CME Proceedings: Cutting edge in lab medicine
Femela M
Consultant- Pathologist, Kauvery Hospital, Radial Road, Chennai
Introduction
Kauvery Hospital, Radial Road, Chennai conducted a CME entitled “CUTTING EDGE IN LAB MEDICINE”, on September 13th 2024. It covered a wide range of topics which are practically applied by Lab professionals including Pathologists, Microbiologists, Biochemists, in day-to-day practice.
The program was conducted on a hybrid platform, catering both onsite and online participants. Over 60 delegates participated in the event.
Dr. Anil. B.G, Medical Director, Kauvery Hospital, Radial Road, welcomed the guests, speakers and delegates. Opening remarks was given by Dr. Femela. M, Consultant Pathologist, Kauvery Hospital, Radial Road. Dr. C.N. Srinivas, Group Director, Lab Medicine, Kauvery group of hospitals inaugurated the scientific session.
Session 1
Dr. Lawrence D’Cruze, Associate Professor, Department of Pathology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, discussed the “CASE STUDIES OF CNS LESIONS: ANALYZING REAL-WORLD SCENARIOS”. The session was chaired by Dr. Krish Sridhar, Director and Group Mentor, Kauvery Institute of Brain and Spine, Kauvery Hospitals, Chennai and Dr. Seethalakshmi. S, Director, Department of Lab Medicine, Mehta’s Multi Speciality Hospital, Chennai.
The excerpts from his lecture are as follows:
Main changes in the new WHO classification of CNS tumours
- 1st edition (1979) to 4th edition (2007) – Classification solely based on morphological features
- 5th edition (2021) – 22 New Entities
- 13 entities revised nomenclature
New approach to grading
Multilayered Reporting Format
WHO CNS 5 – 2021 Classification
Markers recommended for the diagnosis of diffuse gliomas in adult
IDH 1 mutation
ATRX mutation
P53 mutation
1p/19q loss
Markers used for grading diffuse gliomas
CDKN2A/B
Ki-67
Some cases were discussed.
Session 2
Dr. Anupma Kindo, Professor, Department of Microbiology, Sri Ramachandra Institute of Higher Education and Research, Porur, Chennai, discussed the “Stitch in time saves nine – Diagnosis of fungal infections”. The session was chaired by Dr. Priyadarshini Shanmugam, Lab Director, Professor and Head, Department of Microbiology, Chettinad Hospital and Research Institute, Chengalpattu District and Dr. Preethi Kabra, Senior Chief of Lab, Neuberg Diagnostics Pvt. Ltd, Chennai.
Clinical approaches for diagnosis of fungal disease
The characteristics of lesions produced by superficial and subcutaneous mycoses suggest their fungal etiology though it may resemble other diseases too.
There is no particular sign and symptom in the case of systemic mycoses. The infection is similar to bacterial, viral, or parasitic disease.
Modern imaging techniques aid in the early diagnosis of fungal disease.
Relevant samples
Systemic Mycoses
Specimen: Biopsy, pus, , urine, sputum, spinal fluid, blood, scrapings, from the edge of lesions.
The urine taken from the catheter bag and twenty-four hours’ sputum is unsatisfactory, because the commensal yeast can multiply rapidly.
Respiratory samples
- An early morning sputum sample is collected.
- Opportunistic pathogens like Candida can be present in the sputum as the oropharyngeal contaminant.
- The presence of Candida in the respiratory specimens is not clinically significant unless it is found in tissue.
Cerebrospinal Fluid
- For the culture of CSF, it should be centrifuged and the sediment is inoculated in the agar.
- CSF should be kept at room temperature or incubated at 30°C.
Blood culture
- Biphasic brain-heart infusion agar broth can be used.
- Most of the fungi can be detected within the first four days of incubation, an occasional isolate of Histoplasma capsulatum may require 10 to 14 days.
Tissue, Bone marrow and Body fluids
- From the pyogenic and the necrotic areas of the wounds, tissue specimens should be taken.
- Povidone-iodine should not be used as the chances of isolation of fungi decreases. It may be applied after the collection.
- The tissue specimen should be minced before culture. Then it should be inoculated in appropriate culture media and incubated at 37°C for 4 weeks.
- Bone marrow can be directly inoculated on media and incubated.
Urine samples
- Twenty-four hours’ urine is not necessary for fungal culture.
- If a delay is anticipated, it should be refrigerated at 4°C. It can be kept for up to 12 hours.
- Culture media should contain antibacterial agents to prevent bacterial contamination and isolate the fungi in pure form.
Vaginal samples
- Clinical features along with the direct smear of secretions help in the diagnosis of vaginal candidiasis.
- About 20 % of healthy females have yeasts as the normal flora, so culture can be misleading too.
