Preservation of Viscera in Suspected Poisoning Cases

When to Preserve Viscera

1. Death is suspected to be due to poisoning (by police or doctor)
2. Deceased was intoxicated or known to use drugs
3. Cause of death not found even after full autopsy
4. Unusual smell, color, or unidentifiable material detected in stomach contents
5. Anaphylactic deaths
6. Deaths due to burns
7. Advanced decomposition
8. Accidental death involving a driver of a vehicle or machine operator
9. When the investigating officer requests it
10. When medical officer suspects poison by smell or other findings during an injury autopsy

Routine Viscera - Must Be Preserved in All Suspected Cases

Note: GI tract contents must be kept in separate bottles from solid viscera, because the relative amounts of poison in stomach vs. intestine vs. solid organs give information about time since ingestion.

Containers

• Use 1-litre capacity, clean, wide-mouthed, white glass bottles with glass stoppers
• Rubber inserts under caps must NOT be used - they extract certain poisons (chloroform, phenols)
• Clean glass containers with sulphuric acid-chromate solution, rinse with distilled water, and dry
• Polyethylene bags/containers can be used, but volatile poisons may diffuse through plastic
• For lungs or tissues to be analyzed for volatile substances, use nylon bags (impermeable to such substances)
• Blood must be collected in screw-capped bottles of ~30 mL

Preservatives

(A) Saturated Common Salt (NaCl) Solution

• Used in all cases of suspected poisoning
• Exceptions - NOT used in poisoning by:
  • Corrosive acids (except phenol/carbolic acid)
  • Corrosive alkalis
  • Corrosive sublimate (mercuric chloride)
  • Aconite

(B) Rectified Spirit (Alcohol)

• Used when saturated salt is contraindicated (corrosive acid cases, except carbolic acid)
• NOT used in suspected poisoning by:
  • Alcohol, kerosene
  • Chloroform, ether
  • Chloral hydrate
  • Formic acid, formaldehyde, acetic acid
  • Phenol
  • Phosphorus (diminishes phosphorescence)
  • Paraldehyde (organic acids and paraldehyde are soluble in alcohol)

(C) Blood Preservatives (special rules)

• Routine: 10 mg/mL sodium or potassium fluoride (inhibits glycolysis, enzyme enolase, and bacterial growth) + 3 mg potassium oxalate (anti-coagulant) per 10 mL blood
• Fluoride should also be added to urine, CSF, and vitreous humor if alcohol estimation is required, and also for cocaine, cyanide, and CO analysis
• Oxalic acid / ethylene glycol / fluoride poisoning: Use 30 mg sodium citrate instead of potassium oxalate
• CO and CN poisoning: Use airtight caps; add a layer of liquid paraffin (1-2 cm) over blood to prevent exposure to atmospheric oxygen
• Do NOT use heparin or EDTA - they interfere with detection of methanol

(D) Urine Preservatives

• 1 mL concentrated HCl, or 100 mg thymol, or 100 mg sodium fluoride per 10 mL urine
• Alternatively: equal quantity of saturated NaCl or rectified spirit; or toluene (preferred)

(E) When Preservative is NOT Necessary

1. Viscera can be analyzed within 24 hours
2. Sample can be kept in a refrigerator or ice box
3. Bones, hair, nails
4. Lungs for detecting inhaled poisons

Important: Viscera must NOT be preserved in formaldehyde - extraction of poison (especially non-volatile organic compounds) becomes very difficult.

