresponding final contamination volumes of lOpl to O.OOlpl). Samples were then centrifuged, resuspended in lml-2ml of mock CSF (PBS + 2% FBS) and transferred to TransFix tubes. For stability experiments, the samples were stored at 4°C until flow cytometry staining and analysis. On the day of analysis, the sample was stained with an antibody cocktail and acquired by flow cytometry.

[0031] The method of the present invention comprises the following steps:

[0032] A potentially blood-contaminated CSF sample is measured on the Flow cytometer such as Cytek Aurora that allows for distinction of RBC based on the scatter parameter in the blue laser.

[0033] Intact RBCs are identified in an initial gating step based on their scatter parameters as well as their scatter parameters in the blue laser line. An exemplified gating strategy is depicted in Figure 1.

[0034] Event count of RBCs from the instrument are taken and this is used as a reference to calculate estimated WBCs from the blood in the sample. This can be done in several ways.

[0035] Based on average statistics and previously used correction procedures (400:1 - average RBC:WBC ratio in humans).

[0036] Another more accurate way of assessment in clinical study is to collect a blood sample at the same time as the CSF sample and use the blood cell counts (RBCs and WBCs) from the same individual at the same time for the correction.

[0037] Using flow cytometric assessment of a paired blood sample using lineage specific markers such as CD3 (for T-cells, CD 19 for B-cells) to get specific sub-population cell counts, allowing for subset-specific correction.

[0038] Cell counts are then subtracted from the total WBC (subset) count in the CSF sample to provide a corrected CSF WBC (subset) count.

[0039] Calculation for blood WBC correction in CSF in validation experiments:

[0040] Step-1 - RBC events were identified using gating steps including the blue side scatter parameter as outlined in Figure 1. An optional gating step on CD45- for RBCs to exclude any contaminating lymphocytes was performed.Step-2 - Lymphocyte and monocyte events were identified by scatter parameters and CD45 expression. Based on lymphocyte and monocyte events, the fraction of sample acquired was deduced by comparing with the number of cells in the mock CSF sample.

[0041] Step-3 - To estimate the blood volume acquired on the instrument - RBC event count on flow cytometer was normalized to RBC cell density (cells / pl) measured on Sysmex.

[0042] Step-4 - The total volume of blood contamination in the entire sample was calculated (pl of blood) by division with the fraction of sample acquired (as identified in step-2).

[0043] A comparison of measured blood spike against theoretical blood spike was done and the accuracy was within 58-137%. Refer to table-1.

[0044] Table 1: Accuracy across different levels of blood contamination

[0045]

Claims

Claims1. A flow cytometry method for the determination of cross contaminating target cells in a cerebrospinal fluid (CSF) sample which stem from a blood contamination in the CSF sample comprising:a) detecting red blood cells (RBC) in the (CSF) sample by a gating step based on the red blood cell scatter parameters, andb) calculating the number of cross contaminating target cells in the CSF sample stemming from the blood contamination based on the result of step a).

2. A flow cytometry method for the detection of target cells in a cerebrospinal fluid (CSF) sample contaminated with blood comprising:a) detecting red blood cells (RBC) in the cerebrospinal fluid (CSF) sample by a gating step based on the red blood cell scatter parameters,b) calculating the number of cross contaminating target cells in the CSF sample stemming from the blood contamination based on the result of step a).c) correcting the flow cytometry primary results of the target cells in the CSF sample by subtracting the number of target cells calculated in step b).

3. The flow cytometry method of claim 1 or 2, wherein the CSF sample is a sample of a human individual.

4. The flow cytometry method of claims 1 -3, wherein the calculation in step b) is based on average statistics including the average ratio of RBC : target cell ratio.

5. The flow cytometry method of claims 1 -4, wherein the calculation in step b) is based on the RBC : target cell ratio calculated from a blood sample taken from the human individual as the CSF sample.

6. The flow cytometry method of claims 1 - 5, wherein the calculation in step b) involves flow cytometric assessment of a paired blood sample using target cell lineage specific markers to get specific target cell sub-population cell counts, allowing for target cell subset-specific correction.

7. The flow cytometry method of claims 1 - 6, wherein the target cell is a white blood cell, preferably lymphocytes, more preferably CD20 B lymphocyte.

8. The flow cytometry method of claim 5, wherein the blood sample is taken within 0 -14 days before the CSF sample, preferably at the same day as the CSF sample.

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