Stool culture
- Biopsy of tissue is the specimen of choice rather than the culture of stool specimens for the diagnosis of fungal infections in the gastrointestinal tract.
- Since yeast colonizes as the commensals in 40% of healthy individuals and 75% of compromised patients, cultures may grow yeast and could be misleading.
Eye
- Corneal scrapings are taken in the case of keratomycosis.
- Kimura’s spatula is used aseptically to take the sample from the base and margin of the ulcer.
- In keratomycosis, aspiration of hypopyon is done using the sterile needle.
- In the case of fungal endophthalmitis, the posterior chamber fluid may also be aspirated.
Collection and transportation of clinical samples
- The specimen should be collected aseptically, placed in sterile containers, delivered to the laboratory, preferably within 2 hours and processed within a few hours of collection.
- Prompt processing minimizes the loss in viability and enables an accurate estimation of the quantity of fungus, prevents or reduces the overgrowth of bacterial contamination.
- Swabs are not generally acceptable except in cases of ear canal, nasopharynx, vagina and cervix.
- Specimens should be transported in sterile, humidified, leak proof containers.
- Transport media are generally not suitable for transportation of samples from fungal infections.
- Specimens should be processed and inoculated on to isolation media as soon as possible.
Examination of specimen
- Select from caseous, purulent or bloody areas and necrotic material.
- Punch biopsies should be divided vertically and not horizontally so that each layer of tissue is represented in each specimen.
- Specimens other than dermatophytes should be handled in biosafety level 2.
- For evaluation of the viability of fungi, neutral red staining can be used.
Conventional (microbiological) approaches for diagnosis of fungal disease
- a) Direct Microscopic examination:
- The direct demonstration of fungi in the clinical specimen is taken as the “gold mine”.
- Fungi can be observed directly in the clinical specimens by:
- Wet Mounts
- Histopathology
- Frozen-Section Biopsy
- Fluorescent-Antibody staining
Fungal Culture
- Commonly employed medium is Emmons’ modification of Sabouraud’s Dextrose Agar.
- To inhibit the saprotrophic fungi, cycloheximide can be supplemented in the media.
- Some fungi like Cryptococcus, Talaromyces marneffei, Aspergillus, or Scytalidium are sensitive to cycloheximide. So, cycloheximide should not be used in this case.
Scotch tape
Advantages:
- It can be prepared quickly and easily.
- Slide can be preserved for a longer time.
- Fungi can be seen with their own pigmentation.
Disadvantages:
- Sample won’t be adequate if it is not pressed firmly.
Fluorescent-Antibody Staining
- It is used for the detection of fungal antigen in clinical material such as pus, blood, CSF, tissue impression smears, and in paraffin sections of formalin-fixed tissue.
- For the sputum specimen, it is less satisfactory.
- Advantage: detects fungus when few organisms are present.
Serological tests
- Beta D glucan test, Galactomannan assay, Lateral flow assay
- Presence of the polysaccharide capsular antigens of Cryptococcus neoformans in the CSF can be detected by the latex agglutination test.
Recently Developed Techniques for diagnosis of fungal disease
- AccuProbe
- PNA FISH
- MALDI-TOF Mass Spectrometry
- Quantamatrix Multiplexed Assay Platform
Histopathological diagnosis
- If histopathology shows neither the fungal elements nor the tissue reaction, the fungal isolate can be the contaminant.
- For demonstrating fungi in tissue, specific fungal stains, such as Periodic Acid-Schiff (PAS), Grocott-Gomori’s methenamine silver stain, and Gridley stains are widely used.
- For a demonstration of capsular material of Cryptococcus and endospores and sporangia of Rhinosporidium seeberi, Mayer’s mucicarmine can be used.
Frozen section
- This modality is adopted for making an intra-operative diagnosis of suspected malignancy.
- In mucormycosis and fungal rhinosinusitis, good results have been obtained.
- It is a useful tool to guide the extent of surgical debridement and/or onset of antifungal therapy.
Some cases were also discussed.
Session 3
Dr. C. N. Srinivas, Group Director, Lab Medicine, Kauvery Group of Hospitals, Tamilnadu & Karnataka, discussed the “The Versatile “M” protein in laboratory medicine – Approach to Plasma cell dyscrasias”. The session was chaired by Dr. Sandhya Phadke, Consultant Pathologist, MGM Healthcare, Chennai and Dr. Selvi Radhakrishnan, Consultant Biochemist, Neuberg Diagnostics Pvt. Ltd, Perungudi, Chennai.
Detection of paraproteins in the urine or blood is most often associated with MGUS (Monoclonal gammopathy of undetermined significance) and Multiple myeloma. Paraproteins form a narrow band, or ‘spike’ in protein electrophoresis.