Additional Viscera in Specific Poisonings

Instructions for Packing and Dispatch

1. Separate bottles: Stomach + small intestine (one bottle); liver + kidney (second bottle); blood separately; urine separately; brain in its own bottle
2. Processing: Stomach and intestines are opened before preservation. Liver and kidney are cut into 0.5-1 cm pieces or minced
3. Quantity of preservative should be equal to or more than the viscera in bulk
4. Fill only two-thirds of bottle capacity - to avoid bursting from gases of decomposition
5. Sealing: Stoppers well-fitted, covered with cloth, tied with tape/string, ends sealed; seal as soon as possible to prevent loss of volatile substances
6. Labelling - each bottle must carry:
   • Name, age, sex of victim
   • Autopsy number
   • Police station and crime number
   • Organs contained
   • Date and place of autopsy
   • Preservative used
   • Signature of the doctor
   • Infection warning (if contents are infectious, e.g., HIV, HBV, TB, anthrax)
7. Preservative sample: 25 mL rectified spirit or 25 g NaCl separately bottled and sent - to exclude the preservative itself as a contaminant
8. Secured transport: Sealed bottles placed in a locked, sealed box with personal/departmental seal; sample seal and signature in a separate sealed envelope
9. Documentation: Copies of inquest report, PM report, and Magistrate's authorization sent to FSL along with viscera
10. Authorization: Viscera not analyzed without written authorization from a Magistrate, or a police officer not below the rank of Deputy Superintendent
11. Chain of custody: Viscera box handed to police constable with receipt; constable delivers it personally to FSL and obtains a receipt

Why Each Organ is Preserved - Rationale

• Stomach/GI: Most poisons are ingested; highest initial concentration; helps estimate quantity and time of ingestion
• Liver: Concentrates absorbed poisons even when blood levels have declined; major metabolic organ
• Kidney: Organ of excretion; contains large amounts of poison being cleared
• Blood: Reflects systemic levels; must be from peripheral vein (central blood levels may show postmortem redistribution)
• Urine: Proof of absorption and excretion; cannot be added with evil intent as it comes from the body
• Muscle: Drug levels in muscle more accurately reflect blood levels than liver or kidney

Postmortem note: After death, cellular barriers of mucous and serous membranes break down, so substances in the GI tract can migrate to thoracic and abdominal organs - causing a false rise in apparent blood levels. This is why separate bottling of GI contents vs. solid viscera is essential.

Numbered Bottle System for Viscera Preservation

As described in FMT Reddy (The Essentials of Forensic Medicine and Toxicology, 36th Ed.)

Bottle No. 1

• Stomach and intestines are opened before being placed in the bottle
• Preservative: Saturated common salt (NaCl) solution

Bottle No. 2

• Cut into pieces of 0.5-1 cm thickness, or minced in a grinder
• Preservative: Saturated common salt (NaCl) solution

Bottle No. 3

• Preservative: 10 mg/mL sodium fluoride (inhibits glycolysis, enzyme enolase, bacterial growth) + 3 mg potassium oxalate (anticoagulant) per 10 mL blood
• Use screw-capped bottle (~30 mL)

Bottle No. 4

• Preservative: 1 mL concentrated HCl, or 100 mg thymol, or 100 mg sodium fluoride per 10 mL; or equal quantity saturated NaCl or rectified spirit; Toluene is preferred

Bottle No. 5 (Control/Blank)

• Sent separately to exclude the possibility of any poison present in the preservative itself as a contaminant

Additional Bottles (Poison-Specific)

Key Rules for All Bottles (Reddy)

1. Preservative quantity must equal or exceed the bulk of viscera
2. Fill only two-thirds of bottle capacity - space for decomposition gases
3. Stopper: Well-fitted, covered with cloth, tied with tape, ends sealed; seal immediately to prevent loss of volatile substances
4. Label must contain: name, age, sex; autopsy number; police station; crime number; organs contained; date and place of autopsy; preservative used; doctor's signature; infection warning if applicable
5. Do NOT use formaldehyde - makes extraction of poisons (especially non-volatile organics) very difficult
6. Do NOT use rubber inserts under caps - extract chloroform and phenols
7. Sealed bottles placed in a locked, sealed box with personal/departmental seal; sample seal + signature in a separate sealed envelope
8. Copies of inquest report, PM report, and Magistrate's authorization sent to FSL with the viscera
9. Analysis is done only with authorization from a Magistrate or a police officer not below the rank of Deputy Superintendent
10. Viscera box handed to police constable with receipt; constable delivers to FSL personally and obtains a receipt

Preservative Exceptions - Quick Summary

• The Essentials of Forensic Medicine and Toxicology, 36th Edition (Reddy), pp. 131-132

Tardieu Spots

Definition and Eponym

Mechanism of Formation

1. Increased venous stasis - obstruction to venous return causes congestion, raising intracapillary/venous pressure to the point of rupturing small vessels
2. Increased capillary permeability due to hypoxia - anoxia enlarges pores between endothelial cells, allowing blood (plasma and cells) to transudate into tissues

These signs develop when mechanical obstruction to breathing is maintained for about 30 seconds. (Parikh's)

Appearance

• Shape: Usually round, well-defined
• Color: Dark red (may appear purple)
• Size: Usually pinhead size (but vary - can be scanty or so numerous as to resemble a measles rash involving the skin of the face and eyelids)
• When spots aggregate, they can form larger purpura (patches of purplish discoloration)
• Better visible in fair-skinned persons and in fresh bodies
• Tend to disappear as putrefaction sets in

Classic Locations

In carotid artery obstruction: facial, cerebral, and orbital petechiae are LESS prominent than in pure venous obstruction (because arterial supply is also cut off, reducing overall blood flow and pressure). Sudden complete carotid obstruction can cause facial pallor.

In strangling: spots are more pronounced above the level of constriction of the neck.

In traumatic asphyxia: spots are more pronounced in the chest.

Subpleural Tardieu Spots (Visceral Pleura)

Tardieu Spots on Skin (Dependent Areas / Hanging)

True vs. False (Postmortem) Tardieu Spots

Gordon and Mansfield (and later Shapiro, 1955) questioned the antemortem origin of many of these spots. Zaini and Knight (1981) confirmed that only one-third of subpleural hemorrhages are true petechiae and two-thirds are false - due to intrapleural venous channels, air blisters, thickened pleural plaques, and dust pigments. They are difficult to distinguish by naked eye. (Dikshit)

Conditions Where Tardieu Spots are Seen

1. All forms of mechanical asphyxia - hanging, strangulation, smothering, drowning, traumatic asphyxia
2. Hanging - Rosen's Emergency Medicine specifically notes them as "helpful tipoffs to a hanging mechanism"
3. Terminal stages of asphyxia in any cause of death
4. Coronary deaths (differential)
5. Diseases with hemorrhagic tendency (differential) - in disease, spots are smaller, more widely distributed, and more numerous

Important Notes

• Absence of Tardieu spots does NOT exclude death from asphyxia - they are rarely prominent in drowning
• Numerous petechiae can be produced as a common autopsy artifact during reflection of scalp flaps in any type of death - of no significance
• Petechiae seen in the reflected scalp are due to tearing of small vessels during autopsy, NOT asphyxia
• Skin behind the ears is a good site to look for petechiae after blood has drained from the tissues
• When asphyxial signs including Tardieu spots are found, a close look in good light is necessary

Clarifying the Comparison

Tardieu Spots vs. Measles Rash - Differentiation

Key Point for Forensic Context

1. You are examining a dead body - measles is a disease of the living
2. Tardieu spots are petechiae (non-blanching, subepithelial hemorrhages) - confirmed by pressing a glass slide against them: they do not blanch
3. Internal serous surfaces (subpleural, subpericardial, subconjunctival) show the same petechiae - measles rash has no internal equivalent
4. The histology shows extravasated RBCs in the skin/serous surface (Tardieu), vs. inflammatory infiltrate + epidermal changes (measles)

Signs of Mechanical Asphyxia (Other Than Tardieu Spots)

Cardinal Signs (The Classical 4/5)

1. Cyanosis

• Bluish-purple discoloration of skin, lips, tongue, and mucous membranes
• Due to diminished oxygen tension in blood and a rise in reduced haemoglobin
• Becomes apparent only when at least 5 g% of reduced haemoglobin is present
• Seen prominently in the face, lips, fingernails, and organs with rich venous/capillary beds (lungs, meninges, liver, spleen, kidneys)
• In neck compression: cyanosis almost invariably follows congestion of the face because venous blood accumulates and contains much reduced haemoglobin
• Limitations:
  • Terminal cyanosis is common in many forms of death; not specific to asphyxia
  • After 24 hours postmortem, cyanosis may be entirely due to postmortem change
  • Absence of cyanosis does not exclude asphyxia