Classification of monoclonal gammopathies
MGUS (Monoclonal Gammopathy of Undertermined Significance)
- Monoclonal gammopathy without any clinical or radiological abnormalities – Asymptomatic
Clinical characteristics
- Monoclonal protein concentration < 30 g/L
- Clonal bone marrow plasma cells < 10%
- Absence of hypercalcemia, creatinemia
- Normal blood smear
- In serum: Normal concentration of polyclonal Ig
- In urine: absence of Bence Jones protein or present at a low concentration
- Monoclonal protein concentration is stable over the time.
MGUS is a pre-tumoral condition. The transformation into a malignant gammopathy is dependent on:
- Isotype
- M-protein concentration
- Kappa/Lambda ratio
Smoldering Myeloma
It is an asymptomatic, very slow-growing type of myeloma.
Clinical characteristics
Monoclonal protein > 30 g/L
Or clonal bone marrow cells > 10 %
Absence of myeloma-related bone lesions or symptoms.
Multiple Myeloma
2nd most frequent bone marrow malignant disorder after leukemia, constituting 1 % of total cancers. Multiple Myeloma affects the bone marrow due to plasmocytes over-proliferation.
Guidelines – diagnosis and follow-up of MM
In case of a gammopathy:
- Serum and urine should be assessed for monoclonal protein.
- Gel or capillary protein electrophoresis is recommended.
- Immunofixation or immunotyping should be used for the typing of the monoclonal protein.
Normal profile in serum protein electrophoresis
Profile in monoclonal gammopathy
Rules for the identification of a monoclonal protein
- Presence of a peak or a distorsion in gamma
- Presence of an additionnal peak or distorsion in beta-1 or beta-2
- Beta-2 ≥ beta-1 (out of inflammatory context or hepatic disease)
- Increase of beta-1 (out of iron deficiency anemia or hemolyzed sample)
- Isolated increase of alpha-2 (out of inflammatory context).
Following the lecture, some cases were discussed.
Session 4
Dr. Debasis Gochhait, Additional Professor, Department of Pathology, JIPMER, Puducherry discussed the “Approach to Proliferative Glomerulonephritis in Pediatric Patients”. The session was chaired by Dr. Chandramouleeswari, Professor & Head, Department of Pathology, Institute of Child Health (affiliated to Madras Medical College), Egmore, Chennai and Dr. Muthu Kumar. P, Clinical Lead and Senior Consultant, Department of Nephrology, Kauvery Hospital, Radial Road, Chennai.
Syndromes & renal compartment
Nephrotic syndrome: Proteinuria
- Glomerular compartment – MCD/FSGS/MGN/DN/Amyloidosis
Nephritic syndrome: Proteinuria + RBC+ active sediments
- Glomerular compartment – Lupus nephritis, IgA, IRGN, C3 Glomerulopathy
Hematuria: only RBC
- Glomerular compartment – Alport , IgA
Renal dysfunction: Increase serum urea/creatinine
- Tubular & interstitial compartment – ATN/ Interstitial nephritis
RPRF/RPGN: Proteinuria+ active sediments + Renal dysfunction
- Glomerular+ Tubulointerstitial+ Vascular – ANCA , Anti GBM
Processing of kidney biopsy
Transportation:
Normal saline – in house
Mitchels media / Zeus medium – preserved up to 5-7 days
Routine Histopathology: H&E/ PAS
Special stains: GMS/ MT/ Congo red
IHC: if required
Direct Immunofluorescence: Cryostat and Frozen section; process as early as possible; capture images.
Electron microscopy: Thin basement membrane, Alport, Organized deposits.
Patterns of proliferation
Focal (Some):
Diffuse (All):
Segmental (Part):
Global (Full):
Mesangioproliferative:
Membranoproliferative:
Proliferative GN in pediatric patients
- Infection related GN
- IgA related nephropathy
- Lupus nephritis
- C3 Glomerulopathy
Approach to Crescentic Glomerulonephritis
Some cases were discussed following the lecture.
Session 5
Dr. Femela. M, Consultant Pathologist, Kauvery Hospital, Radial Road discussed the “Upper Gastrointestinal Cytology: Discussion of non-neoplastic and neoplastic lesions and demonstration of techniques”. The session was chaired by Dr. Ezhilvizhi Alavandar, Consultant Pathologist, Billroth Hospitals, Chennai and Dr. Vishnu Abishek. R, Lead Consultant, Institute of Gastro Sciences, Kauvery Hospital, Radial Road, Chennai.
Cytologic techniques of sampling
- Brush cytology
- Imprint cytology
- Crush cytology
- Endoscopic ultrasound guided fine needle aspiration cytology (EUS-FNAC)
- Squash cytology
- Balloon sampling
- Lavage cytology
- Salvage cytology
Neoplastic lesions diagnosed by Cytology
Epithelial neoplasms of esophagus, stomach & duodenum – benign & malignant.