2. Congestion of Internal Organs

• Due to obstructed venous return and capillo-venous distension
• Hypoxia causes capillary dilatation → stasis → pooling of dark blood
• The vicious cycle: Asphyxia → capillary dilatation → stasis → reduced venous return to heart → reduced pulmonary blood flow → further asphyxia
• Systemic and pulmonary congestion + dilatation of right side of heart are signs of asphyxial death
• Congestion immediately above a ligature mark on the neck is of great diagnostic significance
• Face, lips, and tongue become swollen and reddened when the neck is compressed
• Limitations: Right ventricular dilatation and pulmonary congestion are common to many forms of death; not specific. Blood redistribution by gravity and rigor mortis also confounds interpretation.

3. Fluidity of Blood (Liquid, Unclotted Blood)

• Blood remains fluid and dark at autopsy; clots do not form normally
• Historically thought to be pathognomonic of asphyxia
• Now known to be due to fibrinolytic enzymes (fibrinolysin) present in the cadaver - amount depends on rapidity of death, not on the nature of death
• High agonal catecholamine levels in rapid deaths may drive fibrinolysis
• Left side of heart is empty; right side contains dark fluid blood (Reddy)
• Limitation: NOT characteristic of asphyxia specifically - can occur in any rapid death

4. Petechial Haemorrhages (Tardieu Spots)

Additional Signs Described in Detailed Classifications

5. Pulmonary Oedema

• Some degree of pulmonary oedema is common but not invariable in asphyxial deaths
• Mechanism: either due to heart failure or increased capillary permeability (debated)
• In anoxic deaths, oedema of more than minor degree indicates death was not very rapid
• Lungs should be weighed at autopsy to quantify oedema
• Limitation: Common in many modes of death; little specific value

6. Pulmonary / Submucosal Haemorrhages

• Large submucosal haemorrhages in the pharynx, especially over the dorsum of the cricoid cartilage (where the cricoid is pressed against the anterior surface of the spine)
• Due to: (a) direct trauma, and (b) rupture of the large thin-walled submucous venous plexus at this site from severe venous congestion
• Also found in a variety of deaths unconnected with trauma

7. Increased Capillary Permeability

• Results from anoxia + stasis; pores between endothelial cells enlarge
• Blood (plasma and cells) transudes into tissues and tissue spaces, leading to:
  • Gelatinous moistening of organs
  • Development of excess fluid in serous sacs (pleura, pericardium)
  • Oedema of mediastinal tissues and lungs (can develop rapidly)
  • Petechial haemorrhages (Tardieu spots) - from further capillary rupture

Summary Table

Important Caveat (DiMaio)

"The classical signs of asphyxia are visceral congestion, petechiae, cyanosis and fluidity of blood. These are nonspecific, however, and can occur in deaths from other causes."

ASPHYXIA

KSN Reddy - The Essentials of Forensic Medicine and Toxicology, 36th Edition

Definition

The term asphyxia indicates a mode of dying, rather than a cause of death.

Physiological Basis (Important Facts)

1. Normal arterial oxygen saturation: 90-100 mmHg in young/middle-aged; 60-85 mmHg in persons above 60 years. In severe to fatal asphyxia, it falls to 20-40 mmHg
2. Brain weight is only 1.4% of body weight but uses 20% of total oxygen. Nervous tissue is the first to be affected
3. In total ischemia of brain:
   • Cerebral cortex: cessation of function begins after 8-15 seconds
   • Brainstem ganglia: after 25-30 seconds
   • Irreparable damage - cortex: ~3 minutes; basal ganglia: 6-7 minutes; vagal center: 9-10 minutes
4. Thumb rule: Breathing stops within 20 seconds of cardiac arrest; heart stops within 20 minutes of stopping breathing
5. If the heart continues for several minutes after breathing stops, lung weight may increase to 450-500 g

Classification / Types of Asphyxia

(1) Mechanical Asphyxia

• (a) Closure of external respiratory orifices - smothering (hand/cloth/mud over nose & mouth)
• (b) Closure of air passages by external pressure on neck - hanging, strangulation, throttling
• (c) Closure by foreign bodies in larynx/pharynx - choking (café coronary)
• (d) Air passages filled with fluid - drowning
• (e) External compression of chest/abdomen interfering with respiratory movements - traumatic asphyxia

(2) Pathological

• Entry of oxygen prevented by disease: bronchitis, acute oedema of glottis, laryngeal spasm, tumours, abscess
• Paralysis of respiratory muscles: acute poliomyelitis

(3) Toxic

• (a) Reduced oxygen-carrying capacity of Hb - CO poisoning
• (b) Enzymatic processes blocked - cyanide poisoning (cytochrome oxidase inhibition)
• (c) Respiratory center paralyzed - opium, barbiturates, strychnine
• (d) Respiratory muscles paralyzed - gelsemium

(4) Environmental

• Insufficiency of oxygen in inspired air (enclosed places, disused refrigerator, trunk)
• Irrespirable gases in atmosphere (sewer gas, CO, CO₂)
• Exposure to high altitude

(5) Traumatic

• Pulmonary thromboembolism from femoral vein thrombosis
• Pulmonary fat embolism from fracture of long bones
• Pulmonary air embolism from incised wound of internal jugular vein
• Bilateral pneumothorax from chest/lung injuries

(6) Postural Asphyxia

(7) Iatrogenic

Pathology of Mechanical Asphyxia

• Obstruction of venous return from the brain → venous congestion
• Obstruction of lymphatic drainage
• Partial or complete obstruction of carotid arteries → cerebral ischaemia
• Pressure on carotid sinus → reflex cardiac arrest

Asphyxia → capillary dilatation → stasis → reduced venous return to heart → reduced pulmonary blood flow → deficient oxygenation → further asphyxia

1. Anoxic anoxia - no oxygen in air breathed (e.g., high altitude)
2. Stagnant anoxia - decreased blood flow, vascular blockage
3. Anaemic anoxia - reduced O₂-carrying capacity (anaemia, CO poisoning, methaemoglobinaemia)
4. Histotoxic anoxia - cells fail to use oxygen (cyanide - blocks cytochrome oxidase)

Symptoms / Stages of Mechanical Asphyxia

Stage 1 - Stage of Dyspnoea

• Excess CO₂ stimulates respiratory centre
• Respiratory rate and amplitude increase; BP and pulse rate increase
• Slight cyanosis

Stage 2 - Stage of Convulsions

• Breathing effort is mostly expiratory
• Face is deeply congested, BP raised, pulse fast
• Neck veins become swollen
• Convulsions occur, then victim becomes insensible and reflexes are abolished

Stage 3 - Stage of Exhaustion

• Respiratory centre is paralyzed
• Complete insensibility; reflexes lost; pupils widely dilated
• Breathing is gasping, mostly inspiratory with long intervals between gasps
• BP falls, muscles relax, respiration ceases → death
• Heart may continue to beat for some minutes after respiration has ceased
• Total duration: 3 to 5 minutes

Postmortem Appearances in Asphyxial Deaths

A. External Findings

• Postmortem hypostasis is well developed
• Face may be pale (slow asphyxia) or distorted, congested, cyanosed, purple, swollen, and oedematous
• Ears and fingernails are bluish
• Eyes are prominent, conjunctivae congested, pupils dilated
• Tongue is protruded in most cases
• Frothy and bloody mucus escapes from mouth and nostrils
• Postmortem discharge of semen from meatus is common (not specific to asphyxia)