Non-neoplastic lesions diagnosed by Cytology
Helicobacter pylori (H. pylori) infection
Gastric tuberculosis
Intestinal metaplasia
Barrett esophagus
Viral (eg. Herpes simplex, Cytomegalovirus) and fungal (eg. Candida) esophagitis
Radiation and chemotherapy induced changes
GI Cytology Brush
Designed in such a way that it remains ensheathed before and after the brushing is performed, in order to avoid contamination and loss of material in the endoscope channel.
Cytologic appearance of the following were discussed.
Normal GI cytology
Normal esophageal epithelium
Normal gastric epithelium
Non-neoplastic lesions
Intestinal metaplasia
Candidiasis
- pylori gastritis
Gastric ulcer
Reactive atypia of gastric epithelium
Barrett esophagus
Neoplastic lesions
Gastric adenomatous polyp
Squamous cell carcinoma – Esophagus
Adenocarcinoma – Stomach – Intestinal and Diffuse types
Radiotherapy-induced changes in a squamous cell carcinoma
Following the lecture, the cytologic techniques were demonstrated to the participants, through live mode and video presentations.
Session 6
Dr. Rajalakshmi, Clinical Embryologist, Kauvery Hospital, Radial Road, Chennai.
Demonstration by: Mr. Mahesh Kumar, National Manager, Medical Electronic Systems (India) Pvt. Ltd. discussed the “Overview and Demonstration of CASA (Computer-Assisted Semen Analysis)”. The session was chaired by Dr. Thendral, Clinical Lead, Consultant Obstetrician & Gynecologist, Kauvery Hospital, Radial Road, Chennai.
Semen analysis
Pre-examination
Patient information
Labelling, Method of collection, abstinence days, avoid using lubricants, other sperm toxic agents and handling of sample after collection.
Sample collection
Should be handed over to proper person in-charge.
Sample reception
After collection maintained at 37°C.
Initial sample handling
Look out for liquefaction, assess the sample 30 -60min after collection.
Examination
Microscopic parameters can be assessed with simple glass slide, Makler’s chamber & Neubauer’s chamber. Concentration < 16 millions/ml is Oligozoospermia.
Assessment guidelines
- Within 1 hour of sample collection, following sample liquefaction, assessment should be done.
- Examine at x200 or x400 magnification.
- Count each motility grade per field of view fast, as motility drops quickly.
- Count 200 cells twice.
- Calculate % of each motility grade out of the total cells counted in each aliquot.
Percentage of total motility
Calculating % Total motility:
No. of motile sperms = No. of total sperms – No. of immotile sperms
% Total motility = (No. of motile sperms / No. of total sperms) x100
Progressive motility
- RAPID: Moving actively in a linear motion or in a large circle with a speed of 25 μm/s.
- SLOW: Moving linearly or in large circle with 5 to < 25 μm/s.
- NON-PROGRESSIVE: All other patterns of movement, except progression < 5 μm/s.
- IMMOTILE: No signs of movement.
Morphology
Biological reference: Sperms with normal morphology should be > 4%.
Other criteria
Sperm vitality: Immotile > 60% – HOS test / E & N staining
Leucoscreen: Round cells > 5 millions/ml – Differentiates WBC & immature sperms
Fructose test: Done to check obstructive pathology.
Sperm DFI- to check integrity of sperm DNA-re-current miscarriages.
Automated Computer Assisted Sperm Quality Analysis – SQA
- Objective and Standardized equipment
- Fast and Accurate
- works as per WHO criteria
- Assess all the Parameters of Sperms in Semen Samples
- More details Parameter of Motility, Velocity and much more.
- Eliminated inter- operator variables from Manual method
- More Clinical Parameters reported
- Accurate Report will help to take appropriate Clinical Decision
- CAP accredited
SQA Technology
A testing capillary is inserted into the SQA-V optical block. Sample concentration is measured by the amount of optical absorption/reflection of light as an infra-red beam traverses the seminal fluid (containing millions of cells). Motility is measured by analyzing modulations (interruptions) in the light source caused by over 10,000 moving sperm cells. Light interruptions are converted into an electronic signals which is displayed as “peaks & valleys”. These signals are averaged and translated into motility based on a proprietary algorithm. Progressive sperms create light modulations that differ from those produced by non-progressive sperms. Immotile sperm does not create any light disturbances at all! Each category of sperms moves in a different unique way and create light modulations which are translated into distinct signals.
Following the lecture, demonstration of the CASA (Computer assisted semen analysis) technology was done.
Session 7
Interesting cases presented by Clinical Lab Technicians, Kauvery Hospital, Radial Road “Interesting cases discussion: Emphasizing the role of laboratory professionals”.
Finally, the program was concluded with vote of thanks by Dr. Femela, Consultant Pathologist, Kauvery Hospital, Radial Road.
Dr. Femela Muniraj
Consultant – Pathology