B. Internal Findings

1. Cyanosis

• Bluish-purple discoloration due to diminished oxygen tension + rise in reduced haemoglobin
• Becomes apparent only when ≥ 5 g/100 mL of reduced haemoglobin is present
• Seen in skin (especially hypostatic areas), lips, ears, tip of nose, fingernails, cheeks; internally in lungs, liver, spleen, kidneys, meninges
• Methhaemoglobin and sulphhaemoglobin also cause cyanosis
• Limitation: Non-specific; common terminal sign; after 24 hours may be entirely postmortem

2. Petechial Haemorrhages - Tardieu Spots

• Most marked where capillary congestion is most prominent - above the level of obstruction in strangulation
• Size: 0.1 to 2 mm (larger = ecchymoses)
• Seen in: skin, sclerae, conjunctivae, outer and inner surfaces of eyelids, oral mucosa
• Internally: visceral pleura (interlobar fissures, around hilum), pericardium, brain (white matter), subarachnoid space, thymus (especially in infants/children)
• When on the visceral pleura = "true Tardieu spots"
• Limitation: Non-specific; can appear postmortem; seen in heart disease, meningococcal septicaemia, blood dyscrasias, coronary thrombosis, severe increase in intrathoracic pressure

3. Congestion of Internal Organs

• Due to obstructed venous return + capillo-venous distension
• Hypoxia → capillary dilatation → stasis → pooling of dark blood
• Lungs, liver, spleen, kidneys, meninges - all congested
• Systemic and pulmonary congestion + right ventricular dilatation are signs of asphyxial death
• Limitation: Non-specific; common to many forms of death

4. Fluidity of Blood

• Blood remains fluid, dark, and unclotted at autopsy
• Due to release of fibrinolysin (fibrinolytic enzymes) from endothelium - a defence mechanism to keep blood flowing during stagnation; depends on rapidity of death, not nature of death
• Left chambers of heart are empty; right side contains dark fluid blood
• Obsolete cardiac findings note (Reddy): Dilatation of the right heart chambers and fluidity of blood are now considered obsolete signs that should be disregarded - seen in any congestive death

• Right lung weight ~300 g = compatible with sudden rhythm disturbance
• Heavier lungs (450-500 g or more) = indicates cessation of respiration with heart continuing to beat for several minutes

Histological Signs of Asphyxia in Tissues

1. Partial disruption of alveolar septa with distinctive haemorrhage within the alveoli and intra-alveolar oedema fluid
2. Brick-red discoloration of nerve cells in the cerebral cortex (stained neurological sections). Pallor and vacuolar degeneration of Purkinje cells in the cerebellum
3. Vacuolar degeneration of liver cells in prolonged suffocation

Asphyxial Stigmata (Important Concept)

• Asphyxia is NOT a pathological entity and cannot be clearly recognized from morbid anatomical findings alone
• The triad of:
  1. Cyanosis
  2. Facial, palpebral, bulbar, subpleural and subepicardial petechiae
  3. Visceral congestion
  • ...are all due to raised venous pressure - merely consistent with, but not diagnostic of asphyxia from anoxic anoxia
• Reliable local indications of fatal obstructing trauma must be demonstrated to establish mechanical asphyxia
• In putrefaction, mechanical asphyxia is indicated by petechiae under eyelids, conjunctivae, sclerae, and facial skin

Variations in Intensity of Asphyxial Signs

• If heart failure occurs before respiratory failure during asphyxia, asphyxial signs may be less marked
• In many cases of foetal hypoxia, asphyxial signs are absent (e.g., sudden blockage of trachea by food, plastic bag over head, entering a space devoid of oxygen)
• Vomiting occurs due to medullary suboxia → air passages may be filled with inhaled vomit at the end of asphyxia. About 20-25% of all individuals aspirate food agonally regardless of cause of death

Delayed Deaths Following Asphyxia

• Higher cortical centres suffer first from hypoxic injury → then basal ganglia → then vital centres
• Delayed deaths follow periods of unconsciousness from anoxic cerebral damage and subsequent hypostatic pneumonia
• Other lethal sequelae: massive subcutaneous and mediastinal emphysema from tracheal/laryngeal lacerations; oedematous swelling of pharyngeal tissues/aryepiglottic folds occluding airway
• When death occurs hours, days, or weeks after the asphyxial episode, the proximate cause of death is the traumatic incident

Medicolegal Importance

1. Asphyxia may be suicidal, homicidal, or accidental
2. Most common form - hanging (almost always suicidal)
3. Most common homicidal form - strangulation
4. Cause of death must be expressed within a broad framework as "consistent with asphyxia" when findings are inconclusive
5. All four classical signs (cyanosis, Tardieu spots, congestion, fluidity of blood) are non-specific - diagnosis requires integration with scene findings and circumstances

Hydrostatic Test (Lung Float Test)

Definition and Eponym

• First described by: Scheyer in 1683
• Also known as: Raygat's test

Note: Do not confuse with Fodere's test / Static test (based on weight of lung relative to body weight) or Plouquet's test - these are different, related tests.

Basis / Principle

• Before respiration: The lungs are unexpanded, collapsed, and liver-like
  • Specific gravity = 1040 to 1050 (heavier than water) → SINK
• After respiration: Alveoli are filled with air, lungs are light and spongy
  • Specific gravity falls to ~940 (lighter than water) → FLOAT

"Even with compression, if the alveoli are aerated at all, they will still contain some residual air after squeezing." (Dikshit)

Procedure (Step-by-Step)

• Lungs are removed at autopsy with the bronchi, trachea, and larynx intact as one unit (the "pluck")

• The entire pluck is placed in a container of water
• If it floats → positive (preliminary)

• Each lung is then separated and placed in water
• Floating → positive for that lobe

• Each lung is cut into 15-20 small fragments
• Each fragment placed in water and tested
• This excludes irregular/partial aeration

• Floating fragments are squeezed between thumb and index finger under the water surface
• Air bubbles escape; if still floats → confirms residual air
• Fragments are then wrapped in cloth and squeezed with a weight
• If pieces STILL float after all this compression → positive test → residual air present → respiration has been established

• Before performing the test, a piece of liver is dropped into water as a control
• If the liver also floats (due to putrefaction), the test result is unreliable and of no use

Interpretation of Results

Differences in Lung Before and After Respiration

Related Tests

Fodere's Test / Static Test (Weight Test)

• Based on increased blood flow to lungs after respiration is established
• Before respiration: Lung weight = 1/70th of body weight
• After respiration: Lung weight = 1/35th of body weight (weight doubles)

Plouquet's Test

• Variant of static test; also uses ratio of lung weight to body weight

Fallacies of the Hydrostatic Test

A. Expanded (Respired) Lungs May SINK (False Negative)

1. Diseases such as:
   • Acute pulmonary oedema
   • Pneumonia
   • Congenital syphilis
2. Atelectasis (non-expansion) due to:
   • Feeble respiration (air not reaching alveoli)
   • Complete absorption of air from lungs by blood (if circulation continued after respiration stopped, or in asphyxia)
   • More air expelled during expiration due to recoil of lung tissue
   • Obstruction by alveolar duct membrane
3. Drowning - when birth occurs in a toilet bowl or bucket of water, the infant may die of drowning; lungs are waterlogged and may sink

B. Unexpanded (Non-respired) Lungs May FLOAT (False Positive)

1. Putrefactive gases - foetal lungs are resistant to putrefaction (due to less blood); soft greenish gas bubbles of different sizes form, which shift under pressure and sink - distinguishing feature is that putrefactive bubbles shift position on pressure and sink when squeezed
2. Artificial inflation - mouth-to-mouth resuscitation or blowing air via tube inflates the lungs partially (stomach also contains air in these cases)
3. Alcoholic fixation

When Hydrostatic Test is NOT Necessary

1. Foetus is < 180 days (not viable)
2. Foetus is a monster (fetal anomaly incompatible with life)
3. Foetus is macerated or mummified
4. Umbilical cord is separated or cicatrized (shows the child lived independently)
5. Stomach contains milk (proof of survival and feeding)
6. Whole of the thoracic contents float (already obvious)

Applications / Significance of the Test

1. If body is not putrefied and all portions float after squeezing → complete respiration established
2. If some fragments float → partial respiration occurred
3. If all pieces sink → lungs unexpanded; infant has not breathed

Medicolegal Value and Limitations (Parikh)

1. Even the slightest degree of decomposition invalidates interpretation
2. Resuscitation attempts (mouth-to-mouth) make evaluation impossible
3. The lungs of a stillborn may float while the lungs of a live-born (even those who lived for days) may sink - this paradox has been documented
4. Foetal trial breathing - some expansion of air sacs occurs in the foetus towards the end of pregnancy as amniotic fluid moves in and out of the bronchial tree; thus an undoubted stillbirth may sometimes reveal extensive alveolar expansion (Meixner found apparently respired alveoli in lung sections from a dead infant taken from the uterus of a dead mother)
5. Shapiro states it is not possible to distinguish histologically the lungs of stillborn infants from those of live-born

"The best way to seek proof of respiration is to look at, feel, and listen to the lungs." (Parikh)

Summary

Gettler's Test (Gettler Chloride Test)

Definition

First published by: Gettler in 1921

Normal Chloride Level

• Normally, chloride content is almost equal in the right and left chambers of the heart
• Normal value: approximately 600 mg per 100 mL
• A difference of ≥ 25 mg/100 mL between the two sides is considered significant and indicates drowning

Physiological Basis

Fresh Water Drowning

• Freshwater is hypotonic relative to blood
• Water is absorbed from the alveoli into the pulmonary circulation (osmosis)
• Blood reaching the left heart is diluted → chloride content is lower on the left than the right
• Blood gets diluted by as much as 72% in 3 minutes (Reddy)
• Chloride in left heart blood: up to 50% lower than normal

Salt Water Drowning

• Salt water is hypertonic relative to blood
• Water is drawn from the pulmonary circulation into the alveolar spaces (reverse osmosis)
• Blood reaching the left heart is concentrated (haemoconcentration) → chloride content is higher on the left than the right
• Volume of fluid drawn out can be up to 42% (Reddy)
• Chloride in left heart blood: increases by 30 to 40%

Interpretation

Historical Development

Other Related Blood Tests in Drowning

Plasma Specific Gravity (Reddy)

• In drowning: specific gravity of plasma from left side < right side
• In non-drowning: the reverse (left > right)

Magnesium Test (Mortiz, 1944)

• In saltwater drowning: magnesium content of the blood on the left side > right side by more than 1.25 mg/1000 mL
• More reliable than chloride for saltwater drowning

Serum Strontium (Abdallah et al., 1985; Azparren et al., 1994)

• Raised serum strontium levels in drowning deaths
• Reciprocally related to the volume of water aspirated
• Can differentiate true drowning from postmortem immersion
• In sea water drowning: difference in strontium concentration between left and right heart blood is always > 75 μg/L

Limitations / Fallacies of Gettler's Test

1. Postmortem diffusion - after death, chloride can diffuse between cardiac chambers, equalising levels and erasing the difference
2. Putrefaction - decomposition alters blood chemistry, making interpretation impossible
3. Negative results are better interpreted than positive results - because the deceased might have consumed the same water while alive (Reddy)
4. Non-specific - similar chloride shifts can occur in other conditions
5. Freimuth's finding - positive specific gravity difference between sides usually indicates death by causes other than drowning
6. Considerable doubt about reliability of all chemical tests exists across the literature (Dikshit)

Medicolegal Significance

• The test, when positive (showing significant left-right chloride difference), supports a diagnosis of drowning
• The type of drowning (fresh vs. salt water) can be inferred from the direction of the difference
• Negative test does not exclude drowning - it may simply mean the test was unreliable in that case
• It must always be corroborated with:
  • Clinical/scene findings
  • Diatom test
  • Autopsy findings (frothy fluid in airways, waterlogged lungs, washerwoman's hands, cutis anserina, etc.)
  • Plasma specific gravity
  • Strontium levels

Summary Table